The evolution of coloration and opsins in tarantulas

  1. Saoirse Foley
  2. Vinodkumar Saranathan
  3. William H. Piel

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Tarantulas paradoxically exhibit a diverse palette of vivid coloration despite their crepuscular to nocturnal habits. The evolutionary origin and maintenance of these colours remains mysterious. In this study, we reconstructed the ancestral states of both blue and green coloration in tarantula setae, and tested how these colours correlate with presence of stridulation, urtication and arboreality. Green coloration has probably evolved at least eight times, and blue coloration is probably an ancestral condition that appears to be lost more frequently than gained. While our results indicate that neither colour correlates with the presence of stridulation or urtication, the evolution of green coloration appears to depend upon the presence of arboreality, suggesting that it ptobably originated for and functions in crypsis through substrate matching among leaves. We also constructed a network of opsin homologues across tarantula transcriptomes. Despite their crepuscular tendencies, tarantulas express a considerable diversity of opsin genes—a finding that contradicts current consensus that tarantulas have poor colour vision on the basis of low opsin diversity. Overall, our findings raise the possibility that blue coloration could have ultimately evolved via sexual selection and perhaps proximately be used in mate choice or predation avoidance due to possible sex differences in mate-searching.

Article activity feed

  1. eLife

    ##Author Response

    We thank the Editor of eLife f or kindly considering our manuscript for publication and for soliciting three peer reviews. We note that the reviews were positive for the most part. We sincerely believe that the key criticisms arise regrettably from a seeming misunderstanding of the motivation and context of our work – one that we hoped was a candid presentation of available data for tarantulas and the methods used. We provide detailed responses to the reviewers’ concerns below. We further note that our manuscript has since been published with minimal changes (Foley et al. 2020 Proceedings of the Royal Society B 287: 20201688, doi:10.1098/rspb.2020.1688).

    Tarantulas belong to an enigmatic and charismatic group with a nearly cosmopolitan distribution and intriguingly show vivid coloration despite being mostly nocturnal/ crepuscular. Using a robust phylogeny based on a comprehensive transcriptomic dataset that includes nearly all theraphosid subfamilies (except Selenogyrinae), we performed both discrete and continuous ancestral state reconstructions of blue and green coloration in tarantulas using modern phylogenetic methods. Using phylogenetic correlation tests, we evaluated various possible functions for blue and green coloration, for instance aposematism and crypsis. Our results suggest green coloration is likely used in crypsis, while blue (and green) coloration show no correlation with urtication, stridulation or arboreality. Our findings also support a single ancestral origin of blue in tarantulas with losses being more frequent than gains, while green color has evolved multiple independent times but never lost. We comparatively assessed opsin expression from the transcriptomic data across tarantulas to understand the functional significance of blue and green coloration. Our opsin homolog network shows that tarantulas possess a rather diverse suite of regular arthropod opsins than previously appreciated.

    While color vision in (jumping) spiders is relatively well studied, to the best of our knowledge, this is the first study to comparatively consider the identity of opsin expression across tarantulas, and in relation to the evolution of coloration. Our study challenges current belief (e.g., Morehouse et al. 2017 doi: 10.1086/693977 and references therein; Hsiung et al. 2015 doi: 10.1126/sciadv.1500709) that tarantulas are incapable of perceiving colors, at least from a molecular perspective and suggests a role for sexual selection in their evolution. This also adds to the growing body of knowledge on the complexity of arthropod visual systems (e.g., see Futahashi et al. 2015 doi:10.1073/pnas.1424670112, Hill et al. 2002 doi:10.1126/science.1076196).

    In short, we believe our results are timely and pertinent broadly to sensory biologists, behavioural ecologists and evolutionary biologists as it is an exhortation for sorely needed behavioural and sensory experiments to understand proximate use of vivid coloration in this enigmatic group.

    ###Summary:

    This study offers some interesting data and ideas on colour evolution in tarantulas, building upon previous work on this topic. However, the reviewers judged that the insights are too taxon-specific and that several key conclusions are too speculative. There were also concerns about the methodology for trait scoring from photographs that the authors might consider going forward.

    ###Reviewer #1:

    This study investigates the evolution of blue and green setae colouration in tarantulas using phylogenetic analyses and trait values calculated from photographs. It argues that (i) green colouration has evolved in association with arboreality, and thus crypsis, and (ii) blue colouration is an ancestral trait lost and gained several times in tarantula evolution, possibly under sexual selection. It also uses transcriptome data to identify opsin homologs, as indirect evidence that tarantulas may have colour vision.

