The Tree Phyllosphere in Urban Environments

Last week the last paper of my phyllosphere project got published at mSystems. It is fair to say that my publishing journey at this journal was, by far, the best yet. Both reviewers gave us great comments and suggestions, which improved the quality of our final work. Trees in urban environments expose their leaves to both chemical contamination and biotic deposition that can come from many unusual sources (i.e. humans, exotic species). This project was aimed to enable comparisons with the more “natural” forest microbiome.

Here is the importance statement we wrote for this project: “In natural forests, tree leaf surfaces host diverse bacterial communities whose structure and composition are primarily driven by host species identity. Tree leaf bacterial diversity has also been shown to influence tree community productivity, a key function of terrestrial ecosystems. However, most urban microbiome studies have focused on the built environment, improving our understanding of indoor microbial communities but leaving much to be understood, especially in the nonbuilt microbiome. Here, we provide the first multiple-species comparison of tree phyllosphere bacterial structures and diversity along a gradient of urban intensity. We demonstrate that urban trees possess characteristic bacterial communities that differ from those seen with trees in nonurban environments, with microbial community structure on trees influenced by host species identity but also by the gradient of urban intensity and by the degree of isolation from other trees. Our results suggest that feedback between human activity and plant microbiomes could shape urban microbiomes.”

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Fig. 4. Nonmetric multidimensional scaling (NMDS) ordination of variation in bacterial community structure of tree phyllosphere along a gradient of urban intensity. Data represent ordination based on Bray-Curtis distances among 108 samples. Samples (points) are colored based on the urban gradient (blue for low intensity, orange for medium intensity, and red for high intensity). In panel a, shapes represent host species identity (squares for Acer platanoides; circles for Acer rubrum; triangles for Acer saccharum; diamonds for Celtis occidentalis; sun crosses for Fraxinus Americana; squares with cross for Fraxinus pennsylvanica; stars for Picea glauca); ellipses indicate 1 standard deviation confidence intervals around samples from urban gradient intensity. In panel b, arrows represent the significant (P < 0.001) correlations between NMDS axes versus the relative abundances of bacterial classes in communities.

Arrieta Lab 2017

Wednesday 13th of July 2017

Lately I have been reflecting on where does my love of science stems from. So far I built this list:

  • Analyzing data
  • Coding (75% == debugging)
  • Reading the papers of others and appreciating the amount of work behind it
  • Spending way too much time making pretty figures
  • Communicating, teaching & sharing my knowledge
  • Lab DNA cuisine

But most of all, getting to meet and work with great human beings. Here is to my new lab in Calgary who have made the last few months a great beginning.

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The end of a beginning

Now this is not the end.
It is not even the beginning of the end.
But it is, perhaps, the end of the beginning.
Sir Winston Churchill, 1942

Yesterday, 24th of May 2017, the third chapter of my thesis got published in Nature: read it! It is so incredible that I didn’t believe it until I saw it on the website…

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Here’s a synthesis:

As recent researches have demonstrated that microbes are crucial actors of human health, microbial organisms could also play a similar role for plants, driving key functions such as productivity. The work of Laforest-Lapointe et al. provides the first evidence that the diversity of microbes on tree leaves influences positively tree community productivity, a finding that has critical implication for forestry and agriculture, as well as for future fundamental research on microbial ecology.

