Protist
Protist, Vol. 152, 265–300, December 2001 © Urban & Fischer Verlag
http://www.urbanfischer.de/journals/protist
ORIGINAL PAPER
Molecular Phylogeny and Taxonomic Revision
of Chlamydomonas (Chlorophyta).
I. Emendation of Chlamydomonas Ehrenberg and
Chloromonas Gobi, and Description of
Oogamochlamys gen. nov. and Lobochlamys gen. nov.1
Thomas Pröscholda,2, Birger Marina, Uwe Gert Schlösserb, and Michael Melkoniana
a
Botanisches Institut, Lehrstuhl I, Universität zu Köln, Gyrhofstr. 15, D-50931 Köln, Germany
Abteilung Experimentelle Phykologie und Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für
Pflanzenwissenschaften der Universität Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
b
Submitted March 27, 2001; Accepted September 18, 2001
Monitoring Editor: Robert A. Andersen
The genus Chlamydomonas (including Chloromonas) is one of the largest green algal genera comprising more than 600 species. To initiate a comprehensive analysis of the phylogeny and systematics
of the genus, we determined nuclear-encoded SSU rRNA sequences from 32 strains of Chlamydomonas, Chloromonas and Chlorogonium with emphasis on oogamous taxa and related strains,
and incorporated these into global molecular phylogenetic analyses of 132 strains of Chlorophyceae.
In addition, we studied the morphology and reproduction of oogamous and related strains by light
microscopy. We recognize and designate 18 monophyletic lineages (clades) within the Chlorophyceae, 11 of which are confined to the CW (basal bodies displaced clockwise) subgroup. The majority of clades recognized within the Chlorophyceae do not correspond to any of the traditional classification systems, which are still largely based on the organization level. Strains assigned to
Chlamydomonas and Chloromonas were found in seven different clades confirming the polyphyly of
the two genera as presently conceived. To initiate the taxonomic revision of Chlamydomonas, C. reinhardtii is proposed as the conserved type of the genus. In consequence, species in clades other than
the clade containing C. reinhardtii must be transferred to other genera, a process initiated in this
contribution. The oogamous strains studied represent a monophyletic lineage, which is described as
Oogamochlamys gen. nov. comprising three species (O. gigantea, O. zimbabwiensis and O. ettlii
spec. nov.). The sister clade to Oogamochlamys consists of isogamous strains characterized by
chloroplasts with incisions and is described as Lobochlamys gen. nov. with two species (L. culleus
and L. segnis). Another clade is characterized by asteroid or perforated, parietal chloroplasts and
contains the type species of Chloromonas (C. reticulata). Thus, the polyphyletic Chloromonas (traditionally defined as “Chlamydomonas without pyrenoids”) can be legitimized as a monophyletic genus
by restriction to this clade and is here emended on the basis of chloroplast characters (the clade contains strains with or without pyrenoids thus rejecting the character “absence of pyrenoids”).
1
In memory of Hanuš Ettl (1931–1997), promoter of the systematics of the genus Chlamydomonas
Corresponding author;
fax 49 221 470 5181
e-mail Thomas.Proeschold@uni-koeln.de
2
1434-4610/01/152/04-265 $ 15.00/0
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T. Pröschold et al.
Introduction
Traditionally the genus Chlamydomonas Ehrenberg
comprises all biflagellate green algae in which the
two flagella are of equal length and emerge in close
proximity to each other, and which contain a single
chloroplast with pyrenoid(s) and a cell wall (Ettl
1976). However, these characters are also found in
other taxa, and are not synapomorphic for Chlamydomonas. Ettl (1976) therefore considered the genus
Chlamydomonas as an artificial taxon, which includes all green flagellates with the characteristics
mentioned above that lack other, specialized characters. The circumscription of species in this genus
is problematic because many original descriptions
were based on light microscope observations of a
few specimens from natural samples without consideration of the variability of morphological characters within the population or of the life history. The
genetic identity of many of the Chlamydomonas
spp. described remains to be determined.
Species of Chlamydomonas have been classified
into ten subgenera or sections, characterized by the
number and position of pyrenoids within the chloroplast (Ettl 1976, 1983a; Pascher 1927). Based on a
single character, i.e. the lack of pyrenoids, several
species were transferred into a new genus,
Chloromonas Gobi emend. Wille (Ettl 1970; Gerloff
1940; Gobi 1899/1900; Wille 1903); however, this
classification has also been considered as artificial
(Ettl 1970, 1979, 1983a).
Chlamydomonas (including Chloromonas) is one
of the largest green algal genera with more than 600
species described (Ettl 1976, 1983a). Fortunately,
more than 300 strains including approximately 100
authentic strains (“type strains”) are available in culture and amenable to experimental, including
molecular analyses. Schlösser (1976, 1984), for example, examined 65 strains of Chlamydomonas for
the presence and cross-reactivity of sporangial cell
wall-lysing enzymes (v-lysins according to Adair and
Snell 1990) and established 15 vegetative lysin enzyme groups (VLE-groups) in the genus. Ettl and
Schlösser (1992) showed, based on the example of
Chlamydomonas applanata Pringsheim, that morphological and reproductive characters of a group of
strains within an autolysin group correlated well,
leading the authors to conclude that strains of a
VLE-group “can be regarded as clones of one
species”. These conclusions were corroborated by
SSU rRNA sequence comparisons of two strains of
the C. applanata group, which were identical (Gordon et al. 1995).
The polyphyly of the genus Chlamydomonas as
well as the genetic distinctness of some VLE-groups
was established by Buchheim et al. (1990, 1996,
1997a, 1997b; see also Nakayama et al. 1996)
based on phylogenetic analyses of partial or complete SSU rRNA sequences. However, a comprehensive phylogenetic analysis of the genus Chlamydomonas including related genera of the Chlorophyceae is still lacking.
In the present communication, we initiate such an
analysis by addressing the phylogenetic history of
the oogamous species of Chlamydomonas (C. gigantea Dill and C. zimbabwiensis Heimke et Starr)
and related isogamous species within the framework of related taxa. Nuclear-encoded SSU rRNA
sequences of 32 strains of Chlamydomonas, Chloromonas and Chlorogonium Ehrenberg were determined and incorporated into global phylogenetic
analyses of 132 strains of Chlorophyceae, which revealed a well-supported molecular phylogeny of the
class. Based on these results and in combination
with detailed investigations of morphology and reproduction we emend the genera Chlamydomonas
(with C. reinhardtii Dangeard as conserved type)
and Chloromonas, and propose the new genera
Oogamochlamys, and Lobochlamys.
Results
Taxon Sampling and Alignment
The initial goal of this study was to present a comprehensive analysis of the phylogeny and taxonomy
of the oogamous Chlamydomonas species (C. gigantea Dill and C. zimbabwiensis Heimke et Starr).
These species have been placed in the section
Pleiochloris based on the presence of more than
two pyrenoids in the chloroplast (Ettl 1976). This
section comprises 36 species (Ettl 1983a; Heimke
and Starr 1979), but only four of the species described are available in culture (C. carrizoensis Deason et Bold, C. rubrifilum Korshikov and the two
oogamous species; strains of C. carrizoensis and C.
rubrifilum have also been designated as C. chlorococcoides Ettl et Schwarz and Chloromonas
clathrata Korshikov, respectively; Schlösser 1994).
Therefore, we examined all available strains of C.
chlorococcoides (3) and Chloromonas clathrata (1).
Since both C. carrizoensis and C. rubrifilum have
elongated chloroplast perforations/incisions, we also
added other Chlamydomonas strains, with chloroplast incisions (including the authentic strains of C.
actinochloris Deason et Bold and two varieties of C.
augustae Skuja). Furthermore, initial molecular phylogenetic studies by Buchheim et al. (1996) suggested that C. gigantea could be related to C.
culleus Ettl (VLE-group 9) and C. segnis Ettl (VLE-
Molecular Phylogeny and Revision of Chlamydomonas
group 10; see Schlösser 1976). We therefore included several strains of these two v-lysin groups in
the analysis (9 of 17 strains available in culture).
C. gigantea is characterized by multiple (> 4) contractile vacuoles, a unique situation within Chlamydomonas; however, multiple contractile vacuoles
are also known in Haematococcus and in seven
species of Chlorogonium (Ettl 1983a; Nozaki et al.
1998). To test the phylogenetic significance of this
character, we determinated SSU rRNA sequences
of three species of Chlorogonium with multiple contractile vacuoles (Chlorogonium euchlorum Ehrenberg, Chlorogonium capillatum Nozaki, M.M.
Watanabe et Aizawa and Chlorogonium elongatum
(Dangeard) Dangeard).
Nuclear-encoded SSU rRNA sequences of 32
strains of the genera Chlamydomonas, Chloromonas and Chlorogonium (see Table 5 for a list of
strains) determined in this study were integrated into
two different alignments: (1) a large data set comprising a total of 152 taxa of the Chlorophyceae, Trebouxiophyceae, and Ulvophyceae with four prasinophyte taxa as outgroups; and (2) a smaller data set
(34 taxa) including all strains of Chlamydomonas
and Chloromonas for which sequences were obtained during this study.
Molecular Phylogenetic Analyses
Phylogenies inferred from these data sets are summarized in Figures 1 and 2; tree topologies shown
were obtained by distance (Fig. 1) or maximum likelihood analyses (Fig. 2); bootstrap percentage values
> 50% are indicated for distance (for two different
models of DNA substitution: HKY85 and TrN+I+G;
see Methods) and maximum parsimony (Fig. 1), and
for maximum likelihood, distance and parsimony
(Fig. 2) analyses. In the figures, the designation of
species of Chlamydomonas and Chloromonas follows the taxonomic rearrangements (including the
new genera Oogamochlamys and Lobochlamys) described at the end of the results.
The global analysis revealed the Ulvophyceae,
Trebouxiophyceae, and Chlorophyceae as separate
lineages within the Chlorophyta in accordance with
earlier studies (reviews by Chapman et al. 1998;
Friedl 1997; McCourt 1995; Melkonian and Surek
1995), but only the Ulvophyceae and Chlorophyceae were supported by moderate bootstrap
values in all analysis (Fig. 1). Within the 132 strains
of Chlorophyceae examined, 18 independent monophyletic clades comprising 122 strains were recognized (these are indicated in Figure 1 and provisionally named after a representative taxon). Ten strains
(marked with an asterisk in Figure 1) could not be al-
267
located to one of the 18 clades, mostly due to their
individual long branches and incomplete taxon sampling. Eleven clades belong to the so-called CW
group of Chlorophyceae (basal bodies clockw ise
displaced; Deason et al. 1991; Nakayama et al.
1996), a well-supported monophyletic lineage
(arrow with encircled CW at the node uniting this
group in Fig. 1). All Chlamydomonas species (including Chloromonas) belong to this lineage, but are distributed over seven different clades (“ Stephanosphaera” -, “ Polytoma”-, “Monadina”-, “Moewusii”-,
“ Oogamochlamys”-, “ Chloromonas”-, and “Reinhardtii”-clade) together with other genera. The polyphyly of the genera Chlamydomonas and Chloromonas was thus clearly revealed (see also previous
studies by Buchheim et al. 1996, 1997b; Hepperle et
al. 1998; Nakayama et al. 1996). The phylogenetic
relationships (i.e. the branching order) between the
11 clades of the CW-group could not be resolved in
this study. However, phylogenetic relationships of
strains within the clades were often resolved (Fig. 1).
Here, we focus on two clades, which contain most
sequences determined in this study including the
two oogamous species, namely the “ Oogamochlamys”-clade and the “ Chloromonas”-clade.
The two oogamous species of Chlamydomonas
(C. gigantea and C. zimbabwiensis) are sisters in a
monophyletic lineage (described below as Oogamochlamys gen. nov.; Fig 1). They form a larger clade
(termed “ Oogamochlamys”-clade) together with two
other Chlamydomonas species, i.e. C. culleus and
C. segnis (previously placed in the sections Chlorogoniella and Euchlamydomonas respectively), which
also form a well-supported lineage (described below
as Lobochlamys gen. nov.; Fig. 1). 17 of the 32
strains investigated belong to the “ Oogamochlamys”-clade, which was well supported in all
types of analysis (Fig. 1). The other two species of
the Pleiochloris section of Chlamydomonas investigated in this study, C. carrizoensis (strain SAG
46.72) and C. rubrifilum (strain SAG 3.85), do not belong to the “ Oogamochlamys”-clade, but are part of
another clade (the “ Chloromonas”-clade; described
below as Chloromonas in emended form) together
with other strains of Chloromonas and Chlamydomonas (the latter again previously placed in different sections). The “ Chloromonas”-clade contains 11
of the 32 strains investigated and was also well supported in all types of analyses (Fig. 1). The global
analysis thus not only revealed the polyphyly of the
genera Chlamydomonas and Chloromonas, but also
the inadequacy of the concept of the subdivision of
the genus Chlamydomonas into sections, as both
the “ Oogamochlamys”- and the “ Chloromonas”clade contain taxa from different sections. The
Molecular Phylogeny and Revision of Chlamydomonas
global analysis also showed that the character state
“multiple contractile vacuoles” is present in three
different clades (“ Stephanosphaera”-, “ Chlorogonium”-, and “ Oogamochlamys”-clade), two of which
(“ Oogamochlamys”-clade and “ Stephanosphaera”clade) also contain taxa in which motile cells have
only two contractile vacuoles, strongly suggesting
that this character has evolved independently in the
respective clades.
To further evaluate the significance of the phylogenetic inferences presented in the global analysis
(Fig. 1), user-defined trees were generated and compared with the topology shown in Figure 1 (tree 1;
“best tree”; Table 1) using Kishino-Hasegawa-tests
by calculating differences in the log-likelihood values [maximum likelihood (TrN+I+G model; see
Methods)] or in the tree lengths (maximum parsimony). In all but two analyses the user-defined trees
were significantly “worse” at the P< 0.05 level than
the “best tree” (Table 1).
Specifying a tree length of zero for the common
branch (i.e., collapsing this branch) of the Chlorophyceae (tree 2) or the common branch uniting the
CW-group (tree 3) resulted in significantly “worse”
phylogenies (Table 1), supporting the robustness of
these lineages. However, when the common branch
uniting the “ Neochloris”-, “ Hydrodictyon”-, “ Scenedesmus”- and “ Bracteacoccus”-clades [these taxa
are sometimes designated as the DO-group (orientation of basal bodies in motile cells directly opposite; Watanabe and Floyd 1989)] was collapsed, the
resulting tree topology (tree 4) using the maximum
likelihood model was not significantly worse than
the best tree (Table 1). This suggests that the phylogenetic relationships between these four clades and
the other clades (“ Chaetophora”-, “ Oedogonium”
and “ Chaetopeltis”-clade), which are not positioned
in the CW-lineage, are not resolved and require further study. User-defined trees were also generated
to test the hypotheses that the section Pleiochloris
of the genus Chlamydomonas is monophyletic (tree
269
5), or that taxa with multiple contractile vacuoles are
monophyletic (trees 6–11); both hypotheses were
rejected (Table 1). Finally, when the common branch
of a clade (“ Dunaliella”-clade sensu Buchheim et al.
1997a and Nakayama et al. 1996; in Fig. 1, this
clade consists of Dysmorphococcus globosus,
Chlamydomonas tetragama, and four clades, i.e. the
“ Dunaliella”-,“ Polytoma”-,“ Stephanosphaera”- and
“ Chlorogonium”-clades), which in our analysis received moderate bootstrap support only in the maximum parsimony analysis (Fig. 1), was collapsed
(tree 12), the resulting tree using the maximum likelihood model was not significantly worse than the
best tree (Table 1). This results suggests that the
phylogenetic relationships between the “Dunaliella” -,
“Polytoma” -, “Stephanosphaera” -, and “Chlorogonium” -clades remain currently unresolved requiring
further study.
In addition to the global phylogenetic analyses
(Fig. 1), we also performed refined phylogenetic
analyses with a reduced taxon sampling comprising
only the strains of the “ Oogamochlamys”- and
“ Chloromonas”-clades (34 strains; Fig. 2) but using
an increased number of positions (1702 compared
to 1642 positions) in the analyses. Because the phylogenetic relationships between the two clades remain unknown and no outgroup could be identified
to either of them (see Fig. 1), unrooted analyses
were performed. Figure 2 shows the maximum likelihood tree (using the TrNef+I+G model of evolution;
see Methods) with bootstrap values referring to
maximum likelihood, distance (using the TrNef+I+G
model) and maximum parsimony analyses.
Within the “ Oogamochlamys”-clade a monophyletic lineage consisting of the oogamous species
(C. gigantea and C. zimbabwiensis) was strongly
supported in all analyses (Fig. 2). Whereas the SSU
rRNAs of the three strains of C. zimbabwiensis are
almost identical (all three originated from Southern
Africa, see Table 5), there is more genetic diversity
among the other five strains. An early diverging
Figure 1. Molecular phylogeny of the Chlorophyta based on nuclear-encoded SSU rRNA sequence comparisons. Four prasinophyte flagellates were used as an outgroup for 152 taxa of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae. The strains for which SSU rRNA sequences were determined in this study are indicated in bold (for strain designations see Tables 4 and 5). The phylogenetic tree shown was inferred by the neighbor-joining method based on distances of 1,642 aligned positions calculated by the HKY85 model (Hasegawa et
al. 1985). Bootstrap percentage values (> 50% ) are given for neighbor-joining [using the HKY85 model (bold,
above branch) and the model of Tamura and Nei (TrN+I+G; not bold, above branch) with estimated shape parameter of the gamma distribution (G = 0.62) and proportion of invariable sites (I = 0.46), which was determined as the
best model for the data set by Modeltest 3.04 (Posada and Crandall 1998)] and unweighted maximum parsimony
(italics, below branch). The monophyletic clades (18) are provisionally named after a representative taxon. Ten
strains (with an asterisk) cannot yet be placed within a clade of the Chlorophyceae. CW (encircled): clade of
Chlorophyceae, in which motile cells have clockwise-displaced basal bodies.
