Transcript Document

The USC
Microbial Observatory
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
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
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
USC Microbial Observatory and San Pedro Ocean Time Series
N
20 km
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
2000
Dec
2003
Whole Bacterial Community Fingerprints
Amplified Ribosomal Intergenic Spacer Analysis (ARISA)
backed by Clone Libraries for ID. Phylogenetic resolution near “species” level
Fluorochrome
16S rRNA gene
ARISA
PCR primers
ARISA
PCR
Intergenic Spacer, Variable Length 23S rRNA gene
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?)
0.1
284 clones
138 from SPOTS
Annual Bacterial Community Reassembly
with Shahid Naeem, Columbia University
Discriminant Function
Analysis
‘Clockfaces’ - Months
are like hours on the
clock.
Chl
max
Radii represent
discriminant function
of taxa (a function of
community
composition).
Central line: mean
Dashed lines: range
over 3 years
1-50
51-100
ARISA Bins
100-150
T-RFLP Eukaryote
Seasonal Pattern
USC M.O.HaeIII
Digest
Typically 40-70 fragments/sample.
USC MO Chl a Max: Protistan HaeIII T-RFLP
350000
300000
250000
200000
150000
100000
50000
Oct-03
Jul-03
0
61
May-03
79
9
12
Jan-03
8
14
4
18
8
19
Aug-02
8
21
1
23
Apr-02
1
26
Fra
77
gm 2 292
4
en
32
tS
0
34
ize
(bp
)
Important tool for
Correlating to ARISAs
(prokaryote community structure)
Jan-02
Oct-01
9
35
5
37
Jul-01
9
44
8
47
Apr-01
4
49
8
50
Jan-01
0
59
6
60
T-RFLP Similarity Matrix Tool Results: RDP II website
HaeIII
TRFs
Date
(74)
Jan
(69)
Apr
0.53
(64)
May
0.54
0.59
(89)
Jul
0.67
0.58
0.63
(44)
Oct
0.55
0.50
0.44
Jan
Apr
May
Jul
Oct
0.53
0.54
0.67
0.55
0.59
0.58
0.50
0.63
0.44
0.56
0.56
Bacterial OTU
Protistan OTU
643
640
646
517
940
607
e76
e135
e129
e500
e276
e302
e362
e335
e481
e130
e73
e74
765
705
e131
e281
475
526
800
478
559
682
e230
655
e126
e502
e198
e182
e272
e493
e496
e341
e338
e498
e485
e488
e597
547
619
770
487
e233
1030
e192
855
e589
610
631
508
532
790
592
805
740
985
1180
538
529
e268
e492
e179
e177
e301
760
e262
e454
e459
e602
481
835
e68
691
e94
e231
e61
e504
571
795
e237
e487
433
649
685
745
850
785
885
e282
700
e325
e327
e196
e195
e331
e228
e402
661
664
1050
568
e501
628
e175
400
424
421
e128
652
e300
472
715
725
775
418
676
679
755
825
469
e261
e324
e598
e451
e600
e603
613
e503
493
e224
e453
e595
905
e274
e336
e340
e497
960
e275
e450
e591
541
625
e181
945
e132
e136
e229
520
616
780
e346
667
e283
e333
e232
710
e332
e236
e234
e486
e235
e277
e339
e590
e606
Bacterial - Protistan Relationships
•Bacterial and Protistan OTU usually cluster
within domains (e.g. protist with protist)
Protistan OTU
Protistan OT
0.1
•But certain Bacterial and Protistan OTU
covary most closely with each other
0.1
Relates Prokaryotic-Eukaryotic Ecology
0.4
Kendall Rank Similarity
0.5
0.6
0.7
0.8
0.9
1.0
547
e481
619
e130
770
e73
487
e74
e233
765
705
1030
e192
e131
855
e281
e589
475
610
526
800
631
478
508
559
532
682
790
e230
592
655
805
e126
740
e502
985
e198
1180
e182
538
e272
529
e493
e268
e496
e492
e341
e179
e338
e177
e498
e301
e485
760
e488
e262
e597
e454
547
e459
619
643
e602
770
640
481
487
835
e233
646
e68
1030
517
691
e192
855
e94
940
e589
e231
607
610
e61
e76
631
e504
508
643
571
e135
532
795
640
e129
790
e237
e500
592
646
e487
805
e276
433
517
740
649
e302
985
940
685
1180
e362
745
607
538
e335
850
e76
529
785
e481
e268
885
e135
e492
e130
e282
e129
e179
e73
700
e177
e500
e325
e74
e301
e327
e276
760
765
e196
e262
e302
705
e195
e454
e331
e459
e131
e362
e228
e602
e281
e335
e402
481
e481
475
661
835
664
e68
526
e130
691
1050
800
e73
e94
568
e501
478
e231
e74
e61
628
559
765
e175
e504
682
571
400
705
795
e230
424
e131
e237
421
655
e487
e128
e281
e126
433
652
649
475
e502
e300
685
472
526
e198
745
715
Rank correlation of occurrence of OTU
Apusomonadidae
Polycystinea
Streptophyta
Euk; env.
Choanoflagellida
Euglenozoa
Apicomplexa
Perkinsea
Glaucocystophyceae
Haptophyceae
Ichthyosporea
Cryptophyta
Cercozoa
Stramenopiles
Phylogenetic
breakdown
of Euks in
18S libraries
from the
time-series.
Acantharea
Dinophyceae
Chlorophyta
M.O. 2001
ARB Tax.
Fungi
Ciliophora
2,224 clones
(400 – 650 bp ea.)
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
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.
(application to real data)
450
99
Results for 970 environmental 18S
clone sequences from a sample in
the Coastal western North Atlantic
Number of Taxonomic Units
400
350
300
98
250
95%, 165 OTUs
200
97
96
95
94
93
92
91
90
89
88
86
87
85
150
100
50
30
0
20
40
40
45
50
55
60
65
70
60
Percent Similarity
75
80
80
100
200
200
Frequency of Taxonomic Unit
180
‘Taxon-level’ distinction
≈1200 18S clones
(Single date, 6 depths,
USC M.O. site)
180
160
160
140
140
120
100
120
80
100
60
80
40
20
60
0
1
40
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
20
0
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
Taxonomic Units
*Large Euk diversity (488 OTUs; 95% similarity: pairwise alignments).
*Most OTUs are rare (large number of ‘background’ of taxa).
Study in Coastal N. Atlantic
72-hr bottle incubation
Natural light; ambient temp.
70
Global distribution
or
Endemism?
Phylotype Abundance
60
50
970 clones analyzed.
165 Total phylotypes (95%).
40
68% (108 out of 165) observed
at only one sampling time.
30
Only 18% observed at all 3
sampling times.
20
10
0
1
11
21
31
41
51
61
71
81
91
101 111 121 131 141 151 161
Phylotype Rank
Countway et al. (2005), GenBank accession AY937465-AY938434
Target Organisms – Caron Lab
Phaeocystis (Haptophyte)
Lingulodinium (Dinoflagellate)
The Daily Breeze: May 12, 2002
Pseudonitzschia
(Diatom)
Chrétiennot-Dinet et al., 1995
Ostreococcus (Chlorophyte)
Ostreococcus T-RFLP signature
at the Chl a Max: July 2001
Percent of total amplified DNA
9.8%
10.7%
Cryptophytes
4%
Stramenopiles
2%
Haptophytes
4%
11.3%
Other
Chlorophytes
9.1%
Dinoflagellates
38.2%
Ostreococcus
9.1%
Unclass. Eukaryotes
14.5%
Ciliates
20.0%
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.