Repeated and widespread evolution of biofluorescence in marine fishes

Domain: earth_science
Published: 2025-05-24
Editorial outcome: minor_revision

Abstract
Biofluorescence, the absorption of high-energy light and its reemission at lower energy wavelengths, is widespread across vertebrate and invertebrate lineages, especially fishes. New observations over the past decade have significantly increased our understanding of the diversity and multifunctionality of fluorescence in fish lineages. In this study, we present a comprehensive account of all known biofluorescent teleosts and estimate the timing and frequency of the evolution of biofluorescence across this diverse group. We show that biofluorescence evolved numerous times in marine teleosts and is estimated to date back ~112 mya in Anguilliformes (true eels). Of the 459 known biofluorescent teleosts reported in this study, the majority are associated with coral reefs. We find that reef-associated species evolve biofluorescence at 10x the rate of non-reef species. Our results suggest that the chromatic and biotic conditions of coral reefs could have provided an ideal environment to facilitate the evolution and diversification of biofluorescence in teleost fishes.

Introduction
Biofluorescence results from the absorption of higher-energy light and its reemission at longer, lower-energy wavelengths by living organisms1. It is a widespread phenomenon across the tree of life and occurs in most major clades of vertebrates1–9. Biofluorescence is found in both terrestrial and marine organisms, although these environments have vastly different lighting conditions. Terrestrial ecosystems are brightly lit by sunlight, which contains a wide spectrum of wavelengths spanning the visible light spectrum. Alternatively, vast portions of the photic ocean are characterized by a relatively monochromatic, blue-shifted environment10. As sunlight hits and enters oceanic waters, longer wavelengths (yellow, orange, red) are rapidly absorbed, resulting in a limited bandwidth of blue light (470–480 nm) by around 150 m depth (frequently much shallower depending on water clarity)10. Thus, the ability to absorb shorter wavelength ambient blue light at depth and reemit it as longer wavelengths through the use of fluorescent compounds may be advantageous to marine organisms to increase visibility and contrast amidst the more monochromatic blue environment of the ocean1.

Biofluorescence is phylogenetically pervasive, yet it remains unknown how much of this potential visual signal is biologically relevant and serves a functional role, versus an anatomical byproduct (e.g., enamel)4,11. For example, carnivorous pitcher plants (Nepenthaceae and Sarraceniaceae) fluoresce along the lip of the pitcher, which attracts insect prey2,12. Sexual dimorphism in green biofluorescence and ultraviolet (UV) reflectance is thought to aid in the mating rituals of jumping spiders (Salticidae)3. In marine fishes, where fluorescent emissions mainly occur in the green to red portions of the visible spectrum, biofluorescence has been implicated in camouflage, communication, species identification, mating, and prey attraction1,13–15. The Pacific spiny lumpsucker (Eumicrotremus orbis) exhibits sexually dichromatic fluorescent emission colors from the body that may enhance mate identification, whereas fluorescence of the pelvic disc in both males and females is thought to be utilized for signaling13. Biofluorescence is also notably prevalent in coral reef ecosystems. Scleractinian corals exhibit red and green fluorescence, which may increase contrast at depth, provide photoprotection for symbionts, and provide visual cues for other reef organisms16–19. Some reef fishes may utilize biofluorescent corals and marine algae for camouflage. Scorpionfishes (Scorpaenidae) and threadfin breams (Nemipteridae) have been observed residing on or near backgrounds with similar fluorescent emission wavelengths to their bodies1. Other reef fishes may be using biofluorescence for intraspecific signaling, including closely related species of reef lizardfishes (Synodontidae) that appear nearly identical under white light, but exhibit significant variation in fluorescent patterning1. The potential multifunctional roles of biofluorescence may be linked to the increased rates of diversification of coral reef fishes20–22.

These potential visual functions of biofluorescence all require that fluorescent emissions lie within the spectral sensitivity of relevant signal-receivers: conspecifics, predators, and/or prey1. Shallow water reef fishes often have relatively good color vision with two or three visual pigments, allowing them to navigate the chromatically complex ecosystem of coral reefs23. Although most reef fishes are sensitive to shorter wavelengths (blue), some species (e.g., Pomacentridae and Labridae) exhibit long-wavelength sensitivity as high as 600 nm (red)24. In addition, members of many families of marine fishes have been reported to possess yellow intraocular lenses that function as long-pass filters and can facilitate the visualization of longer fluorescent wavelengths1,25. Behavioral experiments support fluorescent recognition for sex identification in the fairy wrasse (Cirrhilabrus solorensis)26. Whereas, direct testing of the visual system in the swell shark (Cephaloscyllium ventriosum) and chain catshark (Scyliorhinus rotifer) revealed that fluorescence functions to increase luminosity contrast with the background environment and between skin patches at depth14. However, our knowledge of the visual capabilities of biofluorescent fishes is extremely limited23, as is our understanding of fluorescent molecules. Green fluorescent proteins (GFP), similar to the GFP that was first isolated from the hydrozoan Aequorea victoria27, have only been isolated and characterized in three species of Anguilliformes (true eels)28–30. Smaller fluorescent metabolites were found to be responsible for the green fluorescent emissions in elasmobranchs14, whereas no red fluorescent molecules have yet been isolated from fishes despite the prevalence of red fluorescence across Teleostei.

Given the various proposed functions of biofluorescence and the visual capabilities of marine fishes, further research into its evolution and diversification is crucial. Previous investigations of the phylogenetic distribution of biofluorescence in teleosts found that the phenomenon is both phylogenetically widespread and phenotypically variable across the assemblage1,31,32. However, no recent work has accounted for and incorporated information from the huge increase in biofluorescent teleost diversity that has been documented within the past decade.

