1. Phylogenetic relationships among 28 spirotrichous ciliates
documented by rDNA
Elizabeth A. Hewitt,a
Kirsten M. M€uuller,b
Jamie Cannone,c
Daniel J. Hogan,a
Robin Gutell,c
and David M. Prescotta,*
a
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, USA
b
Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1
c
Institute for Cellular and Molecular Biology and Section of Integrative Biology, University of Texas at Austin, Austin, TX 78712-1095, USA
Received 23 August 2002; revised 5 March 2003
Abstract
The contiguous sequence of the SSU rDNA, ITS 1, 5.8S, ITS 2, and 1370 bp at the 50
end of the LSU rDNA was determined in
25 stichotrichs, one oligotrich, and two hypotrichs. Maximum parsimony, neighbor-joining, and quartet-puzzling analyses were
used to construct individual phylogenetic trees for SSU rDNA, for LSU rDNA, and ITS 1 + 5.8S + ITS 2, as well as for all these
components combined. All trees were similar, with the greatest resolution obtained with the combined components. Phylogenetic
relationships were largely consistent with classical taxonomy, with notable disagreements. DNA sequences indicate that Oxytricha
granulifera and Oxytricha longa are rather distantly related. The oligotrich, Halteria grandinella, is placed well within the order
Stichotrichida. Uroleptus pisces and Uroleptus gallina probably belong to different genera. Holosticha polystylata (family Holosti-
chidae) and Urostyla grandis (family Urostylidae) are rather closely related. These rDNA sequence analyses imply the need for some
modifications of classical taxonomic schemes.
Ó 2003 Elsevier Science (USA). All rights reserved.
1. Introduction
Spirotrichs are a particularly interesting group of
ciliates because of complex evolutionary modifications
of their micronuclear (germline) and macronuclear (so-
matic) genomes and because of the extraordinary ma-
nipulations of DNA required to convert a micronuclear
genome into a macronuclear genome after cell mating
(Prescott, 2000). Micronuclear genes are interrupted by
short, noncoding, AT-rich segments called internal
eliminated segments, or IESs, first discovered in spiro-
trichs (Klobutcher et al., 1984) and subsequently iden-
tified in micronuclear genes in Paramecium (Steele et al.,
1994). IESs divide a gene into segments called macro-
nuclear destined segments, or MDSs. During macro-
nuclear development IESs are spliced out of the DNA,
and MDSs are ligated to form transcriptionally com-
petent genes. In some micronuclear genes in stichotrichs
recombination between IESs within the gene has oc-
curred in the course of evolution, causing MDSs to
become disordered, or scrambled (Prescott, 2000).
Scrambled MDSs become unscrambled and ligated in
the orthodox order during macronuclear development.
Numbers, sizes, sequences, and positions of IESs
within a particular micronuclear gene differ from one
stichotrich to another, reflecting both a high rate in the
accumulation of mutations in IESs (DuBois and Pres-
cott, 1997) and the ability of IESs to migrate along
DNA (DuBois and Prescott, 1995). The IES differences,
in turn, generate differences from organism to organism
in the numbers, sizes and patterns of MDSs within a
particular micronuclear gene. IES/MDS patterns in a
micronuclear gene in a series of stichotrichs instruct us
about the origin and evolution of IESs and MDSs when
they are placed in the context of the phylogenetic rela-
tionships among those organisms (Hogan et al., 2001).
Phylogenetic relationships among stichotrichs have tra-
ditionally relied on morphological features, particularly
Molecular Phylogenetics and Evolution 29 (2003) 258–267
www.elsevier.com/locate/ympev
MOLECULAR
PHYLOGENETICS
AND
EVOLUTION
*
Corresponding author. Fax: 1-303-492-7744.
E-mail address: prescotd@spot.colorado.edu (D.M. Prescott).
