The story of the GLobal Urban Evolution Project, GLUE for short, is not an easy one to tell. It sounds impressive to describe it as the largest scale study of parallel evolution and urban adaptation, and the largest ever collaboration in evolutionary biology. Those descriptors are true, but they miss the point and magic of GLUE. GLUE was and continues to be a meandering odyssey of scientific discovery involving hundreds of scientists, visiting 1000s of places in dozens of countries around the globe to sample thousands of plants, all to answer a seemingly simple question: Can organisms adapt to cities? This blog post takes you behind the scenes to tell the story of where things started, how they developed, took off, sustained and succeeded.
Pre-GLUE
For most of my life I have avoided cities. I am a naturalist who feels more at home paddling the lakes and rivers of Algonquin Park then strolling down the street of a noisy, smelly and crowded city. However, from 2009-2011, I was an Assistant Professor at North Carolina State University in Raleigh NC, USA, with many great colleagues. Two of those colleagues, Profs. Steve Frank and Rob Dunn, were studying urban ecology, and more specifically how urban heat islands influenced insect herbivore population dynamics, and they found big effects. At the time my research focused on plant-herbivore coevolution, and I thought: “Wouldn’t it be cool if plants and insects were rapidly adapting – genetically by natural selection – to urban environments?” We knew almost nothing (but see Cheptou et al 2008) about this question, and it was a fascinating idea for someone that studied plant-herbivore evolutionary ecology.
In 2011, I moved to University of Toronto Mississauga (UTM) and I was still inspired by Steve and Rob’s urban ecology work. I started a project with a former postdoc (Dr. Martin Turcotte) and a couple of undergraduate students to test whether the weedy mustard Capsella bursa-pastoris was adapting to urbanization gradients. The project failed for various reasons, but my interest in using urban areas as a system to study evolution was growing.
Early studies of urban adaptation
In 2014, a very talented student (Ken Thompson) joined my lab to start his M.Sc., now Dr. Ken Thompson after finishing a Ph.D. at UBC. Ken was using white clover (Trifolium repens) as a model to examine whether natural selection on defence genes and traits could also influence the evolution of plant reproductive traits. White clover offered us with a beautiful system to study this problem because it has a simple genetic polymorphism for a potent chemical defence against herbivores - plants either produce hydrogen cyanide (HCN) or they don’t, and because of the meticulous molecular work of Prof. Ken Olsen at Washington University, we know the specific genes that control this variation.
White clover is a classic system for the study of plant defence evolution, with the earliest work dating to the early 1900s. Since that time, generations of scientists have used white clover to understand the ecology and evolution of plant-herbivore interactions. One of the early discoveries was made by Hunor Daday, who showed that plants that lack HCN (acyanogenic genotypes) are less common in cold environments (e.g. high latitudes and the tops of mountains), and plants that produce HCN are most common in warm climates. Daday and many other researchers have since shown that this pattern is found through white clover’s native range (Europe and western Asia), as well as its introduced range, which now includes all inhabited continents because white clover is frequently used in agriculture.
It was based on Daday’s and others’ work that led me to think: Maybe white clover could adapt to urban heat islands just like it does to continental and altitudinal gradients in temperature. If this were true then we’d expect that more plants in urban populations would produce HCN than rural plants. In my excitement to pursue this, I approached Ken with this idea, who was already working on a completely different thesis. I pitched it to him with the overly optimistic idea that we could sample white clover in 3 days and determine the frequency of HCN within each population in less than a week. Ken, being both ambitious and always up to a challenge, agreed and we laid plans to sample plants on three urban-rural transects going east, north and west from the center of Toronto. The initially estimated timeframe proved fairly accurate and the results were fascinating if not shocking (although 7 years later we are still seeking answers). The frequency of HCN within populations changed dramatically as we moved from the center of the city to the outlying rural areas, explaining 25-42% of the variation in HCN frequency depending on the transect. What was most surprising though was that HCN production was most common in rural populations, not in urban populations as we had predicted. Following this, we went onto sample Boston, New York and Montreal, seeing clines in three of the four cities (all but Montreal). We also conducted a bunch of experiments to try and get at the mechanisms. We published these results in Proceedings of the Royal Society of Biology Series B, with the conclusion that clines were common and that the likely mechanism wasn’t urban heat islands, but instead urban cold islands caused by colder nighttime winter temperatures in city environments that often had less snow than rural environments.
