Phylogeny of Ungulates – Studying the Morphological and Molecular

Phylogeny of Ungulates: Studying the Morphological and Molecular Attributes of Hoofed Mammals Jayanth (Jay) Krishnan T.A. Ms. Bianca Pier Lab Partner: Ms. Catherine Mahoney Section 1: Biology September 28 th , 2011

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.

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.

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.

Figures

Figure 1: Phylogenetic Tree for a Variety Organisms – No Consensus no Out- group. As the tree progresses downward the organisms are shown to be more closely related 5

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.

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.

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.

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

Figure 7: Labeled picture of bones in a Human Foot and Ankle

Figure 8: Labeled picture of bones in a Cow Foot and Ankle

Figure 9: Labeled picture of bones in a Horse Foot and Ankle

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 Hindgut True Deciduous Antlers Selenodont Lophodont Lacking Unguligrade Even- Odd- (foregut fermenter Horns horns cusps cusps incisors/canines locomotion toed toed fermenter) in upper jaw

Lion 0 0 0 0 0 0 0 0 0 0 0 (outgroup) Horse 0 1 0 0 0 0 1 0 1 0 1 Pig & 0 0 0 0 0 0 0 0 1 1 0 Peccary Deer 1 0 0 0 1 1 0 1 1 1 0 Pronghorn 1 0 0 1 0 1 0 1 1 1 0 Antelope 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 Horse Pig Deer Pronghorn Cow Sheep Goat Lion XXXX 0 0 0 0 0 0 0 Horse/Donkey XXXX XXXXXX 1 1 1 1 1 1 Pig & Peccary XXXX XXXXXX XXXXXX 2 2 2 2 2 Deer XXXX XXXXXX XXXXXX XXXXXX 5 5 5 5 Pronghorn XXXX XXXXXX XXXXXX XXXXXX XXXXXXXXX 5 5 5 Cown XXXX XXXXXX XXXXXX XXXXXX XXXXXXXXX XXXXXX 6 6 Sheep XXXX XXXXXX XXXXXX XXXXXX XXXXXXXXX XXXXXX XXXXXX 6 Goat XXXX XXXXXX XXXXXX XXXXXX XXXXXXXXX XXXXXX XXXXXX XXXXXX

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 332 XXXXXXXXXXXXXX Goat LDH 332 98.80% Sheep LDH 121 98.30% Pig LDH 332 97.30% Horse LDH 332 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

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

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.

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 out- group. 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

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.

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.

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)

Citations:

[1] Radlick, L. (2011). Determination of Evolutionary Relationships among Ungulates.

[2] Geneious . Computer software. Geneious: Bioinformatics Software for Sequence Alignment .

Vers. 5.5. Biomatters Ltd. Web. .

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.