Introduction

Whales and dolphins are both marine mammals. So are walruses, seals, otters, and manatee. They all have similar morphologies, streamlined bodies, forelimbs have been modified into flipper, a layer of blubber under the and other adaptations for survival in the water. Although the mammal clade evolved on land, these species have returned to the sea. The question is; did they evolve from a single ancestor who returned to the sea, or were there several return events and parallel evolution? We cannot go back in time to observe what happened, but DNA and protein sequences contain evidence about the evolutionary history of an organism and the relationship between other living creatures. By collecting and analyzing the DNA or protein sequences of marine and land mammals, we can infer the phylogeny of marine mammals.

Materials

• Gene Bank a genome browser from the NIH (http://www.ncbi.nlm.nih.gov/) 
• Clustalx and Phylowin (bioinformatics programs used to align sequences and build phylogenetic trees) 
• Google Docs, iWorks Pages, or Microsoft word and Text Edit (used to copy and paste data from genome browser in order to format data for bioinformatics software)
• Instructions

PROCEDURE

In this lab, we will be testing hypotheses about the evolutionary ancestry of different marine mammals. To repeat, we are trying to answer the question: Did marine mammals evolve from a single ancestor who returned to the ocean, or were there distinct return events from separate ancestors? A plausible starting hypothesis is that modern marine mammals have a single common ancestor.

Part A: 

First, we will explore the relationship of the marine mammals to each other vs. their evolutionary relationship to land mammals. To do this, we will test whether seals and whales are more closely related to each other than either of them are to representative land mammals: dogs (land carnivores) or cows (land herbivores). This exercise will mainly train you in using the bioinformatics software.

Organisms to Compare:

• Harbor Seal
• Minke Whale (a baleen whale)
• Dog
• Cow
• Red Kangaroo (outgroup)

Protein used: Hemoglobin B (HBB)

Part B: 

Second, each student will then develop a cladogram which includes a selection of marine mammals and land mammals which represent the major mammalian orders. You will then use this phylogenetic tree to test our hypothesis that all marine mammals have a single common land mammal ancestor.

Organisms to Compare:

Sea mammals groups and representative species

• Pinnepeds: 
• phocids: Harbor Seals, Gray Seals
• otariids: Sea Lions
• odobenids: Walruses
• Cetaceans
• Mysticetes: Minke Whale (or other baleen whales) 
• Odontocetes: Dolphin (or other toothed whales) 
• Sea Otters
• Sirenians: Manatee or dugong

• Carnivora: dog, Canis familiaris 
• Rodentia: rat, Rattus norvegicus 
• Primate: human, Homo sapiens 
• Proboscidea: elephant, Loxodonta africana 
• Artiodactyla: Hippopotamus, Hippopotamus amphibius 
• Perissodactyla: Horse, Equus caballus 
• Chiroptera: Black Flying Fox, Pteropus alecto
• Lagomorpha: Black-tailed jackrabbit, Lepus californicus
• Marsupials: red kangaroo, Macropus rufus
• Monotremes: Platypus, Ornithorhynchus anatinus

Protein or DNA sequence used: Hemoglobin, Myoglobin, CytochromeB (or any of the cytochrome family), gamma-fibrinogen (clotting factor), NADH dehydrogenase, MtDNA sequences... You can use BLAST to help find sequences. These are just a few examples, pick a gene that is robust and which you can find data for all representative groups. You can also concatenate multiple proteins or genes by using this tool to align them properly

Discussion:

You and your partner will produce a standard Google Doc lab report to describe this research. Be sure to include answers to the following questions in your lab report. The questions should guide your analysis and discussion, do not have a questions and answer section in your report

1. Did your tree support your hypothesis? Explain. 
2. What does this phylogenetic tree structure suggest about the evolutionary history of marine mammals? Go into detail here about what parts of the tree lead you to what conclusions about the evolutionary history of the marine mammals. 
3. If marine mammals share common morphological characteristics, what do your conclusions about their evolutionary history imply about these common characteristics? 
4. Why did you choose the protein sequence you did? What other protein sequences or genes might be useful in assessing evolutionary relationships between marine and terrestrial mammals? Show evidence of your research. 
5. Why do you need to align the sequences (with ClustalX) before inputting them into PhyloWin? 
6. What organism served as your outgroup? Why? What function does the outgroup serve?