Exercise: Phylogeny-Answers

Answers to the Phylogeny exercise

Step 8

Answers to "The Phylogeny of HIV" can be found here.

Step 9

• How did you construct the tree? (alignment method, construction of tree, outgroup etc. )

For starters you need to do a multiple alignment of your sequences. A number of different alignment methods can be used (eg. MAFFT or RevTrans). Here you can see an example of a MAFFT alignment.

>Yeast
TACACTTTCTT---AGCTCGTCGTACTGATGCTCCATTCAA------CAAGGTTGTCTTG
AAGGCTTTGTTCTTGTCTAAGATCAACAGACCACCTGTTTCTGTCTCTAGAATTGCTAGA
GCTTTGAAGCAAGAAGGTGC------------------TGCTAACAAGACTGTTGTCGTT
GTTGGTACTGTTACTGACGATGCCAGAATCTTTGAATTCCCAAAGACCACTGTTGCTGCT
TTGAGATTCACTGCTGGTGCCAGAGCCAAGATTGTTAAGGCTGGTGGTGAATGTATCACT
TTGGATCAATTAGCTGTCAGAGCTCCAAAGGGTCAAAACACTTTGATCTTGAGAGGTCCA
AGAAACTCCAGAGAAGCTGTCAGACACTTCGGTATGGGTCC---------ACACAAGGGT
AAGGCTCCAAGAATCTTGTCCACCGGTAGAAAGTTCGAAAGAGCTAGAGGTAGAAGAAGA
TCTAAGGGTTTCAAGGTG
>African_frog
TATCGATTCTT---GGCTCGTCGTACCAACTCCAGTTTCAA------CCGGGTGGTTCTG
AAGCGTCTGTTCATGAGCCGAACCAACAGGCCACCCCTCTCTATGTCCCGTCTTATTCGC
AAAATGAAATTGCAAGGACG------------------TGAAAACAAGACTGCAGTGGTT
GTGGGCTGTATCACAGATGATGTCAGGATCCATGATATCCCCAAACTGAAGGTGTGCGCA
CTTAAAATAACCAGCGGAGCACGTAGCCGAATCCTGAAGTCTGGAGGTCAGATTATGACG
TTTGATCAGCTCGCCCTTGCGGCCCCTAAAGGCCAGAACACTGTTCTTCTTTCAGGACCT
CGTAAGGCCCGTGAAGTATACAGACACTTTGGGAAGGCACCTGGTACTCCACACAGTCGC
ACTAAGCCTTATGTGCTCTCCAAGGGTAGAAAGTTTGAGCGCGCCAGAGGACGCAGAGCC
AGCAGAGGATACAAGAAC
>Pig
TACAGGTTTCT---GGCCAGACGAACCAACTCCACCTTCAA------TCAAGTTGTGCTG
AAGAGGTTGTTCATGAGTCGCACCAACCGGCCACCCCTGTCGCTTTCCCGGATGATCCGG
AAGATGAAGCTTCCTGGCCG------------------GGAAGGCAAGACCGCTGTGGTC
GTAGGGACTATAACCGATGACGTGCGTGTCCAGGAGGTGCCCAAATTGAAGGTGTGCGCT
CTGCGCGTGAGCAGCCGTGCCCGGAGCCGCATTCTCAAGGCCGGGGGCAAAATCCTCACC
TTCGACCAGTTGGCCCTGGACTCCCCCAAAGGCTGTGGCACTGTCCTCCTCTCTGGGCCT
CGCAAGGGCCGCGAGGTGTACAGGCATTTCGGCAAGGCCCCAGGGACCCCGCACAGCCAC
ACCAAACCCTATGTTCGCTCCAAGGGCCGGAAGTTCGAGCGCGCCAGAGGCCGACGTGCC
AGCCGCGGCTACAAAAAC
>Fin_whale
TACAGGTTTCT---GGCCAGGCGAACCAACTCCACCTTCAA------TCAAGTTGTGCTG
