A rapid sequencing method for ribosomal RNA was applied to the resolution of evolutionary relationships among Metazoa. Representatives of 22 classes in 10 animal phyla were used to infer phylogenetic relationships, based on evolutionary distances determined from pairwise comparisons of the 18S ribosomal RNA sequences. The classical Eumetazoa are divided into two groups. Cnidarians arose from a protist ancestry different from the second group, the Bilateria. Within the Bilateria, an early split gave rise to Platyhelminthes (flatworms) and the coelomate lineage. Coelomates are thus monophyletic, and they radiated rapidly into four groups:chordates, echinoderms, arthropods, and eucoelomate protostomes.

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Doilo's law, the concept that evolution is not substantively reversible, implies that the degradation ofgenetic information is sufficiently fast that genes or developmental pathways released from selective pressure will rapidly become nonfunctional. Using empirical data to assess the rate of loss of coding information in genes for proteins with varying degrees of tolerance to mutational change, we show that, in fact, there is a significant probability over evolutionary time scales of 0.5-6 million years for successful reactivation of silenced genes or "lost" developmental programs. Conversely, the reactivation of long (>10 million years)-unexpressed genes and dormant developmental pathways is not possible unless function is maintained by other selective constraints; the classic example of the resurrection of "hen's teeth" is most likely an experimental artifact, and the experimental reactivation of the Archaeopteryx limb developmental program has been shown to be a misinterpretation. For groups undergoing adaptive radiations, lost features may "flicker" on and off, resulting in a distribution of character states that does not reflect the phylogeny of the group.As the history of animal life was traced in the fossil record during the 19th century, it was observed that once an anatomical feature was lost in the course of evolution it never staged a return. This observation became canonized as Dollo's law, after its propounder, and is taken as a general statement that evolution is irreversible (reviewed in ref. Studies of pseudogenes show that, in silenced genes, single base changes will predominate early but frameshifts will eventually occur (6-10). We calculated the time course ofthe degradation of coding information by assuming that the rate of accumulation of nucleotide substitutions in unexpressed genes would occur at rates of neutral substitution and that rates of accumulation of frameshifts would be comparable to observed rates of insertion and deletion in processed pseudogenes.We have considered changes only in the exons because, for the most part, substitutions, insertions, and deletions in introns are not expected to affect the function of gene. Similarly, we have ignored the degradation of intron/exon splice recognition sites in our calculations because these sites represent a tiny fraction of the total length of the exons of a gene. Moreover, there is a degree of redundancy in splice sites (11).The probability that a gene retains a functional sequence is given by P(retain function) = P(survive point substitutions) X P(survive frameshifts) ) [I1 where P(survive point substitutions) is the probability that a substitution will not result in loss of function (f), raised to the number of substitutions incurred in a given period of time: P(survive point substitutions) = (f)rs,[2] where r. is the neutral substitution rate [substitutions per site per million years (Myr)], 1 is the combined length ofthe gene's exons (bp), and t is time (Myr). P(suruiv framcshifts) is the probability that a frameshift wi...

Abstract. We have tested the functional capacity of different beta tubulin isoforms in vivo by expressing B3-tubulin either in place of or in addition to/32-tubulin in the male germ line of Drosophila melanogaster. The testes-specific isoform, B2, is conserved relative to major metazoan beta tubulins, while the developmentally regulated isoform,/33, is considerably divergent in sequence. B3-tubulin is normally expressed in discrete subsets of cells at specific times during development, but is not expressed in the male germ line, /~2-Tubulin is normally expressed only in the postmitotic germ cells of the testis, and is required for all microtubule-based functions in these cells. The normal functions of B2-tubulin include assembly of meiotic spindles, axonemes, and at least two classes of cytoplasmic microtubules, including those associated with the differentiating mitochondrial derivatives. A hybrid gene was constructed in which 5' sequences from the/~2 gene were joined to protein coding and 3' sequences of the ~3 gene. Drosophila transformed with the hybrid gene express B3-tubulin in the postmitotic male germ cells. When exPressed in the absence of the normal testis isoform,/$3-tubulin supports assembly of one class of functional cytoplasmic microtubules. In such males the microtubules associated with the membranes of the mitochondrial derivatives are assembled and normal mitochondrial derivative elongation occurs, but axoneme assembly and other microtubule-mediated processes, including meiosis and nuclear shaping, do not occur. These data show that 133 tubulin can support only a subset of the multiple functions normally performed by/32, and also suggest that the microtubules associated with the mitochondrial derivatives mediate their elongation. When/33 is coexpressed in the male germ line with/32, at any level, spindles and all classes of cytoplasmic microtubules are assembled and function normally. However, when/33-tubulin exceeds 20% of the total testis beta tubulin pool, it acts in a dominant way to disrupt normal axoneme assembly. In the axonemes assembled in such males, the doublet tubules acquire some of the morphological characteristics of the singlet microtubules of the central pair and accessory tubules. These data therefore unambiguously demonstrate that the Drosophila beta tubulin isoforms/~2 and/~3 are not equivalent in intrinsic functional cai~acity, and furthermore show that assembly of the doublet tubules of the axoneme imposes different constraints on beta tubulin function than does assembly of singlet microtubules.

KAP plays an essential role in Kinesin II function, which is required for the axoneme growth and maintenance of the cilia in Drosophila type I sensory neurons. However, the flagellar assembly in Drosophila spermatids does not require Kinesin II and is independent of IFT.