PhylogenyofUngulates:
StudyingtheMorphologicaland
MolecularAttributesofHoofedMammals
Jayanth (Jay) Krishnan
T.A. Ms. Bianca Pier
Lab Partner: Ms. Catherine Mahoney
Section 1: Biology
September 28th, 2011
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Purpose: What did we want to do?
I: Abstract
The primary objective of this lab was to understand and compare the morphological and
molecular attributes for ungulates or hoofed animals. Prior research had determined that each one
of the ungulates studied (viz. horse, pig, deer, pronghorn antelope, cow, sheep and goat)
descended from several common ancestor. By studying different facets of morphology namely
dentition, headgear, foot structures, and digestion we had hoped to first classify these ungulates
into the orders of Perissodactyla and Artiodactyla. These two subclasses have very unique
characteristics, but are primarily distinguished primarily based on their foot morphology whether they are odd toed or even toed hoofed mammals - respectively. After sorting the
organisms we wanted to further classify them by identifying which organisms were closely
related. We did this by expanding our phenotypic observational methodology to further sort the
several species based on genotypic characteristics/genetic similarity.
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II: Methodology - That corresponds with purpose of the experiment
Before the experiment was conducted we choose to do our morphological qualitative
comparisons using the following parameters:
Dentition was studied by determining whether the ungulates and other organisms had
incisors and/or canines in either their top or bottom jaws. The study of ungulate dentition also
included whether there was a diastema, or space between two teeth, among incisors, canines, and
pre-molars. Furthermore the teeth had also been classified as lophodont, selenodont, or bundont
molar cusps, and observed for the possession of hysodont or brachydont cheek teeth.
Headgear of the ungulates indeed varies from one another. These organisms can either
have true horns (permanent), deciduous horns (sheath is temporary), or antlers (temporary).
The ungulates can also perform digestion via hindgut fermentation, foregut fermenter, or
neither.
The inclusion of wet-lab and dry lab technique:
With these observed characteristics along with some bioinformatics techniques [2:
Geneious Software] and wet-lab experimentations of gel electrophoresis we were able to identify
genotypic similarities. Some of the methods of comparative analysis were with the use of tree
diagrams based on amino acid, LDH, COXIII, Cytochrome B sequences and the calculation of
pair-wise identity. With these components of wetlab biology and computational biology, we
were able to succeed in our mission to effectively sort the ungulates based on both molecular and
morphological data.
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All the ungulates studied were artiodactyl with the exception of horses. This species
along with whales and lions served useful as an out-group that led us further analyze our data
using computational and mathematical models.
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Figures
Figure 1: Phylogenetic Tree for a Variety Organisms – No Consensus no Outgroup. As the tree progresses downward the organisms are shown to be more
closely related
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Figure 2: A rooted Phylogenetic Tree based on Cytochrome c oxidase subunit
III (COXIII). The horse is the out-group, no consensus. As the tree progresses
downward, the COX-III sequences share more in common.
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Figure 3: A rooted Phylogenetic Tree based on LDH. The horse is
the out-group, no consensus. As the tree progresses downward, the LDH
isozyme sequences share more in common.
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Figure 4: A rooted Phylogenetic Tree based on Cytochrome B. The Mink
Whale is the out-group (least common animal), no consensus. As the tree
progresses downward, the Cytochrome B sequences share more in common.