1055-7903/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S1055-7903(03)00097-6

2. on the numbers, patterns, and positions of cirri (kin-
eties) and membranelles. These characteristics are not
inherited through nuclear genes and are thus qualitative
and, at best, semiquantitative features that do not ade-
quately define phylogenetic relationships. Therefore, we
have determined nuclear rDNA sequences for 25 sti-
chotrichs, one oligotrich, and two hypotrichs, in which
we are currently studying the structure of several mi-
cronuclear genes, in order to document phylogenetic
relationships more precisely. We can then use these re-
lationships to elucidate the origin and evolution of IESs
and MDS scrambling. Phylogenetic relationships docu-
mented by nuclear rDNA will also help in the appro-
priate classification of spirotrichs, including
unidentified/unnamed spirotrichs, four examples of
which are present among the 28 organisms used in the
present study. Finally, classifications of spirotrichs that
have been based on morphological criteria may need
some adjustment when nuclear rDNA sequences are
available to define phylogenetic relationships.
2. Materials and methods
2.1. Origin of organisms
1. Gastrostyla steineii. A gift from W. Foissner.
2. Sterkiella nova (formerly Oxytricha nova; Foissner
and Berger, 1999). Isolated from a pond in Burling-
ton, North Carolina.
3. Sterkiella sp. (Aspen). Isolated from the Roaring
Fork River, Aspen, Colorado.
4. Pleurotricha lanceolata. Isolated from Ten Mile
Creek, Colorado. Identified by W. Foissner.
5. Unknown F. Isolated from Ten Mile Creek, Colo-
rado.
6. Tetmemena pustulata. Isolated from a pond on the
University of Colorado campus, Boulder, Colorado.
Identified by W. Foissner.
7. Stylonychia sp. (Aspen). Isolated from Ten Mile
Creek, Colorado.
8. Oxytricha sp. (Misty). Isolated from Misty Creek
Pond, Sarasota, Florida.
9. Oxytricha longa. Isolated from Ten Mile Creek,
Colorado. Identified by W. Foissner.
10. Unknown B. Isolated from the Roaring Fork River,
Colorado.
11. Cyrtohymena citrina. Isolated from Maroon Creek,
Aspen, Colorado. Identified by W. Foissner.
12. Paraurostyla weissei. Isolated from Teller Lake,
Boulder County, Colorado. Identified by W. Foiss-
ner.
13. Stylonychia lemnae. Isolated from Teller Lake,
Boulder County, Colorado.
14. Stylonychia mytilus. Isolated in Harbin, China.
Identification confirmed by W. Foissner.
15. Paruroleptus lepisma. Isolated from Ten Mile Creek,
Colorado. Identified by W. Foissner.
16. Uroleptus gallina. Isolated from Teller Lake, Boul-
der County, Colorado.
17. Uroleptus pisces. Isolated from Teller Lake, Boulder
County, Colorado.
18. Urostyla grandis. Isolated from a pond on the Uni-
versity of Colorado campus, Boulder, Colorado.
19. Holosticha polystylata. Isolated from a pond on the
University of Colorado campus, Boulder, Colo-
rado.
20. Oxytricha granulifera. Isolated from the Roaring
Fork River, Aspen, Colorado. Identified by
W. Foissner.
21. Unknown SHS. Isolated from a hot spring (43 °C) in
Steamboat Springs, Colorado.
22. Paraurostyla viridis. Isolated from Misty Creek
Pond, Sarasota, Florida.
23. Halteria grandinella. Isolated from a pond on the
University of Colorado campus, Boulder, Colorado.
24. Engelmanniella mobilis. A gift from W. Foissner
25. Moneuplotes crassus. Macronuclear DNA was a gift
from Carolyn Price, University of Cincinnati.
26. Euplotes aediculatus. Isolated from Teller Lake,
Boulder County, Colorado.
27. Sterkiella histriomuscorum (formerly named Oxytri-
cha trifallax). Isolated from the Jordan River,
Bloomington, Indiana.
28. Unknown FL. Isolated from Misty Creek Pond,
Sarasota, Florida.
