Phylogeny of Geminella (Chlorophyta) and Allies: A Study of 18S rDNA Sequences1
Mark A. Buchheim1* and Julie A. Buchheim2
1Faculty of Biological Science and the Mervin Bovaird Center for Molecular Biology and Biotechnology, The University
of Tulsa, 600 South College Avenue, Tulsa, Oklahoma 74104-3189
2Department of Biological Science, The University of Tulsa, 600 South College Avenue, Tulsa, Oklahoma 74104-3189
*Corresponding Author (mark-buchheim@utulsa.edu)
Running title: Phylogeny of Geminella
Abstract
The phylogeny of pseudofilamentous green algae, like their coccoid relatives, is poorly or ambiguously characterized
by interpretations of gross morphological evidence. Representatives from four pseudofilamentous green algae, Geminella,
Microspora, Planctonema, and Radiofilum, were selected for a molecular phylogenetic investigation using nuclear-encoded,
18S rDNA sequences. Results from phylogenetic analyses indicate that multiple lineages of pseudofilamentous taxa
exist. Furthermore, algal isolates currently ascribed to the genera Geminella, Microspora and Radiofilum did not
form monophyletic groups of generic lineages. Radiofilum transversale and Microspora sp. were allied as a branch
within the Chlorophyceae. In contrast, Radiofilum conjunctivum and Prototheca were resolved as sister taxa in the
Trebouxiophyceae. One of the unnamed Geminella isolates was allied with Klebsormidium in the streptophyte clade.
The remaining Geminella taxa plus Microspora stagnorum formed a robust group (the Geminella clade) at the base
of the chlorophytan branch containing all non-prasinophyte taxa. In some analyses, the basal Geminella clade was
resolved as the sister taxon to the Chlorodendrales. The findings presented here further challenge phylogenetic
concepts based exclusively on gross morphology and emphasize the need for continued taxon and data sampling across
the Chlorophyta.
Key words: 18S rDNA, Chlorophyceae, Geminella, Microspora, Planctonema, Radiofilum Trebouxiophyceae, Ulvophyceae.
Introduction
The green algal genera Geminella, Microspora, Planctonema and Radiofilum form unbranched chains of cells. Each
of these taxa has been described as pseudofilamentous in that the chains of cells are formed through a process
of sporulation rather than cell division (Ettl 1988, Sluiman et al. 1989). The phylogenetic affinities of these
genera remain equivocal. Fritsch (1945), Smith (1950), Bold and Wynne (1985) and Bourrelly (1990) placed Geminella,
Microspora, and Radiofilum in the green algal order Ulothricales. Of these four treatments, only Bourrelly (1990)
addressed the question of Planctonema, also placing it in the Ulothricales. Silva (1982) placed Geminella and Radiofilum
in his order Ulotrichales, but separated Microspora into its own order, the Microsporales. Silva (1982) did not
include Planctonema. In contrast to Silva (1982), van den Hoek et al. (1995) identified Radiofilum and Geminella
as allies in their order Chlorococcales. Neither Microspora nor Planctonema were included in the van den Hoek (1995)
treatment. Graham and Wilcox (2000) included Geminella and Microspora in their treatment of the Chaetophorales,
but they cautioned that the phylogenetic affinities of these taxa are not clear. Neither Planctonema nor Radiofilum
were included in the Graham and Wilcox (2000) summary of the green algae. Given the current understanding of class-level,
green algal diversity based on both ultrastructure (e.g., Mattox and Stewart 1984) and molecular phylogenetics
(e.g., Chapman et al. 1998), the various taxonomic assessments indicate that Geminella, Microspora, Planctonema
and Radiofilum would fall into either the green algal class Chlorophyceae, Trebouxiophyceae, Ulvophyceae, or Charophyceae.
The lack of taxonomic consensus regarding Geminella, Microspora, Planctonema and Radiofilum generally can be attributed
to the absence or paucity of ultrastructural data and to a lack of molecular phylogenetic evidence. Although recent
ultrastructural analysis of the flagellar apparatus in zoospores indicate chlorophycean affinities for Microspora
(Lokhorst and Star 1999), neither Geminella, Planctonema nor Radiofilum are thought to produce motile stages (Smith
1950, Bourrelly 1990). Thus, molecular approaches are needed to clarify the phylogenetic affinities of these enigmatic
green algae. Studies of the nuclear-encoded, 18S rDNA gene have proven useful in elucidating cryptic diversity
among autosporic, coccoid green algae (Huss and Sogin 1990, Huss et al. 1999, Hepperle et al. 2000). Consequently,
the 18S rRNA gene was selected to address the question of phylogeny for these taxa that are, in essence, linear
arrays of coccoid cells. The goal of this investigation is to test hypotheses of relationship (based on traditional
classifications) for selected pseudofilamentous taxa using sequence data from the 18S rRNA gene. Although Geminella,
Microspora, Planctonema and Radiofilum are the focus of the investigation, additional taxon sampling from among
a wide range of chlorophytan green algae was employed to broaden the context in which to interpret 18S rDNA diversity.
