The long... long arc of Neotropical mycology bends toward success
Dearest Patrons,
It's been a criminally, disorientingly long time since we've spoken (4 years, 4 months, and 19 days, to be exact) . Before we get into any details, I want you to know that neither you, nor this project, have been forgotten, though for reasons with which we are all far too familiar, it has been slowed -- sometimes to a crawl, sometimes to a complete standstill -- given the current and recent state of the world in which we live. A lot of things have happened during that time, and it is my intention in writing this update to tell you about all of them, and what it all means for the future. Strap in.
Upon its conclusion, the funds raised from this campaign enabled us to attempt the extraction, amplification, sequencing and analysis of DNA from all ~350 of the specimens documented and collected by Roo and I in our 2014 expedition to Los Cedros. The first gene region to look at in work like ours is ITS (internal transcribed spacer), colloquially known as "the fungal barcode." Armed with ITS sequences, we would be able to see the degree to which our collections matched up with the ITS sequences of all the other fungi to ever have their own ITS sequences uploaded to genetic databases like GenBank and UNITE. This barcoding task, multiplied ~350 times, is where our original ask of $3500 came from, a fundraising goal met and exceeded by this campaign.
In a perfect world, with a magical molecular batting average of 1000, every dollar in excess of that $3500 could go toward the sequencing additional gene regions, particular those which are more informative in determining where a species belongs on the fungal family tree -- RPB1, RPB2, TEF1, β- tubulin, etc. That way, not only could we use molecular data to draw conclusions about the identities of our specimens, but about their phylogenies (relatedness to other fungi) as well.
Shocking to no one at all was our less than 1000 batting average in our sequencing attempts. Instead of that impossibly perfect success rate, we achieved around a 67% success rate, which in the world of fungal barcoding would fall squarely in the category of "ain't half bad!" Those sequences live in a very special, very large, very colorful master spreadsheet (pictured above), alongside the rest of our specimen data, but they have not sat still. On the contrary, as soon as they became available, we started to compare them against the sum total of ITS sequences generated from fungal specimens from all over the world, to see what matched to what, and where, and to what degree, and -- perhaps most importantly -- what to make of those matches, as taking them as gospel truth and on face value can be... problematic.
Another thing that came as no surprise to us was how many of our sequences didn't match anything at all in those databases, or didn't match anything all that closely. We knew this would happen, because there is barely anyone, living or dead, who has seriously studied the fungi of Ecuadorian cloud forests, and of the extremely small number who have (we're talking single digits), they were either alive at a time before sequencing technology existed, or it existed but was prohibitively expensive. Not to mention that DNA degrades over time, making old collections some combination of expensive and difficult-to-impossible to sequence, with even the most cutting edge of current techniques. As such, this project is essentially providing mycological science with the first molecular profile -- the DNA debut -- of kingdom Fungi as it exists in this hyperdiverse, vastly understudied, critically endangered ecoregion. That's huge.
After getting ITS for roughly two thirds of our 2014 collections, we still had $1500 left in the bank to play with, originally earmarked for obtaining additional loci. For a variety of reasons, we decided, for the time being, to keep that money in the bank. Chief among those reasons was our being awarded a National Geographic Explorer grant in the amount of ~$26,000 to mount a return expedition to Los Cedros. We shared this exciting news with you all as the "Special Announcement" referenced here.
It is beyond the scope of this update to describe in detail the amount of work entailed in making our 2018-2019 Los Cedros expedition a success; all the challenges encountered and overcome (keep an eye out for a methods paper sometime in the next 20 years!). Suffice it to say, with limited time and resources, embarking into lots of new territory (literally and metaphorically), a team of ~35 people from a combined four countries successfully mounted and executed a one-month research expedition to a previously uncharted corner of the Los Cedros Reserve for some of most intense (and intensely rewarding) multi-kingdom field work any of us had ever experienced. For many of us, that month represents one of the most profound experiences of our lives. The work was as incredibly hard as it was incredibly fulfilling. It required ingenuity, determination, patience, endurance, blood, sweat, tears, and heart -- from all involved -- and for these virtues we were rewarded ten-fold. The mycology crew generated over 200 new collections, including species never before seen in over a decade of collecting at the reserve, many of which we suspect to be new to science. A few of those collections even came from high up in the treetops, where our botany team's professional tree climbers were daily documenting the many epiphytes, orchids and other plant species that call the cloud forest canopy home. Macrofungal diversity surveys of this unique microhabitat have almost never been conducted, not in Ecuador, not anywhere.
