Photo by Sean Davies- Surfer Magazine

Photo by Sean Davies- Surfer Magazine

prokaryotic

diversity eukaryotic

diversity

One point of view on prokaryotic evolution

How to count the How to count the number number of prokaryotes on of prokaryotes on earth?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.

Numbers of prokaryotesNumbers of prokaryotes in various habitats in various habitats

habitathabitat % of total% of total

marine subsurfacemarine subsurface 6666

terrestrial terrestrial subsurfacesubsurfaceaa

2626

soilsoil 4.84.8

seawaterseawater 2.22.2

freshwater and freshwater and saline lakessaline lakes

0.00430.0043

domesticated domesticated animalsanimals

0.0000800.000080

sea icesea ice 0.0000740.000074

termitestermites 0.0000120.000012

humanshumans 0.00000720.0000072

domesticated birdsdomesticated birds 0.000000040.0000000444

aAverage of range of 0.25-2.5 x 1030

Number and Number and biomass (carbon)biomass (carbon) of prokaryotes in of prokaryotes in the worldthe worldenvironmentenvironment number of number of

prokaryotic cells prokaryotic cells (x 10(x 102828))

Pg of C in Pg of C in prokaryotprokaryot

esesaa

Aquatic habitatsAquatic habitats 1212 2.22.2

Oceanic Oceanic subsurfacesubsurface

355355 303303

SoilSoil 2626 2626

Terrestrial Terrestrial subsurfacesubsurface

25-25025-250 22-21522-215

TOTALTOTAL 415-640415-640 353-546353-546

aPg = 1015 g. For aquatic, subsurface, and soil habitats, prokaryotic cells were estimated to contain 10-20, 86, and 100 fg C, respectively.

Relationship of plant and Relationship of plant and prokaryotic prokaryotic biomass to primary biomass to primary productivityproductivity

ecosysecosystemtem

net net primary primary productiproductivityvity

total carbon content (Pg C)total carbon content (Pg C)

(Pg (Pg C/yr)C/yr)

plantplant soil and soil and aquatic aquatic

prokaryoteprokaryotess

subsurfasubsurface ce

prokaryoprokaryotestes

terrestrterrestrialial

4848 560560 2626 22-21522-215

marinemarine 5151 1.81.8 2.22.2 303303

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.

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 depletedin 13C: δ13C = -25 o/oo

Inorganic carbon is the standard: δ13C = 0 o/oo

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.

Inorganic Cδ13C = 0 o/oo

Organic Cδ13C = -25

Weighed average δ13C = -6 o/oo

Total carbon in biosphere ~7.5 x 1022 g


Geochemical evidence suggests that the total fraction of organic carbon on earth has remained unchanged for billions of years

From Schidlowski et al., 1983


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.


average13 of earth’s carbon

average13 of organic carbon

average13 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. From Schidlowski et al., 1983

average13 of earth’s carbon

average13 of carbonates


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.


first land plants




first metazoa




first multicellular organisms




abundant microfossils




first microfossils

Conclusion

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

Understanding Understanding prokaryotic prokaryotic diversitydiversity

The cellular abundance makes it possible to estimate of cellular production, the number of mutations, and other rare genetic events

Annual cellular production of prokaryotesAnnual cellular production of prokaryoteshabitathabitat PopulatiPopulati

on sizeon sizeTurnover Turnover

time time (da)(da)

Cells yrCells yr-1-1

(x 10(x 102929))

marine marine heterotrophs,heterotrophs,

above 200 above 200 m m

3.6 x 3.6 x 10102828

1616 8.28.2

below 200 below 200 mm

8.2 x 8.2 x 10102828

300300 1.11.1

marine marine autotrophsautotrophs

2.9 x 2.9 x 10102727

1.51.5 7.17.1

soilsoil 2.6 x 2.6 x 10102929

900900 1.01.0

subsurfacesubsurface 4.9 x 4.9 x 10103030

550,000550,000 0.030.03

domestic domestic mammalsmammals

4.3 x 4.3 x 10102424

11 0.020.02

Frequency of rare Frequency of rare genetic eventsgenetic eventsGiven 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.

habitathabitat cells cells produced produced

yryr-1-1 (x (x 10102929))

frequency of four frequency of four simultaneous simultaneous

mutations (hmutations (h-1-1))

marine marine heterotrophs,heterotrophs,

above 200 above 200 mm

8.28.2 2.52.5

marine marine autotrophsautotrophs

7.17.1 2.02.0

soilsoil 1.01.0 0.30.3

subsurfacesubsurface 0.030.03 0.0090.009

domestic domestic mammalsmammals

0.020.02 0.0060.006

yearsyears Total Total number of number of

cells cells producedproduced

Rare Rare genetic genetic eventseventsaa

OneOne 2 x 102 x 103030 44

ThousThousandand

2 x 102 x 103333 55

MillionMillion 2 x 102 x 103636 55

BillionBillion 2 x 102 x 103939 66

aLargest number of mutations likely to have occurred within the same gene and within the same generation

Rare genetic events are common over geological periods of time

Is prokaryotic evolution Is prokaryotic evolution fundamentally different fundamentally different from eukaryotic from eukaryotic evolution?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?