AAGAGGTTGTTCATGAGTCGCACCAACCGGCCACCTCTGTCCCTTTCCCGGATGATTCGG
AAGATGAAGCTTCCCGGCCG------------------GGAAGGCAAAACGGCCGTGGTG
GTGGGGACAGTGACTGATGACGTGCGAGTCCAGGAGGTGCCCAAGCTGAAGGTGTGTGCT
CTCCGGGTGAGCAGCCGCGCCCGGAGCCGCATCCTCAAGGCCGGGGGCAAGATCCTCACC
TTCGACCAGCTGGCCCTGGACTCCCCCAAGGGCTGTGGCACCGTGCTCCTGTCTGGTCCT
CGCAAGGGCCGAGAGGTGTACAGGCATTTCGGCAAGGCCCCAGGAACCCCGCATAGCCAC
ACCAAACCCTATGTACGCTCCAAGGGCCGGAAGTTCGAGCGCGCCAGAGGCCGACGGGCC
AGCCGTGGCTACAAA---
>Human
TACAGGTTTCT---GGCCAGAAGAACCAACTCCACATTCAA------CCAGGTTGTGTTG
AAGAGGTTGTTTATGAGTCGCACCAACCGGCCGCCTCTGTCCCTTTCCCGGATGATCCGG
AAGATGAAGCTTCCTGGCCG------------------GGAAAACAAGACGGCCGTGGTT
GTGGGGACCATAACTGATGATGTGCGGGTTCAGGAGGTACCCAAACTGAAGGTATGTGCA
CTGCGCGTGACCAGCCGGGCCCGCAGCCGCATCCTCAGGGCAGGGGGCAAGATCCTCACT
TTCGACCAGCTGGCCCTGGACTCCCCTAAGGGCTGTGGCACTGTCCTGCTCTCCGGTCCT
CGCAAGGGCCGAGAGGTGTACCGGCATTTCGGCAAGGCCCCAGGAACCCCGCACAGCCAC
ACCAAACCCTACGTCCGCTCCAAGGGCCGGAAGTTCGAGCGTGCCAGAGGCCGACGGGCC
AGCCGAGGCTACAAAAAC
>Monkey_macaque
TACAGGTTTCT---GGCCAGAAGAACCAATTCCACATTCAA------CCAGGTTGTGCTG
AAGAGGTTGTTTATGAGTCGCACCAACCGGCCTCCTCTGTCCCTTTCTCGGATGATCCGG
AAGATGAAGCTTCCTGGCCG------------------GGAAAACAAAACGGCCGTGGTT
GTGGGGACCATAACGGACGACGTGCGGGTTCAGGAGGTGCCCAAACTGAAGGTATGTGCA
CTGCGCGTAACCAGCCGGGCCCGCAGCCGCATCCTCAGGGCAGGGGGCAAGATCCTCACT
TTCGACCAGCTGGCCCTGGACTCCCCCAAGGGCTGCGGCACTGTTCTGCTCTCCGGTCCT
CGCAAGGGCCGAGAGGTGTACCGGCATTTCGGCAAGGCCCCAGGAACCCCGCACAGCCAC
ACCAAACCCTACGTCCGCTCCAAGGGCCGGAAGTTCGAGCGTGCCAGAGGCCGACGGGCC
AGTCGAGGCTACAAAAAC
>Rat
TACAGGTTTCT---GGCCAGACGGACCAACTCCACCTTCAA------CCAGGTTGTGCTG
AAAAGGTTATTTATGAGCCGAACTAACCGGCCACCTCTGTCCCTGTCCCGAATGATCCGG
AAGATGAAGCTTCCTGGTCG------------------GGAGAACAAAACTGCTGTGGTT
GTGGGGACGATCACAGATGATGTGCGGATTCTGGAAGTGCCCAAGCTGAAGGTGTGTGCA
CTGAGGGTGAGCAGCCGGGCCCGAAGTCGGATCCTCAAGGCTGGGGGTAAGATCCTGACC
TTCGACCAGCTGGCCCTGGAGTCTCCCAAGGGCAGGGGCACTGTGCTCTTGTCTGGTCCT
CGGAAGGGCCGAGAGGTGTACCGACACTTTGGCAAGGCCCCAGGAACTCCACACAGCCAC
ACCAAACCCTATGTCCGTTCCAAGGGCCGGAAGTTCGAGCGTGCCAGAGGCCGAAGGGCC
AGCCGAGGCTACAAAAAC
>Mouse
TACAGGTTTCT---GGCCAGACGGACCAACTCCACCTTCAA------TCAGGTTGTGCTG