Those of you following me on Instagram may have caught a glimpse of some of our other impressive fungal finds from the expedition, such as these two strikingly spikey, impressively tiny, entirely unrelated agarics found fruiting from the same accordioned Cyclanthaceae fronds.
Joining us on that expedition was filmmaker, Dylan Stirewalt, and cinematographer, Clayton Kruse, on what would become the first of many shoots for the truly extraordinary, in-progress documentary film, Marrow of the Mountain.
Those of you who have been fortunate enough to see Roo speak at any one of the many mycological societies he's presented at over the last few years will already be familiar with the film and its premise, and the important role he's taken on as the film's producer. For both former audience members of Roo's, and for those altogether unacquainted, I strongly encourage all of you to take a trip over to the film's website, and treat yourself to the many videos and other resources it has to offer, including two beautiful trailers. This, too, has been a labor of love -- with ample labor and ample love -- for both cast and crew. Shooting, editing, funding, and distributing an independent, international documentary is a tremendous undertaking in the best of times, and quite a bit more difficult during a worldwide pandemic. My hat goes off to everyone involved in this project for their perseverance and dedication to tell this critically important story from the underrepresented perspectives of three Ecuadorian women. Having had the super special privilege of seeing a few rough cut fragments some months back (one of the perks of being buddies with the producer!), I have every confidence that all the hard work is going to pay off in a big, big way. The theatrical premiere, whenever the time comes, will be followed by a worldwide streaming release shortly thereafter. Stay tuned.
Immediately following our National Geographic expedition, Roo and I taught a university-level course entitled, Knowing the Ascomycota: Characteristics and Importance, at Universidad San Francisco de Quito (USFQ), the first of its kind in Ecuador. Following the succesful conclusion of that course, I stayed in country for quite a bit longer than other project participants, partly in order to ensure our specimens were properly duplicated, deposited, and packaged for transport to Oregon. If you've never coordinated the building of a custom, reinforced cardboard box with an Ecuadorian packaging and logistics company to house and safely transport ~200 cloud forest fungus specimens to a destination 4100 miles away, you haven't lived! Another perk of staying in Ecuador longer was being present for a victory in the provincial court case brought against the Ecuadorian Ministry of the Environment, the Ecuadorian National Mining Company, ENAMI EP, and the Canadian megamining company, Cornerstone Capital LLC. The lawsuit was filed in November of 2018 on behalf of the autonomous government of Cotacachi and the many impacted localities therein (including Los Cedros), . In their ruling, the court affirmed that the constitutionally protected right to environmental consultation had been violated, and all mining activities in the area were to cease immediately. We had won this battle, but the war would rage on for quite a while longer. Not only would the national and Canadian mining companies wantonly violate the injunction placed on them the provincial court over the ensuing months, but they would have the opportunity to appeal the decision to Ecuador's highest court, and appeal they most certainly did. More on that in a bit.