Most organisms were cultured on Chlorogonium
elongata in open dishes with the following exceptions.
Engelmanniella mobilis was cultured on a mixture of
unidentified bacteria. Urostyla grandis was cultured on
Tetrahymena thermophila. Halteria grandinella was cul-
tured monaxenically on Chlorogonium. Moneuplotes
crassus was not cultured; macronuclear DNA was a gift
of Carolyn Price.
Macronuclei were purified from 10 to 30 packed ml of
cells by the method described previously (Prescott and
Greslin, 1992).
Polymerase chain reactions (PCR) were performed
with macronuclear DNA using terminal SSU rDNA
universal primers (Elwood et al., 1985). Amplified DNA
was electrophoretically analyzed in a 1% agarose gel to
check for appropriate size products. PCR products were
purified (Qiagen, Chatsworth, CA) and directly se-
quenced using internal primers (Elwood et al., 1985) in
the MCDB Departmental sequencing facility.
Sequences of ITS 1, 5.8S, ITS 2 and 1370 bp of the
large subunit rDNA were obtained by TA cloning of
PCR products using either pGEM easy T vector (Pro-
mega, Madison, WI) or pKRX. PCR primers were
designed from SSU sequences of Sterkiella histriomus-
corum and from LSU sequence of Euplotes aediculatus
E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267 259

3. (M98383 and M98377). Plasmid clones containing
inserts of the appropriate size were prepared using the
Qia prep spin miniprep kit (Qiagen, Chatsworth, CA).
Sequencing was performed in the MCDB departmental
sequencing facility using M13 forward and reverse
primers.
2.2. Alignment of nuclear rDNA genes
The nuclear rDNA gene sequences were aligned
manually with the SUN microsystems Unix-based
alignment editor AE2 (developed by T. Macke, see
Larsen et al., 1993). Sequences were juxtaposed in the
alignment to represent, as best as possible, the similar
placement of homologous nucleotides in two and three
dimensional space. Sections of the rRNA with signifi-
cant amounts of sequence identity can be aligned solely
with the sequence information. However, regions with
significant amounts of sequence variation require sec-
ondary structure information to align the sequences
accurately and confidently. Covariation analysis has
been used to accurately predict rRNA secondary
structure and the beginnings of its tertiary structure
(Gutell et al., 1985, 2002). The secondary structure
models for a large phylogenetically diverse collection of
SSU and LSU rRNAs is available at the Comparative
RNA Web site (http://www.rna.icmb.utexas.edu, Can-
none et al., 2002). Usually these variable regions con-
tain a structure that is conserved in the different
sequences, and thus nucleotides can be aligned with
absolute, or near absolute confidence. We are confident
in the alignment of the entire SSU, LSU, and 5.8S
rRNAs.
2.3. Phylogenetic analysis of nuclear rDNA genes
Maximum parsimony, neighbor-joining and quartet-
puzzling analyses were performed using PAUP 4.0 beta
10 (Phylogenetic Analysis Using Parsimony; Swofford,
2002). Parsimony analysis was applied using heuristic
searches under the conditions of random addition of
taxa (100 replicates), steepest descent, tree bisection-re-
connection (TBR) branch swapping and the MUL-
PARS option. The quartet-puzzling method applies
maximum-likelihood tree reconstruction to all possible
quartets that can be formed from all sequences that
serve as starting points to reconstruct a set of optimal
n-taxon trees (Strimmer and von Haeseler, 1996). This
method has been shown to be equivalent or better for
reconstructing the true tree than neighbor-joining
methods (Strimmer and von Haeseler, 1996). The values
represent the percentage of times that a particular
cluster was found among the 1000 intermediate trees
(QPS values). Neighbor-joining analyses were con-
ducted on a matrix of distance values estimated ac-
cording to the Kimura 2-parameter model (Kimura,
1980) using a transition/ transversion ratio of 2.0 and a
single-category substitution rate. Support for nodes on
parsimony and distance trees was established using
bootstrap resampling (1000 replicates). The nuclear
rDNA sequence from T. thermophila (GenBank Acces-
sion No. X54512) was used as an outgroup taxon for all
analyses of the nuclear rDNA genes. Tetrahymena is
distantly related to spirotrichs based on a variety of
taxonomic/genetic criteria.
