14
plant molecular systematics
ACQUISITION OF MOLECULAR DATA . . . . . . . . . . . . . 585
MICROSATELLITE DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
DNA SEQUENCE DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
random amplified polymorphic dna
(rapds) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
Polymerase Chain Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
DNA Sequencing Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
Types of DNA Sequence Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
Analysis of DNA Sequence Data . . . . . . . . . . . . . . . . . . . . . . . . . . 590
AMPLIFIED FRAGMENT LENGTH
POLYMORPHISM (AFLPs) . . . . . . . . . . . . . . . . . . . . . . . 596
REVIEW QUESTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600
RESTRICTION SITE ANALYSIS (RFLPs) . . . . . . . . . . . . . . 592
EXERCISES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600
ALLOZYMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
REFERENCES FOR FURTHER STUDY . . . . . . . . . . . . . . . . 601
Molecular systematics encompasses a series of approaches in
which phylogenetic relationships are inferred using information from macromolecules of the organisms under study.
Specifically, the types of molecular data acquired include that
from DNA sequences, DNA restriction sites, allozymes,
microsatellites, RAPDs, and AFLPs. (The use of data from
other, generally smaller molecules, such as secondary
compounds in plants, is usually relegated to the field of
“chemosystematics” and will not be reviewed here.)
A revolution in inferring the phylogenetic relationships of
life is occurring with the use of molecular data. The following is a review of the types of data, methods of acquisition,
and methods of analysis of molecular systematics.
in a container of silica gel. Alternatively, plant samples
may be frozen or placed in concentrated extraction buffer.
With any of these procedures, DNA is usually preserved
intact. Usable DNA is often successfully isolated from
dried herbarium sheets, attesting to the “toughness” of the
molecule.
DNA SEQUENCE DATA
ACQUISITION OF MOLECULAR DATA
Perhaps the most important method for inferring phylogenetic relationships of life is that of acquiring DNA sequences.
DNA sequence data basically refers to the sequence of
nucleotides (adenine = A, cytosine = C, guanine = G, or
thymine = T; Figure 14.1) in a particular region of the DNA
of a given taxon. Comparisons of homologous regions of
DNA among the taxa under study yield the characters
and character states that are used to infer relationships in
phylogenetic analyses.
The first step of acquiring DNA sequence data is to
identify a particular region of DNA to be compared between
species. Much prior research goes into identifying these regions
and determining their efficacy in phylogenetic analysis.
Plant samples from which DNA is to be isolated may be
acquired by various means. It is vital to always collect a
proper voucher specimen, properly mounted and accessioned
in an accredited herbarium, to serve as documentation for any
molecular systematic study (see Chapter 17). Live samples
may be collected and immediately subjected to chemical
processing, e.g., for allozyme analysis (see later discussion).
For many DNA methods, pieces of leaves (from which chloroplast, mitochondrial, and nuclear DNA can be isolated) are
removed from the live plant and immediately dried, typically
© 2010 Elsevier Inc. All rights reserved.
10.1016/B978-0-12-374380-0.50014-2
POLYMERASE CHAIN REACTION
585
After a gene sequence of interest is identified, the DNA from
a given plant sample is first isolated and purified by various
586
CHAPTER 14
Plant molecular systematics
NH2
O
=
CH 3
N
NH2
N
HN
N
=O
NH
HN
NH
adenine
NH
HN
NH
N
guanine
=
NH2
=
N
O
O
cytosine
thymine
Molecular structure of the four DNA nucleotides. Adenine and guanine are chemically similar purines; cytosine and thymine
are chemically similar pyrimidines.
Figure 14.1
chemical procedures. Following this, the DNA sequences of
interest are amplified using the polymerase chain reaction
(or PCR). The invention of this technology was crucial to
modern DNA sequencing, as it permitted rapid and efficient
DNA amplification, the replication of thousands of copies
of DNA.
The polymerase chain reaction work as follows (see
Figure 14.2). Prior research establishes the occurrence of
relatively short regions of DNA that flank (occur at each
end of) the gene or DNA sequence of interest and that are
both unique (not occurring elsewhere in the genome) and
conserved (i.e., invariable) in all taxa to be investigated.
repeat
cycle
sample DNA
solution
heated
DNA
denatures
3′
5′
5′
3′
temperature
lowered
3′
T-A-G-C-C-A-A-T-C-G-C-T
~
~
T-T-A-A-T-C-G-A-G-G-T-T-A
A-A-T-T-A-G-C-T-C-C-A-A-T
5′
A-T-C-G-G-T-T-A-G-C-G-A
T-A-G-C-C-A-A-T-C-G-C-T
~
~
3′
T-T-A-A-T-C-G-A-G-G-T-T-A
A-A-T-T-A-G-C-T-C-C-A-A-T 5′
5′ A-T-C-G-G-T-T-A-G-C-G-A
primers anneal
to conserved regions
DNA
renatures
3′
5′ A-T-C-G-G-T-T
3′
T-A-G-C-C-A-A-T-C-G-C-T
~
A-A-T-T-A-G-C-T-C-C-A-A-T
5′
A-T-C-G-G-T-T-A-G-C-G-A
~
3′
T-T-A-A-T-C-G-A-G-G-T-T-A
C-T-C-C-A-A-T 5′
3′
5′
DNA strands
replicated
5′
5′ A-T-C-G-G-T-T-A-G-C-G-A
free nucleotides
(catalyzed by DNA polymerase)
bind to primers
Figure 14.2
3′
T-A-G-C-C-A-A-T-C-G-C-T
~
A-A-T-T-A-G-C-T-C-C-A-A-T
5′
A-T-C-G-G-T-T-A-G-C-G-A
~
3′
T-T-A-A-T-C-G-A-G-G-T-T-A
A-A-T-T-A-G-C-T-C-C-A-A-T 5′
Polymerase chain reaction, using cycle sequencing to produce multiple copies of a stretch of DNA.
