Early Photosynthetic Eukaryotes Inhabited Low-Salinity Habitats The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Sánchez-Baracaldo, Patricia, John A. Raven, Davide Pisani, and Andrew H. Knoll. 2017. Early Photosynthetic Eukaryotes Inhabited Low-salinity Habitats. Proceedings of the National Academy of Sciences of the United States of America 114, no. 37: E7737-E7745. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41048975 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA 1 69 2 Early photosynthetic eukaryotes inhabited low salinity 70 3 71 4 habitats 72 5 73 6 Patricia Sánchez-Baracaldo1, John Raven2, Davide Pisani1, Andrew Knoll3 74 7 75 8 1University of Bristol, 2University of Dundee, UK, 3Harvard University 76 9 Submitted to Proceedings of the National Academy of Sciences of the United States of America 77 10 78 11 The early evolutionary history of the chloroplast lineage remains 79 12 an open question. It is widely accepted that the endosymbiosis same time, the ecological setting in which this endosymbiotic 80 13 which established the chloroplast lineage in eukaryotes can be event occurred has not been fully explored (8), partly due to 81 14 traced back to a single event in which a cyanobacterium was phylogenetic uncertainties and preservational biases of the fossil 82 15 incorporated into a protistan host. It is still unclear, however, record. Phylogenomics and trait evolution analysis have pointed 83 16 which cyanobacteria are most closely related to the chloroplast, to a freshwater origin for cyanobacteria (9-11), providing a novel 84 17 when the plastid lineage first evolved, and in what habitats approach to address the early diversification of terrestrial biota 85 18 this endosymbiotic event occurred. We present phylogenomic and for which the fossil record is poor or uncertain. 86 19 molecular clock analyses, including data from cyanobacterial and The earliest widely accepted fossil evidence of photosynthetic 87 20 chloroplast genomes using a Bayesian approach, with the aim eukaryotes is Bangiomorpha, a red alga deposited ∼1.1 Bya (12). 88 21 of estimating the age for the primary endosymbiotic event, the However, recent reports of multicellular photosynthetic eukary- 89 22 ages of crown groups for photosynthetic eukaryotes and the otes at ∼1.6 Bya provide evidence for an earlier establishment of 90 23 independent incorporation of a cyanobacterial endosymbiont by photosynthesis within the eukaryotes (13). Currently, the oldest 91 24 Paulinella. Our analyses include both broad taxon-sampling (119 reliable evidence for eukaryotes as a whole is found in ∼1.7 92 25 taxa) and eighteen fossil calibrations across all cyanobacteria and Bya rocks (14). These cyst-like microfossils occur in low diver- 93 26 photosynthetic eukaryotes.Submission Phylogenomic analyses support the sity assemblages PDF that potentially include stem group eukaryotes 94 27 hypothesis that the chloroplast lineage diverged from its closet or stem representatives of extant major taxa (14,17). Sterane 95 28 relative, Gloeomargarita, a basal cyanobacterial lineage, ∼2.1 bil- biomarkers originally viewed as evidence for 2.7 Ga eukaryotes 96 29 lion years ago (Bya). Our analyses suggest that the Archaeplastida, have now been reinterpreted as younger contaminants (15, 16). 97 30 consisting of glaucophytes, red algae, green algae and land plants, Only around 750-800 Mya do fossils show a major increase in 98 31 share a common ancestor that lived ∼1.9 Bya. Whereas crown eukaryotic diversity that includes recognizable green algae (e.g., 99 32 group Rhodophyta evolved in the Mesoproterozoic Era (1600-1000 Cladophorales) (14, 17, 18), radiations possibly related to the 100 33 million years ago, Mya), crown group Chlorophyta and Strepto- evolution of eukaryovores – eukaryotes that eat other eukaryotes 101 34 phyta began to radiate early in the Neoproterozoic (1000–542 (19). 102 35 Mya). Stochastic mapping analyses indicate that the first endosym- Reconstructing and dating the evolutionary history of early 103 36 biotic event occurred in low salinity environments. Both red and photosynthetic eukaryotes has proven challenging. Most phy- 104 37 green algae colonized marine environments early in their histories, logenetic studies place the divergence of the chloroplast lin- 105 38 with prasinophyte green phytoplankton diversifying 850-650 Mya. eage near the root of cyanobacteria (20-23), although a few 106 39 studies insert chloroplasts higher in the tree (8) or nest them 107 40 Photosynthetic eukaryotes j chloroplast j cyanobacteria j phyloge- within derived clades (e.g., Nostocales (24)). Piecing together 108 41 nomics j relaxed molecular clock 109 42 110 43 Introduction 111 44 Significance 112 45 Life as we know it would not be possible without oxygenic pho- 113 46 tosynthesis. Cyanobacteria were the only prokaryotes to evolve While it is widely accepted that the chrloroplasts in photo- 114 this metabolism, fundamentally changing redox chemistry early in synthetic eukaryotes can be traced back to a single cyanobac- 47 terial ancestor, the nature of that ancestor remains debated. 