PCR & Molecular Systematics! (Science IRL S1 Ep 2.2)

Welcome back to Science In Real Life! In part 1 of this episode, we used PCR to
isolate and amplify a specific region from the genome of 8 species of the carrot family,
and then we sent the PCR product to the magical land of the DNA sequencing facility. You were
probably left wondering “whyy did they do all of that?” Well, you’re about to find out!
Hint: it has to do with molecular systematics! Which is just a fancy way to say that we’ll
be using DNA sequences to understand the tree of life! …The scientific tree of life, that
is, not the Terrence Malick movie that I don’t think anyone really understands. Molly: How does molecular systematics add
to more traditional forms of classifying species? Greg: Well traditional approaches to classifying
species were based mostly on looking at the shapes, the sizes, the colors, numbers of
parts- that’s what we call “morphology.” And also the way the cells are put together, which
is what we call “anatomy.” These systems have been really very successful- I’d say about
80 to 90 percent of traditional classifications have been confirmed by molecular systematics.
The 10 to 20 percent of cases where molecular systematics disagrees with the traditional
systems are usually cases where there was “convergent evolution.” And that’s when species
that are unrelated (don’t share a common ancestry) might have evolved the same kinds of adaptations
in response to similar kinds of environmental pressures. Molly: So for example, species that are all
adapting to a dry, harsh desert environment might all have succulent leaves and spines,
but they don’t share a common ancestor. Greg: Exactly. So making a phylogenetic tree is a little
bit like making a genealogy, or a family tree. In reconstructing family trees we can go to
our parents and grandparents and ask them questions, and we can also go to things like
county courthouses and look up records. But we can’t do that for species of plants, because
they’ve been around for millions of years, so there’s no courthouses that have their
records. So instead instead what we can do is go back into the genome and look, because
the genes have secrets about the way that the plants originated, how they’re related,
and the timing of those things. Molly: Before we jump into data analysis,
here’s a quick reminder of the DNA region we isolated and amplified with PCR. Greg: We’re gonna be amplifying a spacer region
of DNA *between* two genes from the chloroplast genome. And the reason we’re doing that is
that the species that we’re looking at today are very closely related. Their genomes are
going to be very similar, and we need to find a region that’s accumulating mutations much
more rapidly than a gene would. Molly: So if I were building a phylogeny of
distantly related plant groups, I could use a gene accumulated mutations more slowly because
there would still be enough characters to build the tree. Ok so our PCR product, which contains millions
of copies of this chloroplast spacer region, is analyzed by machines at the sequencing
facility that can detect the order of the nucleotide base pairs in the molecules of
DNA. This is the DNA sequence, which we can read like a sentence and use in our data analysis. Greg: What we see here are two different strands,
one’s a forward and one’s a reverse, so they’re both complementary strands of DNA, and if
we zoom in, we can see the actual base pairs, and those base pairs are based on these chromatogram
peaks. These are called chromatograms because the different peaks are different colors,
and the different colors represent the four nucleotides of DNA: Guanine, Adenine, Thymine,
and Cytosine. And, what we’re going to be looking at here is to see if there are any
differences between this sequence from one species, and other species that we’re going
to be comparing it to. So those differences across species are what we call “mutations,” and those mutations are the way we build our phylogenetic tree. So the next step is to take the one species
that we had, and to compare it to all the other species that we’re doing the same thing
for. We have rows, and the rows represent the different species. And then we have columns,
and the columns represent different individual base pairs that make up the gene across all
those different species. So the colors are the same as we saw in the
chromatogram, the four different base pairs. And then differences in the colors represent
the kinds of mutations that we were talking about before. So there can be point mutations,
and those are where there’s just a little bit of difference between whether it’s a,
for example, a cytosine here in the first two, or adenines in the next five. And we
can also have length mutations, and that’s where the sequences are of different lengths
across the entire gene, and we have to line those up. So what we do is we insert gaps,
where we’ve pushed over the sequence so it lines up with the sequences of the other species
that have the extra base pair. Molly: OK so this group of species is missing
that base pair, while this group has it. Greg: Exactly, exactly- and so that sort of
represents kind of a natural grouping of species that you can see there. Molly: OK so how do we get from that alignment
to this tree? Greg: We take the sequences that were in the
alignment, and we analyze them with a computer program that looks for shared similarities,
or shared characters, among these different species. And that computer program then helps
generate this phylogenetic tree. So what we’re seeing here is that the Bowlesia
group represents a single evolutionary lineage. And what that means is that they all share
a single common ancestor. All the Bowlesia group would share a series of characters that
could be mapped out along that branch. Another thing that this tree shows is that
all the species that were originally placed in Bowlesia come out together. So this is
a case where our sort of modern techniques are completely agreeing with, and confirming,
the results of earlier scientific work. This is a tree of related groups, so the same
family, and what we see here is that taxonomy didn’t fare as well. So what we’re seeing
now is, for example: this yellow group is the genus Asteriscium. And so traditionally
we thought that was one genus, so what we would expect is that all the yellow things
would come out together, that there would be a single common ancestor for those. But
instead what we’re seeing, for example, is that some species of Asteriscium in yellow,
and the green ones in Pozoa, share a more recent common ancestor, than other species
of Asteriscium in the same group. So this is showing where our evolutionary trees differ
from the traditional taxonomy, and it gives us a good example of the way that we are able
to improve upon earlier scientific work. Molly: It’s just really amazing to me that
we can go from PCR reactions to understanding the tree of life in a few steps. Greg: Absolutely. PCR has totally revolutionized
biology. We know that it’s really important for things like the Human Genome Project,
you’ve seen how it’s involved in evolutionary biology and reconstructing these evolutionary
trees, and also to give you a sense of some of the research that we do here on plant biodiversity
at the New York Botanical Garden. Molly: Well, thank you so much Greg for joining
us for today’s episode, and thanks so much for watching Science In Real Life, and we’ll
see you next time!


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