J. Phycol. 36, 740–746 (2000)
A MOLECULAR ANALYSIS OF THE EUGLENOPHYTES USING SSU RDNA 1
Eric W. Linton 2
Rutgers University, Department of Cell Biology and Neuroscience, Nelson Biological Laboratories, 604 Allison Road, Piscataway,
New Jersey 08854-8082
Maria Alejandra Nudelman, Visitacion Conforti
Facultad de Ciencias Exactas y Naturales, Departamento de Ciencias Biológicas, Ciudad Universitaria, 1428 Buenos Aires, Argentina
and
Richard E. Triemer
Rutgers University, Department of Cell Biology and Neuroscience, Nelson Biological Laboratories, 604 Allison Road, Piscataway,
New Jersey 08854-8082
(euglenoid movement), or rigid (extent variable).
Three years after the formal diagnosis of Euglena, Dujardin (1841) erected the genus Phacus to contain
those photosynthetic “Euglena species” that were both
rigid and flattened. Unfortunately, the minimal extent of flattening was never defined; consequently,
Phacus contains taxa that are slightly flattened to
highly flattened, practically two-dimensional. Thus began the splitting of “Euglena species” based on observable morphological differences and their regrouping by
similarity, e.g. flat or not flat. This continued with Perty
(1849), who erected the genus Lepocinclis (rigid, not
flattened, and lacking pyrenoids) followed by Pringsheim (1936) with the genus Astasia (osmotrophic, with
and without a stigma) and Jahn and McKibben with the
genus Khawkinea (osmotrophic, with a stigma) (Jahn
and McKibben 1937, Jahn 1946), split from the genus
Astasia.
The splitting and reorganization of taxa based
solely upon morphological similarity is no longer an
acceptable method of phylogenetic reconstruction.
Additionally, given the limited number of morphological characters in unicellular organisms (Triemer and
Farmer 1991a,b), we have employed molecular sequence data, SSU rDNA, using parsimony, maximum
likelihood, and distance in this analysis. Based on
past molecular phylogenies of euglenoids (MontegutFelkner and Triemer 1997, Linton et al. 1999), it
seemed likely that SSU rDNA data would be able to
distinguish whether the genera Phacus, Lepocinclis, Astasia, and Khawkinea are valid or should be returned
to the genus Euglena. This analysis uses the sequences
from 20 euglenoid taxa, including five Phacus species,
and two Lepocinclis species, in an effort to resolve this
issue.
Almost since the creation of the genus Euglena
(Ehrenberg), the taxa assigned to it have been separated, split apart, and reorganized into new genera
based on morphological relationships, resulting in
the creation of the genera Phacus (Dujardin), Lepocinclis (Perty), Astasia (Pringsheim), and Khawkinea
(Jahn and McKibben) based on intuitive methods. In
an effort to assess the validity of these genera, we
have used small subunit (SSU) rDNA data to generate a phylogenetic framework for these genera, with
particular attention to the genus Euglena. Using the
conserved sequence areas, we performed a phylogenetic analysis using parsimony, maximum likelihood,
and distance methods. These different criteria have
resulted in trees of the same topology. The euglenoid clade was composed of phagotrophic euglenoids at the base, which gave rise to phototrophs
that in turn gave rise to osmotrophs. Among the photosynthetic taxa, the biflagellate form diverged prior
to the uniflagellate form. Additionally, the need for
a revision in the taxonomy of some of these genera
was demonstrated. Currently, taxa from the photosynthetic genera Euglena, Phacus, and Lepocinclis do
not form monophyletic clades, but are intermixed
with each other as well as with the osmotrophic taxa,
Astasia and Khawkinea.
Key index words: Astasia; distance; euglenozoa; Khawkinea; Lepocinclis; maximum likelihood; parsimony;
Phacus; phylogeny; taxonomy
The genus Euglena was erected by Ehrenberg in
1830, but was not diagnosed by him until 1838, at
which time it contained photosynthetic as well as colorless taxa. As new euglenoids were described, many
were assigned to the genus Euglena, until it eventually
contained a variety of taxonomic forms: photosynthetic, osmotrophic (with and without a stigma), fusiform, ovoid, flattened (extent variable), metabolic
1
2
materials and methods
Culture conditions. Cultures were obtained from the Culture
Collection of Algae at the University of Texas at Austin (UTEX)
and grown in their respective media. For sources and descriptions of previously sequenced cultures, see Linton et al. (1999)
and Montegut-Felkner and Triemer (1997). Euglena acus
(UTEX 1316) and Euglena spirogyra (UTEX LB 1307) were
grown in soilwater; Euglena anabaena (UTEX 373) was grown in
Received 27 December 1999. Accepted 13 March 2000.