AAGAGGTTGTTCATGAGCCGAACCAACCGGCCACCTCTGTCCCTGTCCCGCATGATCCGA
AAGATGAAGCTTCCTGGCCG------------------CGAGAACAAGACTGCCGTGGTT
GTGGGGACGGTCACAGATGATGTGCGGATTCTGGAAGTTCCCAAGCTGAAGGTGTGTGCA
CTGCGGGTGAGCAGCCGGGCCCGGAGTCGCATCCTCAAGGCTGGGGGTAAGATCCTCACC
TTTGACCAGCTGGCCCTGGAGTCTCCCAAGGGCCGGGGCACTGTGCTCCTGTCTGGTCCT
CGGAAGGGCCGAGAGGTGTACCGACATTTTGGCAAGGCCCCAGGAACCCCACACAGCCAT
ACCAAACCCTATGTCCGTTCCAAGGGCCGGAAGTTTGAGCGCGCCAGAGGCCGAAGGGCC
AGCAGAGGCTACAAAAAC
>Salmon
TATCGTTTACCTGGAAGCAAATGCTCCACTGCTCCCTTCAA------CAAGGTGGTCCTC
AGGAGGCTCTTCATGAGCAGGACCCACAGGCCTCCGATGTCAGTGTCCCGCATGATCCGT
AAGATGAAATTGCCTGGACG------------------TGAGAACAGAACCGCAGTTGTC
GTGGGAACCGTCACTGATGATGTCAGAATTCATGAAATCCCTAATCTGAAGGTCTCGGCA
CTTAAAATAACCAGGCGAAATCGGACGCGAATTCTGAAGTTTGTG---CAGATTATGAGG
TTCGTTGGGCTCGCACTTGCTGCTCCTAATCGGCAGAAGAGTGTTCTTCTTTCCGCCCCC
CGTAACGCGCGTGATGTATCCAGGCACTTTGCCAACGCCCCCAGTATTCC------TCAC
ACTAAGCCTTACGTGCTTTCCAA------CAAGTTACGGCG---CAGAGGCAGCAAGCTC
ACT------TACAACAAC
>Fruit_fly
TACCGCTTCCT---TCAGCGCCGCACCAACAAGAAGTTCAA------CCGCATCATCCTG
AAGCGTTTGTTCATGAGCAAGATCAACAGGCCGCCGCTATCGCTTCAGCGCATCGCTCGC
TTCTTCAAGGCCGCCAACCA------------------GCCGGAGTCTACCATCGTGGTC
GTCGGCACCGTCACCGACGATGCCCGCCTCCTGGTGGTGCCCAAGCTCACCGTGTGCGCC
CTGCACGTCACGCAGACCGCCAGGGAGCGCATCCTGAAGGCCGGCGGTGAGGTCCTGACC
TTCGATCAACTGGCTCTCCGATCGCCCACCGGCAAGAACACGCTGCTGCTGCAGGGCAGG
CGTACCGCCCGCACCGCCTGCAAGCACTTCGGCAAGGCTCCCGGTGTGCCCCACTCGCAC
ACCCGCCCCTATGTCCGCTCTAAGGGACGCAAGTTCGAGCGTGCTCGTGGTCGTCGCTCC
AGCTGCGGCTACAAGAAG
>Arabidopsis
TACCGGTTTCT---GGTAAGGAGAACTAATAGCAAGTTCAA------TGGTGTGATATTG
AAGAGGCTTTTCATGAGCAAAGTCAACAAAGCTCCTCTTTCTCTATCTAGGCTTGTGGAG
TTCATGACTGGCAA------------------------GGAAGATAAGATTGCCGTCTTG
GTTGGAACTATAACTGATGATTTGAGGGTACACGAGATTCCAGCCATGAAAGTGACTGCC
TTGAGGTTCACAGAGAGAGCAAGGGCTCGCATTGAGAAAGCTGGAGGTGAATGCTTAACC
TTTGACCAGCTCGCTCTCAGAGCTCCATTGGGCCAGAACACGGTTCTTCTTAGAGGACCT
AAGAATTCACGTGAAGCAGTGAAGCATTTCGGACCTGCTCCTGGTGTGCCACACAGTCAC
TCCAAGCCATATGTTCGGGCCAAGGGAAGGAAGTTCGAGAAGGCCAGAGGAAAGAGGAAG
AGTCGTGGATTCAAGGTT
>Soy
TATCGCTTCCT---TGTTCGGAGAACTGGCAGCAACTTCAA------TGCTGTTATACTT
