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 1 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. 2 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. 3 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. 4 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. 6 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. 7 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. 8 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 9 Figure 7: Labeled picture of bones in a Human Foot and Ankle Figure 8: Labeled picture of bones in a Cow Foot and Ankle 10 Figure 9: Labeled picture of bones in a Horse Foot and Ankle 11 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 12 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 13 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 14 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. 15 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.