1. MOLECULAR PHYLOGENETICS AND EVOLUTION
Vol. 5, No. 2, April, pp. 391–402, 1996
ARTICLE NO. 0034
Phylogeny of the Chlamydomonadales (Chlorophyceae):
A Comparison of Ribosomal RNA Gene Sequences
from the Nucleus and the Chloroplast
MARK A. BUCHHEIM,* CLAUDE LEMIEUX,† CHRISTIAN OTIS,† ROBIN R. GUTELL,‡
RUSSELL L. CHAPMAN,§ AND MONIQUE TURMEL†
*Faculty of Biological Science and the Mervin Bovaird Center for Molecular Biology and Biotechnology, The University of Tulsa,
Tulsa, Oklahoma 74104-3189; †Faculte´ des Sciences et de Ge´nie, De´partement de Biochimie, Universite´ Laval, Que´bec
(Que´bec) G1K 7P4, Canada; ‡Department of Molecular, Cellular and Developmental Biology, Campus Box 347,
University of Colorado, Boulder, Colorado 80309; and §Department of Plant Biology,
Louisiana State University, Baton Rouge, Louisiana 70803-1705
Received January 19, 1995; revised July 19, 1995
cludes an ecologically, reproductively, and morphologi-
Phylogenetic analyses of nuclear-encoded small-sub- cally diverse array of biflagellate and quadriflagellate
unit rRNA sequences and chloroplast-encoded large- taxa. Two chlamydomonadalean genera, Chlamydomo-
subunit rRNA sequences from flagellate green algae nas and Carteria, have been the focus of recent phyloge-
representing the order Chlamydomonadales were netic investigations. Results from studies of nuclear-
found to show considerable congruence. In general, encoded small-subunit (SSU) rRNA sequence data
the chloroplast data set exhibited more robust support (Buchheim et al., 1990; Buchheim and Chapman, 1991)
for comparable lineages than the nuclear data set. The and chloroplast-encoded large-subunit (LSU) rRNA se-
phylogenetic inferences derived from the independent
quence data (Turmel et al., 1993) have demonstrated
data sets support some, but also challenge many, tradi-
that the genus Chlamydomonas is tremendously di-
tional taxonomic and phylogenetic concepts regarding
verse at the molecular level and probably does not con-
the green flagellates. Results from phylogenetic analy-
stitute a monophyletic group as it is currently de-
ses of both molecular data sets support six distinct lin-
limited (Buchheim et al., 1990). Similar observations,eages that include taxa from the biflagellate genus,
based on studies of nuclear-encoded rRNA data, haveChlamydomonas, and a basal lineage that comprises
been made regarding the quadriflagellate genus Car-taxa from the quadriflagellate genus, Carteria. Both
teria (Buchheim and Chapman, 1992).data sets support the conclusion that Chlamydomonas
In the present investigation, we have addressed sev-is not monophyletic. Although the chloroplast data are
eral unanswered questions from the aforementionedambiguous regarding the question of Carteria mono-
phylogenetic studies. These include the following. (1)phyly, the nuclear data fail to support Carteria mono-
Are hypotheses of nonmonophyly for the genus Chla-phyly. The chlorococcalean genus Chlorococcum was
mydomonas (Buchheim et al., 1990) robust to addi-found to have affinities with the Chlamydomonadales,
indicating that the traditional concepts of both Chlo- tional taxon and character sampling of the nuclear
rococcales and Chlamydomonadales may need revi- genes? (2) Using a broad taxon sampling scheme cov-
sion. The genus Dunaliella is allied within the Chla- ering several chlamydomonadalean genera, do the
mydomonadales, supporting the contention that it chloroplast data corroborate the hypothesis of non-
has lost a typical glycoprotein cell wall. © 1996 Academic monophyly for the genus Chlamydomonas inferred
Press, Inc. from the nuclear data? (3) Do the chloroplast data cor-
roborate the hypothesis of nonmonophyly for the genus
Carteria inferred from the nuclear data? The taxon
sampling scheme used in our collaborative study also
allows us to test provocative hypotheses regarding theINTRODUCTION
alliance of the genus Chlorococcum (Ettl, 1981, 1983)
and to resolve conflicting views on the phylogeny of Du-The green algal order Chlamydomonadales (sensu
Mattox and Stewart, 1984) is an assemblage of unicel- naliella (Floyd, 1978; Ettl, 1983; Mattox and Stewart,
1984; Chappell et al., 1989; Melkonian, 1990). More-lular green flagellates that represent economically and
taxonomically important components of both freshwa- over, our comparative investigation provides the oppor-
tunity to compare two compartmentally and geno-ter and marine environments world wide. The order in-
391
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Copyright © 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.

2. 392 BUCHHEIM ET AL.
mically independent sources of molecular phylogenetic (Buchheim et al., 1990). New sequence data for Chla-
mydomonas monadina, Chlamydomonas mutabilis,evidence for a parallel set of taxa.
Chlamydomonas nivalis, Chlamydomonas radiata,
and Chlorococcum echinozygotum were obtained by
MATERIALS AND METHODS amplifying genomic DNA using the polymerase chain
reaction (PCR). Genomic DNA was obtained using ex-
Taxon Selection traction protocols described previously (Buchheim and
Chapman, 1992). The flanking primers used to amplifyA total of 31 flagellate taxa were included in the phy-
the nuclear SSU rRNA gene, NS-1 and ITS-2, are de-logenetic analyses (Table 1). Twenty-three of these taxa
scribed by White et al. (1990). These two primers am-consist of Chlamydomonas species representing all of
plify a ca. 2.2-kb product that includes most of the SSUthe 15 sporangial wall autolysin groups sensu Schlo¨sser
rRNA gene. DNA sequences were obtained with end-(1984) and eight of the nine morphological groups
labeled primer ([33
P]dATP, NEN DuPont) and double-(Hauptgruppen sensu Ettl, 1976; 1983; see Table 1).
stranded amplification product utilizing the protocolsMany of the isolates of Chlamydomonas currently
and reagents for cycle-sequencing that accompany themaintained by the Sammlung von Algenkulturen Go¨t-
AmpliTaq Cycle Sequencing kit (Perkin–Elmer). Se-tingen (SAG) have undergone a reexamination culmi-
quencing primers for the cycle-sequencing protocol arenating in name changes for a few of the holdings
identical to the sequencing primers used in the RTase(Schlo¨sser, 1994; personal communication; see also
reactions (see Buchheim et al., 1990; Buchheim andBuchheim et al., 1994). Any changes that affect the
Chapman, 1991, 1992).taxa in this study are listed in Table 2. Because the
studies of nuclear and chloroplast data were conducted
Chloroplast Sequence Dataindependently, the issue of comparable taxon sampling
Sequence data from the chloroplast-encoded LSUmust be addressed. The nuclear and chloroplast data
rRNA gene for 13 of 31 ingroup taxa are from previousfrom all but 9 of the 33 taxa (31 ingroup and 2 outgroup
work (see Table 1). These published sequences were ob-taxa) are derived from the same culture isolate (Table
tained using the T7 DNA polymerase (Pharmacia) in1). Of these nine pairs of isolates that differ, five pairs
the presence of recombinant plasmid DNA clones repre-are regarded as cultures related by source of isolate
senting the entire LSU rRNA gene (Turmel et al.,(Starr and Zeikus, 1993; Schlo¨sser, 1994). The sources
1993). New sequence data for each of the remainingof taxa differ between the nuclear and chloroplast data
18 taxa were generated from three overlapping PCR-for only Scenedesmus obliquus (outgroup taxon), Chla-
amplified fragments using the dsDNA cycle sequenc-mydomonas monadina, Dunaliella parva, and Ste-
ing system from Life Technologies and a collection ofphanosphaera pluvialis.
