Transcript Thompson_lecture_July24 - C-MORE

Origins of diversity in the
bacterioplankton
Theory, observations and
evolutionary experiments
7/24/07
Lecture Outline

Diversity 

Generation 

Classification and observing
vertical and horizontal mechanisms
Persistence 
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
Neutral Theory
Niche Theory
Ecological trends in pelagic systems
BIODIVERSITY
A
Marine Assemblage
Fluorescent microscopy
Macro
Diversity: Classification and observing
Micro
Genetic diversity
Bacteria
Archaea
Pace, 1997
Kingdom
Animalia
Nucleic Acids
Diversity: Classification and observing
Eucarya
Ribosomal RNA
-ubiquitous
-conserved function
-conserved+variable sequence
fixed
variable
Image credit:
NIH inside the cell
Areas in red and grey may vary in this molecule, and areas in violet and blue may not.
Quantitatively inferring relationships
“Alignment” of 16S/18S rRNA
Homo sapiens
S. cereviceae
Zea maize
Escherichia coli
Anacystis nidulans
Thermotoga maritima
Methanococcus vannielii
Thermococcus celer
Sulfolobus sulfotaricus
VARIABLE
...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGCTGCAGTTAAAAAG...
...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAG...
...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAG...
...GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCG...
...GTGCCAGCAGCCGCGGTAATACGGGAGAGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCG...
...GTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTACCCGGATTTACTGGGCGTAAAGGG...
...GTGCCAGCAGCCGCGGTAATACCGACGGCCCGAGTGGTAGCCACTCTTATTGGGCCTAAAGCG...
...GTGGCAGCCGCCGCGGTAATACCGGCGGCCCGAGTGGTGGCCGCTATTATTGGGCCTAAAGCG...
...GTGTCAGCCGCCGCGGTAATACCAGCTCCGCGAGTGGTCGGGGTGATTACTGGGCCTAAAGCG...
Bacteria
Archaea
Eucarya
Diversity: Classification and observing
“Domain” Bacteria
1994 - 13 divisions
(all cultured)
2004 - 80 divisions
26/54
BACTERIAL
“PHYLA/CLASS”
~80-90% rRNA
SIMILARITY
Diversity: Classification and observing
RDP Global Census, 2003
FIG. 3. Collector's curve of the Chao1 nonparametric richness estimator for sequences in the RDP-II.
Accession numbers were used to determine the order in which sequences have been sampled. OTUs defined
by a collection of identical sequences reached an estimate of 325,040 different OTUs.
Schloss and Handlesman, 2004
Diversity: Classification and observing
Strategy for exploring
genetic diversity
(Culture-independent)
environment
PCR amplified rRNA genes
identification and
removal of artifacts
clone, sequence and analyze
Chao-1
estimates of
diversity
Diversity: Classification and observing
non-parametric richness estimator
S = Sobs + (a2/2b)
Sobs total # observed species
a species observed once
b species observed twice
Estimating diversity in microbial
ecology
Extrapolated diversity of a sampled
abundance

