Visual adaptation and microhabitat choice in Lake Victoria cichlid fish

When different genotypes choose different habitats to better match their phenotypes, genetic differentiation within a population may be promoted. Mating within those habitats may subsequently contribute to reproductive isolation. In cichlid fish, visual adaptation to alternative visual environments is hypothesized to contribute to speciation. Here, we investigated whether variation in visual sensitivity causes different visual habitat preferences, using two closely related cichlid species that occur at different but overlapping water depths in Lake Victoria and that differ in visual perception (Pundamilia spp.). In addition to species differences, we explored potential effects of visual plasticity, by rearing fish in two different light conditions: broad-spectrum (mimicking shallow water) and red-shifted (mimicking deeper waters). Contrary to expectations, fish did not prefer the light environment that mimicked their typical natural habitat. Instead, we found an overall preference for the broad-spectrum environment. We also found a transient influence of the rearing condition, indicating that the assessment of microhabitat preference requires repeated testing to control for familiarity effects. Together, our results show that cichlid fish exert visual habitat preference but do not support straightforward visual habitat matching.

results are just as good as positive ones and would not want to promote a publication bias in favour of positive results, there is always the possibility that the hypotheses are in fact correct, but the experimental test was not. You discuss that perhaps reward for correct habitat choice was not sufficiently in place, which is important. You discard the difference in light intensity across habitats which was not implemented, for reasons that I didn´t find so convincing. And then there may be another 1000 factors that are imortant for the fish, but that we are not aware of and therefore were not (correctly) manipulated. Perhaps in your paper, mention this possibility -that your experimental test might just not have created the ecological setting that does play out in the field, and therefore that the results are limited. This is fine, that is how science works, and perhaps the hypoteses are indeed wrong. But if you had achieved confirmatory results, the ecological relevance would probably not have to be questioned to the same extent. L 32: add: and thereby increase their ecological performance L 60: is there nothing known about (co)dominance in expression? L 61: as written now, your predictions appear exclusive, which cannot be: they prefer their natural light regime (L 58), and they prefer their rearing light regime. Why not simply state that you will test if, and to what extent, genetic background and rearing regime affect habitat preference? (And then mention the directions re. these effects). L 68: so did you allocate entire families to treatment, or did you split families between treatments (a stronger design)? L 71: is the range 0-5 m the natural range? How do you expose fish to the light of a depth range? It is in the supplement (weighting by depth use), but briefly clarify this here. L 75: this is important, since you might then be testing for novelty avoidance (neofobia), or novelty attraction (neofilia). OK, you discuss this later (L 144). L 92: I was wondering why you didn´t use a binomial model, but there must be temporal autocorrelation in your timing events, so it is hard to know how many independent time event you would have, so the transformation seems indeed better. L 94: activity is an interesting one. Why would activity influence habitat use by itself? More likely, more active fish are exposed to the different environments more, and therefore can choose better (which is probably why you demanded a minimum of four transitions). Or reverse, more active fish move between environments more, therefore not choosing. Either way, I think it is the interactions between activity*species, and activity*rearing environment, that are most interesting, not the main effect of activity. L 94: you don´t include side ("is the shallow light condition on the left or right") as a fixed factor? L 94: what is trial number, and why does this have replicate values (required for a random effect)? Each trial supposedly is unique? If you refer to trial order, then this is not correct -fit it as a fixed effect. L 96: you mention AIC, but then give p-values, which doesn´t make much sense. And what are minimal adecuate models -the outcome of stepwise elimination? Why not just give the results for the full models, testing all effects via loglikelihood ratio tests? L 105: this results sugggests you fitted trail number as a fixed effect, not as a random effect? And you tested an interaction, but you never specified before you would test any interactions -in "data analyses" indicate which interactions you included, and why. L 105: 5 decimals for a p-value is way beyond its reliabilty: I think 3 decimals is precision enough. L 116: I think this is a convoluted way to test an interaction? L 132: genetic signatures? L 138: and perhaps this depends also on competition? It is interesting that in the wild, the deep fish also use shallow waters, but not the reverse, mimicking the overall preference you detected here for shallow conditions perhaps? Might deep fish only use the deep when outcompeted by shallow fish in shallow waters (any field data to test this density-or frequency-dependence?)? If so, and as you already mentioned, perhaps your design was not entirely appropriate to test your hypothesis, as reward was not included. L 149: well, not quite -behavioural preference may depend on several factors (e.g. familiarity versus performance), and may shift over time.
L 151: no hypothesis of why all fish preferred shallow light conditions? L 154: maybe add that future experiments should try to increase the importance of relative ecological performance across test environments, or something like that (i.e. greater reward/punishment). Fig. 2: there seems to be a pattern here, I´m surprised it doesn´t come out? But it actually goes against your prediction, right? The deep species prefers shallow water more? Supplementary materials: L 21: there is a strong correlation between age and genetic background. Could the inclusion of age in your models have removed the effect of genetic background? L 60: I´m not so convinced that average difference in light intensity isn´t important because cloud cover variation creates greater changes. Sun set creates even greater changes, but daylight spectrum differences still matter according to you. Perhaps acknowledge in your paper that this aspect was not manipulated, and might change the results. Someone else may try and replicate your study (in other species perhaps), taking this into account. Just as the reward issue. I hope these comments help to further improve an already fine paper, Pim Edelaar.