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Figure 5: Morphological Cladogram where Ungulates are separated
from other organisms based on their unguligrade locomotion. The
ungulates are then separated by whether they are a perissodactyla or
artiodactyla
Figure 6: Gel Electrophoresis: Presence of different LDH isozymes in
different ungulates. Organisms include Horse, Goat, Sheep, Cow, and
Donkey. Band similarity shows that the horse, goat, sheep, and cow
are more closely related than the donkey
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Figure 7: Labeled picture of bones in a Human Foot and Ankle
Figure 8: Labeled picture of bones in a Cow Foot and Ankle
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Figure 9: Labeled picture of bones in a Horse Foot and Ankle
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Tables:
Table 1: Derived Character Table - The table of the compared
morphological features present in some ancestors of the ungulates are
listed in the first row. Boolean values are then used to illustrate if the
feature is a derived characteristic
Ruminant
(foregut
fermenter)
Hindgut
fermenter
Lion
(outgroup)
0
0
Horse
0
Pig &
Peccary
True
Horns
Deciduous
horns
Antlers
Selenodont
cusps
Lophodont
cusps
Lacking
incisors/canines
in upper jaw
Unguligrade
locomotion
Eventoed
Oddtoed
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
1
0
1
0
0
0
0
0
0
0
0
1
1
0
Deer
1
0
0
0
1
1
0
1
1
1
0
Pronghorn
Antelope
1
0
0
1
0
1
0
1
1
1
0
Cow
1
0
1
0
0
1
0
1
1
1
0
Sheep
1
0
1
0
0
1
0
1
1
1
0
Goat
1
0
1
0
0
1
0
1
1
1
0
Table 2: Matrix of Shared Derived Characteristics –
Illustrates the amount of derived characters in common between two
different ungulates
Lion
Lion
XXXX
Horse/Donkey XXXX
Pig & Peccary XXXX
Deer
XXXX
Pronghorn
XXXX
Cown
XXXX
Sheep
XXXX
Goat
XXXX
Horse
Pig
Deer
Pronghorn
0
0
0
0
XXXXXX
1
1
1
XXXXXX XXXXXX
2
2
XXXXXX XXXXXX XXXXXX
5
XXXXXX XXXXXX XXXXXX XXXXXXXXX
XXXXXX XXXXXX XXXXXX XXXXXXXXX
XXXXXX XXXXXX XXXXXX XXXXXXXXX
XXXXXX XXXXXX XXXXXX XXXXXXXXX
Cow
Sheep
Goat
0
0
0
1
1
1
2
2
2
5
5
5
5
5
5
XXXXXX
6
6
XXXXXX XXXXXX
6
XXXXXX XXXXXX XXXXXX
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Table 3: Pair Comparison of LDH sequences to bovine
sequences – Illustrates pair-wise identity of ungulate LDH
sequences to the bovine sequence
Organism
Number of Amino Acids
Percent Identity
Bovine LDH
Goat LDH
Sheep LDH
Pig LDH
Horse LDH
332
332
121
332
332
XXXXXXXXXXXXXX
98.80%
98.30%
97.30%
96.40%
Table 4: Dentition characteristics of Ungulates –
Table identifies the features of ungulate teeth.
Dentition - Skulls
Incisors/Canines in
Diastema?
Molar cusps?
Cheek teeth?
upper jaw?
Horse
yes
yes
lophodont
hypsodont
Pig
yes
yes
bunodont
brachydont
Cow
no
yes
selenodont
hypsodont
Deer
no
yes
selenodont
hypsodont
Sheep
no
yes
selenodont
hypsodont
Goat
no
yes
selenodont
hypsodont
Pronghorn
no
yes
selenodont
hypsodont
Human
yes
no
bunodont
brachydont
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Results: The Aftermath of Experimentation
Note: These results are given chronologically to how we obtained them that is why numbering of
Figures and Tables are not numerically ascending
Morphological Results:
Figure(s) 7-9: Bone structures of the Horse, Cow, and Human
Table(s): 1, 2, 4
The first pieces of data we collected were purely from observation of the morphology of
the ungulates. We identified bones in several different organisms and looked for similarities
(Figures 7-9) as well as looked for physical specific characteristics in several animals and
compared them to one another. (Tables: 1, 2, 4)
In Figure 7 we first analyzed the human foot which includes calcaneus which is the heal
bone, colored in green. The calcaneum and the talus, colored in purple and yellow are tarsals.
These yellow colored tarsals also contain navicular, medial cuneiform, intermediate cuneiform,
lateral cuneiform, and the cuboid. All five of the metatarsals are labeled in orange while the
phalanges are labeled in blue. The proximal middle phalanx and the distal phalanx are also all
labeled.