5′
Unit III
Systematic evidence and descriptive terminology
These short, conserved, flanking regions are used as a
template for the synthesis of multiple, complementary copies,
known as primers. Primers ideally are constructed such that
they do not bind with one another.
In the polymerase chain reaction, a solution is prepared,
made up of the isolated and purified DNA of a sample;
multiple copies of primers; free nucleotides; DNA polymerase
molecules (typically Taq polymerase, which can tolerate heat);
and buffer and salts. This solution is heated to a point at which
the sample DNA denatures, whereby the two strands of DNA
separate from one another. Once the sample DNA denatures,
the primers in solution may bind with the corresponding,
complementary DNA of the sample (Figure 14.2). Following
binding of the primer to the sample DNA, individual nucleotides in solution attach to the 3′ end of the primer, with the
sample DNA acting as a template; DNA polymerase catalyzes this reaction. A second primer, at the opposite end of
the DNA sequence of importance, is used for the complementary, denatured DNA strand. Thus, the two denatured strands
of DNA are replicated. After replication, the solution is
cooled to allow for annealing of the replicated DNA with the
complementary DNA single strands. This is followed by
heating to the point of DNA denaturation, and repeating the
process. A typical PCR reaction can produce more than a
million copies of DNA in a matter of hours.
DNA SEQUENCING REACTION
After DNA is replicated, it is sequenced. The most common
sequencing technology involves a machine that reads fluorescent dyes with a laser detector. The production of dye-labeled
DNA is very similar to DNA replication using the PCR. The
replicated DNA is placed into solution with DNA polymerase, primers, free nucleotides, and a small concentration of
synthesized compounds called dideoxynucleotides (discussed
later) that are each attached to a different type of fluorescent
dye. As in the polymerase chain reaction, the sample DNA is
heated until the double helix unwinds and the two complementary DNA chains separate (Figure 14.3). At this point,
a primer attaches to a conserved region of one of the strands
of DNA, and free nucleotides in solution join to the 3′ end of
the primer, using the sample DNA as a template and catalyzed by DNA polymerase (Figure 14.3). Thus, a replicated
copy of the DNA strand begins to form. However, at some
point a dideoxynucleotide joins to the new strand instead
of a nucleotide doing so. The dideoxynucleotides (dideoxyadenine, dideoxycytosine, dideoxyguanine, and dideoxythymine)
resemble the four nucleotides, except that they lack a hydroxyl
group. Once a dideoxynucleotide is joined to the chain,
absence of the hydroxyl group prevents the DNA polymerase
from joining it to anything else. Thus, with the addition of
587
a dideoxynucleotide, synthesis of the new DNA strand
terminates (Figure 14.3).
The ratio of dideoxynucleotides to nucleotides in the reaction mixture is carefully set and is such that the concentration
of dideoxynucleotides is always much smaller than that of
normal nucleotides. Thus, the dideoxynucleotides may terminate the new DNA strand at any point along the gene being
replicated. For example, some of the new DNA strands will
be the length of the primer plus one additional base (in this
case the dideoxynucleotide); some will be the primer length
plus two bases (a nucleotide plus the terminal dideoxynucleotide); some will be the primer length plus three bases (two
nucleotides plus the terminal dideoxynucleotide); etc. There
are many thousands, if not millions, of copies of the sample
DNA. Thus, there will be an equivalent number of newly
replicated DNA strands, of all different lengths.
The final step of DNA sequencing entails subjecting the
DNA strands to electrophoresis, in which the DNA is loaded
onto a flat gel plate or in a thin capillary subjected to an electric current. Because the phosphate components of nucleic
acids give DNA a net negative charge, the molecules are
attracted to the positive pole. The DNA strands migrate
through the medium over time, the amount of migration
inversely proportional to the molecular weight of the strand
(i.e., lighter strands migrate further). Each strand is terminated with a dideoxynucleotide to which a fluorescent dye
is attached; each of the four dideoxynucleotides has a different type of fluorescent dye, which (upon excitation) emits
light of a different wavelength. Thus, as the multiple copies
of DNA of one particular length migrate along the gel or
capillary, the wavelength of emitted light is detected and
recorded as a peak, which measures the light intensity.
Because a given emitted wavelength (“color”) is determined
by one of the four dideoxynucleotides, the corresponding
nucleotide can be inferred and its position identified by
the timing of migration of the DNA strands. In this way, the
sequence of nucleotides of the DNA strand can be inferred
(Figure 14.3).
TYPES OF DNA SEQUENCE DATA
For plants, the three basic types of DNA sequence data stem
from the three major sources of DNA: nuclear (nDNA),
chloroplast (cpDNA), and mitochondrial (mtDNA). Nuclear
DNA is, of course, transmitted from parent(s) to offspring
by nuclear division (meiosis or mitosis) via sexual or asexual
(somatic) reproduction. Chloroplasts and mitochondria,
however, replicate and divide independently of the nucleus
and may be transmitted to offspring in a different fashion. For
example, in angiosperms these organelles are usually (with
some exceptions) sexually transmitted only maternally, being
588
CHAPTER 14
Plant molecular systematics
sample DNA (many copies)
add:
primer molecules,
nucleotides,
DNA polymerase,
dideoxynucleotides
new DNA strands denatured
from sample DNA;
after numerous reactions
new DNA strands separated
by electrophoresis (below)
solution heated,
DNA denatures
3’
3’
5’
5’
3’
a single primer anneals
to a conserved region
of one strand of
sample DNA
5’ A-T-C-G-G-T-T-A-G-C*
T-A-G-C-C-A-A-T-C-G-C-A
~
A-A-T-T-A-G-C-T-C-C-A-A-T
5’
at random,
dideoxynucleotide
(C* in this case)
binds to primer strand,
terminating reaction
3’
5’ A-T-C-G-G-T-T-A-G
T-A-G-C-C-A-A-T-C-G-C-A
~
A-A-T-T-A-G-C-T-C-C-A-A-T
5’
primer
3’
5’ A-T-C-G-G-T-T
T-A-G-C-C-A-A-T-C-G-C-A
~
A-A-T-T-A-G-C-T-C-C-A-A-T
5’
second nucleotide
binds to primer strand
sample DNA
first nucleotide
(catalyzed by DNA polymerase)
binds to primer strand
3’
5’ A-T-C-G-G-T-T-A
T-A-G-C-C-A-A-T-C-G-C-A
~
A-A-T-T-A-G-C-T-C-C-A-A-T
5’
(+)
A-T-C-G-G-T-T-A*
A
G
C
G
T
A-T-C-G-G-T-T-A-G*
ELECTROPHORESIS:
electric current applied.
