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NTSYS-pc - Numerical Taxonomy and Multivariate Analysis System

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 NTSYSpc
Numerical Taxonomy
and
Multivariate Analysis
System
Version 2.2

Getting Started Guide

F. James Rohlf
Department of Ecology and Evolution
State University of New York
Stony Brook, NY 11794-5245

EXETER SOFTWARE
47 Route 25A, Suite 2
Setauket, New York 11733-2870
 Information in this document is subject to change. The software described in this
document is furnished under a license agreement (single-user or site license). The software
may be used or copied only in accordance with the terms of the agreement.

Copyright © 2009 by Applied Biostatistics Inc., 10 Inwood Road, Port Jefferson, New
York 11777. All rights reserved worldwide.

ISBN: 0-925031-31-3

Current printing: February 21, 2009

Contents
1. Introduction ................................................................................................................................. 6
1.1 Areas of application ................................................................................................................. 6
1.2 Program modules in NTSYSpc ................................................................................................ 8
1.3 Getting started using NTSYSpc ............................................................................................ 11
1.4 What’s new in version 2.2?.................................................................................................... 13
1.5 What was new in version 2.1? ............................................................................................... 14
2. Modes of operation .................................................................................................................. 16
2.1 Interactive mode .................................................................................................................... 16
2.2 Batch mode ............................................................................................................................ 18
2.3 Both interactive and command modes .................................................................................... 19
3. Menus & related dialogs ......................................................................................................... 20
3.1 Main menu ............................................................................................................................ 20
3.2 Configuration options and file................................................................................................ 21
3.3 Output Listing Window ........................................................................................................ 22
4. Preparation of data files........................................................................................................... 23
4.1 NTS file formats .................................................................................................................... 23
4.2 Examples of NTS files ............................................................................................................ 25
4.3 Interface to other programs .................................................................................................... 27
4.4 Excel files ............................................................................................................................... 28
4.5 Nexus files ............................................................................................................................. 29
5. NTedit ........................................................................................................................................ 29
6. Graphics options & menu ....................................................................................................... 32
6.1 General plot options ............................................................................................................... 32
6.2 Other options ......................................................................................................................... 33
6.3 Plot menu .............................................................................................................................. 33
7. Typical applications ................................................................................................................. 34
iv
7.1 Cluster analysis ..................................................................................................................... 34
7.2 Ordination analyses and biplots ............................................................................................. 35
7.3 Principal components analysis ............................................................................................... 36
7.4 Principal coordinates analysis, PCOORDA ........................................................................... 37
7.5 Nonmetric multidimensional scaling ..................................................................................... 38
7.6 Burnaby's method for size adjustment ................................................................................... 38
7.7 Analysis of shape using landmark coordinates. ...................................................................... 39
7.8 Comparison of dis/similarity matrices .................................................................................... 39
Bibliography .................................................................................................................................... 41
INDEX ............................................................................................................................................... 43
 v

Preface
NTSYSpc was developed originally for use by students in a course “Taxonomia númerica
em microcomputadores” held in September 1985 at the Estação Agronómica Nacional,
Oeiras, Portugal. Many of the programs were written on a portable PC as I worked each
evening on the balcony of a hotel in Estoril—developing the programs needed the next day's
lab projects. The beautiful surroundings and enthusiastic students seemed to have helped.
Most of the design and many programs were developed during the two-week course. It was
quickly recognized that such a program on a personal microcomputer was of general
interest and that even the primitive PC of those days was able to handle most datasets.
NTSYS was originally written in FORTRAN for the IBM 360/50 mainframe computer
at the University of Kansas in 1966. That version was developed with the help of Ron
Bartcher who also converted it for use on a GE-635 computer in 1968. In 1969 John
Kishpaugh and David Kirk helped with the conversion of NTSYS from the GE-635 back to
an IBM 360/50 and then to the Univac 1100 computer system—both at Stony Brook
University. In addition, many others contributed to its development over the years.
NTSYSpc is a new program written in Pascal. Fortunately, after all of the previous
experience with conversions, most of the computational routines in NTSYS were by now
relatively easy to convert to another language. Both the program and the documentation
have greatly benefited over the years by the help of many of the users who have spotted
many “glitches” in the program and the documentation. Drs. Dean Adams, Leslie Marcus,
and Dennis Slice have made a number of important contributions. NTSYSpc will continue
to be developed. New programs and features are planned so that the system can evolve to
better meet your needs. Your comments, suggestions, and criticisms are much appreciated.
NTSYSpc has gone through many revisions. Exeter’s website should be checked for
new releases that can be downloaded. The help file have been expanded and improved. It
contains technical information that was once in the printed documentation.
This Getting Started Guide is intended only as a quick introduction to the use of
NTSYSpc. Details about the many computational modules and examples of their use are
given in the help file. The help file includes technical details about many of the modules.

Port Jefferson, New York F. James Rohlf

6 Introduction

1. Introduction
1.1 Areas of application
NTSYSpc is a system of programs that is used to find and display structure in multivariate
data. For example, one may wish to discover that a sample of data points suggests that the
samples may have come from two or more distinct populations. Of equal interest is the
discovery that some subsets of variables are highly inter-correlated. The program was
originally developed for use in biology in the context of the field of numerical taxonomy
(which explains why the name of the program is NTSYS—for Numerical Taxonomy
SYStem). But the programs have also been widely used in morphometrics, ecology and in
many other disciplines in the natural sciences, engineering, and the humanities. The terms
mathematical taxonomy and automatic classification have also been used to describe this
field of application. The techniques also represent a subset of multivariate data analysis and
have close ties to some methods in the field of pattern recognition.
Within the field of systematic biology, one can distinguish two different approaches to
classification. In phenetics one is concerned with the discovery and description of the
patterns of biological diversity and forming classification based on overall similarity
computed from multivariate data. These methods are commonly used in morphometric
studies. In cladistics one is interested in inferring the evolutionary history of the organisms
under study and using it as a basis for classification. Specialized methods have been
developed to take into account the assumption that the underlying model is of a branching
evolutionary tree. It is expected that the best biological explanation of the observed
diversity of a set of organisms will come in terms of their evolutionary history. The
methods are intended to make the best estimates of the evolutionary tree given a set of
descriptive data on a set of organisms. The most commonly used methods are justified on
the basis of the philosophical principle of parsimony (that the shortest tree that can be fitted
to a set of data should be the best estimate of the true tree) but statistically more powerful
methods based on the principle of maximum-likelihood are increasing in popularity. The
neighbor-joining method is also often used.
Many of the methods furnished in NTSYSpc are associated with the field of phenetics.
However, they are best interpreted as simply methods for multivariate data analysis. There
are programs by others that are specialized for phylogenetic methods. Some of the better
known ones are PAUP 1 and PHYLIP2. However, Saitou and Nei's (1987) neighbor-joining

1 Written by David Swofford, currently distributed by the Illinois Natural History Survey.
 Introduction 7
method of phylogenetic tree estimation is included in NTSYSpc. The UPGMA procedure in
the SAHN module is also often used on molecular data. A unique feature of its
implementation here is that it is able to take ties into consideration rather than simply using
an arbitrary tie-breaking rule. NTSYSpc also contains specialized methods used in geometric
morphometrics to study variation in shapes of objects.
The principal journal devoted to the general theory behind many of these techniques is
the Journal of Classification. It is published for the Classification Society of North America by
Springer. Theoretical papers are also published in many statistical journals. Applications of
these techniques are published in many scientific journals in the areas of application. For
example, Systematic Biology (formerly Systematic Zoology) has published many theoretical
and applied papers with special emphasis to applications in biological taxonomy.
Most users of these techniques begin with a data matrix that contains information
about the properties (features, characters, landmark or outline coordinates, etc.) of a number
objects (individuals, specimens, quadrats, OTUs, etc.). NTSYSpc can then be used to
compute various measures of similarity or dissimilarity between all pairs of objects and then
summarize this information either in terms of nested sets of similar objects (cluster analysis)
or in terms of a spatial arrangement along one or more coordinate axes (ordination analysis or
various types of multidimensional scaling analysis). This User Guide assumes that the reader
has some familiarity with the methods. It does not contain much advice about which
similarity coefficient or which clustering method should be used. It does, however, give
many hints about the use of the methods. To keep the account general, the neutral terms
"object" or "OTU" (for operational taxonomic unit) are usually used to refer to the things
(specimens) being analyzed and the terms "variable" or "character" are used to refer to the
properties used to describe the objects under study.
Users may find the following general references helpful (the complete references are
given in the Bibliography).
• Everitt and Dunn (1992) give a good concise introduction to both cluster analysis and
multidimensional scaling analysis. They furnish examples from biology.
Felsenstein (2004) gives a comprehensive overview of phylogenetic methods.
• Gnanadesikan (1977) describes many methods for detecting patterns in multidimensional
data. Applications are from many fields.
• Hartigan (1975) describes a large number of different clustering methods. Examples (with
test data sets) are from a great many fields.
• Jackson (1991) is an excellent mathematical text on multivariate analysis. It is much more
comprehensive than implied by its title ("A user's guide to principal components").
• Massart et al. (1978) gives a discussion with applications in analytical chemistry.

