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Thank you for clearly addressing the reviewer comments in the text and rebuttal letter. This study is a great contribution to the literature.
# PeerJ Staff Note - this decision was reviewed and approved by Dezene Huber, a PeerJ Section Editor covering this Section #
Please incorporate or address the comments below and thank you for your submission. I look forward to receiving the revised manuscript.
Overall the manuscript is presented clearly and unambiguously. However, see my comments on the experimental design and validity of findings sections below.
Some of the panels in a few figures could be removed as they contribute little to the findings of this study (e.g. Figure 2, panels F and G).
The design of this study was well thought out, but some additional information is required that could influence the interpretation of results. Also, I have a few concerns with some aspects of the design.
Given the diverse visiting fauna observed, it is required that the time of day that these observations were made are reported. The behavior of these taxa can change over the course of the day. For example, many Hoverflies are known to switch from foraging for nectar to pollen around midday. The difference in behavior of nectar vs. pollen foraging can influence flower handling time, and therefore rates of seed set.
In the second experiment, one ramet was bent (lines 126 to 129) close to the ground to produce a “short inflorescence.” How did the orientation of the inflorescences change? Did they now appear more horizontal, and therefore change how pollinators would encounter them? I feel the description of this manipulation should be clarified.
Also, the seed counts from the second experiment may have been confounded by inbreeding. Since three inflorescences per genet were manipulated, pollinator movement between ramets of the same genet would result in geitonogamy, and likely result in reduced seed production. An alternative approach would be to retain a single ramet of desired height from each genet (e.g. cut the remaining ramets) until a desired distribution of single-ramet genets are achieved.
The sentence starting on line 161 states that fitness values were assigned to individual ramets. Fitness is an individual (e.g. genet) level trait, and cannot be assigned to repeated modules on a single individual. Perhaps the authors should revise this to “ramet reproductive performance.” Therefore selection gradients cannot be calculated for individual ramets.
Inflorescence height is just one perceivable trait pollinators use as cues. This study, while undertaking an important and worthwhile question, confounds many of these (e.g. flower size, floral reward availability) cues, but attributes all observed patterns to inflorescence height. Overall, I feel that the wording and interpretation of the findings should be more conservative. For example:
Lines 240 – 241 (and following paragrapsh): I appreciate that the authors whish to place their findings in a broad context, but their finding that certain bee species typically visit certain inflorescence heights does not automatically translate to a taxonomic segregation within inflorescences. What about the movement of insects once within an inflorescence? Many bees typically display an upwards pattern of flower movement within inflorescences, that would alter floral reward characteristics of the entire inflorescences and therefore influence the visitation patterns of subsequently visiting insects.
I also disagree with the sentence ending on line 298. Many factors influence plant fitness besides inflorescence height. Several studies have shown a large influence of resource availability and even genet age (related to resource accumulation in perennials) that strongly influence inflorescence height. While other studies have found increased visitation rates on taller inflorescences, pollinator-mediated selection is not the only influencing factor.
Lines 299 – 314: see my previous comment on why fitness, and therefore selection, cannot be ascribed/estimated on individual ramets, bur rather, is a trait associated with an individual organism.
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This paper provides results of a lovely, balanced experiment manipulating inflorescence height, and relating the treatment to insect visitation (for 2 plant species) and seed set (for 1 plant species). The writing is clear, analyses appropriate, and discussion + literature cited great.
I have 3 general concerns, and a couple of minor ones:
The introduction seems a bit misplaced. It does a great job introducing the adaptive significance of plant height for plants. But it makes a convincing case that plant height also has consequences for pollinator visitation, such that a pollinator perspective is needed, too. But this issue (the pollinator's perspective) is NOT introduced. Presumably, pollinators “care” about rewards (nectar and pollen) and strategies to optimize their foraging gain, based on distance-sensitive foraging costs, and search/sampling costs. None of this is mentioned. It's odd to have a paper that focuses on pollinators pay so little attention to what might explain their behavior. This introduction needs some serious revision, to introduce a pollinator perspective.
The statistics are generally clear, but the conclusions from them often do not follow:
Line 174: “The difference in the shape of the relationship between total visitation and inflorescence height…”. No elements of shape are tested here. What differences the shape? Its height? Its slope? The location of its maximum? We need a more particular test than simply examining the interaction between surrounding veg height * inflorescence height, which should precede such a statement, but needs further qualification.
Line 190: “Inula salicina had a lower visitation rate than Centaurea scabiosa”. This not supported, as no presented model contained a Species term”.
Line 198: “Inflorescences of both species experienced visitation by a very different community of potential pollinators depending on the height of the inflorescence and the height of the surrounding vegetation because different flower visitors responded to both factors in a species-specific way “. There is no presented test of the significance of an interaction between species and surrounding vegetation height, and therefore no support for this statement.
