PROTPARS -- Protein Sequence Parsimony Method

CONTENTS:

• DESCRIPTION
• TEST DATA SET
• CONTENTS OF OUTPUT FILE (with all numerical options on)

The present method insists that any changes of amino acid be consistent with the genetic code so that, for example, lysine is allowed to change to methionine but not to proline. However, changes between two amino acids via a third are allowed and counted as two changes if each of the two replacements is individually allowed. This sometimes allows changes that at first sight you would think should be outlawed. Thus we can change from phenylalanine to glutamine via leucine in two steps total. Consulting the genetic code, you will find that there is a leucine codon one step away from a phenylalanine codon, and a leucine codon one step away from glutamine. But they are not the same leucine codon. It actually takes three base substitutions to get from either of the phenylalanine codons UUU and UUC to either of the glutamine codons CAA or CAG. Why then does this program count only two? The answer is that recent DNA sequence comparisons seem to show that synonymous changes are considerably faster and easier than ones that change the amino acid. We are assuming that, in effect, synonymous changes occur so much more readily that they need not be counted. Thus, in the chain of changes UUU (Phe) --> CUU (Leu) --> CUA (Leu) --> CAA (Glu), the middle one is not counted because it does not change the amino acid (leucine).

To maintain consistency with the genetic code, it is necessary for the program internally to treat serine as two separate states (ser1 and ser2) since the two groups of serine codons are not adjacent in the code. Changes to the state "deletion" are counted as three steps to prevent the algorithm from assuming unnecessary deletions. The state "unknown" is simply taken to mean that the amino acid, which has not been determined, will in each tree that is evaluated be assumed be whichever one causes the fewest steps.

The assumptions of this method (which has not been described in the literature), are thus something like this:

1. Change in different sites is independent.
2. Change in different lineages is independent.
3. The probability of a base substitution that changes the amino acid sequence is small over the lengths of time involved in a branch of the phylogeny.
4. The expected amounts of change in different branches of the phylogeny do not vary by so much that two changes in a high-rate branch are more probable than one change in a low-rate branch.
5. The expected amounts of change do not vary enough among sites that two changes in one site are more probable than one change in another.
6. The probability of a base change that is synonymous is much higher than the probability of a change that is not synonymous.

The input for the program is fairly standard. The first line contains the number of species and the number of amino acid positions (counting any stop codons that you want to include). These are followed on the same line by the options. The only options which need information in the input file are U (User Tree) and W (Weights). They are as described in the main documentation file. If the W (Weights) option is used there must be a W in the first line of the input file. For the U option the tree provided must be a rooted bifurcating tree, with the root placed anywhere you want, since that root placement does not affect anything.

Next come the species data. Each sequence starts on a new line, has a ten-character species name that must be blank-filled to be of that length, followed immediately by the species data in the one-letter code. The sequences must either be in the "interleaved" or "sequential" formats described in the Molecular Sequence Programs documentation. The I option selects between them. The sequences can have internal blanks in the sequence but there must be no extra blanks at the end of the terminated line. Note that a blank is not a valid symbol for a deletion.

The protein sequences are given by the one-letter code used by described in the Molecular Sequence Programs documentation file. Note that if two polypeptide chains are being used that are of different length owing to one terminating before the other, they should be coded as (say)

             HIINMA*????
             HIPNMGVWABT

One will usually want to use "?" after a stop codon, if one does not know what amino acid is there. If the DNA sequence has been observed there, one probably ought to resist putting in the amino acids that this DNA would code for, and one should use "X" instead, because under the assumptions implicit in this parsimony method, changes to any noncoding sequence are much easier than changes in a coding region that change the amino acid, so that they shouldn't be counted anyway!

The options that require information in the input file are the W (Weights) and U (User Tree) options. The form of this information is the standard one described in the main documentation file. For the U option the tree provided must be a rooted bifurcating tree, with the root placed anywhere you want, since that root placement does not affect anything.

The options are selected using an interactive menu. The menu looks like this:

Protein parsimony algorithm, version 3.5c

Setting for this run:
  U                 Search for best tree?  Yes
  J   Randomize input order of sequences?  No. Use input order
  O                        Outgroup root?  No, use as outgroup species  1
  T              Use Threshold parsimony?  No, use ordinary parsimony
  C               Use which genetic code?  Universal
  M           Analyze multiple data sets?  No
  I          Input sequences interleaved?  Yes
  0   Terminal type (IBM PC, VT52, ANSI)?  ANSI
  1    Print out the data at start of run  No
  2  Print indications of progress of run  Yes
  3                        Print out tree  Yes
  4          Print out steps in each site  No
  5  Print sequences at all nodes of tree  No
  6       Write out trees onto tree file?  Yes

Are these settings correct? (type Y or the letter for one to change)

The options U, J, O, T, M, and 0 are the usual ones. They are described in the main documentation file of this package. Option I is the same as in other molecular sequence programs and is described in the molecular sequence programs documentation file. Option C allows the user to select among various nuclear and mitochondrial genetic codes. There is no provision for coping with data where different genetic codes have been used in different organisms.

