Hi Carmelo,
Thank you for those answers.
One more question please:
I know that the number of specimens should exceed the number of the total 
landmarks (fixed-landmarks + semi-landmarks). Is there a rule of thumb of 
by how much or what ratio between specimens to semi-landmarks one should 
keep?

Thank you,

Avi

On Sunday, March 11, 2018 at 5:15:24 PM UTC+2, Carmelo Fruciano wrote:
>
>
>
> Il 6/03/2018 4:44 PM, Avi Koplovich ha scritto: 
> > Hi Carmelo, 
> > Thank you for your answer. 
> > My project tests for the influence of kairomones of a predator fish on 
> > the morphology of Salamander larvae during its development. To do this, 
> > I take pictures every other week of larvae spawned from six different 
> > females and assigned to 3 treatments: No fish, 3 caged fish, 6 caged 
> fish. 
>
> Hi Avi, 
> it sounds like an interesting experiment. I will try to answer to your 
> questions but keeping in mind that I'm not very knowledgeable on 
> salamander development. 
>
> >  1. I intend to use landmark 1 (dorsal connection of the tail fin) as a 
> >     fixed factor. But I thought I may be able to use the tail tip 
> >     (landmark 20) and head tip (landmark 48) as fixed landmarks as well. 
> >     Do you think it's ok in an ontogeny experiment? 
>
> I guess it will depend on how long into ontogeny you will track the 
> larvae and whether or not that point will "disappear" over ontogeny 
> and/or slide unreasonably (depends also on your question). You, being 
> knowledgeable on their biology, are the best judge on that. 
>
> > If not, do you think 
> >     it's ok to slide all semi-landmarks of the tail on landmark 1, and 
> >     all head semi-landmarks on an eye landmark? Since the eye isn't part 
> >     of the head contour, is it ok if I slide one semi-landmark to the 
> >     eye and all rest semi-landmarks of the head one to each other as a 
> >     closed shape? 
>
> The point(s) slid relative to the eye won't be sliding along the 
> direction tangent to the curve you want to approximate (i.e., the 
> curvature of the head). A good starting point on the method could be 
> Gunz & Mitteroecker 2013 - Hystrix 
>
> >  2. Is it ok if landmarks 1 and 39 slid relative to each other as well 
> >     as 41 and 55, since both describe a closed shape? 
>
> It's not particularly desirable (see answer above). 
>
> >  3. Another worry I have is that landmark 40 which I used to create the 
> >     comb fan for both the tail and the head is too far from both of them 
> >     so it doesn't bypass the bending. 
> >  4. I'm affraid I don't fully understand why landmark 40 can not be 
> >     treated as a fixed landmark. In the book of Zelditch 2004, she says 
> >     that one of the basic differences between fixed-landmark and 
> >     semi-landmark is the degree of freedom, while fixed has two because 
> >     it is docked on both X and Y axes while semi only on one of them 
> >     (depending on the nature of the specific fan). Please correct me if 
> >     I'm wrong, but what if I use the side line of the larvae (which is 
> >     an anatomical/homologous feature) as my X axis and use the Y 
> >     component of landmark 1 (dorsal connection of the tail fin) to dock 
> >     landmark 40 on the Y axis? Is it wrong because of the dependency of 
> >     landmark 40 on landmark 1 regarding the Y coordinate? 
>
> I think Don has covered these two very well. 
>
> >  5. Emma Sherratt told me she straightened the bent tail-body using TPS 
> >     software in her paper Sherratt et al. 2017 - Nature ecology & 
> >     evolution. In the supplementary material of her paper she wrote: 
> >     "To correct for dorso-ventral bending in the landmark configurations 
> >     (caused by the joint of the tail with the head/body), we used the 
> >     ‘unbend specimens’ function of tpsUtil v.1.86 (Rohlf 2015). The 
> >     landmark configurations for each specimen were transformed using the 
> >     quadratic approach, straightening from the eye (1) along the 
> >     notochord landmarks (46 to 55) to the tip of the tail (8)." 
> >     Jim mentioned this unbending function here before. I read the help 
> >     about unbending specimens and thought I can use landmarks 20 (tail 
> >     tip), 48 (head tip) and several semi-landmarks I can digitize using 
> >     the comb fan (equally spaced) along the side line of the larvae, in 
> >     order to create the quadratic curve (while the side line "helper" 
> >     semi-landmarks can be later omitted from the dataset - I saw 
> >     Fruciano et al. 2016). Does this sound good? 
> >     I bet that this can basically solve the problems I mentioned in 3 & 
> >     4, since then I can digitized the whole body contour. 
>
> That function is great but, as everything, relies on a set of 
> assumptions (see also Fruciano 2016 - Development Genes and Evolution 
> for a brief discussion). In your case: 
> - that you can consistently identify those points along an hypothetical 
> line (which you would remove after the unbending) 
> - that your arching is well represented by the chosen function 
>
> You are the best judge on whether these assumptions are satisfied in 
> your case or not. 
> I suggested the Valentin et al. (2008 - Journal of Fish Biology) 
> approach because it's more flexible (less stringent assumptions, which 
> obviously doesn't mean assumption-free) and therefore more generally 
> applicable. But, of course, the approach in tpsUtil can be a great 
> solution if it's appropriate to your data. 
>
> Best, 
> Carmelo 
>
>

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