All posts by Cassie Ettinger

Preprint available: Characterization of the mycobiome of the seagrass, Zostera marina, reveals putative associations with marine chytrids

https://www.biorxiv.org/content/10.1101/735050v1

Abstract

Seagrasses are globally distributed marine flowering plants that are foundation species in coastal ecosystems. Seagrass beds play essential roles as habitats and hatcheries, in nutrient cycling and in protecting the coastline from erosion. Although many studies have focused on seagrass ecology, only a limited number have investigated their associated fungi. In terrestrial systems, fungi can have beneficial and detrimental effects on plant fitness. However, not much is known about marine fungi and even less is known about seagrass associated fungi. Here we used culture-independent sequencing of the ribosomal internal transcribed spacer (ITS) region to characterize the taxonomic diversity of fungi associated with the seagrass, Zostera marina. We sampled from two Z. marina beds in Bodega Bay over three time points to investigate fungal diversity within and between plants. Our results indicate that there are many fungal taxa for which a taxonomic assignment cannot be made living on and inside Z. marina leaves, roots and rhizomes and that these plant tissues harbor distinct fungal communities. The most prevalent ITS amplicon sequence variant (ASV) associated with Z. marina leaves was classified as fungal, but could not initially be assigned to a fungal phylum. We then used PCR with a primer targeting unique regions of the ITS2 region of this ASV and an existing primer for the fungal 28S rRNA gene to amplify part of the 28S rRNA gene region and link it to this ASV. Sequencing and phylogenetic analysis of the resulting partial 28S rRNA gene revealed that the organism that this ASV comes from is a member of Novel Clade SW-I in the order Lobulomycetales in the phylum Chytridiomycota. This clade includes known parasites of freshwater diatoms and algae and it is possible this chytrid is directly infecting Z. marina leaf tissues. This work highlights a need for further studies focusing on marine fungi and the potential importance of these understudied communities to the larger seagrass ecosystem.

Marine Fungi Workshop

I just returned from a marine fungi workshop set up by Amy Gladfelter and supported by the Gordon and Betty Moore Foundation. The workshop was from May 7-9th at the Marine Biological Laboratory in Woods Hole, MA. This was actually my second trip to Woods Hole, my first was in summer of 2015 to attend the Microbial Diversity course (click here to read a cheesey poem I wrote about the course).

The workshop started with everyone giving 5 minute lightning talks about their research. It was my first time presenting my research ideas to people outside of UC Davis and even though it was only a 5 minute presentation, I was scared to death. I am pretty sure I was literally shaking in the moments leading up to my talk and my imposter syndrome was yelling at me to run far far away so that the real mycologists (doubly scary since they were mostly all professors) wouldn’t know they’d invited a eco-evolutionary microbiologist / bioinformagician into their midst. I can’t really remember anything that happened in those 5 minutes, but I walked away feeling like I had crushed it (take that imposter syndrome).

After the talks, we discussed what we thought were some big issues in marine mycology as a group before breaking up into 4 smaller groups with the goal of drafting white papers on these issues.

The 4 smaller group topics were:

  1. Who is out there? Identification and isolation of fungi from different parts of the marine environment
  2. How can marine fungi be studied? Establishing model systems to discover new biology
  3. What are fungi doing to influence the geochemical cycle of the ocean? Establishing the function of fungi in chemical cycling and contributions to climate
  4. How are fungi interacting with and shaping the marine biosphere? Identification of fungal interactions across scales of life in the ocean
Some of the dominant themes that resulted from these conversations were (1) a desire to  inform both scientists and non-scientists of the presence of fungi in the ocean; (2) to impart and quantify the importance of the roles of marine fungi in the ocean; (3) the unclear definition of marine fungi and whether or not this definition includes facultative marine fungi, transient terrestrial fungi or freshwater / brackish fungi; (4) our current lack of understanding of the genetic, phylogenetic, functional and ecological diversity of marine fungi and the spatial scales at which they exist in the marine environment; (5) the lack of standardized protocols for the study of fungi more generally and a need for improved / expanded databases for fungal sequence data that potentially incorporate phylogeny.

