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**RAJENDER PARSAD AND L.M. BHAR
**

Indian Agricultural Statistics Research Institute

Library Avenue, New Delhi - 110 012

lmb@iasri.res.in

1. Introduction

In many agricultural experiments, generally the data on more than one character is

observed. One common example is grain yield and straw yield. The other characters on

which the data is generally observed are the plant height, number of green leaves,

germination count, etc. The analysis is normally done only on the grain yield and the best

treatment is identified on the basis of this character alone. The straw yield is generally not

taken into account. If we see the system as a whole, the straw yield is also important either

for the cattle feed or for mulching or manuring, etc. Therefore, while analyzing the data,

the straw yield should also be taken into consideration. Similarly, in varietal trials also the

data is collected on several plant characteristics and quality parameters. In these

experimental situations also the data is generally analyzed separately for each of the

characters. The best treatment or genotype is identified separately for each of the

characters. In these situations, Multivariate Analysis of Variance (MANOVA) can be

helpful. Before discussing about MANOVA, a brief description about Analysis of

Variance (ANOVA) is given in Section 2. A general procedure of performing MANOVA

on the data generated from RCB design is given in Section 3. The procedure of

MANOVA has been illustrated with the help of an example in Section 4.

2. Overview of ANOVA

The ANOVA looks at the variance within classes relative to the overall variance. The

dependent variable must be metric, and the independent variables, which can be many,

must be nominal. ANOVA is used to uncover the main and interaction effects of

categorical independent variables (called "factors") on an interval dependent variable. A

main effect is the direct effect of an independent variable on the dependent variable. An

interaction effect is the joint effect of two or more independent variables on the dependent

variable. Whereas regression models cannot handle interaction unless explicit

crossproduct interaction terms are added, ANOVA uncovers interaction effects on a built-

in basis.

The key statistic in ANOVA is the F-test of difference of group means, testing if the

means of the groups formed by values of the independent variable (or combinations of

values for multiple independent variables) are different enough not to have occurred by

chance. If the group means do not differ significantly then it is inferred that the

independent variable(s) did not have an effect on the dependent variable. If the F test

shows that overall the independent variable(s) is (are) related to the dependent variable,

then multiple comparison tests of significance are used to explore just which values of the

independent(s) have the most to do with the relationship.

3. Multivariate Analysis of Variance

Why do MANOVA, when one can get also much more information by doing a series of

ANOVAs? Even if all our dependent variables are completely independent of one another,

MANOVA

2

when we do lots of tests like that, error inflates. But in many ecological or biological

studies, the variables are not independent at all. Many times they have strong actual or

potential interactions, inflating the error even more highly. In many cases where multiple

ANOVAs were done, MANOVA was actually the more appropriate test.

Consider an experiment conducted to compare v treatments using a randomized complete

block (RCB) design with r replications and the data is collected on p-variables. Let

ijk

y denote the observed value of the k

th

response variable for the i

th

treatment in the j

th

replication p ,..., 2 , 1 k ; r ..., , 2 , 1 j ; v ,..., 2 , 1 i = = = . The data is rearranged as follows:

← ←← ← Replications → →→ →

Treatments

↓ ↓↓ ↓

1 2

…

j

…

r Treatment

Mean ↓ ↓↓ ↓

1 y

11

y

12

…

y

1j

…

y

1r

. 1

y

2 y

21

y

22

…

y

2j

…

y

2r

. 2

y

… …

i y

i1

y

i2

…

y

ij

…

y

ir

. i

y

… …

v y

v1

y

v1

…

y

v1

…

y

v1

. v

y

Replication

Mean→ →→ →

1 .

y

2 .

y …

j .

y …

r .

y

..

y

Here

ij

y = ( ) y ... ...y y y

ijp ijk ij2 1 ij

is a p-variate vector of observations taken from the plot

receiving the treatment i in replication j.

∑

=

=

r

1 j

ij . i

r

1

y y ;

∑

=

=

v

1 i

ij j .

v

1

y y and

∑∑

= =

=

v

1 i

r

1 j

ij ..

vr

1

y y .

