Dramatic diversity of ciliate histone H4 genes revealed by comparisons of patterns of substitutions and paralog divergences among eukaryotes

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.     

Variation in macronuclear genome content of three ciliates with extensive chromosomal fragmentation: a preliminary analysis

The genome architecture of ciliates, including features such as nuclear dualism and large-scale genome rearrangements, impacts gene and genome evolution in these organisms. To better understand the structure of macronuclear chromosomes in ciliates with extensively processed chromosomes, a sample of complete macronuclear chromosomes was sequenced from three ciliate species: Metopus es (Class [Cl]: Armophorea), Nyctotherus ovalis (Cl: Armophorea), and Chilodonella uncinata (Cl: Phyllopharyngea). By cloning whole macronuclear chromosomes into a plasmid vector, we generated nine clones from each of M. es and C. uncinata, and 37 clones from N. ovalis. Analysis of these macronuclear chromosomes provides insight into the evolution of genome features such as chromosome content, gene structure, and genetic code. Phylogenetic patterns can be found in telomere structure and codon usage, which are both more similar in M. es and N. ovalis than C. uncinata. In addition, we provide evidence of lateral transfer of a bacterial endo-beta-mannanase gene onto a M. es chromosome and report the discovery of a 42-bp conserved sequence motif within N. ovalis untranslated regions.     

八种纤毛虫减数分裂基因的分子进化研究

减数分裂是真核生物适应性进化的重要机制,以8种纤毛虫作为实验对象,通过生物信息学方法对其14个减数分裂基因进行了鉴定及分子进化研究。结果表明:(1)不同的纤毛虫种类存在一些特异性的减数分裂基因的丢失与复制现象;(2)减数分裂相关基因在纤毛虫中很保守;(3)纤毛虫减数分裂重要的同源重组过程是在真核生物中不常见的Ⅱ型。本研究表明,纤毛虫减数分裂可能代表了真核生物较原始的减数分裂方式,在进化的过程中很保守,为研究真核生物减数分裂起源与进化提供了重要线索。     

Evolution of Germline-Limited Sequences in Two Populations of the Ciliate Chilodonella uncinata

Ciliates are microbial eukaryotes that separate their nuclear functions into a germline micronucleus and a somatic macronucleus. During development of the macronucleus the genome undergoes a series of reorganization events that includes the precise excision of intervening DNA. Here, we determine the architecture of four loci in the micronuclear and macronuclear genomes of the ciliate Chilodonella uncinata and compare the levels of variation in micronuclear-limited sequences to macronuclear destined sequences at two of these loci. We find that within a population, germline-limited sequences are evolving at the same rate as other putatively neutral sites, but between populations germline-limited sequences are accumulating mutations at a much faster rate than other sites. We also find evidence of macronuclear recombination and incomplete elimination of intervening DNA, which result in increased diversity in the macronuclear genome. Our results support the assertion that the unusual genomic features of ciliates can result in rapid and unpredicted patterns of diversification.     

Non-Mendelian inheritance of paralogs of 2 cytoskeletal genes in the ciliate Chilodonella uncinata

Recognition of the role of non-Mendelian inheritance is on the rise, particularly as epigenetic phenomena are shown to shape the transformation of genomes into phenotypes. Ciliates provide a model system in which to explore the role of epigenetics because ciliates have both a germ line (micronuclear) and somatic (macronuclear) genome within every cell. In the ciliate Chilodonella uncinata, the macronucleus is extensively fragmented such that many genes end up on their own chromosomes. Hence, it is possible to track the fate of unlinked genes within macronuclei of C. uncinata. Here we demonstrate that the pattern of inheritance in isolates of C. uncinata is complex and involves both Mendelian transmission between micronuclei and macronuclei and epigenetic phenomena. The macronuclei from 2 isolates of C. uncinata and their progeny share identical rDNA loci and 2 identical beta-tubulin paralogs, yet have different actin paralogs and some beta-tubulin paralogs that are not shared. We propose a model in which all the divergent paralogs are present in the ciliate micronuclei. Under this model, different paralogs are retained in developing macronuclei following conjugation. We further speculate that an epigenetic mechanism, such as RNA interference, is involved in selective retention of specific paralogs within lines. This system allows the exploration of epigenetic phenomena that shape somatic genomes and provides parallels to studies of the development of somatic nuclei within animals.     

