Dear Brenna,

There is a lot to digest here, so apologies up front if I miss something.  
Also, I’ll keep my response general for the benefit of our colleagues.  We can 
work out specific issues with your data and your analyses via direct email 
communication.

First, you have two sets of shape variables that you present so it is difficult 
to compare your different results and understand exactly the problem, but I see 
a couple of things.  I’m surprised you did not get an error message, as we set 
traps to avoid the type of nested analysis you were trying to perform.  
Currently, nested.update is not that sophisticated to handle a structure like ~ 
a/b/c.  You eluded the trap, somehow (I’ll have to look into this), but if c is 
nested within b and b is nested with a, and one wants to adjust F values such 
that F for b = MSb/MSc and F for a = MAa/MSb, the procedure should be to use a 
recursive strategy with nested.update, to update the fit first by ~b/c, then 
use that update and update it again with ~a/b.  I’m not sure what to tell you 
about the results because I am surprised you got results.

Second, the reason for the same results in your second and third example is 
that bout/rep is the same the error.  In this case, nested.update will not 
accomplish anything, other than identify that there are no residuals after you 
account for your nested effect.

Third, your centroid size (near) invariance appears to have resulted from 
performing GPA on a subset of data that already had GPA performed.  You found a 
significant shape-size association despite what appeared to be little 
variation, but this might just be a scaling issue.  For example, multiply the 
centroid size values by 10000 and re-run the analysis.  The SS and MS will 
change, and the new variable will seem less invariant, but I suspect the 
F-value and P-value will not change.

Good luck and feel free to send me your data-specific questions!

Mike

Michael Collyer

Associate Professor
Biostatistics
Department of Biology
Western Kentucky University
1906 College Heights Blvd. #11080
Bowling Green, KY 42101-1080
Phone: 270-745-8765; Fax: 270-745-6856
Email: [email protected]<mailto:[email protected]>

On Mar 4, 2016, at 2:45 PM, Brenna Hays 
<[email protected]<mailto:[email protected]>> wrote:

Hi All,

I am trying to perform a nested ANOVA in R to test for repeatability and 
subjectivity of landmark placements on three randomly chosen images. I have 
landmarked each image 10 times, in 2 separate bouts (a few months apart).

This is my result from incorporating the entire data set into a nested ANOVA:
#nested ANOVA
error <- procD.lm(f1=ALLlp.shape~ind/bout/rep, iter=999, RRPP=TRUE, data=NULL)
error <- nested.update(error, ~ind/bout/rep)
summary(error)
Call:
procD.lm(f1 = ALLlp.shape ~ ind/bout/rep, iter = 999, RRPP = TRUE,      data = 
NULL)

Type I (Sequential) Sums of Squares and Cross-products
Randomized Residual Permutation Procedure Used
1000 Permutations

                    Df      SS          MS         Rsq           F              
 Z          Pr(>F)
ind                2    0.71265   0.35632  0.98767  1410.6169   19.2446  0.001 
**
ind:bout        1    0.00276   0.00276  0.00383   10.9405      13.4952  0.001 **
ind:bout:rep 36   0.00108   0.00003  0.00150    0.1191        0.2891  1.000
Residuals    20   0.00505   0.00025
Total         59 0.72155
---
Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

I was concerned, however, that these results are comparing between individuals 
(images) which I do not want, since all three images are of different 
individuals. Really, I just want to test variance between bouts and reps, so I 
performed separate nested ANOVAs for each individual. Here is the result for 
one of the individuals (note: it will not let me put the ind factor in since 
for each separate test there is only one category (1 image)):
> error = procD.lm(f1=CochaClp.shape~bout/rep, iter=999, RRPP=TRUE, data=NULL)
> error <- nested.update(error, ~bout/rep)
> summary(error)
Call:
procD.lm(f1 = CochaClp.shape ~ bout/rep, iter = 999, RRPP = TRUE,      data = 
NULL)

Type I (Sequential) Sums of Squares and Cross-products
Randomized Residual Permutation Procedure Used
1000 Permutations

*** F values, Z scores, and P values updated for nested effects

                Df         SS             MS               Rsq          F       
     Z       Pr(>F)
bout          1   0.00238108  0.00238108   0.75651  55.925   9.0398  0.001 **
bout:rep   18  0.00076637  0.00004258   0.24349                1.0534  0.001 **
Residuals  0  0.00000000
Total        19  0.00314745

I also performed an ANOVA on just the bout factor to see. I don't know if the 
residual sum of squares and mean squares should match the bout:rep numbers 
above, but it doesn't seem right to me.
> errorb = procD.lm(CochaClp.shape~bout, iter=999, RRPP=TRUE)
> summary(errorb)
Call:
procD.lm(f1 = CochaClp.shape ~ bout, iter = 999, RRPP = TRUE)

Type I (Sequential) Sums of Squares and Cross-products
Randomized Residual Permutation Procedure Used
1000 Permutations

                 Df         SS            MS                  Rsq      F        
   Z       Pr(>F)
bout           1    0.00238108   0.00238108 0.75651 55.925 9.5151  0.001 **
Residuals 18   0.00076637   0.00004258
Total          19  0.00314745


Additionally, on a simpler note, I performed an ANOVA testing the significance 
of centroid size on shape. The results here do not make sense to me either - it 
states a significant variance but the variance of the centroid sizes is so 
small.
> csize <- centroid.size(CochaClp.shape)
> csize
 [1] 1.000062 1.000067 1.000064 1.000068 1.000059 1.000075 1.000071 1.000074 
1.000079 1.000066
[11] 1.000070 1.000122 1.000065 1.000093 1.000071 1.000098 1.000117 1.000097 
1.000072 1.000083
> var(csize)
[1] 3.170611e-10

Call:
procD.lm(f1 = CochaClp.shape ~ log(csize), iter = 999, seed = NULL,
    RRPP = TRUE, int.first = FALSE, data = NULL)



Type I (Sequential) Sums of Squares and Cross-products
Randomized Residual Permutation Procedure Used
1000 Permutations

           Df         SS         MS     Rsq      F      Z Pr(>F)
log(csize)  1 0.00098216 0.00098216 0.31205 8.1646 4.1957  0.002 **
Residuals  18 0.00216529 0.00012029
Total      19 0.00314745
---
Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1


I believe, as a whole, I am either doing something wrong in my coding with 
procD.lm, or interpreting it incorrectly. Any input or suggestions would be 
extremely helpful. Thank you!

Brenna

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