Dramatic Diversity of Ciliate Histone H4 Genes Revealed by Comparisons of Patterns of Substitutions and Paralog Divergences Among Eukaryotes

Laura A. Katz*,{dagger}, Jacob G. Bornstein{dagger}, Erica Lasek-Nesselquist*,1 and Spencer V. Muse{ddagger}

* Department of Biological Sciences, Smith College, Northampton, Massachusetts
{dagger} Program in Organismic and Evolutionary Biology, University of Massachusetts-Amherst
{ddagger} Bioinformatics Research Center, Department of Statistics, North Carolina State University, Raleigh

Correspondence: E-mail: lkatz{at}smith.edu.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature cited
 
The accumulation of divergent histone H4 amino acid sequences within and between ciliate lineages challenges traditional views of the evolution of this essential eukaryotic protein. We analyzed histone H4 sequences from 13 species of ciliates and compared these data with sequences from well-sampled eukaryotic clades. Ciliate histone H4s differ from one another at as many as 46% of their amino acids, in contrast with the highly conserved character of this protein in most other eukaryotes. Equally striking, we find paralogs of histone H4 within ciliate genomes that differ by up to 25% of their amino acids, whereas paralogs in other eukaryotes share identical or nearly identical amino acid sequences. Moreover, the most divergent H4 proteins within ciliates are found in the lineages with highly processed macronuclear genomes. Our analyses demonstrate that the dual nature of ciliate genomes—the presence of a "germline" micronucleus and a "somatic" macronucleus within each cell—allowed the dramatic variation in ciliate histone genes by altering functional constraints or enabling adaptive evolution of the histone H4 protein, or both.

Key Words: Protein evolution • histone H4 • ciliates • macronucleus • chromosomal rearrangements, fate of paralogs


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature cited
 
The presence of two functionally distinct genomes—the transcriptionally inactive micronucleus and the functional macronucleus—within ciliates may allow the rapid evolution of histone H4 genes in this lineage compared with other eukaryotes. Although both develop from a zygotic nucleus, only micronuclei contain conventional eukaryotic chromosomes and divide by mitosis. In contrast, macronuclei develop through a series of chromosomal rearrangements that include the fragmentation of the genome, into pieces as small as 2 kb in some ciliate classes, and amplification of up to 1,000-fold of some "chromosomes" (Prescott 1994; Katz 2001; Jahn and Klobutcher 2002; Yao, Duharcourt, and Chalker 2002). Not surprisingly, macronuclei divide by amitosis (except within the class Karyorelictea, in which macronuclear division does not occur; reviewed in Raikov [1982], Prescott [1994], and Katz [2001]). The extent of chromosomal processing in macronuclei varies from the relatively limited fragmentation and amplification found in the oligohymenophoreans Tetrahymena and Paramecium, to the extensive processing found in the classes Spirotrichea and Phyllopharyngea (Katz 2001).

In most nonciliate eukaryotes, histone H4 proteins differ at only a few amino acid positions, demonstrating that a limited number of polypeptides can maintain structure and proper gene expression (Luger et al. 1997; Wolffe and Hayes 1999). Histone H4 has the slowest rate of nonsynonymous substitutions among a study of 27 proteins in mammals; further, only three amino acid substitutions were found among comparisons of 16 eukaryotic taxa that included several vertebrates, echinoderms, arthropods, and plants (Graur 1985; Wells and McBride 1989). Exceptions to this conservative pattern of evolution come from taxa in poorly sampled eukaryotic clades, including Giardia (Wu et al. 2000), Leishmania (Lukes and Maslov 2000), and Entamoeba (Binder et al. 1995).

Similarly, paralogs (duplicated copies) of histone H4 within the genomes of most eukaryotes encode for identical or very similar amino acid sequences. With only one exception, the 12 human histone H4 paralogs differ by no more than 2 aa across the entire length of the gene (Piontkivska, Rooney, and Nei 2002) and are identical in amino acid sequence for the region used in this study. The single exception to this pattern (GenBank accession number Z80788) appears to be a highly divergent pseudogene because no expression of this paralog was detected in analyses of several human cell lines (Doenecke, personal communication). The conservation of amino acid but not nucleotide sequences among nonciliate histone H4 paralogs indicates that purifying selection eliminates nonsynonymous substitutions within most eukaryotic genomes (Piontkivska, Rooney, and Nei 2002). In contrast, previous studies suggested that the histone H4 protein diversifies faster in ciliates than in other eukaryotes (Sadler and Brunk 1992; Salvini 1997; Bernhard and Schlegel 1998), although these analyses focused mainly on qualitative assessments of overall patterns of divergence. For instance, there are more amino acid differences in histone H4 between two classes of ciliates (Spirotrichea and Oligohymenophorea) than there are between land plants and animals (Bernhard and Schlegel 1998).