2 Written by Joe Felsenstein, University of Washington.

8 Introduction
• Reyment (1991) gives an overview of the application of multivariate methods and features
discussions of many data sets. The supplement by Marcus gives SAS procedures for the
computations of many of the multivariate analyses discussed in that book.
• Romesburg (1984) gives detailed descriptions of many clustering methods.
• Sneath and Sokal (1973) may be consulted for a general introduction to the field of
numerical taxonomy and for definitions of most of the jargon used in this manual. Most
examples are from biology but extensive references are given to applications in other
fields. The older version, Sokal and Sneath (1963) is still a useful reference as it gives
more complete listings of coefficients.
• Weir (1989) gives a short overview for DNA sequence data.

1.2 Program modules in NTSYSpc

Listed below are short descriptions of the computational modules included in NTSYSpc.
The acronyms under which they are listed, e.g., AUTOREGR, are the codes used in batch
command files. Detailed technical descriptions of the modules (including equations for the
operations and the various coefficients) are provided in the help file. NTSYSpc is not
limited to just the analyses mentioned below. The modules can be used in different
sequences to build many other types of analyses (for example, Gower’s principal
coordinates analysis can be carried out by using the SIMINT, DCENTER, and EIGEN
modules). A list of the names corresponding to the module acronyms is given in the help file
in both its table of contents and in its index. Users experienced with earlier versions of
NTSYSpc may wish to skip to Section 1.4 to see a summary of the new features.
AUTOREGR Fits data using the pure autoregressive model Used in spatial and
phylogenetic autocorrelation analyses.
CANPLS Performs canonical correlation and two-block partial least-squares analyses.
Used to study pattern of correlations between two sets of variables.
COMBINE Combines two or more matrices into one.
CONSENSUS Computes a consensus tree for two of two or more trees (such as multiple
tied trees from SAHN or between two different methods). Several consensus
indices are also computed to measure the degree of agreement between trees.
COPH Produces a cophenetic value matrix (matrix of ultrametric values) from a tree
matrix (produced, e.g., by the SAHN program). Can also compute a matrix of path-
length distances from the results of the NJOIN program. These matrices can be
used by the MXCOMP program to measure the goodness of fit to the similarity or
dissimilarity matrix on which they were based. A phylogenetic covariance matrix
can be computed to use in the MULREG module for comparative studies.
CORRESP Correspondence analysis. This is a useful way to investigate the structure of
2-way contingency tables.
 Introduction 9
CPCA Common principal components analysis. Attempts to fit a single set of
eigenvectors to a series of variance-covariance matrices.
CVA Performs a canonical vectors analysis (a generalization of discriminant function
analysis). It can also be interpreted as a single-classification multivariate analysis
of variance, MANOVA
DCENTER Performs a "double-centering" of a matrix of similarities or dissimilarities among
the objects. The resulting matrix can then be factored to perform a principal
coordinates analysis (a method for displaying relationships among objects in terms
of their positions along a set of axes based on a dissimilarity matrix).
EIGEN Computes eigenvector and eigenvalue matrices of a real symmetric similarity
matrix. This program can be used to perform a principal components or a
principal coordinates analysis by extracting eigenvectors (factors) from a cor-
relation or variance-covariance matrix.
FACTOR Performs the initial step (factor extraction) for a factor analysis of a correlation
or a covariance matrix.
FOURIER Computes Fourier and elliptic Fourier transformations (for both 2D and 3D
curves).
FOURPLOT Plots outlines and estimated outlines produced by the FOURIER module.
FREQ Computes matrices of gene frequencies for input to the SIMINT or SIMGEND
modules.
FROTATE Performs the orthogonal or oblique factor rotation step in a factor analysis.
FSCORES Computes factor scores using a variety of methods.
MDSCALE Nonmetric and linear multidimensional scaling analysis. This can be used as an
alternative to principal components analysis.
MOD3D Plots a 3-way scatter diagram as a 3-D perspective view of a model with t
"objects" at tops of wires attached to a base plane. The view can be rotated
interactively. This program is often used to view the results of a principal compo-
nents or principal coordinates analysis.
MST Computes a minimum-length spanning tree from a similarity or dissimilarity
matrix. This is useful for showing the nearest neighbors of objects based on their
positions in a multidimensional space.
MULREG Performs various types of regression analyses. Includes simple bivariate
regression, multiple regression, multivariate regression, and generalized least-
squares regression (to take into account non-independence of observations).
MXCOMP Compares two symmetric matrices by computing their matrix correlation and
then plotting a scatter diagram. The statistics for a Mantel test are also computed.
It can be used to compute the goodness of fit of a cluster analysis to a dataset (by
10 Introduction
comparing a cophenetic value matrix with a dissimilarity matrix). It can also
compare two matrices with the effects of a third matrix held constant (the Smouse-
Long-Sokal 3-way Mantel test).
MXPLOT Plots 2-way scatter diagrams of rows or columns of a matrix.
NJOIN Implements Saitou and Nei's (1987) neighbor-joining method and Gascuel’s (1997)
unweighted neighbor-joining method to produce estimated phylogenetic trees.
OUTPUT Formats matrices into pages for printing. The files can also be read by most
word processors. This formatted output is also useful for checking to make sure
that an input file has been prepared in the correct format for NTSYSpc.
PLOT Plot one or more columns of a matrix against a selected column. Points can be
connected by lines.
POOLVCV Computes a pooled within-groups variance-covariance matrix from two or
more data matrices. Also performs a test for homogeneity.
PROCPLOT Plots the results of the PROCRUSTES module.
PROCRUSTES Least-squares Procrustes superimposition of the coordinates of points in
two or more objects. Computes the average configuration of points and aligns all
objects to the average.
PROJ Projects a set of objects onto one or more vectors—or onto a space orthogonal to a
set of vectors. In principal components analysis one will project standardized data
onto the eigenvectors of the correlation matrix in order to see the best (in a
least-squares sense) low-dimensional view of a data set. The orthogonal projection
option can be used to implement Burnaby's (1966) method for size adjustment.
RESAMPLE Create samples using bootstrap, jackknife, random permutation, or
random normal deviates).
SAHN Performs the sequential, agglomerative, hierarchical, and nested clustering
methods as defined by Sneath and Sokal (1973). These include such commonly
used clustering methods as UPGMA and single-link. The program can find
alternative trees when there are ties in the input matrix.
SIMGEND Computes matrices of genetic distance coefficients from gene-frequency and
DNA sequence data.
SIMINT Computes various similarity or dissimilarity indices for interval measure
(continuous) data (e.g., correlation, distance, etc. coefficients).
SIMQUAL Computes various association coefficients for qualitative data— data with
unordered states (e.g., simple matching, Jaccard, phi, etc. coefficients).
SPLIT Split (partition) a matrix into two or more matrices in the same file.
 Introduction 11
STAND Performs a linear transformation of a data matrix so as to eliminate the effects of
different scales of measurement.
SUMMARY Summarizes results of a resampling experiment (bootstrap, jackknife, etc.).
SVD Computes a singular-value decomposition of a rectangular data matrix. It allows
you to compute principal axes and projections in a single step.
TPSWTS Computes projections of the 2D or 3D coordinates of objects onto the principal
warps of a thin-plate spline bending energy matrix. This is done to enable a
statistical analysis of the non-affine and uniform components of shape variation.
TRANSF Performs various linear and non-linear transformations of the rows or columns
of a matrix. Can also be used to delete rows or columns and alter the form of
storage of some matrices matrix.
TREE Displays phenetic and phylogenetic trees (e.g., from the SAHN or NJOIN
modules). Options are provided for scaling and scrolling through a tree
interactively.

