Samantha Forde, Ph.DProgram OfficerMarine Microbiology InitiativeGordon and Betty Moore Foundation

Samantha,

Thanks for the response and additional questions about our proposal. Attached is our revised proposal in which we have made some changes relating to the scope and overall budget. We include the proposal and also our answers to some of your previous questions (in the Appendix).

In the proposal we first provide some general background relevant to the project. Then we discuss each Specific Aim. In response to your budget related comments we have decided to reduce the scope of the original proposal in a few ways including a reduction in the scope somewhat in Aims 1 and 2 and also a major reduction in the scope of Aim 3.

We believe that this project is not only important and interesting scientifically but it will also serve to catalyze new interactions both on a small scale (i.e., among our team members, by bringing microbial diversity studies to the field of seagrass ecology) and - we hope - by developing seagrass into a model system for studies of host-microbe interactions.

Sincerely,

Jonathan Eisen, UC DavisJay Stachowicz, UC DavisJessica Green, U. Oregon

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Evolution, ecology, and function ofmicrobial communities associated with seagrasses

A proposal to the Gordon and Betty Moore Foundation

Jonathan Eisen, University of California, DavisJay Stachowicz, University of California, Davis

Jessica Green, University of Oregon

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Summary

Seagrasses are unique in being the only kinds of flowering plants that live entirely in a marine environment. These plants are referred to as “grasses” because they resemble terrestrial grasses in a multiple ways including morphology (e.g., long and narrow leaf blades) and ecosystem characteristics (e.g., they frequently grow in large “meadows” that resemble terrestrial grassland ecosystems). Seagrass meadows are of great scientific and practical interest for many reasons. For example, they provide foraging and nursery habitat for many marine species, including sea turtles, fish, diverse invertebrates, and epiphytic algae (e.g., Harbone et al. 2006). They also serve important roles in nutrient cycling in the ocean (Touchette & Burkholder 2000a, Touchette & Burkholder 2000b) and as a physical anchoring system that protects the coastline from the erosive kinetic energy of waves and tides (Christianen et al. 2013). Many seagrass based ecosystems are under severe threats globally (Orth et al. 2006; Waycott et al. 2009) due to factors such as global climate change, pollution, human encroachment, and invasion of exotic species.

Seagrasses and their ecosystems have been the subject of a great amount of research covering many topics including ecology and biogeography (Williams and Heck Jr., 2001)i (Valentine and Duffy, 2006) ii (Duffy 2006) evolution and systematics (Chen et al. 2012a, Chen et al. 2012b), physiology (Aquino et al. 2005,), morphology and genetics (Hughes et al. 2009), biochemistry, genomics, and even microbiology (Crump & Koch 2008). We are interested in integrating the long interest in seagrass ecology and ecosystem science with more recent work on microbiology to produce a deeper, more mechanistic understanding of the ecology and evolution of seagrasses and the ecosystems on which they depend. More specifically, we propose to carry out detailed studies of the community of microorganisms that live in and on seagrasses – the seagrass “microbiome”. The general justifications for carrying out such studies include (1) all plants exist in tight association with many microbial species (2) such associations likely play critical roles in plant health and functionality (Friesen et al. 2011, Vorholt 2012, Porras-Alfaro & Bayman 2011), (3) prior work on seagrass-associated microbes has identified many potentially important patterns and functions (van der Heide et al. 2012, Raja et al. 2012) and (4) new technologies have revolutionized microbiome studies but have not yet been applied to seagrasses. We propose to answer a series of fundamental questions about seagrass-microbe interactions that should reveal important information about seagrass ecology, evolution and function.

We have divided up our proposed work into three specific aims, detailed below.● Aim 1: How have the microbial communities associated with seagrasses co-

evolved with their hosts and what roles in the past and currently do microbes play in adaptations of plants to fresh and marine water life?

● Aim 2: What drives the community assembly of the seagrass microbiome, and specifically within the Zostera marina model system?

● Aim 3: What role does the microbial community play in the functional ecology of the Zostera marina (with a specific focus on sulfur and nitrogen metabolism and primary production)?

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General Background on Seagrass Associated Microbe StudiesBefore getting into the details of our three specific Aims and our proposed work, we provide first some background information on seagrass-microbe interactions and seagrass-associated microbes. We include information here on Zostera specifically since we propose to work extensively on this one genera. In addition, we provide some background on what is known for other seagrasses. We do not cover all the previous relevant studies here but for those interested we have created a public Mendeley reference collection that anyone can access, with a diverse suite of references on this topic.

There have been diverse studies of microbes associated with Zostera over the last forty years, including general surveys using culture based methods, some culture independent studies, and a variety of functional studies or functional hypotheses about the microbes. We highlight some of the relatively recent papers here:

● Kurilenko et al. 2010 studied individual bacteria from Zostera leaves ● Todorova et al. 2012 studied microbes found in Zostera beds ● Kurilenko et al. 2007 studied the adhesion of bacteria to Zostera leaves.

Other studies of Zostera-microbe interactions include these described below:● Newell 1981 used culture based methods to characterize fungi and bacteria on

Zostera marina leaves. ● Kirchman et al 1984 . showed epiphytic bacteria on Z. marina were able to get all

the carbon they needed from Z. marina. ● Cifuentes et al. 2000 used rRNA PCR to characterize diversity of bacteria and

archaea in sediments colonized by Zostera noltii.● Jensen et al. 2007 showed that there were different bacteria associated with the

roots than the surrounding sediment in Zostera marina (with rRNA PCR).

There are also a diverse array of studies on other seagrasses, again using both culture based and culture independent studies. Examples include those listed below:

● Kurilenko et al. 2010 isolated Granulosicoccus coccoides from Zostera marina leaves. Members of this genus have been found living in association with various other marine organisms (e.g., Forget and Juniper 2013; Miranda et al. 2013)

● Smith et al. 2004 studied the diversity of sulfate reducing bacteria in seagrass bed sediments.

● James et al. 2006 studied the diversity of bacteria in seagrass sediments.● Donnely and Herbert 1999 used microscopy and other methods to compare

bacteria in rhizosphere vs. sediments and in seagrass containing sediments vs. seagrass free ones. They proposed that sulfate reducing bacteria were key players in the nitrogen fixation in sediment and rhizosphere.

● Weidner et al. 2000 used rRNA PCR to characterize bacteria on leaves of seagrass Halophila stipulacea.

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● Crump and Koch 2008 looked at attached bacteria on four aquatic angiosperms: fresh water (Vallisneria americana), brackish (Potomogeton perfoliatus and Stuckenia pectinata), and marine (Zostera marina). They suggested that understanding microbial associations would be critical for restoration efforts. Also, they found 12 OTUs on more than one of the plants.

We are most interested in the prior studies that have either suggested or shown evidence for the functional importance of microbes in nitrogen metabolism of seagrasses, and also studies that have suggested that sulfate reducing bacteria (SRBs) are symbiotic with seagrasses both in terms of detoxifying hydrogen sulfide and also in terms of serving as an intermediary in nitrogen fixation. Examples include Apostolaki et al. 2011 and Apostolaki et al. 2012. More on these functional studies is in the section on Aim 3.