32
P-labeled primers that are complementary to highly
Nuclear Sequence Data conserved regions of the LSU rRNA gene. These prim-
ers allowed us to sequence the entire coding regions ofSequence data from the nuclear-encoded SSU rRNA
gene for 20 of 31 ingroup taxa are from previous investi- the PCR-amplified fragments and part of the introns
that were found. For most taxa, the 5′ and 3′ ends ofgations (Table 1). These published nuclear sequences
were obtained using either rRNA templates (Buchheim the LSU rRNA gene segment amplified correspond to
positions 38 and 2598, respectively, in the Escherichiaet al., 1990; Buchheim and Chapman, 1991, 1992) or
DNA templates (Gunderson et al., 1987; Huss and coli 23S rRNA (Gutell et al., 1994). The PCR amplifica-
tions were carried out using genomic DNA and previ-Sogin, 1990; Lewis et al., 1992). Of those taxa for which
only partial rRNA sequences were obtained, an average ously described conditions (Turmel et al., 1994). Geno-
mic DNA from each taxon was prepared as follows.of 250 bases per taxon (except Chlamydomonas callosa,
Carteria olivieri, and Chlorogonium elongatum, for Cells from a 500-ml culture that was grown in modified
Volvox medium (McCracken et al., 1980) were har-which template was no longer available) were added for
this investigation. These sequence additions are de- vested by centrifugation, washed twice in 500 µl of
buffer A (10 mM Tris–HCl, pH 8.0, 10 mM EDTA, 10rived from the use of the 18P universal eukaryotic
primer (Hamby et al., 1988) as a sequencing primer in mM NaCl), resuspended in 250 µl of buffer A, and
ground with liquid nitrogen with the aid of a mortarthe RTase reactions. The sequence additions roughly
correspond to positions 650 through 950 of the pub- and pestle. As grinding proceeded, the mortar was
slowly warmed; immediately after all of the liquid ni-lished Chlamydomonas reinhardtii sequence (Gun-
derson et al., 1987). New sequence data for Chla- trogen had evaporated, 15 µl of a 1 mg/ml proteinase
K solution and 15 µl of 10% (w/v) sodium dodecyl sul-mydomonas algoe¨formis, Chlamydomonas gigantea,
Chlamydomonas pallidostigmatica, Chlamydomonas fate were added. The resulting mixture was transferred
into a 1.5-ml microtube, incubated at 50°C for 2 h, andpseudopertusa, Chlamydomonas starrii, and Chla-
mydomonas sp. SAG 66.72 were obtained using then extracted successively with 250 µl of phenol, phe-
nol:chloroform:isoamyl alcohol (25:24:1), and chloro-rRNA templates and RTase as described previously

3. PHYLOGENY OF CHLAMYDOMONADALES 393
TABLE 1
List of Taxa Examined in This Study
Sourcec
Autolysin Morphological
groupa
groupb
Nuclear Chloroplast
Outgroup taxon
Ankistrodesmus Corda
stipitatus (Chodat) Korma´rkova´ Legnerova´ NAd
NA SAG 202-5e
SAG 202-5
Scenedesmus Meyen
obliquus (Turp.) Ku¨tz. NA NA SAG 276-3ae
UTEX 393
Ingroup taxon
Carteria Diesing
crucifera Korshikov NA Pseudagloe¨ UTEX 432f
UTEX 432
olivieri West NA Eucarteria UTEX LB 1032f
UTEX LB 1032
radiosa Korshikov NA Eucarteria UTEX LB 835f
UTEX LB 835
Chlamydomonas Ehrenberg
agloe¨formis Pascher NA Pseudagloe¨ UTEX 231 UTEX 231
callosa Gerloff 6 Euchlamydomonas UTEX 624g
SAG 9.72
eugametos Moewus 12 Chlamydella UTEX 9g
UTEX 9k
frankii Pascher 9 Euchlamydomonas SAG 19.72g
SAG 19.72k
geitleri Ettl 14 Chlorogoniella SAG 6.73g
SAG 6.73k
gelatinosa Korshikov 11 Euchlamydomonas SAG 69.72g
SAG 69.72k
gigantea Dill NA Pleiochloris UTEX LB 848 UTEX LB 848
humicola Lucksch 7 Chlorogoniella SAG 11-9g
SAG 11-9k
iyengarii Mitra 5 Euchlamydomonas SAG 25.72h
SAG 25.72k
komma Skuja 2 Euchlamydomonas SAG 26.72g
SAG 26.72k
mexicana Lewin 4 Chlorogoniella SAG 11-60ag
SAG 11-60ak
monadina Stein NA Chlamydella SAG 31.72 SAG 55.72
mutabilis Gerloff NA Pseudagloe¨ UTEX 578 SAG 34.72
nivalis (Bauer) Wille NA Sphaerella UTEX LB 1969 UTEX LB 1969
pallidostigmatica King 10 Chlamydella SAG 9.83 SAG 9.83k
peterfii Gerloff 3 Chlamydella SAG 70.72g
SAG 70.72k
pitschmannii Ettl 13 Chlorogoniella SAG 14.73g
SAG 14.73k
pseudopertusa Ettl NA Amphichloris SAG 42.72 SAG 42.72
radiata Deason et Bold NA Agloe¨ UTEX 966 UTEX 966
reinhardtii Dangeard 1 Euchlamydomonas CC-400i
SAG 11-32bk
starrii Ettl 12 Chlorogoniella SAG 3.73 SAG 3.73
sp. 66.72 8 NA SAG 66.72 SAG 66.72k
zebra Korshikov 15 Euchlamydomonas SAG 10.83g
SAG 10.83k
Chlorococcum Meneghini
echinozygotum Starr NA NA UTEX 118 SAG 213-5
Chlorogonium Ehrenberg
elongatum (Dang.) Dang. NA NA UTEX 11g
UTEX 11
Dunaliella Teodorescu
parva Lerche NA NA UTEX LB 1983j
SAG 19(1)
Haematococcus C. A. Agardh
lacustris (Girod-Chantras) Rostafinski. NA NA UTEX 16h
SAG 34-1b
Stephanosphaera Cohn
pluvialis Cohn NA NA UTEX LB 771h
SAG 78-1a
a
Sporangial wall autolysin group sensu Schlo¨sser (1984).
b
Morphological group sensu Ettl [1976, 1983 (Chlamydomonas taxa); 1979, 1983 (Carteria taxa)].
c
Source of taxa used to generate the data (nuclear or chloroplast): CC, from the Chlamydomonas Genetics Center at Duke University;
SAG, from the Sammlung von Algenkulturen Go¨ttingen; UTEX, from the Culture Collection at the University of Texas at Austin.
d
Not applicable or information not available.
e
Partial sequence extracted from published sequence data of Huss and Sogin (1990).
f
Includes published sequence data of Buchheim and Chapman (1992).
g
Includes published sequence data of Buchheim et al. (1990).
h
Includes published sequence data of Buchheim and Chapman (1990).
i
Partial sequence extracted from published sequence data of Gunderson et al. (1987).
j
Partial sequence extracted from published sequence data of Lewis et al. (1992).
k
Published sequence data of Turmel et al. (1993).

