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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…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
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
Prokaryote and Eukaryote Discovery
Diversity: Short and Long(ish) Time Scales
Defining Relationships among Microbial Taxa
Bacteria by FCM
Viruses by EFM (SYBR Green)
Sept 2000 Dec 2003
Amplified Ribosomal Intergenic Spacer Analysis (ARISA)
backed by Clone Libraries for ID. Phylogenetic resolution near “species” level
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).
Microdiversity - The rule rather than the exception.
ITS shows clusters well. Populations are not clonal.
SAR 11 cluster alone - we estimate ~800 distinguishable sequence types at our coastal study site (Chao 1)- clustered into ~10 groups (ecotypes?)
138 from SPOTS
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
Typically 40-70 fragments/sample.
Important tool for
Correlating to ARISAs
(prokaryote community structure)
Relates Prokaryotic-Eukaryotic Ecology
Rank correlation of occurrence of OTU
Kendall Rank Similarity
(400 – 650 bp ea.)
Protistan taxa are morphologically defined. You might think that would be an advantage,
-Complexity of taxonomy(ies)
multiple fixation procedures
multiple analytical procedures
diverse taxonomic characters
-Deficiencies of taxonomy
small species (few characters)
morphologically amorphous species
-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.
-This will not resolve the issue of the ‘species concept’.
-Ultimately, multiple gene sequences will provide identity.
(application to real data)
Results for 970 environmental 18S
clone sequences from a sample in
the Coastal western North Atlantic
≈1200 18S clones
(Single date, 6 depths, USC M.O. site)
Frequency of Taxonomic Unit
*Large Euk diversity (488 OTUs; 95% similarity: pairwise alignments).
*Most OTUs are rare (large number of ‘background’ of taxa).
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.
Countway et al. (2005), GenBank accession AY937465-AY938434
Ostreococcus T-RFLP signature
at the Chl a Max: July 2001
Percent of total amplified DNA
Caron, Countway & Brown (2004)
(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.