Widespread Distribution of Extensive Chromosomal Fragmentation in
Ciliates
Jennifer L. Riley* and Laura A. Katz*†
*Department of Biological Sciences, Smith College; and †Program in Organismic and Evolutionary Biology, University of
Massachusetts–Amherst
Ciliates are a diverse group of eukaryotes characterized by their division of nuclear function into a ‘‘germ line’’
micronucleus and a ‘‘somatic’’ macronucleus. After conjugation, chromosomes in the transcriptionally active macronucleus develop by fragmentation, elimination, and amplification of germ line chromosomes. Extensive chromosomal processing that generates a macronucleus with gene-sized fragments has thus far been well documented
in members of only one class of ciliates, the Spirotrichea. Here we establish the broad distribution of extensive
fragmentation among members of the class Phyllopharyngea and the genera Metopus (order Armophorida) and
Nyctotherus (order Clevelandellida). Moreover, analyses of small-subunit rDNA genealogies indicate that gene-sized
chromosomes occur in members of the three separate clades: (1) the class Spirotrichea, (2) the class Phyllopharyngea, and (3) the two orders Clevelandellida and Armophorida. Together, these data indicate that the generation
of gene-sized chromosomes is widespread and demonstrate multiple origins of extensive fragmentation within
ciliates.
Introduction
Ciliates challenge traditional models of the structure of eukaryotic genomes with the presence of both a
germ line micronucleus and a somatic macronucleus
within each cell. During conjugation, meiotic products
of the micronucleus are exchanged and fuse to form a
zygotic nucleus. This zygotic nucleus divides mitotically, and at least one daughter nucleus develops into a
macronucleus through a series of processes including
fragmentation, elimination, and amplification of germ
line chromosomes (Orias and Higashinakagawa 1990;
Prescott 1994; Coyne, Chalker, and Yao 1996; Klobutcher and Herrick 1997). Reproduction occurs asexually where, in all but one class of ciliates, each nucleus
replicates independently; the one exception is the class
Karyorelictea, for which the macronuclei do not divide
on their own (Raikov 1982).
The extent of chromosomal fragmentation varies
among ciliates. In the well-studied genera Tetrahymena
and Paramecium (Cl: Oligohymenophorea [O]), fragmentation is limited. For example, in Tetrahymena, the
long chromosomes of the zygotic nucleus are fragmented at several thousand sites to produce approximately
200 different macronuclear chromosomes. Each of these
shorter chromosomes, between 100 and 1,500 kb long,
is then amplified an average of 50 times (Prescott 1994).
In contrast, members of the class Spirotrichea (S) process their approximately 120 micronuclear chromosomes extensively to create as many as 24,000 different
‘‘gene-sized chromosomes’’ (defined here as subchromosomal fragments less than 15 kb in length) in the
macronucleus; each of these chromosomes is replicated
Abbreviation: ssu-rDNA, small-subunit ribosomal DNA.
Key words: chromosomal fragmentation, chromosomal rearrangements, macronucleus, ciliates, Ciliophora, Phyllopharyngea, Armophorida, Clevelandellida.
Address for correspondence and reprints: Laura Katz, Department
of Biological Sciences, Smith College, Northampton, Massachusetts
01063. E-mail: [email protected].
Mol. Biol. Evol. 18(7):1372–1377. 2001
q 2001 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
1372
from 950 to 15,000 times (Klobutcher and Herrick 1997;
Prescott 1994). Prior attempts to identify additional taxa
with extensive fragmentation indicated that, in addition
to members of the class Spirotrichea, two closely related
genera in the class Phyllopharyngea (P; Trithigmostoma
and Chilodonella) have gene-sized macronuclear chromosomes (Lahlafi and Méténier 1991; Steinbrück et al.
1981). Moreover, a single gene, for hydrogenase, in the
macronuclear genome of the ciliate Nyctotherus ovalis
(order Clevelandellida [c]) has also been shown to be
on a gene-sized chromosome (Akhmanova et al. 1998).