    Otherwise, a few comments:

    1. Given that data is limited for the family (only 25% of genera could be included in this study), it seemed a shame not to discuss further the variation in colour and habit within genera. Based on Figure 1 and supplementary tables, the majority of "blue" genera contain a mix of blue and not-blue (and not-photographed) species. Does this mean that blue has been lost many more times in recent evolutionary history? And how often are "losses" on your tree likely to be the result of insufficient sampling for the genus (i.e. you happen not to have sampled the blue species)?

    First, the taxa in our robust and well-resolved phylogeny are representative of the major lineages within Theraphosidae, i.e., we have sampled nearly all theraphosid subfamilies (except Selenogyrinae). Our ideal is also to work with a more complete genus-level molecular phylogeny and corresponding color dataset for theraphosidae. However, this group is generally not well represented in museum collections (let alone in digitized collections), while the pet trade is focussed on only a select number of taxa. While we appreciate the reviewer’s concern that adding more taxa and corresponding data could potentially change the results, we believe that with a strong backbone phylogeny recovering the major branches, the results should not change all that much (For instance, cf. Hackett et al. 2008 10.1126/science.1157704 vs. Prum et al. 2016 10.1038/nature19417, where the initial Hackett et al. backbone is robust to increased sampling). Although the way trait losses are concentrated towards the tip suggests that using a genus-level phylogeny would perhaps show a few more recent trait losses, but unlikely to contradict an ancient origin of blue coloration at the base of this group, especially given the way the outgroups are polarized (i.e., outgroups also exhibit blue).

    1. A key conclusion of the study is that sexual selection should not be discarded as a possible explanation for spider colour. However, there is very little detail given in the discussion to build this case. Do these spiders have mating displays that might plausibly include visual signals? How common are sexually-selected colours in spiders generally? Where on the body is the blue coloration (in cases where it is not whole body)? I also missed whether the images used are of males or females or both, or how many species show sexual dimorphism in colouration (mentioned briefly in the Discussion, but not summarised for species or genera).

    We agree with the reviewer that we should have provided more information regarding sexual dichromatism in tarantulas, and on the images we used in the study (whether male/female). However, the location of blue coloration varies wildly with species – some species have blue chelicerae, blue abdomens, or blue carapaces while others are entirely blue. We also know very little about mating (and selection, if any) strategies in tarantulas, let alone the sensory ecology of this group. However, there is intriguing anecdotal information from one species (Aphonopelma) that they can be active as early as 4pm (Shillington 2002 Canadian J. Zoology, 80: 251-259, doi: 10.1139/z01-227), while some species show an intensification of color upon maturation, often a hallmark of sexual selection. Indeed, we believe that our work will incite broad interest on these intriguing questions.

    1. A quick scroll through the amazing images on Rick West's site suggests that oranges and red/pinks are not rare in tarantulas. Perhaps the data is just not available, but it would be good to mention somewhere the rationale behind the blue/green focus, rather than examining all colours.

    We agree. However, in the present study, we focused on blue and green colors because the data is readily available and we wanted to build upon the previous work by Hsiung et al 2015. Given that violet/blue and likely also some green coloration are structural in origin (Saranathan et al. 2015 Nano Letters, doi: 10.1021/acs.nanolett.5b0020; Hsiung et al. 2015), these hues are unlikely to fade or vary between individuals unlike diet acquired pigmentary coloration. Hence, these colors perhaps better lend themselves to analyses using digital photographs.

    I suggest defining stridulating / urticating setae for non-specialist readers. I had to look these up to understand that they were involved in defence.

    We thank the reviewer for this suggestion.

    I notice the Rick West website says species IDs should not be made from photos alone. Is there a risk of misidentification for any photos?

    We understand the reviewer’s concern. However, Rick West is an experienced arachnologist and quite knowledgeable in tarantula systematics and taxonomy (see https://www.tarantupedia.com/researchers/rick-c-west), which is why we endeavoured to use his website as extensively as possible without resorting to photos from hobbyists. We further validated the IDs with field guides, when in doubt.

    The Results section would benefit from some more clear statements of key results. For example, phrases like "AIC values to assess the relationships between greenness and arboreality are reported in Table 3" could be replaced instead with a summary statement indicating what this table shows.

    We agree and thank the reviewer for this suggestion.