270
T. Pröschold et al.
Figure 2. Molecular phylogeny of the “ Oogamochlamys”- and “ Chloromonas”-clades inferred by SSU rRNA sequence comparisons using 1,702 aligned positions. The unrooted tree shown resulted from a maximum likelihood
analysis [using the model of Tamura and Nei (1993) with estimated gamma shape (G = 0.63), proportions of invariable sites (I = 0.72) and equal base frequencies, TrNef+I+G, calculated as the best model by Modeltest 3.04,
(Posada and Crandall 1998)] of 34 taxa; bootstrap percentage values (> 50% ) were determined for maximum likelihood (using TrNef+I+G; bold) neighbor-joining (using TrNef+I+G; bold italics) and unweighted maximum parsimony (not bold) methods. SSU rRNA sequences determined in this study are indicated in bold, authentic strains
are marked by an asterisk. The strain numbers and the original designations of the strains are indicated as well as
the newly proposed combinations (see also Tables 4 and 5; Cd. = Chlamydomonas, Cm. = Chloromonas).
271
Molecular Phylogeny and Revision of Chlamydomonas
Table 1. Comparisons of the neighbor-joining tree in Figure 1 with user-defined trees by Kishino-Hasegawa-tests
using maximum likelihood (ML) and maximum parsimony (MP) methods.
ML (TrN+I+G)a
Tree topologyb
Diff-lnLc
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
(22322.4)f
27.4 ± 11.3
18.3 ± 8.7
9.5 ± 6.3
98.8 ± 21.8
74.8 ± 15.1
92.3 ± 17.2
52.6 ± 13.9
80.4 ± 16.5
165.6 ± 27.2
130.3 ± 25.1
4.5 ± 3.9
(best tree; Fig.1)
(collap.: Chloroph.)
(collap.: “CW”)
(collap.: “DO”)
(Ccar/Crub; Oochlam)
(Cg/Hp; Ogig)
(Hz/St; Ogig)
(Cg/Hp; Oochl/Lobo)
(Hz/St; Oochl/Lobo)
(Cg/Hp/Hz/St;Ogig)
(Cg/Hp/Hz/St;
(collap.: Dun/Buchheim)
MP
Pd
–
0.0153*
0.0357*
0.1350
<0.0001*
<0.0001*
<0.0001*
0.0002*
<0.0001*
<0.0001*
<0.0001*
0.2491
Diff. lengthe
(3789)g
7
7
5
35
27
29
16
21
57
37
6
P
–
0.0081*
0.0081*
0.0253*
<0.0001*
<0.0001*
<0.0001*
0.0025*
0.0010*
<0.0001*
<0.0001*
0.0143*
a
Maximum likelihood (ML) using the model after Tamura and Nei (1993) with estimated gamma shape and proportions of invariable sites (TrN+I+G).
b
Tree no.1 (best tree) is identical with Fig. 1; modifications in user-defined trees no. 2–12 are indicated by following abbreviations:
Tree 2: collapsed branch of the Chlorophyceae (Chloroph.)
Tree 3: collapsed branch of the “CW”-group (“CW”)
Tree 4: collapsed branch of the “DO”-group (“DO”)
Tree 5: sections Pleiochloris monophyletic (SAG 46.72 Chloromonas carrizoensis [Ccar] and SAG 3.85 C. rubrifilum [Crub] as sister to Oogamochlamys [Oochlam])
Tree 6: “ Chlorogonium”-clade (Cg/Hp) as sister to Oogamochlamys gigantea (Ogig)
Tree 7: Haematococcus zimbabwiensis and Stephanosphaera (Hz/St) as sister to Oogamochlamys gigantea (Ogig)
Tree 8: “ Chlorogonium”-clade (Cg/Hp) to the basis of the “ Oogamochlamys”-clade (Oochl/Lobo)
Tree 9: Haematococcus zimbabwiensis and Stephanosphaera (Hz/St) to the basis of the “ Oogamochlamys”-clade
(Oochl/Lobo)
Tree 10: “ Chlorogonium”-clade (Cg/Hp) and Haematococcus zimbabwiensis and Stephanosphaera (Hz/St) as
sister to Oogamochlamys gigantea (Ogig)
Tree 11: “ Chlorogonium”-clade (Cg/Hp) and Haematococcus zimbabwiensis and Stephanosphaera (Hz/St) to the
basis of the “ Oogamochlamys”-clade (Oochl/Lobo)
Tree 12: collapsed common branch of the “ Stephanosphaera”-, “ Chlorogonium”-, “ Polytoma” and “ Dunaliella”clade (including the single taxa: Chlamydomonas tetragama and Dysmorphococcus globosus; Dun/Buchheim).
c
Difference in -log-likelihood between the best tree (Fig. 1) and the user-defined tree.
d
Probability of obtaining a more extreme T-value under the null hypothesis of no difference between the two
trees (two-tailed test).
e
Difference in tree length between the best tree (Fig. 1) and the user-defined tree.
f
-log-likelihood of the best tree (Fig. 1).
g
Length of the optimal tree in the maximum parsimony analysis.
*User defined tree significantly worse than the best tree at P < 0.05.
strain (C. gigantea UTEX 2218; described below as
a new species) isolated from Zimbabwe was placed
in an intermediate position between the C. zimbabwiensis strains and the other C. gigantea strains.
The later diverging four C. gigantea strains formed
a well-supported monophyletic lineage (Fig. 2).
Among those, strain SAG 21.72, originating from
California, emerged first (Fig. 2). This strain was
a sister to the other three strains of C. gigantea,
which have almost identical SSU rRNA sequences
(although they originated from two continents;
Table 5).
A sister lineage to the oogamous Chlamydomonas species contained nine strains (here assigned to the species C. culleus and C. segnis; Fig.
2). This lineage was also well supported in all analyses (Fig. 2) but in contrast to the C. gigantea/ C. zimbabwiensis lineage did not split up into well-sup-
272
T. Pröschold et al.
ported monophyletic groups (Fig. 2). We tentatively
identify two groups of strains in this lineage: three
early diverging (in a paraphyletic topology) strains
designated C. culleus [including the authentic strain
(SAG 17.73; neotype)], and a later diverging, moderately-supported lineage of six strains, five of which
are authentic strains of previously described
Chlamydomonas species (C. segnis, C. fimbriata
Ettl, C. gymnogama Deason, C. pallidostigmatica
King, C. sajao Lewin). All six strains are here (and in
Schlösser 1994) designated as C. segnis (and described within the new genus Lobochlamys as L.
segnis, see below). Four of the six strains have almost identical SSU rRNA sequences (Fig. 2), although they originated from Europe, North America
and China (Table 5).
The “ Chloromonas”-clade consists of three wellsupported lineages (14 strains) plus three strains,
which are characterized by long branches and
therefore could not be placed with confidence in
one of the three lineages (Fig. 2). One lineage consists of seven closely related strains, which we now
designate as Chloromonas reticulata (Goroschankin)
Wille (type species of Chloromonas; see below). It
contains two strains without pyrenoids previously
listed under the genus Chloromonas [C. rosae (Ettl
H. et O.) Ettl (SAG 51.72; authentic strain) and C.
clathrata (SAG 29.83)], three strains previously designated as Chlamydomonas chlorococcoides including the authentic strain (SAG 15.82), and two
strains previously placed in other species of
Chlamydomonas [C. augustae (SAG 26.86) and C.
macrostellata Lund (SAG 72.81)]. Another lineage of
four strains is genetically more diverse: two strains
referred to Chlamydomonas actinochloris (including
the authentic strain, SAG 1.72) are monophyletic
and sister to a strain designated as Chlamydomonas
bipapillata Ettl (SAG 11-47). These three strains in
turn are a sister to the fourth strain designated as
Chlamydomonas radiata Deason et Bold (UTEX 966:
the authentic strain of this species), which is the earliest-branching taxon in this lineage (Fig. 2). The
third lineage within the “ Chloromonas”-clade is represented by three strains, which we designate as
Chlamydomonas augustae (it contains the authentic
strains of two varieties of the species: SAG 5.73 and
SAG 9.87). The phylogenetic relationships between
the three lineages within the “ Chloromonas”-clade
could not be resolved in this study (Fig. 2). The three
remaining strains of the “ Chloromonas”-clade,
which could not be significantly positioned, have
long individual branches: two species of the section
Pleiochloris, Chlamydomonas carrizoensis (SAG
46.72) and Chlamydomonas rubrifilum (SAG 3.85;
designated as Chloromonas clathrata in Schlösser
1994) as well as Chloromonas serbinowii Wille
(UTEX 492). Bootstrap support for a sister group relationship between C. rubrifilum and C. serbinowii is
moderate in all analyses (Fig. 2).
To further evaluate the phylogenetic position of
several strains in the two clades, user-defined trees
were generated and compared with the topology
shown in Figure 2 (tree 1; “best tree”; Table 2).
Kishino-Hasegawa-tests were performed by calculating differences in the log-likelihood values [maximum likelihood (TrNef+I+G model; for details see
Methods)] or in the tree lengths (maximum parsimony).
The intermediate position of strain UTEX 2218 between C. zimbabwiensis and C. gigantea (see Fig. 2)
was again evident: when UTEX 2218 was placed as
sister to C. zimbabwiensis (tree 3; Table 2) this topology, in both maximum likelihood and maximum parsimony methods, was not significantly “worse” at
the P < 0.05 level than the “best tree”. However,
when UTEX 2218 was placed as sister to the three
closely related C. gigantea strains SAG 44.91, SAG
9.84 and UTEX 1753, disrupting the C. gigantea lineage (Fig. 2), the resulting topology was significantly
“worse” in both analyses (tree 2; Table 2). This suggests that UTEX 2218 is really intermediate and cannot be placed with confidence in either C. gigantea
or C. zimbabwiensis (for morphological observations supporting these conclusions, see below and
Table 3). Both, C. gigantea and C. zimbabwiensis are
robust lineages in accordance with the high bootstrap values obtained for these lineages in all phylogenetic analyses (Fig. 2): for example, when a tree
length of zero was specified for the common branch
uniting the four C. gigantea strains, the resulting
topology was significantly “worse” than that of the
“best tree” (tree 4; Table 2).
The stability of the C. segnis lineage within the C.
segnis/ C. culleus subclade (= Lobochlamys, see
below) was probed by placing the earliest branching
strain SAG 17.72 (authentic strain of “ C. fimbriata”)
of this lineage (Fig. 2) either as sister to the four
closely related strains SAG 1.79, SAG 2.75, SAG
9.83 and SAG 50.84 of C. segnis (tree 5; Table 2) or
as sister to the two C. culleus strains SAG 17.73 and
SAG 18.72 (tree 6; Table 2). Although this strain is
more likely positioned with C. segnis than with C.
culleus, none of the specified topologies is significantly “worse” than that of the “best tree” depicted
in Figure 2, indicating that the C. segnis lineage is
not very robust (in accordance with the only moderate bootstrap support for this lineage in all analyses;
Fig. 2). The branching order of the strains within C.
segnis remains unresolved as well. The three strains
assigned to C. culleus (SAG 18.72, SAG 64.72, SAG
273
Molecular Phylogeny and Revision of Chlamydomonas
Table 2. Comparisons of the maximum likelihood tree in Figure 2 with user-defined trees by Kishino-Hasegawatests using maximum likelihood (ML) and maximum parsimony (MP) methods.
ML (TrN+I+G)a
b
c
Tree topology
Diff-lnL
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
(4840.7)f
28.6 ± 11.6
3.2 ± 3.3
28.6 ± 11.6
2.1 ± 5.9
12.8 ± 8.3
0.9 ± 2.3
1.2 ± 2.0
71.0 ± 13.8
3.8 ± 3.4
10.3 ± 7.6
14.9 ± 7.3
7.3 ± 7.6
2.1 ± 2.6
(Best tree; Fig.2)
(UTEX 2218; Ogig minus SAG 21.72)
(UTEX 2218; Ozim)
(collap.: Ogig)
(SAG 17.72; Lseg minus SAG 52.72)
(SAG 17.72; Lcul)
(SAG 64.72; Lcul)
(SAG 64.72; Lseg)
(Cmrub; Ogig)
(Cmrub; Cmaug)
(Cmrub; Cmret)
(Cmserb; Cmret)
(Cmaug; Cmact)
(Cmcar; Cmret)
MP
P
d
–
0.0137*
0.3291
0.0137*
0.7215
0.1253
0.6797
0.5410
<0.0001*
0.2611
0.1778
0.0208*
0.1706
0.2096
Diff. lengthe
(420)g
8
1
8
0
3
0
1
38
4
9
12
7
1
P
–
0.0046*
0.6549
0.0046*
1.0000
0.0833
1.0000
0.3175
<0.0001*
0.2060
0.0389*
0.0073*
0.0896
0.6549
a
Maximum likelihood (ML) using the model after Tamura and Nei (1993) with estimated gamma shape, proportions
of invariable sites and equal base frequencies (TrNef+I+G).
b
Tree no.1 (best tree) is identical with Fig. 2; modifications in user-defined trees no. 2–14 are indicated by following abbreviations:
Tree 2: Oogamochlamys ettlii (strain UTEX 2218) as sister to the three Oogamochlamys gigantea strains SAG
44.91, SAG 9.84 and UTEX 1753 (Ogig minus SAG 21.72),
Tree 3: Oogamochlamys ettlii (strain UTEX 2218) as sister to Oogamochlamys zimbabwiensis (Ozim),
Tree 4: collapsed branch of Oogamochlamys gigantea (strains SAG 44.91, SAG 9.84, UTEX 1753 and SAG 21.72; Ogig),
Tree 5: Lobochlamys segnis (strain SAG 17.72) as sister to the four Lobochlamys segnis strains SAG 1.79, SAG
2.75, SAG 9.83 and SAG 50.84 (Lseg minus SAG 52.72),
Tree 6: Lobochlamys segnis (strain SAG 17.72) as sister to the two Lobochlamys culleus strains SAG 17.73 and
SAG 18.72 (Lcul),
Tree 7: Lobochlamys culleus (strain SAG 64.72) as sister to the two Lobochlamys culleus strains SAG 17.73 and
SAG 18.72 (Lcul),
Tree 8: Lobochlamys culleus (strain SAG 64.72) as sister to the five Lobochlamys segnis strains SAG 52.72, SAG
1.79, SAG 2.75, SAG 9.83 and SAG 50.84 (Lseg),
Tree 9: Chloromonas rubrifilum (strain SAG 3.85; Cmrub) as sister to Oogamochlamys gigantea (strains SAG
44.91, SAG 9.84, UTEX 1753 and SAG 21.72; Ogig),
Tree 10: Chloromonas rubrifilum (strain SAG 3.85; Cmrub) as sister to Chloromonas augustae (strains SAG 5.73,
SAG 13.89 and SAG 9.87; Cmaug),
Tree 11: Chloromonas rubrifilum (strain SAG 3.85; Cmrub) as sister to Chloromonas reticulata (Cmret),
Tree 12: Chloromonas serbinowii (Cmserb) as sister to Chloromonas reticulata (Cmret),
Tree 13: Chloromonas augustae (Cmaug) as sister to the group Chloromonas actinochloris (strains UTEX 578,
SAG 1.72), C. asteroidea (strain SAG 11-47), and C.radiata (strain UTEX 966) (Cmact),
Tree 14: Chloromonas carrizoensis (Cmcar) as sister to Chloromonas reticulata (Cmret).
c
Difference in -log-likelihood between the best tree (Fig. 2) and the user-defined tree.
d
Probability of obtaining a more extreme T-value under the null hypothesis of no difference between the two trees
(one-tailed test).
e
Difference in tree length between the best tree (Fig. 2) and the user-defined tree.
f
-log-likelihood of the best tree (Fig. 2).
g
Length of the optimal tree in the maximum parsimony analysis.
*User defined tree significantly worse than the best tree at P < 0.05.
17.73) formed a paraphyletic assemblage in the
topology shown in Figure 2. To test for monophyly of
the three strains, strain SAG 64.72 was placed as a
sister to the other two strains (tree 7; Table 2). In the
maximum parsimony method, this topology was as
good (zero difference in tree length) as that of the
“best tree” and in the maximum likelihood method it
was also not significantly “worse” suggesting that
monophyly of the three strains cannot be rejected.
However, when strain SAG 64.72 was placed as sis-
274
T. Pröschold et al.
ter to five strains of C. segnis excluding “C. fimbriata” (tree 8; Table 2) the resulting topology was
again not significantly “worse” than that of the “best
tree”. In conclusion, phylogenetic analyses based
on SSU rRNA sequence comparisons cannot be
used to distinguish C. culleus from C. segnis unequivocally (but see morphological observations
below).
Within the „ Chloromonas“-clade, Chlamydomonas rubrifilum formed a moderately-supported clade
with Chloromonas serbinowii (Fig. 2); however, both
sequences are characterized by long branches. To
evaluate whether a long branch attraction (LBA) artifact is responsible for their apparent relatedness, we
tested the significance of alternative topologies for
C. rubrifilum: placing C. rubrifilum as a sister of
the C. gigantea lineage (disrupting the “ Oogamochlamys”-clade and probing the monophyly of the
section Pleiochloris; tree 9; Table 2) is significantly
“worse” than the topology of the “best tree” (a difference in tree lengths of 38 steps in the maximum
parsimony method!). In contrast, when C. rubrifilum
was placed as sister to C. augustae (tree 10; Table 2)
or as sister to C. reticulata (tree 11; Table 2), both
resulting topologies were not signifcantly “worse”
than that of the “best tree” (with exception of the
C. rubrifilum/ C. reticulata lineage in the maximum
parsimony method; Table 2). Placing C. serbinowii
as sister to the C. reticulata lineage (thus probing the
traditional Chloromonas concept, i.e., absence of
pyrenoids) resulted in a significantly “worse” topology than that of the “best tree” (tree 12; Table 2). In
conclusion, the C. serbinowii/ C. rubrifilum lineage is
not robust (despite its moderate bootstrap support).
