Interspecific Courtship Between Two Endemic Fireflies
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsReview Diversity: Interspecific courtship between two endemic fireflies: A potential case of reproductive interference
This manuscript documents the mating mistakes of male Photinus palaciosi and female Photinus extensus fireflies, and quantifies the occurrence of conspecific (P. extensus females with P. extensus males) versus heterospecific (P. palaciosi males with P. extensus females and P. extensus males) mating attempts over 10 nights in July, when both species are active.
This is a potentially interesting topics for the readers of Diversity, especially for readers interested in male and female mating behaviors, and potential mating mistakes between species that can lead to lost mating opportunities (and in some cases to hybridization).
However, in the present version of the manuscript, a few important pieces of information are missing. By adding these, the authors will be able to give the reader a more complete picture of the mating sequence of these two species, and some of their suggested explanations can be evaluated in more detail and firmly placed into the existing literature.
Strength: The strengths of this manuscript include the use of the individual steps (8 phases) in the mating sequence of two species of Photinus fireflies to document how far conspecific versus heterospecific mating attempts in this study proceeded. These data allow the ranking of weaker towards stronger evidence for potential mating attempts, and also represent a scale of invested time and energy. Another contribution are the quantitative data for 22 P. extensus females, which had an astonishing number of interactions with a total of 74 conspecific males and 45 heterospecific Photinus males over 7 nights. While only one potential (unverified) complete mating sequence with a heterospecific male was recorded and thus actual reproductive investment by female P. extensus is unlikely, there can be -depending on the stage at which the mating attempt is aborted - a considerable investment of (wasted) time and energy, especially by male P. palaciosi. These data represent a valuable contribution to the literature.
Weakness: Light signals play a key role in initiating the mating sequence (phase 1) of Photinus fireflies and the continued flash dialogue (phase 2) maintains both male and female interest. However, no information is given on male light signals or on female response signals in this manuscript. Typically, different Photinus species differ in their male signals (flash duration, number of flashes in a repeated flash pattern, and intervals between flashes and flash patterns). Photinus females typically respond with one or two flashes with a species-specific time delay to the male flash. How do the male flashes and flash patterns and mate search differ between P. extensus and P. palaciosi? Is there any way females could tell the difference? Similarly: how do female flash responses differ? Is there any way males could tell the difference? For example, in North American Photinus species, character displacement causes male flash duration to diverge between sympatric species, which should reduce mating mistakes with heterospecific mates (Stanger-Hall and Lloyd 2015). Lloyd [13] discussed both flash duration, as well as flash rate (in species with multi-flash flash pattern) as a basis for species recognition in his experiments. By including information on the male and female flashes (and their flash dialogue) of the two species in this study, and discussing these in the context of the literature, the conclusions of this manuscript will be based on a much stronger argument, rather than just generally invoking a “selective pressure”.
Please see below for specific suggestions to make the writing in the manuscript more clear (adding context for the readers), and to solidify the arguments made. This study adds valuable information, so please make sure to communicate all aspects of this study and the biology of the fireflies as clearly as possible.
Specific comments, questions and suggestions
Title: Interspecific courtship between two endemic fireflies: A potential case of reproductive interference.
Comment: It seems the title should read: Interspecific courtship between two endemic fireflies: A potential case of reproductive interference. Based on the data in this manuscript, it is not clear what the alternative would be. Rather than framing the manuscript as testing whether it is reproductive interference (what is the alternative tested?), it may be a stronger framework for this study to quantify the observed reproductive interference (preliminary data) in terms of frequency and time investment.
Abstract
Line 23: ”We hypothesize that the intense male competition for mates and the male-biased operational sex ratio, both typical of synchronous fireflies, explains the “indiscriminate” sexual responses of P. palaciosi males and, thus, the frequent interspecific sexual interactions observed, and that these interactions could have negative effects on the fitness of at least P. extensus females.”