In 2016, as Ken Thompson was exiting the lab to start a Ph.D. at UBC, James Santangelo was entering the lab to start a M.Sc., which transitioned into a Ph.D. coadvised by Prof. Rob Ness. James became fascinated with the urban cold island hypothesis and wanted to test it. He proposed to sample 12 more cities along a latitudinal transect. His prediction was that if the urban cold island hypothesis was true then urban-rural clines in HCN production should be common in northern cities and rare in southern cities. To be frank, I thought James was unrealistically ambitious in his proposal to sample 12 cities from Detroit, Michigan, to Tampa, Florida. I couldn’t see how he could triple the sampling Ken and I did in a single summer. But I agreed it was a good idea in principle and I would support it if he was keen to try and do it. James succeeded beyond all of our expectations and further established that clines in HCN were common, even in southern cities that receive little to no snow. This suggested that either the original proposed mechanism for the evolution of urban-rural clines was wrong, or there are different mechanisms in the north and south of eastern North America. James published these results in Evolution Letters in 2020.
In the meantime, we found fairly consistent clines like the type Ken and James found across an additional 20 cities of varying size in southern Ontario, and whenever we found them, they were always in the same direction – HCN production was most common in rural populations, least common in urban populations. Moreover, microsattelite markers suggested these clines couldn’t be explained by non-adaptive processes (genetic drift or gene flow), suggesting natural selection as the most likely explanation. This work was published in Proceedings B in 2018.
Shower inspiration
It was late June 2017 when the idea for GLUE came to me. To be honest, I was taking a shower at the time (yes, sometimes science works that way) when it dawned on me: holy cow, we could use white clover to understand whether organisms are adapting to cities in similar ways throughout the entire world. The thought process that led me to this was that white clover is one of the few plants that occurs in most cities around the world, and through our three previous projects, we had developed the protocols to sample urban-rural gradients quickly and in a consistent way. We had also had a rapid phenotyping protocol for HCN that gives us insight into the underlying changes in allele frequencies. If we could get others around the world to implement our protocols, and use the HCN assay papers and techniques we produce in our lab, perhaps we could study evolution of white clover in any city where it occurs, which really is A LOT of cities throughout the world.
That day I went to Rob Ness’ office to pitch the idea, and to ask him if he thought it would be a good idea to approach our student James Santangelo to ask whether he thought the idea was feasible and if he’d like to be involved. After all, at the time James was already studying urban adaptation in white clover at the time, and it was in part his success in sampling 12 cities that had led to the idea. The plan was that I’d coordinate the project but that we’d all lead it together, since the project was too large for any one person to tackle or lead on their own. Rob was positive about the idea and when I went to James and pitched it, James’ at first intently listened. When I asked, “Do you think it is a good idea?”, James’ answer was immediate: “Of course I think it is a good idea, I suggested it to you six months ago!” This felt like the moment when the student became the master. To my credit, I have no recollection of James’ original suggestion, but given my less than perfect memory and the craziness of the idea, I’m sure I would have counseled James at the time that the idea was too big to take on, especially as a student project. However, clearly James’ earlier idea stuck with me and percolated, so much so that it seemed like brilliant inspiration six months later.
Launching the Global Urban Evolution project
With the idea for the project forged, we needed to work out the difficult details and logistics of how to pull it off. The first meeting took place on Friday, July 7, as noted by the email below.
From: Marc Johnson <marc.johnson@utoronto.ca>
Date: Tuesday, July 4, 2017 at 11:41 AM
To: Rob Ness <rob.ness@utoronto.ca>, James Santangelo <james.santangelo@mail.utoronto.ca>
Subject: Global urban evolution project (GUEP)
Okay, so we need to work on the acronym, but I was hoping to steal your attention on Friday from 11-12 to start talking about the Global Urban Evolution (GUE?) project.