To describe the evolutionary history of extensive
chromosomal fragmentation in ciliates, particularly
among the less well studied lineages, we examined the
relationship between extensive fragmentation and the
presence of the highly polytene ‘‘giant’’ chromosomes
that can be seen in the developing macronuclei of some
ciliates. These giant chromosomes, analogous to the
polytene chromosomes of Drosophila salivary glands,
represent a transient stage in macronuclear development
in which germ line chromosomes replicate without nuclear division (Ammermann 1987). Giant chromosomes
are found in Spirotrichea (Ammermann 1987), the phyllopharyngeans Chilodonella and Trithigmostoma (Radzikowski 1979; Lahlafi and Méténier 1991), the genus
Nyctotherus (c) (Wichterman 1937; Grassé 1952; Golikova 1964), and possibly also Metopus (order Armophorida [a]) (Noland 1927). Smaller oligotenic chromosomes have also been found in members of the order
Suctoria, an additional lineage of Phyllopharyngeans
(Grell 1949). We tested for the presence of gene-sized
chromosomes and characterized ssu-rDNA sequence
data from N. ovalis (c), Metopus palaeformis (a), three
species (Heliophrya erhardi, Tokophrya lemnarum,
Ephelota sp.) of the subclass Suctoria (P), and one species (Chilodonella uncinata) of the subclass Cyrtophoria
(P). As positive controls, we included two members of
the class Spirotrichea, Halteria grandinella and Euplotes crassus, as extensive fragmentation has been well
documented in this class (Steinbrück et al. 1981; Prescott 1994; Klobutcher and Herrick 1997). We also in-
Extensive Chromosomal Fragmentation in Ciliates
1373
Materials and Methods
Ciliate Isolation and Culture
FIG. 1.—Small-subunit ribosomal DNA genealogy generated by
maximum-likelihood analysis using PAUP* 4.0d65 (Swofford 1999)
with parameters calculated in Modeltest (Posada and Crandall 1998).
Asterisks indicate clades whose members contain extensively fragmented chromosomes. Numbers above the branches are bootstrap values based on 100 replicates using heuristic search with LogDet distance settings/faststep bootstrap with ml settings/neighbor-joining bootstrap/heuristic search using parsimony settings. Black dots indicate
100% bootstrap support under all models. Dashes indicate branches
found in maximum-likelihood analysis that were not found in other
analyses. Underlined taxa were characterized for this study.
cluded several oligohymenophoreans, as extensive fragmentation is absent from these lineages.
Despite the fact that phylogenetic relationships
among classes differ depending on the criteria used,
there is no evidence that ciliates with giant chromosomes are monophyletic (fig. 1). Specifically, analyses
of molecular data, morphology, and ultrastructure place
the class Phyllopharyngea in a subphylum separate from
the class Spirotrichea and the orders Clevelandellida and
Armophorida. For example, analysis of infraciliature
places the Phyllopharyngea in the subphylum Postciliodesmatophora and the Spirotrichea (including the orders Armophorida and Clevelandellida) in the subphylum Cyrtophora (Small and Lynn 1985). Likewise, parsimony analyses of combined morphological, nuclear,
and ultrastructure characters place the Spirotrichea and
Clevelandellida (and presumably Armophorida) within
the subphylum Tubulicorticata and the Phyllopharyngea
within the subphylum Epiplasmata (de Puytorac, Grain,
and Legendre 1994). Analyses of ontogenesis also place
the class Spirotrichea in a clade distinct from Phyllopharyngeans (Foissner 1996). Finally, no analyses of
ssu-rDNA in the literature support the monophyly of, or
even a close relationship between, the Spirotrichea and
the Phyllopharyngea (Lynn and Sogin 1988; Leipe et al.
1994; Hammerschmidt et al. 1996; Wright, Dehority,
and Lynn 1997; Hirt, Wilkinson, and Embley 1998).
Cultures of C. uncinata (50194) and T. lemnarum
(50032) were acquired from the American Type Culture
Collection (Manassas, Va.). The Didinium sp. (L11) culture was from Connecticut Valley Biological Supply
Company (Southampton, Mass.), and Prodiscophrya
(1618/2) was obtained from the Culture Collection of
Algae and Protozoa (Cumbria, U.K.). Heliophrya erhardi and M. palaeformis were obtained from Manfred
Hauser at Ruhr-Universitat Bochum, Germany, and
Bland Finlay, Institute of Freshwater Ecology, Cumbria,
U.K., respectively. Ciliates were cultured following established protocols (Lee and Soldo 1992). Nyctotherus
ovalis was isolated by handpicking cells from the guts
of the cockroach Periplaneta americana (Connecticut
Valley Biological Supply Company, #L 3500). Ephelota
sp. was obtained by picking individual cells off of the
hydrozoan Tubularia (Marine Biological Laboratories,
Woods Hole, Mass., #240). We generated clonal lines of
C. uncinata by passing individual ciliates through three
rounds of isolation culture. Similarly, clonal lines of H.
