Snowball Earths, population bottlenecks, and the evolution of marine photosynthetic bacteria

Prochlorococcus are the most abundant photosynthetic organisms in the modern ocean. A massive DNA loss event occurred in their early evolutionary history, leading to highly reduced genomes in nearly all lineages, as well as enhanced efficiency in both nutrient uptake and light absorption. The environmental landscape that shaped this ancient genome reduction, however, remained unknown. Through careful molecular clock analyses, we established that this Prochlorococcus genome reduction occurred during the Neoproterozoic Snowball Earth climate catastrophe. The lethally low temperature and exceedingly dim light during the Snowball Earth event would have inhibited Prochlorococcus growth and proliferation and caused severe population bottlenecks. These bottlenecks are recorded as an excess of deleterious mutations that accumulated across genomic regions in the descendant lineages. Prochlorococcus adaptation to extreme environmental conditions during Snowball Earth intervals can be inferred by tracing the evolutionary paths of genes that encode key metabolic potential. This metabolic potential includes modified lipopolysaccharide structure, strengthened peptidoglycan biosynthesis, the replacement of a sophisticated circadian clock with an hourglass-like mechanism that resets daily for dim light adaption, and the adoption of ammonia diffusion as an efficient membrane transporter-independent mode of nitrogen acquisition. In this way, the Neoproterozoic Snowball Earth event altered the physiological characters of Prochlorococcus, shaping their ecologically vital role as the most abundant primary producers in the modern oceans. Significance Statement Prochlorococcus are the most abundant photosynthetic organisms in the modern ocean, where they support much of the marine and global biological productivity. In this study, we reconstructed a precise timeline of Prochlorococcus evolution and discovered that a major Prochlorococcus genome reduction took place during the Neoproterozoic Snowball Earth events. Since Prochlorococcus are generally adapted to warm waters, the extremely cold conditions during the Snowball Earth drove population bottlenecks, which left strong signatures in genomic DNA through the accumulation of detrimental mutations. The reconstruction of ancestral genome content further unveiled the metabolic strategies that Prochlorococcus evolved to overcome the extreme conditions during the climate catastrophe. These findings show how large changes in Earth’s past climate have left genomic imprints on extant microorganisms, likely shaping their metabolic potential and ecological role in the oceans today.

analyses, we established that this Prochlorococcus genome reduction occurred during the  pigments, light-harvesting systems, and the phycobiliproteins, which allowed for efficient 59 light absorption in the water column (4), and thus increased growth rates and primary 60 production (5). 61 Prochlorococcus genomes have been shaped by stepwise streamlining, including a 62 major genome reduction in their early evolution and a few minor modifications that 63 followed (6, 7). Modern marine Prochlorococcus lineages, in particular those with small 64 genomes, show very low ratios of nonsynonymous (dN) to synonymous (dS) nucleotide 65 substitution rates, suggesting that natural selection is a highly efficient throttle on the 66 accumulation of deleterious mutations (i.e., nonsynonymous mutations) in 67 Prochlorococcus (6, 8). On long time scales, however, nucleotide substitutions at 68 synonymous sites become saturated, invalidating the use of dN/dS to infer selection 69 efficiency in deep time (9). Thus, an alternative approach focuses instead on different 70 types of nonsynonymous substitutions leading to radical versus conservative changes in 71 amino acid sequences, with the former more likely to be deleterious (10,11). Excess 72 radical mutations accumulate from random fixation of deleterious mutations by genetic 73 5 drift (i.e., reduced efficiency of selection). Using this approach reveals that the major 74 genome reduction in Prochlorococcus took place under reduced selection efficiency (9) 75 and implies that the ancient population went through severe bottlenecks as the likely 76 result of environmental catastrophe.

77
The environmental context underlying Prochlorococcus genome reduction remains 78 unknown, however, and precise molecular dating is needed to link this important 79 evolutionary event to its possible environmental drivers. Implementing comprehensive 80 molecular clock analyses, we now link the early major genome reduction event of

91
Prochlorococcus experienced a massive gene loss event on the ancestral branch 92 leading to the last common ancestor (LCA) of clades HL, LLI and LLII/III (6,7,13

146
Among these stresses, the most prominent was likely lethally low temperature.

147
Maintaining membrane fluidity is of paramount importance under low temperature 148 conditions, which is largely achieved by the activities of fatty acid desaturase encoded by 149 desA and desC. As a result, we inferred that these genes were retained in SBE-LCA ( consequence, their circadian clocks rather behave like an "hourglass" which is reset every 232 morning (43-45). Our analysis indicated that kaiA was lost at SBE-LCA (Fig. 1A). This is 233 likely due to the prolonged darkness or low light conditions during the Snowball Earth, 234 rendering the sophisticated circadian clock dispensable. 235 We argue that the genome reduction and metabolic adaptation events discussed

293
Genomic sequences of Cyanobacteria were downloaded from public databases and 294 manually annotated (Table S2; (Table S3) 296 previously proposed to be valuable to date bacterial divergence (56)   60). Our analysis showed that this controversy is likely caused by the inclusion of 301 composition-heterogeneous proteins, and that using composition-homogeneous proteins 302 led to consistent support for the former hypothesis ( Fig. S4; Table S4; see Section 2.2 in 303 Supplemental Methods). Since molecular dating analysis is known to be intrinsically 304 associated with calibration points (61), we adapted multiple calibration sets from 305 previous cyanobacterial studies for the purpose of comparison (Table S1; (Table S5).