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**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**  _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?  _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.