grandinella, isolated from the greenhouse pond at Smith
College, were generated by passing individual cells
through three rounds of isolation. We isolated total DNA
from the ciliates using a standard DNA preparation (Ausubel et al. 1993). Euplotes crassus DNA (strains Ust2 and Lx2-4) was provided by L. A. Klobutcher (Department of Biochemistry, University of Connecticut,
Farmington, Conn.).
Characterization of Extensive Fragmentation by
Southern Hybridizations
We transferred total DNA from 0.5%–0.7% agarose
gels to nylon membranes and used probes generated by
PCR with the colorimetric detection kit and protocols of
Boehringer-Mannheim (Indianapolis, Ind., 1745832).
Probes were generated either by direct incorporation of
DIG-labeled dUTP (Boehringer-Mannheim 1573152)
into PCR products (a-tubulin and histone H4) or by random priming from total E. crassus DNA. The a-tubulin
probes were amplified from a cloned portion of this gene
from H. erhardi and represented amino acids 20–409 of
the a-tubulin gene. Primers for the a-tubulin PCR were
Tub 371 ((CUA)4ATHCANCCNGAYGGNCARATGC
C) and Tub 4092 ((CAU)4CATNCCYTCNCCNACRW
ACCA). Histone H4 probes were generated by combining DIG-labeled PCR products from M. palaeformis, N.
ovalis, E. crassus, and H. erhardi. Primers for these
PCRs were H4F0111 ((CUA)4ggNRTNACNAARCCN
gCNAT) and H4R0112 ((CAU)4TTNARNgCRTANAC
N A C R T C). To label the entire E. crassus genome,
;1mg of total DNA (strain Lx2-4, from L. A. Klobutcher, University of Connecticut) was labeled using a high
prime mix (Boehringer-Mannheim 1585606).
Characterization of Small-Subunit rDNA
Small-subunit rDNA fragments were amplified using eukaryotic-specific primers (Medlin et al. 1988), and
1374
Riley and Katz
Table 1
Sizes of Macronuclear Chromosomes in Ciliates
PROBE
TAXON
CLASS (ORDER)
atub (kb)
H4 (kb)
Ecras (kb)
Paramecium . . . . . . . . . . . . . . .
Tetrahymena . . . . . . . . . . . . . .
Didinium sp. . . . . . . . . . . . . . .
Euplotes crassus . . . . . . . . . . .
Halteria grandinella . . . . . . . .
CHilodonella uncinata . . . . . .
Heliophrya erhardi . . . . . . . . .
Tokophrya lemnarum . . . . . . .
Metopus palaeformis . . . . . . . .
Nyctotherus ovalis . . . . . . . . . .
Oligohymenophorea
Oligohymenophorea
Litostomatea
Spirotrichea
Spirotrichea
Phyllogpharyngea
Phyllogpharyngea
Phyllogpharyngea
(Armophorida)
(Clevelandellida)
.23
.23
.23
1.9, 1.8, 14
2.2, 1.9
1.8
1.8
1.8
2.25
2.2
.23
NA
NA
1.5, 0.7
1.4, 0.8
.23
NA
0.7
.23
.23
18–.23
NA
18–.23
0.5–.23
0.5–.23
0.5–5
NA
0.5–5
0.5–5
0.5–5
NOTE.—Shown are results of Southern hybridizations probed with two genes, a-tubulin (atub) and histone H4 (H4), as well as with the entire E. crassus (S)
genome (Ecras).
all reactions were run with 3 mM MgCl2, 1 3 PCR
buffer, 0.55 U Platinum TAQ DNA Polymerase (GibcoBRL, Grand Island, N.Y., 10966-034), 0.4 mM dNTP,
six pmol primer, for a 25-ml reaction. Resulting PCR
products were cleaned using Qiagen’s PCR kit (Qiagen
Inc, Valencia, Calif., 28106) and cloned with the uracil
DNA glycosylase (UDG) cloning kit and pAMP1 vector
(Gibco-BRL 18381-012). Sequences were generated by
amplifying and sequencing from one to five cloned PCR
products per taxon using the Perkin-Elmer Big-Dye terminator kit (Wellesley, Mass., 4303152) and analyzed
on a Perkin-Elmer ABI-310 or ABI-377 sequencer.
