The USC Microbial Observatory
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The USC Microbial Observatory tment of Biological Sciences tment of Biological Sciences rsity of Southern California rsity of Southern California Trousdale Parkway, AHF 301 Trousdale Parkway, AHF 301 ngeles, CA 900089-0371 ngeles, CA 900089-0371 //www.usc.edu/dept/LAS/biosci/Caron_lab/index.html //www.usc.edu/dept/LAS/biosci/Caron_lab/index.html 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 …at the San Pedro Ocean Time-series Station T. Michaels: B. Jones, W. Berelson M. Neumann, R. Schimmoeller E. Caporelli, J. Herndon, X. Hernandez, G. Smith
description
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: - PowerPoint PPT Presentation
Transcript of The USC Microbial Observatory
The USCMicrobial Observatory
Department of Biological SciencesDepartment of Biological SciencesUniversity of Southern CaliforniaUniversity of Southern California3616 Trousdale Parkway, AHF 3013616 Trousdale Parkway, AHF 301Los Angeles, CA 900089-0371Los Angeles, CA 900089-0371http://www.usc.edu/dept/LAS/biosci/Caron_lab/index.htmlhttp://www.usc.edu/dept/LAS/biosci/Caron_lab/index.html
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
…at the San PedroOcean Time-series Station
T. Michaels:B. Jones, W. BerelsonM. Neumann, R. Schimmoeller E. Caporelli, J. Herndon, X. Hernandez, G. Smith
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
N
USC Microbial Observatory and San Pedro Ocean Time Series
20 km
http://wrigley.usc.edu/data_sys/
Nitrate (µM)
Temperature °C
Phosphate (µM)
Silicate (µM)
Chlorophyll
Oxygen (ml/l)
Temperature
Oxygen
Chlorophyll a
USC Microbial Observatory and San Pedro Ocean Time Series
Sept 2000 Dec 2003
Bacteria by FCM
by EFM
Viruses by EFM (SYBR Green)
Prochlorococcus FCMSynechococcus FCMPicoeukaryotes FCM
USC Microbial Observatory and San Pedro Ocean Time Series
Sept Dec2000 2003
Aloricate ciliates
Dinoflagellates
Diatoms
Whole Bacterial Community FingerprintsAmplified Ribosomal Intergenic Spacer Analysis (ARISA)
backed by Clone Libraries for ID. Phylogenetic resolution near “species” level
Each peak represents an “Operational Taxonomic Unit.”Reference: Fisher and Triplett 1999, others...
ARISA PCR
Run products on a fragment analyzer.
16S rRNA gene
ARISAPCR primers
Fluorochrome
23S rRNA geneIntergenic Spacer, Variable Length
PCR from these primers to make Clone Libraries to identify ARISA OTUs16S 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.
0.1
SAR 11 cluster alone - we estimate ~800 distinguishable sequence types at our coastal study site (Chao 1)- clustered into ~10 groups (ecotypes?)
0.1ITS
16S
284 clones138 from SPOTS
SAR11cluster(in San PedroChannel)
ARISA Bins1-50 51-100 100-150
Discriminant Function Analysis
‘Clockfaces’ - Months are like hours on the clock.
Radii represent discriminant function of taxa (a function of community composition).
Central line: meanDashed lines: range over 3 years
Annual Bacterial Community Reassembly with Shahid Naeem, Columbia University
Chl max
6179
129148
184198
218231
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292324
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Fragment Size (bp)
USC MO Chl a Max: Protistan HaeIII T-RFLPT-RFLP Eukaryote Seasonal Pattern USC M.O.HaeIII Digest
Important tool forCorrelating to ARISAs(prokaryote community structure)
Typically 40-70 fragments/sample.
T-RFLP Similarity Matrix Tool Results: RDP II website
HaeIIIHaeIII
TRFsTRFs DateDate JanJan AprApr MayMay JulJul OctOct
(74)(74) JanJan 0.530.53 0.540.54 0.670.67 0.550.55
(69)(69) AprApr 0.530.53 0.590.59 0.580.58 0.500.50
(64)(64) MayMay 0.540.54 0.590.59 0.630.63 0.440.44
(89)(89) JulJul 0.670.67 0.580.58 0.630.63 0.560.56
(44)(44) OctOct 0.550.55 0.500.50 0.440.44 0.560.56
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Kendall Rank Similarity
e606e590e339e277e235e486e234e236e332
710e232e333e283667
e346780616520
e229e136e132945e181625541
e591e450e275960
e497e340e336e274905
e595e453e224493
e503613
e603e600e451e598e324e261469825755679676418775725715472
e300652e128421424400e175628e501568
1050664661
e402e228e331e195e196e327e325700
e282885785850745685649433
e487e237795571
e504e61
e231e94691e68835481
e602e459e454e262760e301e177e179e492e2685295381180985740805592790532508631610
e589855e1921030e233487770619547
e597e488e485e498e338e341e496e493e272e182e198e502e126655
e230682559478800526475e281e131705765e74e73
e130e481e335e362e302e276e500e129e135e76607940517646640643
Protistan OTU Bacterial OTU
•But certain Bacterial and Protistan OTU covary most closely with each other
•Bacterial and Protistan OTU usually cluster within domains (e.g. protist with protist)
Kendall Rank Similarity
e606e590e339e277e235e486e234e236e332
710e232e333e283667
e346780616520
e229e136e132945e181625541
e591e450e275960
e497e340e336e274905
e595e453e224493
e503613
e603e600e451e598e324e261469825755679676418775725715472
e300652e128421424400e175628e501568
1050664661
e402e228e331e195e196e327e325700
e282885785850745685649433
e487e237795571
e504e61
e231e94691e68835481
e602e459e454e262760e301e177e179e492e2685295381180985740805592790532508631610
e589855e1921030e233487770619547
e597e488e485e498e338e341e496e493e272e182e198e502e126655