Comment: This is a hypothesis that was not tested in this study (nor was any other hypothesis, and this is fine; the present study can stand on its own for providing solid data on conspecific and heterospecific mating attempts). No data were collected (or are cited for these species in the literature) on OSR and the relationship between male competition and indiscriminate mating. In addition, no reference was provided of what is “typical of synchronous fireflies” and which species. Synchronous fireflies like P. carolinus are probably most similar in their behavior to P. palaciosi (please describe this system in the methods section in more detail), and very different from the synchrony in Asian fireflies (male leks attracting females), so referring generally to synchronous fireflies may not be justified. I suggest rephrasing this final sentence as something that should be tested in a future study (but this requires laying out the existing data for this and other species in the discussion and making a case for this connection); right now, this reads more like a speculation than an evidence-based prediction.
Line 35: “Synchronous fireflies are an intriguing problem for scientists…”
Comment: What is the problem? Please explain.
Line 39: “that makes synchronous fireflies a potential problem for the non-synchronous, less abundant, sympatric firefly species.”
Comment: this requires context for the reader. Please explain (a) how bioluminescent fireflies use light signals during their mating dialogue, and (b) how sympatric species can interfere, especially a synchronized species with many signaling males, (c) explain how the synchrony of P. palaciosi works.
Line 43: “decreased male choosiness…”
Comment: Please note: Lloyd [13] also noted that synchronous male P. carolinus are easily decoyed (with flash light). That should be added at least to discussion, since it supports your point.
Line 46: “If these interactions have a negative effect on female (and/or male) fitness, we attest a case of reproductive interference [1,2,4,5], a potentially powerful selective pressure that could affect the evolution of bioluminescent signals and other elements of the mating sequence [11].
Comment: For a direct firefly reference of how character displacement minimizes signal interference in sympatric species during firefly signal evolution see Stanger-Hall and Lloyd 2015.
Line 102: “Although the mating season of the two species starts at different times….”
Comment: Who starts earlier, i.e., which species is later in their season when the other species emerges? Please specifiy. Are there any other firefly species present at the same time?
Line 106: “The nightly mating period of P. extensus starts at about 19:45 h and finishes at about 21:00 h, while that of P. palaciosi starts approximately at 20:15 and finishes at about 21:45 h”
Comment: Was this twilight or completely dark? It seems this should be mostly twilight? Please add.
Line 142: “observers… in the dark at about one meter from the female(s) and registered her/their interactions with males in voice notes in a cell phone”
Comment: Was the cell phone screen completely dark? Also please add to discussion whether or not the close proximity of a human observer could have influenced the observed interaction (i.e. the male approach: some species are more sensitive to observer movements than others), especially since so many conspecific mating sequences were aborted.
Line 146 ff: Mating sequence.
Comment: please add how the male and female signals look like and how they differ between the two species in your study. This is key for a study on mating (and mating mistakes) in bioluminescent fireflies.
Results
Line 172: “The percentage of interactions that started with an exchange of signals with a flying male (Communication in flight) was 29.7% (22/74) in the intraspecific case and 40% (18/45) when the male was P. palaciosi.”
Comments:
(1) Does this mean there was no signal exchanged in the other interactions? Or that the observer did not see it (Line 135: We detected females when they were sending bioluminescent signals or when they were in copula)? Please clarify and provide context for this comparison.
(2) This statement also means that almost the same (total) number (22 vs 18) of observed interactions started with a signal exchange: this makes the description of the signals even more important. Did all these originate with the female responding to the respective male signal? Or did the P. palaciosi males approach females that may have responded to a conspecific male? Or can males find females without a light signal? Did you record this information? Please clarify.
Line 176: Figure 3 (all observed interactions):
Comments:
(1) please change colors of bars (to black and grey? Or solid black and open black?). Pink and blue are hard on the eyes (also for Fig 4).