17-Dec-2018
Dear Mr Mameri, The editors assigned to your paper ("Visual adaptation and microhabitat choice in Lake Victoria cichlid fish") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Dear authors, Your manuscript has been seen by two reviewers. The RSOS format allows you to expand the manuscript, which will help in addressing their concerns in your revision. Reviewer 1 is concerned that light intensity, not ony spectrum, may have played a role in the experiment, and asks how experimental light conditions corresponded to natural conditions. Additionally, more background on cichlid opsin gene expression may help to clarify some issues. Following reviewer 2, the discussion could delve deeper into the various factors (beyond the tested ones) that might have influenced experimental habitat choice. Best regards, Kristina Sefc Comments to Author: Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The authors aimed at testing whether divergent visual sensitivity affects visual habitat preferences. They also aimed at testing whether manipulating visual sensitivity by rearing fish under two different light conditions affects the species preferences for lighting. To answer these questions, the authors used two closely related Lake Victoria cichlids that differ in their depth preferences -one inhabits deeper red-shifted water than the other does. Such depth preference has been previously suggested to correspond to differences in the visual sensitivity of the species. The authors conclude that in contrast to their hypothesis, fish did not show obvious preference to the light conditions that mimicked their typical natural habitat.

Major concerns
In the wild, the species that occupies deeper water is exposed to a red-shifted spectrum but also to an overall lower light intensity. Without knowing the intensity of the two experimental light regimes, it is hard to conclude what aspect of the two light conditions drove the preference to the 'broad spectrum' condition. For example, it might be possible that fish actually preferred the brighter or dimmer light condition.
What are the characteristics of the experimental light environment in terms of spectrum and intensity? Were light intensities matched to those encountered in the natural habitats? It would be useful to present these data relative to the lighting regimes encountered in the wild. What were the estimated quantum catches of the various opsin types expressed in each of the species?
One of the mechanisms of the presumed divergence in visual sensitivity between the species is differential opsin gene expression. However, the authors do not provide any evidence on whether opsin expression differed between the tested species. Is it possible that all fish exhibited similar gene expression as a result of their exposure to the same light condition prior to the beginning of the experiment? How fast such plasticity in opsin gene expression could be?
The authors state (lines 55-57) that they manipulated visual development by raising the fish under different light conditions. However, they provide no evidence for the capacity of such manipulation. In other words, did the different light conditions actually affected the visual sensitivity, and if so, how?
Minor concerns Lines 39-43 -It is not clear what are the differences between P. pundamilia and P. sp. 'pundamilia-like'. The same is also true for P. nyererei. Please clarify.
Line 46 -'They' presumably refers to the two species, but this isn't clear enough.
Line 51 -Please elaborate on how the causality between visual sensitivity and habitat preferences is being determined.
Line 74 -'only group-level data was recorded' should be 'only group-level data were recorded'.
Line 74-75 -What was the light regime before fish were exposed to the experimental light regimes?
Line 86-88 -"After analysing the first two trials, we submitted P. sp. 'pundamilia-like' and P. sp. 'nyererei-like' to a third trial to increase statistical power for testing species differences." Why not simply say that groups were tested three times. Did the third trial differ from the two previous ones? Actually, only when I reached Figure 2 I realized that the third trial differed from the rest in that hybrids were not tested. It might be better to clarify this issue up front.
Line 99 -There is inconsistency in the use of the term 'trial'. Line 87 says that the species were subjected to a third trial. However, in line 99, it is indicated that '75 of the 80 trials were successful'. Please clarify.
Line 119 -'nor trials' should probably be replaced with 'or trials'.