Author for correspondence: email [email protected].
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SSU ANALYSIS OF THE EUGLENOPHYTES
Bristol’s proteose peptone medium; Phacus pusillus (UTEX 1282)
and Phacus megalopsis (UTEX 1284) were grown in soilwater
(GR⫹), and Phacus caudata (UTEX 1285) was grown in soilwater
(GR⫹/NH4). Based on light, TEM, and SEM studies by V. Conforti and M. A. Nudelman, neither P. megalopsis nor P. caudata
conformed to the diagnosis for these species, but appeared to be
Phacus splendens and Phacus oscillans, respectively. Therefore, they
are referred to as P. splendens and P. oscillans in this manuscript.
DNA isolation, amplification, and sequencing. Genomic DNA was
isolated from cells as previously described (Linton et al. 1999),
using either the Chelex procedure (Goff and Moon 1993) with
Chelex 100 resin (Bio Rad, 143-2832, Hercules, CA), or the
Puregene DNA Isolation Kit (Gentra Systems, Inc., D-5500A,
Minneapolis, MN) following the plant tissue protocol, or the
Dynabead DNA Direct System I (Dynal, 630.02, Oslo, Norway)
following the general DNA isolation protocol. The SSU rDNA
sequences were amplified using PCR (Montegut-Felkner and
Triemer 1997), with universally conserved primers, 5⬘ coding
and 3⬘ noncoding (Medlin et al. 1988). The amplified products
were purified and sized on agarose gels then extracted using
the QIAEX II DNA Gel Extraction Kit (Qiagen, 20021, Santa
Clarita, CA) according to manufacturer’s instructions. The purified template was then sequenced with internal primers for
conserved regions (Elwood et al. 1985, Montegut-Felkner and
Triemer 1997) using an ABI 377 dye-terminator cycle sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA).
All sequences were generated using both forward and reverse
strands of the SSU rDNA. Any discrepancies among sequences
from different primers were resolved by reading the ABI chromatogram for each primer.
Sequence alignment. The Genetic Data Environment (GDE 2.2)
program (Smith et al. 1994) was used on a Sparc2 workstation
to enter and align sequences from all taxa. Sequences of Euglena
stellata (GenBank accession AF150936), E. acus (AF152104), E.
spirogyra (AF150935), E. anabaena (AF242548), Phacus oscillans
(AF181968), P. pusillus (AF190815), and P. splendens (AF190814)
were added to previously published sequences: Petalomonas cantuscygni (U84731), Peranema trichophorum (U84733, U84734), Khawkinea quartana (U84732), Eutreptiella sp. (AF112875), Phacus pyrum
(AF112874), Euglena viridis (AF112872), Euglena gracilis (M12677),
Euglena agilis (AF115279), Euglena sp. (AF112873), Lepocinclis
ovum (AF110419), Lepocinclis ovata (AF061338), and Astasia longa
(AF112871). The SSU rDNA sequence of Phacus similis (SAG
58.81) from the Culture Collection of Algae at the University of
Göttingen (Sammlung von Algenkulturen Göttingen) was provided by Dr. A. Preisfeld (University of Bielefeld, Bielefeld, Germany). Sequences were manually aligned, as suggested by Sogin
and Gunderson (1987), with the published E. gracilis sequence
obtained from the database at the University of Antwerp (http://
rrna-www.uia.ac.be). The alignment was improved using the
published secondary structure of E. gracilis, K. quartana, P. trichophorum, and P. cantuscygni SSU rRNA (Van de Peer et al.
1999), as suggested by Kjer (1995). This method was especially
useful in conserved to modestly variable regions. Highly variable regions, helices E 8_1, 10, 11, E 23_1, E 23_5, and E 23_10
could not be unambiguously aligned, and were not used in the
analyses due to the inability to assess homology. A total of 1673
nucleotides were included in the phylogenetic analyses under
parsimony, distance, and maximum likelihood criteria.