4. 394 BUCHHEIM ET AL.
TABLE 2 tively. The alignments are available from the authors
upon request.
List of Nomenclatural and Taxonomic Changes
among SAG Isolates of Chlamydomonas Data Analyses
Original Reason Three methods of phylogenetic reconstruction, maxi-
designation New designation for change
mum parsimony, neighbor-joining (NJ), and maximum
likelihood (ML), were employed in a comparison of theChlamydomonas
agloe¨formis Chlamydomonas debaryana Goroschankin Reexaminationa
two independent molecular data sets. All parsimony
eugametos Chlamydomonas moewusii Gerloff Synonymyb
analyses were conducted using the program PAUP,
frankii Chlamydomonas culleus Ettl Reexaminationa
version 3.1.1 (Swofford, 1993) mounted on a Macin-geitleri Chlamydomonas noctigama Korshikov Reexaminationa
gelatinosa Sphaerellopsis aulata (Pascher) Gerloff Reexaminationa
tosh Quadra (800 or 950) machine. Bootstrap values
humicola Chlamydomonas applanata Pringsheim Reexaminationc
(Felsenstein, 1985) from 100 resamplings were calcu-
iyengarii Chlamydomonas proboscigera Korshikov Synonymyb
lated for each set of data. Decay indices (Bremer, 1988;komma Chlamydomonas debaryana Goroschankin Reexaminationa
mexicana Chlamydomonas oblonga Pringsheim Reexaminationa
Mishler et al., 1991) were also calculated and mapped
nivalis Chlamydomonas augustae Skuja Reexaminationa
to cladograms or consensus trees reconstructed frompeterfii Chlamydomonas asymmetrica Korshikov Reexaminationa
each of the data sets. Tree searches were conductedpallidostigmatica Chlamydomonas segnis Ettl Reexaminationa
sp. SAG 66.72 Chlorococcum novae-angliae Archibald et Bold Reexaminationa
heuristically using the TBR option with MULPARS.
To increase the probability of finding all islands ofa
Schlo¨sser (1994, personal communication).
most parsimonious trees, the order of taxon additionb
Ettl (1983).
was randomized 50 times. All trees were rooted usingc
Ettl and Schlo¨sser (1992).
the outgroup method. Characters were optimized to
branches under the assumption of accelerated transfor-
mation (ACCTRAN). All characters were regarded as
unordered (Fitch, 1971).form:isoamyl alcohol (24:1). Following precipitation
For the NJ analyses (Saitou and Nei, 1987) of thewith ethanol in the presence of 0.2 M NaCl, nucleic
nuclear and chloroplast data sets, 1000 bootstrap repli-acids were dissolved in 100 µl of TE buffer (10 mM Tris–
cates of each data set were generated using SEQBOOT,HCl, pH 8.0, 1 mM EDTA), 2 µl of a 10 mg/ml RNase
and pairwise distances were calculated with DNADISTA solution was added, and the resulting mixture was
using the Kimura (1980) two-parameter model of nucle-incubated at 37°C for 30 min. The DNA was precipi-
otide change. Topologies were reconstructed from thetated three times with ethanol in the presence of 2.5 M
distance matrices using NEIGHBOR. Majority-ruleammonium acetate and was finally dissolved in 10 µl
consensus trees were produced using CONSENSE, andTE.
branch lengths were estimated using FITCH with the
Sequence Alignments user tree option. All of these analyses were carried out
using the PHYLIP package, version 3.5c (Felsenstein,Previous work (Buchheim et al., 1990; Turmel et al.,
1993) served as the starting point for all alignments. 1993) mounted on a Sun SPARCstation 10 Model 40.
Each ML analysis was conducted using the fast-Both sets of sequences, nuclear and chloroplast, were
manually aligned on the basis of conserved primary DNAml software (Olsen et al., 1994) mounted on a Sun
SPARCstation 10 and the fastDNAml boot script pro-and secondary structure models. The alignments of nu-
clear sequences were completed on a VAX-6320 com- vided with this software. Bootstrap values from 100 re-
samplings were calculated for each data set. Majority-puter (University of Oklahoma Genetic Computer
Group) using the LINEUP program from the GCG se- rule consensus trees were produced using CONSENSE,
and branch lengths were estimated using fastDNAmlquence analysis package (Genetics Computer Group,
1991), version 7.3, whereas the chloroplast sequences with the user tree option.
S. obliquus was used as an outgroup taxon to root thewere aligned on a Sun SPARCstation 10 Model 40 us-
ing the Genetic Development Environment program trees calculated with the NJ and ML methods, whereas
both S. obliquus and Ankistrodesmus stipitatus were(Stephen Smith, previously of the Harvard Genome
Laboratory). The secondary structure model for Volvox employed as outgroup taxa to root the trees generated
with the parsimony method. These two autosporiccarteri (Rausch et al., 1989) was used to assist in
aligning the partial nuclear rRNA sequences. The sec- (non-zoospore-producing) taxa are classified in the
green algal order Chlorococcales by most algal taxono-ondary structures of all chloroplast rRNA sequences
were modeled using the program XRNA (B. Wieser, un- mists. The Chlorococcales represent one of seven or-
ders, including the Chlamydomonadales, within thepublished results). For the phylogenetic analyses, re-
gions not clearly alignable for all taxa were excluded. Chlorophyceae (sensu Mattox and Stewart, 1984); thus,
morphological criteria indicate that these two taxa areThe nuclear and chloroplast data sets used in our study
consist of 990 and 2426 aligned nucleotides, respec- not within the group of interest.

