Are Seals Related to Whales? Seals and whales are both marine mammals. So are dolphins, walruses, and manatee. They all have streamlined bodies, legs reduced to flippers, blubber under the skin and other adaptations for survival in the water. Mammals evolved on land, but these creatures have returned to the sea. Did they evolve from a single ancestor who returned to the ocean, or were there different return events and parallel evolution? We can’t go back in time to observe what happened, but DNA sequences contain evidence about the relationships of living creatures. From these relationships, we can learn about the evolutionary history of marine mammals Using sequence information in GenBank and bioinformatics software, determine the relationship between seal, whale, dolphin, walruse, and manatee. Also include in your analysis: human, mouse, dog, horse, bear, and weasel. Use the protein sequence of the hemoglobin beta gene to do your comparisons. Hemoglobin has been studied by many evolutionary biologists, so sequences are available in GenBank from many different organisms. Hemoglobin is a good test molecule since it shows both conservation across species (it performs an essential function), and variation. Species with unique challenges such as holding their breath for long underwater dives, may evolve changes in their hemoglobin to improve their supply of oxygen. Lets start by locating the hemoglobin beta protein sequence from harbor seal. The NCBI also provides a search engine for DNA and protein sequences. Go the home page of the NCBI (http://www.ncbi.nlm.nih.gov) and set the drop-down Search menu at the top to “Protein”, then type “harbor seal beta hemoglobin” in the text box and hit the Go button. This will jump you to a page that lists a few protein sequences including HBB_PHOVI (P09909). That is hemoglobin beta from Phoca vitulina, the harbor seal. If you click on the link to P09909, you will see the full GenBank record for this sequence including the Journal article where it was originally published (in 1986). AUTHORS Watanabe,B., Maita,T., Matsuda,G., Goodman,M. and Johnson,M.L. TITLE Amino-acid sequence of the alpha and beta chains of adult hemoglobin of the harbor seal, Phoca vitulina JOURNAL Biol. Chem. Hoppe-Seyler 367 (12), 1251-1258 (1986) QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Scroll down to the bottom of the page to see the actual protein sequence. To get rid of all the text and just get the protein, you can use the Display menu at the top of the page. Set the pull-down menu to “FASTA” and hit the Display button. Copy the protein sequence including the title line that starts with the greater than sign: “>gi|122664|sp|P09909|HBB_PHOVI Hemoglobin beta chain” One way to get a very quick estimate of evolutionary relationships is to use a sequence similarity search program such as BLAST. BLAST is available as a free tool at the NCBI website (http://www.ncbi.nlm.nih.gov/blast). Go to the protein-protein BLAST page and paste in the harbor seal protein sequence in the Search box. Then just hit the friendly blue BLAST! button. In the new window that pops up, hit the Format button. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. The results are listed in order of similarity, with the best matches at the top of the list. Naturally the HBB_PHOVI sequence matches itself. The next match is to a giant otter, then polar bear, river otter, walrus, ferret, panda, mink. A bit farther down the list are bears, bats, even some primates, but no close matches to whales. Try a protein BLAST search starting with the sperm whale hemoglobin beta sequence. What do you match? From this information, what is the closest land animal relative of the whale? BLAST is a very rough tool for making evolutionary comparisons. More accurate molecular taxonomy requires more careful work. We need to build a multiple alignment and calculate a phylogenetic tree. CLUSTAL is a free tool for making alignments and trees that is powerful, yet fairly easy to use. CLUSTAL is available as free software for your computer, and on a number of web sites including the European Bioinformatics Institute (EBI): http://www.ebi.ac.uk/clustalw/ One challenge in using CLUSTAL is getting your sequences organized in the correct format. CLUSTAL takes a set of sequences uploaded in a single text file, or all pasted into its text box. It is generally easiest to collect your sequences in a text file on your computer. Copy them in FASTA format from NCBI into a text editor (WordPad, TextEdit) or word processing program (MS Word) on your computer. Take care to copy the FASTA header line (with the “>” symbol) along with the sequence information. Also, it is helpful to rename the sequence so that immediately after the > symbol (with no spaces) is the name of the organism rather than the GenBank gi number. In addition to the hemoglobin beta from harbor seal (HBB_PHOVI) and sperm whale (HBB_PHYCA), collect hemoglobin beta (HBB) from other marine mammals, human (HBB_HUMAN), mouse (HBMS), bear, horse (HBB_HORSE), dog (HBB_CANFA), and ferret (HBB_MUSPF). Use the NCBI search engine, Display each sequence in FASTA format, and copy them all into a single text file. When you have all of the sequences organized, just copy and paste into the text box on the EBI CLUSTAL web page and hit the Run button. After a minute or two, you will get a new screen with an alignment and a tree diagram. There is a lot of information in the tree. First, are whale and seal on the same branch? What other species are closest to each? Do other parts of the tree make sense? Are human and mouse really that close? If there are parts of this tree that seem unlikely, what other bioinformatics experiments could you do to verify your results? What are some possible sources of error in this analysis?