The Quest for Phaeocollybia

part 5


DNA Barcoding

“Ron’s favorite Phaeocollybia — the striking P. fallax” – Lorelei Norvell

“Ron’s favorite Phaeocollybia — the striking P. fallax” – Lorelei Norvell

Leon Shernoff: So the next thing I wanted to ask about was what sort of cool things are happening in the field? What do you think is the next exciting thing?

Lorelei Norvell: Well, the next exciting thing probably is the use of bar codes. This is not going to excite field mycologists overmuch, because we like to go out there and look and eyeball and feel at one with the universe by putting a name on something – this is what got me into it, you know? – you’re all-powerful, all-controlling, and you know the name!

You remember that story where Rolf Singer supposedly dreamt that he went to heaven, and it was all ruined because every mushroom had a little tag next to it, telling what it was? (laughing) In other words, all the fun is taken out, all the joy of speculation!

But as you know, there are problems with identifying mushrooms based on the physical features of the fruiting body. First of all, there are some species that are so physically similar that you’d have to culture them – and that takes a long time – to tell them apart. But more seriously, if you want to really find out what’s out there, you don’t want to have to wait for the one week a year that a mushroom fruits – if it fruits, and if you can catch it. Cryptic species have to be distinguished via DNA, if you want to be able to do it in any reasonable length of time; and more importantly if you can do DNA surveys of root tips and other parts of the rhizosphere, you can find the fungi that there are present underground – even if you never see a mushroom from them above ground.

So now mycologists, including the BLM in Oregon, are trying to find out how best to bar code mushrooms. They’re determining what short DNA sequence – the bar code – will identify a species. So that all you need to do is run a ‘quick’ PCR, which is these days done much more cheaply than it used to be, and you’ll have things identified, whether or not a fruiting body is present.

We been trying this for Phaeocollybia out here since 2008. Matt Gordon, who works with the BLM, has been developing DNA bar codes. With Phaeocollybia listed as a genus of concern for the Northwest Forest Plan, it seemed logical to develop bar codes, particularly as the species are difficult to identify. I can identify the species fairly quickly, but… I have a finite existence here, and others have to be able to answer, “Well, is the Phaeocollybia there or not?’

You really can’t depend on somebody who spent… 30 years learning a genus, because when they’re gone, you have to reassemble all those books and everything else, and figure out what they knew. So Matt, Ron Exeter, and the BLM team have sampled all along the places where we previously collected Phaeocollybias; Matt is matching sequences to see what’s present and what’s not. And of course he is finding that there are times when certain species of Phaeocollybias are very evident in the core sample, but are not fruiting so you wouldn’t know it.

I do not use the terms “amateur” and “professional” because I regard them as divisive; they had a splintering effect for years, because you would have this endless battle between ‘amateurs’ and ‘professionals’ over semantics. It’s rather the focus on whether you like going out, being in nature, collecting things, learning about the environment from total immersion that is important. Or whether you’re an academic one who learns about it through books, lab benches and what have you. But now you have so many people in both communities who do so much of both, that that distinction is breaking down and people are able to work together with mutual… appreciation. It’s much better than a focus on “I’m better than you because I have a degree,” or “I’m better than you because I know the beast better than you at its own level.”

And then you introduce commercial mushroom harvesters, who are surprisingly knowledgeable, whom everybody looks down on because they’re getting money for what they’re doing, and yet they know a hell of a lot, if you just talk to them. (laughing)

(laughing) I remember Dick Sieger once called me a ‘good mushroom gone bad,’ because I chose all this book-learning and knowledge over something I already knew well enough as far as he was concerned! I think he was kidding…

Anyway, rather than use ‘amateur,’ I just use the term ‘field mycologist.’ I am a field mycologist. But I’m an ‘also-put-in-my-time-at-the-bench’ mycologist and also spend a lot of time at the microscope – which field mycologists do if they want to get to the end of things.

I think coming up through the ranks of the Oregon Mycological Society and NAMA was an incredible gift, because you learn how people will work together and share knowledge and collaborate. But because of the intertwining of all of these various disciplines in mycology, people are working in collaboration at the academic level too, and it has to be done that way. It’s an extremely exciting time in that regard. The internet has really facilitated communication, so that writing papers with colleagues across continents is no longer a problem. It used to be you would send a letter and then you wait, and meanwhile you lose the thread of what you were talking about.