Alignment and Phylogenetic Analyses
Sequences were aligned using the DCSE software
(De Rijk and De Wachter 1993) with default parameters
and adjusted by eye. Exclusion of ambiguously aligned
regions left 1,225 characters, of which 563 were variable
and 433 were parsimony-informative. Analyses of sequences aligned with CLUSTAL W (Thompson, Higgins, and Gibson 1994) as implemented by Megalign
(DNAStar Inc., Madison, Wis.) with a gap penalty of
10, a gap length penalty of 10, and a pairwise penalty
of 3 gave concordant results (data not shown).
To assess the stability of the topology of the ssurDNA genealogy to different evolutionary models, genealogies were generated using maximum-likelihood
(ML), maximum-parsimony (MP), LogDet (LD) distance, and neighbor-joining (NJ) algorithms using
PAUP* 4.0d65 (Swofford 1999). Parameters for ML
analyses were calculated with hierarchical likelihood ratio tests using Modeltest 3.0 (Posada and Crandall
1998). Based on this analysis, we used a TrN (Tamura
and Nei 1993) model with a proportion of invariable
sites of 0.3388 and a gamma distribution parameter of
0.5422. MP analyses were done using a heuristic search
with 10 random-addition sequences and transversions
weighted twice as much as transitions. NJ analyses used
a Kimura two-parameter correction, a gamma distribution with a 5 0.5, and inverse squared objective weighting. Heuristic searches were also performed using LD
distances to correct for compositional biases and an inverse-squared objective function. Bootstrap support was
calculated using 100 replicates for each model. To explore alternative hypotheses, Kishino-Hasegawa tests
(Kishino and Hasegawa 1989) were performed comparing optimal trees generated in MP and ML analyses with
a topology in which all taxa known to extensively fragment their genomes were constrained to be
monophyletic.
Results
Extensive Fragmentation of Macronuclear
Chromosomes
To detect extensive chromosomal fragmentation,
we performed Southern hybridizations on total DNA
isolated from several taxa and probed these samples
with color-labeled markers for two genes: a-tubulin and
histone H4. Southern hybridization results for M. palaeformis (a), N. ovalis (c), and all of the Phyllopharyngeans studied show that the a-tubulin gene is found on
gene-sized chromosomes less than 2.5 kb in length (table 1 and fig. 2a). Southern hybridizations using histone
H4 probes confirmed the presence of gene-sized chromosomes in H. grandinella (S) and T. lemnarum (P),
whereas the same probes hybridized to larger (.23 kb)
fragments in M. palaeformis (a), N. ovalis (c), and C.
uncinata (P) (table 1 and fig. 2b).
To assess the overall size range of macronuclear
chromosomes, we labeled the entire genome of E. crassus (S), as the genome of this ciliate is composed primarily of highly amplified coding sequences in the macronucleus (Prescott 1994; Klobutcher and Herrick
1997). While these Southern hybridizations provide an
efficient way to assess the distribution of coding sequences in the genomes of ciliates, interpretation of the
results is confounded by the degrees of relatedness
among the ciliates examined: we expect greater hybridization between more closely related ciliates. For this
reason, we took the conservative approach of simply
reporting the range of chromosome sizes evident among
the different ciliates (table 1). These data confirm the
individual gene Southern hybridizations, with Phyllopharyngeans, M. palaeformis (a), and N. ovalis (c) all
showing a preponderance of small macronuclear chromosomes (table 1 and fig. 2c). In contrast, the E. crassus
Extensive Chromosomal Fragmentation in Ciliates
1375
FIG. 2.—Representative Southern hybridizations probed with (a) alpha-tubulin (1, 6, 7 5 molecular weight markers; 2 5 Myrionecta
rubrum [O]; 3 5 Chilodonella uncinata [P]; 4, 10 5 Euplotes crassus [S]; 5 5 Heliophrya erhardi [P]; 8 5 Metopus palaeformis [a]; 9 5
Nyctotherus ovalis [a]) (b) histone H4 (1 5 E. crassus [S]; 2 5 Halteria grandinella [S]; 3 5 Paramecium tetraurelia [O]; 4 5 Didinium sp.