AAGAGATTGTTCATGAGCAAGGTTAACAAACCCCCATTGTCTTTGTCAAGGTTGATTAAG
TATACGAAGGGGAA------------------------GGAAGATAAGATTGCAGTGGTG
GTGGGGTCTATAACCGATGATATTCGTGTTTATGAAGTTCCACCATTGAAAGTTACAGCA
CTCAGGTTTACAGAGACTGCCCGTGCAAGAATTGAGAAGGCAGGCGGTGAATGCTTGACG
TTTGATCAGTTGGCTCTCAGGGCTCCTCTGGGACAGAACACGGTCCTTCTTAGAGGCCCA
AAGAATGCTCGCGAAGCTGTGAAGCACTTTGGTCCTGCTCCTGGTGTCCCTCACAGCCAC
ACCAAGCCTTATGTTCGAGCAAAGGGAAGGAAGTTTGAGAGGGCTAGAGGAAGGAGGAAC
AGCCGAGGATTTAGGGTT
>Rice
TACCGCTTCCT---GGTGCGGAGGACCAAGAGCCACTTCAA------CGCCGTGATCCTG
AAGCGGCTCTTCATGAGCAAGACCAACCGCCCGCCGCTCTCGATGCGCCGTCTCGTCAGG
TTCATGGAGGGGAAGGTACCTGATCGCCATGCCATTTCGGGGGACCAGATCGCCGTGATC
GTGGGCACCGTCACAGATGACAAGAGGATCTATGAGGTGCCGGCGATGAAGGTGGCTGCT
CTCAGGTTCACCGAGACCGCGAGAGCACGGATCATCAATGCCGGTGGCGAGTGCCTCACG
TTCGACCAGCTCGCTCTCCGCGCCCCGCTTGGCCAGAACACGGTCCTCCTGAGGGGTCCC
AAGAACGCTAGGGAAGCTGTTAAGCACTTTGGCCCTGCTCCAGGAGTTCCCCACAGCAAC
ACTAAGCCATATGTTCGCTCAAAGGGAAGGAAATTTGAGAAGGCAAGAGGAAGAAGGAAC
AGCAAGGGCTTCAAGGTA
>Methanocaldococcus_jannaschii
ATTGAGATATTAAAGCAGGAAAGTTATAAAAATCAGGCAAAGATTTGGAAGGATATTGCA
AGAAGGTTAGCAAAACCAAGAAGAAGGAGAGCAGAGGTAAATTTAAGTAAGATAAACAGA
TACACAAA------------------------------AGAAGGAGATGTTGTTTTAGTT
CCTGGTAAAGTTTTAGGAGCTGGGAAGTT------AGAGCACAAGGTTGTCGTTGCTGCA
TTTGCATTCTCAGAAACAGCTAAAAAATTAATTAAAGAAGCTGGAGGAGAAGCAATAACA
ATTGAAGAGCTAATAAAAAGAAATCCAAAAGGTTCAAATGTTAAAATT------------
------------------------------------------------------------
------------------------------------------------------------
------------ATGGCG
>Pyrococcus
ATTCGTTACCTCAGGAAAAAGTCTAATGAAGAGAAAGTTAAGATATGGAAGGACATAGCT
TGGAGACTTGAAAGACCAAGGAGGCAGAGGGCCGAAGTAAACGTCAGCAGGATAAACAGG
TACGCGAA------------------------------GGATGGAGACATGATAGTGGTT
CCAGGGAGCGTTCTTGGGGCCGGCAAGAT------AGAGAAGAAGGTCATTGTAGCTGCT
TGGAAGTTCAGTGAAACTGCAAGGAGAAAAATCGAGGAGGCCGGTGGGGAGGCCATAACG
ATTGAAGAGCTAATTAAGAGGAATCCAAAGGGAAGTGGAGTAATAATT------------
------------------------------------------------------------
------------------------------------------------------------
------------ATGGAG

