Molecular clocks have the potential to shed light on the timing of early metazoan divergences, but differing algorithms and calibration points yield conspicuously discordant results. We argue here that competing molecular clock hypotheses should be testable in the fossil record, on the principle that fundamentally new grades of animal organization will have ecosystem-wide impacts. Using a set of seven nuclear-encoded protein sequences, we demonstrate the paraphyly of Porifera and calculate sponge͞eumetazoan and cnidarian͞bilaterian divergence times by using both distance [minimum evolution (ME)] and maximum likelihood (ML) molecular clocks; ME brackets the appearance of Eumetazoa between 634 and 604 Ma, whereas ML suggests it was between 867 and 748 Ma. Significantly, the ME, but not the ML, estimate is coincident with a major regime change in the Proterozoic acritarch record, including: (i) disappearance of low-diversity, evolutionarily static, pre-Ediacaran acanthomorphs; (ii) radiation of the high-diversity, short-lived Doushantuo-Pertatataka microbiota; and (iii) an orderof-magnitude increase in evolutionary turnover rate. We interpret this turnover as a consequence of the novel ecological challenges accompanying the evolution of the eumetazoan nervous system and gut. Thus, the more readily preserved microfossil record provides positive evidence for the absence of pre-Ediacaran eumetazoans and strongly supports the veracity, and therefore more general application, of the ME molecular clock.Porifera ͉ acritarchs ͉ Ediacaran ͉ coevolution T he sudden appearance of diverse metazoan fossils Ϸ530 million years ago (Ma) is the focus of ongoing and heated debate: Is this recording a true ''Cambrian explosion'' of early metazoan evolution or merely the onset of extensive burrowing and biomineralization (1)? Certainly the invisibility of microscopic, nonburrowing, and͞or nonbiomineralizing metazoans in the fossil record allows for the possibility of deep Proterozoic origins, so independent lines of evidence must be sought. Molecular clocks offer a potentially powerful approach for testing such evolutionary hypotheses (2), but recent analyses have yielded conspicuously discordant results. Pisani et al. (3), for example, estimate that the protostome-deuterostome ancestor evolved 900-1,100 Ma, whereas Douzery et al. (4) place this node at 642-761 Ma and Peterson et al. (5) at Ϸ570 Ma. With estimated divergence times differing by Ͼ500 million years (myr), there is clearly a need to assess both the methods used and predictions made by individual molecular-clocks analyses.Pisani et al. (3) have argued that their deep estimate for metazoan origins is robust because it agrees with a previous analysis calibrated by using different taxa, i.e., between chick and mouse (6) vs. centipedes and millipedes and spiders and horseshoe crabs (3). However, Peterson et al. (5) demonstrated that a significant rate reduction is associated with the vertebrate sequences, such that a vertebrate-calibrated clock produces a spurious 2-fold ove...