1.3 Getting started using NTSYSpc

Installation of NTSYSpc is quite easy since a standard type of installation program is used.
Simply insert the disk and run its setup program. The only decision you will have to make
during installation is to select the name of the directory to be used. A program group will
be created on your startup menu. There will be icons for NTSYSpc, NTedit, help files, and
the readme.txt file. NTSYSpc can be un-installed (e.g., in case you need to move NTSYSpc to
another computer) by using the standard Add/Remove icon from the Windows Control
Panel. When you run NTSYSpc
the first time you will be asked to
provide your name, institution
(optional), and a registration
number.
Once the program is installed,
click on the NTSYSpc icon in the
start menu to see what NTSYSpc
"looks like" (see Figure 1.1). The
NTSYSpc main window is divided
into several regions. At the left is a
bar divided into folders with the
buttons for their computational
modules shown in the second
column. Click on a button (such as Figure 1.1. NTSYSpc main window
“Output”) to load the
corresponding program module.
12 Introduction
A form will be displayed in which
you can specify the input file and
other options for the selected
module (see Figure 1.2). A test
data set, TEST.NTS, is supplied
so you can try a few operations
right away. Click on the cell
opposite “Input file” to bring up a
file open dialog box. Note that by
default the dialog box assumes
that data file names end with the
file extension “.NTS”. For other
types of files, click on the “File of
type:” window at the lower left Figure 1.3. Entry form for the OUTPUT module.
and select “Excel”, “Nexus”, or
“All files.” Use this dialog to
locate the TEST.NTS file in
C:\NTSYS (or wherever you
installed NTSYSpc) and then
click on the “Open” button.
Then click on the “Compute”
button to run the Output
module. The results will be
displayed in the Listing
window (every time you run
a module a new section is
added to the listing
notebook). Note that there
will be nothing in this
window until after you click
the “Compute” button to
actually perform some
computations. An example is
given in Figure 1.3. Press the
1 key or use the Help menu
items to open the help file.
Note that the separate
modules do not provide
complete analyses. A
Figure 1.2. Listing window after running the Output module
sequence of modules will
on the TEST.NTS data file.
usually be used in order to
carry out a complete analysis. This structure makes NTSYSpc more flexible and useful in
 Introduction 13
research applications. Unless batch files are used, this approach also helps one appreciate
the various components making up a standard analysis. See Chaper 7 for a number of
examples.
Next, you should read Chapter 2 on “Modes of operation” to learn how to use
NTSYSpc in both interactive and batch modes. Note that This Style of type is used to
indicate strings of characters that the user is expected to type into the computer, such as file
names. Chapter 4 on “Preparation of input data files” is, of course, essential reading as it
describes the format of the data files. It also describes the use of the special editor, NTedit.
Be sure to check the README.TXT file for any last-minute notes or corrections to this
User Guide. The blue registration card should also be filled-out and returned since this
allows us to notify you of any problems that are discovered with this version of NTSYSpc. It
will also allow us to notify you of the availability of updates as new programs and features
are added to NTSYSpc. Your comments, corrections, and suggestions about the program are
welcomed.

1.4 What’s new in version 2.2?

Some of the new features in version 2.2 are listed below.
Changes to the user interface:
• The design of the user interface has been changed to make NTSYSpc input
dialogs more standard and easier to use.
• Menu of modules is less crowded so that additional modules can be listed.
• Configuration module improved Customization window added to the
configuration window.
• Added CSV and matlab output options to most modules. CSV files can be read
by Excel for further processing.
New modules:
• COMBINE – join two or matrices into one matrix.
• FACTOR – factor analysis (extraction using principal factor or maximum
likelihood methods).
• FROTATE – Factor rotation to attempt to find simple structure solutions. Both
orthogonal and oblique methods are available.
• FSCORES – compute factor scores using several different methods.
• RESAMPLE – generate samples (bootstrap, jackknife, random permutation, and
random normal deviates) from a data matrix
• SPLIT – split (partition) a matrix into two or more matrices in the same file.
14 Introduction
• SUMMARY – summarize results of resampling experiments
Improved modules
• CPCA – Better organization of the results of the statistical tests.
• POOLVCV – Added tests for homogeneity of subsets of covariance matrices.
• TPSWTS – Added new methods for computation of the uniform component.
• CANPLS – Now accepts raw data and creates several new types of plots.
NTedit:
• Can now append matrices from two files or within the same file.
• Drag and drop files from the Windows explorer to the edit window.
• Cells in the data grid are now color-coded.

1.5 What was new in version 2.1?

Changes to the user interface:
• The design of the user interface has been changed to make NTSYSpc easier to
use. The computational modules are now organized in folders in a bar along the
left side of the main window.
• A customize option allows the user to place frequently used modules in a user
defined folder.
• Excel data files can now be read directly by all appropriate NTSYSpc modules
(the less reliable DDE and OLE methods are no longer used).
• Nexus tree files can now be read and the NTS file format has been extended to
allow trees with OTUs at different heights.
• Plot options (choice of fonts, colors, etc.) can now be saved and reloaded in
graphics options files. The options are now organized in a convenient tabbed
notebook display.
New modules:
• PROCRUSTES – Procrustes superimposition of coordinates of 2D or 3D
landmarks on specimens or superimpositions of multivariate ordinations of any
dimension.
• PROCPLOT – 2D and 3D plot of the results of a Procrustes superimposition.
• FOURPLOT – Plot outlines and estimated outlines from the FOURIER module.
• MULREG – generalized multivariate multiple regression. Can be used to
perform simple bivariate regression, multiple regression, multivariate regression,
and generalized least-squares regression (as used for the comparative method).
 Introduction 15
• PLOT – Plot one or more columns of a rectangular matrix against a selected
column. Points can be given different symbols and connected by lines.
Improved modules
• CONSEN – Input trees can be in the nexus file format.
• COPH – Input tree can be in the nexus format. Path length (additive) distances
and Phylogenetic covariances can be computed in addition to the usual
ultrametric distances.
• CVA – Can now compute canonical variates scores for individual observations.
• FOURIER – Many changes were made to make this module more useful. Linked
to the new FOURPLOT module for direct viewing of the results.
• MOD3D – Rewritten to provide interactive 3D rotation (drag with the mouse)
and better axis labeling. Different symbols can be given to different points. 3D
biplots can be produced.
• MXCOMP – The Smouse-Long-Sokal 3-way Mantel test has been added along
with the additional plots it requires.
• MXPLOT – Points can be given different symbols. 2D biplots can be produced.
• NJOIN – Completely rewritten to greatly increase its speed. Large datasets (more
than 500 OTUs) can now be processed. Results can be saved in nexus format or
as an extended NTS tree format that allows for unequal heights of the OTUs in
the tree. An option has been added for unweighted neighbor-joining trees.
• OUTPUT – Supports Excel files and the new tree formats.
• POOLVC – Allows missing values in data when computing the mean and
covariance matrices.
• TPSWTS – For convenience, it now includes the Procrustes superimposition step.
It also now provides an estimate of the uniform component for 3D data.
• TREE – Can now plot trees with unequal heights of the OTUs.
NTedit:
• Can now directly read rectangular matrices from Excel files (the DDE and OLE
methods are no longer used).
• A new text-mode display allows NTedit to be used to edit any ASCII file
(including NTSYSpc batch command files). Files sizes up to 16MB with line
lengths up to 32K can be edited. Standard cut, copy, paste, and other text editing
commands are supported.
• The current matrix or file being edited can now be printed from within the
editor.
16 Modes of operation

2. Modes of operation
There are two modes in which NTSYSpc can be used: interactive and batch. In interactive
mode a module is selected from the main window by clicking on a button which causes a
window showing the various parameters and options for that module to be displayed. After
this form is filled in, click the Compute button to “run” the module and have the results
appear as a new section in the Listing window. Start batch mode by selecting the “Run
batch file…” item on the File menu or by using the convenient speed button on the toolbar.
The batch dialog box will let you select a file containing a sequence of NTSYSpc commands,
specify up to nine parameters, and run the batch file. The batch file contains commands that
call up various modules, supply parameters, and execute them automatically. Batch files are
convenient for the processing of large data sets or for processing a large number of data sets
(perhaps from a simulation).

