Fossil calibrations from Yoon et al. (2004), Berney & Pawlowski (2006): MYA for Bangiomorpha (red alga) MYA for Florideophyte red algae MYA for first land plants MYA for first seed plants MYA for angiosperm vs. gymnosperm split MYA min. for oldest known Nymphaeales MYA for monocot vs. dicot split MYA min. for Proterocladus (Cladophoraceae) Estimation of divergence times in the volvocine algae Matthew D. Herron, Jeremiah D. Hackett, Frank Aylward, and Richard E. Michod Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, USA The volvocine green algae (Volvox and its close relatives) are an important model system for the evolution of multicellularity and cellular differentiation. The evolutionary transition from single cells to completely differentiated multicellular individuals can be describe as a series of small steps, many represented in extant volvocine algae (Kirk 2005). Reconstructing the history of these changes allows us to describe an increase in the level of complexity within a gradualist framework. Rausch et al. (1989) estimated the divergence of colonial volvocine algae from unicellular relatives at MYA using nuclear genes; these estimates assume a constant substitution rate across lineages. We used multiple genes with multiple fossil calibrations to estimate divergence times among unicellular and colonial volvocine algae. Figure 1. Chronogram of red algae, green algae and land plants based on three chloroplast genes (psaA, psaB, and psbC) and one nuclear gene (SSU) using LF. Gray boxes are ranges of estimates across the sample of 300 posterior trees. Outlined boxes are fossil calibrations from Yoon et al. (2004); gray boxes for these nodes are ranges inferred during fossil cross-validation. Bayesian posterior probabilities below 1.0 are shown. Phylogenetic reconstruction of red algae, green algae, and land plants using Bayesian Markov Chain Monte Carlo methods in MrBayes. Three chloroplast genes: P700 chlorophyll a-apoprotein A1 (psaA), P700 chlorophyll a- apoprotein A2 (psaB), and photosystem II CP43 apoprotein (psbC) partitioned by codon. One nuclear gene: small ribosomal subunit (SSU). Divergence times estimated using Langley-Fitch (local molecular clock method; LF), non- parametric rate smoothing (NPRS), and penalized likelihood (PL) in r8s (Figure 1). Sample of 300 posterior trees. Multiple fossil calibrations (see inset, Figure 1). Analyses evaluated by fossil cross-validation. Divergences within the volvocine algae estimated using LF (Figure 2). Sample of 300 posterior trees from the five chloroplast gene analysis of Herron & Michod (2008). Beta subunit of ATP synthase (atpB), large subunit of Rubisco (rbcL), psaA, psaB, and psbC, partitioned by codon. INTRODUCTIONMATERIALS AND METHODS RESULTS CONCLUSIONS Figure 2. Chronogram of volvocine algae based on five chloroplast genes using LF, calibrated with the divergence between Paulschulzia (Tetrasporales) and Volvox carteri derived from analysis in Figure 1. Gray boxes are ranges of estimates across the sample of 300 posterior trees. Bayesian posterior probabilities below 1.0 are shown. * It is unclear if the MRCA of V. aureus and V. carteri had specialized germ cells. The colonial volvocine algae (Tetrabaenaceae, Goniaceae, and Volvocaceae) diverged from unicellular relatives at least 200 MYA. An early, rapid radiation established all of the major colonial lineages (Tetrabaenaceae; Astrephomene; Gonium; Euvolvox; Pandorina + Volvulina; Yamagishiella; Eudorina + Pleodorina + remaining Volvox) by 150 MYA. Most colonial body plans are not recent innovations but ancient adaptations that have been stable over a time scale of tens of millions of years. Several nominal species include divergences over 50 MYA (Astrephomene gubernaculifera, Gonium multicoccum, Volvulina steinii, Pandorina morum, Yamagishiella unicocca, Eudorina elegans) A body plan similar to that of modern Eudorina, with complete inversion and a large volume of extracellular matrix (ECM) was present at least 180 MYA. Sterile somatic cells originated at least 30 MYA in Euvolvox (V. globator + V. barberi + V. rousseletii), at least 80 MYA in the clade including the remaining Volvox and Pleodorina, and at least 125 MYA in Astrephomene. BIBLIOGRAPHY Berney, C. and Pawlowski, J A molecular time-scale for eukaryote evolution recalibrated with the continuous microfossil record. Proc. R. Soc. B 273: Herron, M. D. and R. E. Michod Evolution of complexity in the volvocine algae: transitions in individuality through Darwin's eye. Evolution 62: Kirk, D. L A twelve-step program for evolving multicellularity and a division of labor. BioEssays 27: Rausch, H., N. Larsen, and R. Schmitt Phylogenetic relationships of the green alga Volvox carteri deduced from small-subunit ribosomal RNA comparisons. J. Mol. Evol. 29: Sanderson M. J r8s ("rates"), version 0.9. Software for the analysis of rates of evolution and miscellaneous other stuff. University of California, Davis, California, USA Yoon, H. S., J. D. Hackett, C. Ciniglia, G. Pinto, and D. Bhattacharya A molecular timeline for the origin of photosynthetic eukaryotes. Mol. Biol. Evol. 21: Gains and losses of characters defined by Kirk (2005): 1. 1.Incomplete cytokinesis 2. 2.Partial inversion 3. 3.Rotation of basal bodies 4. 4.Organismal polarity 5. 5.Transformation of cell wall into ECM 6. 6.Genetic control of cell number 7. 7.Complete inversion 8. 8.Increased volume of ECM 9. 9.Sterile somatic cells Specialized germ cells * Asymmetric division Bifurcated cell division program # # Volvocaceae Goniacaceae Tetrabaenaceae Chlorophyta Streptophyta Rhodophyta Euvolvox