Mikaela Salvador: Assessing the Phylogenetic Diversity of nifH-containing Organisms at Deep-sea Methane Seeps and Non-seep Sediments

Authors and Affiliation:

Mikaela L. Salvador, Bennett J. Kapili, Amanda C. Semler, Anne E. Dekas
Department of Earth System Science, Stanford University, Stanford, CA


Methane seeps host methane-cycling and nitrogen-fixing microorganisms, where their activity regulates sediment methane emission and sustains community productivity, respectively. Previous molecular studies and stable isotope tracer experiments have demonstrated that consortia of anaerobic methanotrophic archaea and Deltaproteobacteria are key drivers of both processes. Interestingly, additional nifH sequences from unknown source organisms have also been recovered. Here, we analyze the previously published nifH sequences from eight globally-distributed deep-sea methane seeps and seven non-seep sites using the new R package PPIT (Phylogenetic Placement for Inferring Taxonomy) to assess the full phylogenetic diversity of nifH-containing organisms. We infer a taxonomically diverse community of nifH-containing organisms, including Alphaproteobacteria, Gammaproteobacteria, and Firmicutes that are similar to certain symbionts such as Mesorhizobium, Candidatus Thiodiazotropha endoloripes, and Treponema endosymbiont of Euconympha respectively. We also compare the phylogenetic diversity of nifH sequences recovered from methane seeps to that from non-seeps to understand the distribution of potential methane seep diazotrophs in the broader marine benthos. Our results suggest that methane seeps host a greater taxonomic diversity of diazotrophs than previously recognized and highlights the value of reexamining collections of previously published environmental sequencing data using current tools and updated reference databases.

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15 Comments on “Mikaela Salvador: Assessing the Phylogenetic Diversity of nifH-containing Organisms at Deep-sea Methane Seeps and Non-seep Sediments

  1. Hello, Mikaela!
    Great presentation!
    I was interested if you had an explanation for why diversity was greater at non-seep sites. Could methane seep sites be like sites that are largely anoxic, with the presence of methane or the absence of O2 limiting what bacteria can survive in these environments?

    • Hi Chris!
      Thank you for your question! I’m not entirely sure about why diversity was greater at non-seeps, but one possible explanation could be that overall community diversity at each environment further constrains the diversity of diazotrophs, or nitrogen-fixing organisms. If methane seeps have a small overall diversity compared to non-seeps, it will also correlate to having a small phylogenetic diversity of nitrogen-fixing microorganisms as well.
      In regards to the second part of your question, we don’t think the smaller diversity is due to anoxia, and this is because both sites, methane seep or non-seep, will have less oxygen at greater depths. Thus, phylogenetic diversity probably doesn’t depend on presence or absence of oxygen.

  2. I love your figures! You have so many different types (map, bar graph, diagram, etc.). The visuals on the poster really deepened my understanding of your project.

    • One question, do you believe that there is even more diversity than you found? Would a different technique possible reveal this?

      • I do believe that there is probably more phylogenetic diversity than what is shown because the number in nifH sequences varies a lot from tens to hundreds and in one case, a little over a thousand. For instance, if you look at the Dang (2012) paper from the methane seep stacked barplot as well as the Kapili (2020) paper and Zhou (2016) paper from non-seep stacked barplot, they seem to have way more nifH sequences than the rest of the papers and also seem to have more phyla. (It seems that there is this general trend that with the higher number of nifH sequences, the more diverse phyla that is found.) I think that might have to do with the different DNA sequencing methods the researchers in each paper used, so it is possible that if the other researchers in the papers with less phylogenetic diversity used the DNA sequencing method such as in Zhou (2016), they would find more phylogenetic diversity and that there might be more underlying diversity than what is currently shown in my poster.

  3. Hi Mikaela,

    Great job! I really loved your poster, it looks great, you did a nice job with white space and figures. I also really liked how you highlighted future questions/areas for more exploration.

    It sounds like you did so much this summer! Were there any results that were particularly surprising or unexpected?

    Can you also explain a little behind the motivation of this work and the important behind better understanding methane seeps and their communities/productivity? Are there connections between how deep sea methane seeps work and life on land?

    • Hi Bianca!
      To answer your first question, the results that were particularly surprising were when we discovered that certain groups like the Alphaproteobacteria, Gammaproteobacteria, and Firmicutes were similar to symbionts. Even though past research has shown that symbiosis is key to deep-sea marine sediments and especially to methane seeps (because these environments are more energy expensive), it hasn’t really been looked into that nitrogen-fixing organisms, or diazotrophs, are actually involved in this symbiosis too, so my mentors and I are were really excited about that specific finding!

    • Hi Bianca!
      To answer your second question, understanding the microbial ecology of the methane seeps is important because the microorganisms in these environments serve as a methane cap so that methane doesn’t get into the water column. Looking at their community as well as productivity is critical in understanding processes regulating the climate since methane is a potent greenhouse gas.
      In terms of connections between how deep sea methane seeps work and life on land, there are metabolisms that exist in methane seeps that also exist on land! For instance, the metabolism of methanotrophy happens at methane seeps but also occurs in peat bogs in terrestrial ecosystems.

  4. Great presentation! I’m curious if there’s a connection between the phylogenetic diversity and rate of methane cycling. Do certain phyla impact the biogeochemistry of the seeps more than others?

    • Hi Patrick!
      I don’t necessarily believe that one taxa at phyla is more important or impact than another because they all serve a particular purpose. That being said, the ANME (anaerobic methanotrophic archaea) are a keystone species in that they fix nitrogen and convert sulfate into sulfide, which feeds other community members.
      Regarding your question on a connection between phylogenetic diversity and rate of methane cycling, that’s actually one of the projects my mentors is working on right now and how the community of ANME’s impacts methane cycling! We’re not entirely sure of the answer for that, but we’re definitely looking into it.

  5. Excellent presentation, Mikaela! I particular like how you addressed the overall significance of nifH and nitrogen fixation right at the beginning of your presentation. It looks like the biggest difference between the seep and non-seep diazotrophs is that one cluster of Euryarchaeota found at non-seeps but not at seeps (Fig 8). Do you know which taxa are found in that cluster?

    • Hi Anne! I just looked at the ordination dataframe I created, and they all seem to be from different classes of Euryarchaeota in that extra lineage from the non-seep sites! One of the taxa from the Kapili (2020) is from Methanobacteria, the second taxa from the Kapili and Dekas (in review) paper is from Methanomicrobia, and the third taxa from the Zhou (2016) paper is from Methanococci.

    • Hi Colette!
      Thanks for your question! In terms of how I found the deep-sea symbionts, I looked at which unidentified nifH sequences were placed near symbionts on the overall phylogenetic tree, and it seems that about 21 of them are placed near Gammaproteobacterium symbionts such as Candidatus Thiodiazotropha endoloripes or Candidatus Thiodiazotropha endolucinda, 59 of them are placed near Rhizobiales symbionts such as Mesorhizobium or Rhizobium, and 1 of them was placed near the Treponema symbiont of Euconympha. Hope that answers your question!

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