| ## EMBOSS: PHYLIP

# Original PHYLIP dnainvar documentation.

This is the original PHYLIP documentation and includes information on the old menu driven program which is now replaced with the EMBOSS acd file.

                                                                       version 3.5c

           DNAINVAR -- Program to compute Lake's and Cavender's phylogenetic
                         invariants from nucleotide sequences

    (c) Copyright  1986-1993  by  Joseph  Felsenstein  and  by  the  University  of
    Washington.  Written by Joseph Felsenstein.  Permission is granted to copy this
    document provided that no fee is charged for it and that this copyright  notice
    is not removed.

         This program reads in nucleotide sequences for four species  and  computes
    the   phylogenetic  invariants  discovered  by  James  Cavender  (Cavender  and
    Felsenstein, 1987) and James Lake (1987).  Lake's method is also called by  him
    "evolutionary parsimony".  I prefer Cavender's more mathematically precise term
    "invariants", as the method bears  somewhat  more  relationship  to  likelihood
    methods  than  to  parsimony.  The invariants are mathematical formulas (in the
    present case linear or quadratic) in the EXPECTED frequencies of site  patterns
    which  are  zero for all trees of a given tree topology, irrespective of branch
    lengths.  We can consider at a given site that if there are no ambiguities,  we
    could  have for four species the nucleotide patterns (considering the same site
    across all four species) AAAA, AAAC, AAAG, ...  through TTTT, 256  patterns  in
    all.

         The invariants are formulas in the expected pattern frequencies,  not  the
    observed  pattern  frequencies.   When  they  are  computed  using the observed
    pattern frequencies, we will usually find that they are not precisely zero even
    when  the  model is correct and we have the correct tree topology.  Only as the
    number of  nucleotides  scored  becomes  infinite  will  the  observed  pattern
    frequencies  approach  their  expectations; otherwise, we must do a statistical
    test of the invariants.

         Some explanation of invariants will be found in the above papers, and also
    in  my  recent  review  article on statistical aspects of inferring phylogenies
    (Felsenstein, 1988b).  Although  invariants  have  some  important  advantages,
    their  validity also depends on symmetry assumptions that may not be satisfied.
    In the discussion below suppose that the possible unrooted phylogenies  are  I:
    ((A,B),(C,D)),  II: ((A,C),(B,D)), and III: ((A,D),(B,C)).

    Lake's Invariants, Their Testing and Assumptions

         Lake's invariants are fairly simple to describe: the patterns involved are
    only  those in which there are two purines and two pyrimidines at a site.  Thus
    a site with AACT would affect the invariants, but a site with AAGG  would  not.
    Let  us  use (as Lake does) the symbols 1, 2, 3, and 4, with the proviso that 1
    and 2 are either both of the purines or both of the pyrimidines; 3  and  4  are
    the other two nucleotides.  Thus 1 and 2 always differ by a transition; so do 3
    and 4.  Lake's invariants, expressed in terms of expected frequencies, are  the
    three quantities:

    (1)         P(1133) + P(1234) - P(1134) - P(1233),

    (2)         P(1313) + P(1324) - P(1314) - P(1323),

    (3)         P(1331) + P(1342) - P(1341) - P(1332),

    He showed that invariants (2) and (3) are zero under Topology I,  (1)  and  (3)
    are  zero  under topology II, and (1) and (2) are zero under Topology III.  If,
    for example, we see a site with pattern ACGC, we  can  start  by  setting  1=A.
    Then  2  must  be  G.   We can then set 3=C (so that 4 is T).  Thus its pattern
    type, making those substitutions, is 1323.  P(1323) is the expected probability
    of the type of pattern which includes ACGC, TGAG, GTAT, etc.

         Lake's invariants  are  easily  tested  with  observed  frequencies.   For
    example,  the  first  of  them  is a test of whether there are as many sites of
    types 1133 and 1234 as there are of types 1134 and 1233; this is easily  tested
    with  a  chi-square  test  or, as in this program, with an exact binomial test.
    Note  that  with  several  invariants  to  test,  we  risk  overestimating  the
    significance  of  results  if  we  simply  accept  the  nominal  95%  levels of
    significance (Li and Guoy, 1990).

         Lake's invariants assume that each site  is  evolving  independently,  and
    that  starting from any base a transversion is equally likely to end up at each
    of the two possible bases (thus, an A  undergoing  a  transversion  is  equally
    likely  to  end  up  as a C or a T, and similarly for the other four bases from
    which one could start.  Interestingly, Lake's results do not assume that  rates
    of  evolution are the same at all sites.  The result that the total of 1133 and
    1234 is expected to be the same as the total of 1134 and 1233 is unaffected  by
    the  fact that we may have aggregated the counts over classes of sites evolving
    at different rates.