Reviewer: 2
Comments to the Author(s) In this ms the authors investigate whether genetic background and rearing environment affect preference for light environment in two closely related cichlid species which might have diverged in the wild due to distinct habitat preferences as related to their visual systems. Hence, the study tests a clear speciation scenario, one which so far has almost not been tested. I therefore found the study interesting, relevant and timely. The experimental design is adecuate, as are the analysis. Sample size is perhaps somewhat small (as in many behavioural studies), which may mask small effects. It might be worth to discuss the issue of lack of power, and whether any observed trends at least go in the predicted direction or not. I also have a few suggestions for improvement, or clarification, of analyses. The main predictions are mostly not confirmed. While I´m aware that conceptually negative results are just as good as positive ones and would not want to promote a publication bias in favour of positive results, there is always the possibility that the hypotheses are in fact correct, but the experimental test was not. You discuss that perhaps reward for correct habitat choice was not sufficiently in place, which is important. You discard the difference in light intensity across habitats which was not implemented, for reasons that I didn´t find so convincing. And then there may be another 1000 factors that are imortant for the fish, but that we are not aware of and therefore were not (correctly) manipulated. Perhaps in your paper, mention this possibility -that your experimental test might just not have created the ecological setting that does play out in the field, and therefore that the results are limited. This is fine, that is how science works, and perhaps the hypoteses are indeed wrong. But if you had achieved confirmatory results, the ecological relevance would probably not have to be questioned to the same extent. L 32: add: and thereby increase their ecological performance L 60: is there nothing known about (co)dominance in expression? L 61: as written now, your predictions appear exclusive, which cannot be: they prefer their natural light regime (L 58), and they prefer their rearing light regime. Why not simply state that you will test if, and to what extent, genetic background and rearing regime affect habitat preference? (And then mention the directions re. these effects). L 68: so did you allocate entire families to treatment, or did you split families between treatments (a stronger design)? L 71: is the range 0-5 m the natural range? How do you expose fish to the light of a depth range? It is in the supplement (weighting by depth use), but briefly clarify this here. L 75: this is important, since you might then be testing for novelty avoidance (neofobia), or novelty attraction (neofilia). OK, you discuss this later (L 144). L 92: I was wondering why you didn´t use a binomial model, but there must be temporal autocorrelation in your timing events, so it is hard to know how many independent time event you would have, so the transformation seems indeed better. L 94: activity is an interesting one. Why would activity influence habitat use by itself? More likely, more active fish are exposed to the different environments more, and therefore can choose better (which is probably why you demanded a minimum of four transitions). Or reverse, more active fish move between environments more, therefore not choosing. Either way, I think it is the interactions between activity*species, and activity*rearing environment, that are most interesting, not the main effect of activity. L 94: you don´t include side ("is the shallow light condition on the left or right") as a fixed factor? L 94: what is trial number, and why does this have replicate values (required for a random effect)? Each trial supposedly is unique? If you refer to trial order, then this is not correct -fit it as a fixed effect. L 96: you mention AIC, but then give p-values, which doesn´t make much sense. And what are minimal adecuate models -the outcome of stepwise elimination? Why not just give the results for the full models, testing all effects via loglikelihood ratio tests? L 105: this results sugggests you fitted trail number as a fixed effect, not as a random effect? And you tested an interaction, but you never specified before you would test any interactions -in "data analyses" indicate which interactions you included, and why. L 105: 5 decimals for a p-value is way beyond its reliabilty: I think 3 decimals is precision enough. L 116: I think this is a convoluted way to test an interaction? L 132: genetic signatures? L 138: and perhaps this depends also on competition? It is interesting that in the wild, the deep fish also use shallow waters, but not the reverse, mimicking the overall preference you detected here for shallow conditions perhaps? Might deep fish only use the deep when outcompeted by shallow fish in shallow waters (any field data to test this density-or frequency-dependence?)? If so, and as you already mentioned, perhaps your design was not entirely appropriate to test your hypothesis, as reward was not included. L 149: well, not quite -behavioural preference may depend on several factors (e.g. familiarity versus performance), and may shift over time. L 151: no hypothesis of why all fish preferred shallow light conditions? L 154: maybe add that future experiments should try to increase the importance of relative ecological performance across test environments, or something like that (i.e. greater reward/punishment). Fig. 2: there seems to be a pattern here, I´m surprised it doesn´t come out? But it actually goes against your prediction, right? The deep species prefers shallow water more? Supplementary materials: L 21: there is a strong correlation between age and genetic background. Could the inclusion of age in your models have removed the effect of genetic background? L 60: I´m not so convinced that average difference in light intensity isn´t important because cloud cover variation creates greater changes. Sun set creates even greater changes, but daylight spectrum differences still matter according to you. Perhaps acknowledge in your paper that this aspect was not manipulated, and might change the results. Someone else may try and replicate your study (in other species perhaps), taking this into account. Just as the reward issue. I hope these comments help to further improve an already fine paper, Pim Edelaar.