Does the large Does the large number of number of prokaryotes imply a prokaryotes imply a high diversity?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?

Congruence between DNA pairing and phenetic similarity (Jones and Sneath [1970] Bact. Rev. 34:40-81).

>70 % pairingwithin a species

Definition of species in

prokaryotes

Strains with >70 % hybridization (pairing) of their genomic DNAs or a Tm of hybrids of

their genomic DNAs of <5 oC constitute a species. A large amount of phenetic diversity is included within a species.

Congruence between DNA pairing and phenetic similarity (Jones and Sneath [1970] Bact. Rev. 34:40-81).

>70 % pairing implies large genetic variation

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)

What if we apply this What if we apply this criterion to criterion to eukaryotes?eukaryotes?Most primates would belong to the same

species.

From Sibley and Ahlquist (1984)

one species?

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)

At this rate, most bacterial genera would be about 350 Ma

175

A group of related alpha proteobacteria

crown gall

N2-fixing nodules

Agrobacterium, Sinorhizobium, and Rhizobium represent related organisms with similar life styles with an age of about 350 Ma.

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

Radiolabel released from synthetic ligninafter 30 da.


Silicibacter pomeroyi: a marine organism that transforms dimethylsulfoniopropionate to dimethylsulfide and methane thiol. Gonzalez et al. (2003) IJSB 53: 1261-1269.

Phase contrast and electron micrographs of DSS-3. Arrow indicates surface blebs formed from outer membrane.


In fact they are all: heterotrophs, aerobes, auxotrophic for some amino acids and vitamins, and stain Gram negative

horsemouse

What if we applied these same criteria to eukaryotes?

Mammals and marsupials would be in the same genus

In fact they are all: heterotrophs, aerobes, auxotrophic for some amino acids and vitamins, and stain Gram negative

horsemouse

What if we applied these same criteria to eukaryotes?

Mammals and marsupials would be in the same genus

Time since last common ancestor (Ma)

80 175

How many species and higher taxa in nature?

Use rRNA gene sequence comparisons to get at this.

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 = 0.50S = 0.992 P = 0.95S = 0.986 P = 0.99

Consensus of usage of taxonomic ranks for prokaryotes in Bergey’s

phylum

class

order

family

genus

95%

50%

Cumulative fraction of comparisons at each taxonomic rank with the specified 16S rRNA sequence similarity.

Dyszynski and Whitman, submitted

0.90

0.95

Sequence data summary for an Sequence data summary for an rRNA gene library of soil bacteriarRNA gene library of soil bacteria

Libraries prepared : 42

Clones prepared : 4032Sequences obtained : 3719Chimeric sequences : 12Non-16S rRNA sequences : 1Sequences used for analyses :

3706Mean read length : 842 bpLess 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

0

500

1000

1500

2000

2500

0 1000 2000 3000 4000

Number of Sequences

Nu

mb

er

of

OT

Us

D = 0.01

0.03

0.05

0.10

Rarefaction curves fail to plateau even for deep phylogenetic groups.

Greater than 2000 OTUs more diverse than species. (D=0.01).

Even this fairly large effort on common soil failed to fully sample the bacterial taxa

0

100

200

300

400

500

0 1000 2000 3000 4000

number of sequences sampled

num

ber

of

OT

U's

obse

rved

Prokaryotic diversity in natural habitats

Rarefaction analyses of rRNA gene libraries from soil in Georgia detect >400 OTUs with 0.90 divergence.

0

100

200

300

400

500

0 1000 2000 3000 4000


num

ber

of

OT

U's

obse

rved

Prokaryotic diversity in natural habitats

For 3706 sequences, 400 OTUs detected.Chao1 estimator: 669 (95 % COI 583-797).

0

100

200

300

400

500

0 1000 2000 3000 4000


num

ber

of

OT

U's

obse

rved

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

ConclusionsConclusions

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.

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