Materials and Methods
Taxon Selection
New 18S rDNA sequence data were collected for Cylindrocapsa geminella (GR-32, AF387159), Eremosphaera viridis (UTEX
LB 34, AF387154), Geminella minor (SAG 22.88, AF387150), Geminella minor (GR-36, AF387151), Geminella sp. (SAG
54.81, AF387158), Geminella sp. (SAG 20.84, AF387157), Geminella terricola (SAG 20.91, AF387152), Hazenia mirabilis
(UTEX LB 846, AF387156), Microspora sp. (UTEX LB 472, AF387160), Microspora stagnorum (SAG 51.86, AF387153), Planctonema
sp. (J45-9, AF387149), Planctonema sp. (M110-1, AF387148), Radiofilum conjunctivum (GR-2, AF387155) and Radiofilum
transversale (UTEX LB 1252, AF387161). A total of 80 ingroup taxa representing the classes Chlorophyceae, Trebouxiophyceae,
Ulvophyceae, and Prasinophyceae were included in the phylogenetic analyses (Appendix 1).
DNA Extraction and Preparation of Sequencing Templates
Genomic DNA was obtained using extraction protocols described previously (Buchheim and Chapman 1992). For some
templates, a Mini-Beadbeater (Biospec, Inc., Bartlesville, OK) was used to break open cells. Double-stranded DNA
sequencing templates were obtained by symmetrically amplifying genomic DNA using the PCR. The flanking primers
used to amplify the 18S rRNA gene are described by White et al. (1990). Products from two or more independent amplifications
were pooled to increase template concentration and to allow for the detection of heterogeneity in the 18S rDNA
array.
Automated Sequencing
New sequence data were obtained with the protocols and reagents that accompany the four-color PRISM™ reagent kit
(Perkin-Elmer) designed for use in ABI automated DNA sequencing systems. The eleven sequencing primers for the
18S rRNA gene have been described previously (Hamby et al. 1988, Buchheim et al. 1997).
Sequence Alignments
Previous work (Buchheim et al. 2001 [in press]) served as the starting point for all alignments. SeqApp (Gilbert
1994) and MacClade 4.0 (Maddison and Maddison 2000) were used to manually align the data. A total of 165 sites
were excluded from phylogenetic analyses of the 18S rDNA data because they exhibit questionable homology in expansion
regions that vary in length and exhibit base changes among taxa. The data set has been deposited in TreeBase [NOTE
TO EDITOR & REVIEWERS: TreeBase accepts datasets from published or in press manuscripts; thus, the dataset
will be deposited upon acceptance of manuscript] and an alignment of the new sequences has been deposited in GenBank.
Phylogenetic Analyses of Independent Data Sets
Three methods of phylogenetic reconstruction were employed for independent analyses of 18S rDNA data: character
analysis using maximum parsimony (MP) optimality criteria, a distance matrix approach using minimum evolution (ME)
optimality criteria, and a maximum likelihood (ML) approach. All analyses were conducted using PAUP* (version 4.0b8,
Swofford [2001]). These three methods, which generally represent the spectrum of philosophical approaches to phylogenetic
reconstruction, were employed in order to identify branches that are not robust to method of analysis (Buchheim,
unpublished observations). Groups whose placement is not robust to method of analysis are almost invariably poorly-
or ambiguously-resolved in the independent analyses. Thus, topological differences between multiple approaches
reinforce any observations of topological ambiguity from the independent analyses. Moreover, the application of
different approaches potentially allows the investigator to identify a broader range of alternative hypotheses
for further study and testing.
Maximum Parsimony Method. Tree searches for MP analyses were conducted using the tree-bisection-reconnection (TBR)
option. The order of taxon addition was randomized 50 times. Bootstrap values (Felsenstein 1985) from 1000 resamplings
using nearest-neighbor-interchange (NNI) searches with simple taxon addition were calculated for the data. Fitch
parsimony (Fitch 1971) was invoked for MP analyses and no sites were differentially weighted. Gaps were treated
as missing data.
Distance Matrix Method. Modeltest 3.06 (Posada and Crandall 1998) and PAUP* 4.0b8 (Swofford 2001) were used in
tandem to test the goodness-of-fit of DNA substitution models against the 18S rDNA data. Based on the results of
a hierarchical likelihood ratio test (hLRT), distance matrices for ME analysis were constructed using the Tamura-Nei
(TrN) model as implemented in PAUP* 4.0b8 (Swofford 2001) and I (estimates of the percentage of invariant sites)
and G (estimates of the gamma distribution shape parameter) were estimated from the data. Tree searches for ME
analyses were conducted heuristically using the TBR option. To increase the probability of finding all islands
of optimal trees under the ME criterion, the order of taxon addition was randomized 50 times. Bootstrap values
(Felsenstein 1985) from 1000 resamplings using heuristic NNI searches with simple taxon addition were calculated
for the data. Starting trees for each replicate were obtained by the neighbor-joining method.