The observations can be represented by a two way classified multivariate model Ω ΩΩ Ω

ij j i ij

: e b t µ y Ω + + + = i = 1, 2,…,v; j = 1, 2,…,b, …(3.1)

µ µµ µ = (µ

1

µ

2

… µ

k

… µ

p

)’ is the 1 p × vector of general means, t

i

= (t

i1

t

i2

… t

ik

… t

ip

)’ are the

effects of treatment i on p-characters, and b

j

=(b

j1

b

j2

… b

jk

… b

jp

)’ are the effects of

replication j on p-characters. e

ij

= (e

ij1

e

ij2

… e

ijk

… e

ijp

)’ is a p-variate random vector

associated with y

ij

and assumed to be distributed independently as p variate normal

distribution ) , ( Σ 0 N

p

. The equality of treatment effects is to be tested i.e. H

0

: (t

i1

t

i2

…t

ik

…t

ip

)’ = (t

1

t

2

…t

k

…t

p

)’ (say) p , , 2 , 1 i = ∀ against the alternative : H

1

at least two

of the treatment effects are unequal. Under the null hypothesis, the model (3.1) reduces to

ij j ij 0

: e b α y Ω + + = …(3.2)

where ) t µ ..., , t µ t µ (

p p 2 2 1 1

′ + + + = α .

An outline of MANOVA Table for testing the equality of treatment effects and replication

effects is

MANOVA

3

MANOVA

Source DF SSCPM (Sum of Squares and Cross Product Matrix)

Treatment v-1 = h

H = ( )( )

∑

=

′

− −

v

1 i .. . i .. . i

b y y y y

Replication r-1 = t

B= ( )( )

∑

=

′

− −

b

1 j .. j . .. j .

v y y y y

Residual (v-1)(r-1)

= s

R= ( )( )

∑ ∑

= =

′

+ − − + − −

v

1 i

b

1 j .. j . . i ij .. j . . i ij

y y y y y y y y

Total vr-1

T= ( )( )

∑ ∑

= =

′

− −

v

1 i

b

1 j .. ij .. ij

y y y y =H+B+R

Here H, B, R and T are the sum of squares and sum of cross product matrices of

treatments, replications, errors (residuals) and totals respectively. The residual sum of

squares and cross products matrix for the reduced model

0

Ω is denoted by

0

R and is

given by H R R + =

0

.

The null hypothesis of equality of treatment mean vectors is rejected if the ratio of

generalized variance (Wilk's lambda statistic)

R H

R

+

= Λ is too small. Assuming the

normal distribution, Rao (1973) showed that under null hypothesis Λ is distributed as the

product of independent beta variables. A better but more complicated approximation of

the distribution of Λis

ph

) c ab ( 1

b / 1

b / 1

−

Λ

Λ −

~ F (ph, ab-c)

where

+ −

− =

2

1 h p

s a , ( ) ( ) { } 5 h p / 4 h p b

2 2 2 2

− + − = ,

2

2 ph

c

−

=

For some particular values of h and p, it reduces to exact F-distribution. The special cases

are given below:

For h = 1 and any p, this reduces to

p Λ

) 1 p s ( Λ) 1 ( + − −

~ F (p, s – p + 1)

For h=2 and any p, it reduces to

p Λ

) 1 p s ( ) Λ 1 ( + − −

~ F (2p, 2(s – p + 1))

For p=2 and any h:

h Λ

) 1 s ( ) Λ 1 ( − −

~ F (2h, 2(s – 1)).

For p = 1, the statistic reduces to the usual variance ratio statistics.

The hypothesis regarding the equality of replication effects can be tested by replacing Λ

by

R B

R

+

and h by t in the above.

MANOVA

4

Several other criteria viz. Pillai's Trace, Hotelling-Lawley Trace or Roy's Greatest Root

are available in literature for testing the null hypothesis in MANOVA. Wilks' Lamda is,

however, the commonly used criterion. Here, we shall restrict to the use of Wilks' Lamda

criterion. For further details on MANOVA, a reference may be made to Seber (1983) and

Johnson and Wichern (1988).

Remark 3.1: One complication of multivariate analysis that does not arise in the

univariate case is the ranks of the matrices. The rank of R should not be smaller than p or

in other words error degrees of freedom s should be greater than or equal to p (s ≥ p).