Plant diversity is known to play a crucial role in the functioning of terrestrial ecosystems. Plant ecosystem productivity (the biomass produced by a community of plants) is a key ecosystem function both in natural ecosystems as well as in agriculture and forestry. The productivity of an ecosystem increases when a higher diversity of plants is present in the community. Previously, this correlation between productivity and diversity was attributed to the fact that when more species with different ecological niches are present in a community, the resources available in an ecosystem will be more fully utilized, leading to increased overall productivity. Recent advances in DNA sequencing technology have revealed the incredible diversity of microorganisms living on and in plant tissues – the plant “microbiome” – the combined genetic material of microorganisms living on plants. These plant-associated microbes can have important effects on the growth and health of individual plants, but their importance for ecosystem function is not well understood. In this study, Dr. Isabelle Laforest-Lapointe and colleagues demonstrate that the bacteria living on tree leaves can also influence ecosystem productivity, even after accounting for the role of plant diversity. The research team, including Isabelle Laforest-Lapointe (UQAM), Alain Paquette (UQAM), Christian Messier (UQAM/UQO) and Steven Kembel (UQAM) measured and quantified the diversity of bacteria living on tree leaves in a biodiversity experiment with trees (IDENT) established near Montréal, Canada, where trees were planted in different combinations of species diversity and functional diversity and the productivity of these tree communities was measured after several years of growth. Through sequencing of bacterial barcode genes, the research team quantified the number of different bacterial taxa living on each tree. The principal finding of this study was that tree communities whose leaves host a more diverse set of bacterial taxa were more productive, producing more biomass even after accounting for the importance of plant diversity. This discovery suggests that the plant microbiome could play a key role in terrestrial ecosystem productivity, and that models of the relationship between plant diversity and ecosystem productivity could be extended by adding information on the microbial communities associated with plants. The study also demonstrated that plant diversity influences microbial diversity on leaves, with each tree species possessing a distinctive bacterial community, and with trees growing amongst a diverse set of neighbour trees tending to have a higher diversity of bacteria on their leaves than trees growing among trees from the same species. This study suggests that leaf microbiome diversity could play a key role for terrestrial ecosystem productivity, a discovery having multiple potential beneficial consequences in agriculture and forestry, as well as for fundamental research on the roles of multitrophic networks in terrestrial ecosystems and the theories that attempt to explain relationships between biodiversity and ecosystem function.


Now I am not a Ph.D. student anymore. Today marks the day of my first pay as a Postdoctoral fellow at the University of Calgary, Cumming School of Medicine, Depts. of Physiology and Pharmacology & Pediatrics, under the supervision of the excellent Dr. Marie-Claire Arrieta.

From working on microbes inhabiting the leaves of trees, I will now be looking at samples of infant intestinal microbes or mice microbes! Poop it is!

Of Big Meetings and Me

As I am clearly entering the last four months before giving in my Ph.D. thesis …
I crawl under an unprecedented amount of data and possible projects to put into papers. BUT, the summer 2016 was the perfect timing to present my recent findings at international conferences and I chose to attend The Ecological Society of America Annual Meeting in Fort Lauderdale (ESA2016).

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Here I give you my advices when attending such big meetings:

  • Field trips are awesome especially when you get to see alligators

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  • Arrive early to follow a workshop or organize one 

This year I was contacted by a fellow R instructor to help teach a workshop on How to build functions in R. It was a great experience and I definitely want to organize one myself in the future.

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  • Get involved as a volunteer

It was a great way to get involved and meet people from outside my specific field of research but I recommend making sure that you don’t miss a key session because of your involvement.

  • Contribute to a section

This year I got involved in the Open Science Section. Since the beginning of my Ph.D., my director Steve Kembel has emphasized the importance of Open Science in our lab. We have published our first two papers in Open Access journal, with links to the data, metadata and code. I believe that making your science available improves its quality and holds you accountable to your work. “Open science is a no-barrier approach to scientific research.”

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  • Choose well your talk’s session

This year I was ambivalent between emphasizing the plant or microbial ecology parts of my talk. I ended up choosing to put the microbial ecology forward put after the meeting I reflected on it and decides I should have gone with plant ecology because the talks in this section were much more attended…

  • Be bold, don’t be scared to ask questions, meet people and introduce yourself

Next year’s meeting is in Portland, and I definitely hope to be there!

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Pursuing a Ph.D. is a privilege and a test of character

<< WARNING: I OVERUSE LISTS. >> 

Yesterday I was reading a blog post on the “unspoken dark side” of getting a Ph.D. In the article, the author shared his struggle during his post-graduate studies and described how academia is a hotspot for depression and poor mental health (based on scientific surveys).