Maximum Likelihood Method. The same hLRT data used for ME analysis were also used for the ML analysis. Trees were
compared using the TrN model under ML optimality criteria. The model parameters I, G, a substitution rate matrix,
and nucleotide frequencies were estimated from the data. Tree searches for ML analysis were conducted heuristically
using the TBR option. The optimal tree from ME analysis was used as the starting tree for the ML tree search. Bootstrap
values (Felsenstein 1985) from 100 resamplings using heuristic NNI searches with simple taxon addition were calculated
for the data. Starting trees for each replicate were obtained by the neighbor-joining method.
Rooting. The outgroup method was used to root all trees. Sequence data from Cyanophora paradoxa (X68483) and Glaucocystis
nostochinearum (X70803) were used to root the trees. These glaucocystophyte taxa have been resolved as a sister
group to the green plant lineage in previous studies of 18S rDNA data (Bhattacharya et al. 1995).
Results
Phylogenetic Information Content
A total of 1604 aligned sites were included in the phylogenetic analyses of 18S rDNA data. Aligned sequence data
from all taxa yielded a total of 700 variable sites (excluding gap-only sites) of which 558 were informative for
parsimony analysis.
Phylogenetic Reconstruction
MP Analyses. The MP analysis of 18S rDNA data yielded four optimal trees, which differed from one another in the
relative position of the chaetophoralean clade (Chaetophora, Fritschiella, Stigeoclonium, Uronema, Schizomeris,
Aphanochaete) and the Geminella clade (Fig. 1). Geminella taxa fell into two lineages. One
lineage was comprised of a single Geminella isolate (SAG 20.84) that was resolved as a member of the Streptophyta.
The remaining Geminella taxa formed a group with Microspora stagnorum. The two Microspora taxa fell into two separate
groups, one (M. sp.) allied with Radiofilum transversale and the other (M. stagnorum) allied with the chlorophyte
Geminella clade mentioned previously. The Radiofilum+Microspora clade is placed in a chlorophycean clade that includes
Bracteacoccus (Fig. 1). The remaining Radiofilum taxon (R. conjunctivum) was resolved as the
sister taxon to Prototheca wickerhamii in the Trebouxiophyceae (Fig. 1).
ME Analyses. The optimal tree from ME analysis of 18S rDNA data differs from the MP trees (Fig.
1) in that the Radiofilum+Microspora clade is grouped with Cylindrocapsa and its allies (Fig.
2), and the Streptophyta are resolved as monophyletic (Fig. 2).
ML Analyses. The optimal tree from ML analysis of 18S rDNA data (Fig. 3) differs from the MP
tree (Fig. 1) and ME tree (Fig. 2) in that the Trebouxiophyceae are resolved
as a monophyletic group. The ML tree (Fig. 3) and the MP tree (Fig. 1)
are alike in support of an alliance between the Radiofilum+Microspora clade and the Cylindrocapsa clade. The ML
(Fig. 3) and ME trees (Fig. 2) are alike in support of a monophyletic Streptophyta.
Discussion
The Chlorophyta
The results of the molecular phylogenetic analyses offer several general observations regarding chlorophytan diversity.
These results support previous findings regarding the monophyly of the ulotrichalean ulvophytes (Steinkötter
et al. 1994, Friedl 1997, Marin and Melkonian 1999, Fawley et al. 2000) and the non-monophyly of the prasinophytes,
including the Chlorodendrales (Friedl 1997, Chapman et al. 1998, Nakayama et al. 1998, Fawley et al. 2000). These
data place Hazenia mirabilis as a member of the ulotrichalean ulvophyte group. The results from analyses of 18S
rDNA data are largely ambiguous regarding the strict monophyly of the Chlorophyceae (sensu Mattox and Stewart 1984)
and of the Trebouxiophyceae (sensu Friedl 1995). The results from analyses of the 18S rDNA data regarding the classes
Chlorophyceae and Trebouxiophyceae are discussed below.
Chlorophyceae. The Oedogoniales, a putative member of the Chlorophyceae (sensu Mattox and Stewart 1984), are comprised
of filamentous taxa that may be branched (Bulbochaete) or unbranched (Oedogonium). Results from analysis of 18S
rDNA data reveal that the Oedogoniales are variously resolved as the (1) basal lineage in the Chlorophyceae (Fig.