DNA strands
migrate to (+) pole
(inversely to
molecular weight)
A-T-C-G-G-T-T-A-G-C*
A-T-C-G-G-T-T-A-G-C-G*
DNA strands scanned during migration.
Peaks of wavelengths correspond to
fluorescent dyes attached to specific
dideoxynucleotides
A-T-C-G-G-T-T-A-G-C-G-T*
(–)
Figure 14.3
DNA sequencing reactions. A* = dideoxyadenine; C* = dideoxycytosine; G* = dideoxyguanine; T* = dideoxythymine.
Unit III
Systematic evidence and descriptive terminology
589
U
GG
yc
C- f 6
GC
A
oB
*rp
C1
p
p sbJ
p sbL
ps sbF
o bE
W rf 1
P - -CC03
UG A
G
rp1
20
5’rp
s12
clp
P*
po
*r
2
oC
rp
H
atp
F
*atp
atpA
Chloroplast DNA
rpoA
rps11
rpl36
infA
rps8
rpl14
rpl16 *
rps3
rpl22
rps19
rpl2 *
rpl23
I-CAU
S-GCU
Q-UUG
(Nicotiana tabacum)
JLB
matK
psbA
H-GU
G
rp12
*
rp12
r-CA 3
U
A
IR
orf B
1
F
ndh
rpl3
2
sprA
L -UA
G
ycf 5
ars
iA
Inverted
Repeat A
ori 1
Small Single
Copy Region
or
UU
N -G 75
orf
rrn
23
rrn
4.5
R-Arrn 5
CG
orf3
50
31
rr
3’
f1
*I-
rp
s7
rp
s1
2*
o
L-Crf 115
AA
hB
*
1
lA
nd
or
ycf
ndh
23
rrn
4.5
rrnrrn5
CG
R -A
N
orf -GUU
75
SSC
rps 15
ndhH
or
JSA
B
* ndhA
ndhI
ndhG
ndhE
psaC
ndhD
o
V- rf 7
G 08
A
n1 C
6
*A GA
-U U
GC
JS
2
yc
o f 15
or rf 92
f7
9
08
f 7 AC
or -G 16
V rrn U
A
-G C
*I -UG
*A
15
f1
or CAA
*
LhB
nd 7 2 *
s 1
rp ’rps
3
31
f1
or
B
Inverted
Repeat B
psbI
psbK
*rps16
155,939 base pairs
IR
5
f1
yc rf 92 8
o rf 7
o
U
R-UC
*
G-UCC
*K-UUU
matK
ycf
f2
yc
2
ars
2
rps I
atp
psbN
* petD
E
Y -UU
D -GU C
-G A
U
ps C
bM
LSC
9
JLA
psb8
psbT
psbH
* petB
psaA
ycf 3*
orf 74
rps4
U
T -UG
J
ndh K
ndhh C
nd
C*
UA
V- E
atp B
atp
cL
aI
ps f4
yc 10
f
yc
tA
f9
p
peet L
ps tG
rp aJ
rp 133
s1
8
psaB
rps 14
IM -CAU
orf 1
S-U 05
GA
U
CA
rb
cD
ac
pe
or
T-
G-GCC
ycf 9
psb
C
S -GGA
M-
rbcL
0A
orf 7 AA
*L -U A
A
F -G
atpB
psb
D
Large Single
Copy Region
Figure 14.4 Molecular structure of the chloroplast DNA of tobacco (Nicotiana tabacum). Note large single-copy region (LSC),
small single-copy region (SSC), and the two inverted repeats (IRA and IRB). Also note location of atpB, rbcL, matK, and ndhF genes (see
Table 14.1). (Redrawn from Wakasugi, T., M. Sugita, T. Tsudzuki, and M. Sugiura. 1998. Updated gene map of tobacco chloroplast DNA.
Plant Molecular Biology Reporter 16: 231–241, by permission.)
retained in the egg but excluded in sperm cells. (In conifers,
interestingly, chloroplast DNA is transmitted paternally, not
maternally.)
The use of sequence data from the DNA of chloroplasts
has proven to be very useful in elucidating both lower
and higher level relationships. The basic structure of chloroplast DNA for a flowering plant, with coding genes indicated,
is shown in Figure 14.4. Like all organelle and prokaryotic
DNA, chloroplast DNA is circular. Curiously, most angiosperms have a region of chloroplast DNA known as the
inverted repeat, which is the mirror image of the corresponding region (Figure 14.4). Some of the more commonly
sequenced chloroplast DNA genes are listed in Table 14.1,
although many more have been utilized.
In addition to coding genes of chloroplast DNA, the
sequences between genes, known as intergenic spacers, may
be used in phylogenetic analyses. Intergenic spacer regions
often show a higher degree of variability than the coding
genes, making the former more useful for analyses at a lower
taxonomic level, such as species or infraspecies. A list of
some commonly used chloroplast intergenic spacers is seen
in Table 14.2.
Nuclear DNA sequencing has been used to a lesser degree
in plant systematics. Some nuclear genes such as alcohol
dehydrogenase (Adh), which has traditionally been used in
allozyme studies, are becoming more frequently used.