On a related topic, there have been an expanding number of studies of the microbiomes of marine and freshwater macroalgae. Examples include Miranda et al. 2012, Friedrich 2012, Hollants et al. 2013, Fernandez et al. 2012, and Egan et al. 2013. These studies and any future ones will serve as an important tool for comparison to the microbial communities found in and on seagrasses. Similarly, studies of the microbiomes of terrestrial plants will also serve as an important comparison. There has been an explosion of such studies recently. Examples include Peiffer et al. 2013, Hirsch and Mauchline 2012, Lundberg et al. 2012, and Bulgarelli et al. 2012. Of possible particular interest are those studies of the relation between the microbiome of plants and salt tolerance (e.g., Ruppel et al. 2013).

All of the studies listed in the previous paragraph make use of next generation sequencing as part of the characterization process. There are no published studies we know of using these technologies for studying microbes associated with Zostera or other seagrasses. This is not to discount the earlier work on seagrass-associated microbes. Much of the work is of fundamental importance - characterizing not only “who is there” at some level but providing hypotheses and evidence for functional roles of the microbes that live in and on seagrasses. However, due to limitations in the technology it has not been possible for anyone to address the types of questions we are targeting here.

Thus we believe the time is right for the work we propose. Seagrasses are important for many reasons. The microbiomes of seagrasses are likely to play varied and important roles in the biology of the seagrasses themselves. Prior work has been very suggestive, and has documented some key findings about the diversity and function of microbial communities on seagrasses. Technological changes mean that new questions can finally be addressed in regard to host-microbial community interactions. Application of these technologies to terrestrial plants (and also to animals) has led to a revolution in microbiome studies. Findings showing critical roles for microbiomes in plants and animals stream in daily - these studies appear to be accelerating almost exponentially. Yet seagrasses have been largely left out of this revolution. We propose to correct this and believe the work proposed here will help turn seagrasses into a model system for studying microbiomes in species that play critical roles in coastal marine ecosystems.

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Preliminary Data

We have carried out some preliminary culture-independent DNA based studies of microbes associated Z. marina and are testing both laboratory and computational methods to analyze the Z. marina microbiome. Samples were collected from leaves (approximately 1 inch from the middle of the leaf,) roots (a single root, rinsed in seawater,) sediment (the top 2 inches, with no roots present, homogenized), and bulk seawater (pumped in from the bay and filtered on a 0.2 micron filter). DNA was extracted, and a 380 bp fragment of the V4 region of the 16S rRNA gene was amplified via PCR. This approach targets primarily bacterial DNA in the environment, but some archaeal DNA sequences were also recovered. Amplicon libraries were barcoded and pooled before sequencing (paired end, 150bp) on the Illumina MiSeq. Sequence data were analyzed with QIIME (Caporaso et al. 2010) and principal coordinate analysis (PCoA) was used to visualize the differences between sample sites. We have found, for example, that samples from different parts of the plants (leaves, roots, sediment) and from the surrounding water show pretty clear differentiation (see Figure 1).

Figure 1. PCA plot of microbial diversity in the microbiome of Zostera marina. Samples were collected from leaves, roots, sediment, and seawater from eight genotypes of Z. marina from the Bodega Marine Lab. Microbiome composition was inferred from the sequencing and analysis of 16S rRNA PCR libraries. Different shapes correspond to different categories of samples.

Our results here are mostly presented to show evidence that (1) we can readily obtain information about seagrass microbiomes using rRNA PCR (though not surprising in any way) and that (2) the microbiomes on different parts of the plant are distinct and are also distinct from the surrounding seawater. This suggests that at least Z. marina, like terrestrial plants, lives in association with non-random bacterial assemblages. Whether these interactions are mutualistic or antagonistic and the role they may play in allowing vascular plants to invade the marine environment remains to be seen.

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AIM 1. Characterize the evolutionary history of the microbial communities associated with seagrasses. Develop and test new methods for community phylogenetic analysis and studying the co-evolution of the communities with their hosts and with each other. Apply these and existing methods to address specific questions about seagrass microbiomes such as (1) how microbial communities changed as their hosts transitioned from fresh-water to marine environments; (2) for the separate evolutionary invasions of the marine environment by seagrass ancestors whether convergent evolution occurred for the microbiomes as it did for the hosts.

Background for Aim 1We propose in this Aim to examine the evolutionary history of seagrasses and their microbial communities. To do this we propose in-depth comparative analysis of the microbiomes from across the diversity of seagrasses and also including outgroups that bracket the separate evolutionary invasions of the marine environment by distinct seagrass lineages. To understand some of the key questions we are asking, we must first introduce what is known about the phylogeny of seagrasses and the evolutionary history of some of their traits. Key points of relevance to our proposal are summarized below:

Seagrass diversity

All seagrasses belong to the Alismatidae subclass of plants, which is itself within the monocot class. The seagrasses have been classified into 59 species from four or five families within the Alismatidae: Hydrocharitaceae, Cymodoceaceae, Posidoniaceae, Zosteraceae, and possibly Ruppiaceae (there is disagreement about whether Ruppiaceae, a monotypic family, is distinct from Cymodoceaceae). An excellent overview of the different seagrass families is provided in Papenbrock 2012. We reproduce Table 1 from this paper below:

Table 1. This is from Papenbrock 2012. ISRN Botany: 103892. doi:10.5402/2012/103892. (Note - this is an open access publication and reproduction of the table is allowed as long as the source is cited).

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We note - the taxonomic diversity among seagrasses is considered remarkably low by many researchers (e.g., Orth et al. 2006). Despite global distribution there are only ~60 species.

Polyphyly

Phylogenetic studies of plants reveal that the seagrasses overall are polyphyletic. That is, when one examines the phylogenetic tree of seagrasses and their relatives, the seagrasses map to multiple separate subgroups in the tree with non-seagrasses intermixed between seagrass groups (e.g., see Les et al. 1997 and Les et al. 1993). Overall the families of seagrasses can be grouped into three distinct clusters (i.e., clades or monophyletic groups) in the phylogenetic tree of plants (Figure 2.)

Three invasions

In the long and diverse history of angiosperms (the flowering plants), there have been only three independent transitions from from terrestrial/aquatic habitats to a fully marine existence (Les et al. 1997 and Les et al. 1993). All three of these occurred within the Alismatidae subclass. These invasions are hypothesized to have occurred along the thick red branches of the tree depicted in Figure 2. Thus seagrasses represent something analogous to what whales (and their relatives) represent in mammals - an invasion (over evolutionary time) of the marine environment from the land. These separate evolutionary invasions are a critical component of this Aim for this proposal. They will allow us to address fundamental questions about what role microbes potentially play in adaptation to the marine environment because we can look for common patterns across the three separate invasions.

Convergence.

The fact that only three lineages of angiosperms have managed to evolve the ability to return to the marine environment suggests (to us at least) that the transition to a marine environment is a difficult one. The challenges of life in the marine environment for vascular plants (compared to land or non-marine aquatic systems) include attenuation of light, oxygen deficient sediments, higher salinity, wave and tide action, and reduced CO2 availability. The selective pressures of life for an angiosperm in marine habitats have led to extensive convergent evolution such that the polyphyletic seagrasses are frequently discussed as a group (e.g., see Les et al. 1997 Lothar et al. 2011). There have been a large number of studies utilizing this convergent evolution to study seagrass phenotypes, physiology, and even genomics (see Wissler et al. 2011 for a good review on convergence in seagrasses).

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Figure 2. Phylogenetic tree of major groups of seagrasses highlighting the three main clades under consideration here. This tree is based on an alignment of rbcL, and was produced via maximum likelihood analysis, as implemented in RAxML. Hypothesized re-invasions of the marine environment are shown in the thick red lines.