Once back in the US, our team began work on a conference poster, which was presented at the 2019 Annual Meeting of the Mycological Society of America in Minneapolis, Minnesota. In it we summarize our work at Los Cedros, our preliminary findings thus far, and implications/plans for the future. The poster also serves as the basic outline for a much larger, much more comprehensive publication to come, in which all the data from all 11 years and 6 expeditions will be presented, with corresponding sequences published in the GenBank and UNITE genetic databases, and iNaturalist observations for each of our ~1750 collections, many containing high-resolution, focus-stacked, color-calibrated images of species which have never before been illustrated or photographed. Once completed, this paper will constitute one of the largest single contributions to the knowledge of Ecuadorian fungal diversity in the history of the country. The same publication will also examine fungal community variance between different sampling plots; highlight certain known or suspected rare/endemic species awaiting assessment in the IUCN Fungal Red List; and discuss the past, present and future threats facing Reserva Los Cedros, and how our research and research like it helps offset and protect against them. While polishing and perfecting this great big synopsis has been and will continue to be our primary focus for the time being, we hope to keep engaging the interest of specialists for the production of smaller, more taxonomically focused publications. This is where you'll start to see novel Los Cedros taxa described, baptized into binomial nomenclature for the first time.
The MSA meeting was the actually the halfway point for me in a cross-country drive from Ithaca, New York to Eugene, Oregon, where I would be moving to dedicate more time to this project. It was a time of great anticipation. Roo and I looked forward to spending long days and nights with necks crooked over microscopes, combing through literature, and continuing the sequencing efforts begun as a result of the success of this campaign. I began planning what was slated to become the first Richard P. Korf Memorial North American Ascomycete Foray, to be held the following spring at one of the most special places on Earth, the Opal Creek Ancient Forest Center. I was asked by a local cafe to show my photography for the first time in a gallery setting for two month-long, back-to-back shows.
Then, around six months from the date of this campaign's last lab note, the first cases of COVID-19 started to appear in the United States.
I needn't beleaguer you, dear reader, with too many details of what happened next. We were all there. There were some ways, however, in which Eugene, Oregon was hit particularly hard. That summer, the west coast of the United States experienced one of the most destructive fire seasons on record. One of these fires, the Holiday Farm fire, closed in on the Eugene-Springfield metropolitan area like a fiery noose, giving our small city the month-long distinction of possessing the single worst Air Quality Index rating on all of planet Earth.
By the end of the calendar year, over 10 million acres had burned across California, Oregon and Washington, resulting in nearly $20 billion in damages and upwards of 12,000 deaths. Among the casualties was the Opal Creek Ancient Forest Center, the wilderness it once sat at the center of, and the man who dedicated his life to the unrelenting protection of both, George Atiyeh, whose remains were discovered not far from his forest home.
The job of performing fungal biodiversity research is not an easy one under the best of circumstances, but for us and for countless other scientists around the world, much of our work was rendered all but impossible to continue under the circumstances presented by COVID-19 and its secondary and tertiary effects. Laboratories, herbaria, entire universities and museums and research institutes, were shuttered in observance of public health mandates to avoid the spread of the virus. We continued to do what we could from our homes, shifting to virtual equivalents of in-person courses, seminars and conferences whenever possible, but it wasn't easy. Luckily, a great deal of what remained to be done at that stage of our project could be done from behind a keyboard and through the mail. I finished the entire backlog of expedition photos, our core team members conducted zoom meetings around once a month to discuss our progress and troubleshoot problems, and we generally kept the train chugging on its track. Eventually, as more pandemic dust began to settle, we were able to take that surplus $1500 and apply it to another round of sequencing, further strengthening our data set to now include upwards of 900 ITS sequences, along with some of those additional "stretch goal" loci we'd promised for specimens deserving of that extra molecular attention, and still have money left to spare. What's more, with sequencing costs looking like they're dropping by an order of magnitude courtesy of the breakthroughts brought about by MinION and Nanopore technology, we find ourselves in an unexpectedly comfortable financial position to do even more molecular work than we thought was in the budget.
The last item we have to debrief you all on is something that truly deserves its own lab note and headline, which will immediately follow the posting of this one. If you've waded this far into the weeds of our mega-Ecuador-update, congratulations! Take a bathroom break! Get up and stretch! Drink a glass of water! Have a snack! You've earned it! And while you do, I'll be right over in the next lab note, which will be of a much more merciful length, promise!
Yours in Spores,
-Danny
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