[L], 5 5 molecular weight marker; 6 5 Tokophrya lemnarum [P]), and (c) the E. crassus genome (1 5 P. tetraurelia [O]; 2, 6, 7 5 molecular
weight markers; 3 5 Didinium sp. [L]; 4 5 H. grandinella [S]; 5 5 E. crassus [S]; 8 5 T. lemnarum [P]; 9 5 N. ovalis [c]).
(S) genome hybridizes to larger chromosomes of Paramecium tetraurelia (O) and Didinium sp. (Litostomatea [L]) (fig. 2c).
Characterization of ssu-rDNA
We characterized ;1,650 bp of the ssu-rDNA
gene from one to five clones each of E. crassus
(AY007437, AY007438, AY007439, AY007440), C.
uncinata (AF300281, AF300282, AF300283,
AF300284), H. grandinella (AY007441, AY007442,
AY007443, AY007444), N. ovalis (AY007454,
AY007455, AY007456, AY007457), M. palaeformis
(AY007450, AY007451, AY007452, AY007453), H.
erhardi (AY007445, AY007446, AY007447,
AY007448, AY007449), and Ephelota sp. (AF326357).
The average intraspecific uncorrected distance among
clones within a taxon is 0.0028 (range 0.00178–0.0037).
This variation may be due to PCR/sequencing error or
variation among the amplified copies of the macronuclear rDNA chromosomes.
Phylogenetic Framework
To establish a phylogenetic framework on which to
map the evolutionary history of extensive fragmentation, we constructed genealogies based on variation in
the ssu-rDNA gene. Because of the uneven sampling of
ciliates in GenBank and in our lab, we constructed genealogies using a symmetrical sampling of up to five
sequences (when available) for each ciliate class based
on a revised classification by Lynn and Small (1988).
We excluded the class Plagiopylea, for which there are
only two ssu-rDNA sequences, but included the two orders Armophorida and Clevelandellida, which are both
considered sedis mutabilis, of uncertain taxonomic
placement (Lynn 1997).
Overall, our phylogenetic analyses of ssu-rDNA sequences are consistent with published genealogies (Lynn
and Sogin 1988; Leipe et al. 1994; Hammerschmidt et
al. 1996; Wright, Dehority, and Lynn 1997; Hirt, Wilkinson, and Embley 1998); although we retain many
classes defined by morphology, relationships among
classes depends on the model of evolution used, and
there is only weak bootstrap support for many interclass
relationships (fig. 1). All algorithms and models provide
strong evidence, based on bootstrap values .80%, for
the monophyly of the classes Heterotrichea, Karyorelictea, Litostomatea, Spirotrichea, and Phyllopharyngea, as
well as for the sister status of Metopus (a), Nyctotherus
(c), and Nyctotheroides (c). The monophyly of the orders Armophorida and Clevelandellida is reconstructed
in all analyses except the ML analysis, in which Caenomorpha uniseralis (a) is paraphyletic to the remaining
armophorids and clevelandellids. The classes Oligohymenophorea, Nassophorea, Prostomatea, and Colpodea
either show only weak support for monophyly or do not
appear to be monophyletic based on this taxon sampling
(fig. 1).
1376
Riley and Katz
Also, as in other ssu-rDNA analyses, there is strong
support (100% bootstrap in all analyses) for the ancient
division in ciliates between the classes Karyorelictea
and Heterotrichea (subphylum Postciliodesmatophora)
and the remainder of the ciliate classes (subphylum Intramacronucleata) (Lynn and Sogin 1988; Leipe et al.
1994; Hammerschmidt et al. 1996; Lynn and Small
1997; Wright, Dehority, and Lynn 1997; Hirt, Wilkinson, and Embley 1998). The only other interclass relationship that is constant in all analyses is the grouping
of the classes Phyllopharyngea, Colpodea, Oligohymenophorea, Prostomatea, and Nassophorea. However, relationships among these classes are unstable, and bootstrap support for this node is relatively low (LD, 51%;
ML, 60%; NJ, 63%; MP, ,50%). Finally, the sister status of the class Spirotrichea with the two orders Clevelandellida and Armophorida is only reconstructed in
the NJ and LD analyses with weak bootstrap support
(,50% in both cases). In the ML and MP trees, the class
Spirotrichea and the orders Armophorida and Clevelandellida are polyphyletic.