    In the Figure 1 caption I think there is a typo: 'the proportions of species with images that possess blue colouration (grey = no available images)" but should this say "grey = not blue"?

    We apologize for the confusion. This is not a typo – this is in relation to Trichopelma, for which no images of described species were available, and so we cannot conclude that none of the taxa are blue/green.

    142 - the lengthy discussion here of whether there is one or more mechanisms by which blue is produced in tarantulas, and the detailed criticism of Hsuing SEMs, seems a bit out of place given that the current study does not investigate the proximate mechanism of blue colouration but merely its presence.

    We respectfully disagree. The core support for Hsiung et al.’s (2015) argument against sexual selection as a driver of color evolution in tarantulas comes from their structural diagnoses of the nanostructures responsible for the violet/blue structural coloration and their subsequent argument that a diversity of divergent nanostructures rather than convergence argues against sexual selection. While it is true that we did not investigate the proximate mechanism of blue coloration here, one of us (Saranathan et al. 2015) has already done so elsewhere. It appears that in insects and spiders, the bulk of the nanostructural diversity is across families and not within.

    Table S6 - It is not clear to me how the values for predicted N orthologs were calculated.

    This is mentioned in line 354 of our methods – “Per the ‘moderate’ criteria from the Alliance of Genome Resources (55), hits may be considered orthologous if three or more of the twelve tools in their suite converge upon that result”.

    The Table S7 caption states: "A * indicates currently undescribed species with blue or green colour that can be confidently attributed to corresponding genus. However, as the described species exhibit no blue or green colour, we conservatively scored these as 0." Is this a conservative approach though? If they have been confidently assigned to genus, I don't understand why they would not be included.

    This refers to the cases where a hitherto undescribed species possesses the blue or green color. However, even though the species has not formally been described, its placement in the genus is not in question. We have not included such undescribed species in our tabulated number of species per genus, as it is difficult to express any such undescribed species as a fraction of the total number of species in that genus.

    ###Reviewer #2:

    This paper presents a broad-ranging overview of tarantula visual pigments in relationship with the color of the spiders. The paper is interesting, well-written and presented, and will inspire further study into the visual and spectral characteristics of the genus.

    We thank the reviewer for her/his/their kind words.

    First a minor remark, Terakita and many others distinguish between opsin, being the protein part of the visual pigment molecule and intact light-sensing, so-called opsin-based pigment, often generalized as a rhodopsin. The statement of line 65, 'convert light photons to electrochemical signals through a signalling cascade' is according to that view strictly not correct. Furthermore, the presence of opsins in transcriptomes may be telling, but it is not at all sure that they are expressed in the eyes, if at all. As the authors well know, in many animal species some of the opsins are expressed elsewhere. It may be informative to mention that.

    We thank the reviewer for this clarification. As for the regions of opsin expression, we very much agree – were it not for constraints of sample availability, we would also have preferred to sequence only the eyes and brain of various tarantulas that were all exposed to similar lighting conditions. However, we encouragingly see that our “leg only” transcriptomes have far fewer (often no) opsins as compared to the whole-body data.

    The blueness or greenness feature prominently in the paper, but the criteria used for determining to which class a spider belongs are not at all sure. The Color Survey and Supplementary Table S2 refer to Birdspiders.com, but that requires a donation; not very welcoming. The other used sources are also not readily giving the insight or overview which material was sampled. I therefore think that the paper would considerably gain in palatability by adding a few exemplary photographs as well as measured spectra. Of course, I am inclined to trust the authors, but I would not immediately take color photographs from the web as the best material for assessing color data with 4-digit accuracy. Furthermore, the accessible photographs do not always show nice, uniform colors, so it might be sensible to mention which body part was used to score the animals. And finally, using CIE metric might infer to many readers that the spiders are presumably trichromatic, like us. Any further evidence?

    We refer to the detailed description of our method for scoring blue or green coloration in tarantulas (l. 277-303). Briefly, we calculated ΔE (CIE 1976) difference values using between the images of each taxa against a suitable reference (average of green leaves, or Haplopelma lividum, the bluest taxa in our survey based on the b value of its images). We use the ΔE Lab values to perform quantitative ancestral state reconstruction, while we use ΔE b (for blue) and ΔE a (for green) to discretize the data for understanding trait gains and losses.

