J. Phycol. 39, 226–235 (2003)
PHYLOGENY OF EUGLENOPHYCEAE BASED ON SMALL SUBUNIT rDNA SEQUENCES:
TAXONOMIC IMPLICATIONS 1
María Alejandra Nudelman 2
Laboratorio de Biología Comparada de Protistas, Departamento de Biodiversidad y Biología Experimental,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina; and Pabellón II,
Ciudad Universitaria, Buenos Aires, C1428EHA, Argentina
María Susana Rossi
Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular y Celular,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina; and Pabellón II,
Ciudad Universitaria, Buenos Aires, C1428EHA, Argentina
Visitación Conforti
Laboratorio de Biología Comparada de Protistas, Departamento de Biodiversidad y Biología Experimental,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina; and Pabellón II,
Ciudad Universitaria, Buenos Aires, C1428EHA, Argentina
and
Richard E. Triemer
Michigan State University, Department of Plant Biology, 166 Plant Biology, East Lansing Michigan 48824-1312, USA
Small subunit rDNA sequences of 42 taxa belonging to 10 genera were used to infer phylogenetic relationships among euglenoids. Members of the phototrophic genera Euglena, Phacus, Lepocinclis, Colacium,
Trachelomonas, and Strombomonas plus the osmotrophs
Astasia longa, Khawkinea quartana, and Hyalophacus
ocellatus were included. Six major clades were found
in most trees using multiple methods. The utility of
Bayesian analyses in resolving these clades is demonstrated. The genus Phacus was polyphyletic with taxa
sorting into two main clades. The two clades correlated with overall morphology and corresponded in
large part to the previously defined sections, Pleuraspis Pochmann and Proterophacus Pochmann. Euglena was also polyphyletic and split into two clades.
In Bayesian analyses species with less plastic pellicles
and small disk-like chloroplasts diverged at the base
of the tree. They grouped into a single clade which
included the two Lepocinclis spp., which also are rigid
and bear similar chloroplasts. The metabolic Euglena
species with larger plastids bearing pyrenoids and
paramylon caps arose near the top of the tree. The loricates Strombomonas and Trachelomonas formed two wellsupported, but paraphyletic, clades. The strong support
for the individual clades confirmed the value of using
lorica features as taxonomic criteria. The separation
of the osmotrophic species A. longa, K. quartana, and
H. ocellatus into different clades suggested that the
loss of the photosynthetic ability has occurred multiple times.
Key index words: euglenoids; Euglenophyta; Euglenozoa; molecular phylogeny; SSU rDNA
Euglenoids are a diverse group of unicellular flagellates regularly found in a variety of freshwater and marine environments. Despite the fact that euglenoids
are common, the taxonomy and phylogeny among the
phagotrophic, osmotrophic, and phototrophic species
is still to be resolved. Phagotrophs possess a unique and
complex ingestion apparatus (Leedale 1967, Triemer
and Farmer 1991a,b). Some osmotrophic and phototrophic taxa show relicts of such a feeding structure
(Dynesius and Walne 1975, Willey and Wibel 1985a,b,
Surek and Melkonian 1986, Owens et al. 1988, Willey
et al. 1988, Solomon et al. 1991, Shin et al. 2000, 2001,
Shin and Boo 2001). It is now accepted that a plastid
from a green alga, ingested by a phagotrophic euglenoid, gave rise to the phototrophic forms by means of
a secondary endosymbiosis (Gibbs 1978, 1981). Typically, the storage product is a 1,3-glucan called paramylon. The shape and localization of the paramylon granules in the cytoplasm, and their possible association with
the chloroplasts have been considered key diagnostic
features of green euglenoids. Another important diagnostic feature present in all euglenoids is the pellicle. The pellicular complex consists of the cell membrane underlain by an epiplasmic layer of variable
thickness. This proteinaceous layer forms interlocking bands, giving the cell surface an undulate appearance of alternating strips and striae. The pellicle may
be longitudinally or helically striated. Pre- and postarticular plates or teeth-like projections may be present
1 Received
2 Author
23 May 2002. Accepted 11 November 2002.
for correspondence: e-mail [email protected].
226
PHYLOGENY OF EUGLENOPHYTES
in the articulation zone of two adjacent bands (Leander and Farmer 2001b). The organization and variations of these basic components in the different taxa
result in a pellicle that may range from highly elastic
(or metabolic) to quite rigid. A few forms are surrounded partially or completely by an extracellular envelope, termed a lorica, composed of mineral impregnated mucilage (West and Walne 1980). The shape,
color, and ornamentation of the lorica are the main
taxonomic criteria used to identify species among the
members of the genera Trachelomonas and Strombomonas. However, it has been demonstrated that envelope
characteristics might be strongly influenced by environmental conditions (Pringsheim 1956, Singh 1956).