I got to meet a bunch of awesome people from a variety of fields (including systematics, cell biology, genetics, chemistry, bioinformatics, etc), some of whom I had heard a lot about / seen before on twitter and others who were completely new to me! I only wish it had been 1-2 days longer to further promote networking opportunities and collaborative discussions. Despite the jam-packed workshop schedule, we somehow managed to fit in a boat trip on one of the MBL’s collection vessels, the Gemma.

Throughout the conference, I realized a few things (1) I should probably be going to and giving talks at more conferences; (2) networking skills are extremely important; (3) I need to learn more about fungal taxonomy and systematics; (4) I am now super excited to look at and incorporate fungi in some of my other non-seagrass projects; (5) working on my computer on a bus is not a good idea and makes me extremely motion sick.
20180507_154134
Found some microbes in Woods Hole, but no marine fungi 😦
This workshop served as a breathe of fresh air for me and helped renew my excitement for analyzing my seagrass-associated fungal ITS data. It also gave me a few cool ideas of things to do moving forward. I am extremely grateful that I had the opportunity to attend and that the Moore foundation was able to bring us all together. I can’t wait for the next marine fungi meet-up!

Now out in PeerJ: Microbiome succession during ammonification in eelgrass bed sediments

https://peerj.com/articles/3674/?td=bl

Abstract

Background

Eelgrass (Zostera marina) is a marine angiosperm and foundation species that plays an important ecological role in primary production, food web support, and elemental cycling in coastal ecosystems. As with other plants, the microbial communities living in, on, and near eelgrass are thought to be intimately connected to the ecology and biology of eelgrass. Here we characterized the microbial communities in eelgrass sediments throughout an experiment to quantify the rate of ammonification, the first step in early remineralization of organic matter, also known as diagenesis, from plots at a field site in Bodega Bay, CA.

Methods

Sediment was collected from 72 plots from a 15 month long field experiment in which eelgrass genotypic richness and relatedness were manipulated. In the laboratory, we placed sediment samples (n = 4 per plot) under a N2 atmosphere, incubated them at in situ temperatures (15 °C) and sampled them initially and after 4, 7, 13, and 19 days to determine the ammonification rate. Comparative microbiome analysis using high throughput sequencing of 16S rRNA genes was performed on sediment samples taken initially and at seven, 13 and 19 days to characterize changes in the relative abundances of microbial taxa throughout ammonification.

Results

Within-sample diversity of the sediment microbial communities across all plots decreased after the initial timepoint using both richness based (observed number of OTUs, Chao1) and richness and evenness based diversity metrics (Shannon, Inverse Simpson). Additionally, microbial community composition changed across the different timepoints. Many of the observed changes in relative abundance of taxonomic groups between timepoints appeared driven by sulfur cycling with observed decreases in predicted sulfur reducers (Desulfobacterales) and corresponding increases in predicted sulfide oxidizers (Thiotrichales). None of these changes in composition or richness were associated with variation in ammonification rates.

Discussion

Our results showed that the microbiome of sediment from different plots followed similar successional patterns, which we infer to be due to changes related to sulfur metabolism. These large changes likely overwhelmed any potential changes in sediment microbiome related to ammonification rate. We found no relationship between eelgrass presence or genetic composition and the microbiome. This was likely due to our sampling of bulk sediments to measure ammonification rates rather than sampling microbes in sediment directly in contact with the plants and suggests that eelgrass influence on the sediment microbiome may be limited in spatial extent. More in-depth functional studies associated with eelgrass microbiome will be required in order to fully understand the implications of these microbial communities in broader host-plant and ecosystem functions (e.g., elemental cycling and eelgrass-microbe interactions).