Herein we investigate the early evolution of biofluorescence in Teleostei, determine when the phenomenon first evolved in this group, reconstruct the evolutionary patterns of fluorescent emission colors in various lineages, and report on the number of times the phenomenon is known to have independently evolved across this huge and remarkably diverse (>30,000 species) vertebrate assemblage. We also investigate whether the rise and global expansion of modern coral reefs contributed to the incredible diversity of biofluorescence we observe across modern teleost lineages.

Results
Species-level survey of biofluorescence
We report 48 previously unreported biofluorescent teleost species based on observations of recently collected specimens (Supplementary Data 2), exhibiting red (11 spp.), green (32 spp.), and both red and green (5 spp.) fluorescent emissions. We found 413 currently known biofluorescent teleost species across peer reviewed literature1,13,15,26,28,31–44. Combined with our new records, this results in a total of 459 biofluorescent teleost species spanning 87 families and 34 orders (Supplementary Data 1). Of these, fluorescent emissions are red only in 261 species, green only for 150 species, and both red and green for 48 species. We also recorded a total of 469 species where fluorescence was not observed (Supplementary Data 1).

Ancestral state reconstructions of biofluorescence
Biofluorescence was present in 267 of the 613 total species in the trimmed Rabosky et al.45 time-calibrated phylogeny, spanning 79 families (Fig. 1). The best-fit Mk models were both the equal-rates and the all-rates-different models, hence they were model-averaged during stochastic character mapping proportional to their Akaike model weights (67.9% and 32.1%, respectively). The root node of the tree (192.8 mya) likely exhibited an absence of fluorescence and had a posterior probability of 33.6% for biofluorescence presence. The oldest node with fluorescence (66.8% posterior probability) was the ancestor of all Anguilliformes (~112 mya) (Fig. 1A). The next oldest nodes where biofluorescence is present are ~104 mya in Syngnathiformes and ~87 mya in Perciformes45, which had posterior probabilities of 79.3% and 82.5% for biofluorescence presence, respectively (Fig. 1C,D). From the root, a mean of 178.9 changes occurred between states. Of these, ~101 were from absence to presence of biofluorescence, and ~78 were from presence to absence. These changes were almost proportional to the mean total time spent in each state: 55% (12,921.24 my) and 45% (10,571.26 my) for presence and absence, respectively.

Ancestral state reconstructions of biofluorescent emission color
Of the 267 biofluorescent teleost species in the Rabosky et al. trimmed phylogeny, fluorescent color was red only in 142 species, green only in 99 species, and both red and green in 26 species (Supplementary Data 3, Fig. 2). The evolution of red and green fluorescence was best described by a corHMM model with two rate classes, both with an all-rates-different model and without dual transitions. Green first evolved in the ancestor of Anguilliformes (Fig. 2A), with node likelihoods of 62% for green fluorescence only, 14.6% for red fluorescence only, and 22.2% for both red and green fluorescence. The most recent common ancestor of Synodus (Aulopiformes: Synodontidae; Fig. 2B) exhibited a majority node likelihood for both red and green fluorescence (54.1%), followed by red only (40.3%) and green only (5.1%). The most recent common ancestor of Pseudocheilinus + Cirrhilabrus (Labriformes: Labridae; Fig. 2C) had node likelihoods of 83.4% for red fluorescence only and 15.4% for no fluorescence. However, the most recent common ancestor of Cheilinus (Labriformes: Labridae; Fig. 2D) had a majority node likelihood of 92.0% for only green fluorescence only. The node likelihoods were 94.3% for red fluorescence in Antennariidae (Lophiiformes; Fig. 2E) and 91.2% for green fluorescence in Nemipteridae (Spariformes; Fig. 2F).

In Perciformes45, the ancestor of the entire order (Fig. 2G) exhibited node likelihoods of 68.9% for red fluorescence only and 28.9% for no fluorescence. However, two clades of Perciformes had different majority character states. Node likelihoods for Cyclopteridae + Liparidae (Perciformes; Fig. 2H) were 56.8% for both red and green fluorescence, 39.5% for red fluorescence only, and 3.4% for green fluorescence only, while sculpins (Rhamphocottidae + Agonidae + Psychrolutidae; Fig. 2I) had a 90.9% likelihood for green fluorescence only and 5.2% for no fluorescence. The ancestor of Syngnathiformes (Fig. 2J) had node likelihoods of 57.2% for red fluorescence only, 39.3% for no fluorescence, and 3.4% for green fluorescence only. However, the likelihoods for the most recent common ancestor of Hippocampus (Syngnathiformes; Fig. 2K) was 54.8% for both red and green fluorescence and 45.1% for red fluorescence only. Two clades of Gobiiformes had majority likelihoods for the evolution of red fluorescence only. The node likelihoods were 95.3% for red fluorescence only and 4.6% for no fluorescence in one clade of gobiid genera (Pleurosicya + Gobiodon + Bryaninops + Paragobiodon + Eviota; Fig. 2L), and 68.6% for red fluorescence only and 31.1% for both red and green fluorescence in another clade of Gobiidae (Gobiodon, Fig. 2M). The ancestor of Pleuronectiformes (Fig. 2N) had node likelihoods of 65.5% for red fluorescence only, 22.9% for both red and green fluorescence, 8.7% for no fluorescence, and 2.8% for green fluorescence only.

Diversification analysis of biofluorescence and reef association
The best fitting SSE models were character-independent models, indicating that factors besides fluorescence and reef-association influence lineage diversification. The best fitting models were the MuCID model with seven and eight hidden states. Average net diversification rates, informed by the ancestral state reconstructions, were similar (r = 0.029–0.031) with overlapping standard deviations across all character state combinations (Fig. 3B). However, based on transition rates, the rate of change between character states, teleost fishes on reefs evolved fluorescence at a rate 10x faster than species off reefs (Fig. 3A, bolded arrows; Fig. 4; Supplementary Fig. 3). However, reef fishes also lost fluorescence at a rate 1.38× faster than non-reef fishes. Additionally, fishes with fluorescence transitioned to living off reefs at a rate 2.6× faster than they transitioned on reefs (Fig. 3A).