Depend on several factors:
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450
Number of OTUs observed
400
350
300
250
200
150
100
50
0
0
1
2
3
4
5
6
7
9
11
13
14
16
21
27
32
43
abundance
Diversity: Classification and observing
45
Underlying distribution
Sample size
Sampling strategy
Two types of estimators for diversity
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Non-parametric estimators (Chao and lee, 1992)
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No abundance distribution model is assumed
predict minimum number expected-> not total diversity!
 Chao-1
2
CHAO1: S  Sobserved 
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
S1
2S2
ACE (abundance-based coverage estimator of species richness)
estimates the diversity of rare and abundant taxa separately
Parametric estimators
(see discussion in Hong, PNAS, 2006)
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The abundance distribution is assumed to have a specific form
Observed distribution is fit by maximum likelihood model
 Power-law
 Lognormal
 Pareto
 Gamma
Large potential for error as we really don’t understand the natural
distributions of biodiversity!
Diversity: Classification and observing
How many types coexist in marine microbial
communities? - wide variation in estimates
Environment
Units
Richness
(model)
Reference
Meso and Deep Sea
97% V6 (16S
variable region)
~104 (chao1 and ACE)
Sargasso seawater
-103 L
- all samples
100%16S rRNA
94% rpoA
100% 16S rRNA
1,412 (observed)
~1000 (chao1)
106 (chao1)
Plum Island Sound
seawater - 1L
100% 16S rRNA
99% 16S rRNA
1633 (chao1)
520 (chao1)
Acinas and
Klepac-Ceraj et
al., Nature 2004
Hypersaline
microbial mat
100% 16S rRNA
99-100% 16S rRNA
1,336 (observed)
>104 (chao1 and ACE)
Ley R. E. et al.,
AEM 2006
Salt marsh
sediment - 5g
99% 16S rRNA
Diversity: Classification and observing
2411 ± 542
(pareto distribution)
Sogin et al,
PNAS 2006
Venter et al.,
Science 2004
Hong et al.,
PNAS 2006
Why such variabilities in distribution?
Rare Biosphere
~25%
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Most diversity is rare
New approach: Tag-sequencing
16S allows high-sample number =
118,000 PCR amplicons ~120 bp
hyper-variable region of 16S rRNA
Non-parametric (chao1 and ACE)
estimation of diversity at eight
oceanic sites - meso- and
bathypelagic realms combined
predict ~104 coexisting types per
site (10-fold higher than other
marine estimates)
Relative abundance of OTUs
varies 1000-fold. Most of diversity
is low-abundance populations
Sogin, PNAS 2006
Diversity: Classification and observing
~25%
Q: How is relatedness of 16S rRNA
correlated to genomic similarity?
Sediment actinobacteria
Stackebrandt
and Goebel, 1994
70% DNA-DNA re-association has been the “gold standard” for assigning culturepositive organisms to microbial species. - correlates to 97-100% rRNA identity. The
reciprocal relationship does not hold.
A: Not that well…
Diversity: Classification and observing
How are bacterial genomes differentiated?
e.g. three E. coli strains have in common <40% of total protein genes
Core genome:
- shared by all
(e.g., housekeeping)
Flexible genome:
- strain specific
(e.g., pathogenicity
islands, antibiotic
resistance, integrons)
• strains from different environments
Diversity: Classification and observing
Welch et al. (2002)
Size variation among bacterioplanktonic
“V. splendidus” genomes
Isolates paired by identical
Hsp60 alleles and represent
spectrum of observed diversity
Genome sizes (4.5 to 5.6 Mb)
phylogenetic relationships
of Hsp60 alleles
Suggests some diversification
is due to large-scale genome
changes
Diversity: Classification and observing
What drives genome diversification?
GENOME
Elements In
Duplication
Horizontal gene transfer
-homologous recombination
-non-homologous recombination
Elements Out
Gene Loss
Dynamic genome content
Diversity: Generation
Mechanisms of Lateral Diversification
Foreign DNA uptake:
Conjugation
Transformation
Transduction
Conjugation: A bacterium attaches to another
bacterium and passes a fragment of its DNA
(chromosomal or plasmid) to the recipient cell.
It is not known how many environmental bacterial exchange DNA
through conjugation.
(CAMERA query): What is the ratio of genes encoding sex pili to recA?
Diversity: Generation
Mechanisms of Lateral Diversification
Foreign DNA uptake:
Conjugation
Transformation
Transduction
WH Freeman
Transformation:
Fragments of bacterial DNA are taken up by a cell from the environment.
These genetic fragments may recombine with the host chromosome, permanently
adding new genes.
Diversity: Generation
Mechanisms of Lateral Diversification
Foreign DNA uptake:
Conjugation
Transformation
Transduction
WH Freeman
Transduction:
Phage carry bacterial DNA from one bacterium to another
Diversity: Generation
What happens after DNA uptake?
Destruction
Plasmid replication
Homologous Recombination
Site-specific Recombination
Non-homologous Recombination
Diversity: Generation
What happens after DNA uptake?
Destruction
Plasmid replication
Homologous Recombination
Site-specific Recombination
Foreign DNA recognized by foreign methylation patterns
Restriction endonucleases cleave foreign DNA
G
A
*
*
*
C ATA
G
ATTGCCCGTAATATTACG
TAACGGGCATTATAATGC
Diversity: Generation
G T AC
T TC
DNA as Food?
[DNA]marine >100ug/L
[DNA]sediment >100ug/g
DNA uptake mutants
FIG. 3. Average growth yields
of wild-type (WT) or com
mutant cells in minimal
medium supplemented with
0.1% ultrapure sonicated
salmon sperm DNA as the
sole source of carbon and
energy. Growth yields
(indicated above each bar)
were determined by dividing
the number of cells after 24 h
of incubation by the number of
cells at inoculation.
Palchevskiy and Finkel, J. Bac, 2006
Wild-type E. coli
Diversity: Generation
Mechanisms of Lateral Diversification
Destruction
Plasmid replication
Homologous Recombination
Site-specific Recombination
WH Freeman
Diversity: Generation
What happens after DNA uptake?
Destruction
Plasmid replication
Homologous Recombination
Site-specific Recombination
WH Freeman
Diversity: Generation
Ratio: recombination to mutation
Identify recombination events as deviations from phylogenetic congruency
12 strains of E. coli (method of Wilson et al 1977)
Guttman and Dykhuizen, Science 1994
In E. coli recombination is 50-fold more likely to change a nucleotide site than mutation
Multiple locus sequence typing (MLST) ->
Similar recombination rates in other pathogen populations
Diversity: Generation
What happens after DNA uptake?
Destruction
Plasmid replication
Homologous Recombination
Site-specific Recombination
Mobile genetic elements insert in genome:
• Transposons (IS + transposase)
• Integrons (attI site + integrase + gene
cassette with attC site)
~1% of Vibrio genomes are annotated
as transposases or integrases.
V. vulnificus “super-integron” genecapture system contains 188 attC sites
and 202 orfs
Diversity: Generation
integrase
Mechanisms of Vertical (clonal) Diversification
Vertical inheritance
 Point mutations
 neutral (wobble)
 nonsense/frameshift
 Chromosomal mutations
 Deletion
 Duplication
 Rearrangements
3.5 billion years in the making
Diversity: Generation
How fast does the molecular clock tick?
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Neutral mutation rates:
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E.coli lab cultures: 0.003 mutations per genome
division (Drake, 1991, 1993, 1998)
E.coli natural populations: 0.0001-0.0002
mutations per genome division
Buchnera natural populations: 0.0001-0.0002
mutations per genome division
1-2% 16S rRNA divergence per 50 million years
(Moran et al 1993) based on Buchnera
3.5 billion years of evolution!
Diversity: Generation
QuickTime™ and a
TIFF (Uncompress ed) dec ompres sor
are needed to s ee this pic ture.
Co-evolution of the
buchnera-aphid symbiosis
allows calibration of
microbial evolution rate.
Fitness differences:
Diversification by mutation
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Evolution of growth advantage in stationary phase: older cultures
out-compete younger cultures.
Mapped to mutations in rpoS (stress-response sigma factor).
Finkel, et al PNAS (1999)
Fig. 1. Consecutive generations of GASP mutants arise in the same culture. Progressively aged cultures were mixed. (A) One-dayold in the majority (solid line) vs. 10-day-old in the minority (broken line). (B) Ten-day-old in the majority (solid line) vs. 20-day-old in
the minority (broken line). (C) Twenty-day-old in the majority (solid line) vs. 30-day-old in the minority (broken line). Asterisks indicate
that cfu ml-1 were below the limit of detection (<102 cfu ml-1).
Diversity: Generation
So far:
There is vast (unknown) diversity of 16S rRNA
ribotypes
There is even more genomic diversity associated with
those ribotypes
There are many mechanisms for microbial
diversification…
So, what drives the cohesion of populations into the
recognizable types we can observe?
Diversity: Generation
The frequency of recombination falls off
exponentially with the degree of genomic
DNA sequence divergence.
Fraser, Science 2007
Bacillus, Staphylococcus and E. coli
Is homologous recombination more likely
within phylogenetic clusters?
gene
transfer among
closely related strains
Microbial phylogenetic taxa may show
some degree of biological isolation
(similar to Mayr 1942)