To elucidate the pattern of histone H4 diversity within ciliates and to compare patterns of substitutions with those in other well-sampled eukaryotic clades, we sequenced a portion of the histone H4 gene from 13 ciliate species. Ciliates studied include representatives from six of nine classes of ciliates along with two related orders of uncertain taxonomic position (table 1). We analyzed variation among major eukaryotic clades using a genealogical perspective and compared patterns of nucleotide and amino acid substitutions among histone H4 paralogs.


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Table 1 Divergence Within and Between Ciliate Histone H4 Paralogs Characterized for This Study.

 

    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature cited
 
Isolation of Ciliate DNAs
Sterkiella histriomuscorum DNA was donated by T. Doak and G. Herrick (University of Utah), and both Blepharisma americanum and Stentor coeruleus cultures were from Connecticut Valley Biological Supply Company. Strombidium sp. and Pleuronema sp. cultures were collected in Harbortown, Florida. Details on culturing and isolation of remaining taxa are reported elsewhere (Riley and Katz 2001).

Characterization of Ciliate Histone Sequences
We amplified a portion of histone H4 from total-genomic DNAs using gene-specific primers H4F011+ ([CUA]4 GGNRTNACNAARCCNGCNAT) and H4R011- ([CAU]4 TTNARNGCRTANACNACRTC) and platinum Taq DNA polymerase (GibcoBRL). PCR products purified using the Qiaquick PCR purification (Qiagen) were cloned in pAMP1 vector (GibcoBRL) and miniprepped using the Qiaprep Spin Miniprep kit (Qiagen). Sequences were generated from all clones in both directions using the BigDye terminator RR mix from PE Applied Biosystems. Reactions were cleaned using gel filtration cartridges from Edge Biosystems, and samples were run on either an ABI 3100 or ABI 377 automated sequencer. For paralogs represented by multiple clones, we randomly chose a sequence to represent the paralog for analyses.

Data Analysis
We searched for histone H4 GenBank entries using the Entrez browser through the program Editseq implemented by DNASTAR, Inc. We limited our searches to exclude ESTs, GSSs, patented sequences, and sequences less than 100 nt, and only included sequences from clades containing at least three genera. For the well-sampled taxa—plants + green algae ("greens"), animals and fungi—we only included species with paralogs to maintain a data set of reasonable size (table S1 in Supplementary Material online at www.mbe.oupjournals.org). Multiple sequence alignments, including our contigs assembled in Seqman (DNAStar, Inc), were generated by ClustalW (Thompson, Higgins, and Gibson 1994) as implemented by Megalign (DNAStar, Inc) with a gap penalty of 10 and gap length penalty of 10.

To construct genealogies, we used PAUP* version 4.0b10 software (Swofford 2002), implementing the Neighbor-Joining algorithm. Nucleotide distances were estimated using maximum-likelihood settings in PAUP* (Swofford 2002), with a GTR model and parameters estimated by Modeltest (Posada and Crandall 1998) as implemented in HyPhy (Kosakovsky Pond and Muse 2003). Amino acid distances were calculated in Tree-Puzzle version 5.0 (Strimmer and von Haeseler 1997) using a JTT model, with variation in rates among sites estimated by gamma distribution with six rate classes.

We compared the rates of nonsynonymous and synonymous substitutions within the constrained animal, fungi, "green," and ciliate clades. The dN/dS ratios were estimated by maximum-likelihood methods in HyPhy (Kosakovsky Pond and Muse 2003), with the MG94_3x4 model of substitution on a constrained topology (see below). The ratio of the synonymous and nonsynonymous rates estimated by this model (Muse and Gaut 1994) is very similar to the ratio of the expected number of nonsynonymous substitutions per nonsynonymous site to the expected number of synonymous substitutions per synonymous site (dN/dS [Muse 1996]), and hence we use this term. Values of dN/dS were compared using Kruskal-Wallis tests corrected for ties (P = 0.003) (Kruskal and Wallis 1953), Mood's median test (Mood 1950), and Fisher's exact test implemented by the program RxC (bioweb.usu.edu/mpmbio).

