Origins of diversity in the bacterioplankton

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Origins of diversity in the bacterioplankton

• Theory, observations and evolutionary experiments
• 7/24/07

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Lecture Outline

• Diversity -
• Classification and observing
• Generation -
• vertical and horizontal mechanisms
• Persistence -
• Neutral Theory
• Niche Theory
• Ecological trends in pelagic systems

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BIODIVERSITY
A

• Marine Assemblage
• Fluorescent microscopy

Macro Micro
Diversity Classification and observing
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Genetic diversity
Bacteria
Archaea
Pace, 1997
Eucarya
Nucleic Acids
Diversity Classification and observing
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Ribosomal RNA -ubiquitous -conserved
function -conservedvariable 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.
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Quantitatively inferring relationships
Alignment of 16S/18S rRNA
VARIABLE
Homo sapiens ...GTGCCAGCAGCCGCGGTAATTCCAGCTCC
AATAGCGTATATTAAAGTTGCTGCAGTTAAAAAG... S.
cereviceae ...GTGCCAGCAGCCGCGGTAATTCCAG
CTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAG... Zea
maize ...GTGCCAGCAGCCGCGGTAATTCCAGC
TCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAG...
Escherichia coli ...GTGCCAGCAGCCGCGGTAATACGG
AGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCG... Anacyst
is nidulans ...GTGCCAGCAGCCGCGGTAATACGGGAGAGG
CAAGCGTTATCCGGAATTATTGGGCGTAAAGCG... Thermotoga
maritima ...GTGCCAGCAGCCGCGGTAATACGTAGGGGGCAA
GCGTTACCCGGATTTACTGGGCGTAAAGGG... Methanococcus
vannielii ...GTGCCAGCAGCCGCGGTAATACCGACGGCCCGAGTG
GTAGCCACTCTTATTGGGCCTAAAGCG... Thermococcus
celer ...GTGGCAGCCGCCGCGGTAATACCGGCGGCCCGAGTG
GTGGCCGCTATTATTGGGCCTAAAGCG... Sulfolobus
sulfotaricus ...GTGTCAGCCGCCGCGGTAATACCAGCTCCGCGA
GTGGTCGGGGTGATTACTGGGCCTAAAGCG...
Diversity Classification and observing
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Domain Bacteria
1994 - 13 divisions (all cultured)
BACTERIAL PHYLA/CLASS 80-90 rRNA SIMILARITY
Diversity Classification and observing
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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
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Strategy for exploring genetic diversity (Culture-
independent)
environment
PCR amplified rRNA genes
identification and removal of artifacts
clone, sequence and analyze

• Chao-1 non-parametric richness estimator
• S Sobs (a2/2b)
• Sobs total observed species
• a species observed once
• b species observed twice

estimates of diversity
Diversity Classification and observing
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Estimating diversity in microbial ecology

• Extrapolated diversity of a sampled abundance
• Depend on several factors
• Underlying distribution
• Sample size
• Sampling strategy

Diversity Classification and observing
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Two types of estimators for diversity

• Non-parametric estimators (Chao and lee, 1992)
• No abundance distribution model is assumed
• predict minimum number expected-gt not total
  diversity!
• Chao-1
• 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)
• 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 dont
  understand the natural distributions of
  biodiversity!

Diversity Classification and observing
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How many types coexist in marine microbial
communities? - wide variation in estimates
Diversity Classification and observing
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Why such variabilities in distribution? Rare
Biosphere
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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
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• 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 culture-positive
organisms to microbial species. - correlates to
97-100 rRNA identity. The reciprocal
relationship does not hold. A Not that well
Diversity Classification and observing
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How are bacterial genomes differentiated?
e.g. three E. coli strains have in common lt40 of
total protein genes

• Core genome
• shared by all
• (e.g., housekeeping)
• Flexible genome
• strain specific
• (e.g., pathogenicity islands, antibiotic
  resistance, integrons)

Welch et al. (2002)

• strains from different environments

Diversity Classification and observing
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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
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What drives genome diversification?
GENOME
Elements Out
Elements In
Duplication Horizontal gene transfer -homologous
recombination -non-homologous recombination

• Gene Loss

Dynamic genome content
Diversity Generation
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Mechanisms of Lateral Diversification
Foreign DNA uptake Conjugation Transformation Tr
ansduction
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
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Mechanisms of Lateral Diversification
Foreign DNA uptake Conjugation Transformation Tr
ansduction
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
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Mechanisms of Lateral Diversification
Foreign DNA uptake Conjugation Transformation Tr
ansduction
WH Freeman
Transduction Phage carry bacterial DNA from one
bacterium to another
Diversity Generation
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What happens after DNA uptake?
Destruction Plasmid replication Homologous
Recombination Site-specific Recombination Non-homo
logous Recombination
Diversity Generation
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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
ATTGCCCGTAATATTACG TAACGGGCATTA
TAATGC
A
A
C
T
G
T
G
A
C
T
T
C
Diversity Generation
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DNA as Food?
DNAmarine gt100ug/L DNAsediment gt100ug/g
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.
DNA uptake mutants
Palchevskiy and Finkel, J. Bac, 2006
Wild-type E. coli
Diversity Generation
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Mechanisms of Lateral Diversification
Destruction Plasmid replication Homologous
Recombination Site-specific Recombination
WH Freeman
Diversity Generation
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What happens after DNA uptake?
Destruction Plasmid replication Homologous
Recombination Site-specific Recombination
WH Freeman
Diversity Generation
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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)
-gt Similar recombination rates in other
pathogen populations
Diversity Generation
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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 gene-capture system contains 188
attC sites and 202 orfs
integrase
Diversity Generation
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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
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How fast does the molecular clock tick?