5. PHYLOGENY OF CHLAMYDOMONADALES 395
TABLE 3 mannii, C. geitleri, C. pseudopertusa, Chlamydomonas
sp. SAG 66.72, and Clc. echinozygotum), a ‘‘C. gigantea’’
Comparison of the Primary Structure of the Nuclear
lineage (C. gigantea, C. frankii, and C. pallidostigma-
and Chloroplast Sequence Data
tica), a ‘‘C. radiata’’ lineage (C. radiata, C. nivalis, and
C. mutabilis), and a Carteria lineage (Car. olivieri andNuclear Chloroplast
Category of comparison data data Carteria crucifera). Although congruent regarding
some of the major divergences (see Strict, Fig. 1), the
Total number of sites 990 2426
results from the three distinct methods of phylogeneticTotal variable sites 220 (22.2%) 865 (35.7%)
reconstruction differ from one another in some re-Total binary transitions 106 (10.7%) 345 (14.2%)
Total A ↔ G transitions 51 (5.2%) 182 (7.5%) spects. The relative positions of Carteria radiosa and
Total C ↔ T transitions 55 (5.5%) 163 (6.7%) Chlamydomonas monadina are especially labile across
Total binary transversions 61 (6.2%) 201 (8.3%)
the three methods. The former ranges from an alliance
Total A ↔ C transversions 8 (0.8%) 39 (1.6%)
with the Haematococcus lineage (Pars and NJ, Fig. 1)Total G ↔ T transversions 17 (1.7%) 66 (2.7%)
to a position as the sister group to the C. radiata lin-Total A ↔ T transversions 19 (1.9%) 75 (3.1%)
Total G ↔ C transversions 17 (1.7%) 21 (0.9%) eage (ML, Fig. 1). The position of C. monadina varies
Total multiple-state sites 53 (5.4%) 309 (12.7%) from the sister group to the Haematococcus lineage
Informative sites 126 (12.7%) 586 (24.2%)
(Pars and NJ, Fig. 1) to the sister group of the C. euga-
Informative binary transitions 65 (6.6%) 210 (8.7%)
metos lineage (ML, Fig. 1). In addition, the position ofInformative A ↔ G transitions 28 (2.8%) 106 (4.4%)
the C. radiata lineage relative to the HaematococcusInformative C ↔ T transitions 37 (3.7%) 104 (4.3%)
Informative binary transversions 18 (1.8%) 102 (4.2%) and C. eugametos lineages is either ambiguous (Pars
Informative A ↔ C transversions 2 (0.2%) 18 (0.8%) and NJ, Fig. 1) or is weakly resolved (ML, Fig. 1). Last,
Informative G ↔ T transversions 7 (0.7%) 31 (1.3%)
the C. gigantea lineage varies from a position as theInformative A ↔ T transversions 7 (0.7%) 44 (1.8%)
sister group of the Euchlamydomonas lineage (ParsInformative G ↔ C transversions 2 (0.2%) 9 (0.4%)
and ML, Fig. 1) to a position as the sister group to aInformative multiple-state sites 43 (4.2%) 266 (11.0%)
clade that includes the two Carteria taxa and is basal
to the Haematococcus and C. eugametos lineages (NJ,
Fig. 1).
RESULTS
Phylogenetic Analysis of Chloroplast Data
Structure of the Sequence Data Parsimony analysis of the chloroplast data set re-
sulted in a single minimal length tree (L ϭ 2464, CI ϭA summary of the primary structure of the two in-
0.418, RI ϭ 0.591; see Fig. 2). The results from NJ anddependent data sets is presented in Table 3. The
ML analysis of the chloroplast data are also presentedsummary includes a comparison of variable and in-
in Fig. 2. The chloroplast data consistently resolve theformative sites, transitions and transversions, and
same seven lineages determined from the nuclear datamultistate sites. The chloroplast data are more variable
(i.e., ‘‘Euchlamydomonas’’ lineage, ‘‘C. mexicana’’ lin-than the nuclear data in terms of both number and per-
eage, ‘‘Haematococcus’’ lineage, ‘‘C. eugametos’’ lineage,centage of variable sites as well as number and percent-
‘‘C. gigantea’’ lineage, ‘‘C. radiata’’ lineage, and a ‘‘Car-age of informative sites (see discussion below).
teria’’ lineage). In addition, the chloroplast data are
Phylogenetic Analysis of Nuclear Data more robust across methods of phylogenetic reconstruc-
tion than the nuclear data. The results from the threeParsimony analysis of the nuclear data set resulted
analyses (Fig. 2) differ only in (1) the position of D.in 24 minimal length trees [L ϭ 326, CI ϭ 0.518 (Kluge
parva and H. lacustris within the Haematococcus lin-and Farris, 1969), RI ϭ 0.682 (Farris, 1989)]. A strict
eage, (2) the relative positions of C. gelatinosa, C. cal-consensus analysis (Rohlf, 1982) illustrates the taxo-
losa, C. reinhardtii, and C. zebra within the Euchlamy-nomic congruence between the competing hypotheses
domonas lineage, and (3) the position of Car. radiosa.(Fig. 1). Results from NJ and ML analysis of the nu-
clear data are also presented in Fig. 1. The nuclear data
consistently resolve seven lineages that include a
DISCUSSION‘‘Euchlamydomonas’’ lineage (C. reinhardtii, Chlamy-
domonas komma, C. starrii, C. callosa, Chlamydo-
Strength and Quality of Phylogenetic Signalmonas iyengarii, and Chlamydomonas zebra), a ‘‘Chla-
mydomonas mexicana’’ lineage (C. mexicana and C. The chloroplast phylogenies are clearly more robust
than the nuclear phylogenies. This difference is best ex-peterfii), a ‘‘Haematococcus’’ lineage (Haematococcus
lacustris, C. agloe¨formis, C. humicola, Chlorogonium plained by an examination of the structure of the two
data sets. One possible factor in this difference betweenelongatum, S. pluvialis, and D. parva), a ‘‘Chlamydo-
monas eugametos’’ lineage (C. eugametos, C. pitsch- data sets may be the use of partial nuclear sequences

6. 396 BUCHHEIM ET AL.
FIG. 1. Analyses of nuclear-encoded SSU rRNA sequence data using the methods of maximum parsimony, NJ, and ML. The parsimony
(Pars) tree is a strict consensus analysis of 24 minimal length trees. Bootstrap values for all unambiguously resolved nodes are included
above each internode; corresponding DI values for the parsimony tree are included below each internode. Note that bootstrap values below
50 are not reported for the parsimony tree. A strict consensus tree of the parsimony, NJ, and ML trees is also shown.
chloroplast data have nearly twice the number (as ain comparison with the complete chloroplast sequences.
In addition, the nuclear data have more ambiguous percentage of the total number of sites) of informative
sites compared to that in the nuclear data set (Tablesites (a consequence of sequences derived from reverse
transcriptase sequencing) than the chloroplast data. 3). Moreover, the number of informative transversions
in the chloroplast data set is nearly quadruple that inAlthough the differences in the structure of variation
between nuclear and chloroplast data sets are less dra- the nuclear data set (Table 3). These two observa-
tions are consistent with the results of analyses of thematic than the difference in size (990 sites vs 2426
sites, respectively; see Table 3), the chloroplast data two independent data sets in which the branches on
trees derived from the chloroplast data generally ex-generally have greater percentages of binary transi-
tions, binary transversions, and multistate sites than hibit more robust character support than comparable
branches on trees derived from analysis of the nuclearthe nuclear data. The more important observation, at
least in terms of the parsimony analyses, is that the data. In other words, the chloroplast data are not only

7. PHYLOGENY OF CHLAMYDOMONADALES 397
FIG. 2. Analyses of chloroplast-encoded LSU rRNA sequence data using the methods of maximum parsimony, NJ, and ML. The parsi-
mony (Pars) tree is the single minimal length tree found. Bootstrap values for all unambiguously resolved nodes are included above each
internode; corresponding DI values for the parsimony tree are included below each internode. A strict consensus tree of the parsimony, NJ,
and ML trees is also shown.
comprising biflagellate unicells. Ettl (1976, 1983) hasmore variable, but also have a greater density of phylo-
genetically informative sites than the nuclear data. As organized the members of the genus into nine morpho-
logical Hauptgruppen. The nine Hauptgruppen differwould be predicted from these observations, the chloro-
plast data (CI ϭ 0.418, RI ϭ 0.591) exhibit greater lev- from one another primarily in chloroplast shape, pyre-
noid position, pyrenoid number, and papillum size andels of detected homoplasy than the nuclear data (CI ϭ
0.518, RI ϭ 0.682). We have attempted to minimize shape. Results from analyses of both molecular data
sets strongly support the conclusion that the genusundetected homoplasy in both data sets through the
use of relatively extensive taxon sampling. Chlamydomonas is not monophyletic and, in addition,
they suggest that the morphological criteria used to de-
Taxonomic and Phylogenetic Implications
limit the nine Hauptgruppen (sensu Ettl, 1976, 1983)
Chlamydomonas. Chlamydomonas is a speciose (ca. should be reexamined. In general, the topologies from
450 species sensu Ettl, 1976, 1983), green algal genus both data sets are inconsistent with the morphological

8. 398 BUCHHEIM ET AL.