1), (2) sister taxon to a trebouxiophycean clade that includes Planctonema and Radiofilum (Fig. 2), or (3) sister
taxon to the Ulvophyceae (Fig. 3). None of these placements is robust (>70%) as assessed by the bootstrap. Of
these, only the former reflects a monophyletic Chlorophyceae (sensu Mattox and Stewart 1984). The topological ambiguity
regarding the Oedogoniales is consistent with previous assessments (Booton et al. 1998b, Buchheim et al. 2001 [in
press]) and is likely due to the long branch associated with the group. The remainder of the Chlorophyceae, exclusive
of the Oedogoniales, are resolved as monophyletic by all methods of analysis (Figs. 1-3) with modest bootstrap
support (>70) in both the ME (Fig. 2) and ML (Fig. 3) analyses. These observations suggest that, although the
problem of the Oedogoniales has not yet been solved by enhanced taxon sampling, the 18S rDNA data corroborate the
ultrastructure-based assessment that the remainder of the Chlorophyceae are monophyletic (Mattox and Stewart 1984).
Trebouxiophyceae. The Trebouxiophyceae (sensu Friedl 1995) are resolved as monophyletic in only the ML analysis
of 18S rDNA data (Fig. 3). Neither the monophyly (Fig. 3) nor the non-monophyly (Figs. 1, 2) of the Trebouxiophyceae
have robust support from analysis of these 18S rDNA data. Although several investigations have demonstrated modest
to robust support for a monophyletic Trebouxiophyceae (e.g., Friedl 1995, 1997), other studies revealed only weak
support for a monophyletic Trebouxiophyceae (e.g., Lewis 1997, Hepperle et al. 2000, Marin and Melkonian 1999)
or failed to support trebouxiophycean monophyly (e.g., Buchheim and Lewis 1999). The data presented here do not
resolve the issue of trebouxiophycean monophyly. However, three new taxa are revealed to have trebouxiophycean
affinities. Eremosphaera is strongly supported as a basal member (Figs. 1-3) of the Oocystis clade (see Hepperle
et al. 2000) within a branch of the Trebouxiophyceae. A trebouxiophycean alliance for Eremosphaera was predicted
by Graham and Wilcox (2000). In addition to Eremosphaera, the pseudofilamentous taxa, Planctonema and Radiofilum
conjunctivum are resolved as basal members of the Oocystis clade (Figs. 1-3). A trebouxiophycean placement of Planctonema
and Radiofilum conjunctivum is observed in all analyses, however, the position of the latter exhibits poor bootstrap
support in all analyses (Figs. 1-3).
Geminella
The results presented here indicate that at least two lineages of Geminella taxa exist. One lineage, represented
by a single taxon (Geminella sp. SAG 20.84), is robustly allied with Klebsormidium in the streptophyte group. The
remaining Geminella taxa are robustly grouped in a "Geminella clade" with Microspora stagnorum (see further
discussion of Microspora below). Statistical analysis (Kishino-Hasegawa tests conducted using PAUP* 4.0b8 [Swofford
2001]) reveals that constraint trees forcing the monophyly of all Geminella taxa are significantly different (P<0.0001)
from optimal trees (MP, ME and ML). Clearly, the unnamed SAG 20.84 isolate belongs in a genus separate from Geminella.
Given that the 18S rDNA sequences of Geminella sp. (SAG 20.84) and Klebsormidium are quite distinct (18 nucleotide
substitutions), it remains for future investigation to determine if SAG 20.84 should be regarded as a new species
of Klebsormidium, or placed in a new genus.
The 18S rDNA data are ambiguous regarding the broader alliance of the Geminella clade within the Chlorophyta. Two
alternatives are presented in the various analyses. One alternative hypothesis places the Geminella clade as the
basal member of a large chlorophytan alliance that includes all members of the Trebouxiophyceae, Ulvophyceae, and
Chlorophyceae (Fig. 1). The other alternative hypothesis links the Geminella clade with the Chlorodendrales as
the basal member of the Chlorophyceae-Trebouxiophyceae-Ulvophyceae clade. Neither hypothesis is consistent with
traditional classifications regarding Geminella. Regardless of the broader alliances of the Geminella clade, the
18S rDNA data suggest that this group represents a new lineage within the Chlorophyta. If the interpretation of
the 18S rDNA data presented here is correct, then the Geminella clade represents one of the basal groups within
a radiation that gave rise to three of the class-level lineages within the Chlorophyta.
Microspora
Microspora is not resolved as a monophyletic group by the 18S rDNA data. Statistical analysis (Kishino-Hasegawa
tests conducted using PAUP* 4.0b8 [Swofford 2001]) reveals that the constraint tree forcing monophyly of the two
Microspora taxa is significantly different (P<0.0001) from optimal trees (MP, ME and ML). Given the taxonomic
contexts examined in this investigation, it seems likely that the isolate currently recognized as Microspora stagnorum
will eventually be merged into the genus Geminella. Structural investigations are currently underway to assess
the morphological features that characterize the members of this group.