Over the years, several attempts to incorporate internal cell features (i.e. chloroplast and pyrenoid structure) into a classification scheme for the loricates has
met with only limited success (Pringsheim 1953), and
the limits between the genera are poorly defined.
Since the description of Euglena by Ehrenberg (1838),
various authors have proposed different classification
schemes based largely on the morphology and localization of chloroplasts. Recent molecular evidence suggests that the genus Euglena is polyphyletic (Linton et
al. 1999, 2000, Milanowski et al. 2001, Muellner et al.
2001). Phacus and Lepocinclis were erected from the
genus Euglena by Dujardin (1841) and Perty (1852),
respectively. Phacus was created to include those rigid
flat leaf-like species with many small discoid chloroplasts,
without pyrenoids (Pochmann 1942). Species of the genus Lepocinclis also were rigid and lacked pyrenoids but
were pear-shaped or circular and were not flattened
in cross-section. However, morphological and molecular studies from the few species examined to date suggested the need for a reevaluation of these taxa as separate genera.
Although their relationship with kinetoplastids
(Kivic and Walne 1984, Triemer and Farmer 1991a,b,
Montegut-Felkner and Triemer 1997, Linton et al.
1999, 2000, Preisfeld et al. 2000, Muellner et al. 2001)
and the monophyly of the green euglenoids (Linton
et al. 1999, 2000, Preisfeld et al. 2000, Muellner et al.
2001) and diplonemids (Maslov et al. 1999, Busse and
Preisfeld 2002) is accepted, the evolutionary relationships among taxa is only beginning to emerge. The
aim of the present study is to incorporate additional
small subunit (SSU) rDNA data from key photosynthetic taxa into the previous phylogenetic framework
to resolve the major clades of green euglenoids.
materials and methods
Taxa sequenced. Table 1 provides a list of all organisms used
in this study. Cultures were obtained from the Sammlung von
Algenkulturen (SAG) at Göttingen and the Culture Collection
of Algae at the University of Texas (UTEX) at Austin. Based on
LM, SEM, and TEM studies we determined that the cultures labeled as Trachelomonas hispida and T. oblonga var. punctata contained the same organism. Both taxa were sequenced to confirm this at the molecular level.
DNA isolation, amplification, and sequencing. Total genomic DNA
was isolated from cultures as described previously (Linton et al.
227
1999) or with the Puregene DNA Isolation Kit (D-5500A, Gentra Systems, Inc., Minneapolis, MN, USA), using the plant tissue
protocol. The SSU rDNA sequences were amplified by PCR as
described by Linton et al. (1999) (Table 2). The amplified fragments were purified and sized on agarose gels and then extracted
using the QIAEX II DNA Gel Extraction Kit (20021, Qiagen, Santa
Clarita, CA, USA) according to manufacturer’s instructions. The
purified template was sequenced using an ABI 377 dye-terminator
cycle sequencer (Perkin Elmer Applied Biosystems, Foster City,
CA, USA). All sequences were generated using data from 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 to enter and align sequences
from all taxa. The SSU rDNA sequences obtained as part of this
study have been deposited in GenBank under the accession numbers given in Table 1 and were added to previously published sequences. The SSU rDNA sequences of Phacus orbicularis, P. aenigmaticus, P. pseudonordstedtii, and P. parvulus were kindly provided
by Dr. A. Muellner (Institute of Botany, Vienna, Austria). Sequences were manually aligned using the published Euglena
gracilis sequence from the database at the University of Antwerp
(www.rrna-www.uia.ac.be). The alignment was improved using
the secondary structure of E. gracilis, Khawkinea quartana, Peranema trichophorum, and Petalomonas cantuscygni SSU rRNA (Van
de Peer et al. 1999), as suggested by Kjer (1995). This method was
especially useful in aligning sequences from conserved to modestly
variable regions. Highly variable regions, such as found in helices
E 8_1, 10, 11, E 23_1, E 23_5 and E 23_10, that could not be unambiguously aligned were not used in the analyses due to the
inability to assess homology. A total of 1490 nucleotides were
included in the data set. The data matrix is available through
www.treebase.org (study accession number S800, matrix accession
number M1266).
Phylogenetic analysis. Maximum parsimony (MP) and Bayesian
analyses were performed on the aligned data. Each included nucleotide position was treated as an independent, unordered, multistate character of equal weight, and alignment gaps were treated
as missing characters. 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 phylogenetic signal (Hillis and Huelsenbeck 1992).