Micronuclear and macronuclear forms of beta-tubulin genes in the ciliate Chilodonella uncinata reveal insights into genome processing and protein evolution

Chilodonella uncinata, like all ciliates, contains two distinct nuclei in every cell: a germline micronucleus and a somatic macronucleus. During development of the macronucleus from a zygotic nucleus, the genome is processed in several ways, including elimination of internal sequences. In this study, we analyze micronuclear and macronuclear copies of beta-tubulin in C. uncinata and find at least four divergent paralogs of beta-tubulin in the macronucleus. We characterize the micronuclear version of one paralog and compare its internally eliminated sequences (IESs) with previously described IESs in this species. These comparisons reveal the presence of a conserved sequence motif within IESs. In addition, we compare the sequences of beta-tubulin from C. uncinata with other ciliates and to other alveolates in order to test the hypothesis that the mode of molecular evolution in ciliates obscures phylogenetic signal in protein-coding genes. We find that heterogeneous rates of substitution in beta-tubulin across ciliates result in unstable genealogies that are inconsistent with phylogenies based on small subunit rDNA genes and on ultrastructure. We discuss the implications of our findings for genome processing and protein evolution in ciliates.     

Patterns of protein evolution in Tetrahymena thermophila: implications for estimates of effective population size

High levels of synonymous substitutions among alleles of the surface antigen SerH led to the hypothesis that Tetrahymena thermophila has a tremendously large effective population size, one that is greater than estimated for many prokaryotes (Lynch, M., and J. S. Conery. 2003. Science 302:1401-1404.). Here we show that SerH is unusual as there are substantially lower levels of synonymous variation at five additional loci (four nuclear and one mitochondrial) characterized from T. thermophila populations. Hence, the effective population size of T. thermophila, a model single-celled eukaryote, is lower and more consistent with estimates from other microbial eukaryotes. Moreover, reanalysis of SerH polymorphism data indicates that this protein evolves through a combination of vertical transmission of alleles and concerted evolution of repeat units within alleles. SerH may be under balancing selection due to a mechanism analogous to the maintenance of antigenic variation in vertebrate immune systems. Finally, the dual nature of ciliate genomes and particularly the amitotic divisions of processed macronuclear genomes may make it difficult to estimate accurately effective population size from synonymous polymorphisms. This is because selection and drift operate on processed chromosomes in macronuclei, where assortment of alleles, disruption of linkage groups, and recombination can alter the genetic landscape relative to more canonical eukaryotic genomes.     

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.     

Multiple genes of apparent algal origin suggest ciliates may once have been photosynthetic

Plantae (as defined by Cavalier-Smith, 1981) plastids evolved via primary endosymbiosis whereby a heterotrophic protist enslaved a photosynthetic cyanobacterium. This "primary" plastid spread into other eukaryotes via secondary endosymbiosis. An important but contentious theory in algal evolution is the chromalveolate hypothesis that posits chromists (cryptophytes, haptophytes, and stramenopiles) and alveolates (ciliates, apicomplexans, and dinoflagellates) share a common ancestor that contained a red-algal-derived "secondary" plastid. Under this view, the existence of several later-diverging plastid-lacking chromalveolates such as ciliates and oomycetes would be explained by plastid loss in these lineages. To test the idea of a photosynthetic ancestry for ciliates, we used the 27,446 predicted proteins from the macronuclear genome of Tetrahymena thermophila to query prokaryotic and eukaryotic genomes. We identified 16 proteins of possible algal origin in the ciliates Tetrahymena and Paramecium tetraurelia. Fourteen of these are present in other chromalveolates. Here we compare and contrast the likely scenarios for algal-gene origin in ciliates either via multiple rounds of horizontal gene transfer (HGT) from algal prey or symbionts, or through endosymbiotic gene transfer (EGT) during a putative photosynthetic phase in their evolution.     

The dynamic nature of eukaryotic genomes

Analyses of diverse eukaryotes reveal that genomes are dynamic, sometimes dramatically so. In numerous lineages across the eukaryotic tree of life, DNA content varies within individuals throughout life cycles and among individuals within species. Discovery of examples of genome dynamism is accelerating as genome sequences are completed from diverse eukaryotes. Though much is known about genomes in animals, fungi, and plants, these lineages represent only 3 of the 60-200 lineages of eukaryotes. Here, we discuss diverse genomic strategies in exemplar eukaryotic lineages, including numerous microbial eukaryotes, to reveal dramatic variation that challenges established views of genome evolution. For example, in the life cycle of some members of the "radiolaria," ploidy increases from haploid (N) to approximately 1,000N, whereas intrapopulation variability of the enteric parasite Entamoeba ranges from 4N to 40N. Variation has also been found within our own species, with substantial differences in both gene content and chromosome lengths between individuals. Data on the dynamic nature of genomes shift the perception of the genome from being fixed and characteristic of a species (typological) to plastic due to variation within and between species.     