The longer individual branch of C. rubrifilum (compared to C. serbinowii) makes this strain apparently
more vulnerable to alternative topologies.
To test for the monophyly of the Chloromonas
strains with asteroid chloroplasts (tree 13; see
below) and the position of Chlamydomonas carrizoensis within the „ Chloromonas“-clade, we placed
Chlamydomonas augustae as sister to the lineage
consisting of C. actinochloris, C. asteroidea and C.
radiata (tree 13) and C. carrizoensis as sister to C.
reticulata (tree 14). Both topologies were not significantly “worse” than that of the “best tree”.
Light Microscopy of Oogamous
Chlamydomonas Species and
Comparison with Species Diagnoses
The morphology and life history of C. gigantea (five
strains; Table 5) and C. zimbabwiensis (three strains;
Table 5) have been investigated under standard culture conditions (see Methods) by light microscopy.
The results of the morphological observations on
vegetative cells (young zoospores and mature cells
at the end of the light period; see Methods) are summarized in Table 3. In general, strains fall into three
groups represented by C. gigantea (4 strains), C.
zimbabwiensis (three strains), and a single strain
(UTEX 2218) also previously designated as “ C. gigantea”. Mature cells of C. gigantea and C. zimbabwiensis differ from each other mainly in the number
of contractile vacuoles (two in C. zimbabwiensis and
up to 200 in C. gigantea; Figs 5a,m, 6m–p), cell size,
number of pyrenoids, and in the surface structure of
the parietal, massive chloroplast (Table 3). In all
strains, the chloroplast surface displays ridges/
grooves (shallow incisions), which run mostly parallel to the cell axis (Figs 5, 6a–d). In C. gigantea the
chloroplast surface is coarsely ribbed (Figs 5b,
6a–b), whereas in C. zimbabwiensis the ridges/
grooves are much finer (Fig. 5n). Interestingly, strain
UTEX 2218 occupies an intermediate position between C. gigantea and C. zimbabwiensis as the cell
size and chloroplast surface structure (Figs 5g–h,
6d) are similar to those of C. zimbabwiensis,
whereas the numbers of pyrenoids (3–8) and contractile vacuoles (> 20; Fig. 6 q–t) are more similar to
those of C. gigantea (Table 3).
Asexual reproduction in all strains investigated
starts with the resorption of the flagella. In all strains,
mostly four zoospores (rarely two or eight) were
formed (Fig. 3c, 4c). In C. gigantea (including strain
UTEX 2218) the first division step is a true transverse
division (perpendicular to the cell axis) without prior
rotation of the protoplast (Fig. 3b), whereas in C.
zimbabwiensis the protoplast first rotates by 90º
within the parental cell wall before division commences (“false transverse division” according to Ettl
1988; Fig. 4b); the second division plane in both
species is perpendicular to the first one. Pyrenoids
and eyespots did not disappear during the cell divisions. One zoospore received the parental eyespot
whereas the others formed new eyespots. Liberation of zoospores occurred after swelling of the
parental cell wall and partial dissolution at the flagellar pole (not shown).
Sexual reproduction in all strains is a homothallic
proterandric oogamy (not observed in strains SAG
21.72, and SAG 9.84). Cells of C. gigantea (except
strain UTEX 2218) differentiating into male gametangia became yellowish-green, accumulated starch
grains, dissolved the pyrenoids and divided simultaneously into four protoplasts, followed by successive divisions into 32 or 64 (rarely 128) spermatozoids (Fig. 3d,e). Spermatozoids were released by
partial autolysis of the parental cell wall at the flagellar pole (Fig. 3f). Cells of C. zimbabwiensis and strain
Molecular Phylogeny and Revision of Chlamydomonas
275
Table 3. Comparison of structural characteristics in the genus Oogamochlamys (= oogamous Chlamydomonas
species).
O. giganteaa
SAG 44.91
Figs 3, 5 a-f, 6 a, b, m-p
O. ettlii
UTEX 2218
Figs 5g-l, 6 c, d, q-t
O. zimbabwiensisb
SAG 45.91
Figs 4, 5 m-r
nearly spherical
broadly roundedcylindrical-oviform
nearly spherical
broadly roundedcylindrical-oviform
nearly spherical
broadly roundedcylindrical-oviform
Cell size
in zoospores
in mature vegetative cells
10–15 µm
30–50 × 25–35 µm
10–12 µm
16–27 × 18–22 µm
10–12 µm
15–22 × 15–20 µm
Cell wall
thin
thin
thin
Cell wall papilla
small, rounded
small, rounded
broad, rounded, two
humped or absent
Flagellar length
about as long
as the cell
about as long
as the cell
about as long
as the cell
Chloroplast shape
parietal, massive,
parietal, massive,
surface with coarse ridges surface with fine ridges
parietal, massive,
surface with very fine ridges
4–8
10–16(–20)
discontinuous,
matrix multipartite
3–6
3–8
discontinuous,
matrix multipartite
2–4
2–6
discontinuous,
matrix multipartite
pale red,
elliptic to narrowly
elongate
anterior
pale red,
elliptic to narrowly
elongate
anterior
pale red,
elliptic to narrowly
elongate or punctiform
anterior
Contractile vacuoles
2 apical +
many (>20),
distributed over the
whole cell surface
2 apical +
many (>20),
distributed over the
whole cell surface
2 apical
Nucleus position
central or
slightly anterior
central or
slightly anterior
central or
slightly anterior
Character
Cell shape
in zoospores
in mature vegetative cells
Pyrenoids
number in zoospores
in mature vegetative cells
shape
Eyespot
shape
position in the chloroplast
a
The Oogamochlamys gigantea strains SAG 21.72 and the authentic strains of Chlamydomonas megalis (SAG
9.84) and Chlamydomonas capensis (UTEX 1753) reveal the same morphological characters.
b
The Oogamochlamys zimbabwiensis strains UTEX 2213 and UTEX 2214 reveal the same morphological characters.
UTEX 2218 differentiated into male gametangia
in the same manner as in C. gigantea except that
cell divisions were successive from the beginning
(as in asexual reproduction) and not initially simultaneous as in C. gigantea (Fig. 4d–f). 16 spermatozoids were formed, which were released by partial
autolysis of the parental cell wall at the flagellar pole
as a mucilaginous package of cells (Fig. 4g). The dimensions of the teardrop-shaped, naked spermatozoids were 6–10 × 4–6 µm (C. gigantea) and 4–6 ×
3–5 µm (C. zimbabwiensis and UTEX 2218). Spermatozoids of all oogamous strains were biflagellate
(flagellar length 1.5 times the cell length) with two
contractile vacuoles, a pale-green small chloroplast
with an eyespot but without a pyrenoid and nucleolus (Fig. 3f).
Oogenesis was triggered by the presence of spermatozoids (therefore proterandric); a spermatozoid
attached near the cell wall papilla of a vegetative cell
and induced its protoplast to round up and, after
partial lysis of the wall at the flagellar pole, escape
from the cell wall (Figs 3g,h, 4h). The free protoplasts, in most cases without flagella and enclosed
by mucilage, represent functional egg cells.
Figure 4. Stages of development in Oogamochlamys zimbabwiensis, strain SAG 45.91. a vegetative cell, b divided cell after a 90° rotation of the protoplast, c sporangium with four zoospores, d immature male gametangium, e mature male gametangium, f male gametangium shortly before the liberation of spermatozoids,
g liberation of spermatozoids, h hatching of the egg, i zygote. Bar: 20 µm.
Figure 3. Light microscopy of developmental stages in Oogamochlamys gigantea (strain SAG 44.91). a vegetative cell, b first transverse division during asexual reproduction, c sporangium with four zoospores, d immature
male gametangium, e mature male gametangium, f liberation of spermatozoids, g spermatozoid attached to a
vegetative cell, h hatching of the egg, i-n maturing zygotes, i with developing secondary cell wall, j optical transverse section, k surface view, l primary and developing secondary cell wall, m mature zygote with thickened secondary cell wall and accumulation of starch grains and carotenoids, n nontransparent secondary zygote cell wall.
Bar: 20 µm.
278
T. Pröschold et al.
Figure 5. Schematic drawings of vegetative cells of Oogamochlamys spp . a–f Oogamochlamys gigantea, strain SAG
44.91, g–l Oogamochlamys ettlii spec. nov., strain UTEX 2218, m–r Oogamochlamys zimbabwiensis, strain SAG
45.91, a, g, m optical longitudinal section, b, h, n surface view of the chloroplast with ridges and many contractile
vacuoles scattered between the ridges (only in b and h), c, i, o cell in apical view, d, j, p bipartite pyrenoid from young
cells, e, k, q multipartite pyrenoids from mature cells, f, l, r multipartite pyrenoid broken apart in mechanically
pressed cells. Bar: 20 µm.
In C. gigantea (except strain UTEX 2218), the zygote formed a smooth primary wall after fertilization
(Fig. 3i), which was replaced by a thick secondary zygote wall ornamented with regular projections
(Fig. 3j,k). Zygotes contained many starch grains (Fig.
3i,j) and gradually changed their color from green to
brownish-red (Fig. 3l,m). Zygote size and cell wall ornamentation varied within each strain (Fig. 3l-n).
In C. zimbabwiensis and in strain UTEX 2218, a
smooth primary zygote wall is formed after fertilization (not shown), which was replaced by a thin secondary zygote wall without ornamentations (Fig. 4i).
Figure 6. a–l Light micrographs (Nomarski interference contrast) of chloroplast structures and contractile vacuole
function in vegetative cells of Oogamochlamys, Lobochlamys, and Chloromonas spp. c, e, g, i, k optical section,
a, b, d, f, h, j, l surface view of the chloroplasts, a, b chloroplast with coarse ridges/grooves in Oogamochlamys gigantea (in b, a zoospore), strain SAG 44.91 (fixed cells), c, d chloroplast with fine ridges/grooves in Oogamochlamys ettlii spec. nov., strain UTEX 2218 (live cells), e, f chloroplast with incisions in Lobochlamys segnis, strain
SAG 52.72 (live cells), g, h chloroplast with incisions leading to formation of lobes in Lobochlamys culleus, strain
SAG 17.73 (live cells), i, j asteroid chloroplast in Chloromonas actinochloris, strain SAG 1.72 (fixed cells), k, l parietal chloroplast with perforations in Chloromonas reticulata, strain SAG 29.83 (fixed cells), m–p Four contractile
vacuoles in diastole (black arrows) and systole (empty arrows) in Oogamochlamys gigantea, strain SAG 44.91 (live
cell), q-t Two contractile vacuoles in diastole (black arrows) and systole (empty arrows) in O. ettlii spec. nov., strain
UTEX 2218 (live cell). Bar: 20 µm.
280
T. Pröschold et al.
Zygotes stored starch grains but, in contrast to C.
gigantea, remained green. Zygote germination was
not observed and could not be induced in any oogamous strain.
Several strains, which are collectively assigned
here to C. gigantea, are authentic strains of other
Chlamydomonas species (SAG 9.84: C. megalis
Bischoff et Bold 1963; UTEX 1753: C. capensis
Pocock 1962) or have previously been determined
as C. pseudogigantea Korshikov (SAG 21.72;
Schlösser 1982) or C. gigantea (SAG 44.91;
Schlösser 1994). However, the light microscopical
examination of these strains under defined culture
conditions did not reveal any significant differences
(see above); therefore, we follow a proposal by Ettl
and Gärtner (1995) and recognize only one species,
i.e. C. gigantea (for an emended diagnosis of C. gigantea, see “Taxonomic Revisions and Diagnoses”).
For three strains of C. zimbabwiensis no nomenclatural changes at the species level are required;
nevertheless, an emended diagnosis is provided
below since the original diagnosis (Heimke and Starr
1979) did not include chloroplast characters.
Light Microscopy of Isogamous Chlamydomonas and Chloromonas Strains Studied
and Comparison with Species Diagnoses
26 isogamous strains of Chlamydomonas and
Chloromonas related to the oogamous species (see
Fig. 2) were studied by light microscopy under standard culture conditions (see Methods), their desig-
Table 4. Nomenclatural changes of Chlamydomonas and Chloromonas strains studied after taxonomic revisions
or new determinations.
Strain
SAG 1.72
SAG 34.72
SAG 5.73
SAG 13.89
SAG 9.87
SAG 11-47
SAG 46.72
SAG 47.72
SAG 26.86
SAG 29.83
SAG 72.81
SAG 15.82
SAG 16.82
SAG 12.96
SAG 51.72
SAG 3.85
SAG 17.73
SAG 18.72
SAG 64.72
SAG 52.72
SAG 17.72
SAG 2.75
SAG 9.83
SAG 50.84
SAG 1.79
SAG 44.91
SAG 9.84
UTEX 1753
SAG 21.72
UTEX 2218
SAG 45.91
UTEX 2213
UTEX 2214
(= UTEX 965)
(= UTEX 578)
(= UTEX 968)
(= UTEX 966)
(= UTEX 1969)
(= UTEX 1970)
(= UTEX 1337)
(= UTEX 1057)
(= UTEX 1059)
(= UTEX 1343)
(= UTEX 1349)
(= UTEX 1638)
(= UTEX 1905)
(= UTEX 2277)
(= UTEX 1919)
(= UTEX 1492)
(= SAG 22.98)
(= UTEX 848)
(= SAG 24.98)
Original designation in Schlösser
(1982, 1994), and Starr and Zeikus (1993)
New designation
Chlamydomonas actinochloris
Chlamydomonas mutabilis
Chlamydomonas augustae
Chlamydomonas augustae
Chlamydomonas augustae
Chlamydomonas bipapillata
Chlamydomonas chlorococcoides
Chlamydomonas radiata
Chlamydomonas augustae
Chloromonas clathrata
Chlamydomonas macrostellata
Chlamydomonas chlorococcoides
Chlamydomonas chlorococcoides
Chlamydomonas chlorococcoides
Chloromonas rosae
Chloromonas clathrata
Chlamydomonas culleus
Chlamydomonas culleus
Chlamydomonas culleus
Chlamydomonas segnis
Chlamydomonas fimbriata
Chlamydomonas gymnogama
Chlamydomonas pallidostigmatica
Chlamydomonas sajao
Chlamydomonas segnis
Chlamydomonas gigantea
Chlamydomonas gigantea
Chlamydomonas gigantea
Chlamydomonas gigantea
Chlamydomonas gigantea
Chlamydomonas zimbabwiensis
Chlamydomonas zimbabwiensis
Chlamydomonas zimbabwiensis
Chloromonas actinochloris
Chloromonas actinochloris
Chloromonas augustae
Chloromonas augustae
Chloromonas augustae
Chloromonas asteroidea
Chloromonas carrizoensis
Chloromonas radiata
Chloromonas reticulata
Chloromonas reticulata
Chloromonas reticulata
Chloromonas reticulata
Chloromonas reticulata
Chloromonas reticulata
Chloromonas reticulata
Chloromonas rubrifilum
Lobochlamys culleus
Lobochlamys culleus
Lobochlamys culleus
Lobochlamys segnis
Lobochlamys segnis
Lobochlamys segnis
Lobochlamys segnis
Lobochlamys segnis
Lobochlamys segnis
Oogamochlamys gigantea
Oogamochlamys gigantea
Oogamochlamys gigantea
Oogamochlamys gigantea
Oogamochlamys ettlii
Oogamochlamys zimbabwiensis
Oogamochlamys zimbabwiensis
Oogamochlamys zimbabwiensis
Molecular Phylogeny and Revision of Chlamydomonas
281
nation was reevaluated (Ettl 1983a), and some
nomenclatural changes are proposed (Table 4). Authentic strains were compared with the original diagnoses and descriptions.
Isogamous Strains Assigned to the
“Oogamochlamys”-clade
All six strains (including the authentic strain of C.
segnis, SAG 52.72) assigned here to C. segnis contain a cup-shaped chloroplast with a thickened basal
part extending anteriorly (Figs 6e, 7a). The chloroplast surface exhibits narrow and deep incisions running mostly parallel to the cell axis (Figs 6f, 7b). Incisions in the posterior part of the chloroplast do not
perforate the plastid but can extend toward the single pyrenoid which is located in a basal position (Figs
6e, 7a). These characteristics correspond well with
the original description of C. segnis (Ettl 1965a), especially with the additional description (Ettl 1965b).
Two strains previously designated as C. gymnogama
(SAG 2.75) and C. pallidostigmatica (SAG 9.83)
within the section Chlamydella (with lateral position
of the single pyrenoid; Ettl 1983a) revealed laterally
positioned pyrenoids only in freshly liberated
zoospores, whereas mature vegetative cells contained a centrally placed, basal pyrenoid. The chloroplast shape (tubular with a cross-bridge containing
the pyrenoid; characteristic for the section Pseudagloë) described for C. sajao (Lewin 1984) and the distinctly asteroid chloroplast described for C. fimbriata
(Ettl 1965a) were not observed in the two authentic
strains SAG 50.84 (C. sajao) and SAG 17.72 (C. fimbriata), instead both strains conformed with the diagnosis of C. segnis. Isogamous sexual reproduction
was induced in three strains (SAG 52.72, SAG 2.75
and SAG 17.72) and corresponds with the previous
description given by Deason (1967) for C.
gymnogama (authentic strain SAG 2.75). Based on
these observations the six strains investigated here
were assigned to C. segnis requiring nomenclatural
changes for C. gymnogama, C. pallidostigmatica, C.
sajao, and C. fimbriata (Table 4), and an emended diagnosis, which contains a description of the isogamous sexual reproduction.
The three strains assigned to C. culleus (including
strain SAG 17.73 designated as “authentic strain
(neotype)” in Schlösser 1994) are characterized by a
cylindrical/parietal chloroplast with prominent incisions, running predominantly parallel to the cell axis
but often also branching transversely. The incisions
traverse the chloroplast, leading to the formation of
irregular chloroplast lobes (Figs 6g,h, 7c,d). The
pyrenoid is located laterally in median/submedian
position. For the three strains no nomenclatural
changes are required.
