LATIN SQUARE DESIGN (LS)

Facts about the LS Design

-With the Latin Square design you are able to control variation in two directions.

-Treatments are arranged in rows and columns

-Each row contains every treatment.

-Each column contains every treatment.

-The most common sizes of LS are 5x5 to 8x8

Advantages of the LS Design

1. You can control variation in two directions.

2. Hopefully you increase efficiency as compared to the RCBD.

Disadvantages of the LS Design

1. The number of treatments must equal the number of replicates.

2. The experimental error is likely to increase with the size of the square.

3. Small squares have very few degrees of freedom for experimental error.

4. You can’t evaluate interactions between:

a. Rows and columns
b. Rows and treatments
c. Columns and treatments.

Effect of the Size of the Square on Error Degrees of Freedom

SOV Df 2x2 3x3 4x4 5x5 8x8

Rows r-1 1 2 3 4 7
Columns r-1 1 2 3 4 7
Treatments r-1 1 2 3 4 7
Error (r-1)(r-2) 0 2 6 12 42
Total r2 - 1 3 8 15 24 63

Where r = number of rows, columns, and treatments.

-One way to increase the Error df for small squares is to use more than one square in the
experiment (i.e. repeated squares).

1
Example
Two 4x4 Latin squares.

SOV Df
Squares sq – 1 = 1
* Row(square) sq(r-1) = 6
* Column(square) sq(r-1) = 6
Treatment r-1 = 3
Square x Treatment (sq-1)(r-1) = 3
* Error sq(r-1)(r-2) = 12
Total sqr2 – 1 = 31
*Additive across squares.

Where sq = number of squares.

Examples of Uses of the Latin Square Design

1. Field trials in which the experimental error has two fertility gradients running
perpendicular each other or has a unidirectional fertility gradient but also has residual
effects from previous trials.

Gradient 2

Gradient 1

2. Animal science feed trials.

3. Insecticide field trial where the insect migration has a predictable direction that is
perpendicular to the dominant fertility gradient of the experimental field.

4. Greenhouse trials in which the experimental pots are arranged in a straight line
perpendicular to the glass walls, such that the difference among rows of pots and
distace from the glass wall are expected to be the major sources of variability.

A D C B B C A D D A B C C B D A

2
Randomization Procedure

-Depends on the type of Latin Square you use.

3x3 Latin Square

-Start with the standard square and randomize all columns and all but the first row.

1 2 3 3 1 2
1 A B C C A B
Randomize columns
2 B C A A B C
3 C A B B C A
Standard square

Randomize all but the first row

C A B
B C A
A B C

4x4 Latin Square

-Randomly choose a standard square.

-Randomize all columns and all but the first row.

5x5 Latin Square

-Randomly choose a standard square.

-Randomize all columns and rows.

3
Analysis of a Single Latin Square

Example
Grain yield of three maize hybrids (A, B, and D) and a check (C).

Row Column 1 Column 2 Column 3 Column 4 Row (∑ R)

1 1.640 (B) 1.210 (D) 1.425 (C) 1.345 (A) 5.620
2 1.475 (C) 1.185 (A) 1.400 (D) 1.290 (B) 5.350
3 1.670 (A) 0.710 (C) 1.665 (B) 1.180 (D) 5.225
4 1.565 (D) 1.290 (B) 1.655 (A) 0.660 (C) 5.170
Column total (∑ C ) 6.350 4.395 6.145 4.475 21.365

Step 1. Calculate treatment totals.

Treatment Total
A 5.855
B 5.885
C 4.270
D 5.355

Step 2. Compute the Correction Factor (CF).

Y..2
CF = 2
r

21.3652
=
42

= 28.53

Step 3. Calculate the Total SS

TotalSS = ∑ Yij2 − CF

= (1.64 2 + 1.210 2 + 1.425 2 + ... + 0.66 2 ) − CF

= 1.4139

4
Step 4. Calculate the Row SS

RowSS =
∑ Row − CF
r

(5.62 2 + 5.35 2 + 5.2252 + 5.17 2 )

= − CF
4

= 0.0302

Step 5. Calculate the Column SS.