2.1 Interactive mode

The main program window displays a program bar with folders corresponding to sets
of program modules (see Figure 1.1 above).
Click on a folder at the left to select a section. This displays icons in the next column
that correspond to the modules available. To select a module, click on the corresponding
icon. The selected module will then display a parameter entry form at the right side of the
main program window (such as that for the STAND program shown in Figure 2.1). To run a
program you must enter the required information in the Entry Window (you need to at least
specify the name of an input file). To fill in the entry form, select the desired locations in the
form using a mouse and enter the appropriate information (the method of entering the
information depends on the type of field). The default choices, if there are any, will have
already been entered into the form.
Input or output matrix names: names are any valid Windows file names (including long
names). File names can, optionally, be preceded by a drive specification (e.g.,
a:test.nts) or a path specification (e.g., c:\data\test.nts). If the name
contains either a colon or a backslash character, then the name is used as is.
Otherwise the name will be appended to the current data directory. The program
will remember the drive and directory from previous runs so that you do not have
to enter it every time if all the files are in the same directory. It is easiest to simply
double click on the cell to bring up a file open dialog box where you can select the
file visually.
 Modes of operation 17
Numerical constants: Often
numerical constants
make sense only within
certain limits.
NTSYSpc will not
permit you to enter an
out-of-range value.
Decimal points should
not be typed when
integer numbers are
expected by the
program.
Pick lists: Many of the programs
require one to select
one of several choices
for a field (such as a Figure 2.1. Dialog window for the STAND module.
method of standardiza-
tion or a clustering method). They are indicated by the small upside down triangle
at the right end of the field (there are two examples in Figure 2.1). Click on the
field to display a list of the available choices. Move the cursor or the mouse to
high-light the desired option and then click with the left mouse button. Sometimes
there is a blank entry signifying that this option is to be ignored. The selected code
will then be entered into the form.
Checkboxes: Press the space bar or click with the mouse to alternate between checked (for
yes) and unchecked (for no) states. These are used to indicate, for example,
whether the program should operate on the rows of the input matrix or whether
additional information should be included in the output listing.
Once the fields have been filled--in correctly, click the Compute button to run the
program (since the Compute button has the focus initially, you can also just press the R
key). The Listing window will be opened to a new section and it will show a summary of the
input parameters you specified, information about the input files, and the results of the
computations. If you provided names for output files then these results are stored on disk
and are available as input to other modules. For some programs short-cut graphics speed
buttons will appear on the small toolbar at the bottom left of the parameter entry window.
Pass the mouse over the button to display the hint box describing the type of plot produced
by each button. For example, Figure 2.2 shows the buttons available after running the
EIGEN program.
In case of an error (such as entering the name of a non-existent file for an input data
matrix), the program will "beep" and display a message in an Error Window. Click the OK
button to close this window so that you may correct the problem and try again.
18 Modes of operation
To close the parameter entry window for a module you may either click the Close
button or simply select another module from the NTSYSpc main window.

2.2 Batch mode

In batch mode NTSYSpc will attempt to
directly execute a sequence of modules without
displaying the parameter entry windows for
each. Commands are entered in an ASCII file
which can be prepared with an editor such as
Windows notepad or wordpad (although the
latter has the annoying habit of always placing
the extension .txt at the end of a file name).
The following is a simple example.
' standardize rows of data matrix
*stand o=data r=sdata Figure 2.2. Batch mode window. Click the
' compute distances among the OTUs d b l b hfl d h
*simint o=sdata r=dist
' now perform a UPGMA cluster analysis
*sahn o=dist r=tree
Lines that begin with a quote characters (either single or double) are treated as
comments. Blank lines are ignored. Each command line begins with an asterisk followed by
the name of the desired program. It is followed by parameter=value pairs that may take one
or more lines (lines that do not start with either an asterisk or a quote character are
considered continuation lines). Each parameter is a code for some program parameter.
Value gives the value of the parameter. There must be an "=" sign (and no blanks) between
the parameter and its value. Each such pair must be separated by at least one blank space.
The parameter is usually a one to three letter code (they are given in the help topic for each
module). They can be typed in either upper
or lower case. The values can be file names,
numerical constants, or option codes. The
values are identical to what would be
specified in an entry form in interactive Figure 2.3. Graphic speed buttons for the
mode. The defaults are also the same. For EIGEN module. The first button calls
legibility it is convenient to keep the lines MXPLOT to produce a 2D plot of the
short and use more than one line for each eigenvectors and the second calls MOD3D
command if convenient. The file TEST.NTB for a 3D plot.
on the distribution disk is an example of a NTSYSpc batch file.
To execute a file containing batch commands, click on the batch speed button on the
toolbar or else select the “Run batch file…” item on the File menu of the main window. This
will bring up the batch mode dialog box as shown in Figure 2.3. Click on the “Load” button
to bring up a file open dialog that allows you to specify which file to use. The click on the
 Modes of operation 19
“Run” button to execute the file. While running, this window will display the currently
executing line. If you change your mind you
may click on the “Cancel” button and the run
will be stopped at the next iteration or logical
breakpoint in the currently executing module
(this might take a while for a large matrix).
The results will be sent automatically to the
Listing window where they can be inspected
when the computations are complete.
It is also possible to prepare a batch file
with replaceable parameters. This allows batch
files to be used with more than one data set. Figure 2.4. Batch mode window with the
If the codes %1, %2,…, %9 are found in a test2.ntb file loaded and two replaceable
batch file they will be replaced by the values parameters provided.
of the corresponding replaceable parameter
strings given in the parameter area of the batch mode window. A maximum of 9 replaceable
parameters can be specified. An example is shown below.
*stand o=%1.nts r=sdata.nts
*simint o=sdata.nts r=dist.nts
*sahn o=dist.nts cm=%2 r=tree.nts
If the first replaceable parameter is "mosq" and the second is "single" (as in Figure 2.4),
then this batch file will be interpreted as if it were as follows:
*stand o=mosq.nts r=sdata.nts
*simint o=sdata.nts r=dist.nts
*sahn o=dist.nts cm=single r=tree.nts

2.3 Both interactive and command modes

During execution, the programs echo the input parameters and the comment information
furnished with the input matrices to the Listing window. In addition, a progress bar and a
status panel given an indication of how computations are progressing within each module.
Press the Cancel button if you need to stop the execution of programs that take a long
time to complete. The program should stop once it completes its next iteration or cycle of
computation (it does not check constantly for a keypress since that would slow the program
down). Alternatively, you can hold down the CAD keys to bring up the Windows
“Close Program” dialog box. Select NTSYSpc and then click on the “End task” button. The
program should then stop abruptly — but any information that was in the listing window
will be lost.
20 Menus and related

3. Menus & related dialogs

3.1 Main menu
Across the top of the main window is a menu bar (see Figure 1.1). The various choices are
described below.
File This pulls down a submenu from which you can select “Edit data file,” “View
listing,” “Printer setup,” “Run batch file,” and “Exit.” The edit menu item displays
a file open dialog in which you can specify the name of the NTSYSpc file you wish
to edit. The separate program NTedit (included with NTSYSpc) is then run. If the
file is a valid NTSYSpc file then it will be displayed in a spreadsheet like format. If
there are any errors in reading the file then an alternative ASCII editor (such as the
Windows notepad or some other user selectable editor) will be run. The view
listing item opens the Listing window (see Section 3.3). The printer setup item
opens the standard Windows printer setup dialog box. The run batch file item
brings up the Batch mode dialog box so that a batch file can be run (see Section
2.2). The exit item closes the program (the program can also be closed by clicking
on the Close speed button on the tool bar.
Options This pulls down a submenu from which you can select “Configuration” or
“Restore defaults.” The configuration item will display a parameter entry form for
various program configuration options. The restore defaults item will reset the
configuration parameters back to their original states. The “Customize” option will
allow you to add or remove modules from the User folder. See the examples in the
next two sections.
Help This pulls down a submenu from which you can select “Contents,” “Topic search,”
or “About.” The contents item displays the table of contents for the help file.
Topic search brings up the Help topics dialog box in which you can search for
various terms. You can search both the index and for various words in the help file.
The About item displays the NTSYSpc “about box” showing copyright
information, version number, and the registration number.
 Menus and related 21

3.2 Configuration options and file

There are a number of aspects of how
NTSYSpc operates that can be modified by a
user. These are done from the
Configuration Window (select
“Configuration” under the Option menu on
the main window). This will display a
window like those used for the various
computational modules. The entries here,
however, are system parameters such as file
formats, directory names, and other options.
The information you enter will be saved in
the ntsys.ini file in the same directory as the
ntsys.exe program. The file also includes
coded information about the position and Figure 3.1. Customization window. Drag
size of various windows used by NTSYSpc. module names from the list at the right to the
User folder at the left.
Configuration parameters
Batch code Description
FF File format code.
LF Listing format code.
LI Listing indent when printing.
DD Data directory—the directory to use as the
current directory for data files.
OW File overwrite code. ?=ask, O=overwrite, and
A=append.
ED Editor to be called from the toolbar or the
File|Edit menu. NTedit is the default but you
can specify your favorite ASCII editor.
The “File format” code is used to write results to disk so they can be used as input to
other modules. The default format of “e” ensures these values are saved with maximum
precision. This value should not be changed except possibly when working with very large
matrices and you are low on disk space. On the other hand, the “Listing format” code will
often be changed so that numerical information displayed in the Listing window has an
appropriate level of precision for a given data set. The default is “8.4f” which means that
floating-point numbers should be displayed with four decimal places within a field eight
characters wide. You can also enter the code as “F8.4” as in FORTRAN but the NTSYSpc
will always store the code with the “f” at the end.
The “File overwrite code” is used to determine what should happen when the program
attempts to save a data file with the same name as an existing file. Ask means that a
22 Menus and related
window will pop-up asking what should be done, overwrite means that the existing file
should be deleted, and append means that the new file will be appended to the end of the
existing file.
If you wish, another editor can be substituted for the NTedit program.
Note: the configuration parameters can also be changed through commands in a batch
file. Use CONFIG as if it were a module and use the "batch codes" given above to change
the values of the parameters. as the following:
config LF=9.6f
Because most analyses require one to run modules located in different folders, you may
find it convenient to make use of the “User” folder. You can drag module names from the
list at the right to the User folder on the left. To remove a module from the User folder right-
click on its name and select “Remove module” from the pop-up menu. You can also rename
a module.