One of the more useful types of nuclear DNA sequences
has been the internal transcribed spacer (ITS) region,
590
CHAPTER 14
TABLE 14.1
Plant molecular systematics
Some chloroplast genes that have been used in plant molecular systematics, after Soltis et al. 1998.
CHLOROPLAST GENES
GENE
LOCATION
FUNCTION
atpB
Large single-copy region of chloroplast
rbcL
Large single-copy region of chloroplast
matK
ndhF
Large single-copy region of chloroplast
Small single-copy region of chloroplast
Beta subunit of ATP synthethase, which functions in the synthesis of ATP via proton
translocation
Large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO),
which functions in the initial fixation of carbon dioxide in the dark reactions
Maturase, which functions in splicing type II introns from RNA transcripts
Subunit of chloroplast NADH dehydrogenase, which functions in converting NADH to
NAD + H+, driving various reactions of respiration
which contains multiple DNA copies (as opposed to single
copies found in most protein-coding genes). The ITS region
lies between the 18S and 26S nuclear ribosomal DNA
(nrDNA); the ITS region is divided into two subregions, ITS1
and ITS2, separated by the 5.8S nrDNA (Figure 14.5A). ITS
sequence data has been most valuable for inferring phylogenetic relationships at a lower level, e.g., between closely
related species. However, it has also been used in elucidating
higher level relationships. (See Baldwin et al. 1995.)
A related DNA sequence region is the external transcribed
spacer (ETS) region. The ETS region lies between 26S and 18S
nrDNA, adjacent to the latter (Figure 14.5B). (The entire region,
including both the ETS and the nontranscribed spacer region
(NTS) is known as the intergenic spacer region, or IGS; see
Figure 14.5B.) The ETS region contains even more sequence
variation than ITS and is useful in analyses at lower taxonomic
levels. (See Baldwin and Markos 1998.)
ANALYSIS OF DNA SEQUENCE DATA
DNA sequence data is converted to characters and character
states to be used in phylogenetic analyses. First, the sequences
of a given length of DNA are aligned, in which homologous
nucleotide positions (e.g., corresponding to the same codon
TABLE 14.2
position of a given gene) are arranged in corresponding
columns (Figure 14.6). For some genes that are relatively
conserved, alignment is straightforward, as all taxa have the
same number of nucleotides per gene. For other genes or
DNA segments, some taxa may have one or more additions,
deletions, inversions, or translocations relative to other taxa.
The occurrence of these mutations, and/or the occurrence of
considerable homoplasy among taxa, can make alignment
of DNA sequences difficult. In addition, multiple copies of
a gene can make homology assessment difficult. Various
computer algorithms can be used to automatically align
sequences of the taxa being studied, but these have assumptions that must be carefully assessed.
Generally, in using DNA sequence data in a phylogenetic
analysis, a character is equivalent to the nucleotide position,
and a character state of that character is the specific nucleotide
at that position (there being four possible character states, corresponding to the four nucleotides; see Figure 14.6). A large
number (often the great majority) of nucleotide positions are
generally invariable among taxa, and some of the variable
ones are often uninformative by being autapomorphic for a
given taxon; thus, relatively few sites are informative and
therefore useful in phylogenetic reconstruction (Figure 14.6).
Some chloroplast intergenic spacer regions that have been used in plant molecular systematics, after Shaw et al. 2005,
2007.
CHLOROPLAST INTERGENIC SPACER REGIONS
3’rps16-5’trnK
3’trnK-matK intron
3’trnV-ndhC
5’rpS12-rpL20
atpI-atpH
matK-5’trnK intron
ndhA intron
ndhF-rpl32
ndhJ-trnF
petL-psbE
psaI-accD
psbA-3’trnK
psbB-psbH
psbD-trnT
psbJ-petA
psbM-trnD
rpl14-rps8-infA-rpl36
rpl16 intron
rpl32-trnL
rpoB-trnC
rps16 intron
rps4-trnT
trnC-ycf6
trnD-trnT
trnG intron
trnH-psbA
trnL intron
trnL-trnF
trnQ-5’rps16
trnS-rps4
trnS-trnfM
trnS-trnG
trnT-trnL
ycf6-psbM
Unit III
Systematic evidence and descriptive terminology
591
ITS Region
LEU1 ITS5
18S nrDNA
A
ITS3
5.8S
nrDNA
ITS1
ITS2
ITS2
26S nrDNA
ITS4
C28KJ
ETS-Hel-1
26S nrDNA
NTS
ETS
ETS-Hel-2
26S-IGS
18S nrDNA
18S-E 18S-IGS 18S-ETS
B
IGS Region
A. Internal transcribed spacers (ITSs) of nuclear ribosomal DNA, illustrating the ITS region and flanking subunits, and showing the orientations and locations of primer sites. After Baldwin et al. (1995). B. External transcribed spacer (ETS) of the intergenic spacer
(IGS) region, also showing orientations and locations of primer sites. After Baldwin and Markos (1998).
Figure 14.5
However, a major addition, deletion, inversion, or translocation can in itself be identified as an evolutionary novelty
(apomorphy), used in grouping lineages together. For example, members of the Faboideae (of the Fabaceae) lack, by
deletion, one of the inverted repeats found in the chloroplasts
of most angiosperms (see Figure 14.4). Chromosomal mutations such as these may be coded separately from single base
differences (e.g., as in the example of Figure 14.6) and may
be given relatively greater weight in inferring relationships.
Several types of weighting schemes may be done with
molecular data. For protein encoding genes, the codon position may be differentially weighted. For example, because
of redundancy of the genetic code, the third codon position
is generally more labile (a change more likely to have occurred
randomly) than the second, and the second may be more
labile than the first. Thus, the first and second codon positions may be given relatively greater weight, respectively
(such as a weight of 10 for the first codon position, 5 for the
second position, and 1 for the third position). The logic here
is that a change in codon position 1 or 2 is less likely to
have occurred at random within a taxon and more likely
represents evolutionary novelties that are shared among taxa.
