Reviewer's report 1
Prof. Christian Blouin (nominated by W Ford Doolittle), Dalhousie University Halifax, Nova Scotia, Canada
This reviewer provided no comments for publication.
Reviewer's report 2
Dr Endre Barta (nominated by Sandor Pongor), International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
Gymnosperm plants played an important role in Earth's flora, especially in the prehistoric ages. In this manuscript, the authors use elegant molecular evolutionary analyses to answer some open questions about the evolutionary processes tailoring the chloroplast-coded rbcL gene during the adaptation to the changing CO2 concentration in the atmosphere. The authors use rbcL coding sequences from three gymnosperm families. The rbcL is a very specific and very constrained protein, coded in the chloroplast genome and also being in complex with the rbcS, which is coded in the nucleus. The three gymnosperm families are good subjects for this analysis because i) they represent almost 14% of the gymnosperm diversity and can be found globally on the Earth, ii) we have fossil records allowing to constrain the phylogenetic tree timescale.
The authors present a robust evolutionary analysis based on the multiple alignment of rbcL coding sequences from different gymnosperm species. They found that a complex adaptation process occurred during the evolution of these taxa. They also discussed the structural and functional consequences of these processes and concluded that certain compensatory replacement mutations could play important role in the fixation of the functionally novel mutations. The analysis is very sound and in most cases based on different methods and models. The basic idea and the results can be interesting for the broader community.
I have only some theoretical questions and three minor technical comments.
Are there any known examples for the same compensatory mutation pairs from other plant species (i.e. evidence for convergent evolution)?
Authors' response:Recent report of the modification on both large and small subunits of Rubisco enzyme in Flaveria (Asteraceae) might be an example for the similar patterns . The two subunits are under selection during the evolution from C
photosynthesis. This pattern may be an evidence for convergent evolution under ecological pressures. However, the compensatory mutation pair is not coincided in the study.
How could the geographical isolation of different populations influence the results of this study? Are there any samples (rbcL sequences) from geographically well separated plants from the same species? Do you expect any polymorphisms at any replacement site in the small populations of these gymnosperm species?
Authors' response:If we take the geographical CO
variation into account, the geographical isolation of different populations will significantly influence the results of the present study. As has been reported previously, the rbcL gene has undergone adaptive evolution during the radiation in the Hawaiian endemic genus Schiedea, which demonstrates that rbcL gene evolved under strong positive selection impacted by the geographical isolation . Nonetheless, the present research is mainly focused on the relations above species level, so the samples (rbcL sequences) from the same species are excluded in the analyses. The polymorphisms at those replacement sites probably exist in the small populations of these gymnosperm species.
The inferred tree topology and the taxonomic classification of the genera in Taxaceae seem to be different. How can you explain this?
Authors' response:Since its lower evolutionary rate, the rbcL gene has certain limitations on the deeper phylogenetic levels (e.g. at the genus level) . On the other respect, the molecular adaptation in rbcL gene per se also has impact on the inferring of the phylogenetic trees . The above two factors may be the explanation for the disagreement between the inferred tree topology and the taxonomic classification.
Is it possible to deduce from this analysis the ancestral sequence of the rbcL gene characteristic for the different nodes?
Authors' response:Positive answer. However, more experimental data is required for the inference of the rbcL gene characteristic even though the inferring of the ancestral sequence from the current ones is of statistical efficiency [90, 91]. We believe that much more work have been left for the further research after our computational estimation.
Reading the abstract at a first glance, it is not clear what is the relation between Rubisco and rbcL. Clarifying this would help the readers who are not familiar in plant biology.
Authors' response:We agree with this remark and changed the sentence accordingly. One sentence has been added into the abstract especially for the introduction of the relation between Rubisco and rbcL gene.
It is very difficult to review the tables in general, and especially the Table 3. Using grids, or re-structuring them would help a lot.
Authors' response:We agree with this remark and re-structured Table3(new Table2) accordingly.
Referencing Figure 4 is before the first reference to Figure 3, and no reference for Table 1 in the text.
Authors' response:We appreciate the constructive comment. The order of referencing figures has been re-checked. Since Table1shows the original plant materials of this research, which cannot be omitted, we changed its appearance from the first table (former Table1) into the last one (new Table5).