e230682559478800526475e281e131705765e74e73
e130e481e335e362e302e276e500e129e135e76607940517646640643
Protistan OTU Bacterial OTU
Kendall Rank Similarity
e606e590e339e277e235e486e234e236e332
710e232e333e283667
e346780616520
e229e136e132945e181625541
e591e450e275960
e497e340e336e274905
e595e453e224493
e503613
e603e600e451e598e324e261469825755679676418775725715472
e300652e128421424400e175628e501568
1050664661
e402e228e331e195e196e327e325700
e282885785850745685649433
e487e237795571
e504e61
e231e94691e68835481
e602e459e454e262760e301e177e179e492e2685295381180985740805592790532508631610
e589855e1921030e233487770619547
e597e488e485e498e338e341e496e493e272e182e198e502e126655
e230682559478800526475e281e131705765e74e73
e130e481e335e362e302e276e500e129e135e76607940517646640643
Protistan OTU Bacterial OTU
0.1
Kendall Rank Similarity
e606e590e339e277e235e486e234e236e332
710e232e333e283667
e346780616520
e229e136e132945e181625541
e591e450e275960
e497e340e336e274905
e595e453e224493
e503613
e603e600e451e598e324e261469825755679676418775725715472
e300652e128421424400e175628e501568
1050664661
e402e228e331e195e196e327e325700
e282885785850745685649433
e487e237795571
e504e61
e231e94691e68835481
e602e459e454e262760e301e177e179e492e2685295381180985740805592790532508631610
e589855e1921030e233487770619547
e597e488e485e498e338e341e496e493e272e182e198e502e126655
e230682559478800526475e281e131705765e74e73
e130e481e335e362e302e276e500e129e135e76607940517646640643
Protistan OTU Bacterial OTU
Kendall Rank Similarity
e606e590e339e277e235e486e234e236e332
710e232e333e283667
e346780616520
e229e136e132945e181625541
e591e450e275960
e497e340e336e274905
e595e453e224493
e503613
e603e600e451e598e324e261469825755679676418775725715472
e300652e128421424400e175628e501568
1050664661
e402e228e331e195e196e327e325700
e282885785850745685649433
e487e237795571
e504e61
e231e94691e68835481
e602e459e454e262760e301e177e179e492e2685295381180985740805592790532508631610
e589855e1921030e233487770619547
e597e488e485e498e338e341e496e493e272e182e198e502e126655
e230682559478800526475e281e131705765e74e73
e130e481e335e362e302e276e500e129e135e76607940517646640643
Protistan OTU Bacterial OTU
0.1
Rank correlation of occurrence of OTU
Bacterial - Protistan Relationships
Relates Prokaryotic-Eukaryotic Ecology
2,224 clones
(400 – 650 bp ea.)Ciliophora
Dinophyceae
PolycystineaStreptophyta
Haptophyceae
Stramenopiles
Perkinsea
Euk; env.
Choanoflagellida
ApusomonadidaeEuglenozoa Apicomplexa
Glaucocystophyceae
Ichthyosporea
Cryptophyta
Cercozoa
Fungi
Chlorophyta
Acantharea
M.O. 2001ARB Tax.
Phylogenetic breakdown of Euks in 18S libraries from the time-series.
What about species diversity?Protistan taxa are morphologically defined. You might think that would be an advantage,and yet…
-Complexity of taxonomy(ies)multiple fixation proceduresmultiple analytical proceduresdiverse taxonomic characters
-Deficiencies of taxonomysmall species (few characters)morphologically amorphous speciesconvergent evolution
-Demands of ecological researchhigh sample numbercomplexity of natural assemblages
• 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.
There is a need to develop practical guidelines for defining OTUs for protistan taxa based on rDNA sequence information.
Caveats:-This will not resolve the issue of the ‘species concept’.-Ultimately, multiple gene sequences will provide identity.
Our approach:
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Percent Similarity
Number of Taxonomic Units
95%, 165 OTUs
Consequences of varying percent similarity for OTU calling.(application to real data)
Results for 970 environmental 18Sclone sequences from a sample inthe Coastal western North Atlantic
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1 19 37 55 73 91 109 127 145 163 181 199 217 235 253 271 289 307 325 343 361 379 397 415 433 451 469 487
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‘Taxon-level’ distinction≈1200 18S clones(Single date, 6 depths, USC M.O. site)
Taxonomic Units
*Large Euk diversity (488 OTUs; 95% similarity: pairwise alignments).
*Most OTUs are rare (large number of ‘background’ of taxa).
Fre
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f T
axon
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Phylotype Rank
Phylotype Abundance
Countway et al. (2005), GenBank accession AY937465-AY938434
Study in Coastal N. Atlantic72-hr bottle incubationNatural 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 distributionor
Endemism?
Global distributionor
Endemism?Jury
still
out!
Target Organisms – Caron Target Organisms – Caron LabLab
Phaeocystis (Haptophyte)
Lingulodinium (Dinoflagellate)
Ostreococcus (Chlorophyte)Chrétiennot-Dinet et al., 1995
Pseudonitzschia(Diatom)
The Daily Breeze: May 12, 2002
Cryptophytes4%
Ostreococcus9.1%
Stramenopiles2%
Haptophytes4%
OtherChlorophytes
9.1%
Dinoflagellates38.2%
Ciliates20.0%
Unclass. Eukaryotes14.5%
Ostreococcus T-RFLP signatureat the Chl a Max: July 2001
9.8%
10.7%
11.3%
Percent of total amplified DNA
Caron, Countway & Brown (2004)
Comparison to flow cytometry…Comparison to flow cytometry…
In parting, two popular microbial myths…(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 ecological tools)
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.