Weighting by codon position may be based on empirical data.
For a given data set, the number of changes occurring
for codon positions 1, 2, and 3 may be used (inversely) to
establish the relative weights.
Another weighting parameter that may be used with DNA
sequence data concerns transitions versus transversions.
Transitions are evolutionary changes from one purine to
another purine (A → G or G → A) or from one pyrimidine
to another pyrimidine (C → T or T → C); see Figure 14.1.
Transversions are evolutionary changes from a purine to
a pyrimidine (A → C, A → T, G → C, or G → T) or from
a pyrimidine to a purine (C → A, C → G, T → A, or T → G).
Weighting using transitions versus transversions may be based
on empirical data. For a given data set, the relative frequency
592
CHAPTER 14
Taxon 1
Taxon 2
Taxon 3
Taxon 4
Taxon 5
Taxon 6
Taxon 7
Taxon 8
Plant molecular systematics
DNA Alignment
Character Coding
00000000000000000001111111111111111111111
88888888899999999990000000000111111111122
12345678901234567890123456789012345678901
GCCTAGCCAAAGCTCTTCCAAGGTGACTCTCAGTTCAAGCT
GCCTAGCCAAAGCTCTTCCAAGCTGACTCTCA------GCT
GCCTAGCCTAAGCTCAACCAAGGTGTCTCTCAGTTCAAGCT
GCCTAGCCTAAGCTCTTCCAAGGTGTCTCTCAGTTCAAGCT
GCCTAGCCAAAGCTCTTCCAAGCTGACTCTCA------GCT
CCCTAGCCAAAGCTCTTCCAAGCTGACTCTCAGTTCAAGCT
CCCTAGCCAAAGCTCTTCCAAGCTGACTCTCAGTTCAAGCT
GCCTAGCCTAAGCTCTTCCAAGCTGACTCTCAGTTCAAGCT
1 2 3 4 5 6
2 0 3 2 0 4
2 0 3 1 0 5
2 3 0 2 3 4
2 3 3 2 3 4
2 0 3 1 0 5
1 0 3 1 0 4
1 0 3 1 0 4
2 3 3 1 0 4
Figure 14.6 Example of alignment of DNA sequences of 41 nucleotide sites (positions 81–121) from eight taxa. Variable nucleotide
sites are in bold. Note deletion of six bases in taxon 2 and taxon 5. Possible character coding of variable sites is seen at right. Coding of
nucleotides is as follows: A = state 0; C = state 1; G = state 2; T = state 3. In this example, the deletion is coded as a single binary character
(character 6), coded differently from nucleotides, as state 4 = deletion absent and state 5 = deletion present.
of transitions versus transversions may be used (inversely) to
establish the relative weights. For example, for a given group
under study, if transitions occur 5× more frequently than
transversions, the latter may be given a weight of 5 and the
former a weight of 1, as illustrated in the step matrix of
Figure 14.7.
These weighting schemes may be viewed as a simplified
component of a process that may be quite complex, taking
into account, e.g., rate of base substitution, base frequency,
and branch length in determining an evolutionary model.
Evolutionary models are commonly used in maximum likelihood and Bayesian analyses. (See Chapter 2.)
DNA sequence data can also be used to evaluate the
secondary structure of a molecule. Thus, nucleotide differences that result in major changes in the conformation of the
product (whether ribosomal RNA or protein) may have
a much greater physiological effect than those that do not
and might receive a higher weight. Computer algorithms can
evaluate this to some degree.
A
G
C
T
A
0
1
5
5
G
1
0
5
5
C
5
5
0
1
T
5
5
1
0
Step matrix of nucleotide changes, showing weighting scheme in which transversions are given a weight 5 times greater
than that of transitions.
Figure 14.7
Parsimony, maximum likelihood, and Bayesian methods
are commonly used to infer phylogenetic relationships using
DNA sequence data (Chapter 2). The most robust hypotheses of
relationship are generally those using a large taxon sampling and
sequence data from multiple (e.g., anywhere from 3 to 20+)
genes and/or sequence regions.
RESTRICTION SITE ANALYSIS (RFLPS)
A restriction site is a sequence of approximately 6–8 base
pairs of DNA that binds to a given restriction enzyme.
These restriction enzymes, of which there are many, have
been isolated from bacteria. Their natural function is to inactivate invading viruses by cleaving the viral DNA. Restriction
enzymes known as type II recognize restriction sites and
cleave the DNA at particular locations within or near the
restriction site. An example is the restriction enzyme EcoRI
(named after E. coli, from which it was first isolated), which
recognizes the DNA sequence seen in Figure 14.8 and cleaves
the DNA at the sites indicated by the arrows in this figure.
Restriction fragment length polymorphism, or RFLP,
refers to differences between taxa in restriction sites, and
therefore the lengths of fragments of DNA following cleavage with restriction enzymes. For example, Figure 14.9 shows,
for two hypothetical species, amplified DNA lengths of
10,000 base pairs that are subjected to (“digested with”) the
restriction enzyme EcoRI. Note, after a reaction with the
EcoRI enzyme, that the DNA of species A is cleaved into
three fragments, corresponding to two EcoRI restriction sites,
whereas that of species B is cleaved into four fragments,
corresponding to three EcoRI restriction sites. The relative
Unit III
Systematic evidence and descriptive terminology
G-A-A-T-T-C
C-T-T-A-A-G
A DNA restriction site, cleaved (at arrows) by the
restriction site enzyme EcoRI.
Figure 14.8
locations of these restriction sites on the DNA can be mapped;
one possibility is seen at the bottom of Figure 14.9. (Note that
there are other possibilities for this map; precise mapping
requires additional work.) Additional restriction enzymes
can be used. Figure 14.10 illustrates how each of the DNA
fragments from the EcoRI digests can be digested with the
BAM HI restriction enzyme, yielding different fragments for
the two species. These data can be added to the original in
preparing a map (one possible map is shown in lower part of
Figure 14.10).