3. Results and discussion
Sequence data for all taxa included in this study were
submitted to GenBank and accession numbers are given
in Table 1.
3.1. Phylogenetic analysis of nuclear SSU rDNA genes
The nuclear SSU rDNA genes were amplified by
PCR and sequenced for all taxa included in the present
study. Parsimony analysis of 331 phylogenetically in-
formative characters of the macronuclear SSU rDNA
gene resulted in 180 most parsimonious trees with a
length of 855 and a consistency index (CI) of 0.595. A
strict consensus of these 180 most parsimonious trees is
depicted in Fig. 1. The most parsimonious trees differ in
topology because of unresolved nodes and branches
with less than 50% support, as seen in Fig. 1. Trees
generated from both neighbor-joining and maximum
likelihood quartet-puzzling analyses show similar to-
pologies and hence only the maximum parsimony tree is
shown. However, bootstrap values for the neighbor-
joining and quartet-puzzling analyses are shown on the
tree (see Fig. 1 legend). Resolution is low among the
various clades seen in Fig. 1, and the species in some
genera do not appear to form monophyletic groups
(e.g., Sterkiella, Oxytricha, and Paraurostyla, Fig. 1).
Some groups are well supported, for example, the group
containing Stylonychia mytilus, Stylonychia lemnae, and
Stylonychia sp. Aspen is strongly supported by parsi-
mony (95%) and neighbor-joining bootstrap analyses
(97%) though not well supported by quartet-puzzling
analysis (62%). Among unidentified ciliates, Unknown F
is clearly associated with Pleurotricha lanceolata, and
this relationship is well supported [97% parsimony (MP)
bootstrap support, 93% quartet-puzzling steps (QPS),
and 87% neighbor-joining (NJ) bootstrap support], as is
Unknown SHS with Oxytricha longa [98% (MP), 89%
(QP), and 100% (NJ)]. However, Unknown FL is not
clearly associated with any particular ciliate but is
moderately supported [89% (MP), 87% (QPS), and 91%
(NJ)] within a clade containing taxa from a number of
genera (Stylonychia, Oxytricha, Gastrostyla, Tetmem-
ena, and Pleurotricha) (Fig. 1). Most interesting is that
O. longa and Oxytricha granulifera do not group to-
260 E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267

4. gether in any of the molecular trees, as would be ex-
pected based on classical taxonomy. In fact, these two
taxa differ from each other by 3.39% (corrected sequence
divergence).
The corrected sequence divergence of the macronu-
clear SSU rDNA gene among the various ciliates ranged
from 0.51% (P. lanceolata and Unknown F) to as high
as 22.9% (T. thermophila and M. crassus). E. aediculatus
and M. crassus are well supported as a solid entity in all
phylogenetic trees (see bootstrap values in Fig. 1) and
are positioned on a long branch in the neighbor-joining
tree (not shown). They differ considerably in sequence
divergence from the remaining ciliates (not including
outgroup taxon), ranging from 15.7% (M. crassus and
Unknown F) to 16.6% (M. crassus and H. grandinella).
In terms of the unknown taxa, the sequence divergence
is low between Unknown SHS and O. longa (0.91%),
and between Unknown F and P. lanceolata (0.51%). On
the other hand, Unknown FL, which is moderately
supported as grouping with a number of genera, differs
from these taxa ranging from 1.58% (Oxytricha sp.
Misty) to 2.49% (Tetmemena pustulata). Similarly, Un-
known B, which is moderately supported as grouping
with Cyrtohymena citrina and Paraurostyla weissei dif-
fers from these taxa by 1.75 and 1.41%, respectively.
