Biology 164 Lab
Examining Plant Evolution:
A Comparison of Morphological and Molecular Phylogenies
(Based on a poster presented by Nitya Jacob and Eloise Carter at the 2006 annual meeting of the American Society of Plant Biologists)
In this study you will use skills you have been developing over the past few labs to make a hypothesis
and prediction about the phylogenetic relationships among 11 genera of plants. These genera are (in
alphabetical order): Arabidopsis, Chara, Equisetum, Lilium, Marchantia, Nitella, Pinus, Polypodium,
Polytrichum, Psilotum, and Zamia. In addition to working with these genera, you will need to determine
a genus that will serve as an outgroup for your analysis.
Morphological Phylogeny
In the first part of the study you will develop a character matrix for the 11 genera of plants by making
observations of their morphology and life histories. Detailed digital images, life history diagrams, along
with fresh and dried herbarium specimens, will be available for your observations. Once your character
matrix is completed, MacClade will be used to generate a phylogeny based on your observations. You
will then use your morphological-based phylogeny to make a prediction about what to expect when
constructing a phylogeny based on molecular data.
To help you get started, a partial character list has been assembled for you in Table 1. You will need to
come up with at least as many characters on your own, hopefully more. Collaboration with other lab
teams is encouraged. When choosing characters, keep in mind that the most useful characters for a
cladistic analysis are those that will divide your list of genera into well-defined clades.
Fresh specimens and herbarium sheets of representative plants will be available for observations on
tables placed in the hallway vestibule just outside of lab. A PowerPoint file named
PlantEvolutionImages, which contains images and life history diagrams of the 11 genera, is available
on the computers. Direct observation of the plants will provide some useful characters, but the life
histories of the plants, while a bit difficult to decipher at first, offer a plethora of important characters that
will be valuable in your analysis. Don’t be intimidated by the life histories! They are easy to decipher
once you understand the basic pattern. Ask lots of questions . . . and remember to select an outgroup
genus, and gather data for that genus as well. Use the table on page 4 for your complete character list.
Table 1: Partial list of morphological characters observed in 11 genera of plants and outgroup.
GENERA
Characters Outgrp Arabid Chara Equis. Lilium March. Nitella Pinus Polypod. Polytrich. Psilot. Zamia
Cuticle
Y
N
Y
Y
Y
N
Y
Y
Y
Y
Y
Stomata
Y
N
Y
Y
Y
N
Y
Y
Y
Y
Y
Vascular tissue
Y
N
Y
Y
N
N
Y
Y
N
Y
Y
in stem
True leaves
True roots
Produces
spores
Multicellular
Submergent
Vessel
elements
Life history
(Zygotic/Sporic)
Y
Y
Y
N
N
N
N
Y
Y
Y
Y
Y
N
N
Y
N
N
N
Y
Y
Y
Y
N
Y
N
N
Y
N
N
Y
Y
Y
Y
Y
N
Y
Y
Y
N
Y
N
N
Y
N
Y
Y
N
N
Y
Y
N
Y
N
N
Y
N
N
Y
N
N
Y
N
N
Y
N
N
S
Z
S
S
S
Z
S
S
S
S
S
Examining Plant Evolution by Comparing Morphological and Molecular Phylogenies
Page 1
Molecular Phylogeny
To construct the molecular phylogeny you will obtain DNA sequences for the gene that codes for a
portion of a key chloroplast enzyme, the Rubisco large subunit (rbcL). Rubisco (an abbreviation for
ribulose-1,5-bisphosphate carboxylase/oxygenase) is considered by some to be the most important
protein on Earth. It certainly is one of the most abundant, since it is found in photosynthetic tissues of
all green plants. Rubisco is the Calvin cycle enzyme that provides the means by which inorganic
carbon, in the form of carbon dioxide, enters the Earth’s biosphere. Since it plays such a pivotal role in
the planet’s carbon cycle, Rubisco is of much interest to plant biologists trying to determine if it is
possible to make agronomic plants more efficient (and perhaps global warming less severe) by
enhancing the expression of this important enzyme. Because rbcL is a chloroplast gene, it has been
fairly well conserved over its long evolutionary history for much the same reason as was the
mitochondrial cytochrome B gene we used for the Darwin finch study: genes in organelles are generally
inherited along maternal lines and thus are not subject to recombination.
To help get you started on your molecular phylogeny, a partial list of rbcL sequences is available in a
text file named Initial_rbcLSequences. Figure 1 shows the sequence in FASTA format for one of the
genera. You will need to retrieve appropriate sequences for the balance of the genera and the outgroup
genus. As with the morphological phylogeny, you are encouraged to collaborate with other lab teams to
make your work easier. If you are unsure of anything, please do not hesitate to ask questions!