Figure 7. Schematic drawings of vegetative cells of
Lobochlamys and Chloromonas spp. with emphasis on
chloroplast structures.
a, c, e, g, i, k, m, o optical longitudinal section,
b, d, f, h, j, l, n, p surface view of the chloroplast,
a, b Lobochlamys segnis, strain SAG 52.72,
c, d Lobochlamys culleus, strain SAG 17.73,
e, f Chloromonas actinochloris, strain SAG 1.72,
g, h Chloromonas augustae, strain SAG 5.73,
i, j Chloromonas reticulata, strain SAG 29.83,
k, l Chloromonas reticulata, strain SAG 15.82,
m, n Chloromonas carrizoensis, strain SAG 46.72,
o, p Chloromonas rubrifilum, strain SAG 3.85. Bar: 20 µm.
282
T. Pröschold et al.
Table 5. List of taxa examined in this investigation. The strains are available in the SAG (Sammlung von Algenkulturen Göttingen; Schlösser 1994) and UTEX (Culture Collection at the University of Texas at Austin; Starr and
Zeikus 1993) algal culture collections. The SSU rRNA sequences are available in the EMBL, GenBank and DDBJ
sequence databases under the accession numbers given.
Strain
species
SAG 12-2a
Chlorogonium
capillatum
SAG 12-2e
Chlorogonium
capillatum
SAG 12-2d
Chlorogonium
euchlorum
UTEX 2571
Chlorogonium
elongatum
SAG 1.72
Chloromonas
actinochloris
SAG 5.73
Chloromonas
augustae
SAG 9.87
Chloromonas
augustae
SAG 13.89
Chloromonas
augustae
SAG 46.72
Chloromonas
carrizoensis
SAG 15.82
Chloromonas
reticulata
SAG 16.82
Chloromonas
reticulata
SAG 29.83
Chloromonas
reticulata
SAG 26.86
Chloromonas
reticulata
SAG 12.96
Chloromonas
reticulata
SAG 3.85
Chloromonas
rubrifilum
Original designation
and remarks
authentic strain of
Chlamydomonas
actinochloris
authentic strain of
Chlamydomonas
augustae var. eupapillata
authentic strain of
Chlamydomonas augustae
var. ellipsoidea
isolated as
Chlamydomonas bullosa;
designated as
Chlamydomonas augustae
authentic strain of
Chlamydomonas carrizoensis
(= Chlamydomonas pyrenoidosa);
designated as Chlamydomonas
chlorococcoides
authentic strain of
Chlamydomonas
chlorococcoides
designated as
Chlamydomonas
chlorococcoides
isolated as
Chlamydomonas yellowstonensis;
designated as
Chlamydomonas augustae
isolated as Chlamydomonas
nivalis; designated as
Chlamydomonas augustae
designated as
Chlamydomonas
chlorococcoides
designated as
Chloromonas clathrata
Isolator and origin
Accession
number
E.G. Pringsheim, 1916, Germany,
Berlin-Dahlem; basin with ducks
AJ410441
E.G. Pringsheim, 1936,
Czechoslovakia, pool near Prague
AJ410442
E.G. Pringsheim, 1951, South Africa,
Cape Flats; dry mud from De Klip Vlei
AJ410443
M. Wood, 1916, USA, TX, Austin;
Biology pond
AJ410444
T.R. Deason, 1958, TX, USA;
soil from Caldwell Co.
AJ410445
H. Ettl, before 1973, Czechoslovakia;
mucous covers in swamp
AJ410452
S. Watanabe, 1979, Indonesia;
soil from paddyfield near Lampong/
Sumatra
R.W. Butcher, England;
salt marsh pool
AJ410453
AJ410454
T.R. Deason, 1958, USA, TX;
soil from Caldwell Co.
AJ410446
K. Schwarz, 1975, Yugoslavia,
Dalmatia; soil from Isle Lavsa
AJ410449
K. Schwarz, 1975, Yugoslavia,
Dalmatia; soil from Isle Lavsa
AJ410450
E. Sutton, 1968, USA, OR,
Cascade Range;
snow from Todd Lake
AJ410448
E. Sutton, 1968, USA, OR,
Cascade Mts.; on snow
AJ410447
T. Reiner, 1993, Germany;
soil from Göttinger Wald near
Mackenröder Spitze
M. Melkonian, 1976, Germany,
Pevestorf/Dannenberg;
pond under ice
AJ410451
AJ410455
Molecular Phylogeny and Revision of Chlamydomonas
283
Table 5. (Continued).
Strain
species
Original designation
and remarks
Isolator and origin
Accession
number
SAG 17.73
Lobochlamys
culleus
SAG 18.72
Lobochlamys
culleus
SAG 64.72
Lobochlamys
culleus
SAG 52.72
Lobochlamys
segnis
SAG 17.72
Lobochlamys
segnis
SAG 2.75
Lobochlamys
segnis
SAG 1.79
Lobochlamys
segnis
SAG 9.83
Lobochlamys
segnis
SAG 50.84
Lobochlamys
segnis
UTEX 2218
Oogamochlamys
ettlii
SAG 44.91
Oogamochlamys
gigantea
SAG 9.84
Oogamochlamys
gigantea
authentic strain (neotype) of
Chlamydomonas culleus
F. Hindák, 1969, Czechoslovakia;
pond in Nordmähren
AJ410461
isolated as Chlamydomonas frankii;
designated as
Chlamydomonas culleus
isolated as Chlamydomonas
elliptica var. britannica; designated
as Chlamydomonas culleus
authentic strain of
Chlamydomonas segnis
G.M. Smith, 1946, USA, FL;
field near Maxville
AJ410462
G.M. Smith, 1940, Nicaragua;
soil from hog wallow, Bluefields
AJ410463
AJ410456
authentic strain of
Chlamydomonas fimbriata
H. Ettl, 1960, Czechoslovakia;
soil from beech forest near
Winkelsdorf, Altvater-Gebirge
F. Hindák, 1962, Czechoslovakia;
Doksy pond Máchovo jezero
authentic strain of
Chlamydomonas gymnogama
T.R. Deason, 1965, USA, AL;
soil from Dauphin Island
AJ410458
designated as
Chlamydomonas segnis
S.S. Badour, 1969, Canada,
Manitoba, Delta Marsh
AJ410457
authentic strain of
Chlamydomonas
pallidostigmatica
authentic strain of
Chlamydomonas sajao
J.M. King, USA, TX, McDadeCaldwell; roadside pool
AJ410459
R.A. Lewin, 1980, China; duckwood
growing near Guangzhou, Canton
AJ410460
designated as
Chlamydomonas gigantea
J.W. Heimke and R.C.Starr, 1978,
Zimbabwe, Rhodesia; soil
AJ410469
designated as
Chlamydomonas gigantea
U.G. Schlösser, 1969, South Africa,
Cape Town; soil from Cape Flats
“De Klip”
H.W. Bischoff and H.C. Bold, 1960,
USA, TX, Llano Co.; soil from
Enchanted Rock
AJ410465
UTEX 1753
Oogamochlamys
gigantea
SAG 21.72
Oogamochlamys
gigantea
SAG 45.91
Oogamochlamys
zimbabwiensis
UTEX 2213
Oogamochlamys
zimbabwiensis
UTEX 2214
Oogamochlamys
zimbabwiensis
authentic strain of
Chlamydomonas megalis;
designated as
Chlamydomonas gigantea
authentic strain of
Chlamydomonas capensis;
designated as
Chlamydomonas gigantea
designated as
Chlamydomonas gigantea
AJ410464
AJ410466
R.C. Starr, 1968, Zimbabwe,
Rhodesia; soil
AJ410467
J. Stein, 1951, USA, CA, Lemoncove;
pond soil
AJ410468
AJ410470
authentic strain of
Chlamydomonas zimbabwiensis
U.G. Schlösser, 1969, South Africa,
Cape Town; soil from Cape Flats
“De Klip”
J.W. Heimke and R.C.Starr, 1979,
Zimbabwe, Rhodesia; soil
designated as
Chlamydomonas zimbabwiensis
J.W. Heimke and R.C.Starr, 1979,
Zimbabwe, Rhodesia; soil
AJ410472
designated as
Chlamydomonas zimbabwiensis
AJ410471
284
T. Pröschold et al.
Strains Assigned to the “Chloromonas”-clade
All seven strains assigned here to Chloromonas
reticulata contain cup-shaped, parietal and massive
chloroplasts extending almost to the flagellar poles
with distinct, irregularly branched perforations
(these traverse the chloroplast, but do not result in
the formation of chloroplast lobes; Figs 6k,l, 7i,j). In
the five strains with a pyrenoid, the perforations radiate from the laterally positioned pyrenoid mostly
parallel to the cell axis; in the two strains without a
pyrenoid (SAG 29.83 and SAG 51.72), perforations
also run parallel to the cell axis, but do not radiate
from a distinct center (Figs 6k–l, 7i–j). The morphological characteristics (except for the presence/absence of a pyrenoid) of all strains correspond well
with the description given by Ettl (1983a) for
Chloromonas reticulata. Determination of the strains
with pyrenoids (using the key provided by Ettl
1983a) was not possible, because strains could not
be assigned to one of the two sections Chlamydella
or Chlorogoniella. However, all strains could be
identified (as C. reticulata) using the Chloromonas
key in Ettl (1983a) when the character “presence of
pyrenoid” was ignored. The authentic strain of
Chloromonas rosae (SAG 51.72) was also identified
as C. reticulata because the subdivision of the
chloroplast into separate lobes was not observed in
culture. Based on these observations, it is proposed
to assign all seven strains to the species
Chloromonas reticulata and to emend its diagnosis
(Table 4 and “Taxonomic Revisions and Diagnoses”,
see below).
Chlamydomonas actinochloris (two strains: authentic culture SAG 1.72 and strain SAG 34.72,
which was misidentified as C. mutabilis; Schlösser
1982), C. bipapillata, C. radiata, and C. augustae exhibit an asteroid chloroplast with a central pyrenoid
(incisions to the central pyrenoid; Figs 6i,j, 7e–h).
Chlamydomonas carrizoensis (Fig. 7m,n), Chlamydomonas rubrifilum (Fig. 7o,p; both with several
pyrenoids) and Chloromonas serbinowii (without
pyrenoid) have a cup-shaped, parietal chloroplast
with distinct perforations. In all strains except for
Chlamydomonas carrizoensis and Chlamydomonas
rubrifilum, the observed characteristics correspond
to the original diagnoses. The authentic strain of
C. carrizoensis (SAG 46.72; mislabeled as C. chlorococcoides in Schlösser 1994 and as C. deasonii in
Ettl 1976, 1983a) contains two-four pyrenoids,
which are, however, only visible in mature vegetative
cells (C. chlorococcoides has only one pyrenoid). In
the original diagnosis (Deason and Bold 1960 –
under the illegitime designation C. pyrenoidosa; see
correction in Chantanachat and Bold 1962), al-
though referring to the same strain, the presence of
two-five pyrenoids is considered as a constant character and chloroplast perforations are not mentioned. These discrepancies require an emended diagnosis of C. carrizoensis based on observations
made during this study (see below). Strain SAG 3.85
(mislabeled as Chloromonas clathrata in Schlösser
1994) could be unambiguously identified as C. rubrifilum (Ettl 1983a) and is characterized by a parietal
chloroplast with prominent perforations and 7–11
pyrenoids (Table 4). However, pyrenoids are only visible after staining with azocarmine (see Methods).
Emendation of the Genera Chlamydomonas
and Chloromonas, and Proposal of the New
Genera Oogamochlamys and Lobochlamys
Molecular phylogenetic analyses of strains of
Chlamydomonas and Chloromonas clearly revealed
that both genera are not monophyletic and instead
are distributed over several independent lineages
(Figs 1, 2). Therefore, the classification of these genera cannot be sufficiently clarified by rearrangements at the species level alone (for taxonomy of
species see above). Instead, a comprehensive taxonomic revision at the generic level is required, including new definitions (emendations) of both genera based on monophyletic lineages.
Emendation of Chlamydomonas
Since species of Chlamydomonas form several independent lineages and are found in six major
clades of the Chlorophyceae (+ C. tetragama; see
Fig. 1), it is first necessary to define a single lineage
that includes the type of the genus, Chlamydomonas pulvisculus (O. F. Müller) Ehrenberg
(Monas pulvisculus O. F. Müller). Unfortunately, the
identity of the type is not determinable because of
the lack of detail in Müller’s drawing. Ehrenberg’s
description of the species (Ehrenberg 1838) is
equally ambiguous while his illustrations can be
tentatively referred to C. reinhardtii, C. noctigama or
species of Chlamydomonas in the section Amphichloris. This situation led Ettl (1976) to propose
C. reinhardtii as neotype of Chlamydomonas; but a
neotype may be chosen only when all material on
which the name of the taxon was based is missing.
Uncertainly of identity of a holotype species (in this
case C. pulvisculus) may be overcome only by designating a species of known identity as conserved
type. This procedure requires a formal proposal
(Pröschold and Silva, in preparation). The genus is
emended to include at most only few species in the
“Reinhardtii” -clade (see “Taxonomic Revisions and
Diagnoses”).
Molecular Phylogeny and Revision of Chlamydomonas
Oogamochlamys and Lobochlamys
As a consequence of the emendation of Chlamydomonas, those species, which are not monophyletic with C. reinhardtii, must be assigned to
other genera most of which will have to be newly described – a process, which is initiated in this contribution for the oogamous species and their isogamous relatives. For oogamous species, the new
genus Oogamochlamys is proposed (containing at
present three species: O. gigantea, O. zimbabwiensis
and O. ettlii spec. nov.). The sister clade of Oogamochlamys which contains isogamous species with incised chloroplasts is here designated as Lobochlamys (with two species: L. culleus, L. segnis; see
“Taxonomic Revisions and Diagnoses”).
Emendation of Chloromonas
Although the defining taxonomic character of the
traditional genus Chloromonas (absence of a
pyrenoid) must be rejected (strains without
pyrenoids are not monophyletic to the exclusion of
pyrenoid-bearing strains), Chloromonas can nevertheless be assigned to a single clade (i.e.,
“ Chloromonas”-clade), which contains the type
species of the genus, i.e. Chloromonas reticulata
(Ettl 1970; Wille 1903). Because this clade contains
strains with one to several pyrenoids as well as
strains without pyrenoids, Chloromonas as recognized here is redefined on the basis of chloroplast
characters (asteroid chloroplasts or cup-shaped,
parietal chloroplasts with irregularly branched perforations) and an emended diagnosis is provided (see
“Taxonomic Revisions and Diagnoses”). Chloromonas currently contains eight species: C. actinochloris, C. augustae, C. asteroidea, C. carrizoensis,
C. radiata, C. reticulata, C. rubrifilum, and C. serbinowii.
Taxonomic Revisions and Diagnoses
Chlamydomonas Ehrenberg 1833 emend.
Pröschold, Marin, Schlösser et Melkonian
Emended Diagnosis: Unicellular Chlorophyta
sensu Bremer (1985) with two flagella in clockwise
basal body orientation; cell wall thin, with or without
papilla, the single chloroplast cup-shaped with
thickened basal part, and without incisions, lobes or
perforations; one discontinuous pyrenoid in slightly
basal position of the chloroplast; eyespot in anterior
position; with two apical contractile vacuoles; nucleus in slightly anterior position.
Asexual reproduction by zoospores, rotation of the
protoplast by 90º before first cell division (“false transverse division”); total lysis of the sporangial wall be-
285
fore release of zoospores through by means of a vegetative lysin enzyme (VLE-group 1, Schlösser 1976).
Sexual reproduction by isogamy, homo- or heterothallic; zygotes not ornamented, red.
The genus is supported by molecular phylogenetic analysis using SSU rRNA sequence comparisons.
Proposed conserved type species: Chlamydomonas reinhardtii Dangeard 1888, Ann. Sci.
Nat. Bot. Sér. 7e, 7; pp. 130–136, pl. XII, fig. 29-39
(descr. et ic. prima, iconotypus).
Epitype: The strain UTEX 90 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
Chloromonas Gobi 1899/1900 emend. Pröschold,
Marin, Schlösser et Melkonian
Emended Diagnosis: Unicellular Chlorophyta
sensu Bremer (1985) with two flagella in clockwise
basal body orientation; cell wall thin with or without
papilla, the single chloroplast asteroid (with a central
pyrenoid) or cup-shaped, parietal and massive with
unbranched or branched perforations; one or more
discontinuous pyrenoids present or pyrenoid absent; eyespot in anterior position; with two apical
contractile vacuoles; nucleus in central or slightly
anterior position.
Asexual reproduction by zoospores, rotation of
the protoplast by 90º before first cell division (“false
transverse division”).
Sexual reproduction by isogamy, homo- or heterothallic; zygotes ornamented or not ornamented,
green.
The genus is supported by molecular phylogenetic analysis using SSU rRNA sequence comparisons.
Type species: Chloromonas reticulata (Goroschankin 1891) Wille 1903 emend. Pröschold,
Marin, Schlösser et Melkonian.
Chloromonas reticulata (Goroschankin 1891)
Wille 1903 emend. Pröschold, Marin, Schlösser et
Melkonian. Wille 1903, Nyt Mag. Naturvidensk. 41,
p. 150, pl. IV, fig. 27 (descr. et ic. prima, iconotypus).
Basionym: Chlamydomonas reticulata Goroschankin 1891, Bull. Soc. Imp. Nat. Moscou, N. S. 5,
p. 124–128, pl. I, fig. 1–9 (descr. et ic. prima, iconotypus).