Col .SS =
∑ Col − CF
r

(6.35 2 + 4.3952 + 6.1452 + 4.4752 )

= − CF
4

= 0.8273

Step 6. Calculate the Treatment SS

TrtSS =
∑Y i.
− CF
r

(5.8552 + 5.8852 + 4.270 2 + 5.3552 )

= − CF
4

= 0.4268

Step 7. Calculate the Error SS

Error SS = Total SS – Row SS – Column SS – Trt SS

= 0.1296

5
Step 8. Complete the ANOVA table

SOV Df SS MS F
Row r-1 = 3 0.030
Column r-1 = 3 0.827
Trt r-1 = 3 0.427 0.142 Trt MS/Error MS = 6.60*
Error (r-1)(r-2) = 6 0.129 0.0215
Total r2-1 = 15 1.414

Step 9. Calculate the LSD.

2 ErrorMS
LSD = tα
2 r

2(.0215)
= 2.447
4

= 0.254

Linear Model

Yij (t ) = µ + β i + κ j + τ t + ε ij (t )

where: µ = the experiment mean.

β i = the row effect,
κ j = the column effect,
τ t = the treatment effect, and
ε ij (t ) = the random error.

6
Latin Square - Combined Analysis Across Squares

-The squares can be at the same location, or three different locations, or three different
years, etc.

Example

Three 3x3 Latin squares

Square 1
∑R
41 (B) 25 (C) 15 (A) 81 SS Row1 = 126.89
20 (A) 32 (B) 24 (C) 76 SS Column1 = 89.55
22 (C) 12 (A) 21 (B) 55 SS Treatment1 = 368.22
∑C 83 69 60 212 SS Error1 = 21.56

Square 2
∑R
27 (C) 28 (B) 3 (A) 58 SS Row2 = 130.89
4 (A) 17 (C) 9 (B) 30 SS Column2 = 110.22
22 (B) 4 (A) 17 (C) 43 SS Treatment2 = 534.22
∑C 53 49 29 131 SS Error2 = 14.89

Square 3
∑R
43 (B) 27 (C) 17 (A) 87 SS Row3 = 126.89
22 (A) 34 (B) 26 (C) 82 SS Column3 = 89.55
24 (C) 14 (A) 23 (B) 61 SS Treatment3 = 368.22
∑C 89 75 66 230 SS Error3 = 21.56

Step 1. Test the homogeneity of the Error MS from each square using Bartlett’s Chi-
square test.

Step 1.1 Calculate the Error SS for each square.

Step 1.2 Calculate the Error MS for each square.

7
Step 1.3 Calculate the Log of each Error MS

Square Error SS Error df Error MS Log Error MS

1 21.56 2 10.78 1.0326
2 14.89 2 7.45 0.8722
3 21.56 2 10.78 1.0326
∑ si2 = 29.01 ∑ log si2 = 2.9374
Step 1.4 Calculate the Pooled Error MS (sp2)

s 2
=
∑s i
=
29.01
= 9.67
p
# sq 3

Step 1.5 Calculate Bartlett’s χ 2

χ = 2
[( )
2.3026( Errordf ) sq log s 2p − ∑ log si2 ]
⎡
1 + ⎢
(sq + 1) ⎤
⎥
⎣ 3 * sq * Errordf ⎦

Where Error df = df for one square.

2.3026(2)[(3 log 9.67 ) − 2.9374]

χ2 =
⎡ (3 + 1) ⎤
1 + ⎢
⎣ 3 * 3 * 2 ⎥⎦

0.0869
=
1.2222

= 0.0711

Step 1.6 Look up the Table χ 2 -value at the 99.5% level of confidence and df = #sq-1.

χ 02.005;2df = 10.6

Step 1.7 Make conclusions

Since χ 2 calc < χ table

2
we fail to reject H o : σ 12 = σ 22 = σ 32 at the 99.5% level of
confidence; thus, we can do the combined analysis across squares

8
Step 2. Calculate Treatment Totals for each square.

Treatment Square 1 Square 2 Square 3 ∑TRT

A 47 11 53 111
B 94 59 100 253
C 71 61 77 209
∑ Square 212 131 230 573

Step 3. Calculate the Correction Factor (CF).

Y...2
CF =
sq * r 2

5732
=
3 * 32

= 12,160.333

Step 4. Calculate the Total SS

( )
TotalSS = 412 + 25 2 + 15 2 + ... + 23 2 − CF
= 2,620.67

Step 5. Calculate the Square SS

SquareSS =
∑ Sq − CF
r2

=
(212 2
+ 1312 + 2302 )
− CF
32

= 618.0

Step 6. Calculate the Row(Square) SS (Additive across squares)

Row(Square) SS = Row1 SS + Row2 SS + Row3 SS

= 384.67

9
Step 7. Calculate the Column(Square) SS (Additive across squares)

Column(Square) SS = Column1 SS + Column2 SS + Column3 SS

= 289.32

Step 8. Calculate the Treatment SS

TrtSS =
∑ TRT i
− CF
sq * r

=
(111 2
+ 2532 + 2092
− CF
)
3*3

= 1,174.22

Step 9. Calculate the Square X Treatment SS.

