The USC Microbial Observatory Department of Biological Sciences University of Southern California 3616 Trousdale Parkway, AHF 301 Los Angeles, CA 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

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

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 FCM Synechococcus FCM Picoeukaryotes FCM USC Microbial Observatory and San Pedro Ocean Time Series Sept Dec Aloricate ciliates Dinoflagellates Diatoms

Whole Bacterial Community Fingerprints Amplified 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 ARISA PCR primers Fluorochrome 23S rRNA gene Intergenic Spacer, Variable Length 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. 0.1 SAR 11 cluster alone - we estimate ~800 distinguishable sequence types at our coastal study site (Chao 1)- clustered into ~10 groups (ecotypes?) 0.1 ITS 16S 284 clones 138 from SPOTS SAR11 cluster (in San Pedro Channel)

ARISA Bins 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 Annual Bacterial Community Reassembly with Shahid Naeem, Columbia University Chl max

T-RFLP Eukaryote Seasonal Pattern USC M.O.HaeIII Digest Important tool for Correlating to ARISAs (prokaryote community structure) Typically fragments/sample.

T-RFLP Similarity Matrix Tool Results: RDP II website HaeIII TRFsDateJanAprMayJulOct (74)Jan (69)Apr (64)May (89)Jul (44)Oct

Kendall Rank Similarity 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) 0.1 Rank correlation of occurrence of OTU Bacterial - Protistan Relationships Relates Prokaryotic-Eukaryotic Ecology

2,224 clones (400 – 650 bp ea.) M.O ARB 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 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

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:

Consequences of varying percent similarity for OTU calling. (application to real data) Results for 970 environmental 18S clone sequences from a sample in the Coastal western North Atlantic

‘Taxon-level’ distinction ≈ S 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). Frequency of Taxonomic Unit

Countway et al. (2005), GenBank accession AY AY 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? Global distribution or Endemism? Jury still out!

Target Organisms – Caron Lab Phaeocystis (Haptophyte) Lingulodinium (Dinoflagellate) Ostreococcus (Chlorophyte) Chrétiennot-Dinet et al., 1995 Pseudonitzschia (Diatom) The Daily Breeze: May 12, 2002

Cryptophytes 4% Ostreococcus 9.1% Stramenopiles 2% Haptophytes 4% Other Chlorophytes 9.1% Dinoflagellates 38.2% Ciliates 20.0% Unclass. Eukaryotes 14.5% Ostreococcus T-RFLP signature at the Chl a Max: July % 10.7% 11.3% Percent of total amplified DNA Caron, Countway & Brown (2004)

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.