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####**Aim 1: A global census of microbial eukaryotes found in association with leaves, roots, and sediment of the seagrass _Zostera marina_.**   _Sampling:_ We have, in 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 each sample type. Samples were take from two seagrass 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 use universal microbial eukaryotic primers, targeting the V4 and V9 variable regions of the 18S ribosomal RNA gene. The V4 region will be amplified using the AReuk454FWD1 and TAReukREV3 primers with Illumina adaptors instead of 454 adaptors from \cite{STOECK_2010} and the V9 region will be amplified using the 1391f and EukBr primers according to the 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 \cite{STOECK_2010}. 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 \cite{Smith_2014}. Sequence analysis will follow a similar worflow to \cite{Korajkic_2015} with sequences analyzed using a modified QIIME, Quantitative Insights Into Microbial Ecology \cite{Caporaso_2010}, workflow using UPARSE \cite{Edgar_2013} to pick open reference operational taxonomic units (OTU’s) at 97% and 99% similarity. Taxonomy will be assigned using the RDP Classifier \cite{Wang_2007} with the SILVA rRNA database \cite{Quast_2012} 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 R. Alpha diversity will be calculated using a variety of metrics including the 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 \cite{Anderson_2001} will be used to determine the significance of sample type (ex. sediment), location and environmental measures (salinity, temperature, etc.) on microbial eukaryotic community composition. To visualize and assess patterns of microeukayotic community structure, a nonmetric multidimensional scaling (NMDS) ordination of the Bray–Curtis dissimilarities will be created. Mantel tests will be performed to determine if correlations exist between the bacterial community composition from the 16S rRNA results and the microeukaryotic community composition we determine here. Sequence data will be deposited to NCBI and raw data will be put on figshare (??????).  ####**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**  _Sampling_: We are currently leveraging our ZEN contacts to collect leaves, roots, and sediment from plants from the Order Alismatales and have funds to 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 local collections performed by our lab, it is very likely that we will exceed our minimum collection goal for seagrass and freshwater species. For each plant species, 5 biological replicates of root, leaf and rhizosphere sediment will be collected at a minimum of three geographic locations.   _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 communities are found in association with different host plants. Sequence data will be deposited as in Aim 1.  ####**Aim 3: Establish culture collection of microbial eukaryotes associated with _Zostera marina_ in Bodega Bay, California.** 
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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 \cite{Wurzbacher_2012}. All non-fungal isolates will be stained for alpha tubulin in combination with FISH staining using a general eukaryotic rRNA probe as in \cite{Hirst_2011}. 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 \cite{Pawlowski_2012}. We will employ our published bioinformatic workflow \cite{Dunitz_2015} to infer phylogenetic placement of the isolates in the context of the most closely related type strains available. Sequence data will be deposited as in Aim 1.  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 (???).  All sequence data will be deposited in ..... **add this to above sections too**