• Neutral mutation rates
• 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!

Co-evolution of the buchnera-aphid
symbiosis allows calibration of microbial
evolution rate.
Diversity Generation
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Fitness differences Diversification by mutation

• 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-day-old 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 (lt102
cfu ml-1).
Diversity Generation
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So farThere is vast (unknown) diversity of 16S
rRNA ribotypesThere is even more genomic
diversity associated with those ribotypesThere
are many mechanisms for microbial
diversification So, what drives the cohesion
of populations into the recognizable types we can
observe?
Diversity Generation
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The frequency of recombination falls off
exponentially with the degree of genomic DNA
sequence divergence.
Bacillus, Staphylococcus and E. coli
Fraser, Science 2007
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Is homologous recombination more likely within
phylogenetic clusters?
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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
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A test Do sequence clusters have coherent
environmental dynamics?
Population 2
Population 1
X Y A B C
?
1
2
Cohesive sequence clusters ecotypes?
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Units of Biology
Biological species
Phylotypes
Reproductive Isolation
Single lineage
Pattern Pluralism
Multiple models relevant
Ecotypes
Ecological niche
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Operational Taxonomy

• Phylotypes
• Proxies for evolutionary species
• Single-lineage of ancestral populations/smallest
  diagnostable cluster of individuals
• (Cracraft, 1983 Eldregde and Cracraft 1980)
• Debatable assumption
• The evolutionary history for the biomarker gene
  is a (good) proxy for the evolutionary history of
  the organism.
• Examples
• 16S ribosomal RNA
• Housekeeping genes

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Operational Taxonomy

• Biological Species
• 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

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Operational Taxonomy

• Ecological Species (Ecotype)
• 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

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Operational Taxonomy

• Pattern Pluralism
• Similarities and differences between organisms
  may be accounted for by evolutionary mechanisms,
  however a single-tree 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

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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
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Diversity Metrics
Conclusions subject to debate!
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Lecture Outline

• Diversity -
• definitions and measurement
• Generation -
• vertical and horizontal mechanisms
• Persistence -
• Neutral Theory
• Niche Theory
• Ecological trends in pelagic systems

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Paradox of the plankton

• 1961 G. E. Hutchinson (Am. Nat. 95137-145)
• The problem that is presented by the
  phytoplankton is essentially how is it possible
  for a number of species to co-exist in a
  relatively isotropic or unstructured environment
  all competing for the same sorts of materials?

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Persistence of Diversity

• Neutral Theory
• stochastic interactions and dispersal
• Niche Theory
• Specialization
• Environmental heterogeneity

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Neutral Theories

• Unified Neutral Theory of Biodiversity (Hubble,
  2001)
• 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?
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Neutral Theories

• Island biogeography (MacArthur and Wilson, 1967)
• 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)

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Specialization Niche Theory
Fundamental Niches (Hutchinson, 1958)
Niche 2
pO2
Niche 1
Temperature
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Specialization Niche Theory
Fundamental Niche
Realized Niche
pO2
Niche 3
Niche 1
Competition Niche Overlap
Temperature
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Specialization Niche Theory
e.g. Carbon Source
pO2
Niche 1
Specialization Co-existence
Temperature
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On Competitive Exclusion

• Complete competitors cannot coexist (Hardin,
  1960)
• BUT
• there are innumerable dimensions in which
  differences could be found (Valiela, 1995)

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Sinking Detritus (Marine Snow)Ephemeral
Microenvironments (Niches)
F. Azam, Nature 2001
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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
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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
photo-autotrophs 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
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• 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
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• 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
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Persistence of Diversity

• Neutral Theory
• stochastic interactions and dispersal
• Niche Theory
• Specialization
• Environmental heterogeneity
• Ecological trends in pelagic systems
• Environmental gradients
• Biogeography

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Do genes track environmental gradients?

• 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.

Fig. 1. Temperature versus salinity (T-S)
relations for the North Pacific Subtropical Gyre
at station ALOHA (2245'N, 158W). 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.
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Habitat-enriched gene 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.
Clusters of orthologous groups
Photic Zone Light-driven processes
(KEGG) Motility (KEGG) Iron-transport Deep
sea Transposases and integrases Pilus synthesis
(KEGG) Antibiotic synthesis
DeLong, et al Science 2006
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Gene-based biogeography

• Global Ocean Survey
• 7.6 million random sequence reads (Venter et al
  2007)

GOS transect map (Yutin, EM 2007)
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Gene-based biogeography

• Global Ocean Survey
• PufM subunit of anoxygenic photosynthetic
  reaction center.

PufM-type
Different types associated with different
environments
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)
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Summary

• How do we assess microbial diversity?
• Classification and Observations
• 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

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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 co-existing types
• Linking structure (co-existing diversity) to
  function (e.g. global biogeochemical processes)
  can be approached in a taxa-independent manner
  that relies on the ecologies of genetic systems.

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