Author's Response to Decision Letter for (RSOS-181876.R0)
See Appendix A.

Comments to the Author(s)
The authors have done a thorough job in addressing my concerns and applying my suggestions. I have no further comments on the manuscript.

Recommendation? Accept with minor revision (please list in comments)
Comments to the Author(s) I also was a reviewer for the previous version. The authors have responded well to the comments, and the paper has much improved. There are a few minor things that might still need some attention. L 16: maybe say adaptive genetic differentiation L 180: you still have repeat number as a random effect, so basically you are estimating a variance based on three values (and assuming the underlying distribution is normal, for no real good reason). This isn´t really wrong, and it should not change the results much, but it makes more sense to test it as a fixed effect. Especially since you are interested in the specific effect of each level: you later test whether repeat had a different effect for shallow-reared and deep-reared fish (L 192). So that is similar (but less elegant and less correct) to testing for a repeat*rearing environment effect, which you can easily include in your model if you include repeat as a fixed effect. I mentioned this before in my review, but the authors have not picked up on it (and did not understand the last comment about convoluted interaction testing -I hope it is clear now). L 185: don´t start a sentence with a number: Of the 80 trials, 75 were successfull. L 192: p=0.407? Yes, as in supplement. L 215: perhaps also repeat that such a preference might be a contributing driver to adaptive population divergence in the first place, to make clear why this study is very interesting. L 226: future experiments should provide *scope for* such reward ... Of course we should not reward the fish if they choose what we want ("predict") them to choose, but when the phenotype-environment match somehow affects their ecological performance (i.e. they reward themselves). For example, if food is provided on both sides, but they only see it on one side because of their visual capacities. On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-181876.R1 entitled "Visual adaptation and microhabitat choice in Lake Victoria cichlid fish" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
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We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) I also was a reviewer for the previous version. The authors have responded well to the comments, and the paper has much improved. There are a few minor things that might still need some attention. L 16: maybe say adaptive genetic differentiation L 180: you still have repeat number as a random effect, so basically you are estimating a variance based on three values (and assuming the underlying distribution is normal, for no real good reason). This isn´t really wrong, and it should not change the results much, but it makes more sense to test it as a fixed effect. Especially since you are interested in the specific effect of each level: you later test whether repeat had a different effect for shallow-reared and deep-reared fish (L 192). So that is similar (but less elegant and less correct) to testing for a repeat*rearing environment effect, which you can easily include in your model if you include repeat as a fixed effect. I mentioned this before in my review, but the authors have not picked up on it (and did not understand the last comment about convoluted interaction testing -I hope it is clear now). L 185: don´t start a sentence with a number: Of the 80 trials, 75 were successfull. L 192: p=0.407? Yes, as in supplement. L 215: perhaps also repeat that such a preference might be a contributing driver to adaptive population divergence in the first place, to make clear why this study is very interesting. L 226: future experiments should provide *scope for* such reward ... Of course we should not reward the fish if they choose what we want ("predict") them to choose, but when the phenotype-environment match somehow affects their ecological performance (i.e. they reward themselves). For example, if food is provided on both sides, but they only see it on one side because of their visual capacities. Decision letter (RSOS-181876.R2)

05-Mar-2019
Dear Mr Mameri, I am pleased to inform you that your manuscript entitled "Visual adaptation and microhabitat choice in Lake Victoria cichlid fish" is now accepted for publication in Royal Society Open Science.
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Dear Editorial Board and Reviewers,
We have now concluded the revision of our manuscript entitled "Visual adaptation and microhabitat choice in Lake Victoria cichlid fish". We have considered all comments and suggestions by the referees, and we detail below how they were incorporated into the revision.
The main changes include adding information on opsin expression, moving supplementary methods to the main text (particularly details on light treatments), clarifying our data analyses and expanding our Discussion section to address specific explanations for our findings.
We believe the revised version of the manuscript has greatly improved in terms of contents and readability, and we hope that it now meets the standards for publication in Royal Society Open Science.
On behalf of the authors,