    BirdSpiders.com only requires one to enter names of genera as search terms in order to see photos that we used. However, we agree could have provided some photos of exemplars. We do realise that using pictures is not ideal, as opposed to reflectance spectrophotometry (our ideal as well), which is why we limited ourselves to a single reputable source (BirdSpiders.com) for consistent images, whenever possible. However, acquiring sample material and reflectance of tarantulas is challenging. This group is generally not well represented in museum collections (let along in digitized collections), while the pet trade is focussed on only a select number of taxa and doing field work to collect specimens is fraught with moral and ethical issues (e.g., see https://www.nytimes.com/2019/04/01/science/poaching-wildlife-scientists.html). This study nevertheless represents a substantial improvement upon a recent high-profile work that used the OSX “color picker” function (Hsiung et al. 2015).

    Indeed, available evidence on tarantula vision (including our opsin sequences) suggests tarantulas are likely trichromats (Dahl and Granda 1989 J. Arachnol., Morehouse et al. 2017) similar to jumping spiders (e.g., Zurek et al. 2015, doi: 10.1016/j.cub.2015.03.033), so we consider CIE as an appropriate color space for a putative tristimulus system in tarantulas (see also our response to Reviewer 3). Again, this underscores the need for future studies on the sensory biology and psychophysics of this enigmatic group.

    ###Reviewer #3:

    This neat paper continues the story of structural colour evolution in a group that is rarely appreciated for their ornamentation. The study uses colour & ecological data to model their evolution in a comparative framework, and also synthesises transcriptomic data to estimate the presence and diversity of opsins in the group. The main findings are that the tarantulas are ancestrally 'blue' and that green colouration has arisen repeatedly and seems to follow transitions to arboreality, along with evidence of perhaps underappreciated opsin diversity in the group. It's well-written and engaging, and a useful addition to our understanding of this developing story. I just have a few concerns around methods and the interpretation of results, however, which I feel need some further consideration.

    We thank the reviewer for his/her/their kind words.

    As the authors discuss in detail, this work in many ways parallels that of Hsiung et al. (2015). The two studies seem to agree in the broad-brush conclusions, which is interesting (and promising, for our understanding of the question), though their results conflict in significant ways too. Differences in methodology are an obvious cause, and they are particularly important in studies such as this in which the starting conditions (e.g. the assumed phylogeny or decisions around mapping of traits) so significantly shape outcomes. The current study uses a more recent and robust phylogeny, which is great, and the authors also emphasise their use of quantitative methods to assign colour traits (blue/green), unlike Hsiung et al.

    We thank the reviewer for his/her/their appreciation.

    1. This latter point is my main area of methodological concern, and I am not currently convinced that it is as useful or objective as is suggested. One issue is that the photographs are unstandardised in several dimensions, which will render the extracted values quite unreliable. I know the authors have considered this (as discussed in their supplement), but ultimately I don't believe you can reliably compare colour estimates from such diverse sources. Issues include non-standardised lighting conditions, alternate white-balancing algorithms, artefacts introduced through image compression, differences in the spectral sensitivities of camera models, no compensation for non-linear scaling of sensor outputs (which would again differ with camera models and even lenses), and so on (the works of Martin Stevens, Jolyon Troscianko, Jair Garcia, Adrian Dyer offer good discussion of these and related challenges). Some effort is made to minimise adverse effects, such as excluding the L dimension when calculating some colour distances, but even then the consequences are overstated since the outputs of camera sensors scale non-linearly with intensity, and so non-standardised lighting will still affect chromatic channels (a & b values). So with these factors at play, it becomes very difficult to know whether identified colour differences are a consequence of genuine differences in colouration, or simply differences in white balancing or some other feature of the photographs themselves.

    We thank the reviewer for his/her/their carefully considered thoughts and for drawing our attention to the work of Martin Stevens, Jolyon Troscianko, Jair Garcia, and Adrian Dyer in this regard (e.g. Stevens et al. 2007 Biol. J. Linn. Soc. Lond., doi: 10.1111/j.1095-8312.2007.00725.x). These are fair points raised by the reviewer. We are indeed aware that there are clear drawbacks in working solely with photographs from online sources as opposed to optical reflectance data (our ideal), but we are sure that the reviewer appreciates how challenging it is to source specimens of tarantulas. It is for this reason that we restricted ourselves to photographs from mostly only 1 reputable source (BirdSpiders.com). Furthermore, this is why we chose a perceptual model that permits device independent color representation, one that lets us separate chromatic variables from brightness, keeping in mind the underlying assumptions. However, some recent research suggests that CIELab space can perform reasonably well as compared to the latest algorithms for illuminant-invariant color spaces (Chong et al. 2008 ACM Transactions on Graphics, doi: 10.1145/1360612.1360660). Please also see our response below (to point #2) and also to Reviewer #2 above.