3.3 Output Listing Window

This window uses a notebook metaphor to display listing output from the computational
modules. Each time a module is run a new section is created with an index tab numbered in
sequence and labeled by the
name of the module. An
example is shown in Figure
3.1. A section can be
examined by clicking on a tab
and then moving the
scrollbars. Note that the
entire window can be resized.
The File menu provides
a number of important
operations. The entire
“notebook” can be reloaded
from a previous run, saved to
an ASCII file, cleared (i.e.,
deleted), or printed.
Alternatively, the currently
displayed section of the
notebook can be saved to an
ASCII file, deleted, or
printed.
The Edit menu provides Figure 3.2. Example of the listing window after running the
commands in the test.ntb batch file.
commands to select all the
 Menus and related 23
text in the current section, to cut selected text to the Windows clipboard, to copy selected
text to the clipboard, and to paste in text from the clipboard. These commands permit you
to copy results into other software such as a word processor. They also allow you to delete
unwanted information before printing or saving to a disk file. Keeping such notebooks is a
convenient way to verify which options were used to produce a certain result. The purpose
of reloading a notebook is to allow the appending of new results so that a record of all the
computations for a particular project can be kept together. The file format is simply an
ASCII file with the form feed character separating sections.
The Options menu allows you to change the font used both for the on-screen display
and for printing. The indent item controls the size of the left margin when printing.
The Help menu provides the standard contents, topic search, and about items.

4. Preparation of data files

NTSYSpc NTS files are ordinary ASCII files (txt files, not binary files). A file for a data
matrix may be prepared with an editor or any word processor that has a txt (non-document)
mode. If you try to use a document file there may be invisible binary codes that NTSYSpc
will not know how to interpret. Free-format is used for the entries in the data matrices. This
means that at least one blank space or a comma is required between numbers. The NTedit
program included with NTSYSpc can be used to prepare data files and ensure that they are
in the proper format. In addition, NTSYSpc can also read data matrices from Excel
spreadsheets (*.XLS files) and trees from Nexus format files.

4.1 NTS file formats

A matrix can contain 4 kinds of records. The comment and label lines are optional.
Comments These optional lines are used to include notes with the data. The first character
in each line must be some type of quote character " or '. The information on
these lines will be copied onto comment lines in any matrices based on this input
matrix. In addition, each subsequent program will add an additional comment
line so that the sequence of steps leading to a given matrix can be determined.
Matrix parameter line This line contains 4 integer numbers (The second and third may have
a suffix letter to indicate the presence and location of row and column labels) and
possibly a floating point number. They must be separated by at least one blank
space.
• The first number is a code for the type of matrix.
24 Data files
1 rectangular data matrix,
2 symmetric dissimilarity matrix,
3 symmetric similarity matrix,
4 diagonal matrix,
5 tree matrix for dissimilarity data,
6 tree matrix for similarity data,
7 graph matrix for dissimilarity data, and
8 graph matrix for similarity data.
• The second and third numbers are the numbers of rows and columns in the matrix.
If labels are to be furnished for either the rows or columns (or both) then a letter
must be entered right after the number (with no spaces in between). An “L” is
used to indicate the presence of a list of labels in a separate record placed before
the data. For example, “25L” means that there are 25 rows and labels are
furnished in a separate record. A lower case "l" can also be used—but this is less
desirable since it looks so similar to the number "1". The letter “B” is used to
indicate that row labels are placed as the first item in each row and “E” indicates
that the row labels are placed after the end of each row.
• The fourth number is 0 if there are no missing data in the matrix. If there are
missing data then the fourth number should be a "1" followed by at least one blank
and then the numerical code used to denote the missing values—999 is a popular
choice.
Row and column labels Labels must be furnished if a "B", "E", or "L" is placed after the
numbers of rows or and "L" after the number of columns in the previous line. Row
labels can be placed in one of three locations: as the first element at the beginning
of each row (B), as the last element at the end of each row (E), or as a separate list
of row labels in front of the matrix (L). The column labels if present always consist
of a list of labels with the first label beginning on a new line. Each label consists of
strings of characters (up to 16 letters or digits but no blanks). They are separated by
one or more blanks or by a comma, i.e., the are entered free format. Examples are
given below.
Matrix data lines The elements of the matrix are entered with rows in the input matrix
corresponding to one or more lines in the input file (i.e., matrices are always
entered rowwise). Symmetric matrices are entered as rows beginning with column
1 and ending with the diagonal elements (i.e., the lower half matrix with diagonals
is entered rowwise). If all the elements for a row do not fit on a single line, then
continue typing on as many new lines as needed. It is important that the first
element of a new row starts on a new line—even if the previous line is mostly
empty. The elements themselves are free format. Values must be separated by one
 Data files 25
or more blanks or a comma. Missing values are indicated by the numerical code
provided on the matrix parameter line. They cannot be simple left blank (they can,
however, be indicated by a “.” if a missing value code is provided on the matrix
parameter line).
The lines can be very long (the theoretical limit is 2 GB!)—but it will be easier to work
with them with most editors if you use shorter lines (80 characters or fewer). Blank lines are
ignored.
More than one matrix can be stored in a single file. The records for a second matrix
(starting with the optional comment lines) simply follow after those for the first. Most
program modules in NTSYSpc will perform the selected set of operations on each of the
matrices in an input file. The results for the second and subsequent matrices are simply
appended to the files produced by processing the first matrix. For some programs it is
necessary to put more than one matrix in a single file in order to perform a certain
computation. It is required by programs such as CPCA, CVA, and POOLVCV. It is also
necessary in order to compute the majority rule consensus tree for more than two trees.
Note: if you prepare the original data matrix so that the rows correspond to the
characters (variables) and the columns correspond to the objects being classified (OTUs, data
points, etc.), then you will find that many of the default row/column direction options will
be correct.
Because there is always the chance that there will be an error in the preparation of a
data matrix, it is strongly suggested that you use the NTedit program and that you first try
the OUTPUT module to display your input data matrix. It can be printed out for
convenience in proofing. If there are major problems in the file format you may need to load
the matrix into NTedit in text mode.

4.2 Examples of NTS files

An example of a data matrix file with 3 comment lines and labels for the columns but not the
rows is given below. This set of test data is furnished on the distribution disks for NTSYSpc
and is used for many of the examples given in this manual.
"A sample data matrix to test NTSYSpc
"There are 5 characters (rows) and 10 OTUs (columns)
"The columns are labeled. No missing values.
1 5 10L 0
A B C D E F G H I J
8 7 9 13 6 12 9 7 11 6
5 6 3 3 7 10 5 7 5 7
11 13 12 9 8 18 17 21 22 13
10 18 22 8 7 17 18 26 24 18
11 6 12 10 10 19 13 13 19 14
26 Data files
An example of a data matrix with column labels and with row labels placed at the
beginning of each row:
1 4B 3L 0
c1 c2 c3
r1 1, 3, 4
r2 3, 2, 1
r3 3, 4, 2
r4 2, 1, 2
The computation of several genetic distance coefficients require rectangular matrices
that indicate which alleles correspond to the same locus or sample sizes for different
samples. These are simple matrices with just one row. See the help file for details.
An example of a symmetrical correlation matrix file (note that elements past the
diagonal of a symmetric matrix must not be entered). Labels can only be placed in a list in
front of the data (i.e., only the “L” code is valid).
"A sample correlation matrix with labels
3 5L 5 0
A B C F E
1
0.4 1
0.3 0.4 1
0.6 0.3 0.4 1
0.7 0.3 0.4 0.5 1
In this case the “L” can be appended to either the number of rows, the number of
columns, or to both. But only one set of labels should be furnished. If a symmetric matrix is
the output of some other program it may be stored as a full square matrix. In that case you
should code it as a rectangular matrix and use the SYMD or SYMS options of the TRANSF
program to convert it to the lower half matrix form required by NTSYSpc. See the help file
for more example, especially the matrices needed for the analysis of genetic data
(microsatellite, RAPD, etc.).
Tree matrices (matrix types 5 and 6) are usually produced by programs rather than
entered by a user. The usual exception is when one wishes to enter an expected tree to
compare with the observed results (using the CONSEN program). There are two styles in
which a tree can be entered in NTSYSpc. The format used internally in NTSYSpc is
described at the end of the description of the SAHN program.
In addition, you can describe a tree using nested parentheses as in the NEXUS format
used, for example, in the program PAUP. This option is only available for tree matrices
based on dissimilarities (matrix type code = 5). While complete NEXUS files cannot be read,
the tree descriptions can be processed as long as the OTUs names are given as integer
numbers (corresponding to their position in a data matrix). This format is provided to
enable trees produced by other programs to be entered into NTSYSpc more easily. One can
 Data files 27
also enter trees by hand using this notation -- but it becomes awkward for large trees since it
is easy to miscount parentheses.
In this format nesting is indicated by parentheses, branch lengths (which are optional)
are given in the format ":value" after each OTU name and right parenthesis, and the end of
the tree is indicated by a semicolon. If branch lengths are not provided then NTSYSpc will
generate arbitrary clustering levels consistent with the set relationships given in the tree.
Note that one must either provide branch lengths for all branches or else for none of them.
A mixture will produce unpredictable results.
Example of a NEXUS style tree not using branch lengths:
" NEXUS style input with OTU labels provided.
5 5L 2 0
A B C D E
(((1,3),2),(4,5));
This implies a tree of the following topology:
1--.
3----.
2----L---.
4--. |
5--L-----L---
Example of an input file using branch lengths:
" Example using branch lengths but no OTU labels.
5 5 2 0
(((1:2.1,3:2.5):1.6,2:3.3):0.7,(4:0.5,5:0.3):0.9);
This tree has the same topology as in the previous example. It should be noted, as in
the above example, that the branch lengths may be inconsistent with the levels (heights)
used to describe an ultrametric tree. In the above example the branch length for OTU 1 is 2.1
but the length for OTU 3 is 2.5. The program will use the average (2.4) of these values. An
additional problem is that the raw average of heights of each interior node may not increase
as one goes towards the root. In the above example the height at which the set {1,3,2} joins
the root is 4.3 and the height at which {4,5} joins is 1.3. The average of these two values is 2.8
which is smaller than the level at which {2} joined {1,3}. The program constrains the average
heights to be at least 0.0001 greater than the largest height within the sets being joined. This
preserves the topology indicated by the parentheses but shows the trees graphically as
looking as if there was a multifurcation.