Microbiomes

As outlined above, there have been a variety of studies of microbes found in and on or in association with seagrasses. Prior work has included both culture-based and culture independent work. This work has shown some interesting results and we in no way mean to diminish its importance here. However, there has been in essence no work making use of high throughput sequencing as a component of studies of seagrass microbiomes. In addition, the work that has been done has focused primarily on different questions that we propose to address here.

Fungal component of the microbiome

Plant-fungi symbioses (mycorhiza) are among the most ancient and ubiquitous terrestrial symbioses. The most common form, arbuscular mycorhizae (AM) are found in 70-90% of all terrestrial plants (Parniske, 2008). AM fungi (AMF) have been observed in association with submerged freshwater and marshy plants (Radhika, 2007), but not with seagrasses (although there have been only rare attempts to find seagrass-associated AMF) (see Neilsen, 1999 and Panno, 2013).

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Nevertheless, there are at least 800 species of obligate marine fungi (Raghukumar, 2008) and 88 of them have found in association with the seagrass species Posidonia oceanica based on culture-dependent identification methods (Panno, 2013). Presumably many more fungal associations might be found via the culture-independent approach described here, especially with the increased sampling of seagrass species that we will provide. These fungi could prove to play an important role in nutrient acquisition and protection from pathogens, much like they do with terrestrial plants (Parniske, 2008.) Role of microbes in sulfur and nitrogen metabolism associated with seagrasses.

Multiple studies have shown or suggested a role for microbes in various nitrogen and sulfur metabolic processes in association with seagrasses. More detail on this is under Aim 3 below.

Key questions for Aim 1 In light of their evolutionary history, we believe seagrasses can serve as a fundamentally important model system for understanding evolution of host-microbe interactions and also for better placing studies of plant-microbe interactions into a broader context. We propose in this Aim to study the co-evolution of microbial communities across the diversity of seagrasses. In particular we propose to address the following set of somewhat overlapping questions:

● As the seagrasses themselves evolved and converged upon common physiological and morphological solutions to life in the marine environment, what happened to their microbial communities? For example what happened to the microbiomes on the bold red branches in Figure 1?

● As terrestrial aquatic plants moved into the marine environment, did they bring their microbes with them? Or did the seagrasses develop new associations with microbes that were already adapted to a marine lifestyle? Fortunately there are a large number of projects collecting data on the microbiomes of diverse terrestrial plants which can serve as a comparison.

● How do microbial communities and microbial interactions differ in marine plants versus those for aquatic or terrestrial plants?

● Do the microbial communities in the different parts of seagrasses (e.g., roots vs. leaves) all show the same evidence of co-evolution?

● For the microbiome, has there been a differentiation between different aspects of diversity (e.g., taxonomic vs. phylogenetic vs. functional) in regard to co-evolving with the hosts? In particular, as the hosts converged in association with re-invasion of the sea have the functions of the microbiome converged?

● How have microbiomes changed throughout seagrass history and are any of the changes associated with ecological or biogeographical differentiation of the hosts? For example, how do the microbiomes of rock attached seagrasses compare to the sediment root bound ones?

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● How does the variation in the microbiome within species of seagrasses (at different sites/in different conditions) compare with the variation between species (within sites and between sites)?

We note these studies will also be of value in broader, more general studies of the interaction and co-evolution of microbial communities with hosts. More general questions we will be addressing include:

● When a host changes its niche drastically, what happens to its microbial communities?

● How do communities of microbes co-evolve with a host?● What role do microbes play in evolutionary diversification & transitions in the

host?

General approach for Aim 1For this Aim, we propose to conduct a broad comparative study of the microbiomes of seagrasses and close relatives of seagrasses from non-marine systems. We will also collect “control” samples from the surrounding sediment and water column, and where possible, from nearby plants or macroalgae. The proposed work will largely involve three main steps: collecting samples and metadata, DNA based characterization of microbial communities, and comparative/phylogenetic analysis of communities to answer the questions outlined above. We summarize some of the key issues in our planned approach here.

Full seagrass study

We propose to sample as many of the 59 described species of seagrasses as possible from across the globe. This would generally be very difficult to do completely on our own. Therefore we propose that in addition to our own sampling work, to engage the broader scientific community and even potentially the public in collecting samples. We have been developing a standardized protocol for collection and storage of samples that should make this feasible.

Key sampling locations

In order to, as much as possible, remove the effects of differing environments on measures of microbial communities on different seagrasses, and in order to control for differences in sampling by different people, we have identified four key locations for collections of subset of samples. These include places near the PIs home institutions along the west coast of the United States, as well as near the home labs of east coast colleagues in Florida and Virginia.

Along the west coast of the US we will sample from long-term field sites established by the Stachowicz lab at the UC Davis Bodega Marine Laboratory (detailed in Aims 2 and 3) as well as from a suite of established sites in the Pacific Northwest (briefly detailed here). The Green lab has established collaborations with Francis Chan at Oregon State University who has ongoing field work focused on seagrass ecological interactions in relation to oceanographic variability (Kavanaugh et al. 2009; Evans et al. 2013 ) . Chan

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and collaborators have established 13 sites between Oregon (North of Newport) and Central California (Bodega Marine Lab). In the process of selecting a few key sampling locations from among these, we will draw upon the extensive data sets on seagrass biogeography and associated environmental data available through the ZEN program.

Species to be sampled

As mentioned above, we plan to make use of the broader scientific community and the public in sampling seagrasses so as to obtain as diverse a collection of samples as possible. However, we propose to focus our own sampling work on a subset of all possible species so as to guarantee obtaining samples from each clade as well as from closely related species on non-seagrass plants (that will serve as outgroups and for comparative analyses). The three clades described below are annotated on Figure 2.

For Clade 1, we propose to sample Zostera species that occur both in pure stands (only one species) as well as mixed stands. Zostera marina, the dominant species in the North American Pacific, can form mixed stand populations with Zostera japonica in the northwest and with Ruppia maritima in Baja California. Zostera japonica is a recent exotic species brought over from Japan through oyster farming. We will also sample species of the Phyllospadix, which are unique in that they attach to rocky surfaces rather than soft sediments. As an outgroup to Clade 1, we will sample several members of the Potamogetonaceae, which includes common freshwater and brackish water pondweed species.

For Clade 2, Halodule beaudettei, Syringodium filiforme, and Ruppia maritima will be collected from the Indian River Lagoon, Florida. Halodule wrightii is a subtropical species that will be collected in the Gulf of California off the coast of mainland Mexico. We will also collect Ruppia species where it is co-occurs with Zostera marina in Baja California. Ruppia maritima lives in fresh, brackish and marine habitats, and we will collect Ruppia samples from each of these three habitats. As outgroups to Clade 2 (which will necessarily include species that are basal to Clade 1 + Clade 2), we will collect freshwater and brackish members of the Juncaginaceae, as well as the Scheuchzeria, the most basal lineage of the large clade that includes both Clade 1 and Clade 2.

For Clade 3 Thalassia testudinum, Halophila engelmannii, Halophila decipiens and Halophila johnsonii will be collected at the Smithsonian Marine Station, from the Indian River Lagoon, Florida. As an outgroup to Clade 3, we will sample several species of Vallisneria, which is a member of the widely-distributed, freshwater, sister clade to Clade 3. We will also sample from members of the genus Najas, which is basal to both Clade 3 and its sister clade. Finally, we will sample from among the closest (non-aquatic) relatives of the Alismatidae, a clade containing the Tolfieldia, a family of species endemic to Northern California.

