What Is a Phylogenetic Tree?
A phylogenetic tree is a way for scientists to represent evolutionary relatedness between different organisms. It is composed of nodes (organisms) and branches (a visualization of relatedness among organisms). The more related two organisms are, the closer they will be on a phylogenetic tree. But how do scientists determine where an organism belongs on a phylogenetic tree? Before the advent of bioinformatics, organisms were grouped by common morphological features: the distance between a dog and a cat, for example, is much smaller on a phylogenetic tree, than the distance between a dog and a crocodile. However, a dog and a crocodile are much closer than a dog and a plant. This method, although useful for large, morphologically distinct species, is imprecise and completely inadequate for microscopic organisms that very often have no distinct morphological features. With the advent of bioinformatics, a new method of measuring relatedness was found: DNA similarity. Since, most mutations occur randomly, it can be assumed that the longer two organisms have been evolving separately, the more their DNA will differ. A phylogenetic tree can be made using the Biology Workbench by finding, aligning and comparing protein sequences (which, for our purposes, act just like DNA sequences) of highly conserved genes of several different organisms. We have to use highly conserved genes because, among microorganisms, the rate of evolutionary change is so great that if we were to compare genes that are not highly conserved, we would not see any homology. Remember, the more similar the DNAs of two organisms, the closer they will be on a phylogenetic tree.


A Phylogenetic Tree of the Succinate Dehydrogenase Subunit 2 Gene
To construct our first tree, we are going to use a gene that you are already familiar with: the succinate deH subunit 2 gene. This gene is present in organisms from all 3 domains of life and it is also highly conserved, making it a good candidate for constructing a phylogenetic tree.

1.Select the "R. americana mitochondrial succinate dehydrogenase, subunit 2" sequence.

2. Select BLASTP.

3. In the next window, select the GBBCT database and 250 in the "1-line descriptions" pull-down menu. Click "Submit".


4. To generate a good phylogenetic tree, we need to include organisms that represent all of the different divisions or classes of bacteria. From the search results, select the following sequences:

Alpha, Rickettsia conorii 15619100 Alpha, Rickettsia prowazekii 3860572 Alpha, Agrobacterium tumefaciens 15157847 Alpha, Mesorhizobium loti 14024067 Gamma, Escherichia Coli 42926 Gamma, Vibrio Cholera 3341643 Gamma, Haemophilus influenza 1573838 Beta, Neisseria Gonorrhea 150366 Archae, Methanobacterium thermoautotrophicum 2894537 Epsilon, Helicobacter Pylori 2313275 Bacillus, Staphylococcus Aureus 14246761 Beta, Neisseria meningitidis 7379742 Alpha, Paracoccus denitrificans 975315 Bacillus, Bacillus subtilis 142966 Archae, Archaeoglobus fulgidus 2689375 Archae, Thermoplasma volcanium 14325503

5. "Import" all the sequences selected.

6. These sequences will be listed on the Protein Tools homepage.


7. Select all of the sequences that you just imported, plus the R. americana succinate deH subunit 2 sequence.

8. Select CLUSTALW in the tool menu and click on "Run". Click "Submit" on the next page.

9. The results will be very difficult to interpret at this point, so just click on "Import Alignment." You are now in the Alignment Tools domain of the Workbench.

10. Select the aligned sequences as shown in the image below.

11. Select DRAWGRAM from the tool menu, hit "Run" and click "Submit" on the next page. The phylogenetic tree that you obtain should look something like this:


In order to make sense of this tree, all of the ID numbers need to be substituted with actual bacterial names and the division each bacterium belongs to! The Biology Workbench does not provide a tool to do that, but for the tree above this was done "manually", with the following results:



Now it is easy to see the different divisions (and, hence, groupings) of bacteria. Most importantly, at the very top, notice that R. americana places with the alpha-Proteobacteria, once again indicating the very strong relatedness between them.


A Phylogenetic Tree of the Chloroplast Ribosomal Protein S12 Gene
To construct our second tree, once again we will use a gene that you are already familiar with: the chloroplast S12 ribosomal gene of P. purpurea. As with the succinate deH gene, this gene is present in all organisms and it is also highly conserved,

1. Select the "Chloroplast 30S ribosomal protein S12" sequence.

2. Select BLASTP

3. In the next window, select the GBBCT database and 250 in the "1-line descriptions" pull-down menu. Click "Submit"



4. To generate a good phylogenetic tree, we need to include organisms that represent all of the different divisions or classes of bacteria. From the search results, select the following sequences:

Cyanobacterium, A. nidulans, 11204 Cyanobacterium, Synechocystis, 1653348 Cyanobacterium, Spirulina, 47447 Bacillus, Clostridium, 15026202 Epsilon, Helicobacter, 4155706 Bacillus, Streptococcus, 11493923 Bacillus, Subtilis, 2632267 Bacillus, Aureus, 14246074 Beta, Corrodens, 41653 Chlamydia, Chlamydia, 3328863 Mycobacterium, Leprae, 725269 Mycobacterium, Smegmatis, 511125

5. "Import" all the sequences selected.

6. All of these sequences will be listed on the Protein Tools homepage.



7. Select all of the sequences that you just imported, plus the P. purpurea chloroplast S12 ribosomal gene sequence.

8. Select CLUSTALW in the tool menu and click on "Run". Click "Submit" on the next page.

9. The results will be very difficult to interpret at this point, so just click on "Import Alignment." You are now in the Alignment Tools domain of the Workbench.

10. Select DRAWGRAM from the tool menu, hit "Run" and click "Submit" on the next page. The phylogenetic tree that you obtain should look something like the one shown below:



When the bacterial ID's are substituted with actual names of bacteria and their divisions, we see a clear pattern:



Here we see that the chloroplast S12 ribosomal gene of the simple eukaryote Porphyra purpurea is grouped with the Cyanobacteria, indicating a strong evolutionary relatedness between them.

Conclusion
Using various bioinformatics tools in the Biology Workbench, you have shown that mitochondrial and chloroplast gene sequences from a basic eukaryotic cell are highly homologous to gene sequences in certain groups of bacteria. This type of evidence strongly supports the endosymbiotic theory. The endosymbiotic theory proposes that eukaryotic cells originated from prokaryotic cells that engulfed fellow prokaryotes, and instead of digesting them, formed a symbiotic relationship where one provided the nutrients and shelter, while the other produced more than enough energy for both of them. Specifically, you have found evidence suggesting that mitochondria evolved from alpha-Proteobacteria, while chloroplasts evolved from Cyanobacteria.