Phylogenetic analysis. Parsimony, maximum likelihood, and
distance analyses were performed on the aligned data using
PAUP*4.0d65 (Swofford 1999). Each included nucleotide position was treated as an independent, unordered, multistate character of equal weight, and alignment gaps were treated as a fifth
base. A parsimony (heuristic search with random stepwise addition and 2000 repetitions) search to find a minimum-length
tree(s) was performed using addition sequence set at furthest,
ACCTRAN character state optimization, tree bisection-reconnection (TBR) branch swapping, and MULTREES on. The
maximum likelihood analyses (heuristic search with random
stepwise addition and 100 repetitions, TBR branch swapping,
starting branch length using Rogers-Swofford approximation
method, and MULTREES on) were run using the Kimura two-
741
parameter (K2P) model (Kimura 1980). Distance trees were
generated (heuristic search with random stepwise addition and
2000 repetitions) using the Kimura two-parameter model. Evolutionary distances were also computed in TreeCon Windows
version 1.3b (Van de Peer and De Wachter 1994) using the substitution rate calibration algorithm of Van de Peer (Van de
Peer et al. 1996). P-values (similar to the gamma distance computation of Jin and Nei 1990) from 0.3 to 0.6 were tested. The
left-skewness (g1) test was performed using 10,000 randomly
generated trees under the parsimony criterion (as stated above)
to determine if the data set contained a phylogenetic signal
(Hillis and Huelsenbeck 1992).
The robustness and stability of parsimony and distance trees
were estimated using nonparametric bootstrapping (Felsenstein 1985) with 2000 replicates. Maximum likelihood nonparametric bootstrapping was performed with 100 replicates. The
stability of the most parsimonious tree was also measured in
PAUP* using the decay index (Bremer 1994, Donoghue et al.
1992) with constraint trees generated by the program AutoDecay 2.9.6 (Eriksson 1997). For all parsimony analyses, consistency and retention indices were based only on informative positions.
Outgroup selection. The euglenoids P. trichophorum and P. cantuscygni were chosen as outgroups in this study, based on previous work that clearly demonstrated that they were not members
of the genera Euglena or Eutreptiella (Linton et al. 1999). Also,
their divergence of 26% to 33% from the other taxa in this
study placed them closer to, without being part of, the ingroup
than taxa previously used as outgroups (Montegut-Felkner and
Triemer 1997, Linton et al. 1999).
results
The base composition of the data set was balanced
with a mean GC ratio of 55%; thus, the nucleotide ratios among the taxa were not skewed. The left-skewness
test with 10,000 randomly generated trees resulted in a
g1 equal to ⫺0.79, which indicated that the data matrix
contained a phylogenetic signal, P ⬎ 0.01 (Hillis and
Huelsenbeck 1992). The shortest randomly generated
parsimony trees were at least 532 additional steps in
length.
Parsimony analysis of the complete data set yielded
two most parsimonious trees, strict consensus shown
(Fig. 1) with a length of 2722 steps based on 627 parsimony informative characters. The two trees varied
only in the branching order of E. acus, E. spirogyra,
and L. ovum. Although the node containing these
three taxa was strongly supported (bootstrap 90% and
decay d7) the relationship among these taxa could
not be resolved and was best represented as a trichotomy (Fig. 1). Most of the remaining nodes of the tree
showed high bootstrap and decay values, especially at
its base. The separate divergences of Eutreptiella sp. followed by E. anabaena were both strongly supported by
100% bootstrap values and decay indices of d45 and
d44, respectively. The topology of the parsimony tree has
members of the photosynthetic genera (Phacus, Lepocinclis), and osmotrophic genera (Khawkinea, Astasia) intermixed among members of the photosynthetic genus
Euglena. The osmotrophs, K. quartana and A. longa, did
not form a distinct clade, but were positioned separately within a well-supported (bootstrap 86% and decay d6) monophyletic clade containing taxa from the
photosynthetic genus Euglena. Within this clade, the
grouping of E. stellata and E. viridis as sister taxa was
742
ERIC W. LINTON ET AL.
well supported, while the divergence of K. quartana
had low support (bootstrap 55% and decay index d1).