Restriction site fragment data can be coded as characters
and character states in a phylogenetic analysis. For example,
given that the restriction site maps of Figure 14.10 are
correct, the presence or absence of these sites can be coded as
characters, as seen in Figure 14.11. Restriction site analysis
contains far less data than complete DNA sequencing,
accounting only for the presence or absence of sites 6–8 base
pairs long. It has the advantage, however, of surveying considerably larger segments of DNA. However, with improved
and less expensive sequencing techniques, it is less valuable
and less often used than in the past.
ALLOZYMES
Allozymes are different molecular forms of an enzyme that
correspond to different alleles of a common gene (locus).
(This is not to be confused with isozymes, which are forms of
an enzyme that are derived from separate genes or loci.)
Allozymes are traditionally detected using electrophoresis, in
which the enzymes are extracted and placed on a medium
(e.g., starch) through which an electric current runs (similar
to gel electrophoresis in DNA sequencing). A given enzyme
will migrate toward one pole or the other depending on
its charge. Similarly, different allozymes of an enzyme will
migrate differentially because they differ slightly in amino
acid composition and therefore have somewhat different
593
electrical charges. Allozymes subjected to electrophoresis
are identified with a stain specific to that enzyme and the
bands marked by their relative position on the electrophoresis
medium.
Allozymes have traditionally been used to assess genetic
variation within a population or species, but they can also be
used as data in phylogenetic analyses of closely related species, e.g., species within a monophyletic genus. Figure 14.12A
illustrates an example of electrophoretic allozyme banding
data for five species and an outgroup.
There are several ways to code polymorphic allozyme data.
One way is to code each allele as a character and the presence
or absence of that allele as a character state. A second way
to code allozyme data is to treat the locus (corresponding
to the gene coding for the enzyme) as the character and
all unique combinations of alleles as character states (as in
Figure 14.12B). The number of state changes between these
unique allelic combinations can be a default of one. However,
another method of coding is to treat the loss of each allele as
one state change and the gain of an allele as a separate state
change. Thus, the number of state changes between different
allelic combinations can vary, as seen in Figure 14.12C. Step
matrices (see Chapter 2) are used to code these in a cladistic
analysis.
Yet another way to code allozyme data is to take into account
the frequency of alleles present in a given taxon. For example, by
this method, species A, which has allele X present with a frequency of 95% and allele Y with a frequency of 5%, would
receive a different coding from species B, which has the same
alleles, but in frequencies of 55% and 45%, respectively.
MICROSATELLITE DNA
Microsatellites are regions of DNA that contain short
(usually 2–5) repeats of nucleotides, an example being
TGTGTG, in which two base pairs repeat. The regions are
termed tandem repeats; if they vary within a population
or species, they are called variable-number tandem
repeats (VNTR). (Other designations and acronyms are
used, depending on the particular field of study.) These
tandem repeats can be located all across the genome; at a
given location (locus), the repeat will tend to be of a certain length. However, individuals within or between populations may vary in the number of tandem repeats at a given
locus (or even show allelic variation) because of irregularities
in crossing-over and replication. Thus, variable-number
tandem repeats can be used as a genetic marker.
Microsatellites are identified by constructing primers that
flank the tandem repeats and then using PCR technology.
594
CHAPTER 14
Plant molecular systematics
Species A
Species B
DNA − 10,000 bp long
DNA − 10,000 bp long
+
EcoRI
+
EcoRI
5000 bp
4000 bp
4000 bp
3000 bp
2000 bp
1000 bp
1000 bp
EcoRI EcoRI
Species A
5000
6000
EcoRI
EcoRI EcoRI
3000
5000
Species B
6000
Figure 14.9 Example of restriction site analysis of species A and B, using restriction site enzyme EcoRI. Note differences in fragment
lengths. Possible restriction site maps of species A and B are shown in the lower portion of the figure.
Unit III
595
Systematic evidence and descriptive terminology
Species A
Species B
EcoRI digest
EcoRI digest
5000 bp
4000 bp
4000 bp
3000 bp
+
2000 bp
+
BAM HI
BAM HI
1000 bp
+
+
BAM HI
+
BAM HI
+
BAM HI
BAM HI
+
1000 bp
BAM HI
4000 bp
3500 bp
3000 bp
2200 bp
1800 bp
1600 bp
1500 bp
700 bp
700 bp
300 bp
300 bp
400 bp
EcoRI EcoRI
3500
Species A
5300
5000
BAM HI
EcoRI
Species B
3400
6000
BAM HI
EcoRI EcoRI
5300
7800
3000
5000
BAM HI
BAM HI
6000
BAM HI
Example of restriction site analysis of species A and B, using restriction site enzyme EcoRI, followed by restriction site
enzyme BAM HI. Possible restriction site maps of species A and B are shown in the lower portion of the figure.
Figure 14.10
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CHAPTER 14
Plant molecular systematics
CHARACTERS
EcoRI
BAM
EcoRI
BAM
BAM
EcoRI
BAM
TAXA
3000
3400
3500
5000
5300
6000
7800
Species A
−
−
+
+
+
+
−
Species B
+
+
−
+
+
+
+
Figure 14.11 Character coding of restriction site map data of Figure 14.10, derived by presence or absence of EcoRI or BAM sites at
specific locations along DNA.
(The primers are initially identified for a species by the
time-consuming process of synthesizing genetic probes of a
tandem repeat, screening DNA for binding to these probes,
and sequencing these regions to design primers that flank the
tandem repeats.) Once the primers are identified, PCR can be
used to quickly generate multiple copies of the tandem repeat
DNA, the length of which (for a given individual at a given
locus or allele) can be determined by gel electrophoresis.
(See example in Figure 14.13.)
Microsatellite analysis can generate data quickly and
efficiently (once the primers are identified for a given group)
for a large number of individuals. It is most often used for
population studies, e.g., to assess genetic variation or homozygosity. Its use in systematics is largely in examining
relationships within a species (such as to assess infraspecific
classifications) or between very closely related species.