An MP search (heuristic search with random stepwise addition
and 1000 repetitions) to find a minimum-length tree(s) was performed using addition sequence set at furthest, tree bisectionreconnection branch swapping, and MULTREES option in effect
using PAUP*4.0b5 (Swofford 1999). The robustness and stability
of MP was estimated using nonparametric bootstrapping (Felsenstein 1985) with 1000 replicates. Modeltest (Posada and Crandall
1998) was used to select parameters for the Bayesian analyses (MrBayes version 2.01, Huelsenbeck and Ronquist 2001). The analyses were conducted with Basefreq equal, Nst 6 (GTR model),
Revmat (1.3515, 3.5241, 1.1699, 0.7542, 4.8900, 1.000), rates gamma, shape 0.5358, number of Markov chains 4, number
of generations 500,000, and sample frequency set to 100. This
yields 5000 trees, 1000 of which are required for “burn in,” that is,
the time required for likelihood to converge on a stable value.
Trees shown are the consensus derived from 4000 trees.
Outgroup selection. The colorless euglenoid Peranema trichophorum and the biflagellate photosynthetic taxon, Eutreptiella sp. were
chosen for the outgroup based on previous work that demonstrated that these taxa are closely related to the ingroup taxa
without being part of it (Linton et al. 2000).
results
Forty-two euglenoid SSU rDNA sequences were analyzed (Table 1). A left skewness test was performed,
and the shortest random tree found was 4615 steps compared with 2958 steps in the most parsimonious tree.
The g1 0.90855 (p 0.01) indicated that the data set
228
Table 1.
MARÍA ALEJANDRA NUDELMAN ET AL .
List of organisms studied.
Taxon
Astasia longa Pringsheim
Colacium mucronatum Bourrelly et Chaudefaud
C. vesiculosum Ehrenberg
Euglena acus Ehr.
E. agilis Carter
E. gracilis Klebs
E. mutabilis Schmitz
E. spirogyra Ehr.
E. stellata Mainx
E. viridis Ehr.
Eutreptiella sp.
Hyalophacus ocellatus Pringsh.a
Khawkinea quartana (Moroff) Jahn et McKibben
Lepocinclis buetschlii Lemmerman
L. ovata (Playf.) Conrad
L. ovum (Ehr.) Lemm.
Phacus acuminatus Stokesa
P. aenigmaticus Drezepolskia
P. agilis Skuja
P. alatus Klebsa
P. brachykentron Pochmann
P. orbicularis Huebner
P. oscillans (caudata) Klebs
P. parvulus Klebs
P. pleuronectes Dujardin
P. pseudonordstedtii Poch.
P. pusillus Lemm.
P. pyrum Stein
P. similis Christen
P. splendens (megalopsis) Poch.
Peranema tricophorum (E.) Stein
Strombomonas sp1.a
Strombomonas sp.a
Trachelomonas volvocinopsis var. spiralis Pringsh.a
T. conspersa Paschera
T. echinata Singha
T. hispida (Perty) Stein emend. Deflandrea
T. intermedia Dangearda
T. oblonga var. punctata Lemm.a
T. pertyi Pringsh.a
T. volvocina Ehr.
Source
GenBank accession number
UTEX 516
Unknown
Unknown
UTEX 1316
UTEX 1605
UTEX 753
Unknown
UTEX 1307
UTEX 372
UTEX 85
CCMP 389
SAG 228.80
ATCC 30895
Korean isolate
SAG B 1244-5
SAG 1244-8
UTEX 1288
ASW 08012
SAG 1088
SAG1261-b
UTEX 1317
ASW 08045
UTEX 1285
ASW 08060
Korean isolate
ASW 08010
UTEX 1282
UTEX 2357
SAG 58.81
UTEX 1284
CBS (P7-13-1838)
NJ (US) isolate
Korean isolate
UTEX 1313
SAG 1280-1
SAG 1283-22
UTEX 1326
SAG.205-80
UTEX 1325
SAG 1283-13
Korean isolate
AF112871
AF326232
AF081592
AF152104
AF115279
M12677
AF096992
AF150935
AF150936
AF112872
AF112875
AF445458
U84732
AF096993
AF061338
AF110419
AF283311
AF283313
AY014998
AY014999
AF286209
AF283315
AF181968
AF283314
AF081591
AF283316
AF190815
AF112874
AF119118
AF190814
U84733, U84734
AF445461
AF096995
AY015004
AY015000
AY015001
AF090377
AY015002
AF445462
AY015003
F096995
Cells were grown in the media indicated in the SAG (www.gwdg.de/epsag/phykologia/epsag.html) and UTEX catalogues
(www.bio.utexas.edu/research/utex/).
a This study.
had strong phylogenetic signal (Hillis and Huelsenbeck
1992). The g1 was tested and remained significant after
the deletion of different taxa or sets of taxa from the
analysis. The base composition in the data set was balanced with a mean GC ratio of 54%; thus the nucleotide
ratios were not skewed.
