454 Sequencing of Microbial Communities in Hood Canal

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ICOMM proposal for the application of 454 Tag Sequencing in Hood Canal, WA Rocap & Horner-Devine

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Seattle

Hood

Canal

Sisters Point

Hama Hama

A

B

Seattle

Hood

Canal

Sisters Point

Hama Hama

A

B

Figure 1. A) Location of Hood Canal in Puget

Sound, Washington. B) Oxygen concentrations

in Hood Canal in August, 2006 (measured andinterpolated). Stars indicate proposed sites for

this study. (Figure courtesy of HCSEG and UW

SAL lab).

LIVING IN THE DEAD ZONES:

454 SEQUENCING OF MICROBIAL COMMUNITIES IN HOOD CANAL,WAGabrielle Rocap ([email protected]) and Claire Horner-Devine ([email protected])

University of Washington, Seattle, WA, USA.

ABSTRACTWe propose to use 454 Tag Sequencing in Hood Canal, a narrow fjord in Puget Sound, Washington, to

examine the relative influence of biotic and abiotic parameters (including dissolved oxygen, nutrients,chlorophyll a, salinity, temperature and phytoplankton community composition and abundance) on the

ecology of marine bacteria. This work leverages an ongoing study of spatial and temporal patterns of

bacterial community composition (using ARISA) relative to changing phytoplankton communities andabiotic conditions, including dramatic seasonal shifts in dissolved oxygen. The 454 sequencing approach

will greatly expand our ability to access the diverse estuarine bacterial community in Hood Canal beyond

the dominant and most easily amplified community members. Most importantly, we will be better able toaddress questions about whole community shifts as well as shifts in the abundance of particular taxa over

time and in response to changing environmental conditions.

INTRODUCTIONAlthough there may be millions of bacterial species on Earth,

and as many as 4-6 x 1030 individuals (Whitman et al. 1998),we are only beginning to investigate how bacterial diversity

is distributed in space and time (Horner-Devine et al. 2004;

Martiny et al. 2006). We are also starting to understandfeedbacks among bacterial community composition,

dynamics of individual taxa and environmental conditions

and processes. In particular, there is a growing appreciationof the enormous diversity and role of very rare members of

the community (Sogin et al. 2006).

COASTAL HYPOXIA AND MICROBES The phenomenon of

hypoxia/anoxia is widespread, occurring in the waters of 16

of the 21 coastal United States. In the most well knowncase, the Gulf of Mexico, the hypoxic area along the

Louisiana and Texas coasts measured 20,700 km2

in the

summer of 2001 (Rabalais et al. 2001). Where dissolvedoxygen is greatly reduced (hypoxia, < 2 mg/l) or eliminated

(anoxia, 0 mg/l), these so-called dead zones exclude fish,

shrimp and benthic organisms and eliminate importanthabitat for many commercially and recreationally important

marine species (Officeret al. 1984). Dead-zones, however,

are far from dead. Hypoxic and anoxic zones support an

active community of anaerobic and hypoxia-adaptedorganisms including bacteria, archaea, protists and viruses.

Despite the increase in the number of hypoxic and anoxic

regions world-wide and the impact these low oxygen regionscan have on biological communities and commercial

fisheries, we know relatively little about the microbes

driving these processes and living in the dead zones.Hood Canal is a glacial fjord in Puget Sound located

about 80 miles from Seattle (Fig. 1). The long narrow shape

of Hood Canal results in a classic estuarine circulation pattern and a stratified water column throughoutmuch of the year. Hood Canal has also been shown to be more susceptible to eutrophication than other


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20072006

Hama Hama/Duckabush

200720062005

Sisters Point/Twanoh

20072006

Hama Hama/Duckabush

20072006

Hama Hama/Duckabush

200720062005


200720062005


Figure 2. Continuousoxygen and chlorophyll

data from ORCA buoys at our two study sites.

htt ://orca.ocean.washin ton.edu/index.html

phytoplankton, microbial processes and environmental characteristics. Major lines of inquiry that we willalso assess with data from 454 tag sequencing are as follows: i)How do bacterial richness and

composition vary over time?; ii) Do microbial richness and community composition vary with abiotic and

or biotic factors?; iii) Do some taxa co-occur less often than expected by chance?; and iv) Are specifictaxa associated with changes in dissolved oxygen conditions or other environmental factors?