(2) based on the numbers (totals), these are all interactions with observations starting at different points in the mating sequence, which makes them harder to directly compare. Suggestion: Can you add another figure that shows only the interactions that all started with signals (=potentially complete sequences)? And then show % distribution (phases) of these for each species in a separate (top/bottom) panel? This would allow for a more direct comparison between distributions of conspecific versus heterospecific mating sequences that all started with mating signals.
Line 182: “The duration of most intra and interspecific interactions was less than 20 minutes (70.6% and 73.3% of intra and interspecific interactions, respectively), and the interactions that lasted more than one hour (15.6%) were exclusively with P. extensus males (Figure 4).
Comment: this is important info for quantifying effort and should be specifically discussed in the discussion (relative to total activity period). This limits the wasted effort of P. palaciosi males for heterospecific mating attempts; but I am curious why conspecific mating attempts were also aborted so early. This should be discussed in more detail in the discussion.
Line 196: “we observed five perched P. extensus males … being approached at different times by 14 P. palaciosi males”.
Comment: What did the P. extensus males do? Did they signal at all? If yes, how do their signals differ from P. extensus females? Was it mating signals or other signals (see Faust 2010 for the different signals that P. carolinus use)? If not: were signals not seen by observer? Or absent? If absent: how were they detected by the P. palaciosi males? Please add this to the discussion for a more complete picture.
Line 200: “homo and heterospecific females”
Comment: please change homo to conspecific
Discussion
Line 207: “the motivation of both males and females appears to be purely sexual”.
Comment: You cannot infer “motivation”, this is a cognitive construct. Please rephrase as observation and be more specific, e.g., “Predatory females of many Photuris species mimic the female response from other firefly species to attract male prey [12], but the male and female signals of the two Photinus species in our study are used to attract conspecific mates.”
Line 214: “In four species pairs Lloyd [13] observed interactions similar to courtship, and in three of them there was physical contact between males and females, including one copula between the only female tested of P. granulatus and a P. tenuicinctus male.
Comment: Lloyd [13] also gave information on the signal variable that was/was not used to discriminate/or not between species in his species pairs. Please add this to the discussion and discuss in context of P. extensus and P. palaciosi signals.
Line 219: “Unfortunately, no information about female brachiptery is provided for all species.
Comment: This is incorrect: Lloyd [13] provided this information in the other sections of his paper (please see the information on species groups). In short: the females of P. scintillans, P. brimleyi, P. punctulatus, P. tenuicinctus, P. collustrans, P. tanytoxus, and P. granulatus, are all brachypterous. Also note spelling: brachyptery.
Line 222: “Differences between signals do not explain the results because in eight pairs the signals of each species were different”
Comment: Can you please clarify and be more specific? What about the eight pairs? And what about the signals was different? Flash duration? Flash rate? Flash pattern?
Line 225: “In our opinion, Lloyd’s study [12] suggests that researchers have possibly overlooked interspecific courtship in nature”
Comment: I am not sure what you mean here. His data show that firefly responses are variable, allowing for mistakes. Rather than saying that others (who?) have overlooked something, I would rephrase it as something your study is adding. For example, by quantifying heterospecific versus conspecific mating attempts of P. palaciosi males and measuring lost time, your study is the first to …
Line 228: “We think that the simplest explanation for our behavioural observations is that P. palaciosi males mistakenly courted P. extensus females.”
Comment: I do not quite follow why you need this as an explanation. Did you have an alternative explanation? I would suggest to start with this as a mating mistake, and your big contribution is to quantify its costs and discuss the costs and potential benefits of the observed behaviors.
Line 230: “We suggest that the P. extensus females mistakenly responded to the bioluminescent signals of P. palaciosi, which, in turn, mistakenly interpreted these responses as produced by females of their own species and courted the heterospecific females.
Comment: Rather than restating your findings as a suggestion, I recommend you use your findings and discuss why these mistakes may be happening and are not lost to selection: e.g. are the species recently sympatric and possibly currently under selection to reduce mistakes? Is there any evidence for that?
