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.