The separate divergences of the other members of the
Euglena-Khawkinea-Astasia clade were well supported
by both bootstrap and decay indices. The remaining
taxa formed a well-supported monophyletic sister clade
(bootstrap 84% and decay d11) to the Euglena-Khawkinea-Astasia clade. This clade contained three genera:
Euglena, Phacus, and Lepocinclis, distributed among
three clades, only one of which was mono-generic. A
high bootstrap of 85% and a decay of d6 supported
the Phacus-specific clade, with P. pusillus diverging
first, followed by P. oscillans and P. similis. The sister
clade to the Phacus-specific clade did not contain any
of the remaining members of the genus Phacus, but
was composed of E. acus, E. spirogyra, and L. ovum. Although the support for grouping these three taxa was
strong, parsimony analysis was unable to resolve their
branching order, resulting in a trichotomy. The remaining clade composed of P. pyrum, P. splendens, and
L. ovata was sister to the Phacus-specific and EuglenaLepocinclis clades. This clade was well resolved and
strongly supported (bootstrap 84% and decay d11)
with P. splendens diverging prior to the L. ovata and P.
pyrum group.
Maximum likelihood analysis yielded a single tree
with a topology identical to that found using parsimony. This tree included the unresolved trichotomy,
but had weaker bootstrap support (69%) for the split
between the Euglena-Khawkinea-Astasia clade and the
Phacus-Euglena-Lepocinclis clade (Fig. 2).
Distance analysis using the K2P (Fig. 3) or substitution rate calibration algorithm (P equal to 0.3 or 0.6,
not shown) resulted in trees of identical topologies,
which differed only slightly in branch lengths and
bootstrap support. The topology of the distance trees
was identical to that seen in the parsimony and maximum likelihood trees except that the split between
the Euglena-Khawkinea-Astasia, and Phacus-Euglena-Lepocinclis clades was strongly supported (bootstrap 88%)
as in the parsimony analysis (84%). Additionally, the
trichotomy among E. acus, E. spirogyra, and L. ovum remained unresolved, but the clade had a high bootstrap support of 99%.
discussion
At the present time, there is no single ideal method
available to assess phylogeny, especially for organisms
believed to have a long evolutionary history, such as
the euglenoids. Therefore, parsimony, distance, and
maximum likelihood methods were used to analyze
all sequence data, keeping in mind each method’s advantages and limitations. Additionally, distance analysis using the substitution rate calibration algorithm, as
implemented in TreeCon for Windows (Van de Peer
and De Wachter 1994), estimates large evolutionary
distances more accurately by calculating substitution
rates of individual nucleotides (Van de Peer et al.
1996). However, this method requires at least 30 or
more aligned sequences to estimate a P-value, and sufficient data is not available for the euglenoids at this
time. As an alternative, we used a range of predefined
values to test the effects of different substitution rates
on tree topology. Low P-values indicate a greater
spread in substitution rates (P ⫽ 0.3), while higher
values indicate a similar substitution rate (P ⫽ 0.6)
among the individual nucleotides.
Fig. 1. Strict consensus tree of two
most parsimonious trees for euglenoid species generated with PAUP*.
Numbers above the branches represent the percentage of 2000 bootstrap replications in which the node
was found, and numbers at the nodes
(d) indicate decay indices.
SSU ANALYSIS OF THE EUGLENOPHYTES
The phylogeny presented here is in agreement
with the previous molecular phylogenies of MontegutFelkner and Triemer (1997) and Linton et al. (1999),
which suggest that phagotrophic euglenoids gave rise
to the phototrophs, which in turn, gave rise to the osmotrophs, while phototrophs with two emergent flagella (Eutreptiella sp.) preceded those with one emergent flagellum. Moreover, the current analysis also
demonstrates the need for a revision in the taxonomy
of euglenoids.
Within the crown clade, A. longa remains the sister
taxon to E. gracilis and Euglena sp., while K. quartana is
still the sister taxon to E. agilis, A. longa, E. gracilis, and
Euglena sp., indicating a closely shared evolutionary history among these genera. As discussed previously (Linton
et al. 1999), this close relationship has been evident since
the establishment of the genus Astasia (Pringsheim
1936) and the genus Khawkinea (Jahn and McKibben
1937, Jahn 1946). The separation of Astasia from Euglena, and later the separation of Khawkinea from Astasia, was first recognized as being unnatural by Pringsheim and Hovasse (1950). Pringsheim (1956) later
recognized that the loss of photosynthesis had occurred multiple times in various photosynthetic euglenoid groups, and the validity of separating the colorless
forms with a stigma into a separate genus was doubtful. Although only two osmotrophic euglenoids were
included in our analyses, support for the multiple origins of osmotrophic euglenoids was shown: A. longa
and K. quartana had separate divergences. This appears
to support Pringsheim’s (1956) view that the various
Astasia and Khawkinea species are more closely related
to members of the photosynthetic genus Euglena than
to members of their own genera. The weak support in
743
the parsimony and maximum likelihood analyses for
the positioning of K. quartana may indicate that its
photosynthetic ancestor has not yet been identified.