Methods
Species-level survey of biofluorescence
Research, collecting, and export permits were obtained from the government of the Bahamas, from the Ministry of Fisheries and Ministry of Environment, Honiara, Solomon Islands, and from the Department of Environment, Cayman Islands Government. Additional permits include Florida Atlantic University’s Institutional Animal Care and Use Committee (IACUC, protocols A16‐34 and A19-36), Florida Fish and Wildlife Conservation Commission Special Activity Licenses (SAL-18-1889-SRP and SAL-18-1785A-SRP), and Florida Department of Environmental Protection permit 09101815. All data supporting the findings of this study are available within the paper and its supplementary files.

We compiled a comprehensive list of all known fluorescent teleosts via a literature search from peer-reviewed descriptions of biofluorescence (Supplementary Data 1) in addition to our own observations of recently collected material (Supplementary Data 2). Live and frozen specimens used for examination and imaging (n=107) were collected in the Solomons Islands in 2012, 2013, and 2019, Greenland in 2019, the Bahamas (Exumas) in 2011 and 2012, the Cayman Islands in 2016, Southern California, USA (San Diego) in 2015 and 2016, near-shore locations in Washington, USA in 2022, Florida, USA in 2022, Thailand (Gulf of Thailand and Andaman Sea) in 2024, and obtained through the aquarium trade (Supplementary Data 2). Additional fluorescence imaging was conducted at the Mystic Aquarium, Mystic, CT, USA in 2011 and 2012, and at the Birch Aquarium, Scripps Institution of Oceanography, La Jolla, CA, USA in 2015 and 2016.

For each fluorescent species, we recorded the color of fluorescent emission: either red (590-750 nm), green (515–570 nm), or both. For specimens that we examined directly, fluorescent emission colors were first judged by eye using a variety of blue excitation lights and scientific-grade long-pass filters. Specimens collected in Washington were examined for fluorescence using a NIGHTSEA Stereo Microscope Fluorescence illuminator with a Royal Blue (RB) head (440–460 nm; Supplementary Fig. 1) and a scientific grade long-pass filter (500 nm). Specimens in Florida were examined for fluorescence using a SeaLife Sea Dragon Mini Fluoro light (440–457 nm; Moorestown, NJ, USA; Supplementary Fig. 1) and a Semrock scientific grade 514 nm long-pass filter. For all other specimens, emission spectra were elicited via illumination with Royal Blue LED lights collimated to ensure perpendicular incidence on the scientific grade 450–470 nm interference filter surface (Omega Optical, Inc., Brattleboro, VT, USA) and fluorescence was confirmed via use of either 514 nm or 561 nm long-pass filters (Semrock, Rochester, NY). An Ocean Optics USB2000+ portable spectrophotometer (Dunedin, FL, USA) equipped with a hand-held fiber optic probe (Ocean Optics ZFQ-12135) was then used to record fluorescence emission spectra. Emission spectra were elicited via illumination with Royal Blue LED lights collimated to ensure perpendicular incidence on the scientific grade 450-470 nm interference filter surface (Omega Optical, Inc., Brattleboro, VT, USA; Semrock, Inc., Rochester, NY, USA). Emission spectra were recorded by placing the fiber optic probe near specific anatomical parts of the individual fish specimen exhibiting fluorescence. This was repeated several times for each specimen and each anatomical region to ensure the accuracy of measurements. Peak emissions (lambda-max) were used to confirm fluorescent emission colors (Supplementary Data 2).

Fluorescent macrophotography
For specimen imaging, we placed individual fish in a narrow photographic tank and gently held them flat against the thin glass front. We imaged fluorescent emissions in a dark room using a Nikon D800 or D4 DSLR camera outfitted with a Nikon 60 or 105 mm macro lens, or a Sony A7SII camera outfitted with a Sony 90 mm macro lens. We covered the flashes (Nikon SB910) with blue interference band pass excitation filters (Omega Optical, Inc.) and attached a long-pass (LP) emission filter (Semrock, Inc.) to the front of the camera lens to record any emitted fluorescence. To best capture the fluorescent emissions we tested multiple LP filter pairs. For example, a 514 nm LP filter was used to capture green fluorescence, whereas a 561 nm LP filter was used to image longer-wavelength fluorescence (orange and red) and to block any emitted green fluorescence in species with both fluorescent colors (i.e., overlapping fluorescent pigments).

Phylogenetic comparative methods
To assess the number of times fluorescence has evolved across Teleostei, we used the time-calibrated Rabosky et al.45 phylogeny. This tree includes 32 of the 34 orders of known biofluorescent teleosts45 (Supplementary Data 1). To avoid gaps in fluorescent data, we trimmed this dated phylogeny to only include species that have been investigated for biofluorescence (Supplementary Data 1). The resulting trimmed tree contained 613 species, including 85% of the 87 known biofluorescent teleost families and 58% of the 461 currently known biofluorescent species (Supplementary Data 1, 3; Supplementary Fig. 2).

Ancestral state reconstructions of biofluorescence
The evolution of fluorescence (presence/absence) was first examined in R60 by fitting four extended Mk models of evolution: equal-rates, all-rates-different, and two irreversible trait models (presence to absence only; absence to presence only) using the fitMk function in phytools 2.061 (Supplementary Code 1). Ancestral state reconstructions were then performed with stochastic character mapping (nsim = 1000) under the best-fit Mk model (determined by weighted AIC of the maximum likelihood estimates) using the simmap function61. The reconstructions were summarized to determine transition rates and total time spent in each state using phytools 2.061. We considered an ancestral node to likely be fluorescent if the posterior probability for presence was >60%.