Additional samples can be collected through collaborations in Indonesia, Europe and Australia, once sampling protocols are refined. We are currently designing easy-to-use

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sampling kits that will both be sent to these collaborators as well as made available for citizen scientists who wish to participate.

Characterizing the plants, the microbes, and the environment

For each plant being sampled we propose to characterize the plants themselves, the microbes in, on, and near the plants, and “metadata” data about the sampling location and protocols. For each plant being studied we will collect information that will include location, plant species being sampled, surrounding plants and other local environmental conditions, date, and more. As much as possible we will use standardized metadata ontologies and guides such as that from the Terragenome project for soil). For the plants themselves we will record species identity, collect voucher specimens, tissue punches for DNA analysis, and photographs.

For the microbial studies we will collect samples from the leaves, roots, sediment, and water column. Previous studies, as well as our preliminary data (Figure 1), have shown that there are differences between the microbial communities of the rhizoplane (rhizomes and roots) and the phylloplane (leaves and shoots) associated with aquatic plants. These communities also differ from the surrounding sediment and water column (Jensen et al, 2007, Crump and Koch 2008). Samples will be collected so as to best control for depth, time of day and tide, season, age of plants, and other variables deemed to be possibly important. Detailed information about each sample (e.g., depth, leaf size, etc) will be recorded, again using recommended metadata ontologies and guides as much as possible.

Replicates will be collected for each plant species within each site and when possible we will collect samples from different sites for each species. We note - we are doing extensive testing of protocols using samples from Bodega Bay and thus some of the fine details of sampling protocols will likely change.

DNA sequencing

For this study we propose to focus almost exclusively on DNA based analyses of the microbial communities present in our samples. For each sample, DNA will be extracted using methods we have developed for studying other plant-associated microbial communities. Then for each sample we will conduct bacterial and archaeal 16S rRNA and fungal ITS PCR and sequencing using the standard Illumina-barcode based approach used widely in the community. For fungal studies we may make use of primers from McGuire, 2013 which have been proven to amplify DNA from a broad phylogenetic diversity of fungi, and include the AMF discussed above. We expect to be able to pool hundreds to thousands of samples per run and thus the cost in terms of sequencing per sample should be relatively low. We also propose to do metagenomic sequencing for at least a portion of samples. We have had relatively good success in using Illumina’s Nextera transposon-based library construction methods for metagenomic sequencing of microbial communities and plan to use that here.

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Data analysis

We plan to carry out standard rRNA and metagenomic analyses as we have done in a variety of other studies including both taxonomic and phylogenetic diversity assessments. In addition, we plan to compare and contrast diversity metrics that focus on species with those that measure functional diversity (using methods being developed in the GBMF iSEEM2 project for which Eisen is CoPI). Perhaps most importantly we will use both existing methods and develop new methods to study the evolution of microbiomes through the history of seagrasses. For example, we will use each microbial species as a character and its abundance in each sample as a character state to reconstruct a “phylogenetic” history of microbiomes (as in Ley et al. 2008 ). We can use such a phylogenetic history of microbiomes, as well as the phylogenetic history of seagrasses, to make inferences about the influence of host phylogeny on the microbiome phylogeny. As an alternative approach, we will use microbiome composition as a trait to be overlaid on a phylogeny of the seagrasses. This will allow us to use methods for ancestral character state reconstruction (Felsenstein 1985; Garland et al. 1992) to infer the microbiome composition of ancestral plant species at key transition points along the way to a marine lifestyle.

There are other ways to study cophylogenetic patterns between hosts and their symbionts beyond what is presented in Ley et al. (2008): if we have enough host phylogenetic data we can use event-based or global-fit methods (e.g. Legendre et al. 2002) to test for a cophylogenetic signal. We can also test for specific associations between host and bacterial clades (Legendre et al. 2002). Given that we are sampling from geographically distributed sites we may be able to test if biogeography, rather than strict taxonomic specificity, is the main determinant of the association (Decelle et al. 2012). Finally we could use methods developed by Ives & Godfray (2006) to quantify the degree to which the evolutionary association is due to host evolution, microbiome evolution, or co-evolution of both (which the methods above don’t allow for - they just test for co-evolution of both).

Note – we are interested in looking for both “traditional” convergent evolution of the microbes associated with seagrasses (e.g., has their been any convergent evolution in the genomes of the microbes) and also what can be generally referred to as “convergence” of the entire community structure (this is convergence in the sense of the microbial community being viewed as a trait of the host).

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AIM TWO: Determine what drives community assembly of the seagrass microbiome, specifically within the Zostera marina model system. This will be done using comparative studies from different sites as well as experimental studies at Bodega Marine Lab.

Background for Aim 2The fundamental questions we propose to address in Aim 2 relate to the ecological assembly of microbial communities on seagrasses. Because so little is known about the seagrass microbiome, our research will benefit from a two-step approach that first examines community assembly across the phylogenetic diversity of seagrasses collected in Aim 1. Second, we will narrow our focus to a single host species - Zostera marina - in an experimentally controlled setting. To put our questions into context, we briefly define and summarize concepts relevant to testing hypotheses about the assembly of ecological communities.

Core microbiome

Studies of microbes in various habitats have sought to identify the ‘core microbiome’ – the ecologically important microbial taxa shared among multiple communities sampled from the same habitat. It has been hypothesized that these commonly occurring organisms are critical to the function of the microbial communities they are a part of. To date, most core microbiome research has been focused on mammals (reviewed in Shade & Handelsman 2012). There have been few studies on land plants (Lundberg et al. 2012; Shade et al. 2013; Kembel et al. in review), and to our knowledge none on marine plants.

Co-occurrence patterns

Diamond (1975)iii was the first to introduce the concept of studying co-occurrence patterns among organisms to evaluate community assembly “rules”. The idea is that non-random co-occurrence patterns, where pairs of taxa occur less or more often together than expected by chance, reflect ecological interactions including competition and cooperation. Co-occurrence analyses have become a staple of microbiome research (reviewed in Faust & Raes 2012). It has been argued that cataloging ‘normally’ occurring microbiome co-occurrence and co-exclusion patterns within and among hosts is critical not only for identifying fundamental ecological interactions, but also for understanding potential transitions among ‘healthy’ microbial states into ‘imbalanced’ states following disturbance. While co-occurrence patterns have been explored in the human microbiome (Faust et al. 2012, The HMP 2012), we are aware of only one co-occurrence study on the plant microbiome (Shade et al. 2013), and none on marine plants.

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Community assembly theory

The goal of community assembly theory, in the broadest sense, is to understand and predict networks of interacting organisms. These networks may describe interactions between hosts and their symbionts, or interactions between symbionts within a host. As described in Aim 1, there has been some progress inferring co-evolutionary relationships among mammal hosts and their microbial symbionts. However most research on the assembly of host-associated microbial communities has been focused on understanding the nature of microbe-microbe interactions. There has been a significant push to draw on ecological theory, and in particular metacommunity theory, to evaluate how processes - like environmental selection, dispersal, diversification and stochasticity - contribute to microbiome diversity patterns within individual host species across space and time (Costello et al. 2012, Mihaljevic 2012, Hanson et al. 2012). We envision building on this theoretical foundation to study the assembly of seagrass microbiomes at two scales: across the phylogenetic diversity of seagrasses collected in Aim 1, and within Zostera marina.