4.3 Interface to other programs

Since the matrix files have a simple format (see the previous section), they should be usable
by other programs with very few changes needed. Results from other programs should also
28 Data files
be convertible into the format described above. The largest problems are apt to be due to
different conventions for furnishing labels and for reading symmetric matrices.

4.4 Excel files

NTSYSpc can read rectangular matrices from "worksheet" files compatible with Excel. These
files must have an extension of “XLS.” The Excel program itself need not be present.
NTSYSpc will search the spreadsheet for the matrix parameter line by starting with the first
row. If only cell A1 contains information then its search continues on to subsequent rows
until a row is found with at least four non-blank cells. The information in the rows being
skipped over is taken as comments. Once the matrix parameter line is found, the cell in
column “A” is interpreted
as the matrix type code. It
must be a "1" for a data
matrix. The two cells to
the right (columns B and
C) must be the number of
rows and the number of
columns. Note: these must
be integer numbers. Do
not try to append a code to
indicate the presence of
row or column labels as
one does for an NTS file.
The cell in column D
contains the code
indicating whether or not
there are any missing Figure 4.1. Example of a sample data file in Excel. Note that the
values. Enter a zero or first two lines are actually text in cells A1 and A2.
leave it blank if there are
no missing values. Otherwise, enter the identifying numerical code (e.g., 999) in the cell in
column E.
The next row contains column labels beginning with column B. If any cells are left
blank they will be replaced with column numbers. Column A contains the row labels. If
any cells are blank then will be replaced by row numbers. Thus row and column labels are
in their natural position—not as records in front of the matrix as in as in the “L” option for
NTS files. The row and column labels should not contain any blanks. The matrix itself
begins in column B. See Figure 4.1 for a simple example where the matrix starts in cell B5.
If empty cells are found within the matrix, they are assumed to also correspond to
missing values. Information in the spreadsheet in rows beyond the matrix is ignored and
 Data files 29
can be used to store other information. Only one matrix can be read from each Excel file and
the matrix should be in the first entry in the spreadsheet .

4.5 Nexus files

This format is supported by many programs concerned with estimating phylogenetic
trees. Within NTSYSpc, the NJOIN module can save tree files in this format. Trees in this
format can be read by the COPH, CONSEN, and OUTPUT modules.
An example is given below. The translate section is required. It gives the labels for the
OTUs. There can be one or more tree commands in a file. Each describes a tree using nested
parentheses. The length of each branch on the tree must be provided following the “:”
character. The file is treated a single stream of characters with line breaks provided
wherever convenient.

#nexus
begin trees;
[11 mosq. extracted from Harbach & Kitching (1998)]
translate 1 Anoph1, 2 Toxo12, 3 Wyeo13, 4 Uran17, 5 Culi21, 6
Orth28,
7 Mans29, 8 Psor32, 9 Aede44, 10 Cule101, 11 Dein126;
tree (1:16,(4:2,((6:1,(5:1,2:1):3):3,
((9:1,(8:1,7:6):4):2,((10:1,11:3):1,3:30):1):2):2):8);
end;
The nexus file format is described in: Maddison et al. (1997). The method used for
describing trees is called the “Newick Standard” and was adopted June 26, 1986 by an
informal committee meeting during the Society for the Study of Evolution meetings in
Durham, New Hampshire. James Archie, William H.E. Day, Wayne Maddison, Christopher
Meacham, F. James Rohlf, David Swofford, and Joe Felsenstein were present. The reason for
the name is that the second and final session of the committee met at Newick's restaurant in
Dover, NH. Examples and a simple description of this format are available at
http://evolution.genetics.washington.edu/phylip/newicktree.html.

5. NTedit
The Ntedit program, included with NTSYSpc, is a data editor designed for use with
NTSYSpc data files. For each of the basic file formats (rectangular, symmetric, diagonal,
tree, and graph) it displays an appropriate arrangement of the cells in the spreadsheet.
Using NTedit ensures that the files are formatted correctly.
30 NTedit
The program can be
started in three ways.
1. Click on the
NTedit icon to
start the program.
2. Load this program
from the File|Edit
file data file menu
item or the edit
speed-button on
NTSYSpc’s
toolbar.
3. Use a DOS
command window
and type ntedit Figure 5.1. Example of NTedit with the test.nts file loaded in a
and the name of a grid view.
file and then press
the R key to start the program.
Once the program is started, you can either create a new file or load an existing file.
NTS format files can be loaded either in a spreadsheet like grid (Figure 5.1) or in a plain
ASCII text editing (Figure 5.2) view. Excel files can only be displayed in the grid view and
nexus files can only be displayed in the text view.
In the grid mode you can enter or correct data in any of the cells. You can insert or
delete rows and columns within the table by clicking on the appropriate menu choices or the
speed buttons on the tool bar. You can also add or delete rows and columns from the end of
the table by entering new values in the edit boxes displaying the current numbers of rows
and columns. To change the labels for the rows or columns (given in the first, protected,
row or column of the data table) click on the RowLabs or Col.Labs buttons to unprotect
these entries. You can then type new information in these cells. The new names must not
have any blanks within them. Click these buttons again to re-protect these labels from
accidental change.
To create a new file use the following steps:
1. select “New” from the file menu,
2. select the proper matrix type from the list (you may receive a warning about the
possible loss of data when you change matrix types),
3. enter the correct numbers of rows and columns in the edit boxes labeled “No. rows”
and “No. cols.” (note that the new values do not take effect until your cursor leaves
the edit boxes), and then
4. start entering your data.
 NTedit 31
If there are missing
data the identifying
numerical code needs to be
entered in the edit box
labeled “Missing.” Click
on the “Comments” button
if you wish to add
comments to the matrix.
When you are done you
can use the “Check
matrix” item under the
Edit menu to check that all
data values are properly
formatted numbers. It also
will check to make sure
there are no empty cells. Figure 5.2. Example of NTedit with the test.nts file loaded in
This same check is made
when to attempt to save the matrix to a disk file. You will be given a chance to replace all
the empty cells with whatever code you specified for missing data (if that field is blank then
zeroes will be used).
NTedit can also be used to view and make changes in existing files. Changes have to be
made with care as there is no “undo” feature. A limitation of this mode is that the file must
already be in a proper format. If you try to load an NTS file that is not formatted properly
you will receive an error message and NTedit will try to load the file in ASCII text mode.
More flexibility is provided when editing a file in ASCII text mode (see Figure 5.2). Text
may be freely moved around and cut and pasted from other software. An “undo” and
“redo” feature is implemented (see the Edit menu). This mode is especially useful when a
file has format problems that prevent it from being loaded in the usual spreadsheet mode.
Its use is similar to that of the Windows notepad program. Some advantages include the
ability to load much larger files and it does not append .txt to the ends of file names when
saving.
The NTedit help can be consulted for additional information – including various
keyboard shortcuts for use in text mode.
32 Graphics

6. Graphics options & menu

The plots produced by NTSYSpc can be enhanced in many ways by taking advantage of the
many options available.
Begin by clicking on a plot with the right mouse button or by selecting the “Plot
options” item on the Options menu above the plot. The options available depend upon the
type of plot. Figure 6.1 shows an example for the MXPLOT module. All plots allow the user
to specify a title and a subtitle and the fonts used to display them (select the “Titles” tab).
There is also always a button labeled “General” (select the “Options” tab) that opens the
general plot options dialog box described in the next section.