Ancestral state reconstructions of biofluorescent emission color
We also reconstructed the evolutionary history of red and green fluorescence across teleosts. We used the corHMM R package version 2.162 to incorporate hidden rate classes by leveraging hidden Markov models63 (Supplementary Code 1). These models allow for variation in transition rates throughout the tree and are commonly used for analyses of large clades spanning long evolutionary timeframes63. We fit equal-rate, symmetric, and all-rates-different transition rate models with and without a hidden rate category. We also fit hybrid hidden rates models where one rate class was an equal-rates model and the other was an all-rates different model. We fit all models with and without dual transitions for a total of 14 models. Weighted AICc values were used to determine the best-fitting model and calculate the model-averaged ancestral state likelihood values. The maximum likelihood estimates of some transition rates sat on a likelihood ridge and led to the estimation of unrealistically high transition rates. However, although high transition rates negatively influence stochastic character mapping, they have little effect on marginal ancestral state reconstruction64; as a result, the marginal ancestral state reconstructions were used.

Diversification analysis of biofluorescence and reef association
To investigate whether biofluorescence on coral reefs promotes increased diversification rates in marine teleosts, we implemented several Multicharacter Hidden State Speciation and Extinction (MuHiSSE) models using the hisse R package version 2.1.1165 (Supplementary Code 1). All species with biofluorescence presence/absence data were also coded as being reef associated or not using the classifications from Larouche et al.66, resulting in a total of 479 reef-associated species and 134 non-reef species (Supplementary Data 3). We fit a total of 10 models to the dataset. First, we ran a null MuSSE model with the same turnover parameter for all state combinations. Then we ran a “true” MuSSE model, where the turnover rate parameters differed between each fluorescence-habitat state combination. We also ran one character-dependent (MuHiSSE) and seven character-independent (MuCID) models that incorporated hidden states. Hidden states are used to account for unknown factors besides fluorescence and reef-association that may affect transition rates and lineage diversification. The MuHiSSE model included one hidden state, turnover rates were allowed to vary across the different states, and transition rate parameters were allowed to vary across hidden states. We ran MuCID models for two through eight hidden states, where the turnover rates were unlinked from the observed states and the transition rate parameters were constrained to be identical across hidden states. Note, for all models we used a single extinction fraction parameter, which was kept constant across all observed and hidden states. To account for incomplete phylogenetic sampling, we estimated sampling fractions for each fluorescence-habitat state (non-fluorescent/non-reef, fluorescent/non-reef, non-fluorescent/reef-associated, fluorescent/reef-associated). We first used the proportion of fluorescent fishes in the dataset reported in this study (Supplementary Data 1) to estimate the number of fluorescent and nonfluorescent species in the Larouche et al.66 dataset, which assigns a character state of reef-associated or non-reef for 3339 marine species. We then estimated sampling fractions based on the proportion of species sampled in the trimmed Rabosky et al.45 phylogeny with each fluorescence-habitat state out of the full Larouche et al.66 dataset. We also constrained the root state to be non-reef associated based on the findings of previous studies that marine teleostean fishes originated in non-reef habitats21,66.