Key Questions for Aim 2The first step in understanding the symbiotic relationship between seagrass microbes and their host is to characterize the baseline microbiota and the differences that are associated with host phylogeny, genotype, and environment. This baseline data can be used to test what factors might lead to any observed patterns, and to develop and test mechanistic models of seagrass microbiome assembly. We propose to address the following set of questions:

● Is there a ‘core microbiome’ or set of commonly occurring microorganisms that appear in all assemblages associated with seagrasses?

● Are there significant co-occurrence or co-exclusion relationships between pairs of microbial taxa within the seagrass microbiome?

● How much is microbial community assembly influenced by phylogenetic, genetic, and ecological variation in the host?

● How do local community assembly hypotheses depend on the metacommunity - the taxonomic or phylogenetic scale of the microbial source pools?

Approach for Aim 2To answer the questions above we propose to draw upon the comparative seagrass microbiome data and metadata described in Aim 1, and data from our ongoing Zostera marina experiments as described below. We summarize our key empirical, statistical and theoretical approaches here.

Full seagrass study

In Aim 1 we describe our approach to gathering microbiome data and metadata across the seagrass phylogeny, by sampling as many of the 59 species of seagrasses as possible from across the globe. Some of this information will be critical for comparative purposes here in Aim 2.

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Zostera marina (eelgrass) study

Some aspects of community assembly will be hard to address with the broad phylogenetic and ecological diversity seen across all seagrasses. Therefore we propose for this Aim to also focus in on a single species - Zostera marina. We have chosen to focus in more detail on microbial associations with Z. marina for a number of reasons including the following:

● The importance of Z. marina as a mediator of ecosystem processes and functioning is well established (Hughes et al. 2004). Z. marina has been used as a model organism to study the impact of rising ocean temperatures on population structure and genetic diversity (Alexandre et al. 2012), and to develop strategies for the restoration of coastal habitat that has disappeared due to human impact (Resuch et al. 2005; Franssen et al. 2011; Reynolds et al. 2012).

● Diverse genetic and genomic resources are available for studies of Z. marina and there is a moderately sized community of researchers continuing to develop tools for this species (Hughes 2009). For example there have been genome scans for genetic and population studies (e.g., Oetjen and Reusch 2007; Oetjen et al. 2010), there is an online EST database and a decent amount of EST data (Wissler et al. 2009), and there have been a variety of functional genomic studies (Franssen et al. 2011). We believe our work on the microbiome of Z. marina will further improve the “model organism” status of this species.

● The sequencing of the Z. marina genome has been approved as part of the DOE-JGI Community Sequencing Proposal system.

● This species is distributed globally throughout the Northern hemisphere. We are collaborating with Dr. Pamela Reynolds, once coordinator of The Zostera Experimental Network (ZEN), and now a post-doc at UC Davis. ZEN is a collaboration among many labs involved in a single Z. marina ecology experiment (for study locations see Fig 3). One of us (Stachowicz) is part of the ZEN project. Dr. Reynolds is assisting us with the development of sampling kits and instructions that will be sent to each of the participating sites, distributed throughout the range of Z. marina.

● Z. marina is both easily accessed and well studied locally by our team, as described below.

Figure 3. Locations of the partners in the Zostera Experimental Network (ZEN) who will be asked to provide tissue, sediment, and water samples from their field sites

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Leveraging current and planned common garden experiments in the Stachowicz lab

A key aspect of our plan in Aim 2 is that we will be able to take advantage of ongoing experiments in the Stachowicz lab on the functional ecology of Z. marina. In this work, the Stachowicz lab will be carrying out common garden1 and reciprocal transplant experiments, continuing with a system extensively used in the past (Hughes et al. 2009; Hughes et al. 2009; Tomas et al. 2011). A current NSF grant in the Stachowicz’ lab will fund continued experiments to examine the effect of Z. marina genotype on ecosystem functioning.

We provide a summary of the system being used in the Stachowicz lab here. There are five distinct eelgrass beds in Bodega Bay. From these, the Stachowicz lab collected 250 individuals from intertidal, shallow subtidal, and deeper subtidal locations. Twenty highly polymorphic microsatellite loci specifically developed for Z. marina (e.g. Reusch et al. 2000) were then genotyped and 40 individuals were selected, encompassing a broad range of relatedness, for propagation to be used in common garden experiments. Individual shoots were planted in pots containing homogenized, sieved sediment that was collected from a location without eelgrass and placed in outdoor, flow-through, seawater tanks.

The experimental framework in the ongoing Stachowicz lab experiments is ideal for addressing some of the key Aim 2 questions. For example, the common garden experiments will allow for the reduction of variation due to environmental factors and a focus in on variation due to host genetic or phenotypic features in our community assembly analyses. With support from this proposal, we could take advantage of already planned experiments. The potential synergism between this grant and the funded NSF project is substantial and means that we can develop the microbial dimension of this system without paying again for expensive and time-consuming field and laboratory experiments.

Comparative analyses

Our general approach will be to utilize existing methods (reviewed in Ramette 2007; Lozupone et al. 2012) that are widely used to characterize the main drivers of variation across microbiome samples and to develop new methods when needed. We will begin with the broad-scale comparative seagrass microbiome data from Aim 1 and quantify the core microbiome (Shade & Handelsman 2012) and co-occurrence patterns (Faust et al. 2012) among all sampled microbial taxa. In addition to the co-evolutionary analyses detailed in Aim 1, we will use multivariate analyses of microbial communities (e.g. by using principal coordinates analysis with taxonomic (Bray-Curtis) and phylogenetic measures of community dissimilarity such as UniFrac (Lozupone et al. 2011)), host geography, and additional host attributes or environmental metadata that are consistently available across sample sites to uncover correlative relationships.

Next we will focus in on our Z. marina model system, applying the approaches described above to the Zostera Experimental Network (ZEN) sample data. We will also 1 for some information about the common gardens see Jenna Lang’s blog.

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use the common garden experiments established by the Stachowicz lab to explore drivers of variation in the seagrass microbiome in a more controlled experimental setting.

General methods we will use include:

● Compare the microbial communities in/on different parts of the plants with communities found in surrounding environments (e.g., water, sediment).

● Compare different genotypes to each other within the common gardens to examine the impact of host genetics on community assembly. We note this has been done very recently in a study of the fungal microbiome of poplar (Balint et al. 2013 ).

● Conduct an additional common garden experiment on top of those being done in the Stachowicz lab right now. In this experiment, the soil for the common garden will be a mixture/pool of soils from the source environments of all the plants (rather than soils collected from non-eelgrass sites described above). This will allow us to vary in a more realistic way the source, or metacommunity, that is potentially colonizing the plants.

● Include extensive and detailed environmental measurements with each and every microbome study.

● Examine how the microbiome changes over time within individual plants. This will enable us to ask questions about the temporal stability and dynamics of the core microbiome (Caparoso et al. 2011; Hu et al. 2013; Li et al. 2013).

● Use the results from the community assembly studies to identify a set of “most wanted” taxa for culture-based studies and for genomic analysis. This was done in the Human Microbiome Project and in the JGI Grand Challenge Project on Arabidopsis endophytes. Such work is beyond the scope of this proposal but we will seek separate funding for this.