Zebra x Horse = Hebra
BACTERIAL
“SPECIES”
~97% rRNA
SIMILARITY
HUMAN &
DONKEY
~99% rRNA
SIMILARITY
A test:
Do sequence clusters have coherent
environmental dynamics?
Population 2
Population 1
16
?%
Y
AB C
14
ABUNDANCE
X
12
10
1
2
1463
1470
1477
TIME
Cohesive sequence clusters = ecotypes?
1484
Units of Biology
Biological species
Phylotypes
Reproductive Isolation
Single lineage
Pattern
Pluralism
Multiple models relevant
Ecotypes
Ecological niche
Operational Taxonomy
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Phylotypes
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Debatable assumption:
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Proxies for evolutionary species
Single-lineage of ancestral populations/smallest diagnostable
cluster of individuals
(Cracraft, 1983; Eldregde and Cracraft 1980)
The evolutionary history for the biomarker gene is a (good) proxy
for the evolutionary history of the organism.
Examples
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16S ribosomal RNA
Housekeeping genes
Biological species
Pattern
Pluralism
Ecotypes
Phylotypes
Operational Taxonomy
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Biological Species
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Earnst Mayr, 1942
Groups of organisms that can interbreed
Reproductive isolation
Homologous recombination in microbes may enable bio-species
like evolution
Observed in microbes through multi-locus sequence typing
(MLST) or whole-genome comparisons
Biological species
Pattern
Pluralism
Ecotypes
Phylotypes
Operational Taxonomy
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Ecological Species (Ecotype)
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Lineage that occupies an ecological niche (adaptive zone)
Definition allows for “hybridization” events if niche is unchanged
Observed in microbes through population dynamics
Van Valen, 1976
Biological species
Pattern
Pluralism
Ecotypes
Phylotypes
Operational Taxonomy