MP analysis of the complete data set of 1490 characters resulted in two most parsimonious trees with 2958
steps based on 586 informative characters. The two
trees differed only in the placement of Phacus acuminatus and P. brachykentron (Fig. 1; Only one tree is shown.
In the alternative MP tree these two species form a sister group with the five taxa shown in clade F). Euglena
mutabilis diverges at the base of the tree followed by the
divergence of six major clades (A–F). Only clades B
and F were well supported. Support for clades labeled
A, C, D, and E was low, and the relationships among
the six clades could not be resolved. When illustrated
as a 50% majority rule consensus tree, nearly all the internal nodes collapsed into a large polytomy (tree not
shown). MP analyses were also run considering gaps as
a fifth base and gave identical results.
Because of the inability of parsimony to resolve the
internal nodes on the tree, we took a Bayesian approach
to analyze the SSU rDNA data set. Figure 2 shows the
consensus tree from the Bayesian analysis. We consider
values of 90% or above (p 0.1) as highly supportive.
The species are grouped into six major clades, three
of which are strongly supported (A, C, and F). The genus Euglena is polyphyletic and split between clades A
and F. Clade F, which diverges at the base of the tree,
contains rigid or semirigid taxa and includes E. spirogyra, E. oxyuris, and E. acus. Also included in this clade
are Lepocinclis ovum and L. buetschlii. Euglena mutabilis,
which is positioned at the base of the parsimony tree
(Fig. 1), is now clustered with other metabolic Euglena
species in clade A. The genus Trachelomonas is also polyphyletic and distributed among clades B and C. Most
species fall into clade B, but T. conspersa and T. volvocinopsis var. spiralis group with the two species of Strom-
229
PHYLOGENY OF EUGLENOPHYTES
Table 2.
Primers used for amplification and sequencing.
Primer
1(A)ZB*
B Eug
77 Eug
(300)*
679 (528)*
931 Pha
1031 Evir*
(926)
(960)
(1055)*
(1200)
(1400)
113 Eug*
(300)
(516)*
1434 Pha
(926)
(1055)*
(1200)
1913 Eug*
(1400)
1520 (B) ZB*
1434 Pha
Sequence (5-3)
AAC
TTG
GTG
AGG
CGG
AAC
GGA
AAA
TTT
GGT
CAG
TGT
TGC
CCG
ACC
CTA
CCG
CGG
GGG
CAT
ACG
TGA
CTA
CTG
ATC
GAT
GTT
TAA
CAG
AAA
CTC
GAC
GGT
GTC
ACA
TGA
GAA
AGA
CGG
TCA
CCA
CAT
CTG
GGC
TCC
CGG
GTT
CTG
CTG
CGA
TTC
ATC
CTC
AAA
TCA
GCA
TGT
CAC
TGT
TCG
CTT
AGG
ATT
TGC
CAC
GGA
GGT
TTC
AGG
GAT
CCA
TGA
TTC
CAG
ACA
AGT
TGA
ACA
TGG
GAT
CGC
AAG
AAC
GCC
TGT
CAT
ACC
AGA
GCA
GTG
TGC
TGT
CCT
GTA
ATG
CGG
CTC
CCC
GTG
ATT
CGG
CCG
GCT
CCG
GAG
CCT
CTC
CTA
TTG
ACC
CCT
TCA
TAC
AGG
CTA
GCC AGT
GTC
GCT
AG
C
AG
CTC
GAC GG
G
C
TC
CC
GA
C
ATC
AGT TT
G
CAG AC
TTC ACC TAC
ATC
Annealing direction
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Reverse
Reverse
Reverse
Reverse
Reverse
Reverse
Reverse
Reverse
Reverse
Reverse
Reverse
*, used as internal primers; (), universal primers; Eug, Euglena specific; Evir, Euglena viridis specific; Pha, Phacus specific.