3.2. Phylogenetic analysis of nuclear LSU rDNA genes
Approximately 1370 bp at the 50
end of the nuclear
LSU rDNA genes were amplified by PCR and se-
quenced. Parsimony analysis of 462 phylogenetically
informative characters of the nuclear LSU rDNA genes
resulted in 38 most parsimonious trees with a length of
786 and a consistency index (CI) of 0.495 and for which
a strict consensus is depicted in Fig. 2. The most parsi-
monious trees differed in topology due to unresolved
nodes and branches with 50% bootstrap values. Trees
generated from both neighbor-joining and maximum
likelihood quartet-puzzling analyses showed similar to-
pologies, and hence only the maximum parsimony tree is
shown. Similar to the macronuclear SSU analysis, there
is low resolution among many clades, and several of the
genera also do not form monophyletic clades (Oxytri-
cha, Sterkiella, Uroleptus, Paraurostyla, etc.). In fact, the
topology of the phylogenetic tree in Fig. 2 is nearly
identical to that of the macronuclear SSU rDNA tree
depicted in Fig. 1. The unknown taxa in this analysis
form similar relationships to those seen in Fig. 1 with
the exception that Unknown B is not associated with
C. citrina and P. weissei, and relationships of this
unknown to other ciliates are poorly understood.
Table 1
Lengths (bp) of the SSU rDNA, ITS 1, 5.8S, ITS 2 and LSU rDNA sequenced in 28 Spirotrichs and Tetrahymena thermophila
SSU ITS 1 5.8S ITS 2 26S Accession Nos.
Unknown F 1771 127 153 203 1368 AF508777
Pleurotricha lanceolata 1771 127 153 203 1369 AF508768
Tetmemena pustulata 1771 119 153 203 1367 AF508775
Oxytricha sp. (Misty) 1771 131 153 203 1369 AF508764
Sterkiella nova 1771 131 153 204 1370 AF508771
Gastrostyla stenii 1771 131 153 192 1369 AF508758
Sterkiella sp. (Aspen) 1771 132 153 203 1370 AF508772
Sterkiella histriomuscorum 1771 130 153 203 1369 AF508770
Unknown FL 1771 124 153 203 1369 AF508778
Stylonychia sp. (Aspen) 1769 132 153 203 1367 AF508754
Stylonychia mytilus 1771 124 153 203 1367 AF508774
Stylonychia lemnae 1770 121 153 203 1368 AF508773
Paraurostyla weissei 1771 121 153 203 1366 AF508767
Cyrtohymena citrina 1772 134 153 204 1367 AF508755
Unknown B 1770 132 153 204 1369 AF508776
Unknown SHS 1774 127 153 201 1371 AF508769
Oxytricha longa 1770 128 153 201 1369 AF508763
Oxytricha granulifera 1774 124 153 198 1365 AF508762
Paraurostyla viridis 1774 124 153 198 1365 AF508766
Engelmanniella mobilis 1773 121 154 201 1375 AF508757
Halteria grandinella 1779 125 153 194 1370 AF508759
Uroleptus gallina 1775 129 153 192 1369 AF508779
Paruroleptus lepisma 1772 125 153 192 1365 AF508765
Uroleptus pisces 1772 130 153 200 1327 AF508780
Holosticha polystylata 1769 128 153 192 1356 AF508760
Urostyla grandis 1768 130 153 198 1366 AF508781
Moneuplotes crassus 1890 113 150 182 1379 AF508761
Euplotes aediculatus 1881 67 150 186 1385 AF508756
Tetrahymena thermophilaa
1753 130 154 177 3760 X54512
a
Engberg and Nielsen (1990).