Pattern Pluralism
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Similarities and differences between organisms may be
accounted for by evolutionary mechanisms, however a singletree like pattern (tree of life) is not the expected outcome.
“Different evolutionary models and representations of
relationships will be appropriate, and true, for different taxa or at
different scales or for different purposes”
Doolittle and Bapteste, PNAS 2007
Biological species
Pattern
Pluralism
Ecotypes
Phylotypes
“Pattern Pluralism” in Chimeric Thermotoga
“Different parts of a genome may belong to different biological
Species if our species concept is based on the ability to share
Information by homologous recombination.”
Nesbo, Dlutek and Doolittle, 2006
Diversity Metrics*
Metric
Unit
Pro’s
Con’s
Ribotype (16S rRNA)
phylotype
universal,
Culture-independent
High associated genomic variability
Single-locus
Housekeeping gene
phylotype
Higher phylogenetic resolution,
Culture-independent
Not universal,
High associated genomic variability
Multi-locus sequence
types (MLST)
phylotype
Biospecies?
Can determine recombination
rates and clonality of population
structure
Not universal,
Need cultured isolates*
Environmental
Gene clusters
ecotype
phylotype
Culture-independent and
suggests ecological significance
Must link to environmental data set,
within cluster genomic variation is
unknown.
Metagenome
fragments
ecotypes
phylotypes
Biospecies?
Environmental detection,
presents total genetic diversity
Challenges associated with linking
phylogetically-informative genes to
genetic diversity
Whole Genomes
ecotypes
phylotypes
Biospecies?
Most accurate
Large sequencing effort, Need cultured
isolates*
*Conclusions subject to debate!
Lecture Outline

Diversity 

Generation 

definitions and measurement
vertical and horizontal mechanisms
Persistence 


Neutral Theory
Niche Theory
Ecological trends in pelagic systems
Paradox of the plankton
1961 G. E. Hutchinson (Am. Nat. 95:137-145)
“The problem that is presented by the phytoplankton is
essentially how is it possible for a number of species to coexist in a relatively isotropic or unstructured environment all
competing for the same sorts of materials?”
Persistence of Diversity

Neutral Theory
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stochastic interactions and dispersal
Niche Theory
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Specialization
Environmental heterogeneity
Neutral Theories
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Unified Neutral Theory of Biodiversity (Hubble, 2001)
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Seek to explain community level patterns without (before)
invoking specialization of species.
Highly similar (ecologically-equivalent or functionally-redundant)
species co-exist by stochastic mechanisms.
Diversity is a balance of immigration and local extinction
How strong is “purifying
selection” over the scale of
ocean mixing?
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Neutral Theories

Island biogeography (MacArthur and Wilson, 1967)
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Number of species on a island is determined by effect of distance
from mainland and the island size.
Many types of “islands”: habitat surrounded by an inhabitable
environment (fish, marine snow…)
New species are created by isolation of gene pools and drift
(allopatric speciation)
Specialization: Niche Theory
Fundamental Niches
(Hutchinson, 1958)
Niche
#2
pO2
Niche
#1
Temperature
Specialization: Niche Theory
Fundamental Niche
Realized Niche
pO2
Niche
Niche#3
#1
Competition = Niche Overlap
Temperature
Specialization: Niche Theory
e.g. Carbon Source
pO2
Niche
#1
Specialization = Co-existence
Temperature
On Competitive Exclusion