The 18S rDNA data (Figs. 1-3) indicate that the UTEX isolate of Microspora is closely allied with another pseudofilamentous
taxon, Radiofilum transversale. Although this alliance is robust, the relative position of the Microspora+Radiofilum
clade is not (cf. Figs. 1-3). Invoking structural evidence does not necessarily resolve the issue. Ultrastructural
analysis of the zoospore in Microspora (Lokhorst and Starr 1999) suggests a link with Bracteacoccus. Analysis of
18S rDNA data (Lewis 1997, present investigation) places Bracteacoccus within a chlorophycean clade that includes
Hydrodictyon and Neochloris (Figs. 1-3). Both MP (Fig. 1) and ML (Fig. 3) analyses support a broad alliance of
the Microspora+Radiofilum clade and the Bracteacoccus clade. Moreover, constraining the data to reflect a monophyletic
clade comprised of Bracteacoccus minor and Microspora sp. yields trees from MP, ME and ML analysis that are not
significantly different from optimal trees. The probabilities from Kishino-Hasegawa tests (implemented using PAUP*
4.0b8 [Swofford 2001]) ranged from P=0.07 to P=0.11. Thus, these molecular phylogenetic results appear to corroborate
the ultrastructural interpretation (Lokhorst and Star 1999). However, the optimal tree from ME analysis (Fig. 2)
places the Microspora+Radiofilum clade as the sister group to the Cylindrocapsa clade. Like Microspora and Radiofilum,
Cylindrocapsa is pseudofilamentous (Ettl 1988). Therefore, the ME hypothesis is a better fit with the gross morphological
evidence. If analyses using MP and ML criteria are constrained to reflect an alliance of Microspora sp., Radiofilum
transversale, and the Cylindrocapsa group, the resulting constraint trees are not significantly different from
the optimal trees (P values range from P=0.27 to P=0.49).
An independent source of molecular phylogenetic evidence is clearly needed to resolve this issue regarding placement
of the Microspora+Radiofilum group. Preliminary data from nuclear-encoded 26S rDNA sequences not only corroborate
a close alliance between Microspora sp. and Radiofilum transversale, but also support an alliance of these two
taxa with Cylindrocapsa (Buchheim, unpublished observations). If these observations hold, an apparent conflict
between ultrastructure (Lokhorst and Star 1999) and the molecular phylogeny will need to be addressed.
Planctonema
Planctonema is an obscure member of the green algae, largely ignored by textbook treatments of the Chlorophyta.
Filaments of Planctonema are composed of cells that are widely-separated from neighboring cells, at least in older
cultures (Bourrelly 1990, Buchheim, unpublished observations). The two isolates of Planctonema included in this
investigation are identical at the 18S rDNA level and, thus, form a robust clade. The relative position of the
Planctonema clade is also robust to method of analysis (Figs. 1-3). Bootstrap support for the basal position of
Planctonema within the Oocystis clade is robust (>70%) in both the ME (Fig. 2) and ML (Fig. 3) analyses. Consequently,
the 18S rDNA data indicate that Planctonema is yet another new member of the Trebouxiophyceae, joining other recent
additions such as Oocystis (Hepperle et al. 2000) and Prasiola (Sherwood et al. 2000).
Radiofilum
The two Radiofilum taxa included in this investigation are robustly placed in separate lineages. Statistical analysis
(Kishino-Hasegawa tests conducted using PAUP* 4.0b8 [Swofford 2001]) indicates that a constraint tree forcing monophyly
of the two Radiofilum taxa is significantly different (P<0.0001) from optimal trees (MP, ME and ML). The phylogenetic
position of Radiofilum transversale has already been discussed in the section on Microspora. Radiofilum conjunctivum
in allied in a trebouxiophycean clade comprised of Prototheca wickerhamii and Chlorella vulgaris. Although this
clade is robust to method of analysis, (cf. Figs. 1-3), bootstrap support for the group is weak. Long branch attraction
(Felsenstein 1978) may be confounding resolution in this case as both P. wickerhamii and R. conjunctivum exhibit
long terminal branches. However, a more dense taxon sampling of trebouxiophycean algae fails to reveal an alternative
to that presented here (Buchheim, unpublished observations).
The other question that remains is which isolate is a legitimate species of Radiofilum? Gross morphological assessment
confirms that both isolates demonstrate coccoid-like filament development and possess a demonstrable sheath surrounding
the filament (Buchheim, unpublished observations). The R. transversale isolate demonstrates a tendency to bi- and
pluriseriate filaments which is somewhat unusual among species of Radiofilum. However, in all gross morphological
respects, the two isolates fit the current understanding of the genus Radiofilum. Consequently, it seems likely
that taxonomic revision will be necessary to accommodate the morphological similarities coupled with molecular
diversity exhibited by the two taxa. Additional species of Radiofilum need to be studied in order to begin to resolve
both the phylogenetic and taxonomic problems regarding this enigmatic green algal genus.