A primary goal of the analyses is to identify clades with exceptionally high dN/dS ratios. Because some of our analyses involve averaging dN/dS ratios over lineages, we wanted to limit the effect of outlying observations, which are likely to arise as the result of the sampling properties of ratio estimators. Towards that end, we restricted the range of estimates of dN and dS by setting values less than 10-10 to 0 and estimates of 10 or more to 10. Similarly, dN/dS values were capped at 2, including infinite N/0 ratios, and estimates of dN/dS that were equal to 0/0 were set at a value of 0. We believe these settings cause our analyses to be conservative in identifying clades with high dN/dS values by minimizing the effects of dN/dS ratios with extreme values in either the numerator or denominator that are likely the result of large sampling variation. This procedure is conservative for our purposes because it only reduces large dN/dS estimates, making it more difficult for an estimate to be identified as extreme.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature cited
 
To assess intraspecific as well as interspecific patterns of variation, we sequenced three to 11 clones, each representing roughly half of the histone H4 coding sequence, from 13 species of ciliates. The portion of the gene that we characterized corresponds to most of {alpha}-helix 1 ({alpha}H1), all of {alpha}-helix 2 ({alpha}H2), and loop 1 (L1) and loop 2 (L2) of the histone H4 protein (Luger et al. 1997). The number of histone H4 paralogs varies in ciliates, as we identified between two and six paralogs of histone H4 within species (table 1). Because we lack mapping data on our sequences, we define paralogs as homologous sequences from a species that diverge by 2% or more at the nucleotide level. This operational definition is appropriate for conservative sequences such as these histone genes and should not be considered a general rule. Our definition of paralogs is supported by the observation that variation caused by macronuclear copying, PCR error, and alleles from these same genomic DNAs ranges from 0% to 0.5% (Riley and Katz 2001; Israel et al. 2002). Using this definition, we characterized two paralogs from Chilodonella uncinata, Metopus palaeformis, Sterkiella historiomuscorum, Bursaria truncatella and Monoeuplotes crassus, three paralogs from Heliophrya erhardi and Pleuronema sp., and four, five, and six paralogs from Halteria grandinella, Nyctotherus ovalis, and Blepharisma americanum, respectively (table 1).

We found no evidence of paralogs in the populations of Stentor sp., Strombidium sp., or Tokophrya lemnarum, even though up to 11 clones were sampled from these three taxa (table 1). Clearly, both the number of clones we sampled and the relative amplification of each paralog within the macronuclear genome affect the number of paralogs identified, and more paralogs are likely to exist. Although we did not characterize the termination codon, all but one of our sequences can be translated into an open reading frame using the universal genetic code. The exception is H. grandinella paralog P4 (HgraP4), which has an inframe TAA and needs the ciliate genetic code (NCBI translation table 6) to generate an open reading frame. Clonal lines and population samples showed similar patterns of sequence divergences (table 1).

Genealogical analyses of ciliate histone H4 sequences, along with paralogs from animals, fungi, and "greens," yield topologies that are discordant with both morphology and other molecular genealogies. For example, the fungi and ciliates are polyphyletic in nucleotide and amino acid genealogies generated in a NJ analysis with ML distances (figure S1 in Supplementary Material online). Our genealogies also fail to show evidence of ancient paralogs (figure 1 and figure S1 in Supplementary Material online), inferred when parallel clades are generated for a given set of taxa. Hence, we conclude that the heterogeneity in rates of evolution explain the discordant topologies.