In Figure 8 we analyzed the foot of a cow. Again colored in green is the calcaneus. In
purple is the astragulus, in yellow is the tarsal, in orange is the metatarsal, in red is the coffin
bone and in blue are the phalanges. The tarsal in the cow can be compared to the tarsals in the
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human foot. At this point we started observing the structural and functional similarities between
a cow’s leg and a human foot.
Lastly in Figure 9 we show the identification of the parts of a horse foot and ankle.
Although these bones look very different that the last two figures the same similarities in bone
structure remain and are labeled.
After this exercise in bone identification we began trying to find morphological
relationships among all the ungulates by observing their bone structures. Our parameters
included dentition, horns, digestion and foot structure. We recorded our results in Table 1 and 4.
We were then curious how similar the animals were based on the data we observed. We hence
created a matrix of shared derived characteristics in Table 2. In our results we concluded that the
horse has the least shared derived characteristics with only one similarity. The pig shared two
characteristics with the others. The deer and pronghorn antelope shared as much as five common
characteristics.
Our results in Table 1 showed us that the cow, deer, sheep, goat, and the pronghorn
antelope are all ruminant artiodactyls. These organisms do not have incisors or canines in their
upper jaws, but all do have diastema between their incisors and/or canines. This set of organisms
are also known to have selenodont cusps efficient for the grinding of plant materials, and
hypsodont cheek teeth which are able to withstand the wear of a silica-rich herbaceous diet.
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Molecular Results:
Figure(s) 1-4: Tree Diagrams; Figure 6: Gel Electrophoresis
Table(s): 3
In the first four figures we used phylogenic trees (with a variable condition of COX II,
LDH, or Cytochrome B) to compare amino acid sequences of the various ungulates and drew out
the relationships among the different species. For example, the horse and the donkey are very
closely related, but not nearly as closely related as some of the other organisms. These
relationships are further proven by measuring how high the percent pair identity was among two
comparable organisms or if gel electrophoresis was done to see which organisms are the most
closely related.
Figure 1 is a phylogenetic tree for variety organisms without either a consensus or an outgroup. As the tree progresses downward the organisms are shown to be more closely related.
In Figure 2, the phylogenetic tree with the horse (the only perissodactyla) was selected as
the outgroup and cytochrome c oxidase subunit III (COX-III) sequences were compared. It is
known from prior research that COX-III is extremely important for the process of making ATP.
COX III was important in the transferring of electrons to the oxygen. Based on how close the
nodes are, one can conclude which organisms are related genotypically.
In Figure 3 the enzyme LDH or lactase dehydrogenase is used to find relationships
among the animals genotypically. LDH’s function as an enzyme works to catalyze a reaction
from pyruvate to lactate. Again the horse was chosen as an out-group and relations were
identified among the selected animals when protein alignment was done. In addition to doing
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tree diagrams we also performed the wet lab experiment of gel electrophoresis (Figure 6) to
analyze the LDH isozyme patterns. Analysis of the band patterns told us that all the organisms
(goat, sheep, and cow) were very closely related while the donkey was not. Furthermore in Table
3, we used computation to prove that the LDH sequence of the goat is most similar to bovine.
The goat and the bovine’s sequences had 98.80% pairwise identity. The sheep had the second
highest pair-wise identity with the LDH sequence, 98.3%. The other two organisms had even
lower percent pairwise identity, but the percentages were all still fairly high.
Lastly in Figure 4 we created a tree diagram of Cytochrome b. This protein is highly
involved in respiration (more specifically in the production of ATP). It is a very unique sequence
of amino acids. The bowhead whale is the out group. Some results we observed were that the
horse and tapir are very closely related. Both of these species are also closely related to the pig
and collared peccary which as a pair of animals are related. Similarly the deer and the American
elk are both linked and closely related to the pronghorn antelope. The goat and sheep are also
strongly connected.
Putting it all together: Figure 5 – Cladogram
Using all that we had learned in Figure 5 we created a morphological cladogram where
the ungulates were separated from other organisms based on their unguligrade locomotion. The
ungulates are then separated by whether they are a perissodactyla or artiodactyla, and the further
separated by their dentition and headgear.
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Discussion: Why did we get such results?