I fully agree that getting a Ph.D. (or a Master) can carry you down a road filled with tests and pitfalls. Indeed, the possible issues during post-graduate studies are numerous:

  • STRESS
  • Unsupportive/absent adviser
  • Minimal/absent social interactions at work
  • Uncertainty about the future
  • Daily/frequent/obnoxious (self-)doubt
  • Roller-coaster productivity/motivation
  • Academic experience falling short of expectations
  • MORE STRESS

I did see people not enjoying post-graduate studies and I did experiment first hand some of the above. Indeed, my jaw blocked shut because I processed my stress grinding my teeth in my sleep. Pursuing post-graduate studies is definitely a test of how you address being stressed. However, I don’t think we should “accept depression as part of the course” and I disagree with saying that there is a psychological cost to a Ph.D. You might as well just say there is a psychological cost to life; depression, isolation or struggle is not reserved to highly trained brainy isolated Ph.D. candidates. You might lose someone close to you; fall ill to some terrible sickness; or just stop seeing the light in a perfectly balanced life. Such is the faith of humans, the brain equilibrium is probably our greatest Achilles’ heel.

I can’t speak for all domains, but I thought we were forgetting a massive thing here:

Having the possibility of pursuing a Ph.D. degree is a great PRIVILEGE one should acknowledge and celebrate.

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Art created by vinaquero@gmail.com, https://www.instagram.com/vinaquero/.

What should you celebrate?

  • Work in a field or on a subject that you chose
  • Realize yourself through your work
  • Keep learning
  • Make your own schedule
  • Be in contact with curious, educated, informed human beings
  • Have the opportunity to travel and meet people around the world
  • Have an easy access to further training
  • Make a contribution to society, as tinny as it might be
  • Be paid for all the above (at least in Biology)

As I enter the last part of my post-graduate training, I realize the luck (which I also earned through hard work and discipline #earnednotgiven) I had in the last three years and how I enjoyed it. I also plan to keep remembering this every day until the defense, through good and bad days.

Here is what I am grateful for:

  • A great adviser and co-adviser that complement each other well.
  • An outstanding support system, both at home and with my family and friends.
  • A research center that provides priceless professional services.
  • Healthy finances.
  • Loving what I do: learning, research, analyses, coding, teaching, EVERYTHING!
  • Being disciplined with a flexible schedule.
  • Sleeping easily, always.

You’re starting a Ph.D.? Here are some advices.

Yes, sometimes I shiver contemplating all the uncertainty my future holds. But then I remember the vastness of opportunity tomorrow offers me and I fill electrified and thrilled.

Do you?

Tale of a First International Conference

As a finishing Ph.D. candidate hoping to find a job in research, establishing work contacts and collaborations across the world is one of my main goals this year.

I started 2015 well by spending three months in California and travelling to Oregon and British Colombia. Then this summer I had the chance to go to the 10th International Symposium on Phyllosphere Microbiology (a quinquennial conference directly in my research field) in Switzerland.

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The meeting was organized by Julia Vorholt (http://www.nature.com/nrmicro/journal/v10/n12/execsumm/nrmicro2910.html), the author of the first paper I read on the Phyllosphere and its complexity. As it was a single-session meeting (everybody listened to the same talk), I got to appreciate the multiple aspects of research on leaf microbiology ranging from abstract physics of microbial presence on leaves to applied techniques for the USDA to control Salmonella infections. Oh and someone in the US is trying to increase rainfall by using Pseudomonas bacteria to trigger droplets formation. Not kidding.

Although much progress has been made in the last five years, there are still so many basic questions that need to be answered. Especially in microbial ecology, since our understanding of communities’ dynamics depends on detection technology and our comprehension of host-bacteria interaction, I feel that we are only looking at the tip of an iceberg. Even if this realization seems challenging, it fuels me to do more and understand better the mechanisms I am looking at.

The 10th International Symposium in Phyllosphere Microbiology was a blast for many reasons:

  • I got to meet great researchers from around the world.
  • I learned an awful lot of things through the four days of conferences.
  • I saw how important it is to work with scientists from different fields (i.e. physics).
  • I got an award for BEST CONTRIBUTION sponsored by ISME for my poster.
  • Switzerland is mesmerizing though very warm.

Hope to see you all in five years!

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THE CHANGING FACE OF SCIENCE

I love to listen to elders. Their wisdom, their experience, and the differences between their life and ours; they always initiate a rich reflection in me. Things inside and outside of Science have changed so much for the last 100 years and most of us aren’t able to grasp the implications of these changes.

Not so long ago, I had the pleasure to listen to a great conference by a seasoned researcher that shaped and transformed the field of plant ecology. He started to teach and do research at a time where there were no computers, when reviewing articles was done by hand on paper and transmitted by post.