There also seems to be a missing analysis in Experiment 2. We see measurements of insect visitation, and measurements of seed set, but the two are never related in a statistical model. This seems to be an oversight. I tried this quickly with data sets 3 and 4: calculating sums of treatment seeds produced, and sums of visitors and visits. I fit the model sumseeds ~ totalflowers (a nuisance variable) + InfloresenceHeight:sumflowersvisited, I get a significant positive term for sumflowersvisited, but no effect of height. A virtually identical result obtains from using sumvisitors in place of sumflowersvisited. So while there is strong support for the idea that visitation promotes seed set, there is none for the idea that, independently, height promotes seed set. This conclusion promotes a stronger, more nuanced conclusion: height promotes greater seed set by greater visitation, but height itself has no effect on seed set. Switching to a different model, where Y was sum#ofFlowerswithseeds, the result was similar for sumvisitors, but different for sumflowersvisited (in this latter case, all that mattered were how many flowers were presented). All of this, of course, ignores bee taxonomy.
More minor concerns
Line 129: This experiment is excellent, but more complicated than suggested here. While the only difference between the 3 treatments (positioning inflorescences on a single plant at low, moderate, and high levels) was inflorescence height, this treatment has man y consequences for seed set that are unrelated to bee visitation. First, water transport, and the difficulty of moving it vertically, is a major constraint on plant architecture. Moving water sideways (low inflorescence) is much easier. Second, low inflorescences means shading of their associated photosynthetic structures, and therefore lower rate of local carbon fixation, which should matter when reproductive allocations are made locally. I suggest at least recognizing these complications.
Line 61: Given that high heritability generally implies a weak relationship with fitness (e.g. Price & Schluter 1991), high heritability of plant height suggests the evolutionary non-importance of plant height. Providing a high measure of heritability in the introduction therefore suggests a problem not worthy of study.
Overall, I really enjoyed reading this manuscript. It was well written, the experiments were carefully thought out, and the methods and results were clear and descriptive. I think this paper obviously deserves to be published. My comments below mainly refer to how the paper is framed, not any fundamental flaws.
I do have a few concerns, which are largely in the framing of the paper. It seems to me the theory or background focus of the paper clashes with what the experiment was designed to show. The introduction emphasizes the potential evolutionary implications, but these experiments weren’t really designed to show selection effects (in my opinion). Showing that pollinator preference is driving selection in flower height would require a lot more complexity. As it is, inflorescence height is a plastic trait that changes depending on environmental conditions, likely including the height of surrounding vegetation. This context makes the ultimate effect of pollinator preference much more complex. So saying that you’ve shown “pollinator-mediated selection on inflorescence height” seems to be a stretch to me, given your results.
The last sentence of the abstract seems odd to me as well: even if you show that pollinators are driving inflorescence height, how would that correspond to selection in pollinators? Coevolution requires selection acting simultaneously on both interacting parties.
In this sense, referring to the differing pollinator communities in tropical canopies is also misleading because (as far as I understand) that refers to plants that grow to different heights and have adaptations to different light levels, not the same species that can grow to different heights.
Similarly, some of the conceptual framing seemed misleading to me. For example, the paper mentions in a couple of places that flower visitors “prefer to fly at a certain height” (e.g. lines 39-40), but this experiment doesn’t necessarily show that. It just shows that they prefer to visit flowers at particular heights. Your own study shows that the height at which they visit flowers is context-dependent and not an inherent preference and also that different pollinator groups have different preferences, and are therefore not driving selection for taller flowers (necessarily). Indeed, if you look at the visitation rates for Centaurea scabiosa (going purely on total visitation), there would be selection for an “optimal” height, relative to the surrounding vegetation. In both short and tall surrounding veg, the visitation decreases as the flowers get above 80 cm. (this might very well apply to Salvia above heights evaluated in the study, which were constrained by the maximum stretch of the plants)
I also feel like phenotypic plasticity deserves a description in the introduction, maybe to replace the emphasis on evolution.
Finally, as far as I can tell (I may have missed it), you do not reference the supplemental materials in the main text. It seems they should be cited as the other tables are cited, where relevant.
The only thing I was missing here was the number of bees collected and the number of species identified. It would also be nice just to have a total number of man-hours spent sampling/observing insects visiting flowers. It just gives the reader an idea of sampling effort and how much data are involved in the analysis.
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In general, I feel like modifiers are overused in this ms. For example, "strongly" (8 uses in paper) "very" (7 uses) etc. are usually unnecessary.
The first sentence of the abstract is ambiguous.
Line 53: “There are” instead of “there is”
Line 245: “despite the fact that” instead of “despite that fact that”
Line 246: “Previous studies” instead of “previous studies”
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