RANDOM AMPLIFIED
POLYMORPHIC DNA (RAPDS)
Another method of identifying genetic markers is by using a
randomly synthesized primer to amplify DNA in a PCR reaction.
In this method, the primer will anneal to complementary
regions located in various locations of isolated DNA. If
another complementary site is present on the opposing DNA
strand at a distance that is not too great (i.e., within the limits
of PCR), then the reaction will amplify this region of DNA
(Figure 14.14). Because many sections of DNA complementary to the primer may occur, the PCR reaction will result in
DNA strands of many different lengths, which can be sizeseparated by electrophoresis. Because even closely related
individuals may show some sequence variation that could
determine potential primer sites, these different individuals
will show different amplification products. Thus, RAPD
refers to using randomly generated primers for the
amplification of DNA to identify polymorphic DNA
regions of different individuals or taxa. (See example in
Figure 14.14.)
RAPDs, like microsatellites, may often be used for withinspecies genetic studies, but may also be successfully employed
in phylogenetic studies to address relationships within a
species or between closely related species. However, RAPD
analysis has the major disadvantages in that results are
difficult to replicate (being very sensitive to PCR conditions)
and in that the homology of similar bands in different taxa
may be unclear.
AMPLIFIED FRAGMENT LENGTH
POLYMORPHISM (AFLPS)
This method is similar to that of identifying RFLPs in that a
restriction enzyme is used (Figure 14.15A) to cut DNA into
numerous, smaller pieces, each of which (because of the
action of the restriction enzymes) terminates in a characteristic nucleotide sequence (Figure 14.15B). However, the
numerous, cut DNA fragments are then modified by binding
to each end (using DNA ligase) a synthesized, double-stranded
piece of DNA, known as a primer adapter (Figure 14.15C).
The primer adapters are designed to insert at the cut ends
(corresponding to the complementary sequences of the
restriction enzymes). Primers are then constructed that bind
to the primer adapters and amplify the DNA fragments using
a polymerase chain reaction (Figure 14.15D). Electrophoresis
separates the amplified DNA fragments that exhibit length
polymorphism (hence, AFLP), enabling the recognition of
numerous genetic markers.
AFLP data are more experimentally replicable than are RAPD
data and can be used to identify genetic differences among individuals using large pieces of DNA. AFLP has one disadvantage
in that so many fragments may be generated that it is hard to
distinguish them on an electrophoretic gel. However, a slight
modification of the primers used may limit the number of fragments that are amplified, enabling them to be more easily identified. AFLP is largely used for population genetics studies, but
has been used in studies of closely related species and even, in
some cases, for higher-level, cladistic analyses.
Unit III
Species A
Systematic evidence and descriptive terminology
Species B
Species C
Species D
597
Species E Outgroup
22
GOT 18
21
17
IDH
35
31
27
PGI
A
GOT
A
1
B
1
C
1
D
1
E
0
OUT 0
B
IDH
2
2
1
2
0
0
PGI
0
1
2
3
2
0
GOT
IDH
22
(1)
17
(0)
2 steps
18
(0)
2 steps
PGI
27
(0)
2 steps
35
(1)
1 step
1 step
3 steps
2 steps
2 steps
21 1 step
(1)
17,21
(2)
31
(2)
1 step
27,31
(3)
C
Allozyme data. A. Hypothetical allozyme banding data for taxa A–E and Outgroup and enzymes GOT, IDH, and PGI.
B. Coding of data using the locus as the character and unique allelic combinations as character states. C. One possible coding of data
(after Mabee and Humphries, 1993). Diagrams illustrating number of state changes between character states (state number in parentheses);
each loss or gain of an allele counts as one step change.
Figure 14.12
598
CHAPTER 14
Plant molecular systematics
Species A
Species B
PRIMER
PRIMER
5′ A-T-C-G-G-T-T
5′ A-T-C-G-G-T-T
3′
T-A-G-C-C-A-A-C-A-C-A-C-A
~
C-A-C-A-C-A-C-T-C-C-A-A-T
5′
3′
T-A-G-C-C-A-A-C-A-C-A
~
C-A-C-A-C-T-C-C-A-A-T
5′
A-T-C-G-G-T-T-G-T-G-T-G-T
~
3′
G-T-G-T-G-T-G-A-G-G-T-T-A
T-C-C-A-A-T 5′
PRIMER
5′
A-T-C-G-G-T-T-G-T-G-T
~
3′
G-T-G-T-G-A-G-G-T-T-A
T-C-C-A-A-T 5′
PRIMER
T-A-G-C-C-A-A-C-A-C-A-C-A
~
C-A-C-A-C-A-C-T-C-C-A-A-T
T-A-G-C-C-A-A-C-A-C-A
~
C-A-C-A-C-T-C-C-A-A-T
A-T-C-G-G-T-T-G-T-G-T-G-T
~
G-T-G-T-G-T-G-A-G-G-T-T-A
A-T-C-G-G-T-T-G-T-G-T
~
G-T-G-T-G-A-G-G-T-T-A
5′
Microsatellite data. Primers were constructed to flank regions of tandem repeats. Note that tandem repeat region of species A
is longer than that of species B and is thus a genetic difference between the two.
Figure 14.13
Species A
Species B
Primer X
Primer X
3′
5′
3′
5′
5′
3′
5′
3′
Primer X
Primer X
2400 bp
1560 bp
RAPDs data. In this example the same DNA regions for species A and B anneal to different randomly generated primers,
resulting in amplified DNA of different lengths, a genetic difference between the two taxa.