We started the lab with the goal of studying and classifying ungulates by conducting
morphological analyses. However we soon realized that by differentiating ungulates based on
these characteristics into the orders Perissodactyla and Artiodactyla, we were neglecting
molecular similaritiesand other ways to further classify these hoofed mammals. We then used
computational tools and mathematical models and we able to conclude which species were
closely related based on evolutionary relationships. Only by using comparative methods, along
with phenotypic and genotypic analysis we came up with some predictable even some surprising
results. We could then check back between our morphological conclusions and genetic
conclusions and see if the results jived. If they did we could fathom the question, “why?” certain
species seem related.
Conclusions:
I – Explanation of some Morphological Conclusions: Table 1, Table 3 Table 4
First and foremost we were able to effectively conclude that all ungulates use unguligrade
locomotion – they walk on the tips of their toes. As we moved away from bone structure (Figures
7, 8, 9) and delved further into the phylogeny of these animals we were able to further classify
them.
Example 1: The cows, sheep, and goats had many similarities which corresponded with
the definition of bovids. These even toed ruminants are artiodactyls. They also all have true
horns (permanent) and a permanent layer of keratinized skin.
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Example 2: Pigs, on the other hand are suids. They have incisors and canines which are
used as tusks for digging. They however, have neither hindgut nor foregut fermenter and hence
their stomachs are non-ruminant.
Example 3: Deer must be cervids as they have antlers. Upon closer analysis of dentition
one may notices their selenodont cusps (include pre-molars and molars) used for the grinding of
plant materials. Due to these characteristics they can be indeed classified as both artiodactyls and
ruminants.
With these similarities we can conclude that all ruminant artiodactyls have pre-molars.
Example 4: The deer, pronghorn antelope, cow, sheep, and goat all possess hysodont
cheek teeth. These cheek teeth are able to withstand a herbiferous diet rich in silica. Hence
following previous logic the deer, pronghorn antelope, cow, sheep, and goat are herbivorous,
ruminant, artiodactyls. This also corresponds with the conclusion that herbivorous artiodactyls
are ruminants because foregut fermenters have microbes consume cellulose and produce fatty
acids in order to supply energy to the animals.
II – Molecular Discussion: Table 2
Almost all of our molecular classifications were confirmed by our wetlab and drylab
work (as seen in the Results section). Although phenotypic relations are visually observed while
genotypic relations are not. Both of these methods of observation essentially provide a cause and
effect relationship where the effects (phenotype) were concurrent with the cause (genotype). All
the observational classifications we did were confirmed using gel electrophoresis (Figure 6), tree
diagrams of specific amino acid sequences, proteins, and DNA (Figures 1-4) and mathematical
analysis (Table 2)
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Citations:
[1] Radlick, L. (2011). Determination of Evolutionary Relationships among Ungulates.
<http://rpilms.rpi.edu/webct/ContentPageServerServlet//Lab/1Evolution/Wet/1Pre/2.Lab_Exerci
se.Evolutionary_Relationships__.pdf>
[2] Geneious. Computer software. Geneious: Bioinformatics Software for Sequence Alignment.
Vers. 5.5. Biomatters Ltd. Web. <http://www.geneious.com/>.
Bonus Questions:
1) Why do mutations occur in DNA? Is the DNA sequence of a single species identical?
Why or why not? Be specific.
When DNA replicates there is fairly measurable probability that the proteins involved can make
a replication error or mutation. This results in certain DNA sequences of a single species not
being the same due to several gene mutations spread throughout the genome. We know as
Darwin agrees that mutation is the driving force for natural selection and genetic drift. Only
because of these processes are not all members of the same species the same. This would result
in our evolutionary landscape to remain fairly constant and evolution to perhaps never occur.
2) Which specific cells must contain the mutation in order to have an effect on future
generations?
Mutation can only be passed on to affect future generations, if and only if the mutation occurs in
the genotype of gametes or sex cell. Any mutation particularly point mutations that occur in the
non-coding regions of DNA will not be passed on to the future generations regardless of whether
or not the aftermath is positive or negative.
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