115 48 Earth history (1, 2). Cyanobacteria also had a huge impact on the Chloroplasts have been proposed to derive from either early- 116 49 biological diversity of Earth’s ecosystems, partly due to their abil- or late-branching cyanobacterial lineages, and similarly the 117 50 ity to establish symbiotic relationships with a number of different timing and ecological setting of this event remains uncertain. 118 hosts (3-6). Photosynthesis in eukaryotic organisms stems from Phylogenomic and Bayesian relaxed molecular clock analyses 51 show that the chloroplast lineage branched deep within the 119 52 two primary endosymbiotic events involving a cyanobacterium cyanobacterial tree of life ∼2.1 billion years ago (Bya), and 120 53 engulfed by a protistan host. The older of these events gave rise ancestral trait reconstruction places this event in low salinity 121 54 to the Archaeplastida, a monophyletic group that includes the environments. The chloroplast took another 200 million years 122 Glaucocystophyta (glaucophytes), Rhodophyta (red algae) and to become established, with most extant groups originating 55 much later. Our analyses help to illuminate the little known 123 56 Viridiplantae (green algae and land plants). In turn, secondary evolutionary history of early life on land. 124 57 endosymbioses involving archaeplastid lineages (red or green al- 125 58 gae) spread photosynthesis to the haptophytes, cryptophytes, eu- Reserved for Publication Footnotes 126 59 glenids, chlorarachniophyte rhizarians, dinoflagellates, chromer- 127 60 ans, and stramenopiles. A second primary endosymbiotic event 128 61 established photosynthesis within the rhizarian genus Paulinella. 129 62 As primary producers, photosynthetic eukaryotes now dominate 130 63 most terrestrial (e.g., embryophytes and green algae) and marine 131 64 (e.g., diatoms, mixotrophic dinoflagellates and coccolithophores) 132 65 environments. The timing of the first endosymbiotic event and 133 66 ensuing divergence dates for the three major archaeplastidan 134 67 lineages are still debated, with molecular clock estimates for 135 68 the origin of plastids ranging over 800 million years (7). At the 136 www.pnas.org --- --- PNAS Issue Date Volume Issue Number 1--?? 137 205 138 206 139 207 140 208 141 209 142 210 143 211 144 212 145 213 146 214 147 215 148 216 149 217 150 218 151 219 152 220 153 221 154 222 155 223 156 224 157 225 158 226 159 227 160 228 161 229 162 Submission PDF 230 163 231 164 232 165 233 166 234 167 235 168 236 169 237 170 238 171 239 172 Fig. 1. The origin and diversification of photosynthetic eukaryotes and cyanobacteria as inferred from geologic time. The phylogenetic tree shown was 240 173 estimated based on twenty-six genes from 117 taxa implementing Phylobayes 1.7a (96). Bayesian relaxed molecular clock analyses were carried out in 241 174 Phylobayes 4.1 (39) implementing the UGAM (42) and the CAT-GTR substitution model (Table 2). Five calibration points for cyanobacteria and 13 calibrations 242 175 points for photosynthetic eukaryotes (brown circles) were used (Table 1) for the tree shown and were treated as soft bounds. The root of the tree was set 243 176 with a maximum age of 2.7 Bya (97) and a minimum age of 2.32 Bya (2). Age estimates for the numbered nodes (1–9) indicated are given in Table 1, which 244 177 includes the corresponding values for the posterior 95% confidence intervals. 245 178 246 179 chloroplast genomes have undergone a dramatic reduction in 247 180 size compared to their cyanobacterial relatives (25, 26). Here, we 248 181 have implemented a phylogenomic approach to study the early 249 182 evolutionary history of photosynthetic eukaryotes in the context 250 183 of cyanobacterial evolution. Genomic data were used to carry out 251 184 large-scale multi-gene analyses of cyanobacteria and photosyn- 252 185 thetic eukaryotes. Molecular clock analyses provide new evidence 253 186 indicating when the chloroplast lineage and Paulinella diverged 254 187 from their closest cyanobacterial relatives. A Bayesian approach 255 188 offers insights into the habitat in which the first endosymbiotic 256 189 event took place during the Proterozoic Eon. 257 190 258 191 Results 259 192 260 Phylogenomic analyses 193 261 194 Two data sets were analyzed: a genomic dataset including 262 195 135 highly conserved proteins (9) compiled from a total of 49 263 196 cyanobacterial genomes, and a second dataset including twenty- 264 197 six genes comprising 119 taxa that include both cyanobacteria 265 198 and photosynthetic eukaryotes. The first dataset was analysed 266 199 using Maximum Likelihood (ML) in a two-step process: (1) the 49 267 200 Fig.