3.1 Multivariate Treatment Contrast Analysis

If the treatments are found to be significantly different through MANOVA, then the next

question is “which treatments are significantly different?” This question can be answered

through multivariate treatment contrast analysis. In the literature, the multivariate

treatment contrast analysis is generally carried out using the

2

χ -statistic. The

2

χ -statistic

is based on the assumption that the error variance-covariance matrix is known. The error

variance-covariance matrix is, however, generally unknown. Therefore, the estimated

value of error variance-covariance matrix is used. The error variance-covariance matrix is

estimated by sum of squares and cross products (SSCP) matrix for error divided by the

error degrees of freedom. As a consequence, test based on

2

χ -statistic is an approximate

solution. The procedure using the Wilk’s Lambda criterion is also described in the sequel.

Suppose the hypothesis to be tested is H

0

: '

i

i

t t = against H

1

: '

i

i

t t ≠ . This hypothesis can

be rewritten as

H

0

: = ) ( '

i

i

t t − = 0 against H

1

: = ) ( '

i

i

t t − ≠ ≠≠ ≠ 0, …(3.3)

where ) ( '

i

i

′ −t t = ( )

p i ip k i ik 2 i 2 i 1 i 1 i

t t ... t t ... t t t t

′ ′ ′ ′

− − − − . Here

ik

t denote the

effect of treatment i for the dependent variable k. The best linear unbiased estimate of

) ( '

i

i

t t − is

( ) ( )

p i ip k i ik 2 i 2 i 1 i 1 i

. i

. i

y y ... y y ... y y y y '

′ ′ ′ ′

− − − − =

′

− y y

where

ik

y is the mean of treatment i for variable k.

3.1.1 −

2

χ Test

The statistic based on

2

χ , requires covariance matrix of the contrast of interest. The

covariance matrix, in case of a RCB design for elementary treatment contrast is obtained

by dividing the SSCP matrix for errors obtained in MANOVA by half of the product of

error degrees of freedom and the number of replications. Let this variance-covariance

matrix is denoted by

c

Σ . Under null hypothesis, x = '

. i

. i

y y − follows p- variate normal

distribution with mean vector 0 and variance-covariance matrix

c

Σ . Applying the Aitken's

transformation, it can be shown that x Σ z

2 / 1

c

−

= follows a p-variate normal distribution

with mean vector 0 and variance-covariance matrix I

g

, where I

g

, denotes the identity

matrix of order g. Then using the results of quadratic forms, it can easily be seen that z z′

x Σ x

1 −

′ = follows a

2

χ distribution with p-degrees of freedom.

MANOVA

5

3.1.2 Wilk’s Lambda Criterion

For testing the null hypothesis (3.3), we obtain a sum of squares and products matrix for

the above elementary treatment contrast. Let the SSCP matrix for above elementary

treatment contrast be

p p×

G . The diagonal elements of G are then obtained by

( ) v ,..., 2 , 1 ' i i ; p ,..., 2 , 1 k y y

2

r

g

2

k ' i ik kk

= ≠ = ∀ −

= …(3.4)

and the off diagonal elements are obtained by

( )( )

' k ' i ' ik k ' i ik ' kk

y y y y

2

r

g − − = …(3.5)

The null hypothesis is rejected if the value of Wilk's Lambda

| |

| |

*

R G

R

Λ

+

= is small,

where R is the SSCP matrix due to residuals as obtained through MANOVA. The

hypothesis is then tested using the following F-test statistics based on Wilk's Lambda for h

= 1

p

1 p edf

*

* 1 + − −

Λ

Λ

∼ F(p, s-p+1).

4. Illustration using Multivariate Techniques

In this section, the results obtained from bivariate analysis of variance of the data

generated from the experiments conducted under PDCSR are given, where the data on

grain yield and straw yield were observed.

Illustration 4.1: An experiment entitled Studies on the experimentation on

conservation of organic carbon in the soil to improve soil condition was conducted at

Bhubaneshwar on rice crop. The experiment was initiated in the year 1997. The data on

grain and straw used for the illustration pertains to the Kharif season of 2001. Ten

treatments were tried in the experiment. The details of the treatments are given below:

T1 - Recommended N 100%

T2 - Recommended N 100% out of which 10 Kg at first ploughing

T3 - Recommended N 100% out of which 20 Kg at first ploughing

T4 - Recommended N 100% and add 10 Kg N/ha at first ploughing

T5 - Recommended N 100% and add 20 Kg N/ha at first ploughing

T6 - Recommended N + 10 Kg N/ha

T7 - Recommended N + 20 Kg N/ha

T8 - Recommended N + cellulose decomposing enzyme (FYM)

T9 - Recommended N + FYM 5 t/ha during Kharif

T10 - Recommended N + FYM 5 t/ha during Rabi

The results of multivariate analysis of variance are given in the sequel. First the results for

each of the two characters are presented separately.