Discussion
Mapping the presence of gene-sized chromosomes
onto the ssu-rDNA genealogies indicates that there are
three distinct lineages—(1) the class Phyllopharyngea,
(2) the class Spirotrichea, and (3) the orders Armophorida and Clevelandellida—with extensively fragmented
macronuclear genomes (fig. 1). While both the LD and
the NJ analyses group the Armophorida and Clevelandellida as sister to the Spirotrichea, support for this node
is weak. Regardless of the alignment parameters, phylogenetic algorithm, or model of evolution, no topology
supported the monophyly of the three clades with extensively fragmented genomes.
To test for multiple origins, we compared our genealogies with topologies with only a single origin of
extensive fragmentation. Constraining the three clades
with extensive fragmentation to be monophyletic significantly decreases the likelihood of the genealogy, from
ln L 5 211,524.10404 to ln L 5 211,999.80350 (P ,
0.0001, Kishino-Hasegawa test). Similarly, the constrained topology increases the length of the most parsimonious tree by 16 steps (from 2,869 to 2,885); this
difference is not significant by a Kishino-Hasegawa test,
which is not surprising given the low level of support
for interclass relationships generated using maximum
parsimony (fig. 1).
Hence, a combination of Southern hybridizations
and genealogical analyses strongly support polyphyletic
origins of extensive fragmentation of the macronuclear
genome in ciliates. At a minimum, there are at least two
origins in the lineages, leading to (1) the subclasses Suctoria and Cyrtophoria in the class Phyllopharyngea and
(2) the class Spirotrichea and the orders Armophorida
and Clevelandellida. Moreover, these data are consistent
with the view that the presence of giant chromosomes
during macronuclear development correlates with an extensively fragmented macronuclear genome. As there is
an unconfirmed report of giant chromosomes from the
genus Loxophyllum (L) (Balbiani 1890) and potentially
oligotenic chromosomes in Bursaria (L) (Poljansky
1934; Poljansky and Sergejeva 1981), extensive fragmentation may be even more widespread among nonsister ciliate lineages than we have reported here.
Prior models of the origin of dimorphic nuclei in
ciliates have focused on comparisons between only two
classes, Oligohymenophorea and Spirotrichea, with the
little-understood classes Karyorelictea and Heterotrichea
treated as outgroups (Orias 1991a, 1991b; Herrick
1994). These models fail to account for the dramatic
diversity of macronuclei in ciliates, including the multiple lineages with extensively fragmented macronuclear
chromosomes documented here (Katz 2001). The striking variation in degree of chromosomal fragmentation
among ciliates is undoubtedly related to the specialization of macronuclei as the site of virtually all transcription in ciliates. For example, in Stylonychia mytilus (S),
the development of giant chromosomes is marked by a
period of very high and selectively variable DNA replication followed by dramatic fragmentation of the polytenized chromosomes (Ammerman 1971; Ammerman
et al. 1974). Ciliates in the classes Spirotrichea and
Phyllopharyngea and the genera Metopus and Nyctotherus have met the challenge of producing a streamlined
functional macronucleus by fragmenting germ line chromosomes into gene-sized fragments and presumably
eliminating noncoding sequences. That this has occurred
multiple times indicates that the selective pressure to
extensively fragment chromosomes is strong relative to
constraints on the structure of ciliate genomes.
Acknowledgments
Many thanks to Erica Lasek-Nesselquist, Tovah
Salcedo, and Juliet Christian-Smith for their help in collecting sequence data. Thanks also go to Eduardo Orias
and John Tyler Bonner for insightful and stimulating
discussions on the evolution of ciliates. This work was
supported by A. F. Blakeslee Funds administered
through the National Academy of Sciences and a Howard Hughes Medical Institute grant to support undergraduate research at Smith College.