Conclusions
The results of this investigation reinforce recent studies of coccoid taxa (Huss and Sogin 1990, Lewis et al. 1992,
Wilcox et al. 1992, Huss et al. 1999) indicating that gross morphological features have evolved independently in
multiple lineages. Specifically, this investigation clearly demonstrates parallel evolution of the pseudofilamentous
condition at two different levels. First of all, pseudofilamentous taxa that exhibit sufficient morphological distinction
to be placed in separate genera also form distinct, molecular phylogenetic lineages (e.g., Radiofilum vs. Geminella).
This observation is important information for a better understanding of green algal diversity, but is not entirely
unanticipated. More importantly, molecular phylogenetic evidence for multiple, non-monophyletic lineages within
genera (i.e., Geminella, Microspora and Radiofilum) is especially problematic for the algal systematist. These
observations emphasize the notion that superficial similarities in gross morphology may be phylogenetically misleading.
The discovery, using molecular phylogenetic evidence, that a Geminella clade exists as a basal lineage among the
non-prasinophyte Chlorophyta raises two questions. Do additional lineages of Chlorophyta exist that are currently
"hidden" within poorly sampled groups? The recent discovery of the Cylindrocapsa clade (Buchheim et al.
2001 [in press]) coupled with the results of this investigation regarding Geminella, emphasize the need for continued
sampling across the spectrum of green algal diversity. Another question that remains to be addressed is the taxonomic
status of the Geminella clade. Is the Geminella alliance a new class or a new order within an existing class and,
if so, which class? The answer to both questions awaits additional taxon and data sampling.
Acknowledgments
This research was supported by grants from the National Science Foundation (DEB 9220834 and 9726588), the DOE/NSF/USDA
Joint Program on Collaborative Research in Plant Biology (USDA grant no. 94-37105-0173 and 97-35105-4678), The
University of Tulsa (Faculty Research and Summer Faculty Development grants), and the Mervin Bovaird Center for
Molecular Biology and Biotechnology. EPSCoR support of The University of Tulsa DNA Sequencing Facility is gratefully
acknowledged. Joanna Michalopulos, Matthew Rebstock, and Bryan Fetterley assisted with DNA extractions and amplifications.
Karen Draeger conducted the automated DNA sequencing electrophoresis. We thank David Czarnecki and Marvin Fawley
for providing cultures.
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Figure Legends
Fig. 1. One of four minimal length cladograms from MP analysis of 18S rDNA sequence data (L
= 2925, CI [Kluge and Farris 1969] = 0.3372, RI [Farris 1989] = 0.6733, RC [Farris 1989] = 0.2511). Geminella and
its putative allies are presented in boldface. Branch lengths are drawn proportional to changes along branches
(see scale in lower left). Bootstrap values as percentages are noted for each internal branch. Internodes without
bootstrap percentages were supported in fewer than 70% of all replicates. Nodes that collapse in a strict consensus
analysis of equally-parsimonious trees are denoted by an asterisk. Groups of chlorophytan green algae are identified
by vertical lines to the right of the tree.
Fig. 2. Optimal tree from ME analysis of 18S rDNA (minimal evolution score = 2.04517) using
TrN with I (proportion of sites assumed to be invariant) = 0.3987 and G (assuming a discrete gamma distribution)
= 0.5545 estimated from the data. Geminella and its putative allies are presented in boldface. Branch lengths are
drawn proportional to estimates of evolution along branches (see scale in lower left). Bootstrap values as percentages
are noted for each internal branch. Internodes without bootstrap percentages were supported in fewer than 70% of
all replicates. Groups of chlorophytan green algae are identified by vertical lines to the right of the tree.
Fig. 3. Optimal tree from ML analysis of 18S rDNA (-Ln likelihood = 17572.9812) using TrN with
I (proportion of sites assumed to be invariant) = 0.3987, G (assuming a discrete gamma distribution) = 0.5545,
a substitution rate matrix (A:C=1.0, A:G=2.473, A:T=1.0, C:G=1.0, C:T=3.945, G:T=1.0) and estimates of nucleotide
frequencies (A=0.2519, C=0.2226, G=0.2598, T=0.2657) estimated from the data. Geminella and its putative allies
are presented in boldface. Branch lengths are drawn proportional to estimates of evolution along branches (see
scale in lower left). Bootstrap values as percentages are noted for each internal branch. Internodes without bootstrap
percentages were supported in fewer than 70% of all replicates. Groups of chlorophytan green algae are identified
by vertical lines to the right of the tree.