Sq * TrtSS =
∑ (SqXTrt ) − CF − SquareSS − TrtSS
r

=
(47 2
+ 94 2 + 712 + ... + 77 2 )
− CF − SquareSS − TrtSS
3

= 96.45

Step 10. Calculate Error SS (Additive across squares)

Error SS = Error1 SS + Error2 SS + Error3 SS

Error SS = 58.01

Step 11. Complete the ANOVA Table.

SOV Df SS MS F (Squares and Trt are Fixed effects)

Square Sq-1 = 2 618.0 Non-valid F-test
Row(Sq) Sq(r-1) = 6 384.67 Non-valid F-test
Column(Sq) Sq(r-1) = 6 289.32 Non-valid F-test
Trt r-1 = 2 1174.22 587.11 Trt MS/Error MS = 60.73**
Sq X Trt (sq-1)(r-1) = 4 96.45 24.11 Sq X Trt MS/Error MS = 2.49ns
Error Sq(r-1)(r-2) = 6 58.01 9.67
Total Sqr2-1 = 26 2620.67

10
Conclusions:
1. The non-significant Square X Treatment interaction indicates that treatments
responded similarly in all squares.
Table 1. Mean for the square x treatment interaction.
Treatment
Square A B C
1 15.7 31.3 23.7
2 3.7 19.7 20.3
3 17.7 33.3 25.7
LSD(0.05) -------------------ns--------------------

2. The significant F-test for Treatment indicates that averaged across all squares, there
were differences between treatments.
3.
Table 2. Mean for the treatment main effect averaged
Across squares.
Treatment Mean
A 12.3
B 28.1
C 23.2
LSD(0.05) 3.6

Step 12. Calculate LSD’s

Square X Trt: Normally, you would not calculate this LSD because the F-test for the
interaction was non-significant. However, if it would have been significant, you would
have calculated the LSD using the following method:

2 ErrorMS
LSDSqXTrt = t a / 2;errordf
r

2(9.67 )
= 2.447
3
= 6 .2

This LSD would be used for comparisons only in Table 1.

11
Treatment:

2 ErrorMS
LSDTrt = t a / 2;errordf
sq * r

2(9.67 )
= 2.447
3*3
= 3. 6

This LSD would only be used for comparisons in Table 2.

SAS Commands for the Latin Square (individual squares and combined across
squares).

options pageno=1;
data lscmb;
input square row column trt $ yield;
datalines;
1 1 1 b 41
1 1 2 c 25
1 1 3 a 15
1 2 1 a 20
1 2 2 b 32
1 2 3 c 24
1 3 1 c 22
1 3 2 a 12
1 3 3 b 21
2 1 1 c 27
2 1 2 b 28
2 1 3 a 3
2 2 1 a 4
2 2 2 c 17
2 2 3 b 9
2 3 1 b 22
2 3 2 a 4
2 3 3 c 17
3 1 1 b 43
3 1 2 c 27
3 1 3 a 17
3 2 1 a 22
3 2 2 b 34
3 2 3 c 26
3 3 1 c 24
3 3 2 a 14
3 3 3 b 23
;;
ods graphics off;
ods rtf file='latin.rtf';
proc print;

12
title 'printout of data';
run;
proc sort;
by square;
*Comment The previous statements are needed to do the ANOVA for each
individual square;
run;
proc anova;
by square;
class row column trt;
model yield=row column trt;
title 'anova of each individual square';
run;
proc anova;
class square row column trt;
model yield=square row(square) column(square) trt square*trt;
means trt/lsd;
means square*trt;
*Comment Note that there is no LSD command since SAS will not
calculate the LSD values for interactions. SAS only calculates LSD
values for the main effects;
title 'anova combined across squares assuming square and trt are fixed
effects';
run;
proc anova;
class square row column trt;
model yield=square row(square) column(square) trt square*trt;
test h=trt e=square*trt;
*Comment The previous statement is needed since square is a random
effect and treatment is a fixed effect. Squre*trt is the denominator
of the F-test to test treatment;
means trt/lsd e=square*trt;
means square*trt;
*Comment Note that there is no LSD command since SAS will not
calculate the LSD values for interactions. SAS only calculates LSD
values for the main effects;
title 'anova combined across squares assuming square random and trt
fixed';
run;
ods rtf close;