LITERATURE CITED
AKHMANOVA, A., F. VONCKEN, T. VAN ALEN, A. VAN HOEK,
B. BOXMA, G. VOGELS, M. VEENHUIS, and J. H. P. HACKSTEIN. 1998. A hydrogenosome with a genome. Nature 396:
527–528.
AMMERMANN, D. 1971. Morphology and development of the
macronuclei of the ciliates Stylonychia mytilus and Euplotes
aediculatus. Chromosoma 33:209–238.
———. 1987. Giant chromosomes in ciliates. Results Probl.
Cell Differ. 14:59–67.
AMMERMANN, D., G. STEINBRUCK, L. VON BERGER, and W.
HENNIG. 1974. The development of the macronucleus in the
ciliated protozoan Stylonychia mytilus. Chromosoma 45:
401–429.
AUSUBEL, F. M., R. BRENT, R. E. KINGSTON, D. D. MOORE, J.
G. SEIDMAN, J. A. SMITH, and K. STRUHL. 1993. Current
protocols in molecular biology. Wiley-Liss, New York.
Extensive Chromosomal Fragmentation in Ciliates
BALBIANI, É. G. 1890. Sur la structure intime du noyau du
Loxophyllum meleagris. Zool. Anz. 13:110–115,132–136.
COYNE, R. S., D. L. CHALKER, and M.-C. YAO. 1996. Genome
downsizing during ciliate development: nuclear division of
labor through chromosome restructuring. Annu. Rev. Genet.
30:557–578.
DE RIJK, P., and R. DE WACHTER. 1993. DCSE v2.54, an interactive tool for sequence alignment and secondary structure research. Comput. Appl. Biosci. 9:735–740.
DE PUYTORAC, P., J. GRAIN, and P. LEGENDRE. 1994. An attempt at reconstructing a phylogenetic tree of the Ciliophora
using parsimony methods. Eur. J. Protistol. 30:1–17.
FOISSNER, W. 1996. Ontogenesis in ciliated protozoa with emphasis on stomatogenesis. Pp. 95–178 in K. HAUSMANN and
P. C. BRADBURY, eds. Ciliates: cells as organisms. Gustav
Fisher, Stuttgart, Germany.
GOLIKOVA, M. N. 1964. Polytene chromosomes in the developing macronucleus of an infusorium. Tsitologiya 6:250–
253 [in Russian].
GRASSÉ, P. P. 1952. Généralités. Pp. 37–152 in P. P. GRASSÉ,
ed. Traité de Zoologie. Masson et Cie, Paris.
GRELL, K. G. 1949. Die Entwicklung der Makronucleusanlage
im Exconjuganten von Ephelota gemmipara. R. Hertwig.
Biol. Zentralbl. 68:289–312.
HAMMERSCHMIDT, B., M. SCHLEGEL, D. LYNN, D. D. LEIPE,
M. L. SOGIN, and I. B. RAIKOV. 1996. Insights into the
evolution of nuclear dualism in the ciliates revealed by phylogenetic analysis of rRNA sequences. J. Eukaryot. Microbiol. 43:225–230.
HERRICK, G. 1994. Germline-soma relationships in ciliates protozoa: the inception and evolution of nuclear dimorphism
in one-celled animals. Semin. Dev. Biol. 5:3–12.
HIRT, R. P., M. WILKINSON, and T. M. EMBLEY. 1998. Molecular and cellular evolution of ciliate: a phylogenetic perspective. Pp. 327–340 in G. H. COOMBS, K. VICKERMAN,
M. A. SLEIGH, and A. WARREN, eds. Evolutionary relationships among protozoa. Kluwer Academic Publishers, Dordrecht, The Netherlands.
KATZ, L. A. 2001. Evolution of nuclear dualism in ciliates: a
reanalysis in light of recent molecular data. Int. J. Evol.
Syst. Microbiol (in press).
KISHINO, H., and M. HASEGAWA. 1989. Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data and the branching order in
Hominoidea. J. Mol. Evol. 29:170–179.
KLOBUTCHER, L. A., and G. HERRICK. 1997. Developmental
genome reorganization in ciliated protozoa: the transposon
link. Prog. Nucleic Acid Res. Mol. Biol. 56:1–62.
LAHLAFI, T., and G. MÉTÉNIER. 1991. Low molecular weight
DNA in the heteromeric macronuclei of two cyrtophorid
ciliates. Biol. Cell 73:79–88.
