Change of Leaf Trait Asymmetry Type in Tilia cordata Mill. and Betula pendula Roth under Air Pollution

Round 1

Reviewer 1 Report

I have read this work with great interest and I think it is a nice contribution to the field as the application of asymmetry indexes as indicators for environmental pollution have been for unknown reasons rare in the recent litterature. The. authors have made a robust study with a well structured design and with a sufficient sample size which allow robust conclusions.

I have few minor comments which I think the authors should consider.

The use of regression analyses requires that teh data are normally distributed and given the transitions observed from FA, to DA and AS I can see some clear violations of the assumption. and therefore a non-parametric Spearman rank test could be made at the same time simply to show that the correlation is also high using the non-parametric statistic.

The authors only consider the regression with R2 above 50% as interesting...I will say that also models which explain more than 30% could be interesting

The authors analysised FA, DA, AS and mixture but I do not understand why now that. they have the data they did not also use pghenotypic variability as an estimator of developmental instability and the fact that several leaf belonging to the same trees will make such an index quite intersting...se for example:

o   Developmental instability as an estimator of genetic stress Heredity DOI: 1038/sj.hdy.6800777

Lastly I do not understand why the authors have not used a scaled index as there are variations in the size of the leaf......or they should check for the correlation between the size and asymmetry indexes, see for example

o   A new fluctuating asymmetry index, or the solution for the scaling effect? Symmetry DOI: 3390/sym7020327

said that I will not consider my suggestions mandatory but I simply consider them suggestions for improving teh values of this interesting work.

Author Response

1. The use of regression analyses requires that teh data are normally distributed and given the transitions observed from FA, to DA and AS I can see some clear violations of the assumption. and therefore a non-parametric Spearman rank test could be made at the same time simply to show that the correlation is also high using the non-parametric statistic.

Authors' response. We did not use primary data for regression analysis, but medians of FA integrated index and average proportions of leaves with FA calculated for each of studied plots. The normality of their distributions was checked using Shapiro–Wilk’s test (explanation was added to the text, see p. 5, l. 177). The distribution of medians/averages is usually close to the normal due to central limit theorem. We could not use Spearman's correlation test or any other correlation methods because of nonlinear relationship under study. In this case, correlation analysis can not reveal the relations between the studied parameters.

The authors only consider the regression with R2 above 50% as interesting...I will say that also models which explain more than 30% could be interesting.

Authors' response. In addition to R2, we took into account p-value in the regression analysis. In our case, there were no regression equations with R2<0.5 and p-value<0.05.

The authors analysised FA, DA, AS and mixture but I do not understand why now that. they have the data they did not also use pghenotypic variability as an estimator of developmental instability and the fact that several leaf belonging to the same trees will make such an index quite intersting...se for example:   Developmental instability as an estimator of genetic stress Heredity DOI: 1038/sj.hdy.6800777

Authors' response. We collected only 10-20 leaves from each tree to study tree population state. But this leaf sample for a single tree was insufficient to assess phenotypic variability. This was beyond the purpose of our work. To study this issue, a different design of the research is required.

Lastly I do not understand why the authors have not used a scaled index as there are variations in the size of the leaf......or they should check for the correlation between the size and asymmetry indexes, see for example

A new fluctuating asymmetry index, or the solution for the scaling effect? Symmetry DOI: 3390/sym7020327

Authors' response. FA integrated index used by us takes into account the influence of the leaf size. When calculating this index, the L-R absolute value is normalized (divided) by L+R, which allows to get a dimensionless value and eliminate the influence of the leaf size on the value of this index (this explanation was added to the text, see p. 5, l. 173).

Reviewer 2 Report

Leaf symmetry

Define more terms

This paper describes the evolution of leaf trait asymmetry in two species, over years and in different growth location that differs by their air pollution level. The main conclusion from the authors is that the possible impact of air pollution can differ between species as FA integrated is inversely correlated with air pollution in T cordata while in B pendula the relationship is more complex, as it tend to be positively correlated but in possibly different ways depending on the observation year.  

I have several comments. I found that the conclusions made by authors are much too strong as they strongly suggest a causality between air pollution and change in leaf asymmetry. They should be much more cautious about this.

Although they discuss that other factors could have an effect such as weather conditions, this could be more explored as data for this are likely to be available. In addition, they indicate that the soil in the different plots is simiar, but more data could be provided to support this.

The authors showed the evolution of the proportion of leaves with FA (eg in Fig 3) or of FA integrated (eg in Fig4) in function of the air pollution. Did they test if some specific leaf traits were more or less correlated to the level of pollution ?

Minor comment

How was the length of the veins measured : was it approximated to the length of a segment as suggested for measure 2 in figure 2 or was it measured as the actual length of the vein (which can be not straight) as suggested for measure 5 in figure 1 ?

Some more details should be given in the introduction on the different kinds of (a)symmetry, possibly with some examples.

I also found that the way they can be measured (explained in table 1) comes too late in the manuscript.

Author Response

1. 1. I found that the conclusions made by authors are much too strong as they strongly suggest a causality between air pollution and change in leaf asymmetry. They should be much more cautious about this.

Authors' response. The conclusions were replaced with less strong ones (see p. 14, l.354; l. 363).

2. Although they discuss that other factors could have an effect such as weather conditions, this could be more explored as data for this are likely to be available. In addition, they indicate that the soil in the different plots is simiar, but more data could be provided to support this.

Authors' response. The influence of weather conditions on leaf asymmetry was discussed in detail earlier in our previous publications (references to these publications were added to the text, p. 14, l.356-357.

3. In addition, they indicate that the soil in the different plots is simiar, but more data could be provided to support this.

Authors' response. Additional data were included. In addition, a typo was corrected in the description of the soil (p. 3, l.125-126).

4. The authors showed the evolution of the proportion of leaves with FA (eg in Fig 3) or of FA integrated (eg in Fig4) in function of the air pollution. Did they test if some specific leaf traits were more or less correlated to the level of pollution ?

Authors' response. We tested dependencies of individual leaf traits on pollution (unpublished data). It was showed that these relations were more often revealed for averages of several traits than for individual traits. In this case, random changes in individual traits caused by unrecorded environmental factors are excluded. As a result, the dependence of asymmetry on pollution is found much more often.

5. How was the length of the veins measured : was it approximated to the length of a segment as suggested for measure 2 in figure 2 or was it measured as the actual length of the vein (which can be not straight) as suggested for measure 5 in figure 1 ?

 Authors' response. The veins of the birch leaf are straight so their length was measured as shown in Fig. 2. The veins of the linden leaf are curved, so their length was measured as shown in Fig.1.

6. Some more details should be given in the introduction on the different kinds of (a)symmetry, possibly with some examples.

Authors' response. These details were added to Introduction (see p. 1, l. 36-37; 39-42; 44-47).