Synonyms: Chlamydomonas clathrata (Korshikov)
Pascher 1927, Die Süßwasserflora Deutschlands,
Österreichs und der Schweiz Vol. 4, p. 305, fig. 274b
(descr. et ic. prima, iconotypus). Chloromonas
286
T. Pröschold et al.
clathrata Korshikov in Pascher 1927, Die Süßwasserflora Deutschlands, Österreichs und der Schweiz
Vol. 4, p. 305, fig. 274b. Chloromonas rosae (Ettl H.
et O.) Ettl 1963, Nova Hedwigia 6, p. 397, Chlamydomonas rosae Ettl H. et O. 1959, Arch. Protistenkd. 104, p. 99, fig. 28–30 (descr. et ic. prima,
iconotypus), authentic culture: SAG 51.72 = UTEX
1337. Chloromonas rosae (Ettl H. et O.) Ettl 1963
var. polychloris Ettl 1963, Nova Hedwigia 6, p. 396,
fig. 1 (descr. et ic. prima, iconotypus). Chlamydomonas chlorococcoides Ettl et Schwarz 1981,
Plant Syst. Evol. 138, p. 119, fig. 1, 2 (descr. et ic.
prima, iconotypus), authentic culture: SAG 15.82.
Emended Diagnosis: Cells 11–22 × 5–15 µm, ellipsoid, ellipsoid-cylindrical, cell wall thin, papilla broad,
rounded or absent, with two flagella about as long as
the cell. Chloroplast cup-shaped, parietal and massive with irregular, branched perforations, one discontinuous pyrenoid in lateral position or pyrenoid
absent; eyespot pale red, elliptic to narrowly elongate
in anterior position; two apical contractile vacuoles,
nucleus in central or slightly basal position.
Asexual reproduction by four or eight zoospores,
rotation of the protoplast by 90º before first cell division (“false transverse division”).
Sexual reproduction by isogamy; zygotes ornamented, green.
Epitype: The strain UTEX 1970 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
culture: SAG 5.73. Chlamydomonas augustae
Skuja 1943 var. planctonica Ettl 1976, Beih. Nova
Hedwigia 49, p. 404, pl. 64, fig. 1 (descr. et ic. prima,
iconotypus). Chlamydomonas augustae Skuja
1949 var. ellipsoidea S. Watanabe 1983, Arch. Protistenkd. 127, p. 231, fig. 5 (descr. et ic. prima,
iconotypus), authentic culture: SAG 9.87.
Epitype: The strain SAG 5.73 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
Chloromonas actinochloris (Deason et Bold 1960)
Pröschold, Marin, Schlösser et Melkonian comb. nov.
Chloromonas carrizoensis (Deason et Bold in
Chantanachat et Bold 1962) Pröschold, Marin,
Schlösser et Melkonian comb. nov.
Basionym: Chlamydomonas actinochloris Deason et Bold 1960, Phycol. Stud. I, p. 13, fig. 1–3
(descr. et ic. prima, iconotypus).
Epitype: The strain UTEX 965 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
Chloromonas augustae (Skuja 1943) Pröschold,
Marin, Schlösser et Melkonian comb. nov.
Basionym: Chlamydomonas augustae Skuja
1943, Arch. Protistenkd. 96, p. 370–371, pl. 10, fig.
2–24 (descr. et ic. prima, iconotypus). Chlamydomonas augustae Skuja 1943 var. eupapillata
Ettl 1976, Beih. Nova Hedwigia 49, p. 404–405, pl.
64, fig. 2 (descr. et ic. prima, iconotypus), authentic
Chloromonas asteroidea Pröschold, Marin,
Schlösser et Melkonian nom. nov. replaced synonym Chlamydomonas bipapillata (Bourrelly
1951) Ettl 1976 non Chloromonas bipapillata Ettl
1979, Beih. Nova Hedwigia 60, p. 118, pl. 25, fig. 1
(descr. et ic. prima, iconotypus)
Synonyms: Chlamydomonas bipapillata (Bourrelly
1951) Ettl 1976, Beih. Nova Hedwigia 49, p. 407–408,
pl. 57, fig. 5 (descr. et ic. prima, iconotypus).
Chlamydomons macropyrenoidosa Skuja 1927
var. bipapillata Bourrelly 1951, Hydrobiologia 3, p.
256, fig. 2: 35–37 (descr. et ic. prima iconotypus).
Epitype: The strain SAG 11-47 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
Basionym: Chlamydomonas carrizoensis Deason
et Bold in Chantanachat et Bold 1962; Deason et
Bold 1960, Phycol. Stud. I, p. 16, fig. 8–9 (descr. et
ic. prima, iconotypus).
Synonyms: Chlamydomonas pyrenoidosa Deason et Bold 1960, Phycol. Stud. I, p. 16, fig. 8–9
(descr. et ic. prima, iconotypus) non Chlamydomonas pyrenoidosa Schiller 1952, Österr. Bot.
Z. 99, p. 109–110, fig. 6 (descr. et ic. prima, iconotypus). Chlamydomonas deasonii Ettl 1976, Deason
et Bold 1960, Phycol. Stud. I, p. 16, fig. 8–9 (descr.
et ic. prima, iconotypus).
Emended Diagnosis: Cells 15–28 × 8–25 µm, ellipsoid, ellipsoid-cylindrical, cell wall thin, papilla
broad, rounded or absent, with two flagella about as
long as the cell. Chloroplast cup-shaped, parietal
and massive with irregular, branched perforations;
Molecular Phylogeny and Revision of Chlamydomonas
three to five discontinuous pyrenoids in mature
cells, irregularly distributed, one pyrenoid in
zoospores and young vegetative cells; eyespot pale
red, elliptic to narrowly elongate in anterior position;
two apical contractile vacuoles, nucleus in central or
slightly basal position.
Asexual reproduction by four or eight zoospores,
rotation of the protoplast by 90º before first cell division (“false transverse division”).
Sexual reproduction not observed.
Epitype: The strain UTEX 968 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
287
Oogamochlamys Pröschold, Marin, Schlösser et
Melkonian gen. nov.
Basionym: Chlamydomonas rubrifilum Korshikov
in Pascher 1927, Die Süßwasserflora Deutschlands,
Österreichs und der Schweiz Vol. 4, p. 288–289, fig.
251 (descr. et ic. prima, iconotypus) non Chlamydomonas rubrifilum Korshikov var. africana Bourrelly 1961.
Epitype: The strain SAG 3.85 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
Chloromonas serbinowii Wille 1903, Nyt Mag.
Naturvidensk. 41, p. 151, pl IV, fig. 28 (descr. et ic.
prima, iconotypus).
Diagnosis: Unicellular Chlorophyta sensu Bremer
(1985) with two flagella in clockwise basal body orientation; cell wall thin with or without papilla, the single
chloroplast cup-shaped, parietal and massive with
ridges/grooves running parallel to the cell axis; several discontinuous pyrenoids, irregular distributed;
eyespot in an anterior position; with only two apical
contractile vacuoles or with many (>10) additional
contractile vacuoles, distributed over the cell surface;
nucleus in central or slightly anterior position.
Asexual reproduction by zoospores, rotation of
the protoplast by 90º before first cell division (“false
transverse division”) by species with only two contractile vacuoles or transverse division by species
with many contractile vacuoles; partial lysis of the
sporangial wall before release of zoospores.
Sexual reproduction by oogamy, homothallic,
proterandric; zygotes ornamented or not ornamented, green or brownish-red.
The genus is supported by molecular phylogenetic
analyses using SSU rRNA sequence comparisons.
Latin Diagnosis: Cellulae unae viridiplantae
(Chlorophyta sensu Bremer 1985), flagellis duobus
brevioribus quam corpus cellulae provisae (positio
corporis flagellorum in sensu modi horae); papilla
praesenta vel absenta; cellula membrana tenuis;
chloroplastus unicus, depressionibus fissurisque
multis praeditus, duae vacuolae pulsantes vel multae vacuolae pulsantes inter superficies plastidis
atque membranam plasmaticum; multae pyrenoides
laterales discontinae, per chloroplastum solidum
fortuite distributae, stigmate filiformi, paululum anterior. Nucleus magnus cum nucleolum unum, paululum anterior media cellulae.
Reproductio asexualis per bipartitionem repetitum endogenam plerumque in cellulas filias.
Reproductio sexualis ovogama, spermae, ante
ova evolutae, guttiformes sine membrana, flagellae
duae corpore unus dimidiatusque longae, paulum
sub apice insertae, chloroplastus reducta sine
pyrenoide, stigmate distincta. Ova ex cellulae membrana cellulae elapsa spermis inductis. Zygota
matura levis, viridis vel spinulis ornata, porphyra.
Genus novus confirmatus per SSU rRNA sequentiae geneticae.
Type species: Oogamochlamys gigantea (Dill
1895) Pröschold, Marin, Schlösser et Melkonian
Epitype: The strain UTEX 492 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
Oogamochlamys gigantea (Dill 1895) Pröschold,
Marin, Schlösser et Melkonian comb. nov.
Basionym: Chlamydomonas gigantea Dill, 1895,
Jahrb. wiss. Bot. 28, p. 354, fig. 26–30, 34 (descr. et
ic. prima, iconotypus).
Chloromonas radiata (Deason et Bold 1960)
Pröschold, Marin, Schlösser et Melkonian comb.
nov.
Basionym: Chlamydomonas radiata Deason et
Bold 1960, Phycol. Stud. I, p. 14, fig. 4–5 (descr. et
ic. prima, iconotypus).
Epitype: The strain UTEX 966 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
Chloromonas rubrifilum (Korshikov in Pascher
1927) Pröschold, Marin, Schlösser et Melkonian
comb. nov.
288
T. Pröschold et al.
Synonyms: Chlamydomonas pseudogigantea
Korshikov in Pascher 1927, Die Süßwasserflora
Deutschlands, Österreichs und der Schweiz Vol. 4,
p. 287, fig. 249 (descr. et ic. prima, iconotypus),
Chlorogonium pseudogiganteum (Korshikov) Ettl
1958, Zur Kenntnis der Klasse Volvophyceae. I. in
Komárek et Ettl: Algologische Studien, p. 240.
Chlamydomonas capensis Pocock 1962, Arch.
Mikrobiol. 42, p. 59, fig. 1 (descr. et ic. prima, iconotypus), authentic culture: UTEX 1753 = SAG 22.98.
Chlamydomonas megalis Bischoff et Bold 1963,
Phycol. Stud. IV, p. 19, fig. 8–13, 88–91 (descr. et ic.
prima, iconotypus), authentic culture: SAG 9.84 =
UTEX 1492.
Emended Diagnosis: Cells 30–50 × 25–35 µm,
broad rounded to cylindrical to oviform, cell wall thin
with a small rounded papilla, with two flagella about
as long as the cell. Chloroplast cup-shaped, parietal
and massive, the chloroplast surface with coarse
ridges mostly parallel to the cell axis, 10–16(–20)
discontinuous pyrenoids, irregularly distributed;
eyespot pale red, elliptic to narrowly elongate in an
anterior position; many (up to 200) contractile vacuoles, distributed over the cell surface; nucleus in
central or slightly anterior position.
Asexual reproduction by four (rarely two or eight)
zoospores, first division true transverse without prior
rotation of the protoplast.
Sexual reproduction by oogamy, homothallic,
proterandric; first division in gametangium simultaneously resulting in four protoplasts, later divisions
successive, 32 or 64 (rarely 128) spermatozoids
formed in a gametangium; spermatozoids 6–10 ×
4–6 µm, teardrop-shaped, without a cell wall, with
two flagella nearly 1.5 times as long as the cell, with
two apical contractile vacuoles, chloroplast reduced, pale green, with a distinct eyespot, without
pyrenoid.
Zygotes ornamented with regular, flat-stopped
projections, green to brownish-red.
Epitype: The strain SAG 44.91 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
Oogamochlamys zimbabwiensis (Heimke et Starr
1979) Pröschold, Marin, Schlösser et Melkonian
comb. nov.
Basionym: Chlamydomonas zimbabwiensis
Heimke et Starr, 1979, Arch. Protistenkd. 122, p. 21,
fig. 1 (descr. et ic. prima, iconotypus).
Emended Diagnosis: Cells 15–22 × 15–20 µm,
broad rounded to cylindrical to oviform, cell wall
thin, papilla broad, rounded, two-humped or absent,
with two flagella about as long as the cell. Chloroplast cup-shaped, parietal and massive, the chloroplast surface with very fine ridges mostly parallel to
the cell axis, 2–6 discontinuous pyrenoids, irregularly distributed; eyespot pale red, elliptic to narrowly elongate or punctiform in anterior position;
two apical contractile vacuoles, nucleus in central or
slightly anterior position.
Asexual reproduction into four (rarely two or eight)
zoospores, rotation of the protoplast by 90º before
first cell division (“false transverse division”).
Sexual reproduction by oogamy, homothallic,
proterandric; all cell divisions in gametangium successive, 16 spermatozoids formed in gametangium;
spermatozoids 4–6 × 3–5 µm, teardrop-shaped,
without cell wall, with two flagella nearly 1.5 times as
long as the cell, with two apical contractile vacuoles,
chloroplast reduced, pale green, with a distinct eyespot, without pyrenoid.
Zygotes not ornamented, green.
Epitype: The strain UTEX 2213 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
Oogamochlamys ettlii Pröschold, Marin,
Schlösser et Melkonian spec. nov.
Diagnosis: Cells 16–27 × 18–22 µm, broad rounded
to cylindrical to oviform, cell wall thin with a small
rounded papilla, with two flagella about as long as
the cell. Chloroplast cup-shaped, parietal and massive, the chloroplast surface with fine ridges mostly
parallel to the cell axis, 3–8 discontinuous
pyrenoids, irregularly distributed; eyespot pale red,
elliptic to narrowly elongate in an anterior position;
many (up to 20) contractile vacuoles, distributed
over the cell surface, nucleus in central or slightly
anterior position.
Asexual reproduction by four (rarely two or eight)
zoospores, first division true transverse without prior
rotation of the protoplast.
Sexual reproduction by oogamy, homothallic,
proterandric; all cell divisions in gametangium successive, 16 spermatozoids formed in gametangium;
spermatozoids 4–6 × 3–5 µm, teardrop-shaped,
without cell wall, with two flagella nearly 1.5 times as
long as the cell, with two apical contractile vacuoles,
chloroplast reduced, pale green, with a distinct eyespot, without pyrenoid.
Zygotes not ornamented, green.
Molecular Phylogeny and Revision of Chlamydomonas
Latin diagnosis: Cellulae 16–27 × 18–22 µm, cylindricae usque trunctatae ovataeque; flagellis
duobus brevioribus quam corpus cellulae provisae;
papilla parva truncata; cellula membrana tenuis;
chloroplastus unicus, depressionibus fissurisque
multis praeditus, multae vacuolae pulsantes inter
superficies plastidis atque membranam plasmaticum; 3–8 pyrenoides laterales discontinae, per
chloroplastum solidum fortuite distributae, stigmate filiformi, paululum anterior. Nucleus magnus
cum nucleolum unum, paululum anterior media
cellulae.
Reproductio asexualis per bipartitionem repetitum endogenam plerumque in 4 (2–8) cellulas.
Reproductio sexualis ovogama, spermae 4–6 ×
3–5 µm, ante ova evolutae, guttiformes sine membrana, flagellae duae corpore unus dimidiatusque
longae, paulum sub apice insertae, chloroplastus reducta sine pyrenoide, stigmate distincta. Ova ex
cellulae membrana cellulae elapsa spermis inductis.
Zygota matura levis, viridis.
Holotypus: The strain UTEX 2218 permanently
preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) is the nomenclatural
type of the name Oogamochlamys ettlii. The type
material (holotype) has been deposited in the Culture Collection of Algae at the University of Texas at
Austin (UTEX), Department of Botany, The University of Texas at Austin, Austin, Texas 78713-7640,
USA.
Iconotypus: Fig. 5g–l in this study.
Type locality: col. M. A. Pocock as soil, Zimbabwe.
Etymology: This taxon is named after Dr. Hanuš
Ettl (1931–1997) to honor his contribution to the systematics of the genus Chlamydomonas
Authentic culture: UTEX 2218 = SAG 24.98
Lobochlamys Pröschold, Marin, Schlösser et
Melkonian gen. nov.
Diagnosis: Unicellular Chlorophyta sensu Bremer
(1985) with two flagella in clockwise basal body orientation; cell wall thin with mucilage layer around
the flagellated cell, with or without papilla, the single
chloroplast cup-shaped with thickened basal part
cylindrical/ parietal, the chloroplast surface with incisions, partly lobate; one discontinuous pyrenoid,
in basal or lateral position; eyespot pale red, elliptic
to narrowly elongate in an anterior position; with two
apical contractile vacuoles; nucleus in central or
slightly anterior position.
Asexual reproduction by zoospores, rotation of
the protoplast by 90º before first cell division (“false
transverse division”); sporangial wall with mucilage
layer; total lysis of the sporangial wall before release
289
of zoospores by means of a vegetative lysin enzyme.
Sexual reproduction by isogamy, homo- or heterothallic; zygotes not ornamented, green.
The genus is supported by molecular phylogenetic analysis using SSU rRNA sequence comparisons.
Latin Diagnosis: Cellulae unae viridiplantae
(Chlorophyta sensu Bremer 1985), flagellis duobus
brevioribus quam corpus cellulae provisae (positio
corporis flagellorum in sensu modi horae); papilla
praesenta vel absenta; cellula membrana tenuis
cum velamentum gelatum; duae vacuolae pulsantes
anterior; chloroplastus unicus, fissuris multis praeditus; unum pyrenoide lato discontinum, per chloroplastum solidum lateralis vel media distributa, stigmate filiformi, paululum anterior. Nucleus magnus
cum nucleolum unum, paululum anterior media cellulae.
Reproductio asexualis per bipartitionem repetitum endogenam plerumque in cellulas filias; sporangium membranum tenuis cum velamentum gelatum.
Reproductio sexualis homothallica, per conjunctionem isogametarum nudarum ad zygotas formandas effecta. Zygota matura levis, viridis.
