From populations to genomes: Extensive genotypic diversity in a natural bacterioplankton population Janelle R. Thompson Massachusetts Institute of Technology

Outline Introduction Microbial diversity and organization “Vibrio spendidus” model system Plum Island Sound, MA and Barnegat Bay NJ Population dynamics Genomic Diversity Ecological and evolutionary considerations

We live on a microbial world Credit M.Polz 70% Ocean!! (Whitman et al. 1998) x10 30 organisms Pg carbon Estimates Microbes are the most abundant living organisms on the Earth

Credit M.Polz 70% Ocean!! (Whitman et al. 1998) x10 30 organisms Pg carbon Microbes are the most abundant living organisms on the Earth We live on a microbial world - Diverse metabolism - Global processes Estimates

Complex Microbial Communities Microbial Assemblages A. SEM B. Light microscopy with fluorescent-stain A B System response 1 µm Sanitation P. Franks Harmful algal blooms Nutrient cycling DeLong & Karl Structure and Function Relationship?

Marine Assemblages A. SEM B. Light microscopy with fluorescent-stain A B Community Diversity DNA/RNA Isolates Evolutionary Relationships Biomarker genes e.g. 16S ribosomal RNA (ribotypes) ~100% community <0.1 to 10% community Accessible only by molecular methods

Genetic diversity (e.g. 16S rRNA ribotypes) How do they work together to mediate activities in the environment? Individuals Populations Community Community Organization?

Based on work by F. Cohan Co-existing variation Selective Sweep Diversification fitness advantage Population theory informs search… sequence cluster The challenge: identifying functional units in natural communities Community Biomarker Tree

Organization of a bacterioplankton community Plum Island Sound, MA 0.05 subst/site 0.01 subst/site 0.1 subst/site 16SrRNA clone library Most of the ribotype diversity partitioned into sequence clusters with >99% identity =functional units? Acinas & Klepac-Ceraj, et al, Nature, 2004

Motivating Questions: I) Can we identify populations in microbial communities? II) How diverse are the individuals in microbial populations? III) What are the evolutionary forces that may drive the diversification and cohesion of natural populations?

A Test: Do sequence clusters have coherent environmental dynamics? I) Can we identify populations in microbial communities?

Quantification and Identification of Vibrios *Thompson et al., 2002, 2004 mapquest.com 4˚ to 27.5 ˚C Barnegat Bay, NJ

V.wodanis V. splendidus V. anguillarum V. pectenicida V. parahaemolyticus V. alginolyticus V. carchariea/harveyi V. coralilyticus V. shiloi Thompson, et al, subst/site V.logei/fischeri 16S rRNA Survey of Vibrio Diversity Barnegat Bay, NJ Culture-independent survey matched collections of cultured strains.

V.wodanis V. splendidus V. anguillarum V. pectenicida V. parahaemolyticus V. alginolyticus V. carchariea/harveyi V. coralilyticus V. shiloi Thompson, et al, subst/site >99% Clusters Aug 01 Dec 01 Feb 02 Aug 02 V.logei/fischeri 16S rRNA Survey of Vibrio Diversity Barnegat Bay, NJ Sequence clusters change with season and recur in summer

Vibrio Dynamics Clone Libraries V. pectenicida-like V. logei/wodanis cells/ml V. splendidus-1,2 CV ~10 to 25% Year-Round Vibrios (QPCR)

[Thompson, et al, AEM 2004] V. pectenicida-like V. splendidus-1,2 V. logei/wodanis cells/ml Coral pathogen V. parahaemolyticus V. spp. Vibrio Dynamics CV ~10 to 25% Clone Libraries Late-Summer Vibrios (QPCR)

[Thompson, et al, AEM 2004] Temperature relationship: summer and year-round Vibrios Gulf Stream Degrees C 1.Persistence 2.Currents Barnegat Bay, NJ cells/ml TEMPERATURE

Diversity and dynamics of Vibrio ribotype clusters Year-round and warm-water Vibrio ribotype clusters suggest differentiation with respect to seasonal parameters (e.g. temperature). √ Year-round and warm-water Vibrio ribotype clusters suggest differentiation with respect to seasonal parameters (e.g. temperature). Coherent environmental dynamics in √ Coherent environmental dynamics in V. parahaemolyticus-like cluster (>98% rRNA identity) V. splendidus-like cluster (>98% rRNA identity) --> Closer examination of V. splendidus I) Can we identify populations in microbial communities?

Plum Island Sound Ipswich, MA V. splendidus (>99% 16S rRNA) -one of 500+ microdiverse clusters [Acinas and Klepac-Ceraj, et al 2004] -seasonal population dynamics? -genomic diversity? -Do some genotypes have different dynamics? -1˚ to 16 ˚C II) How diverse are the individuals in microbial populations? I) Can we identify populations in microbial communities?

Isolate and analyze strains in ribotype cluster Internal standard Identification: clusters Higher resolution gene * Thompson et al., AEM 2004 Vibrio selective media Genome Fingerprint DNA Digest Gel

V. splendidus dynamics Plum Island Sound, MA QPCR Isolation Red = V. splendidus strains [Thompson, et al, Science 2005] V.splendidus ribotypes - Detected year round - Dominant isolate in summer - dynamics of genotypes (PFGE and Hsp60)?

- 333 strains isolated - 20 taxa of Vibrio and Photobacterium Vibrio splendidus 16S rRNA ribotypes V. splendidus diversity 99% Hsp60 sequences Genome-typing (PFGE) 87% unique N=206 60% unique N=232 AMOVA: Random distribution of sequence-types

Number of Hsp60 alleles: 141 (of 232 strains) Number of PFGE genotypes: 180 (of 206 strains) Chao-1 estimator: 100 to 300 Hsp60 alleles per month 500 to 900 PFGE genotypes per month at least 1,300 genomes overall What is the estimated diversity of “V. splendidus” genomes in the samples?