ANOVA: Grain Yield (GYLD)

Source DF SS MS F Value Pr > F

Model 12 104.6889 8.7241 5.94 <.0001

Error 27 39.6605 1.4689

Total 39 144.3494

MANOVA

6

R-Square CV Root MSE GYLD Mean

0.7252 12.9895 1.212 9.3305

Source DF SS MS F -ratio Pr > F

REP 3 2.7312 0.9104 0.62 0.6083

TRT 9 101.9577 11.3286 7.71 <.0001

ANOVA: Straw Yield (SYLD)

Source DF SS MS F -ratio Pr > F

Model 12 161.1923 13.4326900 6.88 <.0001

Error 27 52.7144 1.9523851

Total 39 213.9067

R-Square CV RMSE SYLD Mean

0.7536 12.6574 1.39737 11.0393

Source DF SS MS F -ratio Pr > F

REP 3 4.4419 1.4806 0.76 0.5273

TRT 9 156.7503 17.4167 8.92 <.0001

It can be seen that for both the characters, the replication effects are not significantly

different whereas the treatments are significantly different. Therefore, for making all

possible paired comparisons, the least significant difference procedure of multiple

comparisons was used. The results are given in the sequel:

t Tests (LSD) for GYLD

Alpha 0.05

Error Degrees of Freedom 27

Error Mean Square 1.4689

Critical Value of t 2.0518

Least Significant Difference at 5% 1.7584

t Grouping Mean N TRT

A 10.8000 4 8

A 10.6000 4 7

A 10.5575 4 6

A 10.5425 4 9

A 10.5125 4 10

A 9.8325 4 5

A 9.1150 4 4

B C 8.0525 4 3

D C 7.3550 4 2

D 5.9375 4 1

*The treatments with the same alphabet are not significantly different.

MANOVA

7

t Tests (LSD) for SYLD

Alpha 0.05

Error Degrees of Freedom 27

Error Mean Square 1.952385

Critical Value of t 2.05183

Least Significant Difference 2.0273

t Grouping Mean N TRT

A 12.9375 4 7

A 12.7750 4 6

B A 12.4375 4 9

B A 12.3550 4 10

B A 12.2625 4 8

B A C 11.7975 4 5

B C 10.7425 4 4

D C 9.8875 4 3

E D 8.5275 4 2

E 6.6700 4 1

*The treatments with the same alphabet are not significantly different.

It can be concluded that the treatment T8 is at Rank 1 for GYLD and T7 gets rank 1 for

SYLD, although the two treatments are not significantly different among themselves. The

treatments T4 and T2 are not significantly different for GYLD and significantly different

for SYLD. Therefore, to rank the treatments collectively for both the characters, the

multivariate analysis of variance was carried out. The results obtained are given below:

Multivariate Analysis of Variance

E = Error SSCP Matrix

gyld syld

gyld 39.66052 44.60303

syld 44.60303 52.7143975

Partial Correlation Coefficients from the Error SSCP Matrix / Prob > |r|

DF = 27

gyld syld

gyld 1.000000 0.975485

<.0001

syld 0.975485 1.00000

<.0001

MANOVA Test Criteria and F Approximations for the Hypothesis of No Overall TRT

Effects.

Statistic Value F-ratio Num DF Den DF Pr > F

Wilks' Lambda 0.11997 5.45 18 52 <.0001

Pillai's Trace 1.2550 5.05 18 54 <.0001

Hotelling-Lawley Trace 4.2100 5.91 18 39.714 <.0001

Roy's Greatest Root 3.2475 9.74 9 27 <.0001

MANOVA

8

From the above Table, it can be concluded that the treatment effects are significantly

different. Now the next question is that what is ranking of treatments? Which treatments

are significantly different? This can be achieved through multivariate contrast analysis.

However, most of the software, carry out the univariate contrast analysis on the combined

average values of all the dependent variables. To account for the correlation structure

between the two variables, the principal component analysis was carried out. The results

are:

Eigenvalues of the Correlation Matrix

Eigenvalue Difference Proportion Cumulative

1 1.98428028 1.96856056 0.9921 0.9921

2 0.01571972 0.0079 1.0000

Eigenvectors

Prin1 Prin2

gyld 0.707107 0.707107

syld 0.707107 -0.707107

It can be seen that the first principal component explains 99.21% of the variance.