Cheers,
Marc
This email also shows that we narrowly missed shortening the project to GUEP or GUE, which no one would have thanked us for.
Our first meeting quickly worked out some of the essential details we needed to overcome. We would need to identify collaborators around the world to implement identical methods to the ones we had used in eastern North America. For this we’d need iron clad protocols, to produce all of the assay papers, and to send the basic sampling materials to everyone to make sure everything was done in the same way. Initially we set 100 cities as an ambitious target, and for this we’d need a website for people to sign up. To accomplish all of this we’d need some extra help, ideally from a technician. Thankfully I had just been awarded a Canada Research Chair, which provided me with sufficient funds to hire Cindy Prashad for the summer. Cindy became the first member of the GLUE Lead Team (GLUE Lead Team member #4) beyond Rob, James and I. Cindy had worked in our lab for several years as an undergraduate student, and had done some amazing work in the clover system already - she was a coauthor on the Proceeding B 2018 paper. With her help we created the www.globalurbanevolution.com website and started to write up the protocols. Cindy left our lab in the fall of 2017 to start a medical technician training program, but her early contributions were instrumental to laying the project’s foundation.
In the fall of 2017, I hired Simon Innes as a GLUE technician. He had also recently finished his undergrad at UTM. Simon was GLUE Lead Team member #5. Simon started the hard work of continuing to refine all of the written protocols for public consumption, from designing transects, to sampling plants, to assaying HCN. James and I would then incessantly edit them to make sure they were crystal clear for anyone to use. Simon and James also made a number of videos to provide would-be collaborators with visual how-to-guides on how to perform all of the steps of the project. Finally, Simon started to prepare the GLUE kits, which involved making 100s of HCN assay papers, ordering 96-well plates to perform the assays, printing out sampling and data sheets, plus written instructions.
I should remind you that to this point we actually didn’t have any collaborators yet. This was just the lead-up before seeing if anyone wanted to collaborate with us on this ambitious project.
Launching GLUE
By the end of 2017 and early 2018, we were ready to launch GLUE. We initially reached out to a few colleagues by email that we thought might be interested in participating, and we were encouraged that most people we asked were enthusiastic to participate. We next launched a campaign on social media, list-serves (e.g. Evoldir, Eco-log) and many emails, to try and identify collaborators that would help us get to 100 cities. Here is the first Tweet that went out in English and many other languages:
This was complemented with many, many emails, especially to colleagues in regions of the world that restricted Western social media (e.g. China, Russia), or are in regions that are often less included in international collaborations (e.g. African continent, South America).
The reaction was immediate and overwhelmingly positive. Within 12 days we had blown past 100 cities and had set a new goal of 200 cities. This tweet and map from Jan 26, 2018, marks where we were at in building the GLUE collaboration on January 26, 2018:
In the weeks that followed we continued to try and fill holes in sampling with targeted emails to colleagues in Africa, South America, China, Japan and South Asia. We eventually made it to 180 cities, but through personal circumstances, historic droughts (e.g., South Africa), and the discovery that while white clover is in most cities, it isn’t in all cities (e.g. Port Moresby, Papua New Guinea, central Africa), so we ended up with 160 cities.
Now with an ever expanding and ambitious group of collaborators, we needed to get them the materials they needed to sample. This is when Simon kicked into high gear, sending out GLUE sampling kits to collaborators throughout the world.
With the kits in hand our collaborators jumped into action. As the spring and summer rolled around in the northern hemisphere, and fall took hold in the southern hemisphere, collaborators started to post pictures of their activities, including the plants they found, the animals they encountered, the pets and kids involved, and the fun they had.
As the summer of 2018 progressed, the data started to come in through datasheets and Google Forms we had distributed. By this point Simon Innes was traveling through Europe for his M.Sc. in our lab working on a non-GLUE project, and so the hard work fell to the next GLUE technicians – Beata Cohan (GLUE Lead Team member # 6) and then Samreen Munim (GLUE Lead Team member # 7). Beata had also just finished her BSc, which involved several undergrad projects in our lab, including coauthoring the 2020 Evolution Letters paper. Samreen had been a student in one of my classes and had proven herself a talented and utterly reliable research assistant.