Genus novus confirmatus per SSU rRNA sequentiae geneticae.
Type species: Lobochlamys segnis (Ettl 1965)
Pröschold, Marin, Schlösser et Melkonian
Lobochlamys segnis (Ettl 1965) Pröschold, Marin,
Schlösser et Melkonian comb. nov.
Basionym: Chlamydomonas segnis Ettl 1965,
Arch. Protistenkd. 108, p. 336, fig. 42–45, (descr. et
ic. prima, iconotypus).
Synonyms: Chlamydomonas fimbriata Ettl 1965,
Arch. Protistenkd. 108, p. 418, fig. 102, (descr. et ic.
prima, iconotypus), authentic culture: SAG 17.72 =
UTEX 1349. Chlamydomonas gymno-gama Deason 1967, J. Phycol. 3, p. 109, fig. 8–12, (descr. et ic.
prima, iconotypus), authentic culture: SAG 2.75 =
UTEX 1638. Chlamydomonas pallidostigmatica
King 1972, J. Phycol. 8, p. 120, fig. 1–3, 16 (descr. et
ic. prima, iconotypus), authentic culture: SAG 9.83 =
UTEX 1905. Chlamydomonas sajao Lewin 1984,
Chin. J. Oceanol. Limnol. 2, p. 93, fig. 1 (descr. et ic.
prima, iconotypus), authentic culture: SAG 50.84 =
UTEX 2277.
Emended Diagnosis: Cells 10–18 × 5–15 µm, ellipsoid, ellipsoid-cylindrical to oviform or fusiform,
cell wall thin with mucilage layer around the flagellated cell, papilla broad, rounded or absent, with two
flagella about as long as the cell. Chloroplast cupshaped with thickened basal part extending anteri-
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T. Pröschold et al.
orly, the chloroplast with distinct incisions, mostly
parallel to the cell axis, one discontinuous pyrenoid
in central or slightly basal position of the chloroplast;
eyespot pale red, elliptic to narrowly elongate in anterior position; two apical contractile vacuoles, nucleus in central or slightly anterior position.
Asexual reproduction by four or eight zoospores,
rotation of the protoplast by 90º before first cell division (“false transverse division”); sporangial wall
with mucilage layer; total lysis of the sporangial wall
before release of zoospores by means of a vegetative lysin enzyme (VLE-group 10, Schlösser 1976).
Sexual reproduction by isogamy; homothallic; gametes morphologically similar to vegetative cells;
zygotes not ornamented, green.
Epitype: The strain UTEX 1343 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
Lobochlamys culleus (Ettl 1965) Pröschold, Marin,
Schlösser et Melkonian comb. nov.
Basionym: Chlamydomonas culleus Ettl 1965,
Arch. Protistenkd. 108, p. 358, fig. 59, (descr. et ic.
prima, iconotypus).
Emended Diagnosis: Cells 10–18 × 5–15 µm, ellipsoid, ellipsoid-cylindrical to oviform, cell wall thin
with mucilage layer around the flagellated cell,
papilla broad, rounded or absent, with two flagella
about as long as the cell. Chloroplast cylindrical/
parietal, chloroplast surface with distinct incisions in
longitudinal and transverse orientation, partly lobate, one discontinuous pyrenoid in parietal position
of the chloroplast; eyespot pale red, elliptic to narrowly elongate in anterior position; two apical contractile vacuoles, nucleus in central or slightly anterior position.
Asexual reproduction by four or eight zoospores,
rotation of the protoplast by 90º before first cell division (“false transverse division”); sporangial wall
with mucilage layer; total lysis of the sporangial wall
before release of zoospores by means of a vegetative lysin enzyme (VLE-group 9, Schlösser 1976).
Sexual reproduction by isogamy; homo- or heterothallic; gametes morphologically similar to vegetative cells; zygotes not ornamented, green.
Epitype: The strain SAG 17.73 permanently preserved in a metabolically inactive state (cryopreservation in liquid nitrogen) in the Culture Collection of
Algae at the University of Texas at Austin (UTEX),
Department of Botany, The University of Texas at
Austin, Austin, Texas 78713-7640, USA.
Discussion
The Traditional Genera Chlamydomonas
and Chloromonas
In this contribution on the phylogeny and systematics of Chlamydomonas/ Chloromonas we intended
to combine analyses of new and published data
(both molecular and structural) with a critical examination of “historical“ descriptions, diagnoses, figures and taxonomic concepts in order to initiate a
comprehensive taxonomic revision of the genera
Chlamydomonas and Chloromonas.
In the SSU rRNA sequence analyses of this study,
species of the traditional genus Chlamydomonas
(including Chloromonas) occurred in seven clades of
the CW-group within the Chlorophyceae (“ Stephanosphaera”-, “ Polytoma”-, “Monadina”-, “Moewusii”-, “ Oogamochlamys”-, “ Chloromonas”-, and
“Reinhardtii”-clade). In three clades (“ Stephanosphaera”-, “Moewusii”-, and “Reinhardtii”-clade),
they were monophyletic with other genera of colonial, capsal, coccoid or filamenteous green algae
and within these clades, species of Chlamydomonas/ Chloromonas were not monophyletic to
the exclusion of the other genera.
The polyphyly of the genus Chlamydomonas/
Chloromonas was indeed already anticipated by
light microscopists (e.g. Ettl 1976, 1983a), but only
demonstrated convincingly by molecular phylogenetic analyses (Buchheim et al. 1990, 1996, 1997b;
Hepperle et al. 1998; Nakayama et al. 1996). Molecular phylogenetic studies by Nakayama et al. (1996)
identified three principal clades within the CWgroup of Chlorophyceae which contained species of
Chlamydomonas/ Chloromonas together with other
genera and designated them “ Volvox” , “ Tetracystis”
and “ Dunaliella” clades, Buchheim et al. (1997a,
1997b) identified five lineages (clades) containing
species of Chlamydomonas/ Chloromonas (termed
A–E in Buchheim et al. 1997a), and Hepperle et al.
(1998) in their molecular phylogenetic analysis of the
Phacotaceae found a total of six clades containing
species of Chlamydomonas/ Chloromonas.
How do these lineages relate to the clades identified in this study? Two of the clades containing
Chlamydomonas/ Chloromonas species identified
by Nakayama et al. (1996) correspond to the “Reinhardtii”- (“ Volvox”) and “Moewusii”- (“ Tetracystis”)
clades in our analysis. The third clade (“ Dunaliella”),
for which Nakayama et al. (1996) found support only
in a neighbor-joining analysis using a simple model
of evolution (Kimura’s two parameter model), was
not supported in our study using a complex evolutionary model proposed for our data set by Model-
Molecular Phylogeny and Revision of Chlamydomonas
test (Posada and Crandall 1998) and consisted of
three separate clades, the “ Stephanosphaera”-, the
“ Polytoma”-, and the “ Dunaliella”-clade, only the
first two contained Chlamydomonas/ Chloromonas
species. Four of the five clades of Buchheim et al.
(1997a) were recovered in the present analysis, they
correspond to the “Moewusii”-clade (clade A), the
“ Chloromonas”-clade (clade B), the “Reinhardtii”clade (clade D), and the “ Oogamochlamys”-clade
(clade E). Only their clade C (similar to the
Dunaliella-clade of Nakayama et al. 1996) was not
supported in our analysis (see above). It is likely that
the larger taxon sampling used in this study (27 taxa
compared to ten taxa or six taxa in the analyses of
Nakayama et al. 1996 and Buchheim et al. 1997a,
respectively) helped us to resolve the uncertainties
regarding the composition of clade C. The six clades
containing Chlamydomonas/ Chloromonas species
found by Hepperle et al. (1998) were also recovered
in our analysis. They correspond to: (1) the
“ Stephanosphaera”-clade
(their
Haematococcaceae), (2) the “ Polytoma”-clade (Dunaliellales II),
(3) the “Monadina”-clade (Chlamydomonas I), (4) the
“Moewusii”-clade (Chlamydomonas IV), (5) the
“ Chloromonas”-clade (Chlamydomonas II), and (6)
the “Reinhardtii”-clade (Chlamydomonas III).
Overall, the previous molecular phylogenetic investigations using SSU rRNA sequence comparisons already identified ten of the 11 clades within
the CW-group described in this study [among the
clades
without
Chlamydomonas/ Chloromonas
species the “ Phacotus”- and the “ Dunaliella”-clades
were identified by Hepperle et al. (1998; their Phacotaceae and Dunaliellales I), and the “Radiosa”-clade
only by Buchheim and Chapman (1992)]. Only the
“ Chlorogonium”-clade had previously not been recognized. We sequenced four strains of Chlorogonium belonging to three species and found them to
be a sister group to Haematococcus pluvialis; in previous analyses H. pluvialis could not be positioned in
phylogenetic trees of CW-group green algae (Buchheim et al. 1996; Hepperle et al. 1998). It is noteworthy that in our analysis (as in Hepperle et al. 1998) no
sister group to the “ Dunaliella”-clade [in contrast to
previous analyses by Buchheim et al. (1997a,b) and
Nakayama et al. (1996)] could be identified, corroborating the conclusion expressed by Melkonian and
Preisig (1984), based on ultrastructure, that
“ Dunaliella cannot be regarded simply as a “naked”
Chlamydomonas”. It is evident, however, that the
“ Dunaliella”-clade should not be separated at a
larger taxonomic level (e.g. order; Ettl 1983b) from
the other clades of the CW-group (as suggested by
Melkonian 1990).
291
What is Chlamydomonas and Chloromonas?
From this study and earlier molecular phylogenetic
analyses (see above), it is clear that the traditional
genus Chlamydomonas/ Chloromonas is polyphyletic within the CW-group of the Chlorophyceae
and that the genus needs to be revised. On the
species level, such a revision will require much more
experimental work on the numerous strains of
Chlamydomonas in culture as well as careful evaluation of hundreds of descriptions and diagnoses of
Chlamydomonas species. On the genus level, a revision of Chlamydomonas must first clarify the identity
of its type species (C. pulvisculus, see below) and
should take into account that: (1) other morphologically well-circumscribed genera of green algae, e.g.,
species of Volvox, form monophyletic lineages with
species of Chlamydomonas to the exclusion of other
Chlamydomonas species; (2) colorless taxa, e.g.,
Polytoma, form clades with species of Chlamydomonas to the exclusion of other Chlamydomonas
species; and finally (3) taxa (strains) without
pyrenoids cannot be separated from taxa (strains)
with pyrenoids calling into question the justification
for the genus Chloromonas in the traditional sense
(i.e. “ Chlamydomonas without pyrenoid”).
What are the formal type species of Chlamydomonas and Chloromonas? An answer to this
question is crucial because these type species will
define the taxonomic identity of their respective
genera in a revised classification. Only those
Chlamydomonas species, which are monophyletic
with the type species (to the exclusion of other taxa),
will retain their generic identity whereas other
species will have to be transferred to other genera.
The formal type species of Chlamydomonas Ehrenberg 1833 (conserved against the original spelling
Chlamidomonas and two earlier synonyms, Protococcus C. Agardh 1824 and Sphaerella Sommerfelt
1824; see Silva 1980) is Chlamydomonas pulvisculus (O.F. Müller) Ehrenberg 1838, which was described by O.F. Müller (1786) as Monas pulvisculus.
Unfortunately, this species is ambiguous as the descriptions and figures by O.F. Müller and Ehrenberg
cannot be matched with modern species (see Results). It has therefore not been dealt with in recent
taxonomic treatments of Chlamydomonas (Ettl
1976, 1983a), and in fact Ettl (1976) considered this
species as invalid („zu streichende Art“). To solve the
problem, Ettl (1976) proposed to select C. reinhardtii
as the type (neotype) of the generic name instead of
C. pulvisculus. We propose to designate C. reinhardtii as conserved type and provide an emended
diagnosis of the genus Chlamydomonas (see Results).
292
T. Pröschold et al.
The position of C. reinhardtii in molecular phylogenies (Buchheim et al. 1996, 1997b; Hepperle et al.
1998; Nozaki et al. 2000; Fig. 1 in this study) demonstrates that this species is intimately associated with
colony-forming Volvocales within the “Reinhardtii”clade (e.g., Tetrabaena, Gonium, Eudorina, Volvox),
but not closely related to most other species of
Chlamydomonas. In consequence, Chlamydomonas
as emended here (sensu stricto) will be confined to
at most a few (probably only one) species.
In Chloromonas Gobi 1899/1900 emend. Wille
1903, the formal type species, C. reticulata, is a welldescribed taxon (Ettl 1970; Goroschankin 1891) and
in this study is represented by seven strains with almost identical SSU rRNA sequences and, with the
notable exception of the presence (five strains) or
absence (two strains) of a pyrenoid, identical structural characters. C. reticulata is part of one of the
CW-group clades, i.e. the “ Chloromonas”-clade,
currently with eight species (see Results). Six of
these species were previously recognized as
Chlamydomonas because of the presence of one or
several pyrenoids. Obviously, in Chlamydomonas/
Chloromonas the character state “pyrenoid” has no
taxonomic significance (even at the species level; i.e.
C. reticulata) and certainly cannot be used as generic
character to define Chloromonas as in traditional
classifications. It should also be noted that the presence or absence of pyrenoids sometimes depends
on environmental conditions such as photoheterotrophy (see Nozaki et al. 1998 for species of
Chlorogonium). The two “ Chloromonas” species C.
perforata and C. oogama, which were studied by
Buchheim et al. (1997b), and which cluster with the
“ Stephanosphaera”- and the “Reinhardtii”-clade in
our analysis (Fig. 1), reveal pyrenoids, which led
Buchheim et al. (1997b) to conclude that they “have
been misidentified as Chloromonas taxa or they represent contaminated cultures”. Similarly, a Chlamydomonas strain (SAG 3.85) isolated by one of us
(MM) was originally placed in the Pleiochloris section
because of the presence of many pyrenoids. Later, it
was designated Chloromonas clathrata (Schlösser
1994) as it apparently now lacked pyrenoids. When
this strain was reinvestigated during this study, we
identified pyrenoids (but only after staining with azocarmine), corroborating the initial observations when
the alga (now designated Chloromonas rubrifilum)
was isolated. Clearly there is much to be learned
about the significance of the presence or absence of
pyrenoids in the genus Chlamydomonas. We consider it most likely, and find no conflicting evidence in
molecular phylogenetic analyses (this study; Buchheim et al. 1997b; Morita et al. 1999), that strains
without pyrenoids evolved from pyrenoid-containing
strains by multiple independent losses. It may be
possible, however, that such pyrenoid losses occurred more often in some clades than in others. In
the two clades, which we investigated in more detail
in this study, strains without pyrenoids occurred only
in the “ Chloromonas”-clade, not in the “ Oogamochlamys”-clade. One possible consequence of the
rejection of the defining character of Chloromonas
(“no pyrenoid”) would be the invalidation of this
genus. Instead, we prefer to retain the name and validate the taxon Chloromonas (equated with the
“ Chloromonas”-clade), which needs to be defined by
other characters. Fortunately, the investigation of the
chloroplast structure revealed the presence of either
asteroid chloroplasts or parietal chloroplasts with
perforations in all strains of the “ Chloromonas”clade investigated, characters which are presently
unknown in other flagellates of the CW-group of the
Chlorophyceae (see Results). Among approximately
130 described species of Chloromonas (Ettl 1983a),
about 30 species also display asteroid chloroplasts
or parietal chloroplasts with perforations and, though
neither cultures nor sequences are available, can be
expected to belong to the “ Chloromonas”-clade. As
an example, Chloromonas insignis and Chloromonas
palmelloides (no pyrenoid, parietal chloroplasts with
perforations) were positioned within the „ Chloromonas“-clade using rbc L-sequence comparisons
(Morita et al. 1999).
Most of the more than 600 species in the traditional genus Chlamydomonas presumably have no
phylogenetic affinity to C. reinhardtii and consequently, will have to be transferred to other described genera, such as Chloromonas (see above),
or described as new genera. To initiate the description of successor genera of Chlamydomonas (sensu
lato), we propose to raise two independent lineages
to genus level and designate them as Oogamochlamys and Lobochlamys (see Results and below).