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He told us about the time when you took your time to write a manuscript, a time when you would spend your Friday afternoon reading the last interesting paper in your field of research (and didn’t have to pick between 500 hundreds). A time when you could still disconnect from anything when you were in vacation. A time when publishing was at a much more slow pace.

Then he switched to talking about the challenges of Science with the new technologies:

  • The amount of papers being published every year;
  • Trying to keep track of the interesting papers published in your domain;
  • The difficulty of publishing with the increased competition from other countries;
  • The struggle of Journals to select which papers should be published;
  • The issues to find good (and fair) reviewers;
  • The closed (capitalist) access to Science;
  • The limits and burdens of social presence online (blogs, twitter, etc.);
  • Etc.

And then the conference switched from “mighty interesting” to “monumentally depressing”.

I acknowledge the actual limits and pitfalls of Science and the publication system. I know the challenges that await someone that wants to do research in a world where funding comes with profit (especially for the last years in Canada). However, if I only focus on the challenges and the issues that I’ll have to tackle, I might as well stay in bed every morning and never get up.

Yes it will be harder; yes it will require more work. But I won’t be discouraged by the negativity of an elder not able to adapt to a fast changing system. And someone who didn’t achieve his dream job or is frustrated by the challenges encountered along the way won’t dishearten me either.

The key resides in being more strategic and knowing yourself. The first question my supervisor asked me when I started my Ph.D. was: “What do you want to do after your Ph.D.?” After identifying everything I had to do to put the odds in my favor, I have worked (and I am still working) on every aspect of our strategy:

  • I tweet, professionally.
  • I write a blog, occasionally, to practice my writing.
  • I have a personal web site to be present online when a future employer looks for it.
  • I am present on LinkedIn, on Research Gate, and I have a profile on Google scholar.
  • And I work hard to produce good papers.

But I won’t stop to love what I do because it is harder then yesterday.

I’ll try to remember that when I’ll be older and when I’ll get scared of the challenges of the youth.

LORD OF THE SCHOLARSHIPS: THE QUEST (IN CANADA)

Writing about my Ph.D. experience, I can’t leave by my horrible great story with scholarship demands. Each September, I go through the same loop: I write not really effectively the never-ending list of documents needed to apply for scholarships. Scholarships from my University. From Companies. From banks. From the provincial and federal govern. And this is how I felt when waiting for answers at the beginning of my Ph.D.:

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Pulling together a great scholarship application is tricky. You need to hit the buzzwords real quick and be solid at all levels. Here are the fields you will usually be judged on:

  • Past grades as an undergraduate or master student
  • Research aptitudes and experiences
  • Project quality (scientific and economical)
  • Social implication, leadership and communication abilities

I applied many times to multiple scholarships and “YES” didn’t come too often my way. Every time I got a “NO”, I felt really disappointed and disgusted at the amount of work put in for nothing.

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Time passed. I published a paper. I got more experiences in research and gave plenty of talks everywhere. And then one day, it started raining scholarships on me. Thus, here are my advices on how to vanquish Sauron the scholarship committees:

First, identify clearly what are the points you’ll be judged on for each scholarships.

For your grades:

  • I wish I could go back in the past and make myself work harder to get better grades but since this is not possible… yet… if you are still getting grades, make it count!

For your research aptitudes and experiences:

  • You already know this one: try to publish papers. No surprise here…
  • Get involved in many small research projects with your director or fellow students.
  • Offer to give a hand on ongoing projects so that you can learn from this and add it to your curriculum.
  • If you can be the manager of a small intern team during sampling season, this adds definitely to your attractiveness!

For your project:

  • Sadly, these days, scholarships goes with profit, for someone, somewhere. So identify the economical attractiveness of your project and make sure that you make it pop.
  • Make it short, clear, and sound.
  • Identify clearly WHY someone should study that. If you are not able to sell your project and make it sexy, you ain’t trying hard enough.

For your social implication:

  • Get involved in organizations around you. The key here is to find the right amount of time to give. Give too much and it will lessen the energy you’ll have to give on your project.
  • Offer to give conferences at citizen meetings or in schools. This will make you practice your synthesis skills and will improve your quality as a speaker.
  • Use any opportunity to give talks at national or international meetings close-by.