6.1 General plot options

The following options (listed by group) are available for all plots.
General: “Preserve axis aspect” means to preserve the aspect of the x and y axes with
respect to the original units of measurements. This must be kept checked for the 3D and
Tree plots. For 2D scatter plots it should be checked when plotting the results of analyses
such as principal components analysis where the relative lengths of the axes is important.
One will usually not want it checked when plotting raw data. “Center” controls whether the
plot is centered in the window.
 Graphics 33
Frame: Optionally, a line can be drawn around the outside of the plot to frame it.
Options are available to control its size and color.
Background color: The background color in the different regions of a plot can be set
individually.
Margin size: Top, bottom, left, and right margin sizes can be set.
Legend: This group is not used at present but will be used to control how groups of
points or lines are identified.

6.2 Other
options
These depend upon
the plot. For MXPLOT
and MOD3D there are
pick lists for selecting the
variables to be plotted
(select the “Variables”
tab). There are also
choices of whether the
data points should be
identified by sequential
numbers or labeled using
the labels in the input
data. There are also Figure 6.1. Example of graphic options window from the
options to control the MXPLOT module.
various attributes of the
points and lines making
up a plot. There are special dialog boxes to allow you to select colors, plotting symbols,
fonts, etc.

6.3 Plot menu

The File menu contains the following items: “Printer setup” (which allows you to
select a printer, paper size, and orientation), “Print preview” (which changes the plot to a
preview of how it will look when printed), “Print” (to print the plot), and “Close” (to close
the plot window). The Edit menu allows one to copy the current plot to the Windows
clipboard. You can then paste it as a bitmap into a word processor or paint program. The
options menu allows you to save the current state of the plotting options to a graphic
options file. The plot options (colors, point and line options, etc.) can later be restored by
34 Graphics
loading this file. The Help menu leads to the standard Contents, Topic search, and About
items.

7. Typical applications
Furnished below are some examples of typical applications of NTSYSpc. For simplicity, the
required steps are shown as sequences of batch command statements. This is a compact
way to describe the sequence of modules and their parameters. See the help file for more
detailed technical information about each module.
Note lines that begin with a quote character are treated as comment lines and are
ignored by NTSYSpc.

7.1 Cluster analysis

Perhaps the most common use of NTSYSpc is for performing various types of agglomerative
cluster analysis of some type of similarity or dissimilarity matrix. The following is an
example of a batch file that will standardize a data matrix, compute distance coefficients
among the columns of the standardized data matrix (there are several other choices of
coefficients), cluster the distance matrix using the single-link clustering method (there are
other choices, such as UPGMA), compute a cophenetic-value (ultrametric) matrix, compute
the cophenetic correlation as a measure of goodness of fit, and then plot the results in the
form of a phenogram. The distance matrix is also displayed.
" Standardize the variables
*stand o=data.nts r=sdata.nts
" Compute a distance matrix
*simint o=sdata.nts r=dist.nts c=dist
" Do a single-link cluster analysis of the distance matrix
*sahn o=dist.nts r=tree.nts cm=single
" Display phenogram
*tree o=tree.nts
" Compute cophenetic values
*coph o=tree.nts r=coph.nts
" Compute the cophenetic correlation
*mxcomp x=coph.nts y=dist.nts
When working interactively, one can view the tree from within the SAHN module by
clicking on the plot speed button. Note that the Mantel test results displayed by the
MXCOMP module should be ignored since the two matrices being compared are not
independently derived.
 Typical applications 35

7.2 Ordination analyses and biplots

In ordination analyses the goal is to position points along coordinate axes in a low
dimensional space (rather than to form sets of points as in cluster analysis). There are many
different methods depending upon the criteria used to define what is meant by the “best”
low-dimensional representation of the relationships among the points. Several programs in
NTSYSpc can be used to perform these analyses.
When an original data matrix is available it is possible, and usually desirable, to make
plots of both the variables and the points with respect to the same axes. This is called a
“biplot”. This allows one to not only see the patterns, trends, etc. among the points and of
relationships (usually correlation) among the variables, but also the relationships between
the points and the variables — at least to the extent that they can be summarized in a few
dimensions. Unfortunately, there seems to be no strong consensus about how to scale the
two ordinations relative to one another. Gabriel (1968, 1971, 1981) defines a biplot of an n×p
matrix Y as a simultaneous bivariate plot of the n points in each column of a matrix A and of
the p variables in each column of matrix B, where Y=ABt (it would be a bimodel if a 3-
dimensional plot were made). Matrices A and B can be expressed in terms of a singular-
value decomposition of matrix Y: Y=UΛVt. One could set A=UΛ and B=V (called a JK biplot
by Gabriel and Odoroff, 1986). In terms of principal components analysis, this corresponds
to computing normalized eigenvectors from the correlation or variance-covariance matrix
for the variables and then using the PROJ program to project the points onto these vectors.
The rows of A are plotted as points and the rows of B are plotted as vectors. Note that the
matrix Λ of singular values is the square root of the eigenvalue matrix obtained in a
principal components analysis. This type of decomposition of a data matrix is called
preference scaling or repertory grid analysis in psychology and sociology.
One can equally well set A=U and B=VΛ (GH biplot of Gabriel and Odoroff, 1986).
One could also multiply both U and V by Λ0.5 (an SQ biplot). This latter choice would seem
most appropriate in correspondence analysis where both the rows and columns can be
interpreted as variables The matrix Λ could also be apportioned in any other way to U and
V as long as their product yields Y.
A problem with a JK biplot is that the vectors for the variables are of unit length and
thus are not in the same scale as the vectors for the points. Since the vectors for the variables
are dimensionless a plot of them does not indicate how well the variance of each variable is
explained by the number of dimensions used. For this reason one can deviate from a true
biplot and multiply both U and V by Λ. As a consequence, one cannot visually estimate an
element, say yij, of the original data matrix as simply the inner product of row i of A and row
j of B, one must compute the projection of row i of A onto row j of B. One compares the
relationships between the ordinations of the points and of the variables by examining the
angles between them. Jackson (1991) indicates that this alternative is popular “among
French practitioners.”
36 Typical applications
The MXPLOT module can be used to make a biplot. Enter the name of the matrix
giving the coordinates of the points as the primary input matrix and enter the name of the
matrix giving the coordinates of the variables as the secondary input matrix. The secondary
matrix is plotted at the same scale and shown as vectors from the origin. If the vectors are
too short or too long one may use the TRANSF program to scale them to a more convenient
length.

7.3 Principal components analysis

Principal components analysis, PCA, is one of the most important methods of
ordination analysis. It constructs a new set of orthogonal coordinate axes such that the
projection of points onto them have maximum variance in as few dimensions as possible.
While defined in terms of variances and covariances, PCA is often applied to standardized
data because the results are sensitive to the choices of units of measurement and the choice
of units is often arbitrary.
The following batch file will standardize a data matrix by rows, compute a matrix of
correlations among the variables (assumes rows), extract 3 eigenvectors from the correlation
matrix, project the standardized data onto these eigenvectors, and then make a 3-
dimensional plot of the objects. Various matrices are also output to files.
" Standardize variables (rows)
*stand o=data.nts r=sdata.nts
" Compute correlations among variables (rows)
*simint o=sdata.nts c=corr r=corr.nts d=row
" Output the correlation matrix
*output o=corr.nts
" Extract first 3 PCA axes from correlation matrix
*eigen o=corr.nts n=3 r=vect.nts val=val.nts
" Output principal component axes
*output o=vect.nts
" Project objects onto PCA axes
*proj o=sdata.nts d=col f=vect.nts r=proj.nts
" Output projections
*output o=proj.nts
" Display 3D plot of projection of objects
*mod3d o=proj.nts
" Display 3D plot of variables defining the PCA axes
*mod3d o=vect.nts d=col
The last two plots together comprise a three-dimensional biplot (a “bimodel”) for these
data.
An alternative procedure would be to not standardize the data and to use a variance-
covariance matrix rather than a correlation matrix in the above steps. In such a case, the
largest weights are given to those variables with the largest variances. This implies that the
variables were measured in comparable units of measurement. This might be appropriate,
for example, for a matrix of log-transformed variables in a conventional (non-geometric)
 Typical applications 37
morphometric study (perhaps with means subtracted following Darroch and Mosimann,
1985).

