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Microbial Inventory Research Across Diverse Aquatic Long Term Ecological Research Sites

MIRADA LTERs is carrying out a Microbial Biodiversity Survey and Inventory across all the major aquatic (marine and freshwater) Long Term Ecological Research (LTER) sites established by the NSF’s US LTER Program. Our inventory takes advantage of the aquatic sampling locations that are part of the established LTER network of sites and builds on existing infrastructure for coordination at the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts set in place by the Alfred P. Sloan Foundation supported Census of Marine Life (CoML) ocean realm project called the International Census of Marine Microbes (ICoMM).


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Our Microbial Inventory Research Across Diverse Aquatic (MIRADA) LTERs has adopted ICoMM’s massively-parallel, 454-based rDNA tag sequencing strategy that allows extensive sampling of both common and rare members of microbial populations and provides a common metric for integrating studies of microbial diversity across aquatic LTER sites. This methodology not only enables cross-site comparisons, but also provides valuable baseline data for integrating population structures with ecosystem change, and understanding microbially-mediated trophic dynamics and biogeochemical processes - areas of study already underway at many of the LTERs.


To view a status report of the MIRADA LTERs progress click here.


Our specific objectives are to:

     1. Document and describe both microbial (Bacteria, Archaea and Eukarya) baseline diversity and relative abundance data for microbial operational taxonomic units (OTUs) as defined by SSU rDNA hypervariable tags at aquatic LTER sites.

     2. Determine which microbial OTUs are common to both freshwater and marine LTERs.

     3. Determine whether diverse aquatic LTER sites possess “signature” assemblages characterized by space, time and environmental parameters.

     4. Discover novel tag sequences that likely represent novel microorganisms that LTER researchers and students can further characterize and study.


MIRADA Collaborators and Contacts (LTER PIs in bold):

Arctic: John Hobbie (MBL), Byron Crump, Joanna Green (Horn Point Laboratory), George Kling (U Michigan)

California Current Ecosystems: Mark Ohman, Brian Palenik, Farooq Azam (U California, San Diego -UCSD)

Coweeta: Ted Gragson, Catherine Pringle, John Frisch∗ (U Georgia, Atlanta -UGA)

Florida Coastal Everglades: Daniel Childers, Joseph Boyer, Christina Pisani∗ (Florida International University)

Georgia Coastal Ecosystems: Tim Hollibaugh, Mary Ann Moran, Melissa Booth, Scott Gifford∗, Merryl Alber (UGA)

Hubbard Brook: Charles Driscoll (Syracuse University), Don Buso (Institute for Ecosystem Studies), Gene Likens (Cary Institute for Ecosystem Studies)

McMurdo: John Priscu, Amy Chiuchiolo (Montana State U), Brian Lanoil, Chao Tang∗, Hans Sukkyun∗∗ (U California at Riverside-UCR), Michael Madigan (Southern Illinois University)

Moorea Coral Reef: Russell Schmitt, Alice Alldredge, Craig Carlson, Andrew Brooks (U California, Santa Barbara-UCSB), Jan Witting (SEA)

North Temperate Lakes: Stephen Carpenter, Trina McMahon, Todd Miller ∗∗(U of Wisconsin)

Palmer Station: Hugh Ducklow, Kristen Myers∗ (MBL) Alison Murray (Desert Research Institute)

Plum Island: Chuck Hopkinson (UGA), Anne Giblin, Aaron Strong (MBL), Martin Polz, Sarah Preheim∗ Jennifer Benoit∗(MIT)

Santa Barbara: Dan Reed, Craig Carlson (UCSB)

Virginia Coastal Ecosystems:Karen McGlathery, Linda Blum, Aaron Mills (U Virginia)

∗= graduate student collaborators; ∗∗postdoctoral collaborators.