    Given the dearth of tarantula specimens and in the absence of spectrometry, future work will have to try and acquire uncompressed original images (with EXIF data) and could perform image processing such as homomorphic filtering and adaptive histogram equalization (Pizer et al. 1987 Computer Vision, Graphics, and Image Processing; Gonzalez and Woods 2018 Digital Image Processing, Pearson) in order to further mitigate artefacts such as those arising from differences in illumination, especially if using images from a diversity of sources.

    1. The justification for some related decisions are also unclear to me. The CIE-76 colour distance is used, and is described as 'conservative'. But it is not so much conservative as it is an inaccurate model of human colour sensation. It fails to account for perceptual non-uniformity and actually overestimates colour differences between highly chromatic colours (like saturated blues). The authors note they preferred this to CIE-2000, which is a much better measure in terms of accuracy, because the latter was too permissive (line 300). I understand the problem, and appreciate their honesty, but this decision seems very arbitrary. If the goal is to quantitatively estimate colour differences according to human viewers, then the metric which best estimates our perceptual abilities would strike me as most appropriate. Also, the fact that all species would be classified as 'blue' using the CIE-2000, when some of them are obviously not blue by simply looking at them, is consistent with the kinds of image-processing issues noted above. I only focus on this general point because it is offered as a key advance on previous work (L 40-41), but I don't think that is clearly the case (though I agree that the scoring methods of Hsiung et al. are quite vague). I'm generally in favour of this sort of quantitative approach, but here I wonder if it wouldn't be simpler and more defensible to just ask some humans to classify images of spiders as either 'blue' or 'green', since that seems to be the end-goal anyway.

    We agree that CIE 1976 is an inaccurate model of “human color sensation,” but at the same time the degree of their applicability or lack thereof to non-human tristimulus visual systems is not clear. In any case, the digital photographs do not preserve UV information anyway. We hasten to add CIE 1976 is still widely used in color science and engineering research for its simplicity and perceptual uniformity, as a simple Google Scholar search would attest. We believe that the reviewer is perhaps mistaken as to our motivation for choosing the CIE 1976 and the exact nature of the shortcomings of the CIE 1976 model, which it turns out to be an unintended advantage. Our goal was not, as the reviewer suggests, to just “quantitatively estimate color differences according to human viewers,” but to do so in a device independent fashion given the constraints of working with already available digital images, and for a putative trichromat visual system. Given there are technically no limits for a and b values in the CIE 76 space, color patches with high values of chroma are computed to have too strong a difference than in actual fact (Hill et al. 1997 ACM Transactions on Graphics, 16, 109-154). This is precisely the kind of situation that we do not face here, as we are essentially comparing shades of blue rather than for instance, chromatic contrasts between saturated blue vs. green or blue vs. red. Moreover, we only use the rectilinear rather than the polar coordinate representation of the colors (in other words, we do not compute the psychometric correlates, chroma Cab, or the hue angle hab). Contrary to the reviewer’s assertion that the CIE 1976 “overestimates color differences between highly chromatic colors (like saturated blues),” a quick perusal of Table S3 affirms that a comparison of highly saturated blues such as between our “standard” H. lividum and Poecilotheria metallica reveals they are quite close in terms of chromatic contrasts (i.e., small E values). Moreover, CIE 1994 and subsequent revisions rely on a von Kries-type transformation to account for non-uniformity of the perceptual space, but as the reviewer is well aware, without an accurate idea of the illumination conditions, use of CIE 2000 is not justified.

    Lastly, we are sure the reviewer appreciates that asking humans to manually score the colors of images (e.g. Hsiung et al. 2015) is neither reproducible nor enables quantitative analyses of trait evolution.

