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Some species of ciliates undergo massive DNA elimination and genome rearrangement to construct gene-sized ''chromosomes'' in their somatic nucleus. An example is the extensively scrambled DNA polymerase ␣ gene that is broken into 48 pieces and distributed over two unlinked loci in Stylonychia. To understand the emergence of this complex phenomenon during evolution, we examined DNA polymerase ␣ genes in several earlier diverging species, representing evolutionary intermediates. Mapping these data onto an evolutionary tree suggests that this gene became extensively fragmented and scrambled over evolutionary time through a series of steps, each leading to greater complexity. Our results also suggest a possible mechanism for intron loss by deletion of intron sequences as DNA during development of the somatic nucleus.ciliate ͉ DNA polymerase ͉ hypotrich ͉ intron loss ͉ spirotrich C iliates comprise a diverse group of microbial eukaryotes, or protists. These organisms contain two types of nuclei: a somatic macronucleus that is active during vegetative growth and a germ-line micronucleus that is quiescent except during sexual recombination (1). During sexual conjugation, the two mating cells exchange haploid micronuclei, after which the old macronucleus degrades, and the new diploid micronucleus develops into the new macronucleus. Extensive loss of DNA from the long micronuclear chromosomes, coupled to DNA rearrangement, removes transposons and most intergenic spacer DNA between genes, as well as intragenic spacer DNA, known as internal eliminated segments (IESs). In spirotrichous (formerly hypotrichous) ciliates, the remaining DNA sequences, known as macronuclear destined segments (MDSs), can occupy as little as 2-5% of the germ-line genome. These mostly coding regions assemble together to form Ϸ24,000 types of DNA molecules in the somatic nucleus. Because their average size is just 2.2 kbp and they usually contain only one gene, they are sometimes called ''nano-chromosomes.'' Short telomeres are added to both ends before these molecules are amplified several-thousand-fold (1-3).Furthermore, the germ-line order of the coding regions in 20-30% of the genes in stichotrichous ciliates (a subset of spirotrichous ciliates) are permuted, or scrambled, relative to their linear order in the macronucleus. Although the mechanism of reordering these segments is not well understood, short direct repeats, called pointers, may guide the unscrambling process (1, 4). Invariably, one copy of a repeated word pair is present at the 3Ј end of germ-line segment n and at the 5Ј end of segment n ϩ 1, providing the information to link them together, with only one copy of the repeated word retained in the final DNA molecule (1). Although fragmented genes do occur in other organisms [examples include V(D)J recombination in the vertebrate immune system and split ribosomal RNA genes in the mitochondria of fungi (5) and protists (6, 7)], rarely are they sewn back together at the DNA level.Three different scrambled genes, actin I (8-10), ␣ telomer...

In order to re-evaluate the systematics of sessilid peritrich ciliates, small subunit (SSU) rRNA gene sequences were determined for 12 species belonging to five genera: Vorticella, Pseudovorticella, Epicarchesium, Zoothamnium, and Zoothamnopsis. Phylogenetic trees were deduced using Bayesian inference, maximum parsimony, and maximum likelihood methods. The phylogenetic analyses suggest that (1) sessilids which have stalks with continuous myonemes that contract in a zig-zag fashion form a separate clade from those which have stalks that contract independently and in a spiral fashion, supporting the separation of the family Zoothamniidae from the family Vorticellidae and (2) Epicarchesium and Pseudovorticella, both of which have reticulate silverline systems, are more closely related to each other than to other vorticellids, suggesting that differences in the silverline system (i.e. transverse vs. reticulate) may be the result of genuine evolutionary divergence among sessilid peritrichs. However, the newly sequenced Zoothamnopsis sinica, which has a reticulate silverline pattern, nests within the unresolved Zoothamnium species that have transverse silverline patterns. Thus, there were at least two evolutions of the reticulate silverline pattern character state from a plesiomorphic transverse state in the peritrichid ciliates. The molecular work demonstrates the genus Zoothamnium to be paraphyletic in relation to morphological studies, and suggests that Astylozoon, Opisthonecta, and Vorticella microstoma possibly share a SSU rRNA secondary structure in the helix E10-1 region.

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