Hi Anderson,

I don’t think PCA is the tool you want to use in this context. Procrustes anova 
already tells you that your two magnification levels are done differently. 
Since your question is ‘can I combine data from different magnification level 
in the same analysis?’, the answer to that in your current study design (single 
sample measured five times at two different scales) is a very qualified no.

But, what is more relevant, (or what I would have liked to know) the magnitude 
of this error in context of actual biological variation.
For that, you have to have a study design where you have a few samples of 
different sizes measured at different magnification scales a couple times (not 
just measuring the same object repeatedly in different magnifications), or 
something along those lines.

There is a really a lot of literature on this.  But you need to use a 
statistics based framework, not EDA tools like PCA to answer this.

M


From: Anderson Feijo [mailto:andefe...@gmail.com]
Sent: Monday, January 1, 2018 11:02 PM
To: Murat Maga <m...@uw.edu>
Cc: MORPHMET <morphmet@morphometrics.org>
Subject: Re: [MORPHMET] Doubt Scaling photos

Dear Murat,

Thank you very much for your email and time for explore my dataset. To perform 
this simple experiment, given the aim was to explore the magnification issue, I 
chose only three landmarks (as you could see in the tps file) easily to 
replicate. The reason I am trying to combine two magnification is that I will 
work with groups with different sizes; for example, species with 10 cm and 
others with 3 cm of total length of skull. Place the camera at a good distance 
to avoid parallax for the large group will lead a low resolution images of the 
small groups. Thus, my initial idea was to have two standardized distances and 
combine later after accounting the scale effects.

In my short experiment, what intrigues me is the clear groups in PCA (and other 
exploratory analyses) based on the two magnifications (see below). At the 
beginning, I was expecting a great overlap between the two distances and 
minimum differences due to landmarks-placement error as seen within each of the 
group.

Best,
Anderson

[Inline image 1]


On Sat, Dec 30, 2017 at 3:57 PM, Murat Maga <m...@uw.edu<mailto:m...@uw.edu>> 
wrote:
Dear Anderson,

It has nothing to do with the scale of your data (or least not directly).

As you can see, you actually performed those two digitization attempts 
differently. It is maybe 1-2 pixels off, but in a very consistent way (greens 
are from one scale, red is the other scale, and cross is the consensus shape). 
This type of systematic error commonly occur in digitization process, as one 
learns better or alters the way landmarks are digitized/defined along the 
process. In your case, perhaps higher resolution image gave a better definition 
(imaging sense) of landmarks, so you captured them very slightly differently 
than low-res image (but in a very consistent way).

If you are concerned, the first step perhaps is to do the same thing (i.e. 
digitization of same sample with different magnifications several times) with 
several real specimens and get a sense of the magnitude of this error in 
context of the biological variation. Then perhaps you can decide, whether you 
can actually combine digitization’s from different magnification levels. There 
is quite a bit of literature on this,  you can start with a recent review.

https://www.ncbi.nlm.nih.gov/pubmed/27038025

Is there a reason you are not using a single magnification level for all your 
samples?
M

[cid:image004.jpg@01D383B3.2FB87A00]

From: Anderson Feijo [mailto:andefe...@gmail.com<mailto:andefe...@gmail.com>]
Sent: Friday, December 29, 2017 1:35 AM
To: MORPHMET <morphmet@morphometrics.org<mailto:morphmet@morphometrics.org>>
Subject: [MORPHMET] Doubt Scaling photos

Hi everyone,

I am starting a new project using GM working with groups with different sizes 
(eg. rodents and small carnivores). I would like to use the whole dataset 
combined in the analyses, instead of perform set of analyses for each sized 
group. So, I did a test using the same skull and place the camera in two 
distance to the object (~15 cm and ~30 cm). My expectation was after scaling 
(using tpsDig) I wouldn´t detect any meaningful difference in the dataset. 
However, I got two clear groups that were even statistically different. I have 
attached here the tps file that I used (10 copies of the same skull, 5 at ~15 
cm and 5 at ~30cm). My question is how can I combine two set of 2d landmarks 
based on photos taken from different distance to the object. I would greatly 
appreciate any suggestion.

All the Best and Happy 2018!

Anderson
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_____________________________________________

Dr. Anderson Feijó

Key Laboratory of Zoological Systematics and Evolution
Institute of Zoology, Chinese Academy of Science
Beichen West Road, Chaoyang District, 100101
Beijing, China

Curriculum: Lattes<http://lattes.cnpq.br/9406413385468571>; 
ResearchGate<https://www.researchgate.net/profile/Anderson_Feijo>

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