REVIEW—CONTRASTING MITOCHONDRIAL GENOME ORGANIZATIONS AND SEQUENCE AFFILIATIONS AMONG GREEN ALGAE: POTENTIAL FACTORS, MECHANISMS, AND EVOLUTIONARY SCENARIOS

The three green algal mitochondrial genomes completely sequenced to date — those of Chlamydomonas reinhardtii Dangeard, Chlamydomonas eugametos Gerloff, and Prototheca wickerhamii Soneda & Tubaki — revealed very different mitochondrial genome organizations and sequence affiliations. The Chlamydomonas genomes resemble the ciliate / fungal / animal counterparts, and the Prototheca genome resembles land plant homologues. This review points out that all the green algal mitochondrial genomes examined to date resemble either the Chlamydomonas or the Prototheca mitochondrial genome; the Chlamydomonas- like mitochondrial genomes are small and have a reduced gene content (no ribosomal protein or 5S rRNA genes and only a few protein-coding and tRNA genes) and fragmented and scrambled rRNA coding regions, whereas the Prototheca- like mitochondrial genomes are larger and have a larger set of protein-coding genes (including ribosomal protein genes), more tRNA genes, and 5S rRNA and conventional continuous small-subunit (SSU) and large-subunit (LSU) rRNA coding regions. It appears, therefore, that the differences previously observed between the mitochondrial genomes of C. reinhardtii and P. wickerhamii extend to the two green algal mitochondrial lineages to which they belong and are significant enough to raise questions about the causes and mechanisms responsible for such contrasting evolutionary strategies among green algae. This review suggests an integrative approach in explaining the occurrence of distinct evolutionary strategies and apparent phylogenetic affiliations among the known green algal mitochondrial lineages. The observed differences could be the result of distinct genetic potentials differentiated during the previous evolutionary history of the flagellate ancestors and / or of subsequent changes in habitat and life history of the more advanced green algal lineages.     

Rapid diversification of mating systems in ciliates

Ciliates are a diverse group of microbial eukaryotes that exhibit tremendous variety in several aspects of their mating systems. To understand the evolutionary forces driving mating system diversification in ciliates, we use a comparative approach synthesizing data from many ciliate species in light of recent phylogenetic analyses. Specifically, we investigate the evolution of number of mating types, mode of mating type inheritance, and the molecular determinants of mating types across the taxonomic diversity of ciliates, with an emphasis on three well-studied genera: Tetrahymena , Paramecium , and Euplotes . We find that there have been many transitions in the number of mating types, and that the requirement of nuclear reorganization may be a more important factor than genetic exchange in determining the optimum number of mating types in a species. We also find that the molecular determinants of mating types and mode of inheritance are evolving under different constraints in different lineages of ciliates. Our results emphasize the need for further detailed examination of mating systems in understudied ciliate lineages.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 187–197.     

DAPI staining and DNA content estimation of nuclei in uncultivable microbial eukaryotes (Arcellinida and Ciliates)

Though representing a major component of eukaryotic biodiversity, many microbial eukaryotes remain poorly studied, including the focus of the present work, testate amoebae of the order Arcellinida (Amoebozoa) and non-model lineages of ciliates (Alveolata). In particular, knowledge of genome structures and changes in genome content over the often-complex life cycles of these lineages remains enigmatic. However, the limited available knowledge suggests that microbial eukaryotes have the potential to challenge our textbook views on eukaryotic genomes and genome evolution. In this study, we developed protocols for DAPI (4′,6-diamidino-2-phenylindole) staining of Arcellinida nuclei and adapted protocols for ciliates. In addition, image analysis software was used to estimate the DNA content in the nuclei of Arcellinida and ciliates, and the measurements of target organisms were compared to those  of well-known model organisms. The results demonstrate that the methods we have developed for nuclear staining in these lineages are effective and can be applied to other microbial eukaryotic groups by adjusting certain stages in the protocols.     

Genome gymnastics: unique modes of DNA evolution and processing in ciliates

In some ciliates, the DNA sequences of the germline genomes have been profoundly modified during evolution, providing unprecedented examples of germline DNA malleability. Although the significance of the modifications and malleability is unclear, they may reflect the evolution of mechanisms that facilitate evolution. Because of the modifications, these ciliates must perform remarkable feats of cutting, splicing, rearrangement and elimination of DNA sequences to convert the chromosomal DNA in the germline genome (micronuclear genome) into gene-sized DNA molecules in the somatic genome (macronuclear genome). How these manipulations of DNA are guided and carried out is largely unknown. However, the organization and manipulation of ciliate DNA sequences are new phenomena that expand a general appreciation for the flexibility of DNA in evolution and development.     

