Hi Anderson,

I concur with Murat about the usefulness of having actually multiple specimens. Analyses on a single specimen, although in principle attractive (as one "isolates" error from biological variation), are not necessarily informative on a practical level. Also there are two problems, one of magnitude (e.g., how large is variation due to distance/presentation relative to biological variation), one of direction (e.g., if one distance/presentation gets consistently digitised differently from the other, that is, you have non-random error/bias). You can find discussions of these in my recent review which Murat very kindly suggested.



I would add that, in addition to the digitization process, differences might have been caused by factors. However, another thing that I noticed in your TPS file is that you have first all the repetitions at one distance, then all the others. So I assume you digitized them in that order. This suggests that, as already pointed out by Murat, the differences might be due to "learning" or changes over time in the way you digitize.


Most likely, the final error will come from a combination of various sources, as it's generally the case.


In general, I would suggest you to run a larger preliminary analysis with:

- multiple biological specimens

- multiple distances

- multiple digitizations

- digitizing in random order across repetitions (there is a very useful function for this in tpsUtil by F.J. Rohlf)

- using multiple statistical tools to gauge how large is the error relative to biological variation and if there are non-random patterns


I hope this helps,

Carmelo




Il 2/01/2018 7:28 PM, Murat Maga ha scritto:

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

    *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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