bomonas in clade C. Similarly, the genus Phacus is split
between clades D and E. Although clades D and E lack
support, it is clear that the genus Phacus is polyphyletic
and that there are at least two clades of Phacus. The position of the colorless taxon Hyalophacus ocellatus within
Phacus clade E and sister to P. orbicularis is strongly supported. Curiously, the two species representing the genus Colacium are placed in clade D as a sister clade to
the Phacus species. Note however, that the relationship
between Colacium and Phacus is not supported (57%
probability).
discussion
Phylogenetic analyses. MP detected many of the clades
found in the Bayesian tree but was incapable of resolving
relationships among the clades. The large polytomies
and weak bootstrap support that were found in the parsimony tree demonstrates the inability of the method
to resolve the deeper relationships among the taxa using this data set. Therefore, we applied a Bayesian approach to the analysis. Unlike maximum likelihood,
which searches for the single most likely tree, a Bayesian analysis searches for the set of trees, which best
fits the data (Hall 2001). In the course of its searching, maximum likelihood can get “trapped” on a suboptimal island and may not find the most likely tree.
Bayesian analysis uses the Metropolis-coupled Markov
Chain Monte Carlo method, which can best be visualized as “a set of independent searches that occasionally exchange information” (Hall 2001). For our analyses we ran four separate Markov chains with 500,000
generations. Bayesian analysis was performed using
MrBayes version 2.01 (Huelsenbeck and Ronquist 2001).
The Bayesian tree is better resolved with substantial support for many nodes.
Loss of photosynthesis. The phylogenies presented in
this study are in general agreement with the trends
presented in previous works (Montegut-Felkner and
Triemer 1997, Linton et al. 1999, 2000, Preisfeld et al.
2000, Muellner et al. 2001). These authors proposed
that phagotrophic euglenoids gave rise to phototrophs,
which then gave rise to certain osmotrophs through a
loss of chloroplasts. The position of the colorless osmotrophic species (Astasia longa, Khawkinea quartana,
and Hyalophacus ocellatus) on the tree indicates that
they all arose from photosynthetic ancestors. Furthermore, our data indicate that loss of photosynthesis in
these taxa is the result of at least three independent
events. Muellner et al. (2001) had previously demonstrated that the genus Astasia was polyphyletic. It now
seems clear that loss of photosynthesis alone is insufficient to warrant separation as a new genus and Astasia,
Khawkinea, and Hyalophacus as now defined will need to
be revised.
Relationships within and among clades. Clade A includes
the osmotrophs Astasia longa and Khawkinea quartana
and four Euglena spp. The taxa in this clade are highly
metabolic, and most of the photosynthetic taxa have
large disk- or shield-shaped chloroplasts with pyrenoids.
Euglena stellata and E. viridis have large stellate chloroplasts with a central pyrenoid. These two taxa are almost identical in overall appearance and can only be
distinguished by the shape of their muciferous bodies
(Godjics 1953, Pringsheim 1956, Zakrys 1986). The
topology of this part of the tree is in agreement with a
previous phylogeny (Linton et al. 2000). Euglena mutabilis, which bears large, curved, disk-shaped plastids with
a central pyrenoid, is firmly placed in clade A, but its
placement as a sister taxon to E. stellata and E. viridis
lacks support. Resolution of this taxon may require
the sequencing of additional related taxa such as E.
230
MARÍA ALEJANDRA NUDELMAN ET AL .
Fig. 1. One of two most parsimonious trees generated for euglenoid species using PAUP*. Six major lineages are identified. Note
the early divergence of Euglena mutabilis near the base of the tree. Numbers adjacent to the nodes represent bootstrap values (%) of
1000 replicates. Values less than 50% are not shown.
satelles and E. deses. Similarly, the relationship of K.
quartana to the residual taxa remains unresolved.
The loricate genera Trachelomonas and Strombomonas (clades B and C) exhibit the greatest diversity among
the photosynthetic euglenoids. The taxonomy of these
groups is based mainly on the external morphology of
the lorica and the ornamentation pattern, both of
which may be influenced by external factors leading
to misidentifications of some taxa.
Trachelomonas pertyi, T. echinata, T. hispida, and “T.
oblonga var. punctata” form a strongly supported monophyletic group within clade B. Morphologically, these
taxa are united by ovoid spiny loricas. Trachelomonas
volvocina and T. intermedia group together with strong
support in both the Bayesian and MP trees. However,
in the MP tree these species are found in clade C and
in the Bayesian tree they are in clade B. Neither position is well supported by bootstrap or probability, but
in either case they are positioned within the loricates.