    1. L26-27, 53-56, 171-176: This is a more minor point than the above, but some of the discussion and logic around hypothesised functions could be elaborated upon, given it's presented as a motivating aim of the text (52-56). The challenge with a group like this, as the authors clearly know, is that essentially none of the ecological and behavioural work necessary to identify function(s) hasn't been done yet, so there are serious limitations on what might be inferred from purely comparative analyses at this stage. The (very interesting!) link between green colouration and arboreality is hypothesised and interpreted as evidence for crypsis, for example, but the link is not so straightforward. Light in a dense forest understory is quite often greenish (e.g. see Endler's work on terrestrial light environments) including at night which, when striking a specular, structurally-coloured green could make for a highly conspicuous colour pattern - especially achromatically (which is what nocturnal visual predators would often be relying on). This is particularly true if the substrate is brown rotten leaves or dirt, in which case they could shine like a beacon. Conversely, if the blue is sufficiently saturated and spectrally offset from the substrate it could be quite achromatically cryptic at dusk or night. To really answer these questions demands information on the viewers, viewing conditions, visual environment etc. The point being that it is a bit too simplistic to observe that, to a human, spiders are green and leaves on the forest floor may be green, and so suggest crypsis as the likely function (abstract L 22-23). So inferences around visual function(s) could either be toned down in places given the evidence at hand or shored up with further detail (though I'm not sure how much is available).

    We agree. Indeed, we are limited by the absence of rigorous behavioural studies. With this in mind, we have already made every effort to tone down and emphasize that our results might point towards a given function, but we do not claim it outright. It is our fervent hope that these findings will form the basis for future behavioural studies by giving researchers a starting point to test their hypotheses.

    We would like to point out that the association we uncovered is actually between arboreal taxa and the presence of green coloration and not as the reviewer says “spiders are green and leaves on forest floor may be green.” These taxa live in natural crevices on trees, shrubs and essentially spend their lives arboreally. Also, green coloration in tarantulas need not be structural in origin (see e.g., Saranathan et al. 2015) and this is why to test for crypsis against foliage, we used (pigmentary) leaves as the representative model for comparison to tarantula green colors. Although, certain lycaenid butterflies (Saranathan et al. 2010 10.1073/pnas.0909616107; Michielsen et al. 2010 10.1098/rsif.2009.0352), for instance, use structural coloration to better aid in crypsis against foliage.

    Minor comments:

    • I'm not familiar enough with with methods for creating homolog networks to comment in detail, but the use of BLASTing existing opsin sequences against transcriptomes seems straightforward enough. As do the methods for phylogenetic reconstruction.

    We agree this is straightforward.

    • L48: What constitutes a 'representative' species? And how reasonable is it to assign a value for such a labile trait to an entire genus? I understand we can only do our best of course and simplifications need to be made, but I can imagine many cases among insects (e.g. among butterflies and flies) where genus-level assignments would be meaningless due to the immense diversity of structural colouration among species (including in terms of simple presence/absence).

    Please see our response to Reviewer 2 above.

    • Line 168: Wouldn't this speak against a sexual function? Only in a tentative way of course, but the presence of conspicuous structural colouration in juveniles, which is absent in adults, would suggest a non-sexual origin to me.

    The reviewer’s inference is incorrect. We do not suggest that blue coloration is present in juveniles but absent in adults, but only that such conspicuous colors already appear in the penultimate moult right before the male creates a sperm web and is ready for mating.

  2. Version published to 10.1098/rspb.2020.1688
  3. eLife

    ###Reviewer #3:

    This neat paper continues the story of structural colour evolution in a group that is rarely appreciated for their ornamentation. The study uses colour & ecological data to model their evolution in a comparative framework, and also synthesises transcriptomic data to estimate the presence and diversity of opsins in the group. The main findings are that the tarantulas are ancestrally 'blue' and that green colouration has arisen repeatedly and seems to follow transitions to arboreality, along with evidence of perhaps underappreciated opsin diversity in the group. It's well-written and engaging, and a useful addition to our understanding of this developing story. I just have a few concerns around methods and the interpretation of results, however, which I feel need some further consideration.

    As the authors discuss in detail, this work in many ways parallels that of Hsiung et al. (2015). The two studies seem to agree in the broad-brush conclusions, which is interesting (and promising, for our understanding of the question), though their results conflict in significant ways too. Differences in methodology are an obvious cause, and they are particularly important in studies such as this in which the starting conditions (e.g. the assumed phylogeny or decisions around mapping of traits) so significantly shape outcomes. The current study uses a more recent and robust phylogeny, which is great, and the authors also emphasise their use of quantitative methods to assign colour traits (blue/green), unlike Hsiung et al.

