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Danny’s DNA Discoveries – Background and Definitions

by Danny Miller

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. 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 judgement 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.

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, please take good pictures and save it, and contact me at education@psms.org. 

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