If not:
1. what are the costs and the potential benefits of female P. extensus to respond to heterospecific male signals? Could they distinguish (do male signals differ)? What about chances of seeing a conspecific male? Err on the safe side? Costs: It seems they just ignore the small/wrong males (no energy wasted rebuffing them?).
2. what are the costs and the potential benefits for P. palaciosi males to be so responsive to heterospecific female (and male?) signals? Could they distinguish (do female signals differ)? What about chances of seeing a conspecific female? Err on the safe side? Most of them seem to notice their mistake early enough. When did the longer mating attempts occur (night and season)?
3. How closely related are these species to each other? Is that known? Could that add to the confusion?
Line 233: “The males of P. palaciosi, as all synchronous fireflies, experience intense competition for females as a result of the typically large number of males looking for females every night.
Comment: please cite a REF for intense competition in synchronous fireflies, and add Lloyd [13] who noted that P. carolinus males are easily decoyed, to strengthen your point here. If applicable, see Faust 2010 for more information on P. carolinus (with alate females).
Line 238: “Male biased OSR are probably typical of synchronous fireflies.”
Comment: This statement needs a reference (see above), otherwise it is just speculation. Also note: most fireflies tend to have a male-biased OSR, especially as the evening gets later and/or the season progresses and females drop out of the mating pool. Do the mating mismatches occur more likely later in the evening? Please develop an evidence-based argument (using data from your and other studies) and frame it as “next steps”: future study to answer which questions?
Line 256: “Our observations led us to suggest that it is possible that, at least, the fitness of P. extensus females is negatively affected.
Comment: Careful using fitness. Fitness possibly could be negatively affected by ? But note: it is only affected if energy is wasted (reducing female life span and/or egg production) and if conspecific matings are missed. Do you have evidence for that? It seems females are passive (no energy used to reject males). How many signaling conspecifics were ignored while a P. palaciosi male was present? This type of evidence should be added to the results and discussed here. Also, it seems that P. palaciosi males are much more likely to incur fitness costs: they are physically active (and burn energy) while the female seems to just passively sit there.
Line 260: Considering that P. extensus females courted by P. palaciosi males stop signalling and responding to their own males.
Comment: This should be in the results. Did you observe a P. extensus male that signaled and was ignored by the female? How often? Also: please discuss why successful copulation with conspecific males is so low for P. extensus: e.g., observer presence? does previous mating (female) make a difference for male attraction? Can males potentially tell? Is signal different in this (or other) species?
Line 266: “and that males transfer spermatophores during copulation (AV, YM and CC, unpublished observations).
Comment: This is interesting. Usually there is selection for loss of spermatophores in firefly species with brachypterous females. So, this seems like the intermediate stage (South et al. 2011). If they indeed have spermatophores, this would explain why females may be less choosy.
Line 273: “If our suspicions are true, reproductive interference is acting as a selective pressure on at least one of the two fireflies studied.
Comment: Can you be more specific? If what is true? You have shown that there is at least a time loss (and energy loss in the case of the P. palaciosi males), which can be substantial – connect this to their daily activity period of 90 minutes to make that point.
Line 279: “Alternatively, if the communication system of P. extensus evolves to prevent reproductive interference, populations interacting with P. palaciosi could diverge from those not suffering reproductive interference, possibly initiating a speciation process.
Comment: this would be character displacement and it would have to involve their mating signals: please use your knowledge of P. extensus and P. palicosi mating signals and integrate this into the literature on NA Photinus fireflies. What other species are sympatric? Do they have similar signals? Please discuss what is known and think about follow-up studies (next steps) that could address the possibilities.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsWe have some comments and suggestions for changing the manuscript:
- The authors should redo the introduction. It now contains fragments of text that should be included in the Materials and Methods, as well as in the results of their own observations. Clearly state the purpose and objectives of the research.
- In discussing the results, please answer the following questions: What sets of chromosomes do the studied species have? Why is there mating between different species? What is the biological meaning of this?
- Write a conclusion.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors. I thank you for your answers.