Preprint available: Microbiome succession during ammonification in eelgrass bed sediments

https://peerj.com/preprints/2956

Abstract

Background. Eelgrass (Zostera marina) is a marine angiosperm and foundation species that plays an important ecological role in primary production, food web support, and elemental cycling in coastal ecosystems. As with other plants, the microbial communities living in, on, and near eelgrass are thought to be intimately connected to the ecology and biology of eelgrass. Here we characterized the microbial communities in eelgrass sediments throughout an experiment to quantify the rate of ammonification, the first step in early remineralization of organic matter, or diagenesis, from plots at a field site in Bodega Bay, CA.

Methods. Sediment was collected from 72 plots from a 15 month long field experiment in which eelgrass genotypic richness and relatedness were manipulated. In the laboratory, we placed sediment samples (n= 4 per plot) under a N2 atmosphere, incubated them at in situ temperatures (15 oC) and sampled them initially and after 4, 7, 13, and 19 days to determine the ammonification rate. Comparative microbiome analysis using high throughput sequencing of 16S rRNA genes was performed on sediment samples taken initially and at 7, 13 and 19 days to characterize the relative abundances of microbial taxa and how they changed throughout early diagenesis.

Results. Within-sample diversity of the sediment microbial communities across all plots decreased after the initial timepoint using both richness based (observed number of OTUs, Chao1) and richness and evenness based diversity metrics (Shannon, Inverse Simpson). Additionally, microbial community composition changed across the different timepoints. Many of the observed changes in relative abundance of taxonomic groups between timepoints appeared driven by sulfur cycling with observed decreases in sulfur reducers (Desulfobacterales) and corresponding increases in sulfide oxidizers (Alteromonadales and Thiotrichales). None of these changes in composition or richness were associated with ammonification rates.

Discussion. Overall, our results showed that the microbiome of sediment from different plots followed similar successional patterns, which we surmise to be due to changes related to sulfur metabolism. These large changes likely overwhelmed any potential changes in sediment microbiome related to ammonification rate. We found no relationship between eelgrass presence or genetic composition and the microbiome. This was likely due to our sampling of bulk sediments to measure ammonification rates rather than sampling microbes in sediment directly in contact with the plants and suggests that eelgrass influence on the sediment microbiome may be limited in spatial extent. More in-depth functional studies associated with eelgrass microbiome will be required in order to fully understand the implications of these microbial communities in broader host-plant and ecosystem functions (e.g. elemental cycling and eelgrass-microbe interactions).

Now out in PeerJ: Microbial communities in sediment from Zostera marina patches, but not the Z. marina leaf or root microbiomes, vary in relation to distance from patch edge

https://peerj.com/articles/3246/?td=bl

tl;dr – The microbes (bacteria) on plant parts  (root, leaf) and near-by sediment were different from each other. We did not find a difference between the microbes on  eelgrass leaves or roots at the edge of a patch versus the middle of the patch. However, the microbes in sediments from different locations in the patch (middle, edge, outside of the patch) differed and these differences correlated with eelgrass density.

Abstract

Background

Zostera marina (also known as eelgrass) is a foundation species in coastal and marine ecosystems worldwide and is a model for studies of seagrasses (a paraphyletic group in the order Alismatales) that include all the known fully submerged marine angiosperms. In recent years, there has been a growing appreciation of the potential importance of the microbial communities (i.e., microbiomes) associated with various plant species. Here we report a study of variation in Z. marina microbiomes from a field site in Bodega Bay, CA.

Methods

We characterized and then compared the microbial communities of root, leaf and sediment samples (using 16S ribosomal RNA gene PCR and sequencing) and associated environmental parameters from the inside, edge and outside of a single subtidal Z. marina patch. Multiple comparative approaches were used to examine associations between microbiome features (e.g., diversity, taxonomic composition) and environmental parameters and to compare sample types and sites.

Results

Microbial communities differed significantly between sample types (root, leaf and sediment) and in sediments from different sites (inside, edge, outside). Carbon:Nitrogen ratio and eelgrass density were both significantly correlated to sediment community composition. Enrichment of certain taxonomic groups in each sample type was detected and analyzed in regard to possible functional implications (especially regarding sulfur metabolism).

