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###Research 4.2.3 Research  Questions (1/2 and Approach (2 1/4  page) Aim 1: A global census of microbial eukaryotes found in association with leaves, roots, and sediment of the seagrass Zostera marina.  Objectives:  We will investigate the global diversity of microbial eukaryotes found in Z. marina beds. As part of this investigation we seek to answer the following questions: Can we detect a phylogeographic signal of Zostera marina-associated microeukaryotes? What is driving microbial eukaryotic community compositions in Z. marina beds, the prescence of Z. marina (host plant) or environmental factors (geography, pH, temperature, etc)? Or is everything everywhere? Do we see specific patterns in either richness or diversity in these beds and do any of these patterns mimic those of macro-organisms known to frequent Z. marina beds?  **Aim 1: A global census of microbial eukaryotes found Sampling: We have,  in association with hand, DNA that has been extracted from 414 samples of Zostera marina  leaves, roots, and sediment (ZEN samples). These samples include 3 biological replicates  of the each sample type. Samples were take from two  seagrass _Zostera marina_.** beds at each of 22 sites in northern Europe, the Mediterranean, Japan, Australia, Central America, and both coasts of the United States. We have successfully amplified and sequenced the 16S ribosomal RNA genes of the microbial communities in these samples. Analysis of these samples is currently underway, and is showing very exciting results. We have also demonstrated success amplifying, sequencing, and analyzing eukaryotic microbial loci (fungal ITS) from samples prepared in the same way as the ZEN samples. We also have a wealth of metadata associated with each site: 109 measurements of plant traits (including biomass, productivity, and microsatellite loci), water and sediment characteristics, and abundances of invertebrate grazers and predator species.  Molecular Biology/Bioinformatic Anaylsis:  We will investigate use universal microbial eukaryotic primers, targeting  the global diversity V4 and V9 variable regions  of microbial eukaryotes found in _Z. marina_ beds. As part the 18S ribosomal RNA gene. The V4 region will be amplified using the AReuk454FWD1 and TAReukREV3 primers with Illumina adaptors instead  of this investigation we seek 454 adaptors from 17 () and the V9 region will be amplified using the 1391f and EukBr primers according  toanswer  the following questions: Can we detect earth microbiome project protocols. The V4 and V9 regions are standardly used in such inventories and are known to be informative at the Class taxonomic level 17 (). Samples will be multiplexed and the resulting DNA libraries will be sequenced using Illumina MiSeq 250 bp paired end sequencing at the UCD Genome Center. Preprocessing and quality assessment will be performed as described by 18 (). Sequence analysis will follow  a phylogeographic signal of _Zostera marina_-associated microeukaryotes? What is driving microbial eukaryotic community compositions similar worflow to 19 () with sequences analyzed using a modified QIIME, Quantitative Insights Into Microbial Ecology 20 (), workflow using UPARSE 21 () to pick open reference operational taxonomic units (OTU’s) at 97% and 99% similarity. Taxonomy will be assigned using the RDP Classifier 22 () with the SILVA rRNA database 23 () and Genbank searches. Phylogenetic trees will be constructed by placing representative sequences for each de novo OTU into the SILVA reference tree with RAxML. The resulting OTU matrix will be rarefied and subsequent data analysis performed  in _Z. marina_ beds, R. Alpha diversity will be calculated using a variety of metrics including  the prescence Shannon and Simpson indices to determine intrasample diversity. The beta diversity metric, Bray–Curtis dissimilarity, will be calculated for the OTU matrix to determine intersample diversity. PERMANOVA tests 24 () will be used to determine the significance  of _Z. marina_ (host plant) or sample type (ex. sediment), location and  environmental factors (geography, pH, measures (salinity,  temperature, etc)? Or is everything everywhere? Do we see specific patterns in either richness or diversity in these beds etc.) on microbial eukaryotic community composition. To visualize  and do any of these assess  patternsmimic those  of macro-organisms known microeukayotic community structure, a nonmetric multidimensional scaling (NMDS) ordination of the Bray–Curtis dissimilarities will be created. Mantel tests will be performed  to frequent _Z. marina_ beds? determine if correlations exist between the bacterial community composition from the 16S rRNA results and the microeukaryotic community composition we determine here.  **Aim Aim  2: A global census of microbial eukaryotes found in association with leaves, roots, and sediment of the Order Alismatales, which includes three, independent lineages of seagrasses** seagrassesAim 2: A global census of microbial eukaryotes found in association with leaves, roots, and sediment of the Order Alismatales, which includes three, independent lineages of seagrasses  Objectives: Aim 2 seeks to expand Aim 1 to include multiple species of seagrass as well as other aquatic plant species in the Order Alismatales. This expansion will allow for investigation into additional questions including: Are there distinct microeukaryotic communities associating with different host plants? Is there evidence of a phylogenetic signal or pattern between host plant species and their microeukaryotes? For locations with multiple host species co-occurring, do we see the same or different associations? Are there any microeukaryotes that are shared between the three lineages of seagrass or unique to each lineage?  Aim 2 seeks Sampling: We are currently leveraging our ZEN contacts  to expand Aim 1 collect leaves, roots, and sediment from plants from the Order Alismatales and have funds  to include multiple amplify and sequence the 16S ribosomal RNA genes of the microbial communities in these samples. Our goal is to have samples from a minimum of three representative  species from each  of the three  seagrass lineages, three of the closest freshwater relatives for each seagrass lineage and two terrestrial outgroups for a minimum of 20 plant species. However, based on collections from our collaborators  as well as other aquatic plant species in the Order Alismatales. This expansion local collections performed by our lab, it is very likely that we  will allow exceed our minimum collection goal  for investigation into additional questions including: Are there distinct microeukaryotic communities associating with different host plants? Is there evidence of a phylogenetic signal or pattern between host plant species seagrass  and their microeukaryotes? freshwater species.  Forlocations with multiple host species co-occurring, do we see the same or different associations? Are there any microeukaryotes that are shared between the three lineages of seagrass or unique to  each lineage? plant species, 5 biological replicates of root, leaf and rhizosphere sediment will be collected at a minimum of three geographic locations.  **Aim 3: Establish culture collection Molecular Biology/Bioinformatic Anaylsis: The V4 and V9 variable regions  of the 18S ribosomal RNA gene will be targetted for amplification as in Aim 1 and the same general bionifornmatic pipeline and statistical analyses methods will be performed. Addtional statistical analyses will be implemented to determine if distinct  microbial eukaryotes associated with Zostera marina communities are found  in Bodega Bay, California.** association with different host plants.  Environmental molecular barcoding studies offer the benefit of high-throughput biodiversity discovery, especially of organisms that are recalcitrant to laboratory culture. However, they suffer from biases during DNA extraction and PCR, and can exclude important, even dominant, taxa \cite{Bik_2012}. They also fall short of providing the means to link biodiversity discovery with functional, ecological significance. The value Aim 3: Establish culture collection  of DNA sequencing-based studies can be improved by augmenting them microbial eukaryotes associated  with a rich, phylogenetically diverse Zostera marina in Bodega Bay, California.Aim 3: Establish  culture collection. Such collections enable the sequencing collection  ofisolate genomes, providing information about metabolic potential, as well as reference sequences for shotgun metagenomics. They also offer a resource for future experimental work. Culturing diverse  microbial eukaryotes from seagrass beds is a labor-intensive process, requiring much time and multiple sampling efforts returning fresh samples. Therefore, we will focus our initial efforts on _Zostera marina_ beds associated with Zostera marina  in Bodega Bay, California, where we have full access to the Bodega Marine Laboratory and the required permits for collection. All isolates will be characterized using both microscopy and molecular methods (described below). California.  Objectives: Environmental molecular barcoding studies offer the benefit of high-throughput biodiversity discovery, especially of organisms that are recalcitrant to laboratory culture. However, they suffer from biases during DNA extraction and PCR, and can exclude important, even dominant, taxa 16 (). They also fall short of providing the means to link biodiversity discovery with functional, ecological significance. The value of DNA sequencing-based studies can be improved by augmenting them with a rich, phylogenetically diverse culture collection. Such collections enable the sequencing of isolate genomes, providing information about metabolic potential, as well as reference sequences for shotgun metagenomics. They also offer a resource for future experimental work. Culturing diverse microbial eukaryotes from seagrass beds is a labor-intensive process, requiring much time and multiple sampling efforts returning fresh samples. Therefore, we will focus our initial efforts on Zostera marina beds in Bodega Bay, California, where we have full access to the Bodega Marine Laboratory and the required permits for collection. All isolates will be characterized using both microscopy and molecular methods (described below).  