Trachelomonas volvocina and T. intermedia have thick ovoid
to spherical loricas, which are smooth or finely punctuated, but spines have not been observed. All the taxa
in clade B of the Bayesian tree share disk-like chloroplasts with diplopyrenoids (pyrenoids capped with
paramylon on both sides). Pringsheim (1953) described
T. pertyi as having haplopyrenoids (stalked pyrenoids,
each with a single cap of paramylon), but we have observed that a diplopyrenoid is present. Trachelomonas
PHYLOGENY OF EUGLENOPHYTES
231
Fig. 2. Consensus tree generated using Bayesian analysis. Numbers adjacent to each node represent probabilities after 500,000 generations. Morphological character states are mapped onto the tree. The first number (shown in parentheses) indicates the character. The second number indicates the state of the character at that node, without indication of polarity. Character 1: Chloroplast and pyrenoids: absent 0, numerous small disks without pyrenoids 1, few large disks or shields with or without pyrenoids 2, few stellate/axial with pyrenoids 3.
Character 2: Capacity for metaboly: present 0; absent 1. Character 3: Lorica: absent 0, thick with ornamentation 1, thin without ornamentation 2.
232
MARÍA ALEJANDRA NUDELMAN ET AL .
oblonga var. punctata was originally described as being
finely punctuated (Lemmermann 1910, Starmach 1983).
Its position in a clade of spiny Trachelomonas species was
problematic but can be explained. The culture purchased from UTEX (LB 1325) named “T. oblonga var.
punctata” had no ornamentation when we received it.
However, after a short time in culture, it developed
strong conical spines, suggesting that the morphotype
without spines was wrongly identified or mislabeled. In
addition, its SSU rDNA sequence is identical to that of
T. hispida. Therefore, this taxon should be considered
as T. hispida rather than T. oblonga var. punctata. In summary, clade B included loricates with brittle well-organized envelopes and disk-like chloroplasts with diplopyrenoids.
Clade C includes T. volvocinopsis var. spiralis, T. conspersa and two Strombomonas species. The genus Strombomonas was created by Deflandre (1930) to include the
Trachelomonas species that have a clear, hyaline, vaseshaped lorica, which lacks a distinct well-delimited collar but gradually tapers to the anterior pore. This lorica is soft, seems to be less mineralized and without
ornamentation, but usually agglutinates external material that gives the lorica an irregular granulate appearance. This is not exclusive for Strombomonas because some
Trachelomonas spp. adhere particles as well, especially
in immature, active, mucilage-secreting cells. This material may obscure the specific ornamentation, usually
leading to misidentification (Nudelman and Conforti
1997, Nudelman et al. 1998). Since the creation of Strombomonas as a separate genus, the delineation between
the two loricate forms has not been resolved.
The lorica of T. conspersa is irregularly sack to flaskshaped, with a short neck like those found in a typical
Strombomonas. The naked cells of this culture also strongly
resemble those of Strombomonas. They are quite different
from most members of Trachelomonas in being elongate
and spindle-shaped with a long colorless tail-like posterior, pronounced metaboly, and a marked striation.
On the basis of observations of live material under LM
and EM and molecular data, we suggest that T. conspersa be considered as a member of the genus Strombomonas. Unfortunately, giving this taxon a name is problematic at present. Trachelomonas conspersa Pascher was
transferred to S. verrucosa when Deflandre (1930) first
described the genus. Later, Pringsheim (1953) stated
that he would adopt the name S. conspersa for his strain
that resembled T. conspersa Pascher. Leedale’s (1967)
description of the genus includes features noted by
Deflandre and adds other features suggested by Pringsheim. Later, Dunlap et al. (1986) proposed that because there were no major differences in lorica structure or elemental composition between the strain of
S. conspersa they had studied and Trachelomonas spp.,
this species should be returned to the genus Trachelomonas. These actions create a suite of taxonomic issues that need to be dealt with independently before a
proper name can be given to this taxon.
Trachelomonas volvocinopsis var. spiralis is also positioned within clade C. Here again, we find that devel-
opmental stages lead to morphological differences in
the lorica. In young cells, the lorica has strong helical
ribs in agreement with the Pringsheim’s description
(Pringsheim 1953). In older cultures, the surface appears rugose due to the agglutination of exogenous
particulate material. Examination under TEM demonstrated that it did not conform to the species diagnosis.
Trachelomonas volvocinopsis is characterized as having
numerous (15–20, Swirenko 1914, Hüber-Pestalozzi
1955, Kim et al. 2000) small lens- or disk-shaped chloroplasts that lack pyrenoids. Our specimens possessed
three to six large shield-shaped parietal chloroplasts
with haplopyrenoids. A final piece of morphological
evidence supporting the inclusion of T. volvocinopsis var.
spiralis and T. conspersa within the Strombomonas clade is
that the loricas of both taxa possessed a typical Strombomonas structure under TEM examination, with a microarchitecture composed of irregularly arranged needle-like elements rather than the well-organized layers
characteristic of Trachelomonas loricas (Dunlap et al.