In addition to approaches and analyses outlined above, we will draw on quantitative methods being developed by the Green lab and colleagues at the META Center for Systems Biology to understand the emergent properties of host-microbe systems.

Metacommunity theory.

We anticipate the comparative analyses described above will provide insights into the mechanisms that shape the structure, dynamics and function of the seagrass microbiome. We propose to couple our comparative approach with one that is informed by community assembly theory (Costello et al. 2012). In particular, we will use metacommunity and phylogenetic ecology theory tools recently developed by the Green lab (O’Dwyer and Green 2010, Morlon et al. 2011, O’Dwyer et al. 2012 ) to further understand what shapes the seagrass microbiome. We will begin by answering a fundamental question: is the assembly of the seagrass microbiome random (i.e. stochastic) or is it deterministic? The answer to this question will depend on the assumed scale of the microbial source pool - the metacommunity - and the local community under study. For this reason, we will test the null assembly hypothesis across a range of scales, for example by assuming that the metacommunity is the total pool of microbes sampled across all seagrass clades and sites versus the microbes

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found only in a particular clade. We will draw upon a rich history of classical ecological theory (Vellend 2010, Weiher et al. 2011, Hanson et al. 2012) to make inferences about the role of processes including environmental selection and dispersal limitation on the seagrass microbiome.

Insights that arise from these analyses will guide future theory development. For example, recent research on microbial community assembly of the phyllosphere (leaves of land plants) has shown that microbes filter onto leaves as a function of plant phylogeny (Redford 2010; Kembel et al. in review). However current community assembly theory does not explicitly consider assembly onto a host phylogeny. For the Human Microbiome Project, for example, investigators are theoretically treating host genetic variation the same way one would treat environmental variation. It would be exciting to build on community assembly theory developed by our labs (O’Dwyer and Green 2010, Morlon et al. 2011, O’Dwyer et al. 2012) and the foundation of ecological network theory (recently reviewed in Fontaine et al. 2011) to derive a theory of host-microbe community assembly for a co-evolving bi-partite network of interacting individuals.

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AIM THREE: What role does the microbial community play in the functional ecology of Zostera marina, with a focus on sulfur and nitrogen metabolism?

Background for Aim 3In this Aim we propose to focus on the functional role(s) of the microbes associated with Zostera marina. The questions related to co-evolution and community assembly of microbes associated with seagrasses outlined in Aim 1 and Aim 2 can be viewed as basic science questions. We believe it is important to answer those questions to understand the fundamental biology of seagrasses and also to understand how the biology of seagrasses compares to other plants. However, the work proposed in these Aims does not fully address the critical question of “What do the microbes associated with seagrasses do?”

Due to budgetary limits (from discussions with Samantha Forde from the Gordon and Betty Moore Foundation), we have decided to reduce this aspect of the proposal from earlier versions of this proposal. We simply had to choose between the different Aims and we believe the work in Aims 1 and 2 is more critical and novel. However, we believe the functional aspect of the research is critically important so we still propose some parts of this work – in particular components that will generate preliminary information for future studies and future funding applications.

For this Aim we propose to study two general functions of Z. marina that seem likely to be influenced by microbial communities: nitrogen uptake and protection from toxic sulfur compounds in roots. We also are very interested in the role of microbes in primary productivity 2 but have removed this from the proposal for focus and budgetary reasons. Some of the reasons for focusing on these two functions include:

Regarding nitrogen metabolism in seagrasses generally and Z. marina specifically

● Given what is known about how bacteria mediate nitrogen dynamics in terrestrial plants, understanding whether similar processes operate in the sea is important for understanding nitrogen dynamics in coastal systems.

● Prior work on nitrogen metabolism in seagrasses and/or in seagrass ecosystems has suggested possible roles for the microbiome. Examples of studies include those listed below:

● Welsh 2000 . Measured accumulation of nitrogen in plants and found some fraction of the nitrogen in Zostera appeared to be coming via bacterial nitrogen fixation. Proposed that plants were feeding diazotrophs (nitrogen fixing microbes) with sugars and getting usable nitrogen in return. Also

2 Previously, the Stachowicz lab has measured PP in Z. marina using field and lab growth studies, short-term measures of photosynthetic rate, and nutrient uptake, and have linked these metrics to the key roles these species play in ecosystem functioning. We have demonstrated consistent variation among genotypes in functioning, but the extent to which host-specific microbes contribute to this variation is unknown.

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proposed that sulfate reducing bacteria were a key intermediary in this exchange.

● Bagwell et al. 2002 found diverse diazotrophs in seagrass beds. ● Aditya et al. 2006 documented diverse nitrate reductase genes in

epiphytic communities on seagrasses. ● Capone and Budin (1982) showed nitrogen fixation in washed roots and

rhizomes of Zostera marina, suggesting that microbial endophytes were able to carry out the nitrogen fixation.

● Welsh et al. 1996 proposed a mutualism between Zostera marina and nitrogen fixing sulfate reducing bacteria (SRBs). More on the connection between SRBs and nitrogen fixation is in the next section on sulfur metabolism.

● Nitrogen is often the limiting nutrient for plant growth in coastal marine ecosystems, and previous work from the Stachowicz lab shows that Zostera genotypes vary in their use of nitrate vs. ammonium. Although these differences are consistent across environmental contexts, it is not clear what role microbes play either in mediating uptake, producing new nitrogen (nitrogen fixers) or re-mineralizing sources of organic nitrogen.

Regarding sulfur metabolism in seagrasses generally and Z. marina specifically

● Prior studies have suggested multiple ways that sulfur metabolizing microbes influence the ecology and function of seagrasses.

● As discussed above, it has been suggested that sulfate-reducing bacteria (SRBs) play a critical role in nitrogen fixation associated with seagrasses. This role may be direct (i.e., the SRBs may be fixing the nitrogen) or indirect (there may be some type of syntrophic link between SRBs and nitrogen fixing organisms). Related to this there have been a variety of studies of SRBs associated with seagrasses including:

● Cifuentes et al. 2000 isolated and characterized diverse SRBs from the rhizosphere of Zostera noltii.

● Nielsen et al. 1999 isolated a new species (Desulfovibrio zostera) of SRB from sterilized roots of Z. marina suggesting it was an endophyte.

● Nielsen et al. 2001 further characterized the nitrogen fixation and sulfate reduction in association with seagrass roots.

● Sulfide is known to be toxic to many organisms, including plants. Because sediments are permanently waterlogged and often replete with organic matter, they rapidly become hypoxic with depth and sulfide tends to accumulate to toxic levels in these locations. The ability to tolerate these conditions may limit colonization of the marine environment (see Aim 1). As mentioned in the response to questions in the appendix, recent work has suggested that lucinid bivalves that contain symbiotic sulfide oxidizing bacteria might play a key role in facilitating seagrass invasion of the sea (van der Heide et al. 2012). However, we think it more likely that direct symbiosis with free-living bacteria themselves are likely to be more important, especially given that lucinid bivalves are not found

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everywhere, but sulfide oxidizing bacteria likely exist wherever anoxic environments occur. As a side note – one of us (Eisen) has done some prior work on the genomics and evolution of chemosynthetic symbionts of various marine invertebrates (e.g., Eisen et al. 1992, Newton et al. 2007 and Roeselers et al. 2010).