Culturing/Isolation: We will sample rhizospheric sediment as well as Z. marina roots and leaves to use as isolation innoculum. We will initially focus on a targeted list of taxa (subset of clades where we expect to see members associating with seagrass based on a) literature and b) personal microscopy?) and subsequently expand our culturing to include representatives from all of the microbial eukaryotic lineages.  Isolation/Culturing Rationale: Microbial eukaryotes are extremely diverse, made up of multiple lineages with each containing numerous marine taxa (CITE). Because we do not know the true scope of this diversity yet in seagrass beds, we will initially focus isolation and culturing methods on targeted taxa(??) (taxanomic groups?) that we know associate with seagrass and rhizospheric sediment based on microscopy. Following the acquisition of initial sequence data and microscopy we will adjust our isolation and culturing efforts to include representatives from all microbial eukaryotic lineages. Our initial culturing efforts will focus on diatoms, algae, amoeba, and fungi (??). We have seen representatives of each of these groups microscopically in Z. marina samples, and we have had some success isolating amoeba from rhizosphere sediment. Additionally, our initial sequencing results using the ITS region suggest high levels of unidentified fungal diversity from Z. marina leaves, roots and rhizosphere samples, so we have chosen to include them in our initial isolation and culturing efforts. Say something about diatoms and algae?  Epiphitic algae can be visible on the outside of seagrass leaves and have been studied (?)…  Detailed below is description on how we will culture a subset of these based on previous experience and what we have already had success culturing (Amoeba, fungi, ciliates).  Fungal Isolation/Culturing: A variety of media recipes found previously successful at isolating seagrass-associated fungi will be used here initially. These include potato dextrose agar, malt-dextrose agar, glucose peptone yeast agar and cornmeal agar, with all media being supplemented with salt and antibiotics 25 ()26 ()27 (). Media will also be made using Z. marina as the carbon source as was done by Torta el al 25 () using the seagrass, Posidonia oceanica.  Amoeba Isolation/Culturing: To enrich for amoeba, samples will be inoculated onto plates containing standard A, B or C (????) media with a marine bacterium as a food source. Upon formation, plaques will be picked from the plate and inoculated into flasks containing liquid (??) media. Flasks will be monitored for amoeba growth.  Seagrass wasting disease (slime mold) - can be grown on Seawater-agar medium.  -algae + protist media recipes http://www.ccap.ac.uk/pdfrecipes.htm  -amoeba isolation http://www.bms.ed.ac.uk/research/others/smaciver/Protocols/AmoebaProts/isolation_of_amoebae.htm  -other microeuks (Laura?) – We want to use the Protocols in Protozoology (http://protozoa.uga.edu/pub/Protocols_in_protozoology.pdf) for the other marine microeuks. Do we want to list off some of specific interest? I’m not sure we can make a solid argument for choosing some over others.  Phenotypic and Molecular Characterization of Isolates: Microscopy of X-day old cultures will be used to record external morphology, and growth rate will be characterized at 10, 20, 30 deg C. Isolates will be fixed and imaged under natural light (brighfield?). Fungal isolates will be stained for chitin and with fungal specific rRNA probes using flourescence in situ hybridization (FISH) as in as in 28 (). All non-fungal isolates will be stained for alpha tubulin in combination with FISH staining using a general eukaryotic rRNA probe as in 29 (). If necessary, a more specific rRNA probe may also be used based on the group-specific sequence obtained.  Molecular taxonomic classification of isolates will be performed using barcoding, first with V4 18S rDNA, then with group-specific markers, as outlined by the CBOL Protist Working Group 14 (). We will employ our published bioinformatic workflow 30 () to infer phylogenetic placement of the isolates in the context of the most closely related type strains available.   Data Management Plan:  Sequence data will be deposited to NCBI and raw data will be put on figshare (??????).After identification and imaging, isolates will be sent to ??????? where they will be openly available. - Where will we put the isolates (what culture collections?) - atcc? ccap (culture collection of algae and protezoa) Addtionally, we will post the microscopy images, 18S sequences and phylogenies periodically to our website as blog posts (???).