7.4 Principal coordinates analysis, PCOORDA

PCOORDA can be thought of as a computational alternative to PCA. The steps shown
below will give results identical to PCA. One important consideration is that when there are
many fewer points than variables computation time may be much less than for the usual
PCA.
The batch file given below performs the following operations: the data matrix is
standardized by variables (rows), a matrix of distances between the objects is computed, the
distance matrix is double-centered, the double-centered matrix is then factored and a plot is
made showing the objects in a 3-dimensional space.
" standardize data if in different units
*stand o=data.nts r=sdata.nts
" Compute distances among objects
*simint o=sdata.nts r=dist.nts
" Double-center the distance matrix
*dcenter o=dist.nts r=dcent.nts
" eigenvectors correspond to projections of objects
*eigen o=dcent.nts n=3 r=proj.nts
*output o=proj.nts
" Display -- Note that direction is "col"
*mod3d o=proj.nts d=col
PCOORDA can also be viewed as a distinct ordination method since it can also be
applied to various types of similarity and dissimilarity matrices ­ or even to experimentally
determined proximity matrices where there is no original “data matrix.” The computational
steps would then be as follows:
" Double-center the matrix
*dcenter o=dist.nts r=dcent.nts
" Extract eigenvectors
*eigen o=dcent.nts n=3 r=proj.nts
" Output eigenvectors = projections
*output o=proj.nts
" Display -- Note that direction is "col"
*mod3d o=proj.nts d=col
Of course, an arbitrary dissimilarity matrix may not be very compatible with a
Euclidean metric. In such cases many of the eigenvalues may be negative. In performing
such an analysis one hopes that such negative eigenvalues are small and can be ignored.
38 Typical applications

7.5 Nonmetric multidimensional scaling

This method is similar to PCOORDA in that it can be used to represent the relationships
among a set of points in a low dimensional space. The difference is that in non-metric
multidimensional scaling analysis the distances among the points in the final configuration
need only have a monotone relationship to the distances implied by the original data matrix.
This relaxed constraint usually makes it possible to get a much better fit in fewer dimensions
than is possible in PCOORDA.
If possible, one begins with the results of a PCOORDA as an initial configuration since
this usually results in many fewer iterations being necessary in the MDSCALE module.
" Use PCOORDA to obtain an initial configuration
*DCENTER O=dist.nts R=dcent.nts
*EIGEN O=dcent.nts N=2 R=init.nts
" non-metric MDSCALE using initial solution
*MDSCALE O=dist.nts N=2 I=init.nts R=final.nts
" rotate result for ease in viewing
*SIMINT O=final.nts C=varcov R=vcv.nts
*EIGEN O=vcv.nts N=2 R=vect.nts
*PROJ O=final.nts D=row F=vect.nts R=result.nts
" plot the final rotated configuration
*MXPLOT O=result.nts
When viewing the plot be sure to set the option “Preserve axes aspect.” The Procrustes
module can be used to compare the final configuration with another ordination such as the
initial Pcoord solution.

7.6 Burnaby's method for size adjustment

The following batch file shows an example of how the ORTH option of the PROJ program
can be used for Burnaby's method to remove the effect of a vector from a data set. The data
are projected onto the hyperplane orthogonal to the specified vector. In the example given
below the first principal component axis is used as "size”. Other vectors such as the
isometric vector (1,1,...,1) could also be used.
" compute VCV matrix from a data matrix
*simint o=data.nts c=varcov r=vcv.nts d=row
" compute first principal component
*eigen o=vcv.nts n=1 r=pc1.nts
" project data onto hyperplane normal to PC1
*proj o=data.nts f=pc1.nts pt=orth r=bproj.nts
The adjusted data matrix bproj.nts can then be used, for example, to compute a
distance matrix which is then clustered by SAHN. The clusters should then not be
influenced by variation in the original data set that was parallel to the first principal
component—which is often mostly due to size. The first principal component points in the
 Typical applications 39
direction in which there is the most variation. If the organisms sampled happen to be about
the same size, then this vector is apt to represent sexual differences, polymorphisms, etc. In
many cases it may be safer to use an a priori defined isometric vector as a size vector (i.e., the
vector 1,1,1,1…,1) or to use the first principal component based only on a carefully selected
subset of variables.
The adjusted data matrix could also be used as input for a canonical variates analysis or
for the computation of size-free generalized distances (see the CVA module).

7.7 Analysis of shape using landmark coordinates.

In recent years there has been many new developments in the field of geometric
morphometrics. The PROCRUSTES module has been included to provide these methods for
studies using 2 or 3-dimensional landmark coordinates. The PROCRUSTES module will
optimally superimpose specimens and then output the average (“consensus”) configuration
as well as a transformed set of data in which each specimen has been optimally aligned to
the consensus configuration and thus has the effects of variation in location, orientation, and
size removed. This matrix of aligned coordinates can then used as a matrix of shape
variables for principal components analysis, cluster analysis, canonical variates analysis, etc.
One has to be careful with some analyses because a covariance matrix based on these
aligned data will be singular (there will be 4 zero eigenvalues for 2D data and 7 for 3D data).
The CVA program in NTSYSpc can handle this if one changes the value for the “Cutoff for
roots” parameter from zero to a small number (such as 0.000000001).
The TPSWTS module can be used to eliminate the singularity of the aligned data. It
transforms the aligned coordinates to a matrix of partial warp scores. When the alpha
parameter is set equal to zero (the usual choice), this operation can be viewed as a rotation
followed by a projection to eliminate the redundant dimensions. A PCA of a covariance
matrix for the aligned data and a PCA of a covariance matrix based on the partial warp
scores will yield identical results (except for the presence of zero eigenvalues when
analyzing aligned data).

7.8 Comparison of dis/similarity matrices

Often one wishes to test whether one set of relationships among a set of objects is
independent of another. For example one may wish to test whether the degree of
morphological difference between samples is related to the geographical distances between
the sampled populations (see, for example, Sokal, 1979). A simple way to do this is by the
use of the Mantel test (Mantel, 1967). The test assumes that the two matrices have been
obtained independently—one cannot use it to test two matrices where one has been derived
from the other. The steps given below assume that one already has a matrix of geographical
distance, gdist.nts.
" Compute morphological dissimilarity matrix
40 Typical applications
*simint o=data.nts c=dist r=mdist.nts d=row
" Compare mdist with gdist, 250 random permutations
*mxcomp x=mdist.nts y=gdist.nts np=250
The program can also be used to perform a 3-way Mantel test using the Smouse-Long-
Sokal method. This procedure performs a Mantel test two matrices that have been adjusted
for the effects of regression on a third matrix. It allows one to test for the relationship
between two distance matrices when the effects of a third matrix have been held constant.
While less efficient than a specialized program, one can use the MXCOMP module to
perform spatial autocorrelation analyses. The geographical distance matrix is replaced with
a series of matrices corresponding to different geographical distance classes. In each matrix
an entry is 1 if objects i and j are within the desired distance class and is 0 otherwise. To
make a distance correlogram one simply plots the resulting matrix correlations as a function
of geographical distance. The Mantel test can be used to determine which coefficients are
statistically different from zero.
 Bibliography 41

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discriminant functions and generalized algorithm and its unweighted
distances. Biometrics, 22:96-110. version, UNJ. Pp. 149-170 in B.
Darroch, J. N. and J. E. Mosimann. 1985. Mirkin, F. R. McMorris, F. S.
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of shape. Biometrika, 72:241-252. Mathematical hierarchies and
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Hartigan, J. A. 1975. Clustering algorithms.
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Maddison, D.R., D.L. Swofford, and W.P.
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 Index 43

INDEX
association coefficients, 11 PCOORDA, 38
axis aspect ratio, 33 phenetics, 6
Batch mode, 19 preference scaling, 36
bimodel, 36 Principal components analysis, 37
biplot, 36 Principal coordinates analysis, 38
Burnaby's method, 39 RAPD data, 27
canonical correlation, 8 repertory grid analysis, 36
canonical vectors analysis, 9 replaceable parameters, 20
cladistics, 6 single-link, 10
Cluster analysis, 35 singular-value decomposition, 11
Common principal components analysis, 9 size
Configuration window, 22 adjustment, 39
consensus tree, 8 spatial autocorrelation analyses, 41
cophenetic value matrix, 8 thin-plate spline, 11
Correspondence analysis, 9 tree matrix, 27
elliptic Fourier analysis, 9 two-block partial least-squares, 8
Excel, 29 ultrametric, 28
File formats, 24 ultrametric values, 8
File overwrite code, 23 UPGMA, 10
Fourier analysis, 9 XLS, 29
homogeneity of covariance matrices, 10
Installation, 11
isometric vector, 40
line limit, 26
Mantel test, 10, 41
matrix comments, 32
microsatellite, 27
minimum-length spanning tree, 9
missing data code, 25, 32
multidimensional scaling, 39
multidimensional scaling analysis, 9
neighbor-joining method, 10
NEXUS format, 27
Ordination analysis, 36
Output Listing Window, 23
PCA, 37

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