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FIG. 1. Genealogy of histone H4 proteins in major clades, all drawn to the same scale to reveal the considerably greater protein evolution in ciliates. Bootstrap values greater than 70% are indicated by the symbol ·. "Greens" = green algae + plants; C/A = Clevelandellida + Armophorida; CONP = Colpodea + Oligohymenophorea + Nassophorea + Prostomatea; S = Spirotrichea; P = Phyllopharygnea, and H = Heterotrichea

 
To examine patterns of substitutions in histone H4 protein evolution, we constrained nodes for the major clades animals, fungi, "greens," and ciliates (fig. 1). Within the ciliates, we further constrained five clades to be monophyletic, reflecting the topologies generated using small subunit ribosomal DNA (e.g., Riley and Katz [2001]): (1) Heterotrichea (H); (2) Phyllopharyngea (P); (3) Spirotrichea (S); (4) the four classes Colpodea + Oligohymenophorea + Nassophorea + Prostomatea (CONP); and (5) the two clades Clevelandellida + Armophorida (C/A). Comparisons of branch lengths in the resulting genealogical analyses reveal accelerated rates of protein evolution in ciliates compared with those of other well-sampled eukaryotic clades (fig. 1). There is considerably more conservation of amino acid sequence (short branches) among "greens," animals, and, to a lesser extent, fungi, than is present among ciliate histone H4s.

The ratio of nonsynonymous to synonymous substitution rates, estimated by maximum likelihood using a codon-based model of sequence evolution (Muse and Gaut 1994) on the constrained topology, is higher in ciliates than in other eukaryotic clades. Comparisons of binned dN/dS estimates reveal that ciliates have significantly greater than expected numbers of values of 0.2 or more compared with other clades (Fisher's exact test, P < 0.001 [table 2]). Similarly, 10% trimmed mean dN/dS (trimmed to reduce the impact of outliers) is highest in ciliates (dN/dS = 0.045) and is at least twice that found in animals (dN/dS = 0.000), "greens" (dN/dS = 0.002), and fungi (dN/dS = 0.021 [table 2]). Nonparametric tests for comparing the distributions of dN/dS values among the four clades are highly significant (P < 0.001 in Kruskal-Wallis adjusted for ties, and P = 0.006 in median test), and both tests indicate that ciliate dN/dS values are the largest.


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Table 2 Estimates of dn/ds from Major Clades Reported in Bins and As 10% Trimmed Means.

 
Moreover, the highest dN/dS values within ciliates are found in lineages that extensively process their macronuclear genomes (P = 0.008 [table 3]), although not all clades with extensive fragmentation have the highest values. The 10% trimmed means among ciliates are significantly higher for two of the clades with extensively fragmented macronuclear genomes, C/A (0.458) and Phyllopharyngea (0.088), than for the remaining clades: CONP (0.037), Spirotrichea (0.012). and Heterotrichea (0.012; P = 0.003 in Kruskal-Wallis adjusted for ties, and P = 0.002 in median test [table 3]). Using the unconstrained topology did not affect the overall pattern of these results.


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Table 3 Estimates of dn/ds from Ciliate Clades Reported in Bins and As 10% Trimmed Means.

 
The fate of duplicated histone H4 genes also differs between ciliates and other eukaryotes. Although the average nucleotide divergences are similar among cladesGo, the amino acid divergences are significantly greater among ciliate paralogs than paralogs in the other clades (P < 0.001 [fig. 2]). In fact, the highest levels of amino acid divergence (>15%) within the ciliates are found among four taxa, H. grandinella (Hgra, Cl: Spirotrichea), N. ovalis (Nova, Order: Armophorida), H. erhardi (Herh, Cl: Phyllopharyngea), and C. uncinata (Cunc, Cl: Phyllopharyngea [fig. 3]), all of which are known to extensively fragment their macronuclear genomes, generating "chromosomes" with only a single gene (Steinbrück et al. 1995; Riley and Katz 2001). In contrast, the average pairwise amino acid differences among paralogs of nonciliate taxa examined are 4% or less, despite the considerable nucleotide divergence among many of these sequences.



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FIG. 2. Average nucleotide and amino acid divergences among paralogs for the major clades. Whereas divergences at the nucleotide level do not differ among major clades, amino acid divergences in ciliates are significantly higher (***P < 0.001, based on both a Kruskal-Wallis Z test [Kruskal and Wallis 1953] and a median test [Mood 1950]). Divergences are from comparisons of six, seven, seven, and 18 paralogs in "greens," fungi, animals, and ciliates, respectively, and were estimated as uncorrected distances in PAUP* (Swofford 2002)

 


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FIG. 3. Average nucleotide (white bars) and amino acid (black bars) divergences among ciliate paralogs estimated as uncorrected distances in PAUP* (Swofford 2002). Inferred amino acid sequences across the region studied are identical between paralogs in eight of the 18 ciliate species. In the remaining 10 ciliates, amino acid divergences among paralogs range from 2% to 25%, with the highest values found in taxa with extensively fragmented genomes (see text)