New insights from 454 tag sequencing We will use data from the pyrosequencing approach to address

four additional lines of inquiry that will greatly enrich the above framework.

1) 454 sequencing may allow us to examine whether abiotic and biotic environmental changes in spaceand time are associated with particulartaxa shifting from rare to dominant and vice versa. When such

shifts occur, are they associated with large community shifts or of only a subset of taxa at a given time?

2) We will examine whether ecological patterns differ for abundant versus rare taxa. We plan to use a

clustering algorithm to designate taxa as rare versus abundant in each sample (alternatively, should distinctcluster not be discernible, we will use a range of cutoffs in abundance to define rarity) and then compare

patterns for each community component. In other words, we will address our ongoing questions listed

above for whole community data as well as for the rare community and the abundant community. We willalso address hypotheses that predict survival advantages for rare species due to decreased predation

pressure and competition. For example, we plan to use measures of co-occurrence to detect possible

signals of competition on different subsets of the community (Horner-Devine et al. 2007).

3) We will also use the new datasets to examine the influence of undersampling, and thus the exclusion ofrare taxa, on observed ecological patterns. We will sub-sample the 454 sequence sets from each sample insilico to simulate a range of degrees of undersampling and repeat the analyses proposed here on these

subsets (see Horner-Devine et al. 2007 for an example on well-sampled macrobial communities).

4) The comparison of the complete 454 dataset with ARISA datasets from the same samples will allow us

to assess the influence of more thorough sampling on observed patterns and potentially give us insight inhow best to interpret the current literature. We hope that this comparison will allow us to generate

hypotheses from the subset of sixteen 454 samples to test in future work from the larger set of samples.

EXPERIMENTAL PLAN

Field site Our two sampling locations in Hood Canal,Hama Hama and Sisters Point (Fig.1 B), are two of thesix locations targeted by an intensive three year study by

the Hood Canal Dissolved Oxygen Program (HCDOP,

http://www.hoodcanal.washington.edu/). This partnershipbetween state, federal, tribal, local and university

scientists was created in 2003 in response to the lowdissolved oxygen conditions. Thus we have access to

several years of data (collected on weekly and monthly

frequency) to inform our choice of four time periods overthe annual cycle to target for 454 sequencing. In addition,

our sampling plan (Table 1) takes advantage of anupcoming collaborative cruise (September, 2007) with Dr.

Andrew Allen of the J. Craig Venter Institute, who will becollecting samples in Hood Canal for a high-throughput

shotgun sequencing library. The opportunity to use 454

tag sequencing to exhaustively sample bacterialcommunity diversity on the same samples used for

creation of a metagenomic library will be invaluable.

Physical, Chemical and Biological data The two

sampling locations have an outstanding amount of


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environmental data available due to the presence of an ORCA buoy at each site (Dunne et al. 2002). Thesemoored profilers provide a continuous record of temperature, salinity, chlorophyll and oxygen throughout

the water column (Fig. 2). Thus we are able to examine conditions before and after our sampling periods.

In general, Hama Hama deep waters are depleted in oxygen, but levels do not fluctuate greatly over theyear. In contrast, Sisters Point exhibits a more pronounced seasonal cycle of severe hypoxia (


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similarity in bacterial community composition, phytoplankton community composition and similarity inenvironmental conditions between pairs of samples (Legendre 2000). Before analysis, data will be

examined for normality. Where appropriate, we will transform data or use non-parametric statistics. We

will assess co-occurrence using Stone and Roberts (1990) C-score (e.g., Horner-Devine et al. 2007).