Future investigation should include the addition of
more phototrophic and osmotrophic euglenoids to
the data set, especially additional Khawkinea and Astasia species, to examine this hypothesis more fully.
Euglena stellata and E. viridis are members of the
grouping Radiatae (Pringsheim 1956), and the subgenus Euglena (Zakryś and Walne 1994). Members are
characterized by ribbon-shaped chloroplasts radiating
from a paramylon center in a star-like pattern. These
two taxa are very similar in appearance, but can be differentiated by the shape of the mucus bodies, made
visible after staining with neutral red, between the pellicle strips. In E. stellata, the mucus bodies are fusiform, whereas in E. viridis they are spherical. Although these taxa are nearly indistinguishable, their
SSU rDNA sequences are distinct, having diverged by
13% from each other, 3% and 10% more than A.
longa and K. quartana have diverged from E. gracilis,
respectively (data not shown). Thus, E. stellata and E.
viridis appear to be distinct species, while the subgenus Euglena and the group Radiatae seem to be phylogenetically valid, i.e. monophyletic as defined.
The group Catilliferae (Pringsheim 1956), subgenus Calliglena (Zakryś and Walne 1994) are represented by E. gracilis, Euglena sp., and E. agilis at the
crown of the tree and E. anabaena near the base of the
tree. These taxa are grouped by their possession of
shield-shaped chloroplasts with double pyrenoids covered with a lens-shaped cap of paramylon. The large
separation of E. anabaena from the other morphologically similar members of the genus Euglena indicates
Fig. 2. Maximum likelihood tree
generated for euglenoid species using
the Kimura two-parameter model
with PAUP*. Numbers above the
branches represent the percentage of
100 bootstrap replications in which
the node was found. Scale bar ⫽ 10%
divergence.
744
ERIC W. LINTON ET AL.
Fig. 3. Distance tree generated for
euglenoid species using the Kimura
two-parameter model with PAUP*.
Numbers above the branches represent the percentage of 2000 bootstrap
replications in which the node was
found. Scale bar ⫽ 10% divergence.
three possibilities. First, a shield-shaped chloroplast containing a double pyrenoid and lens-shaped paramylon
caps has arisen more than once, thereby making this
character homoplastic. Second, it may have been modified in some of the descendants of the last common ancestor, thereby making this character plesiomorphic.
Third, even though the position of E. anabaena is well
supported, our analyses are based on a single gene (SSU
rDNA), and therefore, could be misleading. This issue
may be resolved by the sequencing of more genes, e.g.
LSU rDNA, actin, tubulin, or of SSU rDNA genes from
additional Euglena species, particularly those that belong to the group Catilliferae and the subgenus Calliglena. Additionally, these sequences will be necessary
to assess the validity of the group Catilliferae and the
subgenus Calliglena. At present, SSU rDNA trees suggest that neither is monophyletic, and therefore not a
valid phylogenetic group.
The remaining clades in this analysis are composed
of a combination of Euglena, Lepocinclis, and Phacus
species located within the larger Euglena clade. Initially, these taxa may appear to have little in common,
but they do have similar morphological traits: all are
nearly rigid with disc- or lens-shaped chloroplasts, lack
pyrenoids, and are usually associated with two large
paramylon grains. Additionally, these genera have a related taxonomic history with Phacus and Lepocinclis being
separated from the genus Euglena. The genus Euglena was
erected by Ehrenberg (1830, formally in 1838), but by
1841, the number of Euglena species was becoming quite
large, so Dujardin erected the genus Phacus to contain
those members that were rigid, flattened (amount of flattening varies), and without pyrenoids in “most” species.