Some of our results bear on the question of the
species concept in the genus Chlamydomonas. All
of the Chlamydomonas (including Chloromonas)
species described until now have been established
using a morphological species concept (see Ettl
1976, 1983a). Unfortunately, the variability of the diagnostic features used, was not sufficiently appreciated: most descriptions were based on observations of material from natural samples, and often
only a few cells were examined. In addition, rigorous
discrimination against known species was seldom
made when new taxa were described. Another possible reason for inflation in the number of species,
ironically, relates to a diagnostic character introduced to subdivide the genus into more managable
groups (sections, sometimes termed subgenera):
Molecular Phylogeny and Revision of Chlamydomonas
the number and position of pyrenoids in relation to
the shape and position of the chloroplast (see Ettl
1976, 1983a; Pascher 1927). We and others (Buchheim et al. 1996) have shown that the sections of
Chlamydomonas have little meaning in phylogenetic
terms. As an example, we note that in the authentic
strain of Lobochlamys segnis (= C. segnis), the position of the pyrenoid successively changes during
the cell cycle (from zoospore to mature vegetative
cell) to reflect the characteristics of three sections,
namely Chlorogoniella, Chlamydella and Euchlamydomonas (unpublished observations). It is thus not
surprising that authentic strains of Chlamydomonas
species, which have been assigned to different sections, may be genetically (and morphologically) so
similar that they should be placed in a single
species: e.g. C. gymnogama and C. pallidostigmatica (section Chlamydella), C. sajao (section Pseudagloë), C. segnis and C. fimbriata (section Euchlamydomonas) in the species Lobochlamys segnis (this
study). Furthermore, the number of pyrenoids per
cell is a homoplasy among the strains studied (as in
the section Pleiochloris) and can also vary during
development within a single strain (as in Chloromonas carrizoensis, see Results). Interestingly, in the
majority of the sections of Chlamydomonas the
same diversity of chloroplast characters such as different shapes (cup-shaped, asteroid) or different
chloroplast surfaces (with or without incisions, perforations, or ridges/grooves) occur in each section,
suggesting to us that the concept on which the sections are based, i.e. the number and position of
pyrenoids, obscures other taxonomically more informative morphological characters. This conclusion is
corroborated by our observations that some aspects of the shape and surface structure of the
chloroplast correlate well with results obtained from
the molecular phylogenetic analyses: all strains of
the “ Oogamochlamys”-clade (Oogamochlamys and
Lobochlamys) have cup-shaped/parietal chloroplasts (except for L. culleus, see below) with either
surface ridges (Oogamochlamys) or incisions (Lobochlamys) running mostly parallel to the cell axis, and
strains of the “ Chloromonas”-clade have either asteroid or cup-shaped/parietal chloroplasts with perforations (the latter not resulting in the formation of
chloroplast lobes) but no surface ridges/grooves or
incisions (see Results). We can also extend these
observations to groups of strains within clades. In
strains assigned to Lobochlamys segnis, the chloroplast is cup-shaped with thickened basal part, the
chloroplast surface reveals incisions which (at least
in the chloroplast posterior) do not traverse the
chloroplast. In Lobochlamys culleus, the chloroplast
is cylindrical/parietal, the chloroplast surface ex-
293
hibits incisions which lead to the formation of distinct chloroplast lobes. In strains assigned to the
oogamous species Oogamochlamys gigantea,
Oogamochlamys zimbabwiensis and Oogamochlamys ettlii, the chloroplasts are cup-shaped and
parietal with ridges/grooves running parallel to the
cell axis, which in O. zimbabwiensis and O. ettlii
(perhaps due to the smaller cell size) are less coarse
than in O. gigantea. In the “ Chloromonas”-clade,
strains assigned to C. reticulata have cup-shaped
and parietal chloroplasts with perforations, whereas
strains assigned to C. actinochloris (two strains), C.
augustae (three strains), C. asteroidea, and C. radiata have asteroid chloroplasts. How the two types of
chloroplasts in the “ Chloromonas”-clade relate to
each other, cannot yet be decided, because the
branching order within this clade could not be resolved using SSU rRNA sequence comparisons (see
Fig. 2, Results). The functional significance of the
various chloroplast shapes and surface structures
also remains to be elucidated.
In summary, structural features of the chloroplast
seem to be of taxonomic value in the Chlamydomonas/ Chloromonas complex and in general lend
support to the results obtained by molecular phylogenetic analysis. However, additional critical observations of a variety of morphological characters over
the whole range of strains in this complex are necessary before any firm conclusions can be drawn.
Surprisingly, Pascher (1927) avoided the use of
structural features of the chloroplast for his definition
of subgenera. Instead, he used the homoplasious
chloroplast characters (i.e. presence, number and
position of pyrenoids in relation to chloroplast shape),
which apparently led to the description of artificial
subgenera. Another result of this approach was that
isolates, which differed only in pyrenoid characters
but were otherwise identical, were placed in different
subgenera and consequently also separated at the
species level. However, these isolates as shown in
this study represent only one (morpho)species (e.g.,
Lobochlamys segnis; Chloromonas reticulata). In
conclusion, a questionable subgenus/section-concept in Chlamydomonas led to an inflation in the
number of (morpho)species described, many of
which will have to be abandoned in the future.
Ettl and Schlösser (1992) made a start in this direction by reducing three species of Chlamydomonas to synonyms of a fourth species, C. applanata. In addition, they noted that six further
species described only from natural samples could
not be distinguished morphologically from C. applanata and suggested that they should no longer
be recognized as such. Their conclusions were
based on careful light microscope observations of
294
T. Pröschold et al.
strains of the various species, and comparison with
published diagnoses. Their study was facilitated
because the strains selected for light microscopy
belonged to a common vegetative lysin enzyme
group (VLE-group 7, Schlösser 1976). V-lysins are
proteases, which dissolve the sporangial cell walls
to allow liberation of zoospores (Jaenicke et al.
1987; Matsuda et al. 1995). Schlösser (1976, 1984)
described methods for obtaining v-lysins from
strains for cross-specificity testing. From more than
100 strains of Chlamydomonas tested, v-lysins
were recovered from 65 strains belonging to 42 different species and were assigned to 15 VLE-groups
based on cross-reactivity within groups combined
with non-reactivity between different groups (three
exceptions were noted revealing non-reciprocal
cross-reactivity between different VLE-groups;
Schlösser 1984). Based on their analysis of VLEgroup 7 (C. applanata) Ettl and Schlösser (1992)
concluded that strains of a VLE-group should be regarded as clones of one (morpho)species. In two
strains of this VLE-group (included the authentic
strain of “ C. humicola”), SSU rRNA sequences have
been analysed and shown to be identical (Gordon
et al. 1995). In their molecular phylogenetic analyses, Buchheim et al. (1997a) have shown that all
members of VLE-group 14, which include the authentic strains of C. geitleri, C. hindakii, C. monoica
and C. pinicola, also reveal almost identical SSU
rRNA sequences and can be regarded either as distinct isolates of a single species (C. noctigama) or
as a group of sibling species [because of the nonviability of most offspring following “interstrain”
crosses (Burrascano and Van Winkle-Swift; Genetics 107:s15, 1984)].
How do our results relate to the VLE-groups of
Schlösser (1976, 1984)? V-lysins are not known for
the oogamous species (perhaps their concentration
is too low for the cross-reactivity tests as only a
small hole develops in the sporangial wall) or for
strains of the “ Chloromonas”-clade; however, the
three strains of Lobochlamys culleus belong to VLEgroup 9 and the six strains assigned to
Lobochlamys segnis (including the authentic strains
of Chlamydomonas segnis, C. fimbriata, C. gymnogama, C. pallidostigmatica, and C. sajao) belong to
VLE-group 10. Although our molecular phylogenetic
analyses based on SSU rRNA sequence comparisons could not with confidence separate L. culleus
from L. segnis (see Results; less conserved molecular markers such as the internal transcribed spacers
of the rRNA operon could be useful), in general,
VLE-groups known to be closely related (one-sided
cross-reactivity) were also the closest relatives
in the SSU rRNA phylogeny [e.g. VLE-groups 1
(C. reinhardtii), 2 (C. debaryana) and 15 (C. zebra)
in the “Reinhardtii”-clade, and VLE-groups 12
(C. moewusii) and 13 (C. pitschmannii) in the
“Moewusii”-clade; Fig. 1]. It appears that the phylogenetic signal of the SSU rRNA (at least in Chlamydomonas) is informative almost to the level of the
(morpho)species (VLE-group). This is exemplified by
strain UTEX 2218, which we could neither place in
C. zimbabwiensis nor in C. gigantea based on SSU
rRNA sequence comparisons (see Results). Morphologically, this strain is intermediate between the
two oogamous species. Our unpublished observations show that spermatangia from strain UTEX
2218 cannot induce egg release from vegetative
cells of the two other species (strains SAG 44.91
and SAG 45.91 respectively), and conversely, that
spermatangia from the other two species cannot induce egg release from vegetative cells of UTEX
2218, suggesting that indeed strain UTEX 2218 represents a new (morpho)species, which is here described as Oogamochlamys ettlii.
It has been argued that a biological species concept cannot be applied to the genus Chlamydomonas, because sexual reproduction is unknown
for more than 80% of the species described (Ettl
and Schlösser 1992). However, in the (few) cases
where sexual reproduction, molecular phylogeny
(based on SSU rRNA sequence comparisons), morphology (based on light microscopy) and cross-reactivity of v-lysins has been studied in the same
Chlamydomonas strains almost complete congruence between these characters has been observed:
strains of one VLE-group are morphologically indistinguishable, and they usually represent a group of
biological sibling species (the latter separated from
each other mainly by postzygotic barriers). Strains
of a VLE-group also reveal the most closely related
sequences in the SSU rRNA phylogeny and cannot
be split into different, significantly supported monophyletic lineages (this study). However, it appears
that in the SSU rRNA phylogeny a single VLE-group
is not always monophyletic to the exclusion of another (presumably closely related) VLE-group (e.g.
VLE-groups 9 and 10 in Lobochlamys; Figs 1, 2), indicating that the phylogenetic signal of the SSU
rRNA in such cases is not informative at the (morpho)species level. Nevertheless, the SSU rRNA sequences between strains in the same VLEgroup/(morpho)species often differ from each other
(as in L. segnis or in L. culleus). This situation sheds
some light on the “phylogenetic species concept”
(Baum 1992): if the identification of the smallest diagnosable units is the primary criterion for species
delimitation, then any base difference between two
sequences could lead to the description of a new
Molecular Phylogeny and Revision of Chlamydomonas
species. However, when no sister group relationship
can be identified between such strains (see above),
the sequence difference is obviously not phylogenetically informative. Internal transcribed spacers (in
particular ITS-2) of the ribosomal RNA operon have
been used successfully in recent years to address
the question of the relationship between sequence
divergence and species identity in Chlamydomonas
and in the colonial Volvocales (Coleman and Mai
1997; Coleman et al. 1998; Fabry et al. 1999). One
outcome of these studies has been the recognition
of a correlation between the absence of compensatory base changes (CBC) in conserved stem regions of the ITS-2 and the ability of gametes of different strains to form zygotes (Coleman 2000).
Groups of strains without CBCs readily mate and
form zygotes, i.e., a CBC-clade conforms to a Z (Z
for zygote formation) clade. It is likely, but remains to
be investigated in more detail, that Z-clades and/or
CBC-clades correspond to VLE-groups.
If one accepts the notion that a VLE-group corresponds to a (morpho)species of Chlamydomonas/
Chloromonas (in the traditional sense), then the
number of (morpho)species can be roughly estimated: the recently emended Chlamydomonas
species include about three to five synonymous
species based on comparison with authentic strains
(Ettl and Schlösser 1992; and this study). If this remains the trend for the future, then the number of
species is only about 25% of the known. In addition,
there appear to be numerous incompletely described species. Ettl and Schlösser (1992) in their
revision of C. applanata noted six incompletely described species (compared to C. applanata and its
three synonymous species). If this can be generalized, then of the 600 known species of traditional
Chlamydomonas/ Chloromonas only 50–100 species
will remain (distributed over several genera, see
above). A similar figure is obtained, when we calculate the number of VLE-groups: Schlösser (1976,
1984) found 15 VLE-groups in 42 species. When the
incompletely described species are taken into consideration, a maximum of about 70 VLE-groups/
(morpho)species are to be expected. The present
study of the “ Chloromonas”- and the “ Oogamochlamys”-clades identified 13 (morpho)species (five
in the “ Oogamochlamys”- and eight in the “ Chloromonas”-clade).
The Oogamous Species
(Oogamochlamys gen. nov.)
The four strains designated here Oogamochlamys
gigantea, including the authentic strains of Chlamydomonas capensis and C. megalis (Table 5, Results),
295
are very similar to each other in morphology, reproduction and in their SSU rRNA sequences (Fig. 2).
Many contractile vacuoles scattered over the cell
surface is a feature, which has been previously observed in C. pseudogigantea (Geitler 1954), in C.
megalis (Bischoff and Bold 1963), C. capensis
(Heimke and Starr 1979; not mentioned by Pocock
1962), and in all strains investigated by us. In contrast, only two apical contractile vacuoles were described for C. gigantea (Dill 1895). The two apical
contractile vacuoles occur in all strains investigated
in this study, but the numerous others, which are
smaller and hidden in the grooves of the chloroplast
surface can be overlooked easily. It is thus most
likely that Dill (1895) either did not notice them or, alternatively, that the multiple empty circles scattered
over the chloroplast surface, depicted in his type figure, represent contractile vacuoles and not starch
grains.
The first division step of Oogamochlamys gigantea is always, according to our observations, a „true
transverse division“ without rotation of the protoplast, as also described for C. pseudogigantea (Ettl
1988; Geitler 1954). The first division step in most
Chlamydomonas species is either longitudinal or
transverse after a 90° rotation of the protoplast
within the cell wall (“false transverse division”).
Heimke and Starr (1979) indicated for the type material of C. capensis that the initiation of zoosporogenesis begins with a 90°rotation of the protoplast. F or
the same strains, however, we found only true transverse divisions. Dill (1895) described, but did not illustrate, the first division step in C. gigantea as longitudinal. In the same publication, however, he described a true transverse division for C. reinhardtii
although for this species, only a longitudinal division
of the protoplast after 90° rotation is known
(Schlösser 1972). We suppose that Dill had no possibility for long-term observations of dividing cells,
and do not put undue emphasis on his statements
concerning cell division.
Sexual reproduction in Oogamochlamys gigantea
is a true oogamy characterized by the differentiation
of small, flagellate male gametes with reduced
chloroplasts and large immobile female gametes,
which escape from their cell walls, becoming naked
protoplasts. This was not recognized in some of the
earlier descriptions: Pocock (1962) described different stages of this mode of sexual reproduction in C.
capensis, but did not observe the release of the egg
cell protoplast; she therefore labeled the mode of
sexual reproduction in this species heterogamous.
Dill (1895) depicted a protoplast of C. gigantea released from its cell wall and also cells with the typical zygote walls; however, he did not recognize
296
T. Pröschold et al.
them as stages of the sexual cycle. A complete description of true oogamy was first presented by
Geitler (1954) for C. pseudogigantea and later confirmed by Heimke and Starr (1979). Sexual reproduction is triggered by special inducing conditions
(Heimke and Starr 1979), which were presumably
not met by the authors of C. megalis (Bischoff and
Bold 1963). As a result of this comparison, we integrated the three species discussed above (C. pseudogigantea, C. megalis, and C. capensis) into the
species first described: Oogamochlamys gigantea
Dill (an emended diagnosis is given in the Results
section). In O. zimbabwiensis, the first cell division in
asexual reproduction is a “false transverse division”
unlike the situation in O. gigantea (Heimke and Starr
1979; and this study). Why two closely related taxa
such as O. gigantea and O. zimbabwiensis should
differ in the mode of cell division during asexual reproduction is not known, however a correlation appears to exist between multiple contractile vacuoles
and a true transverse division. We note that this correlation holds for the new species Oogamochlamys
ettlii as well as for unrelated taxa with multiple contractile vacuoles (e.g. in the Chlorogonium-clade;
Ettl 1988; Hoops and Witman 1985).
Conclusions
Based on a molecular phylogentic analysis using
SSU rRNA sequence comparison and a reevalution
of light microscope characters, the traditional concepts of the genera Chlamydomonas and Chloromonas have been revised. Two monophyletic lineages (clades), which contain species previously assigned to Chlamydomonas, are now recognized as
new genera (Oogamochlamys and Lobochlamys).
This is a first contribution towards the taxonomic revision of Chlamydomonas/ Chloromonas using a
combination of molecular phylogenetic analyses
and investigations into the morphology and life history of this important and ubiquitous assemblage of
“green protists”.
Methods
Cultures and light microscopy: Strains were obtained from the Sammlung von Algenkulturen, Universität Göttingen, Germany (SAG; Schlösser 1994)
and the Culture Collection of Algae, University of
Texas at Austin, USA (UTEX; Starr and Zeikus 1993)
and are listed in Table 5. The algae were grown in
modified “Bold Basal Medium” (3N-BBM+V, medium
26a in Schlösser 1997). The cultures of the ooga-
mous Chlamydomonas species (C. gigantea and
C. zimbabwiensis) were grown in 100 ml Erlenmeyer
flasks at 20 ºC at a photon fluence rate of 50
µEm–2s–1, and a light/dark-cycle 14/10 h. Sexual reproduction was induced by the following method:
10 ml of a dense cell suspension from a 14 day-old
culture in Erlenmeyer flasks were sedimented by
low speed centrifugation and the pellets were resuspended in 5 ml 3N-BBM+V or in the same
medium without sodium nitrate (BBM-N+V) to induce gametogenesis. These suspensions were incubated in shallow watch glasses, which rested on
a glass triangle in a petri dish (Starr 1955). The bottom of the petri dish contained 20 ml distilled water
in order to reduce evaporation. Samples were controlled microscopically each day. For the continuous observation of single cells (up to two weeks) we
used a microchamber (Heunert 1973) containing
3N-BBM+V agar medium (1.5% w/v). For species
determination, strains were grown under the same
culture conditions as above. After 14 days of culture, mature vegetative cells (near the end of the
light period) were observed by light microscopy. Micrographs were made using oil immersion lenses
and Kodak Ektachrome (160 ASA) or Fuji RD 135
(100 ASA). The cells were observed in the living
state, either free swimming or immobilized by removal of nutrient solution with filter paper or transfer of cells into a gel (Protoslo, No. 88-5141, Carolina Biological Supply Company, USA, or ultralow
gelling agarose according to Reize and Melkonian
1989). For staining of pyrenoids, azocarmine was
used (5% w/v in 45% v/v acetic acid; see Ettl 1976).
For photographic documentation of the chloroplast
structures, cells (500 µl algal suspension) were either fixed (50 µl 2% OsO4, 20 µl 25% glutaraldehyde, 430 µl 3N-BBM+V-culture medium, for 30
minutes on ice and washed once in culture medium)
or studied alive after they had settled on the slide.
Observations were made using Nomarski interference contrast with a Zeiss inverted microscopy
(IM35). For documentation of contractile vacuole
number and activity, live cells were photographed
using an electronic flash attached to a Zeiss inverted microscope (IM).
DNA isolation, PCR and sequencing: DNA isolation was performed using a CTAB protocol
(Rogers and Bendich 1985; modified by Friedl
1996) followed by the amplification of nuclear-encoded SSU rRNA regions by polymerase chain reactions (PCR; Saiki et al. 1988) using thermocycling protocols and 5-biotinylated PCR-primers
(biot.) as described previously (Marin et al. 1998).
Amplications of SSU rRNA regions were performed using oligonucleotide primers A (biotiny-
Molecular Phylogeny and Revision of Chlamydomonas
lated) (Medlin et al. 1988) and ITS055 (Marin et al.
