Danny's DNA Discoveries
by Danny Miller, firstname.lastname@example.org
This project will attempt to document, over the years, which mushrooms species actually occur in the Pacific Northwest based on DNA analysis of projects funded by PSMS and other entities. I define the PNW as Washington, Oregon, Idaho and southern British Columbia, although MycoMatch includes species found in northern BC as well.
Other free resources: click here to download MycoMatch (MatchMaker) for PC and MycoMatch Mobile (Beta) for iPhone.
If you have any information to add, or corrections to make, please contact me above!
In this series, I will be analyzing the results of several different projects that have funded the DNA sequencing of our local mushrooms. These sequencing efforts have provided a valuable database of information, but analyzing the data properly can be difficult as there are few, in any, textbooks and courses that teach these new techniques. Graduate students in university labs are often having to teach each other tricks and tips they have picked up because some of these techniques are too new to have been incorporated into school curricula. My goals include the following:
These articles will not just be the dry details about the new things we have learned. It is my intention to make them fun to read little ID classes with interesting stories and tips for learning about the group of mushrooms that the exciting new information is about.
PSMS (the Puget Sound Mycological Society, http://www.psms.org) has funded the sequencing of all the collections saved at the NAMA (the North American Mycological Association, http://www.namyco.org) foray near Mt. Rainier WA in 2014. PSMS and NAMA co-funded the sequencing of the collections saved at the NAMA foray near Salem OR in 2018. PSMS is also funding the sequencing of newer collections made at PSMS forays and events. These projects contributed greatly to this and further articles I will write about other genera. This was all done with the generous sequencing help of Stephen Russell of Purdue University, the Hoosier Mushroom Society of Indiana and a founding board member of the North American MycoFlora Project.
The non-profit "North American MycoFlora project" (http://www.mycoflora.org) was recently founded by members of the continent-wide "North American Mycological Association" of amateur mycologists partnered with the "Mycological Society of America" (the association of professional mycologists) to create an organization that could help all of the dozens of mushroom clubs across the continent study their local mushrooms. Specifically, they help educate clubs about how to do DNA sequencing projects and provide low cost services and funding assistance so that each club doesn't have to figure out how to do it on their own. It's also a way to make sure that all of the experts in the various groups of mushrooms have access to the data discovered by every club on the continent to make sure that the information learned gets disseminated and written up in papers for the greater good.
Matt Gordon is doing the very important work of sequencing our "type" collections (see definitions below), so we actually know what our mushrooms are and have something to compare future collections to. I consider this the very important first step of any genetic work that is done, upon which everything reliable will be based.
I am a co-author of the free PNW mushroom identification program “MycoMatch (MatchMaker)” (http://www.mycomatch.com) and its pictorial key, and the results of these studies will be making their way into these programs.
What follows is a definition of some of the terms I will be using throughout the article when debating how confident I am that a certain specimen is really a certain species or not, as well as a bit of background on how one identifies a mushroom after getting a DNA sequence.
The DNA of a mushroom is something on the order of 50 million base pairs (called nucleotides) long. That’s much shorter than human DNA, which has about 3 billion base pairs. It’s still too expensive to sequence (and unwieldy to analyze) that much DNA, so the scientific community has agreed on some short areas that can be sequenced that can more easily provide a good deal of valuable information. One of the most popular regions to sequence, called ITS (Internal Transcribed Spacer), is only about 700 nucleotides long, which is quite manageable. It is sometimes called a “junk” area, meaning it doesn’t really do anything (or at least its integrity is not as important to the survival of the organism as other critical areas). It is thought that it is allowed to mutate quicker than other genes, as a mutation in ITS has little effect on the organism. Mutations in other critical areas might kill the oganism, so those areas resist mutation. With ITS free to mutate as much as it wants, you see smaller differences between individuals, making it a great choice to try and tell if your two specimens are the same species or not. It is not a good region to determine how two organisms are related from a larger perspective (same genus or family), only for determining the more subtle differences that differentiate two species, which is what I am mostly trying to accomplish. ITS has been nicknamed the “barcode” area because you can imagine scanning a mushroom to read its ITS DNA and comparing it to a database so that this hypothetical barcode scanner could then tell you what mushroom you have like produce in the grocery store.
The biggest, most loaded question of all time is: how much DNA difference does there have to be for something to be considered a different species? There is no good answer to that, and there may never be. When comparing ITS regions, although some people say that a difference of 3% probably indicates a different species, it very often turns out that a difference of 0.5% is enough to indicate a different species. That means 4 characters out of the 700 characters of an ITS sequence being different might be enough. 10 differences almost certainly does. But sometimes two mushrooms might have the exact same ITS DNA and still be different species, because, as luck would have it, other regions mutated faster than ITS did, even though that is not typically going to be the case. ITS is divided into 2 parts, ITS1 (up to 300 or so characters) and ITS2 (up to 400 or so characters). Ideally, we are sequencing both parts, but for Russula, we mostly only have ITS2 data available for our local specimens. Any more than 2 differences in ITS2 means the DNA is >0.5% different. So as I make judgment calls below as to whether or not our local species are unique, I will call out any species with more than 2 ITS2 differences as potentially being different.
Complicating things further, mushrooms are diploid organisms like humans, meaning that there are two complete sets of DNA inside them, different from each other. When sequencing, you may get a sequence of the first version, or “allele”, the second version, or both, with more than one possibility for a nucleotide at several locations. Thus, DNA that looks very different (much > 3%) might be caused by two sequences of different alleles, and still represent the same species, until you analyze many different sequences of the mushroom to determine all the places where there may be more than one valid choice of nucleotide.
