Peer review of: The morphology, molecular development and ecological function of pseudonectaries on (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this manuscript, the authors describe a detailed and diverse study of the pseudonectaries of Nigella 
petals. These intriguing structures are glossy and seemingly attractive, but offer no reward to 
pollinators. Functionally similar structures have evolved several times independently in the 
Ranunculaceae, as well as other angiosperm lineages, but their molecular control has not been 
investigated in any system. The authors present data derived from SEM, histology, RNAseq, VIGS and 
ecological pollination studies. Overall, the data is excellent and clearly presented, but I do have some 
suggestions to improve the work. 

Major revisions: 

Lines 137-152: This section lacks some experiments that I might expect while also including material 
whose purpose is not entirely clear. I do not doubt that there is more cell proliferation in the area of 
the pseudonectaries but they also clearly have greater cell expansion. I might have expected the 
authors to perform in situ hybridization with a marker of cell division such as HIS4 or a cyclin to 
clearly demonstrate the longer persistence of cell division in this region. On the other hand, I am 
unclear as to the purpose of Fig. 3s. This appears to be purely an animation of an inferred 
developmental process. It is not based on live imaging or actual collection of data on cell numbers or 
size. The grids in the images do not appear to reflect actual relative cell sizes. There is no explanation 
of how this was generated in the materials and methods. While I find it a reasonable animation of the 
idea of pseudonectary growth, I’m not sure that it adds to the data presented in the rest of the figure. 

Lines 215-218: I’m not sure the UV and bee vision images are that informative. In particular, I’m 
wondering if they could actually measure reflectance, as is often done in other papers on epidermal 
cell types. 

Lines 277-279: I found this part a bit confusing. What do they mean by “…pseudonectaries function to 
distract desirable visitors from rewards intended for pollinators.” Aren’t pollinators desirable visitors? 
Do they mean undesirable? 

Lines 302-331: I think the finding that NidaYAB5 is necessary for pseudonectary is a fascinating 
result. It is clear that it is more than just redeployment of abaxial identity simply to promote 
outgrowth since knockdown of NidaYAB5 eliminates all cell types associated with the pseudonectary. It 
is possible that the gene was initially ectopically expressed on the adaxial surface to promote 
outgrowth but it would appear that it now controls all aspects of pseudonectary development. Overall, 
I found this description a bit muddled and I think it can be improved. 

Later in this section, I thought the statement that “if ectopic expression of YAB5 can explain the 
reason why pseudonectaries of one species are formed, it can explain the formation of 
pseudonectaries in other species.” Do they just mean in Nigella? If so, that’s fine but I think some 
evidence could be presented. Alternatively, are they suggesting that YAB5 has convergently been 
recruited to control pseudonectary formation in other Ranunculaceae or angiosperms? No evidence to 
support that has been presented, so this statement needs to be clarified. 

Minor revisions: 

Overall, the text is clear and well written but it could use revision by a technical editor. For instance, 
in line 53, the authors should say either “…, it is widely accepted that the nectary has…” or “…, it is 
widely accepted that nectaries have…”. In line 77, I would suggest “…lack of suitable study systems…” 
In line 238, I would suggest “…it usually flew away after brief attempts.” 

Fig. 3 Legend: An explanation of panels 1-3 in relation to panels (i) and (m) should be added to the 
legend. 

Reviewer #2 (Remarks to the Author): 

Major claim: Liao et al. report that pseudonectaries in the flowers of Nigella damascena contribute to 
the attraction of pollinators and their orientation towards the nectar-secreting ‘true’ nectaries. The is 
conclusion is supported by experiments with plants that lacked pseudonectaries, which were created 
using VIGS to knock down a gene (NidaYABB5). This paper represents an outstanding contribution to 
the field. Hundreds of studies have reported ultrastructural and developmental aspects of nectaries, 
but fewer studies focused on pseudonectaries, and the potential function of these structures remained 
at the level of speculations. 

Here, the authors start with a thorough ‘classical’ morphological and developmental study of the 
pseudonectaries. Then, they use transcriptomic analyses to discover genes of potential importance for 
the formation of pseudonectaries and they knock down a candidate gene. The corresponding 
phenotype lacks pseudonectaries, which clearly supports the key role of NidaYABB5 in their formation, 
and behavioural studies show that flowers without pseudonectaries received fewer visits by pollinators 
than wildtype plants and that pollinators spend less time on nectar probing in these flowers. 