Complete competitors cannot coexist (Hardin, 1960)
BUT
 “…there are innumerable dimensions in which
differences could be found” (Valiela, 1995)
Sinking Detritus (Marine Snow):
Ephemeral Microenvironments (Niches)
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
F. Azam, Nature 2001
Phytoplankton Niche Axes
Fig. 3. Optimum temperature and light
intensity for growth, (Topt) and (Iopt), of all
initialized Prochlorococcus analogs (all
circles) from the ensemble of 10 model
integrations. Large circles indicate the
analogs that exceeded a total biomass of
106 mol P along AMT13 in the 10th year.
Colors indicate classification into model
ecotypes. Bold diamonds indicate real-world
Prochlorococcus ecotypes.
Follows, et al Science 2007
Emergent biogeography parallels
observations
Figure 1. Annual mean biomass and
biogeography from single integration. (A) Total
phytoplankton biomass (µM P, 0 to 50 m
average). (B) Emergent biogeography:
Modeled photo-autotrophs were categorized
into four functional groups; color coding is
according to group locally dominating annual
mean biomass. Green, analogs of
Prochlorococcus; orange, other small photoautotrophs; red, diatoms; and yellow, other
large phytoplankton. (C) Total biomass of
Prochlorococcus analogs (µM P, 0 to 50 m
average). Black line indicates the track of
AMT13.
Units of selection are Light and
Temperature optima - not taxa
Follows, et al Science 2007
…a study of biogeography on the basis of the global
distribution of genes and their alleles and their patterns
of divergence and dispersal. This should be a central
guiding principle for the new science of
metagenomics….
Nesbo, Dlutek and Doolittle, 2006
…a study of biogeography on the basis of the global
distribution of genes and their alleles and their patterns
of divergence and dispersal. This should be a central
guiding principle for the new science of
metagenomics….
Nesbo, Dlutek and Doolittle, 2006
Recent studies suggest gene-ecologies may circumvent much of the confusion
around trying to link the activities of microbial communities to their
phylogenetic structure
After all - functional genes, not ribosomes drive niche-partitioning
Persistence of Diversity
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Neutral Theory
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Niche Theory
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stochastic interactions and dispersal
Specialization
Environmental heterogeneity
Ecological trends in pelagic systems
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
Environmental gradients
Biogeography
Do genes track environmental gradients?
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Hawaii Ocean Time series
Depth variability of gene distributions examined by
end-sequencing ~5000 fosmids from each depth
Look for specific genes and metabolic traits that were
differentially distributed in the water column.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Fig. 1. Temperature versus salinity (T-S) relations
for the North Pacific Subtropical Gyre at station
ALOHA (22°45'N, 158°W). The blue circles indicate
the positions, in T-S "hydrospace" of the seven
water samples analyzed in this study. The data
envelope shows the temperature and salinity
conditions observed during the period October 1988
to December 2004 emphasizing both the temporal
variability of near-surface waters and the relative
constancy of deep waters.
Habitat-enriched gene groups
Clusters of orthologous groups
Fig. 4. Cluster analyses of COG
annotated photic zone and deep
water sequence bins versus depth.
Yellow shading is proportional to
the percentage of categorized
sequences in each category.
Photic Zone:
Light-driven processes (KEGG)
Motility (KEGG)
Iron-transport
QuickTime™ and a
ompressed) decompressor
ded to see this picture.
DeLong, et al Science 2006
Deep sea:
Transposases and integrases
Pilus synthesis (KEGG)
Antibiotic synthesis
Gene-based biogeography

Global Ocean Survey:

7.6 million random sequence reads (Venter et al
2007)
GOS transect map
(Yutin, EM 2007)
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Gene-based biogeography

Global Ocean Survey

PufM: subunit of anoxygenic photosynthetic reaction center.
PufM-type
Different types associated with different environments
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Cosmopolitan: G Oligotrophic: A,B Coastal: E,K Offshore: C,D
Fig. 4. Anoxygenic photosynthetic population compositions along the GOS transect. Colours used to
represent different types of environments … colours representing the eight major phylogroups …Note
that samples 5, 6 and 7 are different size fractions from the same station.
(Yutin, EM 2007)
Summary

How do we assess microbial diversity?

Classification and Observations
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Contributions of 16S ribotyping to understanding the potential
scale of global microbial diversity
Use of statistical estimators to attempt total diversity estimates
Microbial species concepts - can we define a unit of selection?
How is this diversity generated?

horizontal and vertical mechanisms



3.5 million years of vertical evolution
Mobile genetic elements and LGT
Homologous recombination as a population-cohesive force
Summary

How is this diversity maintained in the oceans?

Neutral Theory: Diversity can partially be explained by
stochastic environmental interactions that drive immigration and
local extinctions

Niche Theory: Diversity is explained by the specialization of coexisting types

Linking structure (co-existing diversity) to function (e.g. global
biogeochemical processes) can be approached in a taxaindependent manner that relies on the ecologies of genetic
systems.
The last slide