Widespread Distribution of Extensive Chromosomal Fragmentation in Ciliates

Jennifer L. Riley and Laura A. Katz

Department of Biological Sciences, Smith College
Program in Organismic and Evolutionary Biology, University of Massachusetts–Amherst


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
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 1990Citation ; Prescott 1994Citation ; Coyne, Chalker, and Yao 1996Citation ; Klobutcher and Herrick 1997Citation ). 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 1982Citation ).

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 1994Citation ). 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 from 950 to 15,000 times (Klobutcher and Herrick 1997Citation ; Prescott 1994Citation ). 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 1991Citation ; Steinbrück et al. 1981Citation ). 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. 1998Citation ).

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 1987Citation ). Giant chromosomes are found in Spirotrichea (Ammermann 1987Citation ), the phyllopharyngeans Chilodonella and Trithigmostoma (Radzikowski 1979Citation ; Lahlafi and Méténier 1991Citation ), the genus Nyctotherus (c) (Wichterman 1937Citation ; Grassé 1952Citation ; Golikova 1964Citation ), and possibly also Metopus (order Armophorida [a]) (Noland 1927Citation ). Smaller oligotenic chromosomes have also been found in members of the order Suctoria, an additional lineage of Phyllopharyngeans (Grell 1949Citation ). 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. 1981Citation ; Prescott 1994Citation ; Klobutcher and Herrick 1997Citation ). We also included 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 1985Citation ). 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 1996Citation ). 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 1988Citation ; Leipe et al. 1994Citation ; Hammerschmidt et al. 1996Citation ; Wright, Dehority, and Lynn 1997Citation ; Hirt, Wilkinson, and Embley 1998Citation ).



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Fig. 1.—Small-subunit ribosomal DNA genealogy generated by maximum-likelihood analysis using PAUP* 4.0d65 (Swofford 1999Citation ) with parameters calculated in Modeltest (Posada and Crandall 1998Citation ). 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

 

    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Ciliate Isolation and Culture
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 1992Citation ). 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. 1993Citation ). Euplotes crassus DNA (strains Ust-2 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 ({alpha}-tubulin and histone H4) or by random priming from total E. crassus DNA. The {alpha}-tubulin probes were amplified from a cloned portion of this gene from H. erhardi and represented amino acids 20–409 of the {alpha}-tubulin gene. Primers for the {alpha}-tubulin PCR were Tub 37+ ((CUA)4ATHCANCCNGAYGGNCARATGCC) and Tub 409- ((CAU)4CATNCCYTCNCCNACRWACCA). 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 H4F011+ ((CUA)4ggNRTNACNAARCCNgCNAT) and H4R011- ((CAU)4TTNARNgCRTANACNACRTC). To label the entire E. crassus genome, ~1µg 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. 1988Citation ), and all reactions were run with 3 mM MgCl2, 1 x PCR buffer, 0.55 U Platinum TAQ DNA Polymerase (Gibco-BRL, Grand Island, N.Y., 10966-034), 0.4 mM dNTP, six pmol primer, for a 25-µl 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 1993Citation ) 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 1994Citation ) 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 ssu-rDNA 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 1999Citation ). Parameters for ML analyses were calculated with hierarchical likelihood ratio tests using Modeltest 3.0 (Posada and Crandall 1998Citation ). Based on this analysis, we used a TrN (Tamura and Nei 1993Citation ) 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 {alpha} = 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 1989Citation ) 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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
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: {alpha}-tubulin and histone H4. Southern hybridization results for M. palaeformis (a), N. ovalis (c), and all of the Phyllopharyngeans studied show that the {alpha}-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 ).


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Table 1 Sizes of Macronuclear Chromosomes in Ciliates

 


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Fig. 2.—Representative Southern hybridizations probed with (a) alpha-tubulin (1, 6, 7 = molecular weight markers; 2 = Myrionecta rubrum [O]; 3 = Chilodonella uncinata [P]; 4, 10 = Euplotes crassus [S]; 5 = Heliophrya erhardi [P]; 8 = Metopus palaeformis [a]; 9 = Nyctotherus ovalis [a]) (b) histone H4 (1 = E. crassus [S]; 2 = Halteria grandinella [S]; 3 = Paramecium tetraurelia [O]; 4 = Didinium sp. [L], 5 = molecular weight marker; 6 = Tokophrya lemnarum [P]), and (c) the E. crassus genome (1 = P. tetraurelia [O]; 2, 6, 7 = molecular weight markers; 3 = Didinium sp. [L]; 4 = H. grandinella [S]; 5 = E. crassus [S]; 8 = T. lemnarum [P]; 9 = N. ovalis [c])

 
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 1994Citation ; Klobutcher and Herrick 1997Citation ). 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 (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 1997Citation ).

Overall, our phylogenetic analyses of ssu-rDNA sequences are consistent with published genealogies (Lynn and Sogin 1988Citation ; Leipe et al. 1994Citation ; Hammerschmidt et al. 1996Citation ; Wright, Dehority, and Lynn 1997Citation ; Hirt, Wilkinson, and Embley 1998Citation ); 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 ).

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 1988Citation ; Leipe et al. 1994Citation ; Hammerschmidt et al. 1996Citation ; Lynn and Small 1997Citation ; Wright, Dehority, and Lynn 1997Citation ; Hirt, Wilkinson, and Embley 1998Citation ). 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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
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 = -11,524.10404 to ln L = -11,999.80350 (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)Citation and potentially oligotenic chromosomes in Bursaria (L) (Poljansky 1934Citation ; Poljansky and Sergejeva 1981Citation ), 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, 1991bCitation ; Herrick 1994Citation ). 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)Citation . 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.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
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.


    Footnotes
 
Geoffrey McFadden, Reviewing Editor

1 Abbreviation: ssu-rDNA, small-subunit ribosomal DNA. Back

2 Keywords: chromosomal fragmentation chromosomal rearrangements macronucleus ciliates Ciliophora Phyllopharyngea Armophorida Clevelandellida Back

3 Address for correspondence and reprints: Laura Katz, Department of Biological Sciences, Smith College, Northampton, Massachusetts 01063. E-mail: lkatz{at}smith.edu Back


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Accepted for publication March 24, 2001.