Figure 14.14
Unit III
A
Systematic evidence and descriptive terminology
599
Add restriction enzyme (e.g., EcoRI) to cleave isolated DNA
G-A-A-T-T-C
C-T-T-A-A-G
B
G-A-A-T-T-C
C-T-T-A-A-G
G-A-A-T-T-C
C-T-T-A-A-G
DNA cleaved into fragments
A-A-T-T-C
G
A-A-T-T-C
G
G
C-T-T-A-A
C
G
C-T-T-A-A
Add primer adapters (+ DNA ligase)
N~N-G-A-A-T-T-C
N~N-C-T-T-A-A-G
G-A-A-T-T-C-N~N
C-T-T-A-A-G-N~N
N~N-G-A-A-T-T-C
N~N-C-T-T-A-A-G
D
G-A-A-T-T-C-N~N
C-T-T-A-A-G-N~N
Amplify with PCR primers
5′ N~N-C-T-T-A-A-G
N~N-G-A-A-T-T-C
N~N-C-T-T-A-A-G
G-A-A-T-T-C-N~N
C-T-T-A-A-G-N~N
G-A-A-T-T-C-N~N 5′
5′ N~N-C-T-T-A-A-G
N~N-G-A-A-T-T-C
N~N-C-T-T-A-A-G
Figure 14.15
G-A-A-T-T-C-N~N
C-T-T-A-A-G-N~N
G-A-A-T-T-C-N~N 5′
AFLP technique. The letters “N~N-” represent a length of nucleotides.
600
CHAPTER 14
Plant molecular systematics
REVIEW QUESTIONS
1. Name the specific types of data used in studies of molecular systematics.
2. How are samples used to acquire molecular data typically processed?
3. Why is collection of a voucher specimen in molecular studies essential?
4. What does DNA sequence data refer to?
5. Explain the polymerase chain reaction and its importance in molecular systematics.
6. What is a primer?
7. Explain the basic process of automated DNA sequencing. What is the significance of dideoxynucleotides?
8. What are the three major types of DNA used in DNA sequence (and other molecular) studies?
9. In chloroplast DNA, what are the large single-copy region, small single-copy region, and inverted repeats?
10. Name some useful chloroplast genes used in plant molecular systematics.
11. What is the internal transcribed spacer region (ITS) and what is its efficacy in plant molecular systematics?
12. How does the external transcribed spacer region (ETS) differ from ITS and what is the advantage of these data?
13. What is DNA alignment, and what are potential problems with this?
14. In general, what are the characters and character states for DNA sequence data?
15. Name the ways that DNA sequence data may be weighted in a cladistic analysis.
16. What factors do models of evolution take into account as used in maximum likelihood or Bayesian analyses?
17. What is a restriction site?
18. What does restriction fragment length polymorphism (RFLP) refer to?
19. How is RFLP data acquired and how is it used in a cladistic analysis?
20. What is an allozyme?
21. How are allozyme data acquired?
22. Explain the different ways to code allozyme data in a cladistic analysis.
23. What are microsatellites and how are these data obtained?
24. What are random amplified polymorphic DNAs (RAPDs) and how are these data obtained?
25. Describe the technique for generating amplified fragment length polymorphisms (AFLPs), citing how this differs from that
of generating RFLPs.
EXERCISES
1. If possible, get a demonstration of the various techniques of molecular systematics, e.g., DNA extraction and sequencing.
Consider a special topics project in which you define a problem and use these techniques to acquire the data to answer the
problem.
2. Access GenBank (http://www.ncbi.nih.gov/Genbank) and acquire molecular data on a particular group of choice. Consider
analyzing these data using phylogenetic inference software (see Chapter 2).
3. Peruse journal articles in plant systematics, e.g., American Journal of Botany, Annals of the Missouri Botanical Garden,
Systematic Botany, or Taxon, and note those that describe the use of molecular data in relation to systematic studies. Identify
the techniques used, data acquired, and problems addressed.
Unit III
Systematic evidence and descriptive terminology
601
REFERENCES FOR FURTHER STUDY
Baldwin, B. G., M. J. Sanderson, J. M. Porter, M. F. Wojciechowski, C. S. Campell, and M. J. Donoghue. 1995. The ITS region of nuclear
ribosomal DNA: A valuable source of evidence on angiosperm phylogeny. Annals of the Missouri Botanical Garden 82: 247–277.
Baldwin, B. G., and S. Markos. 1998. Phylogenetic utility of the External Transcribed Spacer (ETS) of 18S-26S rDNA: Congruence of ETS
and ITS Trees of Calycadenia (Compositae). Molecular Phylogenetics and Evolution 10: 449–463.
Hillis, D. M., C. Moritz, and B. K. Mable. 1996. Molecular Systematics, 2nd ed. Sinauer, Sunderland, Massachusetts.
Mabee, P. M., and J. Humphries. 1993. Coding polymorphic data: Examples from allozymes and ontogeny. Systematic Biology 42: 166–181.
Shaw, J., E. B. Lickey, J. T. Beck, S. B. Farmer, W. Liu, J. Miller, K. C. Siripun, C. T. Winder, E. E. Schilling, and R. L. Small. 2005. The
tortoise and the hare II: Relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. American Journal of Botany
92: 142–166.
Shaw, J., E. B. Lickey, E. E. Schilling, and R. L. Small. 2007. Comparison of whole chloroplast genome sequences to choose noncoding
regions for phylogenetic studies in angiosperms: The tortoise and the hare III. American Journal of Botany 94: 275–288.
Small, R. L., J. A. Ryburn, R. C. Cronn, T. Seelanan, and J. F. Wendel. 1998. The tortoise and the hare: Choosing between noncoding plastome and nuclear Adh sequences for phylogenetic reconstruction in a recently diverged plant group. American Journal of Botany 85:
1301–1315.
Soltis, P. S., D. E. Soltis, and J. J. Doyle (eds.). 1992. Molecular Systematics of Plants. Chapman and Hall, New York.
Soltis, D. E., P. S. Soltis, and J. J. Doyle (eds.). 1998. Molecular Systematics of Plants II: DNA Sequencing. Kluwer Academic, Boston.