Next you need to construct the tree. This can be done using the Treehugger tool: https://services.healthtech.dtu.dk/services/TreeHugger-1.0/ where you simply paste in your multiple alignment.

• A picture of the tree

Now the tree is ready to be opened in FigTree. All the sequences, except two, are from eukaryotes. The last two (Pyrococcus and Methanocaldococcus jannaschii) are both archaea and we therefore choose those two to be our outgroup (Notice that you can easily choose more than one sequence as outgroup, just choose the branch that are connecting both organisms to the rest of the tree and press "Reroot").

• A comparison of your tree with NCBI taxonomy. Are there any taxa that are not placed correctly on your tree?

On the whole, the structure of this tree is exactly as we would expect it, based on the known phylogeny. However, the placement of salmon and frog together in a monophyletic group is not correct. The correct species phylogeny would have salmon branching out below frog, which would branch out below the group of mammals (see illustration below).

There are two additional errors, which are not as easy to detect but can be seen if all the taxa are compared using NCBI Taxonomy's "Common Tree" function (see illustration below).

First, the group of Human+Macaque is placed as a sister group to Pig+Whale, which is not correct. Human+Macaque should have been a sister group to Rat+Mouse, since primates and rodents belong together in the group Euarchontoglires.

Second, yeast is placed further from the animals than the plants are — that is also not correct. Yeast (and indeed all Fungi) actually belong together with the animals in the group Opisthokonta.

It is often seen that a phylogeny based on a single gene differs from the real phylogeny of the species. There are a number of reasons for why this happens, but one important one is simply the stochastic nature of mutations: Occasionally a gene will be most similar to the gene from a non-sister species, for entirely random reasons. This phenomenon tends to disappear as more sequence data is included in the analysis (the law of large numbers).

Step 10

1. 52 results.
   Search string: (protein_name:"ribosomal protein l3") AND (taxonomy_id:2759) AND (fragment:false) AND (reviewed:true)
2. 8 and 26 results, respectively.
   Search strings: (protein_name:"ribosomal protein l3") AND (taxonomy_id:2759) AND (fragment:false) AND (reviewed:true) AND (cc_scl_term:SL-0173)
   and (protein_name:"ribosomal protein l3") AND (taxonomy_id:2759) AND (fragment:false) AND (reviewed:true) AND (cc_scl_term:SL-0086)
   Under the Download tab in UniProt, select "Download all", "FASTA (canonical)" and "Uncompressed".
3. Then use jEdit (or another text editor) to combine them. Combined FASTA file is here: Ribosomal_proteins_34.fasta.txt
4. Go to EBI's MAFFT server, choose "Protein", upload the combined FASTA file, and let all other options be default. When the alignment is done, click "Download Alignment File" and save the file. Then upload the result to TreeHugger and save the resulting Newick file.
5. Yes, it is possible to reroot the tree so that all the cytoplasmic sequences (RL3_*) and all the mitochondrial sequences (RM03_*) are in two separate monophyletic groups. After increasing the font size of the tip labels, the tree looks like this:
6. The mitochondrial proteins are more closely related to each other than to their respective cytoplasmic counterparts. This could indicate that mitochondria have appeared only once in evolution.
7. There is one difference: In the mitochondria, Bovine (cow) is the sister group to Human, while in the cytoplasmic proteins, Mouse+Rat comprise the sister group to Human+Macaque. The cytoplasmic tree is more correct.
8. There are more mutations per time unit in the mitochondrial part of the tree. This is evident from the mitochondrial branches being longer (the mitochondrial tips are further away from the root).