We identified the best-fitting models using weighted AIC values, which estimated the transition rates between character states. We then used the transition rates to conduct marginal ancestral state reconstructions with the hisse R package. The results of the ancestral state reconstructions were summarized by calculating the tip-associated net diversification rates by model-averaging parameters from all models that contributed >5% of the AICc weight. Transition rates measure how frequently a trait is gained or lost (i.e., the rate of change between character states). A higher transition rate towards the loss of a character state, for example, indicates that a lineage is more likely to lose that character over evolutionary time. Diversification rates indicate the rate that new species evolve. If diversification rates are higher in lineages with a certain character state, that character state is likely contributing to the proliferation of species over time. The net diversification rates were visualized using the gghisse package version 0.1.1 (https://github.com/discindo/gghisse). We plotted the number of biofluorescent lineages through time in R60 by assigning character states (reef-associated vs non-reef) based on majority node probabilities.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The study by Carr and colleagues documents all know instances of fluorescence in fishes across the fish tree of life, and examines it in a phylogenetic context. Their ultimate conclusions are that coral reefs facilitated the diversification of fluorescent acanthomorphs and furthermore that fluorescence could be a key innovation leading to the diversification of these acanthomorph species when used for intraspecific signaling.\nAfter examining their provided supplemental files and re-reading their publication, I have several concerns for this manuscript that I will outline here. My primary concern deals with the sampling approach, both literature and new species. The majority of existing literature on fluorescent fishes used in this study focus on coral reef fishes already (for example Anthes et al. 2016 and De Brauwer et al. 2017 either primarily focused on reefs or exclusively worked in the coral triangle for the latter), and their additional sampling approaches primarily involve coral reef regions and taxa (Solomon Islands, Bahamas, Cayman Islands, Florida, and the aquarium trade - which based on their supplemental files appears reef fish focused). While there is some effort to include non-reef taxa, it pales in comparison to the amount of effort put in to examining reef taxa. Therefore, it should not be surprising then that the authors primarily find fluorescence in reef taxa, as those are primarily the taxa they sample. As a simple thought experiment, say the authors examined an equal number of freshwater fishes (some included here, but very few) and found it prevalent in those groups. Then the overall outcome would look very different and it would not appear as though reefs were special in this regard. However, the list of definitive non-fluorescent fishes is still focused on many reef fish taxa, and is not widespread throughout the tree of life that would give greater confidence to their findings. It is likely that fluorescence is far more common than appreciated in fishes, but that it is seldomly examined due to the cost of equipment to do so and the need to examine it prior to preservation.\nAnother concern of mine is the construction of the phylogeny used. There is little justification to why you are building your own phylogeny using 7 loci versus using many of the more comprehensive phylogenies available, either in terms of taxa or amount of loci, especially as you are primarily collapsing branches down to the family level, as many other trees could be adapted for this purpose even if they don't contain the species of interest as tips. As the construction of this phylogeny is critical for your inference of the evolution of this trait, this is something that is very important. The dates on the tree that you recover, which directly impact the interpretation of your result, are strange compared to those published in the literature. For example, you recover Labridae (an important family in your study that you mention several times) with a crown age of nearly 150 million years ago. This is far older than many other studies (Betancur-R et al., 2017 estimates less than 100mya; Ghezelayagh et al., 2022 estimates less than 80mya), and is far older than the fossil record of this family (see Bellwood et al., 2019). Unfortunately, without an accurately dated tree it is difficult to conclude with confidence when fluorescence arose and diversified. While the authors do state that they examine their data in the context of the Rabosky tree, they do not show how this impacted (or didn't) their accumulation over time analysis.\nOther minor concerns of mine include a lack of explanation for why they assume an equal-rates model of evolution for this trait (might be accurate, but there should be some justification), why they only examined fluorescence via Royal Blue LED lights (other excitation lighting will also trigger fluorescence in fishes and many fishes live at depths where more than just blue wavelengths of light occur), and why the authors don't examine their data with a more complex model, such as a Misse model which could take into account fluorescence and presence/absence of reef dwelling within a species. I think that the approach taken over-simplifies fluorescence in fishes, which is far more complex with many family containing a mixture of species that have or do not have it, and some species have ontogenetic shifts in the color and/or presence/absence of this trait. Lastly, one of the authors concluding statements is that fluorescence could serve as a key innovation for diversification of reef fishes, especially when associated with infraspecific communication, yet they don't examine when it's used in the context of communication vs camouflage or some other purpose at all, puzzling me to why they state that in the communication context this would be a key innovation.\nI think that this manuscript overall highlights how little we ultimately know about fluorescence in fishes. While I appreciate the effort to further document this phenomenon across many species, there is still a long way to go before we can identify causal patterns and attribute function to this. A broader sampling and strong effort to examine fluorescence in non-reef habitats and taxa would certainly be welcome.", "reviewer_2": "I recommend that this manuscript be accepted, pending minor revisions. This is an informative update to Sparks et al. 2014 and gives valuable new information on the presence and type of biofluorescence in a number of taxa previously not reported. Most of my comments are minor. The only serious issue I have is that the claim that their results demonstrate a significant rise in reef associated biofluorescent species in parallel with a rise in coral species. This increase coincided with an increase in reef associated fishes of all types after the K-T extinction, so the proliferation of biofluorescent species may just be the result of a general burst of diversification and not specifically tied to biofluorescence. The authors need to show that this rise is statistically distinct from the overall increase of speciation on reefs that occurred since the start of the Tertiary. Without this, it would be premature to say that biofluorescence is a key innovation driving this rapid diversification.\nMethods\nIn addition to the percentages given for how many families/species of biofluorescent taxa were represented in the dataset, it would be helpful to list raw numbers of both what was sampled and how many are known to be biofluorescent. Although this information is in Table S1 and some appears later in the Results, a summary at this point in the manuscript would be useful. Also, state how many orders show biofluorescence and provide a citation.