Key questions for Aim 3 ● Does variation in the microbiome (taxonomic, phylogenetic and functional

potential) correlate with environmental parameters, especially those related to nitrogen and sulfur?

● Measure functional potential of microbiome generally (by studying entire metagenomic data sets) and for specific candidate functions (e.g., sulfur and nitrogen metabolism related gene families).

● Does variation in the microbiome correlate with functional ecology measurements of Z. marina (e.g., the nitrogen accumulation and primary productivity already being done as part of the Stachowicz lab experiments; also some sulfur measurements for specific plots).

● What is the effect of changing nitrogen availability levels on the microbiome and on the plants?

● Are the SRBs and the nitrogen fixing functions present in the same organisms or different ones?

○ Assess via metagenomic sequencing and assembly○ Assess via culturing (e.g., isolate nitrogen fixing microbes and screen

isolates for sulfate reduction, and vice versa).

General approach in Aim 3For this Aim we propose to focus primarily on utilizing metagenomic sequencing as a method of characterizing functional potential of the microbiomes of seagrasses. We have removed proposed work involving culturing and experimental studies of the microbes themselves (for budgetary reasons). However, we believe the metagenomic analysis will still be of great value as we will be able to place it in detailed context from the work in Aims 1 and 2 and from the other experimental work being carried out in the common garden experiments in the Stachowicz lab.

Thus for this Aim the primary source of data will be metagenomic sequencing of samples described in detail in Aims 1 and 2. Our analysis will focus on relating functional profiles in the metagenomes with environmental data and with experimental information from the common garden experiments. We will look both broadly at all protein families and potential functions and also focus specifically on protein families known to be connected to nitrogen and sulfur metabolism.

Leveraging the existing and planned common garden experiments in the Stachowicz lab.

A key aspect of our revised plan in Aim 3 is to leverage extensively the planned work in the common garden experiments in the Stachowicz lab that were discussed in Aim 2. In

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the currently planned work in the Stachowicz lab, we will be characterizing uptake rates of nitrate and ammonium for 40 unique genotypes of Z. marina. Adding measurements of microbial communities and their functional potential to these ongoing experiments would allow us to at least correlate function with microbial community.

We note – as a means of gathering preliminary data for future projects, we propose to gather microbiome data in all the common garden experiments being conducted as part of the Stachowicz lab project. Experiments being carried out include examination of the effects of light and temperature on Z. marina and have controlled mixtures of different genotypes to allow teasing apart of environmental and genetic effects.

To add information of relevance to the microbiome studies we propose adding the following to the common garden experiments:

more detailed collection of information on environmental conditions in the common gardens

measurement of sulfur compounds in a select set of plants and plots a small amount of culturing of microbes and genome sequencing to provide

reference genomes for metagenomic analysis experimental manipulation of nitrogen availability in a small number of common

gardens followed by examination of the effect of this on the nitrogen accumulation in the plants and on microbiomes

Overall the existence of the common garden experiments allow us to do more than just characterize the microbiome of Zostera. It allows us to gather microbial data alongside data on both ecological and biochemical functioning. This resulting data will produce a rich set of hypotheses for further genomic analysis that could be targeted at standard funding agencies such as NSF by us, or by other members of the community.

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Budget Information

Rough Budget EstimatesYear 1 Year 2 Year 3 Total

Personnel salaries and benefits

112,500 219,651 229,005 561,157

Supplies and sequencing 30,000 20,000 20,000 70,000Equipment 7,000 7,000 6,000 20,000Publishing 2,000 3,000 3,000 8,000

Travel and field expenses 10,000 10,000 10,000 30,000

Direct costs 161,500 259,651 268,005 689,157Indirects 19,380 31,158 32,161 82,699

Total 180,880 290,810 300,166 771,855

Personnel Small amount of support for summer salaries for PIs for Years 1-3. 100% support for Jenna Lang in Eisen lab. She will be the project coordinator for Years

1-3. 100% support for two post doctoral scientists for Years 2-3. One will be at UC Davis and

one will be at U. Oregon.

Supplies Supplies for field collection and processing of samples. Supplies and reagents for molecular analysis and sequencing.

Equipment Funds for computers and related equipment for key personnel

Travel and Field Expenses. Funding for travel to field sites, conferences, and for travel between UC Davis and U.

Oregon for joint meetings.

Publication Funds for Open Access publishing fees.

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Appendix 1. ANSWERS TO QUESTIONS FROM SAMANTHA FORDE.

Question 1. Do you foresee applying for NSF funds for any part, or a related part, of the project?

We foresee in the long run that some research on seagrass-microbe systems will be fundable at NSF. Right now we are not planning on applying for NSF funding in this area for multiple reasons including those outlined below:

1. This project sits at the interface of multiple NSF programs but does not fit in well with any. For example, much of the work on seagrass in general and Zostera specifically is funded through oceanography related programs. Although much interesting work has been supported through these projects, the oceanography programs have some areas they have not covered well. Unfortunately what we believe needs to be done to make significant advances in this emerging field includes many topics including community assembly, phylogenetic ecology, host-microbiome interactions, and co-evolution. There are multiple programs at NSF that have funded reasonably cutting edge studies of microbial communities and how they interact with a host but almost all of these are focused on “model organisms” like Arabidopsis or Drosophila or systems where it is also “known” that the microbial community has some important ecological roles (e.g., ant farming, termite gut function, amphibian resistance to chytridiomycosis) or systems where there are only a few symbionts (legume-Rhizobia, invertebrates-chemosymbionts, aphid-Buchnera, and so on).

2. We have surveyed current and past NSF funded projects and although there have been an incredible diversity of projects on microbiomes of animals and terrestrial plants, there are none we could find on marine plants. The most similar funded projects we could find were ones on pathogens of seagrass (i.e., they were studying microbes associated with seagrasses but not anything about the microbome that we could find) and one on seagrass interactions with Lucinid bivalves (which have been proposed to be symbiotic with the seagrasses in terms of sulfur detoxification see http://www.nsf.gov/awardsearch/showAward?AWD_ID=1041941&HistoricalAwards=false ).

3. Much of the work we are proposing here is highly interdisciplinary and such projects are always a challenge to get supported. Our proposed work covers phylogenetics, co-evolution of hosts and microbial communities, community ecology (of microbes and plants), functional ecology, microbiology, biodiversity theory, and genomics. We believe the combination of the proposed Aims (1-3) is important in order to really understand the interactions between seagrasses and their microbiome. Unfortunately, if we were to propose this work to NSF or other agencies we would likely have to split it up into separate subprojects on each topic and this would be less than ideal.

4. We would like to continue to build upon the collaborations that started in the iSEEM project which was funded by GBMF. One focus on the iSEEM project was on

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biogeography of taxa and two of the PIs on the iSEEM project (Eisen and Pollard) are building on that work to study the biogeography of functions. Another area of focus of iSEEM was on phylogenetic approaches to studies of microbial diversity. Eisen and Green developed multiple tools and approaches from that project and in extensive discussions between our labs we decided that it was critical to find a model system to both test those methods and make further developments.

We identified seagrass-microbe interactions as the ideal system for this work for multiple reasons including (1) there is very little work in this area (2) marine plants are critical to the ecology and function of marine ecosystems (3) by studying marine plants and their interactions with microbes we can learn about the general rules of how plants interact with microbes which would be a good complement to work done on terrestrial and aquatic plants (4) the unusual evolutionary history (multiple separate invasions of the marine environment) will allow us to both test and develop methods on co-evolution of communities with a host but also to infer fundamental principles of how plants interact with their microbial communities.