_____________________________________________________________________________
Appendix 1. List of taxa, source of culture material, sequence accession number, and original literature citation.
_____________________________________________________________________________
Taxon Sourcea Citation & Accession
Acrosiphonia sp.
SAG 127-80 Zechman and Chapman, unpublished (U03757)
Aphanochaete magna Godward
UTEX B 1909 Buchheim et al. 2001 [in press] (AF182816)
Bracteacoccus minor (Chodat) Petrova
UTEX 66 Lewis 1997 (U63097)
Bulbochaete hiloensis (Nordstrom) Tiffany
UTEX 952 Booton et al. 1998a (U83132)
Carteria crucifera Korshikov
NIES 421 Nakayama et al. 1996 (D86501)
Carteria eugametos Mitra
UTEX 233 Buchheim et al. 2001 [in press] (U70595)
Carteria olivieri G. S. West
UTEX LB 1032 Buchheim et al. 2001 [in press] (U70596)
Chaetophora incrassata (Hudson) Hazen
UTEX
LB 1289 Booton et al. 1998a (U83130)
Characiopodium hindakii (Lee & Bold) Floyd & Watanabe
UTEX 2098 Lewis et al. 1992 (M63000)
Chlamydomonas moewusii Gerloff
SAG 11-11 Buchheim et al. 1997 (U70786)
Chlamydomonas noctigama Korshikov
SAG 33.72 Buchheim et al. 1997 (U70782)
Chlamydomonas reinhardtii Dangeard
CC-400 Gunderson et al. 1987 (M32703)
Chlamydopodium vacuolatum (Lee & Bold) Floyd & Watanabe
UTEX 2111 Lewis et al. 1992 (M63001)
Chlorella ellipsoidea Gerneck
SAG 211-1a Krienitz et al. 1996 (X63520)
Chlorella mirabilis Andreeva
SAG 38.88 Krienitz et al. 1996 (X74000)
Chlorella vulgaris Beijerinck
SAG 211-11b Sogin, unpublished (X13688)
Chlorococcum echinozygotum Starr
UTEX 118 Buchheim et al. 1996 (U57698)
Coleochaete orbicularis Pringsheim
Linda Graham Wilcox et al. 1993 (M95611)
Cylindrocapsa geminella Wolle
LC GR-32 Present investigation (AF387159)
Cymbomonas tetramitiformis Schiller
Shizugawa Nakayama et al. 1998 (AB017126)
Dunaliella parva Lerche
UTEX LB 1983 Lewis et al. 1992 (M62998)
Elakatothrix viridis (Snow) Printz
LC CH-30 Buchheim et al. 2001 [in press] (AY008844)
Eremosphaera viridis DeBary
UTEX LB 34 Present investigation (AF387154)
Fritschiella tuberosa Iyengar
UTEX 1821 Booton et al. 1998a (U83129)
Fusochloris perforata (Lee & Bold) Floyd & al.
UTEX 2104 Lewis et al. 1992 (M62999)
Geminella minor Heering
SAG 22.88 Present investigation (AF387150)
Geminella minor Heering
LC GR-36 Present investigation (AF387151)
Geminella sp.
SAG 20.84 Present investigation (AF387157)
Geminella sp.
SAG 54.81 Present investigation (AF387158)
Geminella terricola Boye-Petersen
SAG 20.91 Present investigation (AF387152)
Gloeotilopsis planctonica Iyengar & Philipose
SAG 29.93 Friedl and Zeltner 1994 (Z28970)
Glycine max (Linneaus) Merrill
not cited Eckenrode et al. 1984 (X02623)
Hazenia mirabilis Bold
UTEX LB 846 Present investigation (AF387156)
Hydrodictyon reticulatum (Linneaus) Lagerheim
CBS Wilcox et al. 1992 (M74497)
Klebsormidium flaccidum (Kützing) Silva, Mattox & Blackwell
UTEX 2017 Wilcox et al. 1993 (M95613)
Mamiella sp.
Shizugawa Nakayama et al. 1998 (AB017129)
Mantoniella antarctica Marchant
Not cited Nakayama et al. 1998 (AB017128)
Mantoniella squamata (Manton & Parke) Desikachary
CCAP 1965/1 Kranz et al. 1995 (X73999)
Microspora sp.