>Arabidopsis gi|27752802|gb|AY174634.1| Arabidopsis thaliana isolate
KNO10 ribulose 1,5-bisphosphate carboxylase/oxygenase large chain
(RBCL) gene, partial cds; chloroplast gene for chloroplast product
ATAAATTGACTTACTATACTCCTGAATATGAAACCAAGGATACTGATATCTTGGCAGCATTCCGAGTAAC
TCCTCAACCTGGAGTTCCACCTGAAGAAGCAGGGGCTGCGGTAGCTGCTGAATCTTCTACTGGTACATGG
ACAACTGTGTGGACCGATGGGCTTACCAGCCTTGATCGTTACAAAGGACGATGCTACCACATCGAGCCCG
TTCCAGGAGAAGAAACTCAATTTATTGCGTATGTAGCTTATCCCTTAGACCTTTTTGAAGAAGGTTCGGT
TACTAACATGTTTACCTCGATTGTGGGTAATGTATTTGGGTTCAAAGCCCTGGCTGCTCTACGTCTAGAG
GATCTGCGAATCCCTCCTGCTTATACTAAAACTTTCCAAGGACCACCTCATGGTATCCAAGTTGAAAGAG
ATAAATTGAACAAGTATGGACGTCCCCTATTAGGATGTACTATTAAACCAAAATTGGGGTTATCCGCGAA
AAACTATGGTAGAGCAGTTTATGAATGTCTACGTGGTGGACTTGATTTTACCAAAGATGATGAGAATGTG
AACTCCCAACCATTTATGCGTTGGAGAGACCGTTTCTTATTTTGTGCCGAAGCTATTTATAAATCACAGG
CTGAAACAGGTGAAATCAAAGGGCATTATTTGAATGCTACTGCGGGTACATGCGAAGAAATGATCAAAAG
AGCTGTATTTGCCAGAGAATTGGGAGTTCCTATCGTAATGCATGACTACTTAACAGGGGGATTCACCGCA
AATACTAGTTTGTCTCATTATTGCCGAGATAATGGCCTACTTCTTCACATCCACCGTGCAATGCACGCTG
TTATTGATAGACAGAAGAATCATGGTATGCACTTCCGTGTACTAGCTAAAGCTTTACGTCTATCTGGTGG
AGATCATATTCACGCGGGTACAGTAGTAGGTAAACTTGAAGGAGACAGGGAGTCAACTTTGGGCTTTGTT
GATTTACTGCGCGATGATTATGTTGAAAAAGATCGAAGCCGCGGTATCTTTTTCACTCAAGATTGGGTCT
CACTACCTGGTGTTCTGCCTGTGGCTTCAGGGGGTATTCACGTTTGGCATATGCCTGCTTTGACCGAGAT
CTTTGGAGATGATTCTGTACTACAATTCGGTGGAGGAACTTTAGGCCACCCTTGGGGAAATGCACCGGGT
GCCGTAGCCAACCGAGTAGCTCTGGAAGCATGTGTACAAGCTCGTAATGAGGGACGTGATCTTGCAGTCG
AGGGTAATGAAATTATCCGTGAAGCTTGCAAA
Figure 1. Arabidopsis rbcL gene nucleotide sequence obtained from GenBank at NCBI.
Examining Plant Evolution by Comparing Morphological and Molecular Phylogenies
Page 2
Lab Report
Each lab team will prepare one report that includes a Results and Discussion section of the study.
Your Results section will include:
1) one properly labeled table that includes your character matrix (see page 4),
2) two properly labeled figures (your phylogenies),
3) a print out of the text file in FASTA format of the rbcL sequences you downloaded from NCBI
(include accession numbers and other descriptive info for the sequences as in the example
provided in Fig 1, and please use a small font size to save paper), and
4) text that summarizes the groupings of genera that exist within each phylogeny.
Your Discussion section should address the following questions:
1) What criteria did you use for selecting an outgroup genus?
2) Before looking at the molecular phylogeny, how confident were you in the characters you chose
for your morphological phylogeny?
3) How closely correlated are the two phylogenies?
4) Can you offer any explanation for the differences between the two phylogenies?
5) Does the genetic distance data of the molecular phylogeny shed any light about the evolution of
plants that was not revealed in the morphological phylogeny?
6) Was there anything that surprised you about either of the phylogenies?
Examining Plant Evolution by Comparing Morphological and Molecular Phylogenies
Page 3
Biology 164 Lab
Examining Plant Evolution:
A Comparison of Morphological and Molecular Phylogenies
Names of team members: ________________________________________________
Name of outgroup genus: _________________________________________________
Table 1: Complete list of morphological characters observed in 11 genera of plants and
outgroup genus.
GENERA
Characters Outgrp Arabid Chara Equis. Lilium March. Nitella Pinus Polypod. Polytrich. Psilot.
Cuticle
Y
N
Y
Y
Y
N
Y
Y
Y
Y
Stomata
Y
N
Y
Y
Y
N
Y
Y
Y
Y
Vascular tissue
Y
N
Y
Y
N
N
Y
Y
N
Y
in stem
True leaves
True roots
Produces
spores
Multicellular
Submergent
Vessel
elements
Life history
(Zygotic/Sporic)
Zamia
Y
Y
Y
Y
Y
Y
N
N
N
N
Y
Y
Y
Y
Y
N
N
Y
N
N
N
Y
Y
Y
Y
N
Y
N
N
Y
N
N
Y
Y
Y
Y
Y
N
Y
Y
Y
N
Y
N
N
Y
N
Y
Y
N
N
Y
Y
N
Y
N
N
Y
N
N
Y
N
N
Y
N
N
Y
N
N
S
Z
S
S
S
Z
S
S
S
S
S
Examining Plant Evolution by Comparing Morphological and Molecular Phylogenies
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