QPCR estimation of “V. splendidus” population size: - summer months is 640 to 1,890 cells/ml Genome concentration* = (population size/diversity) - each month 2 to 15 cells/ml share identical Hsp60 alleles - on average <1 cell/ml identical genome* *Conservative estimate: - Chao-1 predicts minimum diversity (richness) - “culture biases” would underestimate diversity (richness) What is the estimated diversity of “V. splendidus” genomes in the samples?

* Based on the Chao Genomes* 100 Hsp60 types* 1 16S rRNA cluster 1 ml What is the estimated diversity of “V. splendidus” genomes in the samples? Abundance: 10 3 cells/ml Diversity: 10 3 genome types/ml

Genome sizes (4.5 to 5.6 Mb) ~1000 genes Isolates paired by identical Hsp60 sequences Some diversification is due to large-scale genome changes Size variation among “V. splendidus” genomes Relationships of Hsp60 sequences spectrum of observed diversity How are genomes differentiated?

What drives genome diversification? Elements In Elements Out Duplication Horizontal gene transfer -homologous recombination -mobile genetic elements: -phage-related gene clusters ->1% ORFs are integrases or transposases in strain 12B01 Gene Loss Dynamic genome size & content GENOME

What drives population cohesion? Considerations: (A) Frequency of recombination increases with sequence similarity (B)“Microdiverse” organisms may have access a shared genetic pool. (A)(B) Are microbial populations genetically cohesive via biospecies-like evolution? gene transfer among closely related strains Fraser, Science 2007

Mechanisms for co-existing diversity 1) Genomic variants represent ecologically distinct populations consistent with niche theory. 2) Genome variation does not confer a time-averaged fitness advantage in a stochastic environment. Variation is neutral Variation is neutral Variation is contextually-neutral i.e. it may be under selection in alternate unknown environments Variation is contextually-neutral i.e. it may be under selection in alternate unknown environments Variation affects fitness; is maintained by balancing selection Variation affects fitness; is maintained by balancing selection (e.g. kill the winner, environmental heterogeneity) ---> no single genotype may gain a lasting growth advantage.

Conclusions I) Can we identify populations in microbial communities? II) How diverse are individuals in microbial populations? III) What are the evolutionary forces that may drive the diversification and cohesion of natural populations?

Conclusions I) Can we identify populations in microbial communities? Microdiverse ribotype clusters Coherent environmental dynamics II) How diverse are individuals in microbial populations? III) What are the evolutionary forces that may drive the diversification and cohesion of natural populations?

II) How diverse are individuals in microbial populations? Conclusions I) Can we identify populations in microbial communities? V. splendidus 10 3 genome types/10 3 cells ml -1 heterogeneity up to ~1000 genes Microdiverse ribotype clusters Coherent environmental dynamics III) What are the evolutionary forces that may drive the diversification and cohesion of natural populations?

II) How diverse are individuals in microbial populations? Conclusions I) Can we identify populations in microbial communities? V. splendidus 10 3 genome types/10 3 cells ml -1 heterogeneity up to ~1000 genes Microdiverse ribotype clusters Coherent environmental dynamics - Diversity within a population: balance of HGT & cohesion by recombination. - Vast genomic variation may be contextually neutral or adaptive III) What are the evolutionary forces that may drive the diversification and cohesion of natural populations?

Collaborators Dr. Martin Polz Polz Lab: Sarah Pacocha Vanja Klepac-Ceraj Chanathip Pharino Dana Hunt Jennifer Benoit Ramahi Sarma-Rupavtarm Dr. Luisa Marcelino Dr. Aoy Tomita-Mitchell Dr. Ee Lin Lim (Temple University) Dr. Daniel Distel (Ocean Genome Legacy, New England Biolabs) Dr. William Thilly (MIT) Acknowledgements Funding National Science Foundation Seagrant Department of Energy Joint Genome Institute

Questions?

EnvironmentUnits Richness (model) Reference Human GI tract - per individual 99% 16S rRNA 164 to 332 (chao1) Eckburg et al. Science 2005 Sargasso seawater L 100%16S rRNA 94% rpoA 1,412 (observed) ~1000 (chao1) Venter et al., Science 2004 Plum Island Sound seawater - 1L 100% 16S rRNA 99% 16S rRNA 1633 (chao1) 520 (chao1) Acinas and Klepac-Ceraj et al., Nature 2004 Hypersaline microbial mat 100% 16S rRNA % 16S rRNA 1,336 (observed) >10 4 (chao1 or ACE) Ley R. E. et al., AEM 2006 Salt marsh sediment - 5g 99% 16S rRNA 2411 ± 542 (pareto distribution) Hong et al., PNAS 2006 Marine sediment Forest soil genomesgenomes 1.1 x (DNA reassociation) Torsvik et al. J. Biotech 1998 Soil - 10g genomes 8.3 x 10 6 (DNA reassociation & power law distribution) Gans et al. Science How diverse are natural microbial communities?

Biological species Evolutionary species Ecological species Speciespluralism Units of Biology Reproductive Isolation Ecological niche Single lineage (Multiple definitions relevant!)

Three E. coli strains share <40% of total protein genes in genomes Welch et al. (2002) strains from different environments Would strains co-exist in nature outside a human host? Core genome: - shared by all (e.g., housekeeping) Flexible genome: - strain specific (e.g., pathogenicity islands, integrons) Are co-occurring genomes with same “ribotype” ecologically-equivalent?

Stackebrandt and Goebel, S rRNA similarity Genome similarity Challenge: To identify ecologically-differentiated populations Correlation of ribotype to genome similarity 99% 16S rRNA 70% DNA-DNA hybridization