groups. The only morphological alliance that demon- can be demonstrated between flagellate genera. For ex-
ample, both the nuclear and chloroplast data indicatestrates any degree of cohesiveness when tested against
the molecular data is the Euchlamydomonas Haupt- a close alliance between Clc. echinozygotum and either
C. eugametos and/or C. pitschmannii. Are the autoly-gruppe. A number of taxa ascribed to this group, includ-
ing C. reinhardtii, form a monophyletic assemblage sins of either of these Chlamydomonas taxa similar to
that of Clc. echinozygotum? If autolysin data are to(Figs. 1 and 2). However, the alliance of C. starrii (Chlo-
rogoniella sensu Ettl, 1976, 1983) among these euchla- have relevance for green algal systematics in general,
then the scope of comparison must be broadened be-mydomonads (Figs. 1 and 2) represents an exception to
this generalization. Assuming that the two molecular yond the genus Chlamydomonas given the compelling
evidence for nonmonophyly within the genus.data sets are accurately recovering historical informa-
tion, the inconsistencies between molecular-based phy-
logenies and morphological-based classifications may Is Carteria monophyletic? Although not nearly as
speciose, the quadriflagellate, unicellular genus Car-be due to homoplasy in the morphological data. Given
that Ettl’s (1976) classification is based extensively on teria parallels Chlamydomonas in morphological vari-
ability (Ettl, 1979, 1983). Lembi (1975) has demon-chloroplast morphology, one could conclude that vari-
ability in this character may be plastic or exhibit non- strated that at least two distinct, ultrastructurally
defined lineages exist among species of Carteria. Thehomology. However, it must also be noted that the in-
consistencies between molecules and morphology may two lineages have recently been examined using nu-
clear-encoded rRNA sequence data (Buchheim andsimply be a consequence of comparing a classification
based on a subjective evaluation of evidence (i.e., mor- Chapman, 1992). These molecular data corroborate the
ultrastructural interpretation and, furthermore, theyphological) with a phylogenetic interpretation of evi-
dence (i.e., molecular). The morphological criteria need indicate that these lineages are not monophyletic
(Buchheim and Chapman, 1992). Results of analysesto be reexamined to identify potential plasticity or non-
homology of characters. Furthermore, these morpho- from the present investigation support the distinction
of two Carteria lineages (Car. olivieri ϩ Car. cruciferalogical data need to be interpreted within a phyloge-
netic framework. vs Car. radiosa). The nuclear data remain consistent
in failing to support monophyly of the genus CarteriaDiversity within the genus Chlamydomonas has also
been demonstrated at the biochemical level. Schlo¨sser (Fig. 1), whereas the chloroplast data are ambiguous
regarding the question of Carteria monophyly. The par-(1984) presented evidence for the existence of 15 dis-
tinct lineages within the genus Chlamydomonas based simony and ML analyses of the chloroplast data sup-
port a monophyletic Carteria (88 and 52% bootstrapon differences in enzymatic specificity of sporangial
wall autolysins. Because the taxon sampling scheme values, respectively), while NJ analysis supports a
paraphyletic Carteria (54% bootstrap value; see Fig. 2).for both the nuclear and chloroplast data sets was pri-
marily based on autolysin diversity, the phylogenetic Although error due to limited taxon sampling in the
genus Carteria may be influencing the results of analy-analyses do not provide the opportunity to thoroughly
test the autolysin classification. However, both sets of ses from both data sets, the inclusion of sequence data
for an additional Carteria taxon (Car. lunzensis SAG 8-molecular data presented here are consistent with two
inferences from the autolysin evidence and inconsis- 3) in the chloroplast data set does not resolve the issue.
The parsimony and ML analysis support monophyly,tent with another. For example, C. peterfii and C. mexi-
cana produce distinct sporangial wall autolysins (types whereas the NJ analysis supports paraphyly (data not
shown). While failing to support Carteria monophyly,three and four, respectively); however, Schlo¨sser (1984)
demonstrated that the type three autolysin is capable the nuclear data are ambiguous, under different meth-
ods of phylogeny reconstruction, regarding the place-of lysing the type four sporangial wall. In addition, the
type 14 autolysin (e.g., C. geitleri) is capable of lysing ment of Car. radiosa (Fig. 1). This observation suggests
that the nuclear data may be influenced by inadequatethe type 8 sporangium (Chlamydomonas sp. SAG
66.72). These autolysin cross-reactivities are sugges- taxon sampling in the genus Carteria.
Aside from the apparent differences between molecu-tive of a phylogenetic link that is corroborated by mo-
lecular evidence (Figs. 1 and 2). In contrast, while C. lar data sets regarding the Carteria question, both are
congruent in supporting a basal lineage within the or-starrii and C. eugametos are reported to share the type
12 autolysin (Schlo¨sser, 1984), molecular data do not der Chlamydomonadales that includes the quadriflag-
ellates Car. olivieri and Car. crucifera. In the absencesupport the alliance inferred from the biochemical evi-
dence (Figs. 1 and 2). Clearly, C. starrii and C. euga- of any other information, one would conclude that the
quadriflagellate condition is the ancestral state for themetos are candidates for a reexamination of autolysin
data. Another new line of investigation is suggested by Chlamydomonadales (see also Buchheim and Chap-
man, 1992). Thus, the quadriflagellate condition, whichthe observation that at least some of the distinct autol-
ysin groups appear to have allies other than Chlamydo- appears to be the single synapomorphy for the genus
Carteria, may be plesiomorphic and, therefore, cannotmonas. Specifically, it would be of interest to green al-
gal phylogeneticists to determine if autolysin similarity be used to diagnose a taxon (see also Buchheim and

9. PHYLOGENY OF CHLAMYDOMONADALES 399
Chapman, 1992). Despite what are compelling hen- halophilic, unicellular, biflagellate green algae that
have been characterized as wall-less (e.g., Smith,nigian arguments, the limited taxon sampling forces us
to conclude that the issue of Carteria monophyly re- 1950). However, careful examination has shown that
Dunaliella exhibits an extracellular, wall-like layermains unresolved.
(Oliveira et al., 1980, Melkonian and Preisig, 1984).
Chlorococcum and the class Chlamydophyceae.