 
Despite the high level of divergence among ciliate histone H4 genes, there is conservation at amino acid sites known to contact other proteins or DNA in nucleosomes (fig. 4). This is consistent with the conclusions of Bernhard and Schlegel (1998), that ciliate sequences are evolving under functional constraint and are not pseudogenes. Ciliate histone H4s generally exhibit a conserved amino acid sequence in loops L1, L2, and {alpha}H1, represented by the first 14 and last eight amino acids in our analysis (fig. 4). Five of the 17 amino acid positions involved in protein-protein and protein-DNA interactions are identical in all ciliates (fig. 2). Furthermore, 96.8% and 83.9% of the substitutions among residues that directly contact other histone proteins or DNA are effectively conservative, based on measures of change in charge, or polarity and volume, respectively (Zhang 2000). The highest numbers of amino acid states within ciliates are found at positions with no known contacts in the nucleosome, including the 10 amino acid states at positions 23 and 27. In contrast, the histones of nonciliate eukaryotes display a different overall pattern. Fewer positions vary within the "greens," animal, and fungi clades, and these variable positions have fewer amino acid states. There are only two amino acid states at variable sites within animals and "greens," and no more than five states for positions within fungi (fig. 4).



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FIG. 4. Amino acid polymorphisms across histone H4 compared among major clades. The number of states refers to the number of amino acid states inferred at each position along the gene minus one. Diamonds along the lower axis represent sites with known protein-protein or protein-DNA interactions in the yeast nucleosomes (Luger et al. 1997). All analyses used 151 bp of an H4 sequence that corresponds to amino acids 35 to 86 of a full length H4 sequence of Homo sapiens (Luger et al. 1997)

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature cited
 
We demonstrate that ciliate histone H4 proteins have acquired more amino acid substitutions when compared with those from other well-sampled eukaryotic clades, and that ciliate lineages with highly processed genomes have more divergent histone H4 sequences. Quantitative analyses of dN/dS ratios (tables 2 and 3) and pairwise differences between paralogs (figs. 2 and 3), coupled with a qualitative assessment of branch lengths (fig. 1), reveal the highly divergent nature of ciliate histone H4 proteins. Within ciliates, the paralogs with the highest amino acid divergences are found in species with extensively fragmented macronuclear genomes (fig. 3), and the highest average dn/ds ratios are from two of these clades, the Phyllopharyngea and Clevelandellida/Armophorida (table 3).

The diversity of histone H4 proteins in ciliates may be related to the presence of two functionally distinct genomes within each cell: the transcriptionally-inactive micronucleus and the functional macronucleus. We propose three hypotheses for the dramatic protein evolution in ciliate histone H4s: (1) as previously suggested, relaxed functional constraint on the nucleosomes of amitotic macronuclei allows for an increase in the ratio of nonsynonymous substitutions to synonymous substitutions in comparison to other eukaryotes (Sadler and Brunk 1992; Salvini 1997; Bernhard and Schlegel 1998); (2) adaptive substitutions accumulate in micronuclear copies of paralogs that are essentially hidden from selection in the macronucleus during asexual divisions; and (3) the divergence reflects the accumulation of deleterious mutations. It is likely that all three hypotheses interact to produce the observed pattern. However, we believe that the third hypothesis alone, the accumulation of deleterious mutations, cannot account for the observed level of variation given the tremendous genetic load that this hypothesis would suggest has accumulated within the approximately 850 to 2,200 Myr old ciliate clade (Knoll 1992; Wright and Lynn 1997; Cavalier-Smith 2002).

If relaxed functional constraints explain most of the observed pattern, then we expect that ciliate histone H4 proteins will not function in nonciliate eukaryotes. Surprisingly, the Tetrahymena thermophila histone H4 protein is viable when transformed into yeast (Fogel and Brunk 1997), despite 12 amino acid substitutions across the region we analyzed between these two proteins. This transformation result indicates that at least this one divergent ciliate protein has retained its essential function when present in the yeast nucleosome. A further prediction of a hypothesis of relaxed functional constraints on the macronuclear genome is that all ciliates will contain a conserved histone H4 protein that allows the condensation of chromatin during mitosis in micronuclei, as this nucleus contains a "conventional" (unprocessed) eukaryotic genome. We do not find any evidence for a conserved histone H4 gene within any of our taxa; for example the predicted histone H4 proteins of all phyllopharyngean ciliates are highly divergent (clade "P" [fig. 1]).