13
 03:49 Sunday, October 20, 2013 1
Printout of Data for the Latin Square

Obs square row column trt yield

1 1 1 1 b 41
2 1 1 2 c 25

3 1 1 3 a 15

4 1 2 1 a 20

5 1 2 2 b 32
6 1 2 3 c 24

7 1 3 1 c 22

8 1 3 2 a 12

9 1 3 3 b 21

10 2 1 1 c 27

11 2 1 2 b 28

12 2 1 3 a 3

13 2 2 1 a 4

14 2 2 2 c 17

15 2 2 3 b 9

16 2 3 1 b 22

17 2 3 2 a 4

18 2 3 3 c 17

19 3 1 1 b 43

20 3 1 2 c 27

21 3 1 3 a 17

22 3 2 1 a 22

23 3 2 2 b 34

24 3 2 3 c 26

25 3 3 1 c 24
26 3 3 2 a 14

27 3 3 3 b 23
 03:49 Sunday, October 20, 2013 2
ANOVA of each individual square

The ANOVA Procedure

square=1

Class Level
Information
Class Levels Values
row 3 123
column 3 123

trt 3 abc

Number of Observations Read 9

Number of Observations Used 9
 03:49 Sunday, October 20, 2013 3
ANOVA of each individual square

The ANOVA Procedure

Dependent Variable: yield

square=1

Sum of
Source DF Squares Mean Square F Value Pr > F
Model 6 584.6666667 97.4444444 9.04 0.1029

Error 2 21.5555556 10.7777778

Corrected Total 8 606.2222222

R-Square Coeff Var Root MSE yield Mean

0.964443 13.93706 3.282953 23.55556

Source DF Anova SS Mean Square F Value Pr > F

row 2 126.8888889 63.4444444 5.89 0.1452

column 2 89.5555556 44.7777778 4.15 0.1940

trt 2 368.2222222 184.1111111 17.08 0.0553

 03:49 Sunday, October 20, 2013 4
ANOVA of each individual square

The ANOVA Procedure

square=2

Class Level
Information
Class Levels Values
row 3 123
column 3 123

trt 3 abc

Number of Observations Read 9

Number of Observations Used 9
 03:49 Sunday, October 20, 2013 5
ANOVA of each individual square

The ANOVA Procedure

Dependent Variable: yield

square=2

Sum of
Source DF Squares Mean Square F Value Pr > F
Model 6 775.3333333 129.2222222 17.36 0.0555

Error 2 14.8888889 7.4444444

Corrected Total 8 790.2222222

R-Square Coeff Var Root MSE yield Mean

0.981159 18.74508 2.728451 14.55556

Source DF Anova SS Mean Square F Value Pr > F

row 2 130.8888889 65.4444444 8.79 0.1021

column 2 110.2222222 55.1111111 7.40 0.1190

trt 2 534.2222222 267.1111111 35.88 0.0271

 03:49 Sunday, October 20, 2013 6
ANOVA of each individual square

The ANOVA Procedure

square=3

Class Level
Information
Class Levels Values
row 3 123
column 3 123

trt 3 abc

Number of Observations Read 9

Number of Observations Used 9
 03:49 Sunday, October 20, 2013 7
ANOVA of each individual square

The ANOVA Procedure

Dependent Variable: yield

square=3

Sum of
Source DF Squares Mean Square F Value Pr > F
Model 6 584.6666667 97.4444444 9.04 0.1029

Error 2 21.5555556 10.7777778

Corrected Total 8 606.2222222

R-Square Coeff Var Root MSE yield Mean

0.964443 12.84634 3.282953 25.55556

Source DF Anova SS Mean Square F Value Pr > F

row 2 126.8888889 63.4444444 5.89 0.1452

column 2 89.5555556 44.7777778 4.15 0.1940

trt 2 368.2222222 184.1111111 17.08 0.0553

 03:49 Sunday, October 20, 2013 8
ANOVA combined across squares assuming square and trt are fixed effects

The ANOVA Procedure

Class Level
Information
Class Levels Values
square 3 123

row 3 123

column 3 123
trt 3 abc

Number of Observations Read 27

Number of Observations Used 27

 03:49 Sunday, October 20, 2013 9
ANOVA combined across squares assuming square and trt are fixed effects