Like Chlorococcum, Dunaliella has been the subject of
Both molecular data sets include sequences from two
debate regarding its phylogenetic position. Ettl (1981,
members of the genus Chlorococcum, Clc. novae-
1983) considered the absence of a typical cell wall as
angliae (originally identified as Chlamydomonas sp.
evidence against the inclusion of Dunaliella with other
SAG 66.72, see Table 2) and Clc. echinozygotum. This
anteriorly biflagellate unicells that he placed in the
genus has been the subject of some debate among algal
Chlamydophyceae (sensu Ettl, 1981; 1983). Conse-
systematists. Chlorococcum is characterized by a life
quently, Ettl separated Dunaliella from the walled
history that includes a zoospore (motile) stage and a
flagellates (i.e., order Chlamydomonadales, class Chla-
unicellular, nonmotile, vegetative stage. Both the zoo-
mydophyceae, sensu Ettl, 1981) into the order Dunali-
spore and the nonmotile vegetative stage possess cell
ellales (class Chlorophyceae sensu Ettl, 1981, 1983). Al-
walls. Although the zoospore stage is strongly reminis-
though Melkonian (1990) did not adopt the broad
cent of a chlamydomonad vegetative cell (which are
concept of the classes Chlorophyceae and Chlamydo-
normally motile), the nonmotile stage has been cited as
phyceae, he did recognize an order Dunaliellales as sep-
evidence of a link with other nonmotile, unicellar algae
arate from the Chlamydomonadales, within a class
which lack any motile stages. Traditionally, unicellular
Chlorophyceae (sensu Melkonian, 1990), arguing that
organisms with a nonmotile, walled, vegetative stage
ultrastructural differences indicate that Dunaliella is
have been placed in the order Chlorococcales by algal
not a wall-less equivalent of Chlamydomonas. In con-
systematists (e.g., Smith, 1950; Prescott, 1951; Mattox
trast, Floyd (1978), Mattox and Stewart (1984), and
and Stewart, 1984; Bold and Wynne, 1985; Melkonian,
Chappell et al. (1989), citing similarities in cell division
1990). However, the observation that the zoospores
and flagellar apparatus architecture and interpreting
from some members of the Chlorococcales (e.g., Chlo-
the absence of a typical cell wall as loss or extreme mod-
rococcum) exhibit a glycoprotein cell wall like that ob-
ification, argued for an alliance of Dunaliella with other
served on the motile vegetative cells of the Chlamydo-
chlamydomonadalean flagellates. Using complete nu-
monadales (Miller, 1978) has been acknowledged to be
clear SSU rRNA sequences, Lewis et al. (1992) showed
of potential phylogenetic significance (Melkonian,
that D. parva is allied with Characium vacuolatum and
1990). Both Clc. novae-angliae (Ettl and Ga¨rtner, 1988)
Ettlia minuta, both of which are chlorococcalean taxa
and Clc. echinozygotum (Deason, 1983) have been
that produce walled zoospores. The D. parva sequence
shown to exhibit cell wall features similar to those of
published by Lewis et al. (1992) was used in the nuclear
the Chlamydomonadales. Ettl (1981) has argued that
data set of the present investigation. Our results,
the presence of a glycoprotein cell wall on the motile
which corroborate the findings of Lewis et al. (1992)
stages of Chlamydomonas, Chlorococcum, and other
and support the conclusions of Floyd (1978), Mattox
unicellular green algae is an important, class-level phy-
and Stewart (1984), and Chappell et al. (1989), demon-
logenetic marker. Specifically, Ettl has erected a new
strate that D. parva is allied with the chlamydomona-
class of green algae, the Chlamydophyceae, that in-
dalean (walled) flagellates. Thus, in the case of Duna-
cludes many taxa that produce zoospores possessing a
liella, the molecular data are inconsistent with the
glycoprotein cell wall. Although Ettl’s classification has
concept of the classes Chlamydophyceae and Chloro-
been criticized for being inconsistent in applying diag-
phyceae (sensu Ettl, 1981, 1983) and do not support the
nostic criteria (Mattox and Stewart, 1984), our molec-
concept of a separate order Dunaliellales (sensu Mel-
ular data suggest that at least two species of Chloro-
konian, 1990). If we assume that D. parva is typical of
coccum have chlamydomonadalean sister taxa. As a
the genus (see Lerche, 1937) and if the cell surface coat
consequence, the results from analyses of molecular
of Dunaliella is indeed glycoprotein in nature (Oliveira
data offer two alternatives for the status of the genus
et al., 1980), then the results from analyses of molecu-
Chlorococcum. Either Chlorococcum does not comprise
lar evidence presented here lead us to conclude that the
a natural group or the genus should not be allied with
extracellular layer in Dunaliella is modified from the
other chlorococcalean taxa (sensu Smith, 1950; Mattox
glycoprotein cell wall of an ancestral chlamydomonada-
and Stewart, 1984; Bold and Wynne, 1985; Melkonian,
lean taxon.
1990), but rather should be allied with the Chlamydo-
monadales. Only additional taxon sampling within the
The Haematococcus lineage. Of the core taxa
genus Chlorococcum will allow us to resolve this issue.
within the Haematococcus lineage, four (Dunaliella,
Nonetheless, the results from the present investigation
Haematococcus, Chlorogonium, and Stephanosphaera)
have profound implications for our concept of ordinal
possess morphological features that may represent syn-
classification within the green algae.
apomorphies of the group or for a subset of taxa within
the group. Specifically, all four exhibit a persistence ofDunaliella. Members of the genus Dunaliella are

10. 400 BUCHHEIM ET AL.
motility or sporangial flagella during sporulation of the C. radiata, C. eugametos, and Haematococcus lin-
eages, and the other major clade composed of the Eu-(Droop, 1956a, b; Pocock, 1960; Ettl, 1983). In addition,
Chlorogonium, Haematococcus, and Stephanosphaera chlamydomonas and C. gigantea lineages (Fig. 2). The
nuclear data are ambiguous, under different methodsexhibit multiple (Ͼ2) contractile vacuoles (CVs) scat-
tered throughout the cytoplasm. Two apical CVs is the of phylogeny reconstruction, regarding the position of
the C. radiata lineage within the major clade (Fig. 1),typical condition observed for virtually all other chla-
mydomonadalean taxa (Ettl, 1983; Melkonian, 1990). whereas the chloroplast data consistently place the C.
radiata lineage at the base of the clade (Fig. 2). BothBecause both multiple and scattered CVs and persis-
tent motility/flagella during cell division appear to rep- data sets strongly suggest that the C. mexicana lineage
is the sister group to the Euchlamydomonas lineage.resent apomorphic states, it follows that these char-
acters represent evidence of congruence with the Under all three methods of phylogeny reconstruction,
the chloroplast data strongly suggest that the C. gigan-molecular data. However, both data sets indicate that
C. agloe¨formis and C. humicola are allied within the tea clade is the sister group to the Euchlamydomonas
plus C. mexicana clade. Parsimony and ML analysis ofHaematococcus lineage. Neither of these taxa are re-
ported to exhibit either persistence of motility/flagella the nuclear data also support this alliance.