Major concerns
Referee #1: In the wild, the species that occupies deeper water is exposed to a red-shifted spectrum but also to an overall lower light intensity. Without knowing the intensity of the two experimental light regimes, it is hard to conclude what aspect of the two light conditions drove the preference to the 'broad spectrum' condition. For example, it might be possible that fish actually preferred the brighter or dimmer light condition.
Reply: Indeed, the deep-water habitat is darker than the shallow-water habitat. This information was included in the supplementary information, but we have now incorporated it in the main ms (lines 146-150): "At Python Islands, light intensity in the deeper P. sp. 'nyererei-like' habitat is about 34% of that in the shallow P. sp. 'pundamilia-like' habitat ( Figure 1). We did not adjust the experimental light spectra to reproduce this difference (intensity in the deep condition was 70% of that in the shallow condition." It is very well possible that the behaviour of the fish, and their preference for either environment, was influenced by this difference light intensity. We did not address this possibility explicitly in the text, but we have now incorporated a few sentences in the Discussion (lines 260-267): "Our light treatments differed in both spectral composition and intensity. Therefore, we cannot establish which of these aspects, or a combination of both, was responsible for the observed variation in visual habitat preference. Previous studies [20,24] have recorded fish preferences for darker or brighter environments, but these employed larger differences between habitats (ranging from two-fold to 20-fold) than we used in the present study (light intensity in the red-shifted condition was 70% of that in the broad-spectrum condition). Independently manipulating both spectral composition and light intensity is feasible, and would constitute a logical next step." Referee #1: What are the characteristics of the experimental light environment in terms of spectrum and intensity? Were light intensities matched to those encountered in the natural habitats? It would be useful to present these data relative to the lighting regimes encountered in the wild. What were the estimated quantum catches of the various opsin types expressed in each of the species?
Reply: Following the answer to the previous comment, we also moved to the main text the verification of the spectra by estimating photon capture (lines 140-145; the Figure is still presented in supplementary material, Figure S2).

Referee #1:
One of the mechanisms of the presumed divergence in visual sensitivity between the species is differential opsin gene expression. However, the authors do not provide any evidence on whether opsin expression differed between the tested species. Is it possible that all fish exhibited similar gene expression as a result of their exposure to the same light condition prior to the beginning of the experiment? How fast such plasticity in opsin gene expression could be?
The authors state (lines 55-57) that they manipulated visual development by raising the fish under different light conditions. However, they provide no evidence for the capacity of such manipulation. In other words, did the different light conditions actually affected the visual sensitivity, and if so, how?
Reply: Opsin expression was not measured in the individuals used for this study. However, we have documented changes in opsin expression in other individuals raised in these same light treatments. We now incorporate this information in the text: Introduction: (lines 61-63): "We have previously shown that these light treatments induce changes in opsin expression in Pundamilia [15]. We therefore predict that the light regime during development influences visual habitat preference as well." Discussion (lines 246-252): "We have previously shown that the light treatments we used here induce changes in opsin expression in Pundamilia [15]: deep-reared fish express less short-wavelength-sensitive opsin (SWS2a) and more long-wavelength-sensitive opsin (LWS). Yet, we did not observe a sustained effect of the rearing environment on preference. Possibly, the induced changes in opsin expression (5-15%) were too subtle to cause behavioural effects. More extreme rearing environments can generate larger changes (e.g. [25]) that would potentially influence visual habitat preference in a more persistent way." We do not know how fast opsin expression can change in response to altered light conditions. We speculate that it takes longer than the 1-hour duration of the experiments, but we cannot rule out that exposure to the experimental conditions had an effect.
We have added a section to the Discussion to address these points (lines 253-259): "An alternative explanation for the lack of a sustained effect of the rearing light treatment, is that the differences in opsin expression were erased during the experimental trials, as fish were exposed to both light conditions during this time. It is unknown how quickly opsin expression can change in response to altered light conditions. Studies in killifish [26] and cichlids [27] suggest that changes can occur within a few days, but most studies have used much longer exposure times (weeks to months). We expect that exposure of 1 hour is too short to induce significant changes, but we cannot rule this out."