The ANOVA Procedure

Dependent Variable: yield

Sum of
Source DF Squares Mean Square F Value Pr > F
Model 20 2562.666667 128.133333 13.26 0.0021
Error 6 58.000000 9.666667

Corrected Total 26 2620.666667

R-Square Coeff Var Root MSE yield Mean

0.977868 14.65033 3.109126 21.22222

Source DF Anova SS Mean Square F Value Pr > F

square 2 618.000000 309.000000 31.97 0.0006

row(square) 6 384.666667 64.111111 6.63 0.0183

column(square) 6 289.333333 48.222222 4.99 0.0357

trt 2 1174.222222 587.111111 60.74 0.0001

square*trt 4 96.444444 24.111111 2.49 0.1522
 03:49 Sunday, October 20, 2013 10
ANOVA combined across squares assuming square and trt are fixed effects

The ANOVA Procedure

t Tests (LSD) for yield

Note: This test controls the Type I comparisonwise error rate, not the experimentwise error rate.

Alpha 0.05

Error Degrees of Freedom 6

Error Mean Square 9.666667

Critical Value of t 2.44691

Least Significant Difference 3.5863

Means with the same letter

are not significantly
different.
t Grouping Mean N trt
A 28.111 9 b

B 23.222 9 c

C 12.333 9 a
 03:49 Sunday, October 20, 2013 11
ANOVA combined across squares assuming square and trt are fixed effects

The ANOVA Procedure

yield
Level of Level of
square trt N Mean Std Dev
1 a 3 15.6666667 4.0414519

1 b 3 31.3333333 10.0166528

1 c 3 23.6666667 1.5275252
2 a 3 3.6666667 0.5773503

2 b 3 19.6666667 9.7125349

2 c 3 20.3333333 5.7735027

3 a 3 17.6666667 4.0414519

3 b 3 33.3333333 10.0166528

3 c 3 25.6666667 1.5275252
 03:49 Sunday, October 20, 2013 12
anova combined across squares assuming square random and trt fixed

The ANOVA Procedure

Class Level
Information
Class Levels Values
square 3 123

row 3 123

column 3 123
trt 3 abc

Number of Observations Read 27

Number of Observations Used 27

 03:49 Sunday, October 20, 2013 13
anova combined across squares assuming square random and trt fixed

The ANOVA Procedure

Dependent Variable: yield

Sum of
Source DF Squares Mean Square F Value Pr > F
Model 20 2562.666667 128.133333 13.26 0.0021
Error 6 58.000000 9.666667

Corrected Total 26 2620.666667

R-Square Coeff Var Root MSE yield Mean

0.977868 14.65033 3.109126 21.22222

Source DF Anova SS Mean Square F Value Pr > F

square 2 618.000000 309.000000 31.97 0.0006

row(square) 6 384.666667 64.111111 6.63 0.0183

column(square) 6 289.333333 48.222222 4.99 0.0357

trt 2 1174.222222 587.111111 60.74 0.0001

square*trt 4 96.444444 24.111111 2.49 0.1522

Tests of Hypotheses Using the Anova MS for square*trt

as an Error Term
Source DF Anova SS Mean Square F Value Pr > F
trt 2 1174.222222 587.111111 24.35 0.0058
ANOVA combined across squares assuming square random and trt fixed

The ANOVA Procedure

t Tests (LSD) for yield

Note: This test controls the Type I comparisonwise error rate, not the experimentwise error rate.

Alpha 0.05

Error Degrees of Freedom 4

Error Mean Square 24.11111

Critical Value of t 2.77645

Least Significant Difference 6.4268

Means with the same letter

are not significantly
different.
t Grouping Mean N trt
A 28.111 9 b
A
A 23.222 9 c

B 12.333 9 a
ANOVA combined across squares assuming square random and trt fixed

The ANOVA Procedure

t Tests (LSD) for yield

yield
Level of Level of
square trt N Mean Std Dev
1 a 3 15.6666667 4.0414519

1 b 3 31.3333333 10.0166528

1 c 3 23.6666667 1.5275252

2 a 3 3.6666667 0.5773503

2 b 3 19.6666667 9.7125349

2 c 3 20.3333333 5.7735027

3 a 3 17.6666667 4.0414519

3 b 3 33.3333333 10.0166528

3 c 3 25.6666667 1.5275252