E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267 261

5. Unknown FL is also observed not to be associated with
any particular ciliate, and like Unknown B, is moder-
ately supported [75% (MP), 80% (QPS), and 98% (NJ)]
as grouping within a clade containing a number of
genera (Stylonychia, Gastrostyla, Pleurotricha, Tet-
memena, etc.) (Fig. 2). Similar to the macronuclear SSU
rDNA gene tree, O. longa and O. granulifera are not
observed to group with each other. In fact, O. granu-
lifera is again supported as grouping with Paraurostyla
viridis [100% (MP), 52% (QPS), and 100% (NJ)], and
O. longa is again observed to group with the Unknown
SHS [75% (MP), 94% (QPS), and 94% (NJ) (Fig. 2)], as
seen in the SSU rDNA gene analyses (Fig. 1).
The corrected sequence divergence of the macronu-
clear LSU rDNA gene is higher than that for the mac-
ronuclear SSU rDNA gene and ranged from 0.220%
[Sterkiella sp. (Aspen) and Sterkiella histriomuscorum]
to 30.6% (T. thermophila and E. aediculatus). Similar to
the SSU analysis, E. aediculatus and M. crassus are well
supported as a solid entity in all phylogenetic trees
Fig. 1. Strict consensus of 180 most parsimonious trees from analysis of 331 phylogentically informative characters of the nuclear SSU rDNA gene in
the spirotrichous ciliates. The first of the three numbers above internal branches represent bootstrap resampling results (% of 1000 replicates) for
maximum parsimony analysis. The second number represents the support based on quartet-puzzling steps (QPS, % of 1000 replicates). The third
number is the bootstrap value using the neighbor-joining algorithm (% of 1000 replicates). An asterisk or branch lacking values had less than 50%
bootstrap support.
262 E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267

6. [100% (MP), 82% (QPS), and 99% (NJ)] and are posi-
tioned on a long branch in the neighbor-joining tree (not
shown) and differ considerably in sequence divergence
from the remaining ciliates (18.6–23.2%), not including
T. thermophila, from which they differ by 29.4–30.6%.
The sequence divergence between O. longa and
O. granulifera is considerably higher (10.5%) than that
observed for the macronuclear SSU rDNA gene. The
unknown taxa were also observed to have higher se-
quence divergence values than that noted for the mac-
ronuclear SSU rDNA gene. For example Unknown
SHS and O. longa differ by 5.3%; Unknown F and
P. lanceolata by 1.24%. Unknown B and Unknown FL,
which are observed to group with a clade containing a
number of genera, differed from these taxa by 6.16–
9.18% and 7.34–10.3%, respectively.
3.3. Combined analysis of SSU, LSU, and 5.8S rDNA
genes
Sequences of the macronuclear SSU, LSU, and 5.8S
rDNA genes were combined for phylogenetic analyses.
Fig. 2. Strict consensus of 38 most parsimonious trees from analysis of 462 phylogenetically informative characters of the nuclear LSU rDNA gene in
the spirotrichous ciliates. The first of the three numbers above internal branches represent bootstrap resampling results (% of 1000 replicates) for
maximum parsimony analysis. The second number represents the support based on quartet-puzzling steps (QPS, % of 1000 replicates). The third
number is the bootstrap value using the neighbor-joining algorithm (% of 1000 replicates). An asterisk or branch lacking values had less than 50%
bootstrap support.
E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267 263

7. Parsimony analysis of 838 phylogenetically informative
characters from the three rDNA genes resulted in eight
most parsimonious trees with a length of 2789 and
a consistency index of 0.525, one of which is shown in
Fig. 3. The topology of the neighbor-joining and quar-
tet-puzzling trees were similar and hence only the par-
simony tree is shown (Fig. 3). The topology of this tree is
similar to that seen in the previous two analyses, how-
ever there is more resolution of the various clades. For
example, grouping of Unknown F with P. lanceolata
and Unknown SHS with O. longa are well supported.
Similar to the macronuclear SSU rDNA gene analyses
(Fig. 1), Unknown B is moderately supported in a clade
with C. citrina [71% (MP), 84% (QPS), and 70% (NJ)]
(Fig. 3); however this relationship was not reflected in
the LSU rDNA gene analyses (Fig. 2).
