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The USC Microbial Observatory. …at the San Pedro Ocean Time-series Station. D. Caron, J.Fuhrman: P. Countway, M. Brown, I. Hewson, P. Savai, A. Schnetzer, S. Moorthi, J. Rose, J. Steele, I. Gilg, M. Schwalbach, R. Schaffner, E. Brauer, L. Farrar, B. Strachan, P. Vigil. T. Michaels:

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The USC

Microbial Observatory

…at the San Pedro

Ocean Time-series Station

D. Caron, J.Fuhrman:

P. Countway, M. Brown, I. Hewson, P. Savai, A. Schnetzer, S. Moorthi, J. Rose, J. Steele, I. Gilg, M. Schwalbach, R. Schaffner, E. Brauer, L. Farrar, B. Strachan, P. Vigil

T. Michaels:

B. Jones, W. Berelson

M. Neumann, R. Schimmoeller E. Caporelli, J. Herndon, X. Hernandez, G. Smith

Department of Biological Sciences

University of Southern California

3616 Trousdale Parkway, AHF 301

Los Angeles, CA 900089-0371

http://www.usc.edu/dept/LAS/biosci/Caron_lab/index.html


The USC M.O.

Broad Objectives/Directions

Prokaryote and Eukaryote Discovery

Diversity: Short and Long(ish) Time Scales

Characterizing Distributions

Defining Relationships among Microbial Taxa

Autecological studies



Temperature °C

Nitrate (µM)

Phosphate (µM)

http://wrigley.usc.edu/data_sys/


Silicate (µM)

Chlorophyll

Oxygen (ml/l)


USC Microbial Observatory and San Pedro Ocean Time Series

Temperature

Oxygen

Chlorophyll a

Bacteria by FCM

by EFM

Viruses by EFM (SYBR Green)

Sept 2000 Dec 2003


USC Microbial Observatory and San Pedro Ocean Time Series

Prochlorococcus

FCM

Synechococcus

FCM

Picoeukaryotes

FCM

Aloricate ciliates

Dinoflagellates

Diatoms

Sept Dec

2000 2003


Whole Bacterial Community Fingerprints

Amplified Ribosomal Intergenic Spacer Analysis (ARISA)

backed by Clone Libraries for ID. Phylogenetic resolution near “species” level

ARISA

PCR primers

ARISA PCR

Fluorochrome

16S rRNA gene

23S rRNA gene

Intergenic Spacer, Variable Length

Run products on a fragment analyzer.

Each peak represents an “Operational Taxonomic Unit.”

Reference: Fisher and Triplett 1999, others...

PCR from these primers to make Clone Libraries to identify ARISA OTUs

16S sequence provides ID, ITS sequence provides length and very high resolution phylogenetic information (ca. “strain” level).


SAR11

cluster

(in San

Pedro

Channel)

Microdiversity - The rule rather than the exception.

ITS shows clusters well. Populations are not clonal.

16S

ITS

0.1

SAR 11 cluster alone - we estimate ~800 distinguishable sequence types at our coastal study site (Chao 1)- clustered into ~10 groups (ecotypes?)

284 clones

138 from SPOTS

0.1


Annual Bacterial Community Reassembly

with Shahid Naeem, Columbia University

Discriminant Function Analysis

‘Clockfaces’ - Months are like hours on the clock.

Radii represent discriminant function of taxa (a function of community composition).

Central line: mean

Dashed lines: range over 3 years

Chl max

1-50

51-100

100-150

ARISA Bins


T-RFLP Eukaryote Seasonal Pattern USC M.O.HaeIII Digest

Typically 40-70 fragments/sample.

Important tool for

Correlating to ARISAs

(prokaryote community structure)



Bacterial - Protistan Relationships

Bacterial OTU

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Protistan OTU

  • Bacterial and Protistan OTU usually cluster within domains (e.g. protist with protist)

  • But certain Bacterial and Protistan OTU covary most closely with each other

Relates Prokaryotic-Eukaryotic Ecology

Rank correlation of occurrence of OTU

Kendall Rank Similarity


Phylogenetic breakdown of Euks in 18S libraries from the time-series.