UTEX LB 472 Present investigation (AF387160)
Microspora stagnorum (Kützing) Lagerheim
SAG B 51.86 Present investigation (AF387153)
Microthamnion kuetzingianum Nägeli
UTEX 1914 Friedl and Zeltner 1994 (Z28974)
Myrmecia biatorellae (Tschermak-Woess & Pless) Petersen
UTEX 907 Friedl and Zeltner 1994 (Z28971)
Myrmecia israelensis (Chantanachat & Bold) Friedl
UTEX 1181 Lewis et al. 1992 (M62995)
Neochloris aquatica Starr
UTEX 138 Lewis et al. 1992 (M62861)
Nephroselmis olivacea Stein
SAG 40.89 Nakayama et al. 1998 (X74754)
Nephroselmis pyriformis (Carter) Ettl
CCMP 717 Nakayama et al. 1998 (X75565)
Oedogonium cardiacum Wittrock
UTEX 40 Booton et al. 1998a (U83133)
Oocystis heteromucosa Hegewald
SAG 1.99 Hepperle et al. 2000 (AF228689)
Oocystis solitaria Wittrock f. maior Wille
SAG 83.80 Hepperle et al. 2000 (AF228686)
Parietochloris pseudoalveolaris (Deason & Bold) Watanabe & Floyd
UTEX 975 Lewis et al. 1992 (M63002)
Pediastrum duplex Meyen
UTEX LB 1364 Wilcox et al. 1992 (M62997)
Planctonema sp.
J45-9b Present investigation (AF387149)
Planctonema sp.
M110-1b Present investigation (AF387148)
Prasiola fluviatilis (Summerfelt) Areschoug
R Hodgsonc Sherwood et al. 2000 (AF189072)
Prasiola mexicana J. A. Agardh
MEX12c Sherwood et al. 2000 (AF189075)
Protoderma sarcinoidea (Groover & Bold) Tupa
UTEX 1710 Friedl 1996 (Z47998)
Protosiphon botryoides (Kützing) Klebs
UTEX 99 Nakayama et al. 1996 (U41177)
Prototheca wickerhamii Tubaki & Soneda
SAG 263-11 Huss, unpublished (X74003)
Pseudendoclonium basiliense Vischer
UTEX 2593 Friedl 1996 (Z47996)
Pseudoscourfieldia marina (Throndsen) Manton
K-0017 Fawley et al. 2000 (AF122888)
Pterosperma cristatum Schiller
Yokohama Nakayama et al. 1998 (AB017127)
Pycnococcus provasolii Guillard
CCMP 1199 Fawley et al. 2000 (AF122889)
Pyramimonas parkeae Norris & Pearson
Hachijo Nakayama et al. 1998 (AB017124)
Radiofilum conjunctivum Schmidle LC
GR-2 Present investigation (AF387155)
Radiofilum transversale (de Brébisson) Christensen
UTEX LB 1252 Present investigation (AF387161)
Scenedesmus obliquus (Turpin) Kützing
SAG 276-3a Huss and Sogin 1990 (X56103)
Scherffelia dubia (Perty) Pascher
SAG B 17.89 Steinkötter et al. 1994 (X68484)
Schizomeris leibleinii Kützing
UTEX LB 1228 Buchheim et al. 2001 [in press] (AF182820)
Stigeoclonium helveticum Vischer
UTEX 441 Booton et al. 1998b (U83131)
Tetracystis aeria Brown & Bold
UTEX 1453 Nakayama et al. 1996 (U41175)
Tetraselmis sp.
RG-07 Cavalier-Smith and Chao, unpublished (U41900)
Tetraselmis striata Butcher
CCAP 443 Steinkötter et al. 1994 (X70802)
Trebouxia impressa Ahmadjian
UTEX 892 Friedl and Zeltner 1994 (Z21551)
Trebouxia magna Archibald
UTEX 902 Friedl and Zeltner 1994 (Z21552)
Trochiscia hystrix (Reinsch) Hansgirg
UTEX LB 606 Buchheim et al. 2001 [in press] (AF277651)
Uronema belkae Mattox & Bold (O'Kelly & Floyd)
UTEX 1179 Buchheim et al. 2001 [in press] (AF182821)
Ulothrix zonata (Weber & Mohr) Kützing
SAG 38.86 Friedl, unpublished (Z47999)
Volvox carteri Iyengar
UTEX 1885 Rausch et al. 1989 (X53904)
Zamia pumila Linneaus
not cited Nairn and Ferl 1988 (M20017)
Zea mays Linneaus
not cited Messing et al. 1984 (K02202)
_____________________________________________________________________________
aSource of taxa used to generate published and new 18S rDNA data: CBS = from Carolina Biological Supply, CC = from
the Chlamydomonas Genetics Center at Duke University, CCAP = from Culture Collection of Algae and Protozoa (Plymouth),
CCMP = Provasoli-Guillard National Center for Culture of Marine Phytoplankton, LC = from the Loras College Collection
of Freshwater Diatoms (David Czarnecki, Curator), NIES = from National Institute of Environmental Science (Tsukuba),
SAG = from the Sammlung von Algenkulturen Göttingen, UTEX = from the Culture Collection at the University
of Texas at Austin.
bCulture provided by Marvin Fawley.
cSee Sherwood et al. 2000 for the collection information.