    1. This latter point is my main area of methodological concern, and I am not currently convinced that it is as useful or objective as is suggested. One issue is that the photographs are unstandardised in several dimensions, which will render the extracted values quite unreliable. I know the authors have considered this (as discussed in their supplement), but ultimately I don't believe you can reliably compare colour estimates from such diverse sources. Issues include non-standardised lighting conditions, alternate white-balancing algorithms, artefacts introduced through image compression, differences in the spectral sensitivities of camera models, no compensation for non-linear scaling of sensor outputs (which would again differ with camera models and even lenses), and so on (the works of Martin Stevens, Jolyon Troscianko, Jair Garcia, Adrian Dyer offer good discussion of these and related challenges). Some effort is made to minimise adverse effects, such as excluding the L dimension when calculating some colour distances, but even then the consequences are overstated since the outputs of camera sensors scale non-linearly with intensity, and so non-standardised lighting will still affect chromatic channels (a & b values). So with these factors at play, it becomes very difficult to know whether identified colour differences are a consequence of genuine differences in colouration, or simply differences in white balancing or some other feature of the photographs themselves.

    2. The justification for some related decisions are also unclear to me. The CIE-76 colour distance is used, and is described as 'conservative'. But it is not so much conservative as it is an inaccurate model of human colour sensation. It fails to account for perceptual non-uniformity and actually overestimates colour differences between highly chromatic colours (like saturated blues). The authors note they preferred this to CIE-2000, which is a much better measure in terms of accuracy, because the latter was too permissive (line 300). I understand the problem, and appreciate their honesty, but this decision seems very arbitrary. If the goal is to quantitatively estimate colour differences according to human viewers, then the metric which best estimates our perceptual abilities would strike me as most appropriate. Also, the fact that all species would be classified as 'blue' using the CIE-2000, when some of them are obviously not blue by simply looking at them, is consistent with the kinds of image-processing issues noted above. I only focus on this general point because it is offered as a key advance on previous work (L 40-41), but I don't think that is clearly the case (though I agree that the scoring methods of Hsiung et al. are quite vague). I'm generally in favour of this sort of quantitative approach, but here I wonder if it wouldn't be simpler and more defensible to just ask some humans to classify images of spiders as either 'blue' or 'green', since that seems to be the end-goal anyway.

    3. L26-27, 53-56, 171-176: This is a more minor point than the above, but some of the discussion and logic around hypothesised functions could be elaborated upon, given it's presented as a motivating aim of the text (52-56). The challenge with a group like this, as the authors clearly know, is that essentially none of the ecological and behavioural work necessary to identify function(s) hasn't been done yet, so there are serious limitations on what might be inferred from purely comparative analyses at this stage. The (very interesting!) link between green colouration and arboreality is hypothesised and interpreted as evidence for crypsis, for example, but the link is not so straightforward. Light in a dense forest understory is quite often greenish (e.g. see Endler's work on terrestrial light environments) including at night which, when striking a specular, structurally-coloured green could make for a highly conspicuous colour pattern - especially achromatically (which is what nocturnal visual predators would often be relying on). This is particularly true if the substrate is brown rotten leaves or dirt, in which case they could shine like a beacon. Conversely, if the blue is sufficiently saturated and spectrally offset from the substrate it could be quite achromatically cryptic at dusk or night. To really answer these questions demands information on the viewers, viewing conditions, visual environment etc. The point being that it is a bit too simplistic to observe that, to a human, spiders are green and leaves on the forest floor may be green, and so suggest crypsis as the likely function (abstract L 22-23). So inferences around visual function(s) could either be toned down in places given the evidence at hand or shored up with further detail (though I'm not sure how much is available).

    Minor comments:

    -I'm not familiar enough with with methods for creating homolog networks to comment in detail, but the use of BLASTing existing opsin sequences against transcriptomes seems straightforward enough. As do the methods for phylogenetic reconstruction.

    -L48: What constitutes a 'representative' species? And how reasonable is it to assign a value for such a labile trait to an entire genus? I understand we can only do our best of course and simplifications need to be made, but I can imagine many cases among insects (e.g. among butterflies and flies) where genus-level assignments would be meaningless due to the immense diversity of structural colouration among species (including in terms of simple presence/absence).

    -Line 168: Wouldn't this speak against a sexual function? Only in a tentative way of course, but the presence of conspicuous structural colouration in juveniles, which is absent in adults, would suggest a non-sexual origin to me.

  4. eLife

    ###Reviewer #2:

    This paper presents a broad-ranging overview of tarantula visual pigments in relationship with the color of the spiders. The paper is interesting, well-written and presented, and will inspire further study into the visual and spectral characteristics of the genus.

