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.
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
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.
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.