I have only one, major comment and some very minor remarks. I hope that these comments help the 
authors to optimize the potential impact of their work. 

Major comment: 
The pollination experiment represents the strongest but also the weakest part of the study. First, from 
the methods (line 393-394) it does not become clear where the experiment has been carried out (in 
the wild, in a botanical garden, or just in front of the greenhouse?). It does also not become clear how 
the “effective pollinators” (line 228) were discriminated from the nectar robbers (line 397 says “nectar 
rubbers”, but I assume the authors refer to nectar robbers) and how the authors define “inefficient 
data” (line 397) which - apparently - were removed from the dataset. Due to these issues, I don’t see 
how the authors tested the distraction hypothesis (line 288 states “the distraction scenario, however, 
was not supported in this study”). 

I strongly recommend to provide a much more detailed description of this part, particularly concerning 
the study site and the identification of efficient pollinators. Furthermore, the authors should state 
clearly whether Fig. 6h/i reports only visits of bees, or of all pollinators. They also should state in form 
of concrete numbers what a “substantial contribution of bumblebees and wasps” (line 228/229) 
means. 

Minor remarks: 
Perhaps I missed that detail, but what kind of gene is NidaYABB5? The authors characterise this gene 
as an “abaxial gene”, but I think that some more explanations will be required to make its function 
comprehensible for a general readership. 

The authors use several terms or expressions that do not meet current scientific standards. I already 
mentioned ‘inefficient data’ and ‘substantial contribution’. Further examples: 
Line 34: ..silence of it (the gene) 
Line 68: …, pseudonectaries have not attracted enough attention 
Line 96: …we carefully studied… 
Line 98: … pseudonectaries are totally different 

Referee: Martin Heil 

Reviewer #3 (Remarks to the Author): 

Pseudonectaries represent an extremely interesting flower feature that have received very little 
attention in the recent years. So, I warmly welcome this timely study representing the first attempt to 
investigate pseudonectaries with a multidisciplinary perspective. The authors employed a 
morphological, developmental, molecular and ecological investigation (including a manipulative 
experiment) for identifying the function of pseudonectaries in favoring the attraction of suitable 
pollinators and for marking the entrance of the nectar chamber, thereby guiding the pollinator to the 
hidden nectar. Results provided a strong support to the function of pseudonectaries in rewarding 
flowers in contrast to what has been found and discussed in carnivorous plants and deceptive flowers 
where pseudonectaries have a more obvious deceptive function. 
I think the MS is very well written and data properly presented and discussed (even sometime Results 
anticipate some Discussions). Below I list a few minor comments/issues I suggest incorporating before 
the acceptance of this interesting paper 

Introduction: I think, as pseudonectaries have been often found and described both in carnivorous 
plants and deceptive flowers (where their functions of deceiving the visiting insect is forthright) this 
should be mentioned and cited in the Introduction also because the conclusions of this MS point 
instead to a “positive” function of pseudonectaries in driving the pollinator to the hidden reward. 

Line 140. A reference for the petal developmental stages is needed here. 
Line 157: ….172 and 652 are specifically and preferably expressed in the pseudonectary-containing 
Part III …compared to what?? Something wrong in this sentence. 

Line 184: ……Of the genes that are 185 up-regulated in Part III one… NidaYAB5 was not mentioned 
before. 

Lines 257-268: All this descriptive section should be moved to the introduction 

Lines 222-224. The description provided here is not very clear by looking at picture 6. 

Lines 233-235. We do not know whether the TRV2-NidaYAB5-treated flowers also differ in scent 
emission so that other factors may reduce treated flower attractivity. A cautionary note should be 
applied. 

Line 288. I simply do not think they TESTED this alternative hypothesis. 

Line 300. I do not think this study provides evidence that pseudonectaries favor the attraction of more 
specific, preferred pollinators. And, in particular in this specific case, as the very generalist honey bee 
was the most frequent pollinator. 

Lines 307-308. Is there any reference supporting this statement? 

Line 394-396. Eighteen mocks and 18 TRV2-NidaYAB5-treated flowers with strong phenotypes were 
arrayed side by side (Fig. 6g). This does not correspond to what is showed in the Fig. 6g (where 16 
flowers are showed)