    First a minor remark, Terakita and many others distinguish between opsin, being the protein part of the visual pigment molecule and intact light-sensing, so-called opsin-based pigment, often generalized as a rhodopsin. The statement of line 65, 'convert light photons to electrochemical signals through a signalling cascade' is according to that view strictly not correct. Furthermore, the presence of opsins in transcriptomes may be telling, but it is not at all sure that they are expressed in the eyes, if at all. As the authors well know, in many animal species some of the opsins are expressed elsewhere. It may be informative to mention that.

    The blueness or greenness feature prominently in the paper, but the criteria used for determining to which class a spider belongs are not at all sure. The Colour Survey and Supplementary Table S2 refer to Birdspiders.com, but that requires a donation; not very welcoming. The other used sources are also not readily giving the insight or overview which material was sampled. I therefore think that the paper would considerably gain in palatability by adding a few exemplary photographs as well as measured spectra. Of course, I am inclined to trust the authors, but I would not immediately take color photographs from the web as the best material for assessing color data with 4-digit accuracy. Furthermore, the accessible photographs do not always show nice, uniform colors, so it might be sensible to mention which body part was used to score the animals. And finally, using CIE metric might infer to many readers that the spiders are presumably trichromatic, like us. Any further evidence?

  5. eLife

    ###Reviewer #1:

    This study investigates the evolution of blue and green setae colouration in tarantulas using phylogenetic analyses and trait values calculated from photographs. It argues that (i) green colouration has evolved in association with arboreality, and thus crypsis, and (ii) blue colouration is an ancestral trait lost and gained several times in tarantula evolution, possibly under sexual selection. It also uses transcriptome data to identify opsin homologs, as indirect evidence that tarantulas may have colour vision.

    Otherwise, a few comments:

    1. Given that data is limited for the family (only 25% of genera could be included in this study), it seemed a shame not to discuss further the variation in colour and habit within genera. Based on Figure 1 and supplementary tables, the majority of "blue" genera contain a mix of blue and not-blue (and not-photographed) species. Does this mean that blue has been lost many more times in recent evolutionary history? And how often are "losses" on your tree likely to be the result of insufficient sampling for the genus (i.e. you happen not to have sampled the blue species)?

    2. A key conclusion of the study is that sexual selection should not be discarded as a possible explanation for spider colour. However, there is very little detail given in the discussion to build this case. Do these spiders have mating displays that might plausibly include visual signals? How common are sexually-selected colours in spiders generally? Where on the body is the blue coloration (in cases where it is not whole body)? I also missed whether the images used are of males or females or both, or how many species show sexual dimorphism in colouration (mentioned briefly in the Discussion, but not summarised for species or genera).

    3. A quick scroll through the amazing images on Rick West's site suggests that oranges and red/pinks are not rare in tarantulas. Perhaps the data is just not available, but it would be good to mention somewhere the rationale behind the blue/green focus, rather than examining all colours.

    Minor comments:

    I suggest defining stridulating / urticating setae for non-specialist readers. I had to look these up to understand that they were involved in defence.

    I notice the Rick West website says species IDs should not be made from photos alone. Is there a risk of misidentification for any photos?

    The Results section would benefit from some more clear statements of key results. For example, phrases like "AIC values to assess the relationships between greenness and arboreality are reported in Table 3" could be replaced instead with a summary statement indicating what this table shows.

    In the Figure 1 caption I think there is a typo: 'the proportions of species with images that possess blue colouration (grey = no available images)" but should this say "grey = not blue"?

    142 - the lengthy discussion here of whether there is one or more mechanisms by which blue is produced in tarantulas, and the detailed criticism of Hsuing SEMs, seems a bit out of place given that the current study does not investigate the proximate mechanism of blue colouration but merely its presence.

    The Table S7 caption states: "A * indicates currently undescribed species with blue or green colour that can be confidently attributed to corresponding genus. However, as the described species exhibit no blue or green colour, we conservatively scored these as 0." Is this a conservative approach though? If they have been confidently assigned to genus, I don't understand why they would not be included.

    Table S6 - It is not clear to me how the values for predicted N orthologs were calculated.

  6. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    This study offers some interesting data and ideas on colour evolution in tarantulas, building upon previous work on this topic. However, the reviewers judged that the insights are too taxon-specific and that several key conclusions are too speculative. There were also concerns about the methodology for trait scoring from photographs that the authors might consider going forward.

  7. Version published to 10.1101/2020.04.25.061366v1 on bioRxiv