3.4. Phylogenetic analysis of the 5.8S rDNA gene and
ITS regions
Phylogenetic analyses were carried out on 162 nu-
cleotides in the 5.8S rDNA, however, phylogenetic trees
Fig. 3. One of eight most parsimonious trees from combined analysis of 838 phylogenetically informative characters of the nuclear SSU, LSU, and
5.8S rDNA genes in the spirotrichous ciliates. The first of the three numbers above internal branches represents bootstrap resampling results (% of
1000 replicates) for maximum parsimony analysis. The second number represents the support based on quartet-puzzling steps (QPS, % of 1000
replicates). The third number is the bootstrap value using the neighbor-joining algorithm (% of 1000 replicates). An asterisk or branch lacking values
had less than 50% bootstrap support.
264 E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267

8. showed little or no resolution with the exception of
E. aediculatus and M. crassus, which were well sup-
ported as a solid entity by parsimony (100%) and
neighbor-joining (100%) bootstrap resampling (not
shown). In addition, sequences of the internal tran-
scribed spacers (ITS 1 and 2) were also obtained, and
phylogenetic trees were constructed (Fig. 4). Alignment
of these sequences was difficult because of the consid-
erable variability, and, hence, phylogenetic trees do not
include M. crassus, E. aediculatus, and T. thermophila.
Parsimony analysis of 92 phylogenetically informative
characters of ITS 1 and 2 resulted in 257 most parsi-
monious trees with a length of 252 and a consistency
index (CI) of 0.600. A strict consensus of these 257 most
parsimonious trees is depicted in Fig. 4; as in the case of
the previous analyses only the parsimony consensus is
shown with bootstrap and QPS values included. The tree
depicted in Fig. 4 is similar in topology to the other
figures (Figs. 1–3) but had less resolution with the
exception of a few taxa. For example, Unknown F is well
supported in grouping with P. lanceolata [100% (MP),
82% (QPS), 99% (NJ)], and Unknown SHS, is strongly
associated with O. longa [100% (MP), 89% (QPS), 100%
(NJ)]. These relationships were also noted in the analyses
Fig. 4. Strict consensus of 257 most parsimonious trees from analysis of 92 phylogenetically informative characters of ITS 1 and 2 of the nuclear
rDNA cistron in the spirotrichous ciliates. The first of the three numbers above internal branches represent bootstrap resampling results (% of 1000
replicates) for maximum parsimony analysis. The second number represents the support based on quartet-puzzling steps (QPS, % of 1000 replicates.)
The third number is the bootstrap value using the neighbor-joining algorithm (% of 1000 replicates). An asterisk or branch lacking values had less
than 50% bootstrap support.
E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267 265

9. of SSU, LSU, and combined nuclear rDNA gene anal-
yses (Figs. 1–3).
Our work expands the seminal report of Bernhard
et al. (2001) on phylogenetic relationships among spi-
rotrichs based on SSU rDNA to include sequences of
SSU, LSU, 5.8S rDNA and ITS 1 and 2, and we have
extended the analysis to 28 spirotrichs. Nine of these
organisms were part of the analysis by Bernhard et al.
(2001), and our results on phylogenetic relationships are
in overall agreement with theirs.
The phylogenetic relationships among the 28 spiro-
trichs documented by SSU, LSU, 5.8S, and ITS 1 and 2
are, in most cases, consistent with classical taxonomy
but in other cases, sharply disagree. Classical taxonomy
of spirotrichs is based on morphological characteristics,
depending particularly on such features as the number
and patterns of cirri and membranelles. Such features
are quite useful in identifying different organisms, but
alone they may be of somewhat limited value for de-
fining phylogenetic relationships. Combining classical
taxonomic criteria with molecular and gene analyses
may ultimately provide the best approximation of phy-
logenetic/evolutionary relationships (see for example,
Bernhard et al., 2001).