\nWhen 89% is given for how complete the matrix is, please elaborate on how that was calculated. Was it by aligned base pairs, by gene, etc? For example if only 200 bp of cytb was available for a species, was cytb considered “complete” for that species?\nIf the journal follows the formatting of the software makers, then IQ-TREE is all caps with a hyphen. Also please be more specific about which version of the software was used (e.g. the version as of June 26 is 2.3.5). The citation (33) for iqtree2 should appear with its first mention, after talking about ModelFinder.\nResults\nWhen listing the number of species with different kinds of biofluorescence, some more details are necessary. Based on the numbers, it seems like the 45 species that have both red and green are each also included in the red biofluorescence category and again in the green category (316+198=514 and 514-45=469). That makes sense but is not immediately clear. It might be better to enumerate those that are red only, green only, and both, so that each category is mutually exclusive.\nRelated to the above, the numbers don’t add up for the number of biofluorescent families in the Ancestral State Reconstruction section, where 58 families show biofluorescence, of which 35 are red, 48 are green and 27 are both. Using the same math as above, 35+48-27=56, not 58. If this is accurate, please explain the difference of two families.\nThe Phylogenetic Analysis section only discusses the monophyly of one family (Aulopidae), two orders (Anguilliformes and Aulopiformes), and two supraordinal groups, Acanthomorpha and Percomorpha. This seems arbitrary and too brief. The authors should mention the monophyly or non-monophyly of other major groups.\nWhen discussing orders and other higher level groups, especially Perciformes, it is necessary to cite which classification is being followed. There are different fish classifications in the last few years that have significantly altered the limits of many traditional taxon names like Perciformes.\nDiscussion\nIt would be better to reword the number of biofluorescent taxa as “occurring in 26 orders, 96 families, and over 460 species” so that “over” unambiguously applies to just species.\nExplain what is meant by assemblage in “underestimate the true phylogenetic diversity within the assemblage” (biofluorescent reef fishes?)\nExplain how “highly prevalent and exceptionally phenotypically variable in four diverse assemblages” was determined. Was there a percentage or threshold that warranted this designation?\nCyclopteridae is said to be in Fig. 1E but I could not find it in the figure and no representative of the family is listed in Table S3.\nWhile some species occur in intertidal pools, Liparidae is not exactly a family that comes to mind as an example of shallow water fishes.\nBlenniiformes are said to be in Fig. 1C “Gobiiformes, Blenniiformes, and Syngnathiformes (Fig. 1C).” but that would contradict an earlier statement: “The third major clade includes Pseudochromidae, Gobiescocidae, and Blenniformes (Fig. 1D).” Also, that order appears to be in 1D.\nPlural fishes should be used for “Thus, many reef fishes inhabit areas”\nAs adjectives, scorpaenids and nemipterids should not be capitalized\nPlease cite some examples to support the statement “Whereas Synodontidae is recovered as nonmonophyletic in our phylogenetic reconstruction, this topology is common in studies of Aulopiformes” and explain what “this topology” means. Does that mean the non-monophyly of Synodontidae is common in other studies?\nRefer to them as the Rabosky et al. dataset and Rabosky et al. phylogeny, not just Rabosky dataset or Rabosky phylogeny.\nWhen discussing the distribution of red vs. green vs. both “Green fluorescence occurs independent of red in 16 groups, whereas red fluorescence is only independent of green in five groups”, it would help the reader keep track if you explained what these groups are (from the figures, they seem to vary in taxonomic rank with some being single families, others being multiple families, others being orders, etc.) and state how many biofluorescent groups there are of each. From the manuscript, there are 16 green only groups, 5 red only groups, but how many have both and how many total?\nDelete “of” in “Many of coral reef fishes that possess opsins” or add “the” after “Many of”\nWith the number of authors, please specify who is responsible for a pers. obs.\nProvide a reference for the first evolution of bioluminescence in the following: “predating the evolution of bioluminescence in marine fishes by over 30 million years.”\nWhen making the following statement (We have shown that a significant increase in biofluorescent reef-associated families and species parallels the rise and diversification of modern coral reefs beginning in the early Eocene), it implies that there is statistical evidence showing the increase is significant, but no statistical tests were run to demonstrate that the increase in biofluorescence is more than the general background increase in actinoptergyian diversity that coincided with the start of the Cenozoic, where there was an explosion of reef associated fishes in general.\nReferences\nCapitalization of article titles is inconsistent, for example (1) Sparks et al. 2014 vs. (2) Kurup et al. 2013.\nDOIs are only given for (10) and (57)\nMany scientific names in titles should be italicized: (7) (26) (29) (46) (47) (48) (49) (53) (65) (67) (68) (69)\n(9) Citation looks like it’s incomplete\n(35) Citation looks incomplete or copy and pasted from Google\n(58) Article title is in all caps\nOther\nAdd Table before S1, S2, S9\nAdd Figure before S5, S8, S10\ntime calibrated should be hyphenated in “time-calibrated phylogeny”\nreef associated should be hyphenated in “reef-associated acanthomorph families”\nBlenniiformes is misspelled as “Blenniformes” twice\nFigure 1\nWhy is there a large gap in fish photos between Chlorophthalmidae and Aulostomidae? There are two biofluorescent families (Zeidae and Holocentridae) in that part of the tree that could have been included.\nFigure 1 legend\nI do not know if the journal style requires it, but the statement starting “Major clades” is not a complete sentence.\nFigure 5 legend\nAs a proper noun with an adjective form, Scleractinia should not be used as an adjective, instead it should be “scleractinian coral”. As an adjective, “acanthomorph” should not be capitalized.\nTable S1\nFistulariidae is misspelled as “Fistulariiidae”\nGobiesocidae is misspelled as “Gobiescocidae”\nEuprymna berryi (Sepiolidae) is a cephalopod\nTable S3\nTypeface is inconsistent, especially COI column\nSeveral taxa (Lateolabrax_japonicus, Lepisosteus_oculatus, Mugil_cephalus, Oncorhynchus_mykiss) are not italicized. If using italics, remove underscore between genus and species names.\nFigure S10\nThe typeface is inconsistent between the x-axis and y-axis. The right edge of the parts A and B, representing the present (0 mya) are not aligned.\nIn part A, the x-axis extends beyond the y-axis and the right edge of the graph.\nIn part B, the shaded gray area representing cumulated species is separated from the x-axis by a small gap and the dotted line representing the K-T boundary extends past the x-axis. The shaded area does not start at the origin of the y-axis. The tick marks for the y-axis increments are misaligned and extend into the figure space.", "reviewer_3": "Emily Carr and co-authors present a highly intriguing and well-founded study on the evolutionary history and phylogenetic distribution of 'green' and 'red' biofluorescence among ray-finned fish. This study achieves unprecedented coverage across fish phylogeny, establishing a critical reference point for future research to better understand the evolution and adaptive functions of biofluorescence within a phylogenetic framework. Notably, it is the first to estimate the timing of biofluorescence origins in various fish lineages and connects these with a compelling hypothesis: that the greatest diversification of biofluorescence may have coincided with the emergence and diversification of shallow water reefs.