Discussion

Our results are mostly consistent with prior work on seagrass associated microbiomes with a few differences and additional findings. From a functional point of view, the most significant finding is that many of the taxa that differ significantly between sample types and sites are closely related to ones commonly associated with various aspects of sulfur and nitrogen metabolism. Though not a traditional model organism, we believe that Z. marina can become a model for studies of marine plant-microbiome interactions.

Now out in AEM: Global-scale structure of the eelgrass microbiome

Ashkaan’s paper was accepted in AEM!

https://www.ncbi.nlm.nih.gov/pubmed/28411219

ABSTRACT

Plant-associated microorganisms are essential for their hosts’ survival and performance. Yet, most plant microbiome studies to date have focused on terrestrial species sampled across relatively small spatial scales. Here we report results of a global-scale analysis of microbial communities associated with leaf and root surfaces of the marine eelgrass Zostera marina throughout its range in the Northern Hemisphere. By contrasting host microbiomes with those of surrounding seawater and sediment, we uncovered the structure, composition and variability of microbial communities associated with eelgrass. We also investigated hypotheses about the assembly of the eelgrass microbiome using a metabolic modeling approach. Our results reveal leaf communities displaying high variability and spatial turnover, that mirror their adjacent coastal seawater microbiomes. In contrast, roots showed relatively low compositional turnover and were distinct from surrounding sediment communities — a result driven by the enrichment of predicted sulfur-oxidizing bacterial taxa on root surfaces. Predictions from metabolic modeling of enriched taxa were consistent with a habitat filtering community assembly mechanism whereby similarity in resource use drives taxonomic co-occurrence patterns on belowground, but not aboveground, host tissues. Our work provides evidence for a core eelgrass root microbiome with putative functional roles and highlights potentially disparate processes influencing microbial community assembly on different plant compartments.

IMPORTANCE Plants depend critically on their associated microbiome, yet the structure of microbial communities found on marine plants remains poorly understood in comparison to terrestrial species. Seagrasses are the only flowering plants that live entirely in marine environments. The return of terrestrial seagrass ancestors to oceans is among the most extreme habitat shifts documented in plants, making them an ideal test bed for the study of microbial symbioses with plants that experience relatively harsh abiotic conditions. In this study, we report results of a global sampling effort to extensively characterize the structure of microbial communities associated with the widespread seagrass species, Zostera marina or eelgrass, across its geographic range. Our results reveal major differences in the structure and composition of above- versus belowground microbial communities on eelgrass surfaces, as well as their relationships with the environment and host.

Preprint Available: Global-scale structure of the eelgrass microbiome

Abstract

Plant-associated microorganisms are essential for their hosts’ survival and performance. Yet, most plant microbiome studies to date have focused on terrestrial species sampled across relatively small spatial scales. Here we report results of a global-scale analysis of microbial communities associated with leaf and root surfaces of the marine eelgrass Zostera marina throughout its range in the Northern Hemisphere. By contrasting host microbiomes with those of their surrounding seawater and sediment communities, we uncovered the structure, composition and variability of microbial communities associated with Z. marina. We also investigated hypotheses about the mechanisms driving assembly of the eelgrass microbiome using a whole-genomic metabolic modeling approach. Our results reveal aboveground leaf communities displaying high variability and spatial turnover, that strongly mirror their adjacent coastal seawater microbiomes. In contrast, roots showed relatively low spatial turnover and were compositionally distinct from surrounding sediment communities – a result driven by the enrichment of predicted sulfur-oxidizing bacterial taxa on root surfaces. Metabolic modeling of enriched taxa was consistent with an assembly process whereby similarity in resource use drives taxonomic co-occurrence patterns on belowground, but not aboveground, host tissues. Our work provides evidence for a core Z. marina root microbiome with putative functional roles and highlights potentially disparate processes influencing microbiome assembly on different plant compartments.