Later, Perty (1849) erected the genus Lepocinclis to
contain those members of the genus Euglena that were
rigid, not flattened, and without pyrenoids in all species. With the similarity in morphology and the nature
of their taxonomic history, it is not unreasonable to
find these taxa intermixed.
The genus Phacus forms two separate nonsister
clades in the middle of the tree; whereas one clade is
composed of Phacus species only, the other clade is a
mixture of Phacus and Lepocinclis species. Phacus similis, P. oscillans, and P. pusillus form the monophyletic
clade and differ from the other two Phacus species in
that they have a much flatter body with a “fold.” Phacur pusillus has a wide oblique furrow in the anterior
one-third to one-half of its body; the sides of P. oscillans are often rolled up forming a central groove,
whereas P. similis has one complete twist (Pockman
1942, Huber-Pestalozzi 1955, Tell and Conforti 1986).
Whether these features prove to be diagnostic will require the analysis of additional flat and/or “folded”
Phacus species. The second Phacus clade contains P.
pyrum, L. ovata, and P. splendens, all of which are ovoid
or elliptical in shape, with thick pellicle strips and a
pointed tail (Pockman 1942, Huber-Pestalozzi 1955,
Tell and Conforti 1986). Phacus splendens was originally
described as Phacus species cf. pyrum ([Ehrenberg]
Stein, by Chaudefaud 1937) and is very similar in appearance to P. pyrum but has the distinguishing feature
of chloroplast with pyrenoids, while pyrenoids are lacking in all other Phacus species in this analysis. Lepocinclis ovata is also similar in appearance to P. pyrum, and
was originally described as P. pyrum var. ovata (Playfair
1921) until Conrad (1934) moved it to the genus Lepocinclis. Finally, P. pyrum has been moved twice; it was
originally described as Euglena pyrum (Ehrenberg
SSU ANALYSIS OF THE EUGLENOPHYTES
1831) and was later moved to the genus Lepocinclis by
Perty (1849) before finally being placed in the genus
Phacus by Stein (1878). Pringsheim (1956) and others
have criticized the change of E. pyrum to P. pyrum;
based on the evidence presented here, their assessment appears to be valid not only for P. pyrum but possibly for other Phacus species as well.
The remaining clade on this tree is sister to the two
Phacus clades, and contains E. spirogyra, E. acus, and L.
ovum, which form a trichotomy. This indicates that
there is insufficient phylogenetic signal among these
taxa to definitively resolve their branching order. The
inclusion of additional taxa may resolve this node. Euglena spirogyra and E. acus represent the group Rigidae
(Pringsheim 1956) and the subgenus Discoglena (Zakryś
and Walne 1994). Although all members of the Rigidae
are contained within the subgenus Discoglena, the
Rigidae are limited to nearly rigid, elongated cells with
a posterior spine, whereas Discoglena is more broadly
defined, and contains members that can be either
highly metabolic, shorter, or lacking a spine. Their
unifying morphological feature is the chloroplast,
characterized as being numerous, small lens- or discshaped, and lacking pyrenoids. The chloroplasts of L.
ovum also fit this characterization, as well as being
nearly rigid. The taxonomic history of L. ovum has
been one of reinterpretation; it was originally classified as Euglena ovum by Ehrenberg (1838), then
moved to the genus Phacus by Klebs (1883), before finally being placed into the genus Lepocinclis by Lemmermann (1910). This close association of L. ovum
with E. spirogyra and E. acus, suggested by molecular
data and chloroplast structure, indicates that the original Ehrenberg classification may have been correct.
The use of molecular SSU rDNA sequence data has
led to phylogenetic trees that are well resolved and
stable, with high bootstrap support at most nodes.
Trees generated using parsimony, distance, and maximum likelihood agree on a single topology indicating
a robust phylogeny. This tree places osmotrophs Astasia and Khawkinea among the phototrophic genus Euglena, making Euglena paraphyletic. Also, taxa from
the three phototrophic genera, Phacus, Lepocinclis,
and Euglena, are intermixed on this tree, making all
three genera paraphyletic. The topology of this tree
implies that Phacus and Lepocinclis do not represent
monophyletic lineages, and given their taxonomic histories, it is likely that both genera are artificial groupings based on similarity. Moreover, if these genera are
not natural (“natural” meaning monophyletic in the
cladistic sense) but are, in fact, merely phenotypic
variations, a more phylogenetically accurate scheme
would move these genera back into the genus Euglena,
making Euglena monophyletic. This move into the genus Euglena would appear to be valid also for A. longa
and K. quartana, although whether valid for both genera in toto, will require additional sequence data.
