A molecular timescale of eukaryote evolution and the rise of complex multicellular life

“Sequences were aligned [ 36 ] and regions of ambiguous alignment were removed when necessary.”

“Gene-specific and supergene gamma shape parameters (α) were calculated [40] and used for distance and time estimation [45].”

“These concatenated datasets were analyzed using maximum likelihood (JTT + gamma model, quartet puzzling with 1000 steps) [38], minimum evolution (Neighbor-joining, Poisson + gamma model, 2000 bootstraps, complete deletion) [37], and Bayesian Inference (JTT + gamma model, 50,000 generations, 4 chains with starting temp…”

“The SGLPL method was performed in R8S version 1.6 [48] with maximum likelihood branch lengths calculated under a PC+gamma model [40].”

“Because the coefficient of variation of time estimates is large for small numbers of genes [14], we used a minimum of 20 genes for each divergence.”

“6 [48] with maximum likelihood branch lengths calculated under a PC+gamma model [40].”

“We chose eighteen divergences among major lineages of eukaryotes, including some analyzed previously [24].”

“Organisms with more than 2–3 cell types (the maximum in prokaryotes) appeared relatively early (~2000 Ma), soon after the surface environment became oxygenated at 2300 Ma (Great Oxidation Event; [34]).”

“All proteins were tested for rate constancy [45, 46 ]; those rejected at the 5% significance level were excluded from timing analyses.”

“The origin of life and divergence of archaebacteria and eubacteria were set at 4000 Ma and the origin of eukaryotes at 2700 Ma [27,28], although earlier values for those events would not affect the overall trend, showing a baseline of about 2 cell types in prokaryotes.”

“We used a consensus phylogenetic framework based on a diversity of molecular and morphological studies [21,28].”

“The SGLDT method was performed using Divtime5b [15]; maximum likelihood branch lengths were calculated under a JTT model using an accompanying program, ESTBRANCHES.”

“The most frequently used measure of organismal complexity has been the number of cell types [1,2,32].”

“Molecular clocks have yielded earlier times for the origin of animal phyla [6-9], but the methods have received criticism [10,11].”

“These dates were similar to previous estimates using fewer proteins and different methods [8], and as secondary calibration points were found to be consistent in tests of reciprocity (see next section).”

“The maximum numbers of cell types in major groups of living organisms were obtained from the literature [13,50]: Mammalia (120), Reptilia (120), Amphibia (120), Actinopterygii (120), Arthropoda (69), Agnatha (67), vascular plants (44), mosses (26), Cnidaria (22), Porifera (16), red algae (14), alveolates (14), Pyrenom…”

“The origin of life and divergence of archaebacteria and eubacteria were set at 4000 Ma and the origin of eukaryotes at 2700 Ma [27,28], although earlier values for those events would not affect the overall trend.”

“Prokaryotes and many unicellular eukaryotes have only one or a few cell types, but vertebrates have more than 100 [1].”

“Molecular clocks have yielded earlier times for the origin of animal phyla [6-9], but the methods have received criticism [10,11].”

“Our results with nuclear proteins agree with earlier ribosomal RNA trees [22] in supporting a root near the excavates (e.g., diplomonads) rather than on the opisthokont-amoebozoan branch (e.g., animals, fungi, and amoebas) [ 23 ].”

“To avoid any potential artifacts arising from analysis of multiple alignments [17,18], we have also used concatenated datasets [19].”

“The most frequently used measure of organismal complexity has been the number of cell types [1,2,32].”

“Background Organismal complexity can be defined in many ways, although the most common measure is the number of cell types [1-4].”

“To avoid any potential artifacts arising from analysis of multiple alignments [17,18], we have also used concatenated datasets [19].”

“Times of divergence derived from the fossil record are always underestimates of the true divergence [11,41].”

“A molecular clock study of prokaryote and eukaryote genomes [35] arrived at a similar date (1840 ± 200 Ma) for the symbiotic event leading to the mitochondrion, using different data, methods, and approach.”

“The constraints were Drosophila-Anopheles (250 Ma), Homo-Mus (65 Ma), vertebrate-arthropod (540 Ma), Saccharomyces-Shizosaccharomyces (400 Ma) and animal-plant (1200 Ma) [12,25,26].”

“size, genome size) or do not yet have sufficient data available from a diversity of eukaryotes (e.g., number of genes, proteins, transcription factors, introns/exons) for this analysis [32, 33 ].”

“Molecular clocks have yielded earlier times for the origin of animal phyla [ 6-9 ], but the methods have received criticism [10,11].”

“For some nodes, linear parsimony yields a range of values; in those cases we followed Webster and Purvis [52] in using the midpoint of the range.”

“To avoid any potential artifacts arising from analysis of multiple alignments [17,18], we have also used concatenated datasets [19].”

“A more conservative estimate of 288 Ma [42] was used as the lower bound for the mammal-bird divergence in the Bayesian and penalized likelihood analyses; the upper bound was defined by the presence of stem amniotes in the Mid-Late Visean (~345 Ma) [43].”

“Our results with nuclear proteins agree with earlier ribosomal RNA trees [22] in supporting a root near the excavates (e.”

“Molecular clocks have yielded earlier times for the origin of animal phyla [ 6-9 ], but the methods have received criticism [10,11].”

“We have tested our calibrations for reciprocity [20] and have used both vertebrate and non-vertebrate fossil calibrations and constraints.”

“This would argue against the interpretation of the older (>1600 Ma) fossils of "Grypania" as photosynthetic eukaryotic algae [30] and supports their interpretation as colonial prokaryotes [ 31 ].”

“The mode was used as the measure of central tendency in the multigene analyses due to the sensitivity of the mean to extreme values [47]; standard errors of the mode were obtained with bootstrapping (10,000 replications); outliers were trimmed for the supergene data sets.”

“Times of divergence derived from the fossil record are always underestimates of the true divergence [11,41].”