In contrast, there is evidence to support the second hypothesis that adaptive evolution contributes to the variation observed among ciliate histone H4. Under this hypothesis, divergent paralogs evolve because the dual nature of ciliate genomes enables ciliates to maintain copies of paralogs in their micronuclei while "hiding" them from selection, or at least reducing the impact of selection, in their transcriptionally active macronuclei. All ciliates exhibit some degree of fragmentation and amplification of chromosomes during the development of macronuclei (Jahn and Klobutcher 2002; Yao, Duharcourt, and Chalker 2002). Selection on macronuclei differs from that of conventional nuclei, as processed macronuclear genomes have the potential to: (1) break up linkage groups such that the fate of paralogs is less affected by polymorphisms at linked loci; (2) allow assortment and recombination of paralogs during asexual divisions of the macronucleus, possibly resulting in the removal of deleterious mutations from the macronucleus that are still present in the micronucleus; (3) redefine "genetic load" through the differential amplification of macronuclear "chromosomes," thus making it relatively inexpensive for ciliates to carry duplicated genes and deleterious mutations; and/or (4) enable the accumulation of mutations in the unexpressed micronucleus until conjugation. In effect, during the asexual divisions that follow conjugation, a duplicated copy of a gene can experience reduced selection in the processed macronuclear genome (e.g., if it contains fewer [or no] copies of a deleterious gene), while potentially acquiring compensatory substitutions in the micronucleus. After conjugation, the parental macronucleus is replaced by a new macronucleus that essentially develops from the micronucleus, enabling the expression of any gene that has acquired compensatory changes. Such a mechanism may enable ciliates to explore protein space in a manner that is unique among eukaryotes.

This second hypothesis is further supported by the fact that the most divergent histone H4 genes are found in ciliates with extensively fragmented macronuclear chromosomes (members of the classes Phyllopharyngea and Spirotrichea, and the related orders Clevelandellida and Armophorida). Macronuclear "chromosomes" from ciliates in these three clades often contain only a single gene (Riley and Katz 2001). This extensive processing effectively breaks up linkage groups and potentially reduces the impact of deleterious mutations in macronuclei through assortment or recombination during asexual divisions more than in other ciliates.

Finally, if the dual nature of ciliate genomes allows the rapid evolution of histone H4 proteins, then we expect elevated rates of amino acid substitutions to also be found in other ciliate proteins. In fact, fast protein evolution has been reported for several ciliate genes, including elongation factor 1{alpha}, heat shock protein 70, actin, and eukaryotic release factor 1 (Bhattacharya and Ehlting 1995; Budin and Philippe 1998; Moreira, Le Guyader, and Philippe 1999; Moreira et al. 2002), and divergent paralogs of {alpha}-tubulin have been found in taxa with extensively processed macronuclear genomes (Israel et al. 2002). In most cases, elevated rates of protein evolution in ciliates are accompanied by the presence of divergent paralogs, suggesting that the potential of adaptive evolution through gene duplication (Ohno 1970; Hughes 1994; Force et al. 1999; Hughes 2000; Lynch and Conery 2000) may interact with the dual nature of ciliate genomes to explain the divergence among ciliate histone H4s.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature cited
 
This work was support by a grant from the Tomlinson Fund at Smith College to E.L.N., and NSF grants DEB-0092908 and DEB-0079325 to L.A.K. and DBI-0096033 to S.V.M. Thanks to Tovah Salcedo for her work in isolating N. ovalis cells from cockroach guts, Juliet-Christian Smith for her M. palaeformis H4 sequences, and Steven Williams and Louie Bierwert for support in generating sequences. Finally, we are grateful to Wayne Coats, who helped obtain funding from the Smithsonian Marine Station at Fort Pierce for us to collect ciliates in Florida (contribution no. 583).


    Footnotes
 
1 Present address: Marine Biological Laboratory, Woods Hole, Massachusetts. Back

Mark Ragan, Associate Editor Back


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 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
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Accepted for publication October 27, 2003.