\nGeneral Comments\n(a) Origins and Timing of Fluorescence\nThe reconstructed phylogenetic origins for red and green fluorescence are largely convincing, but I have some concerns regarding the earliest estimated origins (Synodontidae at 184 mya—origin B in Figure 1, followed by Albulidae and Labridae). I was surprised by the high confidence in placing the origin of biofluorescence at the base of these branches rather than somewhere along the branch leading to the single descendant ‘biofluorescent clade’, each containing only a single OTU (family). Can we confidently exclude the possibility that the origin of biofluorescence occurred (much) later along these terminal branches in such cases? Moreover, what ages would be estimated for the earliest origin of biofluorescence using the Rabosky phylogeny?\n(b) Frequency of Independent Evolution Events\nThe study estimates 35 independent evolutions, with an even higher count using the Rabosky dataset. Could the authors evaluate / discuss whether these estimates may be biased upwards due to sampling gaps? For instance, in the large clade containing Acanthuridae, Nemipteridae, Antennariidae and others, with many independent evolutionary transitions, how might the picture change if more extensive sampling in families like Leiognathiidae, Zanclidae, Lophidae or others would reveal new instances of biofluorescence?\n(c) Methods for Detecting Biofluorescence\nThe criteria for distinguishing fluorescence from faint residual reflection during flash photography could benefit from further clarification. For example, did the authors use lambda-max emission peaks to identify cases with peak fluorescence within the defined red or green spectral ranges? Where emission spectra standardized relative to a reflection standard?\nAs a suggestion, I would find it useful if the supplement could contain spectra of the light sources and filters for future comparison. Moreover, would it be possible (and useful?) to publish lambda-max values for each measured species in the online supplement?\n(d) Fluorescence in Shallow Waters\nI agree with the discussion's assertion that shallow waters offer particularly favorable conditions for biofluorescence compared to deep waters. However, to avoid misinterpretation by readers, I suggest noting that fluorescence is likely overwhelmed by full-spectrum sunlight in the upper few meters, likely limiting its contribution compared to reflection. Fluorescence may primarily offer advantages under specific conditions such as for short-distance communication, in shaded habitats, or around dusk and dawn. Earlier visual models for just noticeable differences (JND) in achromatic contrast suggested that red fluorescent signals for intraspecific communication only become detectable at depths below approximately 10 m depth in a triplefin species (ref. 69).\n(e) Biofluorescence as a Driver of Diversification\nWhile biofluorescence is a strong candidate as a driver of fish diversification, I suggest a word of caution when comparing it to bioluminescence. Bioluminescence can generate quite strong contrasts in deep-sea environments or shallow waters at night, whereas the use of fluorescence is inherently restricted to diurnal conditions within the photic zone (see comment (d) above).\nMinor Comments\n- Introduction (earlier work): The study context and the current understanding of biofluorescence functions are well framed. One section touches on earlier work examining the phylogenetic diversity and distribution of biofluorescence among fish. For my taste, this part could integrate refs. 42 and Michiels et al. 2008 (https://link.springer.com/article/10.1186/1472-6785-8-16), which, along with refs. 1 and 13, are the few studies to date to examine fish biofluorescence and its potential functions on a broader phylogenetic scale.\nIntroduction (last paragraph): Review the use of the word 'incredible'—it appears redundant and could be removed.\n- Discussion: Regarding the variation in red emission wavelengths within lineages, this has been documented extensively in ref. 42, which shows evidence for up to three distinct emission peaks within single species and significant variation among individuals.\nSigned: Nils Anthes"}, "author_response": "Response to Reviewers\nThe authors would like to thank the reviewers for their helpful feedback on our\nmanuscript. Per your suggestions, we have made major changes to our analyses\nsummarized here:\n1) We now focus our study on the time calibrated phylogeny from Rabosky et al.\n2018. This tree has much greater taxonomic sampling (>30,000 tips) than our\nprevious tree and is informed by 139 fossil calibrations compared to the 19 fossil\ncalibrations used in our previous topology. The Rabosky phylogeny also has\nmore accurate dating of major clades and provides a better estimate for the\norigin of biofluorescence, which we now place at the ancestor of all\nAnguilliformes (~112 mya).\n2) We replaced the three figures showing the evolution of each emission color\n(former Figs. 2-4) with one figure (Fig. 2), analyzing red only, green only, and\nboth red and green fluorescent emissions as a polymorphic trait. We find that\ngreen fluorescence evolves first in Anguilliformes, but red fluorescence is much\nmore prevalent across the teleost phylogeny.\n3) We replaced the less robust Acanthomorpha figure (former Fig. 5) with a\ndiversification analysis (MuHisse) of the traits biofluorescence and reef\nassociation (Fig. 3). Whereas we find that neither of these characters is the main\ndriver of diversification in our tree, reef-associated teleost species evolve\nbiofluorescence at 10x the rate of non-reef species. This supports our hypothesis\nthat reef habitats could have provided the chromatic and biotic conditions\nnecessary to facilitate the evolution of fluorescence in marine teleosts.\nReviewer #1 (Remarks to the Author):\nThe study by Carr and colleagues documents all know instances of fluorescence in\nfishes across the fish tree of life, and examines it in a phylogenetic context. Their\nultimate conclusions are that coral reefs facilitated the diversification of fluorescent\nacanthomorphs and furthermore that fluorescence could be a key innovation leading to\nthe diversification of these acanthomorph species when used for intraspecific signaling.\nAfter examining their provided supplemental files and re-reading their publication, I have\nseveral concerns for this manuscript that I will outline here. My primary concern deals\nwith the sampling approach, both literature and new species. The majority of existing\nliterature on fluorescent fishes used in this study focus on coral reef fishes already (for\nexample Anthes et al. 2016 and De Brauwer et al. 2017 either primarily focused on\nreefs or exclusively worked in the coral triangle for the latter), and their additional\nsampling approaches primarily involve coral reef regions and taxa (Solomon Islands,\nBahamas, Cayman Islands, Florida, and the aquarium trade - which based on their\nsupplemental files appears reef fish focused). While there is some effort to include non-\nreef taxa, it pales in comparison to the amount of effort put in to examining reef taxa.\nTherefore, it should not be surprising then that the authors primarily find fluorescence in\nreef taxa, as those are primarily the taxa they sample. As a simple thought experiment,\nsay the authors examined an equal number of freshwater fishes (some included here,\nbut very few) and found it prevalent in those groups. Then the overall outcome would\nlook very different and it would not appear as though reefs were special in this regard.