The authors wish to acknowledge financial support provided by
the National Science Foundation PEET (Partnership for En-
745
hanced Expertise in Taxonomy) Grant No. DEB 4-21348. The
authors wish to thank Carole L. Lewandowski for her technical
assistance and for critically reading the manuscript, and the reviewers and editor of this manuscript. The research was submitted by E. W. Linton in partial fulfillment of the requirements
for the Ph.D. degree, Rutgers University, New Brunswick, NJ.
Bremer, K. 1994. Branch support and tree stability. Cladistics
10:295–304.
Chaudefaud, M. 1937. Anatomic comparée des Eugléniens. Le Botaniste 28:85–185.
Conrad, W. 1934. Matériaux pour une monographie du genre
Lepocinclis Perty. Arch. Protkde. 82:203–49.
Donoghue, M. J., Olmstead, R. G., Smith, J. F. & Palmer, J. D. 1992.
Phylogenetic relationships of Dipscales based on rbcL sequences. Ann. Mo. Bot. Gard. 79:249–65.
Dujardin, F. 1841. Histoire Naturelle des Zoophytes Infusoires.
Roret, Paris, 684 pp.
Ehrenberg, C. G. 1830. Neue Beobachtungen über blutartige Erscheinungen in Ägypten, Arabien, und Sibirien, nebst einer
Übersicht und Kritik der früher bekannten. Pogg. Ann. Physik.
Chem. 94:477–514.
———1831. Über die Entwicklung und Lebensdauer der Infusionsthiere: nebst ferneren Beiträgen zu einer Vergleichung ihrer
organischen Systeme. Phys. Abh. Berl. Akad. Wiss. 1831:1–154.
———1838. Die Infusionsthierchen als Vollkommene Organismen. Verlag von Leopold Voss, Leipzig.
Elwood, H. J., Olsen, G. J. & Sogin, M. L. 1985. The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates
Osytricha nova and Stylonychia pustulata. Mol. Biol. Evol. 2:399–410.
Eriksson, T. 1997. AutoDecay ver. 2.9.6 (Hypercard stack distributed
by the author). Botaniska Institutionen, Stockholm University,
Stockholm.
Felsenstein, J. 1985. Confidence limits on phylogenies: an approach
using the bootstrap. Evolution 39:783–91.
Goff, L. & Moon, D. 1993. PCR amplification of nuclear and plastid
genes from algal herbarium specimens and algal spores. J. Phycol. 29:381–4.
Hillis, D. M. & Huelsenbeck, J. P. 1992. Signal, noise, and reliability
in molecular phylogenetic analyses. J. Hered. 83:189–95.
Huber-Pestalozzi, G. 1955. Das Phytoplankton des Süsswassers. In
Thienemann, A. [Ed.] Die Binnengewässer Stuttggart, 16(4). E.
Schweizerbart’che Verlagsbuchhandlung, Stuttgart, 1135 pp.
Jahn, T. L. 1946. The euglenoid flagellates. Q. Rev. Biol. 21:246–74.
Jahn, T. L. & McKibben, W. R. 1937. A colorless euglenoid flagellate
Khawkinea halli n.gen., n. sp. Trans. Am. Microsc. Soc. 56:48–54.
Jin, L. & Nei, M. 1990. Limitations of the evolutionary parsimony
method of phylogenetic analysis. Mol. Biol. Evol. 7:82–102.
Kimura, M. 1980. A simple method for estimating evolutionary rate
of base substitutions through comparative studies of nucleotide
sequences. Proc. Natl. Acad. Sci. USA 63:1181–8.
Kjer, K. M. 1995. Use of rRNA secondary structure in phylogenetic
studies to identify homologous positions: an example of alignment and data presentation from the frogs. Mol. Phylogenet.
Evol. 4:314–30.
Klebs, G. 1883. Über die Organisation einiger Flagellaten-Gruppen
und ihre Beziehungen zu Algen und Infusorien. Untersuchung
Botanisches Inst. Tübingen 1:233–62.
