Department of Biological Sciences, Smith College
Program in Organismic and Evolutionary Biology, University of MassachusettsAmherst
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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 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 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
).
|
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (-tubulin and histone H4) or by random priming from total E. crassus DNA. The
-tubulin probes were amplified from a cloned portion of this gene from H. erhardi and represented amino acids 20409 of the
-tubulin gene. Primers for the
-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. 1988
), 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 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 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 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
= 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
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 0.001780.0037). 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
).
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)
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.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
1 Abbreviation: ssu-rDNA, small-subunit ribosomal DNA.
2 Keywords: chromosomal fragmentation
chromosomal rearrangements
macronucleus
ciliates
Ciliophora
Phyllopharyngea
Armophorida
Clevelandellida
3 Address for correspondence and reprints: Laura Katz, Department of Biological Sciences, Smith College, Northampton, Massachusetts 01063. E-mail: lkatz{at}smith.edu
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akhmanova A., F. Voncken, T. van Alen, A. van Hoek, B. Boxma, G. Vogels, M. Veenhuis, J. H. P. Hackstein, 1998 A hydrogenosome with a genome Nature 396:527-528[ISI][Medline]
Ammermann D., 1971 Morphology and development of the macronuclei of the ciliates Stylonychia mytilus and Euplotes aediculatus. Chromosoma 33:209-238[ISI][Medline]
. 1987 Giant chromosomes in ciliates Results Probl. Cell Differ 14:59-67[Medline]
Ammermann D., G. Steinbruck, L. von Berger, W. Hennig, 1974 The development of the macronucleus in the ciliated protozoan Stylonychia mytilus. Chromosoma 45:401-429[ISI][Medline]
Ausubel F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl, 1993 Current protocols in molecular biology Wiley-Liss, New York
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, M.-C. Yao, 1996 Genome downsizing during ciliate development: nuclear division of labor through chromosome restructuring Annu. Rev. Genet 30:557-578[ISI][Medline]
De Rijk P., R. De Wachter, 1993 DCSE v2.54, an interactive tool for sequence alignment and secondary structure research Comput. Appl. Biosci 9:735-740[Abstract]
de Puytorac P., J. Grain, P. Legendre, 1994 An attempt at reconstructing a phylogenetic tree of the Ciliophora using parsimony methods Eur. J. Protistol 30:1-17[ISI]
Foissner W., 1996 Ontogenesis in ciliated protozoa with emphasis on stomatogenesis Pp. 95178 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].
Grass P. P., 1952 Généralités Pp. 37152 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, 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[ISI][Medline]
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, T. M. Embley, 1998 Molecular and cellular evolution of ciliate: a phylogenetic perspective Pp. 327340 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., 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[ISI][Medline]
Klobutcher L. A., G. Herrick, 1997 Developmental genome reorganization in ciliated protozoa: the transposon link Prog. Nucleic Acid Res. Mol. Biol 56:1-62[ISI][Medline]
Lahlafi T., G. Méténier, 1991 Low molecular weight DNA in the heteromeric macronuclei of two cyrtophorid ciliates Biol. Cell 73:79-88[ISI]
Lee J. J., A. T. Soldo, eds. 1992 Protocols in protozoology Allen Press, Lawrence, Kans
Leipe D. D., D. Bernhard, M. Schlegel, M. L. Sogin, 1994 Evolution of 16S-like ribosomal RNA genes in the Ciliophoran taxa Litostomatea and Phyllopharyngea Eur. J. Protistol 30:354-361[ISI]
Lynn D. H., 1997 A revised classification of the phylum Ciliophora Doflein, 1901 Rev. Soc. Mex. Hist. Nat 47:65-78
Lynn D. H., E. B. Small, 1988 An update on the systematics of the phylum Ciliophora Doflein, 1901: the implications of kinetid diversity BioSystems 21:317-322[ISI][Medline]
Lynn D. H., M. L. Sogin, 1988 Assessment of phylogenetic relationships among ciliated protists using partial ribosomal RNA sequences derived from reverse transcripts BioSystems 21:249-254[ISI][Medline]
Medlin L., H. J. Elwood, S. Stickel, M. L. Sogin, 1988 The characterization of enzymatically amplified eukaryotes 16S-like ribosomal RNA coding regions Gene 71:491-500[ISI][Medline]
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[ISI][Medline]
. 1991b. On the evolution of the karyorelict ciliate life cycle: heterophasic ciliates and the origin of ciliate binary fission BioSystems 25:67-73[ISI][Medline]
Orias E., T. Higashinakagawa, 1990 Genome organization and reorganization in ciliated protozoa Zool. Sci 7:59-69[ISI]
Poljansky G., 1934 Geschlechtsprozesse bei Bursaria truncatella O.F. Müll Arch. Protistenkd 81:420-545
Poljansky G., 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[ISI]
Posada D., K. Crandall, 1998 MODELTEST: testing the model of DNA substitution Bioinformatics 14:817-818[Abstract]
Prescott D. M., 1994 The DNA of ciliated protozoa Microbiol. Rev 58:233-267[Abstract]
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., D. H. Lynn, 1985 Phylum Ciliophora, Dolfein 1901 Pp. 393575 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, D. Ammermann, 1981 Characterization of macronuclear DNA in five species of ciliates Chromosoma 83:199-208[ISI][Medline]
Swofford D., 1999 PAUP* Phylogenetic analysis using parsimony (*and other methods). Version 4.0d65. Sinauer, Sunderland, Mass
Tamura K., 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[Abstract]
Thompson J. D., D. G. Higgins, 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[Abstract]
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, 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[ISI][Medline]