PROJECT MANAGEMENTAll samples and ancillary data for our April and June 2007 time points have been collected, although DNA

has not been extracted from the filters. Samples and ancillary data for September and December 2007 will

be collected as part of our HCDOP microbial study, regardless of the outcome of this proposal. Thus earlyin 2008 we will have DNA in hand to send to MBL for library construction. Once pyrosequencing is

completed, Rocap will be responsible for bioinformatics to create a pipeline for initial analysis of sequence

tags, and Horner-Devine will take the lead on the statistical analyses of community data.

SUMMARYThe work proposed here will allow us to assess the impact of the rare biosphere on observed community

patterns for marine bacteria. Our four lines of inquiry focus on assessing the dynamics and patterns of rare

versus abundant taxa and allow us to compare our results with our larger ongoing study of microbialdiversity. Hood Canal and its low oxygen conditions render it an especially exciting marine environment

in which to examine microbial communities. In addition, any insight into the bacterial dynamics in Hood

Canal can be placed in the larger context of chemical, physical and other biological data and models tohelp us understand the impact of microbial communities on the system as a whole. Finally, the questionsand approaches we propose here apply to other microbial systems and can be used to generate hypotheses

about the influence of the rare biosphere on microbial ecological theory.

REFERENCESClarke K.R. & Warwick R.M. (2001) Change in Marine Communities: an Approach to Statistical Analysis

& Interpretation. 2 edn. PRIMER-E Ltd, Plymouth Marine Laboratory, UK.

Collias E.E., et al. (1974) Atlas of Physical & Chemical Properties of Puget Sound & its Approaches. In:Washington Sea Grant Publication. University of Washington Press, Seattle, WA

Dunne J.P., et al. (2002) The Oceanic Remote Chemical/Optical Analyzer (ORCA) - An autonomous

moored profiler.J. of Atmos. & Oce. Tech., 19, 1709-1721

Fisher M.M. & Triplett E.W. (1999) Automated approach for ribosomal intergenic spacer analysis ofmicrobial diversity & its application to freshwater bacterial communities.AEM, 65, 4630-4636

Horner-Devine M.C., et al. (2004) An ecological perspective on bacterial biodiversity. PNAS, 271, 113-122

Horner-Devine M.C., et al. (2007) A comparison of taxon co-occurrence patterns for macro- &microorganisms.Ecology, 88, 1345-1353

Legendre P. (2000) Comparison of permutation methods for the partial correlation & partial Mantel tests.J. of Statistical Comp. & Sim., 67, 37-73

Magurran A.E. (2004) Measuring Biological Diversity. Blackwell Science Ltd, Malden, MA.

Martiny J.B., et al. (2006) Microbial biogeography: Putting microorganisms on the map.Nature Rev.Micro., 4, 102-112

Newton J.A., et al. (1995) Dissolved oxygen concentrations in Hood Canal: are conditions different than

forty years ago? In:Puget Sound Research '95 Proceedings (ed. Robichaud E). Puget Sound WaterQuality Authority, Olympia, WA

Officer C.B., et al.(1984) Chesapeake Bay Anoxia-Origin, Development,& Significance.Science,223,22-27Rabalais N.N., et al. (2001) Hypoxia in the Gulf of Mexico. J. of Envir. Quality, 30, 320-329

Sogin M.L., et al. (2006) Microbial diversity in the deep sea & the underexplored "rare biosphere". PNAS,

103, 12115-12120Stone L. & Roberts A. (1990) The checkerboard score & species distributions. Oecologia, 85, 74-79

Webb C.O. (2000) Exploring the phylogenetic structure of ecological communities: An example for rain

forest trees.American Naturalist, 156, 145-155Whitman W.B., et al. (1998) Prokaryotes: The unseen majority.PNAS, 95, 6578-6583

454 Sequencing of Microbial Communities in Hood Canal

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