Photo by Sean Davies- Surfer Magazine

1. One point of view on prokaryotic evolution prokaryotic diversity eukaryotic diversity Photo by Sean Davies- Surfer Magazine

2. How to count the number of prokaryotes on earth? Because prokaryotes are everywhere, it might seem impossible to count all of them. Fortunately, most of the earth’s prokaryotes are found in a few large habitats. Whitman, W.B., D.C. Coleman, and W.J. Wiebe (1998) Proc. Natl. Acad. Sci. USA 95: 6578-6583.

3. Numbers of prokaryotes in various habitats aAverage of range of 0.25-2.5 x 1030

4. Number and biomass (carbon) of prokaryotes in the world aPg = 1015 g. For aquatic, subsurface, and soil habitats, prokaryotic cells were estimated to contain 10-20, 86, and 100 fg C, respectively.

5. Relationship of plant and prokaryotic biomass to primary productivity

6. If the abundance of prokaryotes is high today, what has it been like in the past? Geochemical evidence suggests that the total amount of organic carbon on earth has remained unchanged for billions of years. If this is true, it is likely that the biomass has also been about the same.

7. Stable isotope ratio is evidence of the source of carbon: δ13C = [(13C/12C)sam/ (13C/12C)std-1] x 1000 [o/oo,PDB] 13C:12C ~ 1:89 Organic carbon is typically depleted in 13C: δ13C = -25 o/oo Inorganic carbon is the standard: δ13C = 0 o/oo

8. Where is the carbon on earth? Mostly in rocks. Of the carbon in the biosphere, 80 % is inorganic carbonate, 20 % is organic carbon in soils, sediments, and biomass. Organic C δ13C = -25 Inorganic C δ13C = 0 o/oo Weighed average δ13C = -6 o/oo Total carbon in biosphere ~7.5 x 1022 g

9. If the abundance of prokaryotes is high today, what has it been like in the past? Geochemical evidence suggests that the total fraction of organic carbon on earth has remained unchanged for billions of years From Schidlowski et al., 1983

10. If the abundance of prokaryotes is high today, what has it been like in the past? Modern organic carbon is depleted in 13C relative to modern carbonates carbon, and modern carbonates are enriched in 13C. However, the δ13C of the organic carbon is less stable than that of the carbonate. average d13 of carbonates average d13 of earth’s carbon average d13 of organic carbon From Schidlowski et al., 1983

11. If the abundance of prokaryotes is high today, what has it been like in the past? average d13 of carbonates The enrichment of ancient carbonates in 13C is a measure of the fraction of the total biosphere carbon that is in organic carbon and hence an indirect measure of biomass. average d13 of earth’s carbon From Schidlowski et al., 1983

12. If the abundance of prokaryotes is high today, what has it been like in the past? first land plants Ancient carbonates are enriched in 13C, suggesting that the biomass of the earth has remained unchanged over geological time scales and predating the origin of most modern life forms. From Schidlowski et al., 1983

13. If the abundance of prokaryotes is high today, what has it been like in the past? first metazoa Ancient carbonates are enriched in 13C, suggesting that the biomass of the earth has remained unchanged over geological time scales and predating the origin of most modern life forms. From Schidlowski et al., 1983

14. If the abundance of prokaryotes is high today, what has it been like in the past? first multicellular organisms Ancient carbonates are enriched in 13C, suggesting that the biomass of the earth has remained unchanged over geological time scales and predating the origin of most modern life forms. From Schidlowski et al., 1983

15. If the abundance of prokaryotes is high today, what has it been like in the past? abundant microfossils Ancient carbonates are enriched in 13C, suggesting that the biomass of the earth has remained unchanged over geological time scales and predating the origin of most modern life forms. From Schidlowski et al., 1983

16. If the abundance of prokaryotes is high today, what has it been like in the past? first microfossils Ancient carbonates are enriched in 13C, suggesting that the biomass of the earth has remained unchanged over geological time scales and predating the origin of most modern life forms. From Schidlowski et al., 1983

17. Conclusion • The biomass and numbers of prokaryotic cells are large today and have been large for billions of years.

18. Understanding prokaryotic diversity The cellular abundance makes it possible to estimate of cellular production, the number of mutations, and other rare genetic events

19. Annual cellular production of prokaryotes

20. Frequency of rare genetic events Given high rates of production of prokaryotic cells, rare genetic events would be common. For instance, if the mutation frequency is 4 x 10-7 cell-1 generation-1, the frequency of four simultaneous mutations in any gene shared by the population becomes high.

21. Rare genetic events are common over geological periods of time aLargest number of mutations likely to have occurred within the same gene and within the same generation

22. Is prokaryotic evolution fundamentally different from eukaryotic evolution? Most studies of evolution are performed with eukaryotes, which possess very small populations and long generation times. For prokaryotes, the timescale and the numbers of individuals are much larger. Could these factors lead to a fundamentally different process?