during cell division or multiple, scattered CVs (Ettl,
Summary and Conclusions
1976, 1979, 1983). In fact, C. humicola and several re-
The two data sets are congruent for those aspects oflated isolates in autolysin group seven have been re-
the respective topologies that are relatively robust andcently reexamined (Ettl and Schlo¨sser, 1992) and
differ for those parts of the topologies that are not. Bothshown to exhibit two apical CVs and flagellar resorp-
data sets fail to support a monophyletic Chlamydomo-tion prior to the onset of zoosporogenesis. Thus, C. hu-
nas, resolving at least six lineages that include speciesmicola represents an exception to the morphological
of this genus. Neither data set is consistent with thetrends exhibited by other taxa in the Haematococcus
Hauptgruppen scheme for the genus Chlamydomonaslineage. One explanation of this observation is to raise
(sensu Ettl, 1976, 1983). The issue of monophyly/pa-the possibility that some of the putative synapomor-
raphyly of the genus Carteria has not been resolved.phies may not be homologous across taxa. For example,
The nuclear data set fails to support a monophyleticthe form of persistent flagella that characterizes Duna-
Carteria, whereas the chloroplast data set is ambigu-liella may be fundamentally different from that in the
ous regarding the question of monophyly. The allianceother taxa in the Haematococcus lineage. Although Du-
of Chlorococcum among the Chlamydomonadales notnaliella is not truly wall-less, it exhibits binary fission
only represents support for concluding that the Chloro-which is typical of other wall-less flagellates. In con-
coccales is not a natural group (Fritsch, 1945; Mattoxtrast, multiple fission is typical of most chlamydomo-
and Stewart, 1984; Melkonian, 1990), but also chal-nad flagellates. Thus, it is logical to assume that binary
lenges the traditional concepts of both the Chlorococ-fission in Dunaliella is coupled with the loss of a typical
cales and Chlamydomonadales. Both data sets supportchlamydomonad cell wall. Nonetheless, these morpho-
the halophilic Dunaliella as a member of the Chlamy-logical differences indicate we must be cautious in our
domonadales, suggesting that the unusual cell surfaceinterpretation of character homology. At the very least,
coating of Dunaliella is modified from the typical gly-these observations indicate that persistent motility/
coprotein wall of other chlamydomonadalean taxa.flagella take different forms within the Haematococcus
Within the Haematococcus lineage, evidence of congru-lineage. Expanded taxon sampling may help resolve
ence between molecular and morphological data issome of these issues, but a careful comparison of cell
found in the case of taxa that exhibit multiple and scat-division characteristics will be needed to reconcile the
tered CVs (Haematococcus, Chlorogonium, and Ste-apparent differences between our phylogenetic analy-
phanosphaera) and possess flagellated division stagessis of molecular data and the interpretation of morpho-
(Dunaliella, Haematococcus, Chlorogonium, and Ste-logical evidence presented above.
phanosphaera). However, C. agloe¨formis and C. hu-
Relationships among the Lineages micola represent exceptions to this list of congruences.
Despite differences in detail and robustness, the twoThe nuclear and chloroplast data sets are generally
independent data sets exhibit considerable taxonomiccongruent in resolving six lineages that include Chla-
congruence regarding the relationships of flagellatemydomonas taxa as well as a basal Carteria lineage.
taxa representing the order Chlamydomonadales.Furthermore, the two data sets are congruent in recog-
nizing a clade that includes the C. radiata, C. euga-
metos, and Haematococcus lineages. The chloroplast
ACKNOWLEDGMENTS
data are consistent under different methods of phylog-
eny reconstruction in support of a fundamental dichot- We thank U. G. Schlo¨sser for his generous gift of green algal
omy among the six chlamydomonad lineages. This di- strains and for sharing unpublished results. The Oklahoma Univer-
sity Genetic Computer Group is supported by grants from the Centerchotomy is characterized by one major clade composed

11. PHYLOGENY OF CHLAMYDOMONADALES 401
of Excellence in Molecular Medicine and the Oklahoma Center for Fitch, W. M. (1971). Toward defining the course of evolution: Minimal
change for a specific tree topology. Syst. Zool. 20: 406–416.Academic Excellence in Science and Technology. This research was
supported by grants from the National Science Foundation (BSR- Floyd, G. L. (1978). Mitosis and cytokinesis in Asteromonas gracilis,
8918564 and DEB 9220834 to M.A.B. and R.L.C.), the Natural Sci- a wall-less green monad. J. Phycol. 14: 440–445.
ences and Engineering Research Council of Canada (GP0003293 to
Fritsch, F. E. (1945). ‘‘The Structure and Reproduction of the Algae,’’
M.T. and GP0002830 to C.L.), the Mervin Bovaird Center for Molecu-
Vol. I, Cambridge Univ. Press, London.
lar Biology and Biotechnology (to M.A.B.), ‘‘Le Fonds pour la Forma-
Genetics Computer Group. (1991). ‘‘Sequence Analysis Softwaretion de Chercheurs et l’Aide a` la Recherche’’ (93-ER-0350 to M.T. and
Package.’’ Version 7.3, Genetics Computer Group, Madison, WI.C.L.), and the National Institutes of Health (GM 48207 to R.R.G.).
Gunderson, J. H., Elwood, H., Ingold, A., Kindle, K., and Sogin,R.R.G. is an Associate and C.L. and M.T. are Scholars in the Evolu-
M. L. (1987). Phylogenetic relationships between chlorophytes,tionary Biology Program of the Canadian Institute for Advanced Re-
chrysophytes, and oomycetes. Proc. Nat. Acad. Sci. USA 84: 5823–search.
5827.
Gutell, R. R., Larsen, N., and Woese, C. R. (1994). Lessons from an
REFERENCES evolving ribosomal RNA: 16 S and 23 S rRNA structure from a
comparative perspective. Microbiol. Rev. 58: 10–26.
Bold, H. J., and Wynne, M. J. (1985). ‘‘Introduction to the Algae,’’ Hamby, R. K., Sims, L., Issel, L., and Zimmer, E. A. (1988). Direct
Prentice–Hall, Englewood Cliffs, NJ. ribosomal RNA sequencing: Optimization of extraction and se-
Bremer, K. (1988). The limits of amino acid sequence data in angio- quencing methods for work with higher plants. Plant Mol. Biol.
sperm phylogenetic reconstruction. Evolution 42: 795–803. Rep. 6: 175–192.
Buchheim, M. A., and Chapman, R. L. (1991). Phylogeny of the colo- Huss, V. A. R., and Sogin, M. L. (1990). Phylogenetic position of some
nial green flagellates: A study of 18S and 26S rRNA sequence data. Chlorella species with the Chlorococcales based upon complete
BioSystems 25: 85–100. small-subunit ribosomal RNA sequences. J. Mol. Evol. 31: 432–
442.Buchheim, M. A., and Chapman, R. L. (1992). Phylogeny of the genus
Carteria (Chlorophyceae) inferred from organismal and molecular Kimura, M. (1980). A simple model for estimating evolutionary rates
evidence. J. Phycol. 28: 362–374. of base substitutions through comparative studies of nucleotide se-
quences. J. Mol. Evol. 16: 111–120.Buchheim, M. A., Turmel, M., Zimmer, E. A., and Chapman, R. L.
(1990). Phylogenetic systematics of Chlamydomonas based on cla- Kluge, A. G., and Farris, J. S. (1969). Quantitative phyletics and the
distic analysis of nuclear 18S rRNA sequence data. J. Phycol. 26: evolution of Anurans. Syst. Zool. 18: 1–32.
689–699.
Lembi, C. A. (1975). The fine structure of the flagellar apparatus of
Buchheim, M. A., McAuley, M. A., Zimmer, E. A., Theriot, E. C., and Carteria. J. Phycol. 11: 1–9.
Chapman, R. L. (1994). Multiple origins of colonial green flagel-
Lerche, W. (1937). Untersuchungen u¨ ber Entwicklung und Fort-
lates from unicells: evidence from molecular and organismal char-
pflanzung in der Gattung Dunaliella. Arch. Protistendk. 88: 236–
acters. Mol. Phylogenet. Evol. 3: 322–343.