MIRADA Methods and Protocols:

Our sampling protocol involves selecting 8 sites per LTER and sampling these in duplicate for each of the three domains of life- for a total of 48 samples per run of the GS-FLX 454 machine. Sampling for MIRADA involves sterile collection of 1 or more liters of water followed by processing of the samples onto 0.22 µm sterivex filters for DNA extraction, filtration of a portion of the sample onto membrane filters to provide an archive sample for future Fluorescence In Situ Hybridization (FISH), and archival cryopreservation of a portion of the samples in both DMSO and glycerol for future culturing. Click on the links below to view protocols we have established for our collaborators to help collect samples for the MIRADA project:

Sterivex filtration

Sterivex filtration movie

Sterivex manifold components

Sterivex DNA extraction

All MIRADA voucher DNA samples and cryopreserved samples are available upon request to the lead PI. MIRADA cryopreserved samples are being deposited with Dan Distel at the Ocean Genome Legacy.
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Sampling descriptions:


Arctic (ARC)
Samples for the Arctic LTER will explore the major imageseasonal bacterioplankton communities in Toolik Lake, an ultraoligotrophic tundra lake on the North Slope of Alaska. These seasonal communities comprise “winter” communities collected through the ice in April/May, early summer communities collected soon after the spring thaw period, and late summer/fall communities. Samples are being collected near surface in the epilimnion (3 m depth) and also from the hypolimnion (16m depth).
Also included are two samples from a major stream in the watershed of Toolik lake that arises from the tundra but does not include upstream lakes. This will capture the diversity of microbes advected to lakes from the watershed.


California Current Ecosystem (CCE)
Samples for MIRADA will be obtained in Auguimagest this year on a California Cooperative Oceanic Fisheries Investigations (Calcofi) cruise. These samples will capture spatial diversity of microbes from coastal to open ocean along a productivity gradient.

Coweeimageta (CWT)
 One of the oldest continuous environmental studies in North America, Coweeta is based in the eastern deciduous forest of the southern Appalachian Mountains. This LTER is the centerpiece of a longterm cooperation between the Univeristy of Georgia and the USDA Forest Service. MIRADA will sample watershed sites within Coweeta.

Florida Coastal Everglades (FCE)
FCE-LTER sites have been chosen to best imagerepresent sub-ecosystem types. The Everglades have two main watersheds: the large Shark River Slough (SRS-2 and 4) which empties into the Gulf of Mexico and the much smaller Taylor Slough (TS-2 and 6) which drains the SE Everglades and drains into Florida Bay. We selected 2 sites in each watershed; one was located in the freshwater sawgrass marsh while the other was in the mangrove ecotone (oligohaline zone). These 4 sites are being sampled in the wet and dry seasons to get a representative sampling of FCE microbial diversity over two seasons.

Georgia Coastal Ecosystems (GCE)
The GCE sites were chosen to encompass the imagebreadth of ecological and hydrological characteristics of the GCE-LTER system, and hopefully the broadest diversity of microorganisms. Within this sampling scheme, there are two themes: one relating to differences at high and low tide, and a second to differences in the nature and flow of water within the GCE-LTER site. Four sites (Doboy sound, Tolomato development, Altamaha River and Sapelo Sound) were sampled at high and low tide on the same day for the MIRADA project sample set.

Hubbard Brimageook (HBR)
The Hubbard Brook Ecosystem study sustains long-term ecological research within the Hubbard Brook Experimental Forest, a 3,160 hectare reserve in the White Mountain National Forest, New Hampshire, managed by the USDA Forest Service Northern Research Station. On-site research has produced some of the most extensive and longest continuous databases on the hydrology, biology, geology and chemistry of a forest and its associated aquatic ecosystems. MIRADA sampling at Hubbard will take place in July of 2008 and will focus on Mirror Lake and nearby Cone Pond.




McMurdo (MCM)
Samples for 454 sequencing for the Dry Valleys of imagethe McMurdo LTER included Lake Fryxell at 6 and 9 meters depth and West Lobe Bonney at depths of 13 and 18 meters in November of 2007 and April, 2008. Samples from these sites were coupled with samples collected in conjunction with the Dry Valley Lakes Microbial Observatory. The west lobe of Lake Bonney (WLB) is characterized by high concentrations of DMSP- values that are over 100 times the average for seawater. Unlike Lake Bonney, Lake Fryxell (LF) has a highly reducing environment below the chemocline, with evidence for sulfate reduction, methanogenesis, and anoxygenic photosynthesis.

