Dear reader,

As PLoS One was not able to post my reviewer comments alongside the final published paper, I would like to post them here as a comment. Some points might not be very informative, because the page and line number are not present in the final paper, but I would like to highlight one point that I think has been neglected in the final decision:
It is still not clear if and how the authors visualised the Invitrogen molecular weight marker. It was not DIG labelled and therefore can not be detected on the final blot. Because the telomere signals in this study exceed the range of the regular (DIG labelled) molecular weight marker provided by the kit and the study uses data from multiple blots, this is an important issue that affects the reliability of the data this study is based on.

Sincerely

Dr. Thorsten Horn



Reviewers comments to the revised version of :

Ujvari et al. : Short Telomeres in Hatchling Snakes: Erythrocyte Telomere Dynamics is not Associated with Fitness in Tropical Pythons


Although I believe that the manuscript improved significantly, there are still some open questions/issues that I would like to see answered before publication:


P2 L37: I still believe that the causality of this sentence is wrong.

P4 L49: Maybe change to “... ageing, and longevity are also (or substantially?) governed...” as many extrinsic factors are known to have an effect on ageing.

P5 L79:
“< one to >20” does not make sense, as it means from smaller as 1 to bigger than 20, which would be everything. I guess it should be between 1 and 20.

P6 L98: I still would like to see some discussion of this point. I earlier suggested “seasonal differences” (not written in the manuscript!!!) as a possible reason for that. TL is known to be influenced by a variety of environmental factors. Were there years when TL decreased faster than in other years? Was there any event (e.g. drought) that could impose higher stress levels on the snakes in this particular year?

P6 L104: The new sentence does not make sense: non-recaptured snakes had higher TL than not recaptured ones. Both are non-recaptured. Which group had longer TL and what are the possible explanations? From the discussion I assume the non-recaptured ones had higher TL, as this would argue against an influence of TL on survival. However, in terms of fitness it could also mean that the ones with longer telomeres are fitter and therefore have a higher chance of avoiding to be captured or have more energy for dispersal.

P6 L110/11:
To my knowledge Haussmann & Mauck (2008) do suggest that the longer telomeres in adults found in their study was due to selective mortality, as they found evidence for the selection hypothesis that predicts that individuals with short telomeres are culled from the population.

P9 L163: do not suggest or do suggest that not? Maybe “Four of our results do suggest that telomere dynamics did not have a significant impact on python longevity”?
I also would like to see more discussion on the 4 findings (also see above):
(2) How does the variation suggest no effect of TL on longevity? TL attrition rates could change due to environmental factors but could still be linked to longevity.
(3) Still this depends on how good recapture rates correlate with survival. In addition, the link between TL and longevity might lay in the ability of individuals to maintain their TL and not be apparent in hatchlings.
(4) Discussed the comment for P6 L104.
I know it is difficult to explain the observed pattern, but if the authors raise this points I would like to see some discussion why this points support an independence of TL and longevity.


P9: Study Species
I had some trouble understanding the structure of the samples. If I got it right, there are 20 hatchlings hatched in the lab or collected in the wild. Eight of them have been recaptures after 6 or 10 years. In addition there are 50 samples from juvenile and adult snakes, 13 of whom have been captured more than once. Is that correct?
I would like to know how the age of the snakes older than one year have been determined. Were they marked as hatchlings? If so how do you mark a snake for over 15 years and does this have an effect on fitness/survival?

P10: DNA extraction and TL measurement
The mentioned molecular weight marker (Invitrogen) is not DIG-labelled and therefore not visible on the film. How did the authors determine the position of the MWM and transferred it to the blot ? Using constant field electrophoresis, high molecular weight fragments like python telomeres are difficult to measure and reproducibility depends strongly on the resolution of the MWM. I would like to see one lane of the MWM. In this context, it is meaningless to mention one sample that was run on each blot without any measurement error (SD or CV). I would insist in seeing one of these measures as a correlation between replicates only provides limited information about the magnitude of measurement error.

P10 L205:
What does “ahead” mean? Ahead in the direction of migration (i.e. at lower molecular weight) or ahead in the sense of above the smear on the gel (i.e. at higher molecular weight)


Sincerely,

Thorsten Horn