268.
Chappell, D. F., Hoops, H. J., and Floyd, G. L. (1989). Strip-like cov-
Lewis, L. A., Wilcox, L. W., Fuerst, P. A., and Floyd, G. L. (1992).
ering revealed on ‘‘wall-less’’ Asteromonas (Chlorophyceae). J. Phy-
Concordance of molecular and ultrastructural data in the study of
col. 25: 197–199.
zoosporic chlorococcalean green algae. J. Phycol. 28: 375–380.
Deason, T. (1983). Cell wall structure and composition as taxonomic
Mattox, K. R., and Stewart, K. D. (1984). Classification of the green
characters in the coccoid Chlorophyceae. J. Phycol. 19: 248–251.
algae: A concept based on comparative cytology. In ‘‘Systematics
Droop, M. R. (1956a). Haematococcus pluvialis and its allies. I. The of the Green Algae’’ (D. E. G. Irvine and D. M. John, Eds.), pp. 29–
Sphaerellaceae. Rev. Algol. 2: 53–71. 72, Academic Press, London.
Droop, M. R. (1956b). Haematococcus pluvialis and its allies. II. No- McCracken, D. A., Nadakavukaren, M. J., and Cain, J. R. (1980). A
menclature of Haematococcus. Rev. Algol. 2: 182–192. biochemical and ultrastructural evaluation of the taxonomic posi-
Ettl, H. (1976). Die Gattung Chlamydomonas Ehrenberg. Nova Hed- tion of Glaucosphaera vacuolata Korsh. New Phytol. 86: 39–44.
wigia 49: 1–1122. Melkonian, M. (1990). Phylum Chlorophyta Class Chlorophyceae. In
Ettl, H. (1979). Die Gattungen Carteria Diesing emend. France´ und ‘‘Handbook of Protoctista’’ (L. Margulis, J. O. Corliss, M. Melko-
Provasoliella A. R. Loeblich. Nova Hedwigia 60: 1–82. nian, and D. J. Chapman, Eds.), pp. 608–616, Jones & Bartlett,
Boston, MA.Ettl, H. (1981). Die neue Klasse Chlamydophyceae, eine natu¨rliche
Gruppe der Gru¨ nalgen (Chlorophyta). Plant Syst. Evol. 137: 107– Melkonian, M., and Preisig, H. R. (1984). An ultrastructural com-
126. parison between Spermatozopsis and Dunaliella (Chlorophyceae).
Plant Syst. Evol. 146: 31–46.Ettl, H. (1983). ‘‘Su¨ßwasserflora von Mitteleuropa. Band 9: Chlo-
rophyta I. Phytomonadina,’’ VEB, Jena. Miller, D. H. (1978). Cell wall chemistry and ultrastructure of Chlo-
rococcum oleofaciens (Chlorophyceae). J. Phycol. 14: 189–194.Ettl, H., and Ga¨rtner, G. (1988). ‘‘Su¨ßwasserflora von Mitteleuropa.
Band 10: Chlorophyta II. Tetrasporales, Chlorococcales, Gloeoden- Mishler, B. D., Donoghue, M. J., and Albert, V. A. (1991). The decay
drales,’’ VEB, Jena. index as a measure of relative robustness within a cladogram (ab-
stract). Willi Hennig Society Meeting, Toronto, Ontario.Ettl, H., and Schlo¨sser, U. G. (1992). Towards a revision of the sys-
tematics of the genus Chlamydomonas (Chlorophyta). 1. Chlamy- Oliveira, L., Bisalputra, T., and Antia, N. J. (1980). Ultrastructural
domonas applanata Pringsheim. Bot. Acta 105: 323–330. observation of the surface coat of Dunaliella tertiolecta from stain-
ing with cationic dyes and enzyme treatment. New Phytol. 85: 385–Farris, J. S. (1989). The retention index and the rescaled consistency
392.index. Cladistics 5: 417–419.
Olsen, G. J., Matsuda, H., Hagstrom, R., and Overbeek, R. (1994).Felsenstein, J. (1985). Confidence limits on phylogenies: an approach
fastDNAml: A tool for construction of phylogenetic trees of DNAusing the bootstrap. Evolution 39: 666–670.
sequences using maximum likelihood. CABIOS 10: 41–48.Felsenstein, J. (1993). PHYLIP: Phylogenetic Inference Package, ver-
sion 3.5c. Distributed by the author, Dept. of Genetics, Univ. of Pocock, M. A. (1960). Haematococcus in Southern Africa. Trans. R.
Soc. S. Afr. 36: 5–55.Washington, Seattle.

12. 402 BUCHHEIM ET AL.
Prescott, G. (1951). ‘‘Algae of the Western Great Lakes Area,’’ Cran- Starr, R. C., and Zeikus, J. A. (1993). UTEX—The culture collection
of algae at the University of Texas at Austin 1993 list of cultures.brook Inst. of Sci., Bloomfield Hill, MI.
J. Phycol. 29(Suppl.): 1–106.
Rausch, H., Larsen, N., and Schmitt, R. (1989). Phylogenetic rela-
Swofford, D. L. (1993). ‘‘PAUP: Phylogenetic Analysis Using Parsi-tionships of the green alga Volvox carteri deduced from small-sub-
mony,’’ Version 3.1, Computer program distributed by the Illinoisunit ribosomal RNA comparisons. J. Mol. Evol. 29: 255–265.
Natural History Survey, Champaign, IL.
Rohlf, F. J. (1982). Consensus indices for comparing classifications.
Turmel, M., Gutell, R. R., Mercier, J.-P., Otis, C., and Lemieux, C.Math. Biosci. 59: 131–144.
(1993). Analysis of the chloroplast large subunit ribosomal RNA
Saitou, N., and Nei, M. (1987). The neighbor-joining method: A new
gene from 17 Chlamydomonas taxa. Three internal transcribed
method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:
spacers and 12 group I intron insertion sites. J. Mol. Biol. 232:
406–425.
446–467.
Schlo¨sser, U. G. (1984). Species-specific sporangium autolysins (cell- Turmel, M., Choquet, Y., Goldschmidt-Clermont, M., Rochaix, J.-D.,
wall-dissolving enzymes) in the genus Chlamydomonas. In ‘‘Sys- Otis, C., and Lemieux, C. (1995). The trans-spliced intron 1 in the
tematics of the Green Algae’’ (D. E. G. Irvine, and D. M. John, psaA gene of the Chlamydomonas chloroplast: A comparative anal-
Eds.), pp. 409–418, Academic Press, London. ysis. Curr. Genet. 27: 270–279.
Schlo¨sser, U. G. (1994). SAG—Sammlung von Algenkulturen at the White, T. J., Bruns, T., Lee, S., and Taylor, J. (1990). Amplification
University of Go¨ttingen: Catalogue of strains, 1994. Bot. Acta 107: and direct sequencing of fungal ribosomal RNA genes for phyloge-
113–186. netics. In ‘‘PCR Protocols’’ (M. A. Innis, D. H. Gelfand, J. J. Snin-
sky, and T. J. White, Eds.), pp. 315–322, Academic Press, SanSmith, G. M. (1950). ‘‘Freshwater Algae of the United States,’’
Diego, CA.McGraw–Hill, New York.