23. Does the large number of prokaryotes imply a high diversity? Many microbiologists have longed believed that most of the biodiversity on earth was prokaryotic. Qualitative evidence for this opinion comes from the tremendous diversity observed in energy metabolism, motility, and other prokaryotic processes. The abundance of prokaryotes and high cellular production rates supports the hypothesis that rapid evolution and diversification is possible in the prokaryotes. How can we compare the biodiversity between prokaryotes and eukaryotes?

24. Definition of species in prokaryotes Strains with >70 % hybridization (pairing) of their genomic DNAs or a DTm of hybrids of their genomic DNAs of<5 oCconstitute a species. A large amount of phenetic diversity is included within a species. >70 % pairing within a species Congruence between DNA pairing and phenetic similarity (Jones and Sneath [1970] Bact. Rev. 34:40-81).

25. Definition of species in prokaryotes From analyses of genomic sequences, this definition corresponds to >95 % ANI (average nucleotide identity) for >69 % of the DNA or >85 % of the protein encoding genes (Goris et al. 2007. IJSEM 57:81) >70 % pairing implies large genetic variation Congruence between DNA pairing and phenetic similarity (Jones and Sneath [1970] Bact. Rev. 34:40-81).

26. What if we apply this criterion to eukaryotes? Most primates would belong to the same species. one species? From Sibley and Ahlquist (1984)

27. Rate of 16S rRNA divergence Based upon the divergence of 16S rRNA genes of bacterial endosymbionts of insects, 0.05 divergence is about 175 Ma (Ochman et al. 1999. PNAS USA 96:12638) 175 At this rate, most bacterial genera would be about 350 Ma

28. A group of related alpha proteobacteria crown gall Agrobacterium, Sinorhizobium, and Rhizobium represent related organisms with similar life styles with an age of about 350 Ma. N2-fixing nodules

29. A group of related alpha proteobacteria Radiolabel released from synthetic lignin after 30 da. Sagittula stellata: a marine organism that transforms lignin as well as binds lignocellulose particles. Gonzalez et al. (1997) IJSB 47: 773-780. Cells bound to lignocellulose particles

30. A group of related alpha proteobacteria Phase contrast and electron micrographs of DSS-3. Arrow indicates surface blebs formed from outer membrane. Silicibacter pomeroyi: a marine organism that transforms dimethylsulfoniopropionate to dimethylsulfide and methane thiol. Gonzalez et al. (2003) IJSB 53: 1261-1269.

31. What if we applied these same criteria to eukaryotes? Mammals and marsupials would be in the same genus horse mouse In fact they are all: heterotrophs, aerobes, auxotrophic for some amino acids and vitamins, and stain Gram negative

32. What if we applied these same criteria to eukaryotes? Mammals and marsupials would be in the same genus 175 80 horse mouse Time since last common ancestor (Ma) In fact they are all: heterotrophs, aerobes, auxotrophic for some amino acids and vitamins, and stain Gram negative

33. How many species and higher taxa in nature? Use rRNA gene sequence comparisons to get at this.

34. In prokaryotes, the relationship of 16S rRNA sequence similarity (S) and DNA hybridization (D) can be described by a complementary log log plot (r=0.79; Keswani and Whitman, 2001). These results suggest that D>0.70 when S = 0.998 P = 0.50 S = 0.992 P = 0.95 S = 0.986 P = 0.99

35. Consensus of usage of taxonomic ranks for prokaryotes in Bergey’s Cumulative fraction of comparisons at each taxonomic rank with the specified 16S rRNA sequence similarity. Dyszynski and Whitman, submitted 95% 0.95 phylum class family 50% order 0.90 genus

36. Sequence data summary for an rRNA gene library of soil bacteria Libraries prepared : 42 Clones prepared : 4032 Sequences obtained : 3719 Chimeric sequences : 12 Non-16S rRNA sequences : 1 Sequences used for analyses : 3706 Mean read length : 842 bp Less than 0.2 % of the clones possessed >99.8 % similarity to a described species. Less than 2.5 % of clones possessed >99 % sequence similarity to a described species. From Jangid et al., unpublished

37. Even this fairly large effort on common soil failed to fully sample the bacterial taxa Rarefaction curves fail to plateau even for deep phylogenetic groups. Greater than 2000 OTUs more diverse than species. (D=0.01). D = 0.01 0.03 0.05 0.10

38. Prokaryotic diversity in natural habitats Rarefaction analyses of rRNA gene libraries from soil in Georgia detect >400 OTUs with 0.90 divergence.

39. Prokaryotic diversity in natural habitats For 3706 sequences, 400 OTUs detected. Chao1 estimator: 669 (95 % COI 583-797).

40. Prokaryotic diversity in natural habitats Implies that soil contains large numbers of prokaryotic groups each with a diversity comparable to the placental mammals.

41. Conclusions The high number of prokaryotes predicts a high capacity for genetic variation. Although the biodiversity of the prokaryotes is not known, it is likely to be very high, possibly exceeding that of eukaryotes.