For all your application:

  • Find yourself a good friend (from outside your field of research), that knows you well, and go through your application together. This person will help you proofread your text and can tell you if your project is clear. This person will also be key to add activities or experiences to your curriculum that you could have forgotten.

Armed with all these advices, you can now feel like that when sending your application next fall. Good luck!

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ONE MORE QUESTION: WHAT ABOUT THE “RARE” OTUs?

As I am going through my Ph.D., I cannot stress enough the importance of having a critical mind and using it often.

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When analyzing a database of 15 Millions of 16S sequences, I arrived to the point where I had picked OTUs (Operational Taxonomic Unit) and got an impressively high number (45580) from which 28113 were represented by singletons (present once) or doubletons (present twice). More then half of my bacterial community from 200 samples relied on the presence of one or two sequences. It made me think about these “rare” sequences, their significance and reliability.

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SOURCES OF ERRORS

Although they are getting sexier and cheaper every day, the next-gen. sequencing techniques are not free of errors (see Schloss et al. 2011 for strategies to reduce them). Indeed, many sequences might be the result of PCR bias or sequencing errors and therefore be misleadingly assigned as a unique OTU afterwards. Our lack of knowledge on environmental bacterial communities reduces further our ability to distinguish between real “rare” sequences and errors/artifacts. The presence of these artifacts adds another challenge to the already technical task of analyzing high-throughput sequencing datasets.

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Agreeing with Zhan et al. (2014), I found that filtering processes had more influence on low abundance OTUs. Indeed, quality and chimera filtering removed most of the sequences that were unique from my database (excluding 66% of singletons, doubletons, and tripletons). Another thing to think about is the 16S copy number in one bacterial cell. As shown by Lee et al. (2009) and Rastogi et al. (2009), it is highly common that one cell will hold multiple copies of the 16S sequence (see Kembel et al. 2012 for techniques to control for it). Therefore, to find only one copy of that sequence even through a cell holds multiple version of that amplicon makes me doubt the value of such “rare” sequence. And knowing about the flaws of OTU picking, I am eager to see some new techniques that minimize clustering errors or noise.

Should one consider excluding rare OTUs (singletons, doubletons, tripletons, etc.) from their community analysis? And where to stop the cut?

THUS WHAT THE HELL SHOULD I DO?

Although being more stringent with your database impedes you to study the “rare” members of the bacterial communities, I personally prefer to be overly strict (and miss some OTUs) than be overly negligent (and draw false conclusions). Additionally, one must consider his study’s objectives when deciding what to do with the “rare” sequences. You should be extremely careful when using statistical analyses or estimators that are sensitive to the presence of “rare” sequences. For example, diversity will definitely be over-inflated by the “rare” OTUs (Kunin et al. 2010; Bokulich et al. 2013). A strategy could be to use multiple thresholds of sequence number to accept an OTU as real and then repeat your analyses to see if the results change. In conclusion, although it is tempting to try to study the “rare” microbiome, it remains a hard challenge to distinguish between sequencing artifacts and real “rare” sequences.

 

REFERENCES

Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI, et al. (2013) Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat Meth 10: 57–59. doi: 10.1038/nmeth.2276

Kembel, S. W., Wu, M., Eisen, J. A., & Green, J. L. (2012). Incorporating 16S gene copy number information improves estimates of microbial diversity and abundance. PLoS computational biology8(10), e1002743.

Kunin V, Engelbrektson A, Ochman H, Hugenholtz P (2010) Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates. Environ Microbiol 12: 118–123. doi: 10.1111/j.1462-2920.2009.02051.x

Lee ZM-P, Bussema C, Schmidt TM (2009) rrnDB: documenting the number of rRNA and tRNA genes in bacteria and archaea. Nucleic Acids Res 37: D489–93 doi:10.1093/nar/gkn689.

Rastogi R, Wu M, DasGupta I, Fox GE (2009) Visualization of ribosomal RNA operon copy number distribution. BMC Microbiol 9: 208 doi:10.1186/1471-2180-9-208.

Schloss, P. D., Gevers, D., & Westcott, S. L. (2011). Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PloS one6(12), e27310.

Zhan, A., Xiong, W., He, S., & MacIsaac, H. J. (2014). Influence of artifact removal on rare species recovery in natural complex communities using high-throughput sequencing. PloS one9(5), e96928.