It fell to Beata and Samreen to keep distributing kits to collaborators that were ramping up their sampling in the southern hemisphere, and to check every piece of data coming in. They checked every plant assayed, and every data point entered, and then followed-up with collaborators whenever there were questions or errors that required an answer.
We also started to receive boxes and boxes of tissue from collaborators, which were needed for genomic analyses to test how urbanization was affecting gene flow, genetic drift and selection on HCN and its underlying alleles, not to mention the rest of the genome. This is where post-doctoral researcher Dr. Lindsay Miles (GLUE Lead Team member # 8), newly minted Ph.D. from my lab Dr. Connor Fitzpatrick (GLUE Lead Team member # 9), and James started to troubleshoot tissue preservation methods and DNA extraction methods to try and get high quality DNA for genomic sequencing. Beata, Samreen and Connor did a lot of DNA extractions in these early days, but it was a trip by Lindsay to the lab of Jason Munshi-South, with the help of his student Liz (now Dr. Elizabeth Carlen), that helped us understand that our genomic DNA extractions were not of sufficient quality and we needed a new approach.
This approach was eventually solved by James and Lindsay. With the addition of some extra steps to our CTAB extraction protocol, including an extra round of phase extraction, the addition of RNAse (that was a huge oversight the first time around!) and the absolute need for fluorometric quantification of the DNA (we had previously used a nanodrop), we were able to get sufficiently high-quality DNA.
While we had originally intended to use a ddRAD approach to sequence many small portions of the genome from each plant, James convinced Rob and I that a low coverage whole genome sequencing approach was superior and still cost effective. Low coverage data (1-5X) combined with genotype likelihood methods would still give us the ability to estimate all of the population genomic statistics. However, the advantage of low coverage WGS was that it would give us the added benefit of being able to measure selection and linkage disequilibrium across the genome. The problem then came down to money and brute force to create the genomic libraries, sequencing and dealing with bioinformatics of genomics data from 100s and then eventually 1000s of samples. Thankfully, Rob and James had the skills needed for dealing with such a massive dataset, and I was fortunate to receive an NSERC Steacie Fellowship, which funded much of the genomic library preps and sequencing.
While we solved the genomics issues, we also wanted to understand the environmental drivers of any evolution we saw in HCN along the urban-rural clines. Extracting environmental data from specific locations is a tricky business that requires a seasoned hand familiar with GIS and remote sensing. To tackle this project I hired Alex Tong (GLUE Lead Team member #10), who had recently finished an M.Sc. with Prof. Yuhong He at UTM. Over several months, Alex developed pipelines, downloaded satellite images, accessed curated databases, and extracted environmental data for a large slough of environmental variables for each of the >6000 populations sampled by GLUE’s collaborators. These data ended up being instrumental to understanding how urbanization affects environmental change in cities that then drives adaptive evolution – see below and James’ BLOG post.
Playing the long game
By the summer of 2019 most of the data was in while a few additional cities in key locations were being added in the northern and austral summer of 2019. The initial results were already becoming clear – white clover frequently exhibits urban-rural clines on all continents sampled.
It was around this time that we were presenting preliminary results at conferences, and some of my colleagues at these conferences were saying: “You have an incredible story already, why don’t you publish it now”?
However, there were two outstanding questions we really wanted to answer before publishing our results:
Were the clines caused by natural selection, genetic drift or gene flow?
If they were caused by selection, what were the environmental drivers of clines?