LEE, J. J., and A. T. SOLDO, eds. 1992. Protocols in protozoology. Allen Press, Lawrence, Kans.
LEIPE, D. D., D. BERNHARD, M. SCHLEGEL, and M. L. SOGIN.
1994. Evolution of 16S-like ribosomal RNA genes in the
Ciliophoran taxa Litostomatea and Phyllopharyngea. Eur. J.
Protistol. 30:354–361.
LYNN, D. H. 1997. A revised classification of the phylum Ciliophora Doflein, 1901. Rev. Soc. Mex. Hist. Nat. 47:65–78.
LYNN, D. H., and E. B. SMALL. 1988. An update on the systematics of the phylum Ciliophora Doflein, 1901: the implications of kinetid diversity. BioSystems 21:317–322.
LYNN, D. H., and M. L. SOGIN. 1988. Assessment of phylogenetic relationships among ciliated protists using partial
1377
ribosomal RNA sequences derived from reverse transcripts.
BioSystems 21:249–254.
MEDLIN, L., H. J. ELWOOD, S. STICKEL, and M. L. SOGIN.
1988. The characterization of enzymatically amplified eukaryotes 16S-like ribosomal RNA coding regions. Gene 71:
491–500.
NOLAND, L. E. 1927. Conjugation in the ciliate Metopus sigmoides. J. Morphol. Physiol. 44:341–361.
ORIAS, E. 1991a. Evolution of amitosis of the ciliate macronucleus: gain of the capacity to divide. J. Protozool. 38:
217–221.
———. 1991b. On the evolution of the karyorelict ciliate life
cycle: heterophasic ciliates and the origin of ciliate binary
fission. BioSystems 25:67–73.
ORIAS, E., and T. HIGASHINAKAGAWA. 1990. Genome organization and reorganization in ciliated protozoa. Zool. Sci. 7:
59–69.
POLJANSKY, G. 1934. Geschlechtsprozesse bei Bursaria truncatella O.F. Müll. Arch. Protistenkd. 81:420–545.
POLJANSKY, G., and G. I. SERGEJEVA. 1981. Autoragiographic
investigation of the DNA synthesis during development of
the new macronucleus of the ciliate Bursaria truncatella.
Tsitologiya 23:666–673.
POSADA, D., and K. CRANDALL. 1998. MODELTEST: testing
the model of DNA substitution. Bioinformatics 14:817–818.
PRESCOTT, D. M. 1994. The DNA of ciliated protozoa. Microbiol. Rev. 58:233–267.
RADZIKOWSKI, S. 1979. Asynchronous replication of polytene
chromosome segments of the new macronucleus anlage in
Chilodonella cucullulus O.F. Muller. Protistologica 4:521–
526.
RAIKOV, I. B. 1982. The protozoan nucleus: morphology and
evolution. Springer-Verlag, Vienna.
SMALL, E. B., and D. H. LYNN. 1985. Phylum Ciliophora, Dolfein 1901. Pp. 393–575 in J. J. LEE, S. H. HUTNER, and E.
C. BOVEE, eds. An illustrated guide to the protozoa. Society
of Protozoologists, Lawrence, Kans.
STEINBRÜCK, G., I. HAAS, K.-H. HELLMER, and D. AMMERMANN. 1981. Characterization of macronuclear DNA in five
species of ciliates. Chromosoma 83:199–208.
SWOFFORD, D. 1999. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4.0d65. Sinauer,
Sunderland, Mass.
TAMURA, K., and M. NEI. 1993. Estimation of the number of
nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol.
10:512–526.
THOMPSON, J. D., D. G. HIGGINS, and T. J. GIBSON. 1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680.
WICHTERMAN, R. 1937. Division and conjugation in Nyctotherus cordiformis, with special reference to the nuclear phenomena. J. Morphol. 60:563–611.
WRIGHT, A.-D., B. A. DEHORITY, and D. H. LYNN. 1997. Phylogeny of the rumen ciliates Entodinium, Epidinium and
Polyplastron (Litostomatea: Entodiniomorphida) inferred
from small subunit ribosomal RNA sequences. J. Eukaryot.
Microbiol. 44:61–67.
GEOFFREY MCFADDEN, reviewing editor
Accepted March 24, 2001