    Cavender's Invariants, Their Testing and Assumptions

         Cavender's invariants (Cavender and Felsenstein, 1987) are for the case of
    a  character  with  two  states.   In  the  nucleic  acid  case we can classify
    nucleotides into two states, R and Y (Purine and Pyrimidine) and then  use  the
    two-state  results.   Cavender starts, as before, with the pattern frequencies.
    Coding purines as R and pyrimidines as Y, the patterns types  are  RRRR,  RRRY,
    and  so on until YYYY, a total of 16 types.  Cavender found quadratic functions
    of the expected frequencies of these 16 types that were  expected  to  be  zero
    under  a  given  phylogeny,  irrespective  of  branch  lengths.  Two invariants
    (called K and L) were found for each  tree  topology.   The  L  invariants  are
    particularly  easy  to understand.  If we have the tree topology ((A,B),(C,D)),
    then in the case of two symmetric states, the event that A and B have the  same
    state  should  be  independent  of  whether C and D have the same state, as the
    events determining these happen in different parts of the tree.  We can set  up
    a contingency table:

                                     C = D         C =/= D
                               ------------------------------
                              |
                       A = B  |   YYYY, YYRR,     YYYR, YYRY,
                              |   RRRR, RRYY      RRYR, RRRY
                              |
                     A =/= B  |   YRYY, YRRR,     YRYR, YRRY,
                              |   RYYY, RYRR      RYYR, RYRY

    and we expect that the events C = D and A = B will be independent.   Cavender's
    L  invariant  for this tree topology is simply the negative of the crossproduct
    difference,

     P(A=/=B and C=D) P(A=B and C=/=D) - P(A=B and C=D) P(A=/=B and C=/=D).

         One of  these  L  invariants  is  defined  for  each  of  the  three  tree
    topologies.  They  can obviously be tested simply by doing a chi-square test on
    the contingency table.  The one corresponding to the correct topology should be
    statistically indistinguishable from zero.  Again, there is a possible multiple
    tests problem if all three are tested at a nominal value of 95%.

         The K invariants are differences between the L invariants.   When  one  of
    the  tables is expected to have crossproduct difference zero, the other two are
    expected to be nonzero, and also to be  equal.   So  the  difference  of  their
    crossproduct  differences  can be taken; this is the K invariant.  It is not so
    easily tested.

         The assumptions of Cavender's  invariants  are  different  from  those  of
    Lake's.   One  obviously  need not assume anything about the frequencies of, or
    transitions among, the two different purines or the two different  pyrimidines.
    However  one does need to assume independent events at each site, and one needs
    to assume that the Y and R states are symmetric, that the probability per  unit
    time  that  a  Y  changes  into  an  R is the same as the probability that an R
    changes into a Y, so that we expect equal frequencies of the two states.  There
    is  also  an assumption that all sites are changing between these two states at
    the same expected rate.  This assumption is not needed for  Lake's  invariants,
    since  expectations  of  sums  are  equal  to  sums  of  expectations,  but for
    Cavender's it is, since products of expectations are not equal to  expectations
    of products.

         It is helpful to have both sorts of  invariants  available;  with  further
    work  we  may  appreciate what other invaraints there are for various models of
    nucleic acid change.

                                     INPUT FORMAT

         The input data for DNAINVAR is standard.  The first line of the input file
    contains  the  number  of  species  (which must always be 4 for this version of
    DNAINVAR) and the number of sites.  If the Weights option is being used,  there
    must  also be a W in this first line to signal its presence.  There is only one
    option requiring information to be present in the input file, W (Weights).  All
    options other than W are invoked using the menu.

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

    Nucleic acid sequence Invariants method, version 3.5c

    Settings for this run:
      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 out the counts of patterns  Yes
      3              Print out the invariants  Yes

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

    The user either types "Y" (followed, of course, by a  carriage-return)  if  the
    settings  shown  are to be accepted, or the letter or digit corresponding to an
    option that is to be changed.
         The options M and 0 are the usual ones.   It  is  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 documentation file for  the  sequence
    programs.

                                       OUTPUT FORMAT

         The output consists first (if option 1 is selected) of a reprinting of the
    input data, then (if option 2 is on) tables of observed pattern frequencies and
    pattern type frequencies.  A table will be printed  out,  in  alphabetic  order
    AAAA  through  TTTT  of  all  the  patterns that appear among the sites and the
    number of times each appears.  This table will be invaluable for computation of
    any other invariants.  There follows another table, of pattern types, using the
    1234 notation, in numerical order 1111 through 1234, of  the  number  of  times
    each type of pattern appears.  In this computation all sites at which there are
    any ambiguities or deletions are omitted.  Cavender's invariants could actually
    be  computed from sites that have only Y or R ambiguities; this will be done in
    the next release of this program.

