Danny's DNA Discoveries

What follows are definitions of the terms I will be using throughout the article when discussing 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 genes 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 organism, 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. Sometimes two mushrooms might have the exact same ITS DNA and still be different species. This can happen if 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 sequence both parts, but we often only have ITS2 data available for our local Russulas. 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.

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.

Like humans, mushrooms are diploid organisms with two complete distinct sets of DNA. We 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, apparent differences between two sequences may be explained by different alleles of the same species. We may need to analyze more sequences of the mushroom to determine all the places where there may be more than one valid choice of nucleotide.

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 comprehensive when only one short region is sequenced. Obtaining an accurate picture of how a group of sequences relate to each other may require sequences six or more different genes. But even if you could look at the entire genome of 50,000,000 nucleotides, that won't answer all the questions. 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.

What kind of research do I do before I declare that we probably have a mushroom of a certain name in the PNW? If our DNA matches the DNA of an official type collection of a species, that's some good evidence. But there usually isn't an official type sequence to compare with (one of the most important areas of study right now is getting type sequences of as many species as possible). So what I usually have to do is look at sequences from the type area, where the one official mushroom of that species was picked. If a bunch of mushrooms from the same area that people think is that species were sequenced and they all have the same sequence, and that matches our PNW sequence, it might be correct to say we have that mushroom here. But if there are other sequences that don't match ours that people have thought was that mushroom, then it's tricky to know which one is right. And if somebody gave a mushroom with the same sequence a different name, that also casts doubt on things.

This is only the barest of overviews of this process, for more information please read my papers and watch my videos at the top left of this page under "Under Free Resources".

Any results from a single gene region, like ITS that I use, 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 morphological and ecological 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.

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.

The species epithet has to agree with the gender of the genus if it is an adjective, so when a mushrooms changes genus, it may require a slight spelling change to the end of the species epithet, such as Gymnopus peronatus becoming Collybiopsis peronata.

When I talk about a clade of mushrooms, I mean a group of species that are all each other's closest relatives. Other mushrooms may look just like the mushrooms in a clade, but be only distantly related, so they don’t count. Closely related mushrooms may share the same environmental benefits, health benefits and poisons, so it’s important to know how closely mushrooms are related to each other, not simply which 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 education@psms.org.

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.

Monophyletic - you can find a node in the binary tree where every mushroom with that name (at whatever level - species, genus, family, sub-order, order, class, etc.) is past that node, and nothing with a different name is past that node. For instance, the genus Amanita is a monophyletic genus if there is a node past which everything is called Amanita, and no Amanita occur anywhere else in the tree of life. This is a requirement for considering that mushrooms have been properly named without controversy. But another requirement is that doing so does not keep any other name at the same level from being monophyletic (see paraphyletic).

Paraphyletic - Almost monophyletic, but there are one or more nodes inside your tree that contain names that are not the same. For instance, inside the tree for the genus Leucoagaricus, there is a node past which are found all of Leucocoprinus. Leucocoprinus is monophyletic, but it is keeping Leucoagaricus from being monophyletic because it is "inside" it instead of "beside" it where it wouldn't affect it at all. Leucocoprinus is rendering Leucoagaricus paraphyletic - there are one or more branches you have to prune from Leucoagaricus to make it monophyletic. Sticking to the rule that nothing be paraphyletic can be problematic because all of Leucocoprinus would have to be renamed to Leucoagaricus, and you won't have a good name for those very distinct mushrooms, and all just because they evolved at an inconvenient time from a Leucoagaricus ancestor instead of from an extinct ancestor of all Leucoagaricus.

Polyphyletic - species with that level name are found past multiple nodes of the tree. A node that had all of the species with that name past it would also include other names that couldn't be pruned away. Species that are only distantly related have been incorrectly given the same name, probably because they superficially looked the same. This is never allowed.

Russula s.l. (sensu lato) - meaning "in the wide sense" refers to all mushrooms once or currently called Russula including some that don't really belong there based on DNA evidence. Expect some of them to get a new genus name.

Russula s.s. (sensu stricto) - meaning "in the narrow sense" refers to only those Russula mushrooms that form a monophyletic clade together and properly belong in Russula. None of them should need a genus name change.

Basal clade - one of the earliest branches from a certain node in a tree. It's tempting to say it is the "oldest" or "most primitive" group of species, but that's not entirely correct. The extant species in the basal branch are around today, and may have evolved recently from other similar species, just like every other species on a supposedly "more evolved" branch. But there is a more direct line from the species in a basal clade back to the node that is the common ancestor of every species past that node, and the species in the basal clade may be more likely to share traits with the ancient species from which everything past that node evolved.

Ancestral trait - when some species past a node have one character yet others have a different one, the ancestral trait is the one that the (probably extinct) species back at the node had, implying that the species past the node with the different character evolved a change.

Derived trait - the other character, the one that wasn't true of the ancestor back at the node. When deciding which of two traits is ancestral or derived, we usually assume the fewest number of required mutations back and forth to explain the tree.

And remember, when I talk about the taste of a 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.