Reviewer's report 3
Dr Nicolas Galtier, CNRS-Université Montpellier II Laboratoire "Genome, Populations, Interactions, Adaptation", Montpellier, France
This manuscript analyses the molecular evolution of the essential rbcL gene in three gymnosperm families. The functional relevance of amino-acid sites detected as positively selected or co-evolving is discussed. Here are my major comments:
The text is quite affirmative regarding divergence dates, and their relationship with atmospheric CO2 abundance. I am not sure that molecular dating is that trustable, even with the use of clock-relaxed models, as illustrated by many controversies in the recent literature (e.g. Graur & Martin 2004 Trends Genet, Douzery et al. 2004 PNAS, Peterson et al. 2004 PNAS, Roger & Hug 2006 Philos Trans, Emerson 2007 Syst Biol), owing to paleontological uncertainty and tricky rate/time decoupling [92–96]. Some prudence would appear required here, and the uncertainty of date estimates could be discussed. This is especially true knowing that the uncorrelated model in BEAST was used here, an approach which was criticized in the recent past (Lepage et al. 2007 Mol Biol Evol) .
Authors' response:Due to the paleontological uncertainty and trick rate/time decoupling, it is still an unresolved scientific theme whether the modern molecular clock has the ability to reconcile the fossil evidence and the time estimation . However, as indicated in many other literature (e.g. Welch & Bromham 2005 Trends Ecol Evol, Ho 2007 J Avian Biol, Ho 2009 Biol Lett), lots of recent methodological advances have been carried out focusing on the topic [99–101]. Specifically, although correlated model (also known as correlated-rates model, or CR model) outperforms the uncorrelated model (also known as independent-rates model, or IR model) in the instance provided by Lepage et al (2007) , several authors have noticed that the uncorrelated model is better than the correlated model during estimating the dynamics of evolutionary rates in other instances [22, 102–104]. Moreover, Zhong et al. (2009) argued quite recently that the uncorrelated model is superior to the correlated model in guesstimating the episodic rate acceleration in ancestral plant lineages . Collectively, all the above conclusions indicate that the modern molecular clock relied on uncorrelated model is applicable for our present study on the gymnosperm plants.
The reason for species sampling in this study is not obvious. Just three families were (thoroughly enough) sampled, when the focus of the study is on rbcL adaptation during the > 200 Mya of gymnosperm evolution. A more balanced sampling across gymnosperm families might help corroborate some of the results reported here.
Authors' response:The three families (Podocarpaceae, Taxaceae and Cephalotaxaceae) represent over 14% of the gymnosperm diversity and can be found globally on the Earth [23, 24]. Moreover, reliable fossil records can be obtained to calibrate molecular clock for dating the time of the phylogenetic trees . So the thoroughly enough sampled species in the three families could partially represent adaptive and coevolutionary patterns of rbcL gene in the related gymnosperms under geological timeline. And we also believe that further research including other families will shed new lights on the big thesis.
Along the same lines, it would be good to know whether the sites identified as positively selected or coevolving in gymnosperms behave similarly in angiosperms (and perhaps other groups of plants), for which a huge database of rbcL sequences is available.
Authors' response:As far as we can see, the atmospheric CO
concentration is one important factor (also known as ecological pressure) related to the adaptation of Rubisco enzyme [14, 60]. Nevertheless, other factors also have impact on the evolution of this enzyme. For instance, the C
photosynthesis in angiosperms have effects on the modification of rbcL gene and rbcS gene . The comparison analyses merely along the identical timeline, ignoring other ecological pressures, may mislead the conclusions. Since the above reasons, we only focus our sampling on the present families.
The discussion emphasizes potential adaptive processes, in possible connection to CO2 availability across time. I note that if rbcL evolution was related to atmospheric CO2 variations, we would expect adaptive evolution to occur simultaneously in contemporary branches of the tree, in line with sudden RCO2 changes. Such a pattern is not clearly detected, so I wonder what in the data makes the author link rbcL evolution to atmospheric CO2 concentration, especially knowing that the adaptative signal is not prominent.
Authors' response:The ability to undertake adaptive evolution depends on several factors. Gymnosperm plants played an important role in Earth's flora, especially in the prehistoric ages. This implies that the species of the three gymnosperm families have a higher feasibility to undergo adaptation in the prehistoric branches. Along with the rising of angiosperms, members from the contemporary branches of gymnosperm plants are characterized by their small population sizes, which make them feasible for genetic drift. The current analysis results and the biological background drew us a big imagination of the ancestral rbcL gene adaptation associating with the variations of the atmospheric CO