Macronuclear gene-sized molecules of hypotrichs.

The macronuclear genome of hypotrichous ciliates consists of DNA molecules of gene-sized length. A macronuclear DNA molecule contains a single coding region. We have analyzed the many hypotrich macronuclear DNA sequences sequenced by us and others. No highly conserved promoter sequences nor replication initiation sequences have been identified in the 5' nor in the 3' non-translated regions, suggesting that promoter function in hypotrichs may differ from other eukaryotes. The macronuclear genes are intron-poor; approximately 19% of the genes sequenced to date have one to three introns. Not all macronuclear DNA molecules may be transcribed; some macronuclear molecules may not have any coding function. Codon bias in hypotrichs is different in many respects from other ciliates and from other eukaryotes.     

Extremely intron-rich genes in the alveolate ancestors inferred with a flexible maximum-likelihood approach

Chromalveolates are a large, diverse supergroup of unicellulareukaryotes that includes Apicomplexa, dinoflagellates, ciliates(three lineages that form the alveolate branch), heterokonts,haptophytes, and cryptomonads (three lineages comprising thechromist branch). All sequenced genomes of chromalveolates haverelatively low intron density in protein-coding genes, and fewintron positions are shared between chromalveolate lineages.In contrast, genes of different chromalveolates share many intronpositions with orthologous genes from other eukaryotic supergroups,in particular, the intron-rich orthologs from animals and plants.Reconstruction of the history of intron gain and loss duringthe evolution of chromalveolates using a general and flexiblemaximum-likelihood approach indicates that genes of the ancestorsof chromalveolates and, particularly, alveolates had unexpectedlyhigh intron densities. It is estimated that the chromalveolateancestor had, approximately, two-third of the human intron density,whereas the intron density in the genes of the alveolate ancestoris estimated to be slightly greater than the human intron density.Accordingly, it is inferred that the evolution of chromalveolateswas dominated by intron loss. The conclusion that ancestralchromalveolate forms had high intron densities is unexpectedbecause all extant unicellular eukaryotes have relatively fewintrons and are thought to be unable to maintain numerous intronsdue to intense purifying selection in their, typically, largepopulations. It is suggested that, at early stages of evolution,chromalveolates went through major population bottlenecks thatwere accompanied by intron invasion.     

Eusociality Shapes Convergent Patterns of Molecular Evolution across Mitochondrial Genomes of Snapping Shrimps

Eusociality is a highly conspicuous and ecologically impactful behavioral syndrome that has evolved independently across multiple animal lineages. So far, comparative genomic analyses of advanced sociality have been mostly limited to insects. Here, we study the only clade of animals known to exhibit eusociality in the marine realm—lineages of socially diverse snapping shrimps in the genus Synalpheus. To investigate the molecular impact of sociality, we assembled the mitochondrial genomes of eight Synalpheus species that represent three independent origins of eusociality and analyzed patterns of molecular evolution in protein-coding genes. Synonymous substitution rates are lower and potential signals of relaxed purifying selection are higher in eusocial relative to noneusocial taxa. Our results suggest that mitochondrial genome evolution was shaped by eusociality-linked traits—extended generation times and reduced effective population sizes that are hallmarks of advanced animal societies. This is the first direct evidence of eusociality impacting genome evolution in marine taxa. Our results also strongly support the idea that eusociality can shape genome evolution through profound changes in life history and demography.     

A simple and rapid PCR-based method to isolate complete small macronuclear minichromosomes from hypotrich ciliates: 5S rDNA and S26 ribosomal protein gene of Oxytricha (Sterkiella) nova

Hypotrich ciliates present a macronuclear genome consisting of gene-sized instead of chromosome-sized DNA molecules. Exploiting this unique eukaryotic genome feature, we introduce, for the first time in ciliates, a rapid and easy PCR method using telomeric primers to isolate small complete macronuclear DNA molecules or minichromosomes. Two presumably abundant macronuclear DNA molecules, containing ribosomal genes, were amplified from the Oxytricha (Sterkiella) nova complete genome after using this method, and then were cloned and sequenced. The 5S rDNA sequence of O. (S.) nova is the third one reported among hypotrich ciliates; its primary and secondary structure is compared with other eukaryotic 5S rRNAs. The ribosomal protein S26 gene is the first one reported among ciliates. This "End-End-PCR" method might be useful to obtain similar gene-sized macronuclear molecules from other hypotrich ciliates, and, therefore, to increase our knowledge on ribosomal genes in these eukaryotic microorganisms.