M.O. 2001

ARB Tax.

2,224 clones

(400 – 650 bp ea.)


What about species diversity? time-series.

Protistan taxa are morphologically defined. You might think that would be an advantage,

and yet…

-Complexity of taxonomy(ies)

multiple fixation procedures

multiple analytical procedures

diverse taxonomic characters

-Deficiencies of taxonomy

small species (few characters)

morphologically amorphous species

convergent evolution

-Demands of ecological research

high sample number

complexity of natural assemblages


There is a need to develop practical guidelines for defining OTUs for protistan taxa based on rDNA sequence information.

Our approach:

  • Select complete 18S sequences of ‘well-defined’ (i.e. morphologically-defined) protistan species from GenBank.

  • Perform all pairwise comparisons of full-length sequences.

    • examine intra-species (strain-strain) sequence variability.

    • examine inter-species sequence variability.

  • Attempt to determine logical demarcation (% similarity) for species-level distinction.

  • Apply criteria to environmental sequence databases for assessing microbial eukaryote diversity.

Caveats:

-This will not resolve the issue of the ‘species concept’.

-Ultimately, multiple gene sequences will provide identity.


Consequences of varying percent similarity for OTU calling. OTUs for protistan taxa based on rDNA sequence information.

(application to real data)

Results for 970 environmental 18S

clone sequences from a sample in

the Coastal western North Atlantic


‘Taxon-level’ distinction OTUs for protistan taxa based on rDNA sequence information.

≈1200 18S clones

(Single date, 6 depths, USC M.O. site)

Frequency of Taxonomic Unit

Taxonomic Units

*Large Euk diversity (488 OTUs; 95% similarity: pairwise alignments).

*Most OTUs are rare (large number of ‘background’ of taxa).


Global distribution OTUs for protistan taxa based on rDNA sequence information.

or

Endemism?

Jury still out!

Study in Coastal N. Atlantic

72-hr bottle incubation

Natural light; ambient temp.

970 clones analyzed.

165 Total phylotypes (95%).

68% (108 out of 165) observed at only one sampling time.

Only 18% observed at all 3 sampling times.

Global distribution

or

Endemism?

Countway et al. (2005), GenBank accession AY937465-AY938434


Target organisms caron lab

Phaeocystis OTUs for protistan taxa based on rDNA sequence information.(Haptophyte)

Lingulodinium (Dinoflagellate)

The Daily Breeze: May 12, 2002

Pseudonitzschia

(Diatom)

Chrétiennot-Dinet et al., 1995

Ostreococcus (Chlorophyte)

Target Organisms – Caron Lab


Haptophytes OTUs for protistan taxa based on rDNA sequence information.

Cryptophytes

4%

4%

Stramenopiles

2%

Other

Chlorophytes

9.1%

Dinoflagellates

Ostreococcus

38.2%

9.1%

Unclass. Eukaryotes

14.5%

Ciliates

20.0%

Ostreococcus T-RFLP signature

at the Chl a Max: July 2001

Percent of total amplified DNA

9.8%

10.7%

11.3%

Caron, Countway & Brown (2004)


Comparison to flow cytometry
Comparison to flow cytometry… OTUs for protistan taxa based on rDNA sequence information.


In parting, two popular microbial myths… OTUs for protistan taxa based on rDNA sequence information.

(and their corollaries)

The ‘age of discovery’ in oceanography is over.

(if you believe this, you’ve come to the wrong workshop)

C1: We have accurate estimates of protistan diversity.

We know a lot of common morphotypes, but...

(There is genetic diversity we don’t understand)

(Relationship between morphology, sequence identity and physiology is poorly known; we lack ecologicaltools)

We can forget about (or ignore) the species concept.

C1: The ‘omes’ (genome, transcriptome, proteome, metabolome)

will ‘tell all’.

The species (however defined) is the evolutionary unit; not the gene, not the assemblage, not the community.

The problem (sp. concept) is different for proks and euks.


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