         If option 3 is on the invariants are then printed out, together with their
    statistical tests.  For Lake's invariants the two sums which are expected to be
    equal are printed out, and then the result of an one-tailed exact binomial test
    which  tests  whether  the  difference is expected to be this positive or more.
    The P level is given (but remember the multiple-tests problem!).

         For Cavender's L invariants the contingency tables  are  given.   Each  is
    tested  with  a  one-tailed  chi-square test.  It is possible that the expected
    numbers in some categories could be too small for valid use of this  test;  the
    program does not check for this.  It is also possible that the chi-square could
    be significant but in the wrong direction; this is not tested  in  the  current
    version  of the program.  To check it beware of a chi-square greater than 3.841
    but with a positive invariant.  The invariants themselves are computed, as  the
    difference of cross-products.  Their absolute magnitudes are not important, but
    which one  is  closest  to  zero  may  be  indicative.   Significantly  nonzero
    invariants  should  be negative if the model is valid.  The K invariants, which
    are simply differences among the L invariants, are also printed out without any
    test  on  them being conducted.   Note that it is possible to use the bootstrap
    utility SEQBOOT to create multiple data sets, and from the output from  sunning
    all of these get the empirical variability of these quadratic invariants.

                                   PROGRAM CONSTANTS

         The constants that are defined at the beginning  of  the  program  include
    "maxsp",  which  must always be 4 and should not be changed, and "nmlngth", the
    name length.

         The program is very fast, as it  has  rather  little  work  to  do;  these
    methods  are  just a little bit beyond the reach of hand tabulation.  Execution
    speed should never be a limiting factor.

                                 FUTURE OF THE PROGRAM

         In a future version I hope to allow for Y and R codes in  the  calculation
    of  the  Cavender  invariants,  and  to check for significantly negative cross-
    product differences a future in them, which would  indicate  violation  of  the
    model.   By  then there should be more known about invariants for larger number
    of species, and any such advances will also be incorporated.

    ----------------------------TEST DATA SET--------------------------

       4   13
    Alpha     AACGTGGCCAAAT
    Beta      AAGGTCGCCAAAC
    Gamma     CATTTCGTCACAA
    Delta     GGTATTTCGGCCT

    --------------------------TEST SET OUTPUT--------------------------

    Nucleic acid sequence Invariants method, version 3.5c

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

    Alpha        AACGTGGCCA AAT
    Beta         ..G..C.... ..C
    Gamma        C.TT.C.T.. C.A
    Delta        GGTA.TT.GG CC.

       Pattern   Number of times

         AAAC         1
         AAAG         2
         AACC         1
         AACG         1
         CCCG         1
         CCTC         1
         CGTT         1
         GCCT         1
         GGGT         1
         GGTA         1
         TCAT         1
         TTTT         1

    Symmetrized patterns (1, 2 = the two purines  and  3, 4 = the two pyrimidines
                      or  1, 2 = the two pyrimidines  and  3, 4 = the two purines)

         1111         1
         1112         2
         1113         3
         1121         1
         1132         2
         1133         1
         1231         1
         1322         1
         1334         1

    Tree topologies (unrooted):

        I:  ((Alpha,Beta),(Gamma,Delta))
       II:  ((Alpha,Gamma),(Beta,Delta))
      III:  ((Alpha,Delta),(Beta,Gamma))

    Lake's linear invariants

     (these are expected to be zero for the two incorrect tree topologies.
      This is tested by testing the equality of the two parts
      of each expression using a one-sided exact binomial test.
      The null hypothesis is that the first part is no larger than the second.)

     Tree                             Exact test P value    Significant?

       I      1    -     0   =     1       0.5000               no
       II     0    -     0   =     0       1.0000               no
       III    0    -     0   =     0       1.0000               no

    Cavender's quadratic invariants (type L) using purines vs. pyrimidines
     (these are expected to be zero, and thus have a nonsignificant
      chi-square, for the correct tree topology)
    They will be misled if there are substantially
    different evolutionary rate between sites, or
    different purine:pyrimidine ratios from 1:1.

      Tree I:

       Contingency Table

          2     8
          1     2

       Quadratic invariant =             4.0

       Chi-square =    0.23111 (not significant)

      Tree II:

       Contingency Table

          1     5
          1     6

       Quadratic invariant =            -1.0

       Chi-square =    0.01407 (not significant)

      Tree III:

       Contingency Table

          1     2
          6     4

       Quadratic invariant =             8.0

       Chi-square =    0.66032 (not significant)

    Cavender's quadratic invariants (type K) using purines vs. pyrimidines
     (these are expected to be zero for the correct tree topology)
    They will be misled if there are substantially
    different evolutionary rate between sites, or
    different purine:pyrimidine ratios from 1:1.
    No statistical test is done on them here.

      Tree I:              -9.0
      Tree II:              4.0
      Tree III:             5.0