 

http://biorxiv.org/content/early/2016/11/28/089797

MBL Microbial Diversity Summer Course

I spent this past summer at the MBL Microbial Diversity summer course and I’ve written a post about it which can be found here: http://microbe.net/2015/09/28/mbl-microbial-diversity-summer-course/

While at the course, I also cultured a handful of microbes from seagrass leafs/roots and several different amoeba from seagrass bed sediments including one really cool “follow the leader” snail slime like amoeba (slime mold?).

Visualizing Microbiome Data: Choropleth Style

A Pipette and an Open Mind

Recently I’ve needed to visualize spatial changes in my microbiome data that are easily interpretable by other people. The best solution I’ve come across is simply projecting the data onto a drawing of my organism. Like this:

Zostera marina high resolution Alpha diversity of samples from pieces of a seagrass plant projected onto a drawing of that plant.

I’ve used SitePainter to produce these in the past, and in some ways it’s great. I got the figure I wanted and I’ve received a lot of positive feedback about it. The only problem is it has a steep learning curve and is the most dysfunctional GUI I’ve ever come across (generating the figure above took several days of my time, the first time). So when it became necessary to produce over forty more images like it I decided to search for a better way.

My first instinct was that there should be an easy way…

View original post 694 more words

ASM Highlights

The Seagrass Team (Hannah, Jenna & I) hit up ASM this year which was in New Orleans. In case you missed ASM, Jonathan took the effort to compile all the tweets together (#ASM2015) and his efforts can be found: here.

However in case you don’t want to wade though thousands of tweets I’ve included some some brief highlights:

NCBI’s Targeted Loci Blast (MOLE-BLAST)

  • Using MOLE-BLAST you can blast specific 16S or ITS sequences against NCBI currated databases for those marker genes. This seems like it could be really useful if you want to identify bacterial or fungal taxonomy using a marker gene approach. Also, MOLE-BLAST appears to use a tree based approach to help you find the nearest neighbor for taxonomy assignment.

Carl Zimmer’s Talk on Microbiomes and the Hyperbolome

The Session that kept Redefining the Tree of Life (aka Unearthing the Dark Matter of Microbial Metabolism and Diversity)

  • Unfortunately, I missed this session, but apparently both Brett Baker (@archaeal) and Laura Hug (@LAHug_) shook things up
  • Brett Baker brought Thorarchaeota to the party, a monophyletic group that looks like it branches between Lokiarchaeota and Eukaryotes
  • Then Laura Hug showed up with Woesearchaeota and Pacearcheoata, not sure where they fit in, but cool

Contributions to “Extreme” Microbiology by Female Scientists Session

  • The whole session was awesome (and extreme), but Emmie de Wit gave a heartfelt (and tear producing) talk on being a first responder to the Ebola outbreak in Liberia
  • Also, I now really want to go to the Bonneville Salt Flats that Betsy Kleba talked about

Honorable mentions: John Zehr (Talk on open ocean Nitrogen fixing symbionts), David Baltrus (Talk on fungal endophytes with bacterial symbionts in their hyphae), Michael Wagner (Talk on syntrophy between nitrite and ammonia oxiders and alternative substrates for nitrification), Tom Marshburn (A real life astronaut!), Tom Sharpton (A Shotgun Metagenome Annotation Pipeline (ShotMAP))

Hannah and I presented posters at the meeting, her poster (left, #1237) and my poster (right, #1236) can be seen below:

20150601_162116 20150601_162124

Of course, Team Seagrass wasn’t the only ones from the Eisen lab presenting at ASM. David Coil (@davidacoil), Srijak Bhatnagar (@srijakbhatnagar) and Megan Krusor (@MKrusor) also presented posters this year! See tweets with photos below.

Some might say that the real highlight of ASM was New Orleans itself; it was my first time there and I really enjoyed being immersed in the culture (the music!), city and food (especially the food). Below is a picture of me touching the Mississippi River (my first time!). Unfortunately, the water was too murky to search for any seagrass or seagrass relatives.

11391124_10205818157466724_2465408434093120848_n