So in that regard, it is quite a vital and growing time. Nothing is done in a vacuum. It’s worrisome in a way, because I think there are times when the ability to express what you think has been made so easy, that… conclusions are dispensed before they have been tested. And so there is an intermediate misinformation stage these days. But that is evident everywhere – not just with mycology. It is unsettling, particularly if you’re looking at a chronic cancer, to read a news item announcing that a new treatment has been discovered. Unfortunately it is the publication of a new breakthrough that gets press. But what does not get press is the discovery “Whoops! It’s not repeatable. It’s not reproducible. It didn’t work!” The ‘breakthrough’ vanishes and simply does not get talked about. And you don’t hear about this until you pursue it to its logical end. Which means that there is not sufficient duplication of effort where there ought to be: “You need to check this, and see if you can reproduce the result.”

But you know, if you’re going down a blind alley, if you have a hundred people going down the same blind alley and needing to turn back again, I think the irreproducible results should be published more quickly than they are. You’ve got a growing, healthy science, but a certain amount of caution probably is advised.

The founders of the chanterelle collection study project looking over their location: Lorelei Norvell, Frank Kopecky, Maggie Rogers and Janet Lindgren.

The founders of the chanterelle collection study project looking over their location: Lorelei Norvell, Frank Kopecky, Maggie Rogers and Janet Lindgren.

Collaboration is the thing that I would like to stress. Oregon’s chanterelle study was a phenomenal effort. We had so many great volunteers (plus the hardy few who stuck with it for the whole 20 years and supervised by Judy Rogers from 1992–2006). Although the data still need to be statistically evaluated, we found pretty much what they are finding elsewhere: that you’re not going to overharvest if you simply pick the fruiting body of an ectomycorrhizal fungus, whereas you might if you were doing it with a saprophytic fungus – it’s a whole different beast.

It was a really interesting group of folks who worked together well. A massive amount of work was done with volunteer labor— the sort of thing that gives people hope that people can make a solid contribution without waiting for a government or industry grant – and that you can do an awful lot just with manpower and curiosity.

19 The Internal Transcribed Spacer region of ribosomal DNA. Because it mutates rapidly, it has proved uniquely useful in distinguishing closely related species.

So this is all exciting, though of course DNA and barcoding may take some of the excitement out of the in-the-field experience. The work now is on what part of the genome to use, and Conrad Schoch is doing some beautiful work on that. We’re finding that for almost all fungi, it’s the ITS,19 which is really nice, as that is the region I used (during 1993–95) and found that worked really well for Phaeocollybia. (laughs) I had samples for all my specimens, yet DNA that told me, “No, no, no! This sample is not scatesiae! This is oregonensis.” The RFLP identified the proper fungus immediately on the gel – which I confirmed when I went back to the dried specimens and discovered that an oregonensis cap had gotten mixed in with scatesiae on the drier! With Phaeocollybia the ITS is really good at differentiating species, although not perfect –because you do have these clusters of species – olivacea is one confusing group (which includes gregaria) where there’s a lot of ‘noise.’ And the kauffmanii group has problems where for certain species something is interfering with the DNA, so there have been problems in extracting and sequencing the DNA properly.

But what we try to do with bar code is identify a very short sequence that will immediately tell you what the name of this is. So that’s exciting from a knowledge standpoint. It’s an exciting time. People are finally really learning the blending of what we see in the field with the knowledge of the genome, and finding out how to see visibly what the relationships are; looking at the evolution there in biology and how things have evolved over time.

That is all exciting stuff – and never-ending!

Lorelei and a trillium, in ‘Oz’, “our old growth (>250 yrs) chronosequence study plot in Polk County.”

Lorelei and a trillium, in ‘Oz’, “our old growth (>250 yrs) chronosequence study plot in Polk County.”

Further Reading

Lorelei Norvell, “Melbourne approves a new Code”
This is Lorelei’s article explaining the changes in the code for nomenclature.

Susan Milius, “The name of the fungus”
An article that discusses many of the issues with the new Melbourne nomenclature rules. Milius talks with several interesting people connected with them.

Steven Trudell, Paul Rygiewicz and Robert L. Edmonds “Patterns of nitrogen and carbon stable isotope ratios in macrofungi, plants and soils in two old-growth conifer forests”
A discussion of the issues regarding carbon and nitrogen isotopes in fungi.