Moorea Coral Reef (MCR)
Moorea sampliimageng included both off-shore and near-shore sampling to evaluate the change in microbial diversity and community structure along a gradient of DOC and nutrient concentrations. Near shore sampling encompassed 5 of the LTER sampling locations that are regularly monitored by the MCR LTER program. Off-shore samples extended the gradient into more oligotrophic waters via samples collected off of the Sea Education Association’s sailing vessel the Robert C. Seamans. MIRADA postdoctoral fellow Elizabeth McCliment participated directly in the off-shore sampling in conjunction with the sea-going component of an undergraduate field program sponsored through the University of San Diego at the GUMP station on Moorea in January of this year. The MIRADA data will complement TRFLP and clone library data available from collaborator Craig Carlson and colleagues, as well as metagenomic library data for Moorea samples collected during the Venter GOS expedition.


North Temperate Lakes (NTL)
 NTL will be collecting samples at a single time imagepoint from five lakes in Wisconsin in early July 2008. The lakes were chosen because they span several important limnological gradients such as size, depth, trophic status, landscape position, and pH. MIRADA samples will be collected from the epilimnia of four lakes and the hypolimnia of four lakes. One lake is always mixed. These lakes are core to the North Temperate Lakes Long Term Ecological Research (NTL-LTER) site. There are extensive long-term datasets (10-20 years or more) for physical, chemical, and biological limnological parameters. Four of the lakes have instrumented buoys on them as part of the GLEON project (CB, TB, TL, ME). Two of the humic lakes (CB and TB) and one eutrophic lake (ME) have been studied extensively by the NTL-MO group.

Palmer Station (PAL)
Samples for Palmer Station targeted the 200 and image600 grid lines at both an off- and near-shore station. The rationale for choosing these corners was to capture the potential spatial variability in the communities of the northern and southern ends of the grid, as well as the variability in the off- and near-shore stations. The near-shore stations typically have higher primary and bacterial productivity associated with them. The near-shore stations of the 200 line are off the southern end of Adelaide Island extending into Marguerite Bay, which is very highly productive, often much more than the near-shore 600 line. Each station was sampled at the surface and a depth of ~100 m to capture the variability in the communities over depth.

Plum Island (PIE)
MIRADA sampling from the Plum Island imageSound Estuary enable a spring (April) and fall bloom (September) comparison along an estuarine salinity gradient. Four samples collected in replicate from the spring 2006 and fall of 2007 are being employed for tag sequencing. Members of the Polz Laboratory have already carried out one of the most comprehensive studies of bacterioplankton diversity in this estuary (~1,000 clones from a 16S rRNA library constructed from a single sample) and showed that about half of these sequences were unique.

Santa Barbara Coastal (SBC)
MIRADA sampling at the Santa Barbarimagea LTER maximizes the seasonal gradients in productivity associated with the physical transitions i.e. upwelling, summer conditions and autumn DOM gradients inshore to offshore. Samples are being collected during three separate cruises as part of the SBC near shore transect campaign under our SBC-LTER program led by Craig Carlson. This campaign was designed to assess inshore to offshore gradients in a variety of biogeochemical parameters and was motivated by the strong gradient in DOM concentrations observed in the autumn from inshore into the SB channel. The sampling stations consist of 5 stations extending approximately 1.5 miles from a kelp bed into the SB channel. MIRADA samples are collected from two stations one close to the kelp forest and one extended into the SB channel.
Virginia Coastal Reserve (VCR)
VCR sites chosen for MIRADA are sampled each imagemonth and analyzed for 13 water quality variables. Two of the sites have been sampled since 1992, while the other two sites have been sampled since 2004. The specific sites selected represent a transect from the mainland to an ocean inlet in the Machipongo watershed. One of the questions VCR addresses is “How do fluxes of organisms and materials across the landscape influence ecosystem dynamics and state change?”. WQ monitoring along this transect directly addresses this question by measuring the concentration of nutrients, sediments, and cholorphyll. The ability to calculate water flux enables the calculation of flux of these materials to the coastal ocean.

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