1983, 1986).
This study demonstrates that the use of the lorica
in taxonomy is complicated by the fact that the overall
appearance is highly influenced by the age of the cell,
the water chemistry (color and mineral composition),
and protoplast movement (lorica shape). This has led
to a proliferation of species names for what are probably phenotypic variants. However, some features, such
as overall shape of the lorica and the ability to form
spines and collars, seem to be quite stable and species
specific in mature envelopes (Pringsheim 1953, Singh
1956, Rosowski et al. 1975a,b, Donnelly Barnes et al.
1986, Dunlap and Walne 1987) and define highly supported groups in our analyses. Although all euglenoids
secrete mucilage at some point in their life cycles, the loricate genera, Trachelomonas and Strombomonas, acquired
the ability to retain mucilage and mineralize it on the cell
surface, leading to the appearance of the lorica. The
ancestor of clades A, B, and C had the ability to mineralize a lorica, and this ability was later lost in clade A
where mucilage secretion now plays a role in “encystment.” Although both Trachelomonas and Strombomonas form loricas, the ultrastructural features of the loricas are quite different, and the phylogeny still supports
the traditional separation of these genera based on the
characteristics of the lorica. This study has demonstrated
that the loricates form two distinct, but not monophyletic, clades. It is clear that more loricate taxa must be
added to elucidate phylogenetic relationships among
them.
In the MP tree, clade D contains only Phacus spp.
and Lepocinclis ovata, but in the Bayesian tree it forms
a poorly supported clade (57% probability) that includes
the Colacium spp. and will collapse into a polytomy. Clade
D is best represented as at least two clades, one containing Colacium spp. and a second containing the Phacus
spp. and L. ovata. Phacus agilis, which diverges at the
base of the Phacus group is weakly supported, could
also collapse into a polytomy. This taxon has two parietal chloroplasts associated with paramylon caps. The
PHYLOGENY OF EUGLENOPHYTES
remaining taxa form a well-supported clade and share
many traits: all are ovoid in shape, with thick, helically
arranged, raised pellicle strips and a well-developed
pointed tail. The clade is characterized by rigid cells
with disk- or lens-shaped parietal chloroplasts that are
usually associated with large paramylon grains. All but
P. splendens lack pyrenoids (unpublished results). In all
trees, P. splendens clades with P. aenigmaticus. Despite the
lack of pyrenoids in the chloroplast of the latter taxon,
only about half a dozen base differences separate the
SSU rDNA sequences of these taxa demonstrating the
need for an ultrastructural examination of the sequenced
strain of P. aenigmaticus to confirm the morphological features used in identification. The Phacus species in clade D
are ovoid in shape and not as flat and leaf-like as most of
the species of this genus. Most taxa belong to Section
Pleuraspis Pochmann, with the exception of P. agilis
and P. aenigmaticus (Section Proterophacus Pochmann).
The genera represented here share a common taxonomic history. Both Phacus and Lepocinclis were established to accommodate taxa that were formerly in the
genus Euglena. Dujardin (1841) created the genus Phacus
to contain all those members that were rigid, flat, and
mostly without pyrenoids. Later, Perty (1852) suggested
the genus Lepocinclis for those Euglena that were rigid
but not flat and lacked pyrenoids. None of the Phacus
spp. in this clade are flat, and P. splendens has a single
chloroplast with at least one haplopyrenoid. Linton
et al. (2000) previously described the taxonomic history of L. ovata, P. pyrum, and P. splendens. With the additional data presented here, it now seems clear that
L. ovata (Conrad 1934) should be considered as a synonym for P. pyrum var. ovata, originally described by Playfair (1921). The data presented here from some of the
more atypical Phacus spp. (P. aenigmaticus, P. pseudonordstedti, and P. agilis) support earlier arguments by Pringsheim (1956) and Linton et al. (2000) questioning the
previous shifting of these taxa among Phacus, Lepocinclis and Euglena.
Clade E contains typical, rigid, flat, leaf-like species
of Phacus. This group of taxa is present in MP and Bayesian analyses but receives weak support. All the taxa
have small disk-shaped chloroplasts, without pyrenoids
and with large paramylon grains that are ring- or bobbinshaped. The paramylon grains are never associated
with the chloroplast.