Output is standard: if option 1 is toggled on, the data is printed out, with the convention that "." means "the same as in the first species". Then comes a list of equally parsimonious trees, and (if option 2 is toggled on) a table of the number of changes of state required in each position. If option 5 is toggled on, a table is printed out after each tree, showing for each branch whether there are known to be changes in the branch, and what the states are inferred to have been at the top end of the branch. If the inferred state is a "?" there will be multiple equally-parsimonious assignments of states; the user must work these out for themselves by hand. If option 6 is left in its default state the trees found will be written to a tree file, so that they are available to be used in other programs.

If the U (User Tree) option is used and more than one tree is supplied, the program also performs a statistical test of each of these trees against the best tree. This test, which is a version of the test proposed by Alan Templeton (1983) and evaluated in a test case by me (1985a). It is closely parallel to a test using log likelihood differences due to Kishino and Hasegawa (1989) , and uses the mean and variance of step differences between trees, taken across positions. If the mean is more than 1.96 standard deviations different then the trees are declared significantly different. The program prints out a table of the steps for each tree, the differences of each from the best one, the variance of that quantity as determined by the step differences at individual positions, and a conclusion as to whether that tree is or is not significantly worse than the best one.

The program is derived from MIX but has had some rather elaborate bookkeeping using sets of bits installed. It is not a very fast program but is speeded up substantially over version 3.2.

TEST DATA SET

     5    10
Alpha     ABCDEFGHIK
Beta      AB--EFGHIK
Gamma     ?BCDSFG.??
Delta     CIKDEFGHIK
Epsilon   DIKDEFGHIK

Protein parsimony algorithm, version 3.5c

Name          Sequences
----          ---------

Alpha        ABCDEFGHIK
Beta         ..--......
Gamma        ?...S...??
Delta        CIK.......
Epsilon      DIK.......

     3 trees in all found

     +--------Gamma
     !
  +--2     +--Epsilon
  !  !  +--4
  !  +--3  +--Delta
--1     !
  !     +-----Beta
  !
  +-----------Alpha

  remember: this is an unrooted tree!

requires a total of     14.000

steps in each position:
         0   1   2   3   4   5   6   7   8   9
     *-----------------------------------------
    0!       3   1   5   3   2   0   0   0   0
   10!   0

From    To     Any Steps?    State at upper node
                             ( . means same as in the node below it on tree)

         1                ANCDEFGHIK
  1      2         no     ..........
  2   Gamma        yes    ?B..S...??
  2      3         yes    ..?.......
  3      4         yes    ?IK.......
  4   Epsilon     maybe   D.........
  4   Delta        yes    C.........
  3   Beta         yes    .B--......
  1   Alpha       maybe   .B........

           +--Epsilon
        +--4
     +--3  +--Delta
     !  !
  +--2  +-----Gamma
  !  !
--1  +--------Beta
  !
  +-----------Alpha

  remember: this is an unrooted tree!

requires a total of     14.000

steps in each position:
         0   1   2   3   4   5   6   7   8   9
     *-----------------------------------------
    0!       3   1   5   3   2   0   0   0   0
   10!   0

From    To     Any Steps?    State at upper node
                             ( . means same as in the node below it on tree)

         1                ANCDEFGHIK
  1      2         no     ..........
  2      3        maybe   ?.........
  3      4         yes    .IK.......
  4   Epsilon     maybe   D.........
  4   Delta        yes    C.........
  3   Gamma        yes    ?B..S...??
  2   Beta         yes    .B--......
  1   Alpha       maybe   .B........

           +--Epsilon
     +-----4
     !     +--Delta
  +--3
  !  !     +--Gamma
--1  +-----2
  !        +--Beta
  !
  +-----------Alpha

  remember: this is an unrooted tree!

requires a total of     14.000

steps in each position:
         0   1   2   3   4   5   6   7   8   9
     *-----------------------------------------
    0!       3   1   5   3   2   0   0   0   0
   10!   0

From    To     Any Steps?    State at upper node
                             ( . means same as in the node below it on tree)

         1                ANCDEFGHIK
  1      3         no     ..........
  3      4         yes    ?IK.......
  4   Epsilon     maybe   D.........
  4   Delta        yes    C.........
  3      2         no     ..........
  2   Gamma        yes    ?B..S...??
  2   Beta         yes    .B--......
  1   Alpha       maybe   .B........