Therefore, the principal component scores of the observations for the first principal

components are obtained and the univariate analysis of variance was carried out. The

results obtained are:

ANOVA: Principal Component Scores

Source DF SS MS F -ratio Pr > F

Model 12 261.2948 21.7746 6.48 <.0001

Error 27 90.7905 3.3626

Corrected Total 39 352.0854

R-Square CV Root MSE Prin1 Mean

0.7421 12.7311 1.8337 14.4036

Source DF SS MS F -ratio Pr > F

REP 3 6.7790 2.2597 0.67 0.5767

TRT 9 254.5158 28.2795 8.41 <.0001

It can be seen that the treatments are highly significantly different. Therefore, multiple

comparisons using the least significant difference procedure was used.

t Tests (LSD) for prin1

Alpha 0.05

Error Degrees of Freedom 27

Error Mean Square 3.3626

Critical Value of t 2.0518

Least Significant Difference 2.6605

MANOVA

9

t Grouping Mean N TRT

A 16.644 4 7

A 16.499 4 6

A 16.308 4 8

A 16.249 4 9

A 16.170 4 10

B A 15.295 4 5

B A 14.041 4 4

B C 12.685 4 3

D C 11.231 4 2

D 8.915 4 1

The treatment T7 gets the first rank and is non-significantly different from T8. The

treatments T4 and T2 are significantly different among themselves. This procedure

answers the question to some extent. But a multivariate contrasts analysis is the best

answer for this situation. The results of multivariate treatment contrast analysis for making

all possible paired comparisons of the treatments are given in the sequel.

Probabilities of Significance of All Possible Paired Treatment Comparisons using

Wilks' Lamda Criterion

Treats

1 2 3 4 5 6 7 8 9 10

1 .

2

0.1525 .

3

0.0006 0.0388 .

4

0.0010 0.0938 0.1673 .

5

0.0001 0.0055 0.1352 0.3945 .

6

0.0001 0.0004 0.0270 0.0631 0.5497 .

7

0.0001 0.0001 0.0194 0.0253 0.3271 0.8742 .

8

0.0001 0.0020 0.0006 0.0531 0.0200 0.0058 0.0017 .

9

<0.0001 0.0023 0.0181 0.2604 0.5636 0.3653 0.1657 0.1264 .

10

<0.0001 0.0030 0.0159 0.2904 0.4866 0.2667 0.1113 0.1828 0.9755

.

*bold face type shows the treatment pairs that are not significantly different.

From the above results, it is seen that treatments T7 and T8 are significantly different

where as they were found to be not significantly different when analyzed for individual

characters or 1

st

principal component score was used.

Note: The MANOVA described in Sections 2 and 3 can usefully be employed for the

experimental situations where the experiment is continued for several years/ seasons with

same treatments and same randomized layout. For a detailed discussion on this one may

refer to Parsad et al. (2004).

MANOVA

10

SPSS Commands for MANOVA

1. Enter the data

2. ClickAnalyzeGeneral Linear ModelMultivariate

3. Put dependent variables (grain, straw) in “Dependent Variables” box and independent

variables (treat., rep) in “Fixed Factors” box

4. Then Click model Custom and bring the independent variables in model box . Then

click continue

MANOVA

11

5. If you want contrast analysis, then click ‘contrast’ and mark the variables for which

you want contrast analysis otherwise click continue

6. If you want post-hoc analysis, then click ‘post hoc…’ and bring the required variables

in ‘Post hoc test for’ box and then click continue

7. For other statistics, then click ‘Options’ and for diagnostics results, then click ‘Save’

MANOVA

12

8. Finally click ‘OK’ button and get the results

References and Suggested Reading

Johnson, R.A. and Wichern, D.W. (1988). Applied Multivariate Statistical Analysis, 2

nd

Edition. Prentice-Hall International, Inc., London.

Parsad, R., Gupta, V.K., Batra, P.K., Srivastava, R., Kaur, R., Kaur, A. and Arya, P.

(2004). A diagnostic study of design and analysis of field exeriments. Project

Report, IASRI, New Delhi.

Rao, C.R.(1973). Linear Statistical Inference and Application. Wiley Eastern Ltd., New

Delhi.

Seber, G.A.F.(1983). Multivariate Observations. Wiley series in Probability and Statistics.

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