To answer the 1st question, we needed the genomic data to test whether the clines could be explained by changes in genetic drift or gene flow, and ideally whether there was any evidence of direct selection on HCN and its underlying alleles. James adapted a DIY protocol to make the genomic libraries in our labs for white clover, which would have been too expensive to outsource. To accomplish the task of making >2000 genomic libraries for whole genome sequencing we eventually hired the next GLUE technician Sophie Koch (GLUE Lead Team member #11), who over many months before and during the pandemic would make about 600 genomic libraries, which sequenced fairly well, although with huge variation in quality and coverage. A subsequent technician (Inder Sheoran) made the next ~1900 genomic libraries. Our initial submission of the paper included 520 genomic libraries and convincingly showed that the presence of clines couldn’t be explained by genetic drift or gene flow, leaving only selection as a reasonable explanation. However, an astute and stickler of a reviewer insisted on a direct test of selection using the genomic data. Thankfully, the remaining 2000 genomes had just been sequenced, which provided us with a lot more power to perform direct tests of selection on HCN and its underlying alleles – there was selection! All of these genomic analyses were performed by James Santangelo and Rob Ness, and involved developing some pretty clever new bioinformatic methods for genotyping plants based on read counts, as well as new tests for estimating selection on epistatically interacting genes with recessive deletion alleles.
To answer the 2nd question we needed to link the environmental data with the HCN cline data, which would involve some complicated multivariate statistics. James and I muddled our way through some initial tests based on PCA, but eventually we invited Prof. Pedro Peres-Neto of Concordia University (GLUE Lead Team member #12) to collaborate with us on these analyses. Pedro has extensive experience with multivariate analyses of environmental data, and through his guidance we were able to identify the environmental predictors of adaptive HCN clines along urban-rural gradients.
In the end we had connected most of the dots that told a compelling story – cities drive environmental change throughout the world, which frequently causes a cosmopolitan plant to adaptively evolve, sometimes in contrasting ways. You can read about this in Science, with a summary from James Santangelo.
The Unintended Contribution of GLUE
The original motivation for GLUE was to push the boundaries of what we know about parallel evolution and adaptation to cities at a scale never attempted before. The contribution we didn’t anticipate were the many positive broader impacts of GLUE. GLUE was designed to be inclusive. If you wanted to be involved and had access to a lab with basic equipment then we were excited to have you on board. This attracted 144 female and 143 male collaborators. Many of these collaborators were undergraduate students doing their very first project. We heard a lot of inspiring stories about the importance of GLUE for students (look for a future blog post on the students of GLUE!), including the following one which is emblematic of many of these stories (anonymized for confidentiality):
I'm sure you have other stories like this, but for my student, a first generation college student from an immigrant Latina background, this GLUE work was the deciding factor in her acceptance to graduate school! This opened the gate for her career in science. This was truly a gift to her and so many others.
THANK YOU!!!!
In other cases undergraduate classes sampled and assayed plants, as a way to train them about the scientific method, field research, data collection and analysis.
In at least one case, a high school science experience program (SHAD Canada) used GLUE to train high school kids about the scientific method and evolutionary and plant biology.
I know of four students who are not yet university students that were involved as collaborators. Two of these kids were my own, Mae and Oscar, who helped sample 5 cities – Buenos Aires, Santiago, Kyoto, Hiroshima, and Sapporo.
There were many graduate students or post-docs, interested in collaborative science, for which this is there very first paper ever – what a first paper!
As a whole, GLUE brought together 287 scientists working in 26 countries. The logistics of managing this size of a project were challenging, but the level of international cooperation to help achieve a common set of research goals were inspiring and well worth the effort.
GLUE 2.0
The first GLUE study (GLUE 1.0) came out online in Science on March 17th, 2022, St. Patrick’s Day, which also happens to be my birthday. The print version was released on March 18th, which is Rob’s birthday. Could we have asked for a better birthday present?
However, our work on this topic is not stopping at GLUE 1.0. We have made all of the data available to our collaborators and asked them to propose new projects using these data – GLUE 2.0. They have risen to this challenge with the same enthusiasm they brought to the first project, proposing an additional 8 projects – GLUE 2.1-2.8. These new projects will examine how invasive species spread across the world and adapt to new climates and environments as they experience them. They will test how cities are affecting the ecological changes in populations through time, and whether other parts of the genome are adapting at the same time. One project has even called for collaborators to return to the original populations to resample them, to better understand how changes in herbivore communities are affecting the evolution of plant defences in white clover. You’d think that people would have had enough with GLUE 1.0, but already >50 people have signed up to resample >40 cities.
For Rob, James, the rest of the Lead Team and myself, GLUE has been a special labour of love that we feel lucky and honoured to have led. Thanks to all that contributed to its success.
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