Clade F is a well-supported clade that appears in
MP and Bayesian trees. It includes Euglena and Lepocinclis spp. The polytomy of E. acus, E. spirogyra, and L.
ovum obtained previously (Linton et al. 2000) was resolved with the addition of L. buetschlii and E. oxyuris.
All of the members of this clade are rigid or semirigid
cells with many small disk-shaped chloroplasts lacking
pyrenoids with paramylon grains as links, rings, or rods.
The molecular data and chloroplast features indicate
the close relationship between these taxa, and it could
be argued that the original assignation of L. ovum and
L. buetschlii as Euglena may have been correct (Linton
et al. 2000). However, this would still leave Euglena spp.
split between clades A and F. In any case, the data
233
strongly support the grouping of these Euglena and Lepocinclis species into a single taxon. The pellicular substructure of these taxa reinforces this position (Leander
and Farmer 2001a, Leander et al. 2001). One additional
feature that joins these taxa is the presence of rows of
mineral deposits (ferric oxide?) on the pellicle in both
L. buetschlii and E. spirogyra (Dawson et al. 1988).
If one views the entire tree, several patterns emerge.
All the taxa in clades E and F are rigid cells, with numerous, small, discoidal chloroplasts lacking pyrenoids.
Paramylon grains are bobbin-like rings or rods and are
never associated with the plastids. It is worthy to note
that although we lack information on pellicle substructure of some of the members of these two groups, in
those species that have been studied (E. acus, E. spirogyra, P. alatus, P. oscillans, P. pusillus, P. brachykentron, L.
buetschlii), the pellicle showed strong frames, with a
thick submembranous layer and well-developed teethor plate-like pre- and postarticular projections corresponding to the rigidity of the cells (Leedale 1964,
Mignot 1965, Suzaki and Williamson 1985, 1986,
Bricheux and Brugerolle 1986, 1987, Leander and
Farmer 2001a,b, Leander et al 2001, our unpublished
observations). On the basis of these observations and
on results from similar organisms not included in the
present study (P. triqueter and E. tripteris, Leander and
Farmer 2001a) we expect these pellicle features will
be shared by all future taxa that join the tree at group
E or F.
In summary, this study incorporated new taxa into
the current evolutionary scheme of euglenoids that
enabled us to resolve some previous ambiguous relationships. After the acquisition of chloroplasts, the
pressure for maintaining metaboly may have lessened
among the green euglenoids. Rigidity was acquired
through acquisition of a thick frame and pre- and postarticular projections in the pellicle strips of groups E and F.
The Phacus spp. in group D are also rigid, but the pellicle
layer is thinner and does not have articular teeth or
plates, suggesting that more than one mechanism is
involved in rigidity.
Three osmotrophs were included in this study. Hyalophacus ocellatus grouped with the typical Phacus spp.
within clade E, whereas K. quartana and A. longa showed
separate origins in clade A, further supporting the contention that osmotrophy arose multiple times in euglenoids (Linton et al. 1999, Muellner et al. 2001). Nevertheless, this hypothesis should be tested further with
ultrastructural analyses and molecular studies that may
demonstrate the presence of some “vestigial” structures and/or chloroplast genes.
This study demonstrated the presence of two strongly
supported clades containing Euglena species, confirming
the polyphyly seen in previous studies (Linton et al.
1999, Muellner et al. 2001). Like Euglena, the genus
Phacus also was split into two distinct clades that, taken
along with existing morphological data, suggests that the
genus might need to be redefined as two separate genera. Clade E would then retain the name of Phacus as described by Dujardin (loc.cit. 1841) and the diagnostic
234
MARÍA ALEJANDRA NUDELMAN ET AL .
features would be those traditionally assigned to Phacus.
Clade D requires a redefinition, but as only a small percentage of the taxa have been sequenced, we believe it is
premature to do so at this time.
We appreciate the thoughtful comments of two anonymous reviewers and suggestions by Dr. W. T. Stam that improved the
manuscript. We thank Mrs. Carole Lewandowski, Mrs. Stacy
Brosnan, and Lic. Claudio Slamovits for their endless and kind
assistance and logistic and technical collaboration. We are also
grateful to the Laboratorio de Fisiología y Biología Molecular
(FCEyN, UBA). We acknowledge financial support from the
National Science Foundation PEET (Partnership for Enhanced
Expertise in Taxonomy) grant DEB 9712241 and the ANPCyT,
PICT 588/97. M. A. N. is a Research Graduate Fellow of UBA
(University of Buenos Aires), and this research was submitted
in partial fulfillment of the requirements for the Ph.D. degree.
M. S. R. and V. C. are members of CONICET (National Research
Council of Argentina).
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