In this context the 28 spirotrichs present examples of
varying contrasts. For example, S. mytilus, S. lemnae,
and Stylonychia sp. (Aspen) are morphologically very
similar; one may be easily mistaken for another when
the living organisms are viewed microscopically. The
nuclear rDNA sequences agree about their close kin-
ship. Similarly, S. histriomuscorum and Sterkiella sp.
(Aspen) are virtually indistinguishable morphologically.
Their nuclear rDNA sequences reflect a close relation-
ship, but show that they are unquestionably different
taxa. This is affirmed by the micronuclear gene encoding
actin I. In S. histriomuscorum the micronuclear actin I
gene is divided by nine noncoding IESs (internal elimi-
nated segments) into 10 highly scrambled MDSs (mac-
ronuclear destined segments), but this gene in Sterkiella
sp. (Aspen) is interrupted by eight IESs, creating only
nine MDSs (DuBois and Prescott, 1995). Moreover,
although the scrambling patterns of MDSs are strikingly
similar, corresponding IESs are of different lengths and
contain completely different nucleotide sequences.
In contrast to these agreements O. granulifera and
O. longa are morphologically similar and therefore have
been placed in the same genus. Their nuclear rDNA
sequences indicate a much more distant phylogenetic
relationship, which is supported by a phylogenetic
analysis of the actin I gene (Croft et al., 2003). Another
major contradiction is the position of H. grandinella
in the nuclear rDNA tree. By morphological criteria
H. grandinella is placed in an entirely different order,
Halteriida (Petz and Foissner, 1992), but the nu-
clear rDNA sequences place this organism well within
the order Stichotrichida with a relatively close kinship to
O. granulifera, Paraurostyla viridis, and E. mobilis. Shin
et al. (2000) previously found that H. grandinella be-
longs to the Oxytrichidae based on SSU rDNA. Our
results with SSU, LSU, and 5.8S nuclear rDNA are in
agreement, and moreover, show the close relationship
between H. grandinella and O. granulifera based on SSU
rDNA reported by Bernhard et al. (2001). Further, the
amino acid sequence of actin I also places H. grandinella
solidly within the order Stichotrichida (Croft et al.,
2003).
The rDNA tree suggests that U. pisces and U. gallina
may not belong to the same genus. Oxytricha sp. (Misty)
probably belongs to the family Oxytrichidae (genera
Gastrostyla, Sterkiella, Oxytricha, Pleurotricha, Styl-
onychia, and Tetmemena), but it is distantly separated
both from O. longa and O. granulifera. Holosticha
polystylata has been placed in the family Holostichidae
and U. grandis in the family Urostylidae (Lynn and
Corliss, 1991), but the nuclear rDNA analysis indicates
a close phylogenetic relationship between these two or-
ganisms.
Among the 28 organisms represented in the nuclear
rDNA tree four are as yet unidentified organisms, Un-
known F, Unknown B, Unknown SHS, and Unknown
FL. We prefer at this juncture to refer to these organ-
isms by the accession numbers for their nuclear rDNA.
Unknown F may belong to the genus Pleurotricha and
Unknown SHS may share the same genus with O. longa.
The rDNA tree does not suggest possible genus assign-
ments for Unknown B and Unknown FL to any of the
genera represented in this analysis.
Acknowledgments
This work is supportedby NIGMS Research Grant
#R01GM 56161 and NSF Research Grant MCB-
9974680 to D.M. Prescott. K.M. M€uuller is supported by
start up funds from the University of Waterloo. D.J.
Hogan was partially supported by the Hughes Under-
graduate Biological Science Education Initiative, the
University of Colorado Undergraduate Research Op-
portunities Program, and the University of Colorado
Cancer Center. Robin Gutell was supported by NIH
R01 GM 48207. We are grateful to Gayle Prescott for
preparation of the manuscript. We thank Laura Land-
weber for SSU primers.
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