In other words, identical DNA does not mean your species is the same, and vastly different DNA does not necessarily mean that your species is different.
It’s important to remember that DNA is not a magic bullet but only one tool to be used with other, more conventional research tools. DNA results are not reliable with only one short region sequenced, studies may sequence up to six different genes to get a better picture. But just looking at the DNA, even the entire genome of 50,000,000 nucleotides, will never be enough. If there are differences in the way the mushroom looks, or microscopic or ecological differences, a few differences in DNA may be another clue that our mushroom is a unique species. But if there are no other differences, a few differences in DNA may not be significant. Conversely, if there are ecological, morphological and microscopic differences, but no DNA differences in certain genes, it may still be a separate species. You might have to sequence the entire genome to find the DNA differences. Ultimately, it’s a matter of opinion, and the more information we have, the better we can make an informed opinion.
As you might expect, there is an internet database that most people use to store their DNA sequences, called GenBank, so you can compare your sequence to this giant database to get an idea of what it might be. However, this is not very useful at all, as it turns out most GenBank entries are identified incorrectly, with the wrong mushroom name. Most of them! This always surprises people to learn. GenBank might be able to tell you what parts of the world your DNA was found in (without being able to identify it) but unfortunately, it often can’t even do that. Until recently, it has not been common for people to record in GenBank where their mushroom was found. This is a huge oversight that just goes to show how new and imperfect our technology and techniques are.
Only a small percentage of species have their official “type” specimen sequenced, which is a definitive way of knowing how your mushroom compares to the official “real” thing. One of the most important pieces of work being done is to sequence as many types as possible. This is a necessary first step before we’ll be able to make any definite conclusions on a large scale. But many of them are hundreds of years old or don’t exist anymore, so people will have to designate new types, or “neotypes” and make their best guess as to what the original mushroom was. From then on, the neotype will be the official specimen, and it will have to be forever assumed, rightly or wrongly, that it is the same as the original type.
So then, how do you figure out what your mushroom is? It is not easy. You have to look at every part of the world your sequence is found, and every sequence with an identification of the species you think you have, all over the world. You will often find that a half dozen or so vastly different DNA sequences have come out of mushrooms that people thought were the same thing. At most one of them can be right! If every recorded specimen from the type area has the same DNA, and there are no specimens that look like it with different DNA, you might have found a reliable sequence of that species. This is only the barest of overviews of this process, a whole text book could be written on how to determine what species you have from a DNA sequence, and unfortunately, to my knowledge, none have yet so there’s nowhere you can go to learn these techniques yet.
But any results from a single gene region, like this, can only be considered preliminary. Definitive answers of whether or not our local species are the same or different from other species around the world must wait until more gene regions are sequenced and non-genetic studies are done as well to corroborate what we find here.
You can download all of my sequences, plus rudimentary trees created from those sequences, but do not read too much into the trees, they are full of inaccuracies. ITS DNA cannot show accurate relationships between anything but closely related species, and the BLAST algorithm I mostly used to make them, while quick and convenient, does not align very well and does not use the best construction algorithm, and therefore can do an especially poor job of making trees. I recommend using my sequences and better software to make your own trees before making any conclusions.
A few other terms I use:
Russula cf emetica – cf is “confer” in Latin, meaning “compare”. I use this term to mean the mushroom looks like R. emetica, but might not be. It makes no judgement as to whether it is genetically related to R. emetica, only that it looks like it.
Russula aff emetica – aff is “affinis” in Latin, meaning it has an affinity to it. I use this term to mean the mushroom is very closely genetically related to R. emetica, but may or may not be close enough to actually be R. emetica. There is a distinct possibility that it will turn out to be a different species in need of its own new name.
Russula 'xerampelina' - if I put single quotes around a name, it means that is the name we've been using for the mushroom, but it may not be correct, for one of the above reasons. In other words, it would be more correct to call it Russula cf xerampelina or Russula aff xerampelina, depending on whether or not it is actually closely related or just looks similar.
When I talk about a clade of mushrooms, I mean a group of species that are all related to each other. Other mushrooms may look just like the mushrooms in a clade, but be unrelated, so they don’t count. Related mushrooms may share the same environmental benefits, health benefits and poisons, so it’s important to know if mushrooms are actually related to each other, not simply look the same to the untrained eye. These articles will include mushrooms that there is genetic evidence for. We no doubt have additional species that I will not be mentioning, but are either rare or have not been part of a genetic study. If you think you have found a specimen of any of the species that I say we need more information about, or anything that I haven't mentioned, please take good pictures and save it, and contact me at email@example.com.
When I talk about a group of mushrooms, I might mean mushrooms that look the same, even though it's possible they may not be all closely related. A group is not as specific as a clade.
When I talk about comparing DNA, I am specifically talking about comparing the short ITS regions of DNA unless I specifically say otherwise.
And remember, when I talk about the taste of mushroom for identification purposes, some people are comfortable tasting a small piece for 30 seconds and then spitting it out, if they are sure it is not a dangerous species. Do not swallow.
Eventually, there will be a report for every genus. Links that have been written so far and can be clicked on begin with a • and are underlined. You can expand and contract branches of the tree by clicking on an entry with an arrow. Some interesting features that clades have evolved are noted in bold, like spore colour. I've seen similar trees where people have shown which branches evolved to be mycorrhizal, which is neat to see.