1998). PCR products were sequenced with sequencing methods and primers (82F, 528F, 920F,
BR, 920R, 536R) as described by Marin et al.
(1998) and Marin and Melkonian (1999).
The nucleotide sequence data are avaiable in the
EMBL, GenBank and DDBJ sequence databases
under the accession numbers listed in Table 5. SSU
rRNA sequences were manually aligned with homologous sequences from other Chlorophyta using the
Olsen Multiple Sequence Alignment Editing program with respect to primary and secondary structural conservation. Strains designations and
EMBL/GenBank accession numbers of published
sequences are given in Figures 1 and 2. The alignment is available from the authors upon request.
Phylogenetic analyses: Phylogenetic trees were
inferred using distance, parsimony, and maximum
likelihood criteria using PAUP* versions 4.0b2,
4.0b6, and 4.0b8 (Swofford 1998). Two data sets
were used: a large alignment of 156 taxa of Chlorophyta with 1,642 unambiguously aligned positions,
and a smaller data set consisting of 34 taxa of
Chlamydomonas/ Chloromonas and 1,702 unambiguously aligned positions. To decide on the evolutionary model, which best fitted our data, we used
the program Modeltest 3.04 (Posada and Crandall
1998), which employs two statistics, the likelihood
ratio test (LRT) and the Akaike information criterion
(AIC; Akaike 1974). Based on the results of these
tests, the model selected by the hierarchical LRT
was the Tamura-Nei model (TrN; Tamura and Nei
1993), with the proportion of invariable sites (I) and
the gamma shape parameter (G) for among site variation calculated from the data set (TrN+I+G for the
global data set and TrNef+I+G for the smaller data
set; base frequencies and substitution parameters
were estimated by Modeltest). Maximum likelihood
with the Tamura-Nei model with invarint sites and
gamma parameter was used as the distance measure in inferring trees using the distance optimality
criterion. The same model was invoked in maximum
likelihood analyses. For comparison with the
TrN+I+G model in the global analysis we also employed the simpler HKY85 model of evolution (without I and G; Hasegawa et al. 1985). Maximum parsimony analyses used a heuristic search with a
branch-swapping algorithm (tree bisection-reconnection algorithm; 10 replicates with random order
of sequence addition).
The confidence of branching in all methods was
assessed using 1000 bootstrap resamplings of the
data set (except for ML analysis [Fig. 2] was assessed using 100 bootstrap resamplings; Felsenstein 1985).
297
User-defined trees were generated by modifying
the treefile of the “best tree” using TreeView (version
1.6.2; Page 1996). To compare user-defined topologies with the “best tree“ the sequence data file and
the treefiles were loaded into PAUP* and used for
Kishino-Hasegawa-tests (Kishino and Hasegawa
1989); comparisons were based on maximum likelihood (ML; model of evolution selected by Modeltest
3.04, see above) and maximum parsimony criteria
(see Tables 1 and 2).
Acknowledgements
We like to thank Barbara Surek (Cologne) for help
with photomicrography, Paul C. Silva (Berkeley), and
Vincent Demoulin (Liège) for advice in nomenclatural
problems, Robert A. Andersen (Bigelow) for valuable
suggestions to improve the manuscript and
Siegfried Werth (Cologne) for help with image analysis. This research was supported by grants from the
Deutsche Forschungsgemeinschaft to B. Marin, U.
G. Schlösser and M. Melkonian.
References
Adair WS, Snell WJ (1990) The Chlamydomonas reinhardtii Cell Wall: Structure, Biochemistry and Molecular Biology. In Adair WS, Mecham RP (eds) Organization and Assembly of Plant and Animal Extracellular
Matrix. Academic Press, San Diego, pp 15–84
Akaike H (1974) A new look at the statistical model
identification. IEEE Trans Contr 19: 716–723
Baum D (1992) Phylogenetic species concepts.
Trends Ecol Evol 7: 1–2
Bischoff TR, Bold HC (1963) Phycological studies IV.
Some soil algae from Enchanted Rock and related
algal species. Univ Texas Publ 6318: 1–95
Bremer K (1985) Summary of green plant phylogeny
and classification. Cladistics 1: 369–385
Buchheim MA, Chapman RL (1992) Phylogeny of
Carteria (Chlorophyceae) inferred from molecular and
organismal data. J Phycol 28: 362–374
Buchheim MA, Buchheim JA, Chapman RL (1997a)
Phylogeny of the VLE-14 Chlamydomonas (Chlorophyceae) group: a study of 18S rRNA gene sequences.
J Phycol 33: 1024–1030
Buchheim MA, Buchheim JA, Chapman RL (1997b)
Phylogeny of Chloromonas (Chlorophyceae): a study
of 18S ribosomal RNA gene sequences. J Phycol 33:
286–293
Buchheim MA, Turmel M, Zimmer EA, Chapman RL
(1990) Phylogeny of Chlamydomonas (Chlorophyta)
298
T. Pröschold et al.
based on cladistic analysis of nuclear 18S rRNA sequence data. J Phycol 26: 689–699
Buchheim MA, Lemieux C, Otis C, Gutell RR, Chapman RL, Turmel M (1996) Phylogeny of Chlamydomonadales (Chlorophyceae): A comparison of ribosomal RNA gene sequences from the nucleus and the
chloroplast. Mol Phylogenet Evol 5: 391–402
Chantanachat S, Bold HC (1962) Phycological studies II. Some algae from arid soils. Univ Texas Publ
6218: 1–74
Chapman RL, Buchheim MA, Delwiche CF, Friedl T,
Huss VAR, Karol KG, Lewis LA, Manhart J, McCourt
RM, Olsen JL, Waters DA (1998) Molecular Systematics of the Green Algae. In Soltis DE, Soltis PS, Doyle JJ
(eds) Molecular Systematics of Plants II. Kluver Academic Publishers, Boston, pp 508–540
Coleman AW (2000) The significance of a coincidence
between evolutionary landmarks found in mating affinity and a DNA sequence. Protist 151: 1–9
Coleman AW, Mai JC (1997) Ribosomal RNA ITS-1
and ITS-2 sequence comparisons as a tool for predicting genetic relatedness. J Mol Evol 45: 168–177
Coleman AW, Preparata RM, Mehrotra B, Mai JC
(1998) Derivation of the secondary structure of the ITS1 transcript in Volvocales and its taxonomic correlations. Protist 149: 135–146
Deason TR (1967) Chlamydomonas gymnogama, a
new homothallic species with naked gametes. J. Phycol 3: 109–112
Deason TR, Bold HC (1960) Phycological studies I.
Exploratory studies of Texas soil algae. Univ Texas
Publ 6022: 1–72
Deason TR, Silva PC, Watanabe S, Floyd GL (1991)
Taxonomic status of the species of the green algal
genus Neochloris. Plant Syst Evol 177: 213–219
Dill EO (1895) Die Gattung Chlamydomonas und ihre
nächsten Verwandten. Jahrb Wiss Bot 28: 323–358
Ehrenberg CG (1838) Die Infusionsthierchen als vollkommene Organismen. Voss, Leipzig
Ettl H (1965a) Beitrag zur Kenntnis der Morphologie
der Gattung Chlamydomonas Ehrenberg. Arch Protistenkd 108: 271–430
Ettl H (1965b) Untersuchungen an Flagellaten. Österr
Bot Z 112:701–745
Ettl H (1970) Die Gattung Chloromonas Gobi emend.
Wille (Chlamydomonas und die nächstverwandten
Gattungen I). Beih Nova Hedwigia 34: 1–284
Ettl H (1976) Die Gattung Chlamydomonas Ehrenberg
(Chlamydomonas und die nächstverwandten Gattungen II). Beih Nova Hedwigia 49: 1–1122
Ettl H (1979) Die Gattungen Carteria Diesing emend.
Francé und Provasoliella A. R. Loeblich (Chlamydomonas und die nächstverwandten Gattungen III).
Beih Nova Hedwigia 60: 1–226
Ettl H (1983a) Chlorophyta I- Phytomonadina. In Ettl H,
Gerloff J, Heynig H, Mollenhauer D (eds) Süßwasserflora von Mitteleuropa, Bd. 9. Gustav Fischer,
Stuttgart, New York
Ettl H (1983b) Taxonomische Namensänderungen und
Neubeschreibungen unter den Phytomonadina (Chlorophyta). Nova Hedwigia 35: 731–736
Ettl H (1988) Unterschiedliche Teilungsverläufe bei den
Phytomonaden (Chlorophyta). Arch Protistenkd 135:
81–101
Ettl H, Gärtner G (1995) Syllabus der Boden-, Luftund Flechtenalgen. Gustav Fischer, Stuttgart-JenaNew York
Ettl H, Schlösser UG (1992) Towards a revision of the
systematics of the genus Chlamydomonas (Chlorophyta). 1. Chlamydomonas applanata Pringsheim. Bot
Acta 105: 323–330
Fabry S, Kohler A, Coleman AW (1999) Intraspecies
analysis: comparison of ITS sequence data and gene
intron sequence data with breeding data for a worldwide collection of Gonium pectorale. J Mol Evol 48:
94–101
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:
783–791
Friedl T (1996) Evolution of the polyphyletic genus
Pleurastrum (Chlorophyta): inferences from nuclearencoded ribosomal DNA sequences and motile cell ultrastructure. Phycologia 35: 456–469
Friedl T (1997) The Evolution of the Green Algae. In
Bhattacharya D (ed) The Origin of Algae and Plastids.
Springer-Verlag, Wien, pp 87–101
Geitler L (1954) Echte Oogamie bei Chlamydomonas.
Österr Bot Z 101: 570-578
Gerloff J (1940) Beiträge zur Kenntnis der Variabilität
und Systematik der Gattung Chlamydomonas. Arch
Protistenkd 94: 311–502
Gobi C (1899/1900) Über einen neuen parasitischen
Pilz, Rhizidiomyces ichneumon nov. sp., und seinen
Nährungsorganismus Chloromonas globulosa (Perty).
Scr Bot Horti Univ Imp Petropol 15: 251–272
Gordon J, Rumpf R, Shank SL, Vernon D, Birky Jr
CW (1995) Sequences of the RRN18 genes of Chlamydomonas humicola and C. dysosmos are identical, in
agreement with their combination in the species C. applanata (Chlorophyta). J Phycol 31: 312–313
Goroschankin JN (1891) Beiträge zur Kenntnis der
Morphologie und Systematik der Chlamydomonaden.
II. Chlamydomonas reinhardi Dang. und seine Verwandten. Bull Soc Imp Nat Moscou NS 5: 101–142
Greuter W, Brummitt RK, Farr E, Kilian N, Kirk PM,
Silva PC (1993) NCU-3 Names in Current Use for Extant Plant Genera. Koeltz Scientific Books, Königstein
Molecular Phylogeny and Revision of Chlamydomonas
Hasagawa M, Kishino H, Yano T (1985) Dating the
human-ape splitting by a molecular clock of mitochondrial DNA. J Mol Evol 22: 160-174
Heimke JW, Starr RC (1979) The sexual process in
several heterogamous Chlamydomonas strains in the
subgenus Pleiochloris. Arch Protistenkd 122: 20-42
Hepperle D, Nozaki H, Hohenberger S, Huss VAR,
Morita E, Krienitz L (1998) Phylogenetic position of
the Phacotaceae within the Chlamydophyceae as revealed by analysis of 18S rDNA and rbcL sequences. J
Mol Evol 47: 420-430
Heunert H-H (1973) Microtechnique for the observation of living microorganisms. ZEISS Information 79:
74–85
Hoops HJ, Witman GB (1985) Basal bodies and associated structures are not required for normal flagellar
motion or phototaxis in the green alga Chlorogonium
elongatum. J Cell Biol 100: 297–309
Jaenicke L, Kuhne W, Spessert R, Wahle U, Waffenschmidt S (1987) Cell-wall lytic enzymes (autolysins) of
Chlamydomonas reinhardii are (hydroxy)proline-specific proteases. Eur J Biochem 170: 485–491
Kishino H, Hasegawa M (1989) Evaluation of the maximum likelihood estimate of the evolutionary tree
topologies from DNA sequence data, and the branching order of the Hominoidea. J Mol Evol 29: 170-179
Lewin RA (1984) Chlamydomonas sajao nov. sp.
(Chlorophyta, Volvocales). Chin J Oceanol Limnol 2:
92–94
Marin B, Melkonian M (1999) Mesostigmatophyceae,
a new class of streptophyte green algae revealed by
SSU rRNA sequence comparisons. Protist 150:
399–417
Marin B, Klingberg M, Melkonian M (1998) Phylogenetic relationships among the Cryptophyta: Analyses
of nuclear-encoded SSU rRNA sequences support the
monophyly of extant plastid-containing lineages. Protist 149: 265–276
Matsuda Y, Koseki M, Shimada T, Saito T (1995) Purification and characterization of a vegetative lytic enzyme responsible for liberation of daughter cells during
the proliferation of Chlamydomonas reinhardtii. Plant
Cell Physiol 36: 681–689
McCourt RM (1995) Green algal phylogeny. Trends
Ecol Evol 10: 159–163
Medlin L, Elwood HJ, Stickel S, Sogin M (1988) The
characterization of enzymatically amplified eukaryotic
16S-like rRNA-coding regions. Gene 71: 491–499
Melkonian M (1990) Phylum Chlorophyta. Class
Chlorophyceae. In Margulis L, Corliss JO, Melkonian
M, Chapman DJ (eds) Handbook of Protoctista. Jones
and Bartlett Publ, Boston, pp 608–616
Melkonian N, Preisig HR (1984) An ultrastructural
comparison between Spermatozopsis and Dunaliella
(Chlorophyceae). Plant Syst Evol 146: 31–46
299
Melkonian M, Surek B (1995) Phylogeny of the
Chlorophyta: congruence between ultrastructural and
molecular evidence. Bull Soc Zool Fr 120: 191–208
Morita E, Abe T, Tsuzuki M, Fujiwara S, Sato N, Hirata A, Sonoike K, Nozaki H (1999) Role of pyrenoids
in the CO2-concentrating mechanism: comparative
morphology, physiology and molecular phylogenetic
analysis of closely related strains of Chlamydomonas
and Chloromonas (Volvocales). Planta 208: 365–372
Müller OF (1786) Animalcula infusoria fluviatilia et marina. Hauniae
Nakayama T, Watanabe S, Mitsui K, Uchida H, Inouye I (1996) The phylogenetic relationship between
the Chlamydomonadales and Chlorococcales inferred
from 18S rDNA sequence data. Phycol Res 44: 47–56
Nozaki H, Ohta N, Morita E, Watanabe MM (1998)
Toward a natural system of species in Chlorogonium
(Volvocales, Chlorophyta): A combined analysis of
morphological and rbcL gene sequence data. J Phycol
34: 1024–1037
Nozaki H, Misawa K, Kajita T, Kato M, Nohara S,
Watanabe MM (2000) Origin and evolution of the colonial Volvocales (Chlorophyceae) as inferred from multiple chloroplast gene sequences. Mol Phylogen Evol
17: 256–268
Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comp
Appl Biosci 12: 357–358
Pascher A (1927) Volvocales. In Pascher A (ed)
Süßwasser-Flora Deutschlands, Österreichs und der
Schweiz, Heft 4 Gustav Fischer, Jena, pp 1–506
Pocock MA (1962) Algae from De Klip soil cultures.
Arch Mikrobiol 42: 56–63
Posada D, Crandall KA (1998) Modeltest: testing the
model of DNA substitution. Bioinformatics 14: 817–818
Reize IB, Melkonian M (1989) A new way to investigate living flagellated/ciliated cells in the light microscope: immobilization of cells in agarose. Bot Acta
102: 145–151
Rogers SO, Bendich AJ (1985) Extraction of DNA
from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5: 69–76
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi
R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491
Schlösser UG (1972) Chlamydomonas reinhardtii
(Volvocales) Asexuelle Fortpflanzung. Film E1318, Institut für den wissenschaftlichen Film, Göttingen.
Schlösser UG (1976) Entwicklungsstadien- und sippenspezifische Zellwand-Autolysine bei der Freisetzung von Fortpflanzungszellen in der Gattung Chlamydomonas. Ber Dt Bot Ges 89: 1–56
300
T. Pröschold et al.
Schlösser UG (1982) Sammlung von Algenkulturen,
Pflanzenphysiologisches Institut der Universität Göttingen. Ber Dt Bot Ges 95: 181–276
Schlösser UG (1984) Species-Specific Sporangium
Autolysins (Cell-Wall-Dissolving Enzymes) in the
Genus Chlamydomonas. In Irvine DEG, John DM (eds)
Systematics of the Green Algae. The Systematics Association Special Volume 27, Academic Press, London, pp 409–418
Schlösser UG (1994) SAG- Sammlung von Algenkulturen at the University of Göttingen. Catalogue of
strains 1994. Botanica Acta 107: 113–186
Schlösser UG (1997) Additions to the Culture Collection of Algae since 1994. Bot Acta 110: 424–429
Silva PC (1980) Remarks on algal nomenclature VI.
Taxon 29: 121–145
Starr RC (1955) Isolation of sexual strains of placoderm desmids. Bull Torrey Bot Club 82: 261–265
Starr RC, Zeikus JA (1993) UTEX- the culture collection of algae at the University of Texas at Austin. J Phycol Suppl 29: 1–106
Swofford DL (1998) PAUP*, phylogenetic analysis
using parsimony (* and other methods), version 4.0b2.
Sinauer Associates, Sunderland, MA
Tamura K, Nei M (1993) Estimation of the number of
nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol
Evol 10: 512–526
Watanabe S, Floyd GL (1989) Comparative ultrastructure of the zoospores of nine species of Neochloris
(Chlorophyta). Plant Syst Evol 168: 195–219
Wille N (1903) Algologische Notizen IX.-XIV. Nyt Mag
Naturvidensk 41: 89–185