5. We believe the focus of the Gordon and Betty Moore Foundation in multiple areas fits nearly perfectly with this project in that (a) it is about marine microbiology (and thus connected to the MMI program) (b) it focuses on interactions between multiple domains of life (and thus fits with one of the new areas of focus of MMI), (c) it addresses some fundamental questions about plant biology that are not being addressed by other agencies and thus fits in well with the GBMF Plant Biology programs (e.g., the HHMI collaboration), and (d) it will expand knowledge about seagrass ecology; given the critical role seagrasses play in marine system health, the project also relates to the GBMF Marine Conservation Initiative.

6. We want to engage a broader community of scientists and citizens in studies of plant-microbe interactions (e.g., by having scientists from around the globe help us collect samples and by engaging with the public as well in a Citizen Science project). We believe seagrasses are an ideal system to help engage the public and other scientists in studies of marine microbes and plant-microbe interactions and we do not think that it would be possible to obtain funds from NSF to do this work.

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Question 2. Can you expand upon the research directions outlined in Aims 1 and 3 to clarify why and how this project will move the eelgrass system forward, beyond looking at the microbial communities that no one else has yet? In other words, these Aims are still at a relatively high level and more detail would help in evaluating the proposed work. The appendix that is mentioned was not included in the email – this might help in understanding the details.

We note - we wrote a much larger and more detailed proposal which included an Appendix with methods and details on study sites. However, we thought it would be better to first give you the general schema and to make sure we were on the right track before including all the details. So we made a reduced version of the larger document and that is what we sent you. At the end of this document we have included the proposal we sent you with additional details added back in.

Some key points in relation to your question.

In relation to Aim 1 - There have been basically no studies of the evolution of host-microbiome interactions plants. There have been some studies of variation within plant species and how that variation relates to the genetic diversity of that plant species (e.g., see Peiffer et al. 2013 http://www.pnas.org/content/110/16/6548.short ) And there have been some comparisons between the microbiomes of different plant species but what we are proposing here would be the first study to trace and study the evolution of host-microbiome associations in plants.

We note - there have been a few very high profile studies of evolution of host-microbiome associations in animals - especially in relation to humans. For example see Ochman et al. 2010 (http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1000546).and Muegge et al. 2011 (http://www.sciencemag.org/content/332/6032/970.short). We would like to in essence conduct similar studies in plants and the seagrass system is ideal because of the multiple separate invasions of the marine environment. This will allow us to ask questions about repeatability of the changes that occur on particular branches and thus allow a better coupling of functional changes and evolution. We note - the phylogenetic studies of the microbiomes in animals have been quite useful in identifying key features of the microbiomes of particular taxa. For example, the fact that across mammals and even across vertebrates there are key aspects of the microbiome that are conserved has helped guide research on the factors that might be regulating the assembly of the microbiomes. Also - comparative analysis of animals with different diets niches and immune systems has helped guide research on determining which factors that regulate the microbial assembly.

Furthermore the comparative data we propose to generate will also be useful in functional studies of the microbiomes of seagrasses because we will look for correlations between microbial communities and the environmental parameters of the plants from which samples were taken.

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In relation to Aim 2, we are proposing to study the factors that govern community assembly in the seagrass microbiome with an emphasis on Zostera marina. This zooming in on a specific species is important for a few reasons. First Z. marina is a critical member of the coastal ecosystem in many regions because it provides habitat and supports the needs of many other species. Second, it is an established model species for a variety of ecological and functional studies. And third, the focus on a single species will allow us to better determine the specific factors that govern microbial community assembly. Similar studies have been done in Arabidopsis, corn and poplar and are being done in a few other terrestrial plants. And similar studies have been done in a variety of animals. But no such work has been done on any marine plants and such information will of great value in functional and ecological studies of marine systems. In addition, the timing is perfect for doing the proposed work for two reasons. First, one of us (Stachowicz) is setting up a NSF-funded project designed to examine Zostera marina ecological genetics. By adding a microbial component to this work we will gain added value. Second, PI Green has recently been granted NIH funds to develop host-associated microbial community assembly theory for the zebrafish and stickleback model systems. While these model systems are fundamentally different from Z. marina, many aspects of the work will relate and contribute to the proposed research.

In relation to Aim 3, there have been a diversity of studies over the last 10+ years that have suggested a functional connection between seagrasses and microbes in regard to nitrogen assimilation (and nitrogen fixation) and sulfur metabolism including sulfate reduction and sulfide oxidation . For example, in a high profile paper in Science last year (van der Heide et al. 2012 and also see accompanying news story) it was proposed that there was a three way symbiosis “between seagrasses, lucinid clams, and bacteria living in the clams—that likely keeps toxic sediments from building up and killing the seagrass”. Specifically they proposed that sulfide, which is normally toxic, was being oxidized by the bacterial symbionts of these clams and that this allowed the seagrasses to live in places where otherwise the sulfide levels would be too high. It has also been proposed that there is some amount of nitrogen fixation by microbes associated with seagrasses and that in turn the seagrasses are able to assimilate some of this nitrogen. Furthermore it has been shown that there is a metabolic connection between sulfate reduction and nitrogen fixation in these seagrass-associated microbes (e.g., see Welsh 2000).

We provide more detail about the possible connection between seagrasses and microbes in regard to nitrogen and sulfur metabolism in the section on Aim 3 below. We believe that funding for future work on seagrass-microbe associations will require some information on the functional role of microbes and we believe that nitrogen and sulfur functions are the best candidates for such studies.

Furthermore, we note that the Eisen lab has been experimentally studying very similar (at the big picture level) processes in other plants and/or other microbial communities. For example, we are working on the functional role of the microbiome in nitrogen assimilation in maize (with funding from the MARS corporation, and have a paper in revision at Nature on this topic). We are also working on the functional role of sulfur metabolizing microbes in a microbial consortium in collaboration with Victoria Orphan

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and others. In addition, the Eisen lab is also working on multiple metagenomic and genomic studies of nitrogen metabolism and sulfur metabolism including of the genomes of the kinds of chemosynthetic symbionts that have been proposed to be involved in the seagrass system (e.g., Newton et al. 2007 http://www.sciencemag.org/content/315/5814/998.short and Roeselers et al. 2010 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3035367/) (though we note - we believe it is much more likely that free-living sulfide oxidizers are the key players in the detoxification of sulfide that enables seagrasses to survive in otherwise toxic environments).

We therefore believe we have the expertise for working on the functional role of microbes in nitrogen and sulfur metabolism in association with seagrasses and that with initial funding from the Gordon and Betty Moore Foundation we could develop enough of a story to obtain funding from other sources for such studies.

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Endnotes – References for which there was no obvious web link

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i Williams, S.L.; Heck Jr., K.L. (2001). Seagrass community ecology, in: Bertness, M.D. et al. (Ed.) (2001). Marine community ecology. pp. 317-33ii Valentine, J., and J. Duffy. 2006. The central role of grazing in seagrass ecology. Seagrasses: Biology, ecology, and conservation. Springer:463–501iii Diamond JM (1975) Assembly of species communities. In: Cody ML, Diamond JM, editors. Ecology and Evolution of Communities. Cambridge, MA: Harvard University Press. pp. 342–444. pp.