Lemmermann, E. 1910. Kryptogamenflora der Mark Brandenburg
und angrenzender Gebiete (herausgegeben von dem Bot. Ver.
der Prov. Brandenburg). Algen I (Schizophyceen, Flagellaten, Peridineen). Eugleninen, vol. 3. Borntraeger, Leipzig, pp. 484–562.
Linton, E. W., Hittner, D., Lewandowski, C., Auld, T. & Triemer, R. E.
1999. A molecular study of euglenoid phylogeny using small
subunit rDNA. J. Eukaryot. Microbiol. 46(2):217–23.
Medlin, L., Elwood, H. J., Stickel, S. & Sogin, M. L. 1988. The characterization of enzymatically amplified eukaryotic 16S-like
rRNA coding regions. Gene 71:491–9.
Montegut-Felkner, A. E. & Triemer, R. E. 1997. Phylogenetic relationships of select euglenoid genera based on morphological
and molecular data. J. Phycol. 33:512–9.
Perty, M. 1849. Über verticale Verbreitung mikroskopischer Leb-
746
ERIC W. LINTON ET AL.
ensformen. Mittheilungen der Naturforschenden Gesellschaft in Bern
(No. 146–149). pp. 17–45.
Playfair, G. J. 1921. Australian freshwater flagellates. Ibid 46:99–146.
Pockman, A. 1942. Synopsis der Gattung Phacus. Arch. Protistenk.
95:81–252.
Pringsheim, E. G. 1936. Zur Kenntnis saprotropher Algen und
Flagellaten, I: Über Anhäufungskulturen polysaprober Flagellaten. Arch. Protistenk. 87:43–96.
———1956. Contributions towards a monograph of the genus Euglena. Nova Acta Leopoldina 125:1–168.
Pringsheim, E. G. & Hovasse, R. 1950. Les relations de parenté entre Astasiacées et Euglenacées. Arch. Zool. Exp. Gén. 86:499–549.
Smith, S. W., Overbeek, R., Woese, C. R., Gilbert, W. & Gillevet, P.
M. 1994. The genetic data environment: an expandable GUI
for multiple sequence analysis. Comput. Appl. Biosci. 10:671–5.
Sogin, M. L. & Gunderson, J. H. 1987. Structural diversity of eukaryotic small subunit ribosomal RNAs: evolutionary implications.
Ann. NY Acad. Sci. 573:125–39.
Stein, F. R. 1878. Der Organismus der Infusionsthiere. III. Abt. der
Organismus der Flagellaten, William Engelmann, Leipzig, 154 pp.
Swofford, D. L. 1999. PAUP*: Phylogenetic Analysis Using Parsimony
(and Other Methods), version 4.0. Sinauer Associates, Sunderland, Massachusetts.
Tell, G. & Conforti, V. 1986. Euglenophyta pigmentadas de la Ar-
gentina. In Cramer, J. [Ed.] Bibliotheca Phycologica. J. Cramer,
Berlin, 301 pp.
Triemer, R. E. & Farmer, M. A. 1991a. The ultrastructural organization of the heterotrophic euglenids and its evolutionary implications. In Patterson, D. J. & Larsen, J. [Eds.] The Biology of Free-Living Heterotrophic Flagellates. Clarendon Press, Oxford, pp. 186–204.
———1991b. An ultrastructural comparison of the mitotic apparatus, feeding apparatus, flagellar apparatus and cytoskeleton in
euglenoids and kinetoplastids. Protoplasma 164:91–104.
Van de Peer, Y. & De Wachter, R. 1994. TREECON for Windows: a
software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput.
Appl. Biosci. 10:569–70.
Van de Peer, Y., Rensing, S. A., Maier, U.-G. & De Wachter, R. 1996.
Substitution rate calibration of small subunit ribosomal RNA
identifies chlorarchniophyte endosymbionts as remnants of
green algae. Proc. Natl. Acad. Sci. USA 93:7732–6.
Van de Peer, Y., Robbrecht, E., de Hoog, S., Caers, A., De Rijk, P. &
De Wachter, R. 1999. Database on the structure of small subunit ribosomal RNA. Nucleic Acids Res. 27:179–83.
Zakryś, B. & Walne, P. L. 1994. Floristic, taxonomic and phytogeographic studies of green Euglenophyta from the Southeastern
United States, with emphasis on new and rare species. Algol.
Stud. 72:71–114.