\nHowever, the list of definitive non-fluorescent fishes is still focused on many reef fish\ntaxa, and is not widespread throughout the tree of life that would give greater\nconfidence to their findings. It is likely that fluorescence is far more common than\nappreciated in fishes, but that it is seldomly examined due to the cost of equipment to\ndo so and the need to examine it prior to preservation.\nThe reviewer brings up a valid concern regarding sampling technique. The authors have\ninvestigated numerous freshwater families/species and almost all are not fluorescent,\nwith the few exceptions reported in Table S1. However, because time in the field is\nlimited and fluorescent proteins/molecules begin to breakdown after collection (i.e.,\nfluorescent species must be imaged and have emission spectra taken soon after\ncollection), nonfluorescent species are often not collected or given species IDs. We\nhave, however, investigated many freshwater groups for biofluorescence, and\naccordingly have added a list of nonfluorescent freshwater teleost families to Table\nS1(Freshwater nonfluorescent groups) based on previous observations by John Sparks.\nCoral reefs are more often investigated for biofluorescence because freshwater systems\nare simply lacking in fluorescent taxa, especially fishes. This is not a sampling bias, but\na well-known trend in biofluorescent fishes.\nAnother concern of mine is the construction of the phylogeny used. There is little\njustification to why you are building your own phylogeny using 7 loci versus using many\nof the more comprehensive phylogenies available, either in terms of taxa or amount of\nloci, especially as you are primarily collapsing branches down to the family level, as\nmany other trees could be adapted for this purpose even if they don't contain the\nspecies of interest as tips. As the construction of this phylogeny is critical for your\ninference of the evolution of this trait, this is something that is very important. The dates\non the tree that you recover, which directly impact the interpretation of your result, are\nstrange compared to those published in the literature. For example, you recover\nLabridae (an important family in your study that you mention several times) with a crown\nage of nearly 150 million years ago. This is far older than many other studies (Betancur-\nR et al., 2017 estimates less than 100mya; Ghezelayagh et al., 2022 estimates less\nthan 80mya), and is far older than the fossil record of this family (see Bellwood et al.,\n2019). Unfortunately, without an accurately dated tree it is difficult to conclude with\nconfidence when fluorescence arose and diversified. While the authors do state that\nthey examine their data in the context of the Rabosky tree, they do not show how this\nimpacted (or didn't) their accumulation over time analysis.\nWe originally constructed our phylogeny based on the methods of Davis et al. 2016 to\ncompare the origin/evolution of biofluorescence to bioluminescence, which was the\nfocus of the Davis et al. 2016 study. However, we agree that the dates of some groups,\nincluding Labridae, are older than reported in more recent studies. To provide more\naccurate dates for the evolution of biofluorescence, we now use a trimmed Rabosky\ntree for the main analyses of this study. This tree also has much more comprehensive\nsampling with over 30,000 tips and 139 fossil calibrations. To avoid gaps in\nbiofluorescent sampling, we trimmed the Rabosky tree to include only species that have\nbeen investigated for biofluorescence. The resulting phylogeny includes 267 fluorescent\nand 346 nonfluorescent teleost species (Fig. 1).\nOther minor concerns of mine include a lack of explanation for why they assume an\nequal-rates model of evolution for this trait (might be accurate, but there should be\nsome justification),\nPlease refer to our new methods where this concern is addressed:\n“The evolution of fluorescence presence/absence was first examined by fitting four\nextended Mk models of evolution: equal rates, all-rates-different, and two irreversible\ntrait models (presence to absence only; absence to presence only) using the fitMk\nfunction in phytools 2.035. Ancestral state reconstructions were then performed with\nstochastic character mapping (nsim=1000) under the best fit Mk model (determined by\nweighted AIC of the maximum likelihood estimates) using the simmap function35.”\nwhy they only examined fluorescence via Royal Blue LED lights (other excitation lighting\nwill also trigger fluorescence in fishes and many fishes live at depths where more than\njust blue wavelengths of light occur),\nBlue light is a standard excitation wavelength for biofluorescence in teleosts (Anthes et\nal. 2016, Sparks et al. 2014, Cohen and Summers 2022). While some species do\nexhibit fluorescence under UV excitation light (Marshall et al. 2019), this is rare and\nbeyond the scope of our study, which highlights the significance of a blue ambient\nenvironment for the evolution and diversification of biofluorescence in marine teleosts.\nSome studies that focus solely on red fluorescence do use green excitation light\n(Michiels et al. 2008). However, this overlaps with any green fluorescent emission\nwavelengths and is not useful for a study that includes both red and green fluorescent\nwavelengths. Thus, using blue excitation light is appropriate and common for wide scale\nstudies on biofluorescence in fishes and will elicit fluorescence for all longer\nwavelengths (green-red).\nand why the authors don't examine their data with a more complex model, such as a\nMisse model which could take into account fluorescence and presence/absence of reef\ndwelling within a species. I think that the approach taken over-simplifies fluorescence in\nfishes, which is far more complex with many family containing a mixture of species that\nhave or do not have it, and some species have ontogenetic shifts in the color and/or\npresence/absence of this trait.\nThank you for your comment, which makes a valid point. To investigate the evolution of\nbiofluorescent color (wavelength), we now use hidden Markov models. See new\nmethods:\n“We also reconstructed the joint evolutionary history of red and green fluorescence\nacross teleosts. We used the corHMM R package version 2.136 to incorporate hidden\nrate classes by leveraging hidden Markov models37. We fit equal rate, symmetric, and\nall-rates different transition rate models with and without a hidden rate category. We\nalso fit hybrid hidden rates models where one rate class was an equal rates model, and\nthe other was an all-rates different model…”\nTo investigate the relationship between biofluorescence and reef association, we added\na new analysis using a MuHisse model. For this analysis, please see new methods\nunder “Diversification Analysis of Biofluorescence and Reef Association”\nLastly, one of the authors concluding statements is that fluorescence could serve as a\nkey innovation for diversification of reef fishes, especially when associated with\ninfraspecific communication, yet they don't examine when it's used in the context of\ncommunication vs camouflage or some other purpose at all, puzzling me to why they\nstate that in the communication context this would be a key innovation.\nThis statement was based on the previous hypothesis that bioluminescence is a key\ninnovation in the diversification of deep-sea fishes when used for communication via\nspecies-specific signaling (Davis et al. 2016). However, after adding the new\ndiversification analysis with biofluorescence and reef association, we find that another\nfactor besides these two traits is driving diversification in our teleost phylogeny. The text\nstating that biofluorescence could serve as a key innovation has been removed and\nreplaced with the following statement based on the resulting transition rates from the\ndiversification analysis: “We show that reef-associated species evolve fluorescence at\n10x the rate of non-reef species and, therefore, that the rise of modern coral reefs in the\nearly Eocene could have provided the abiotic and biotic conditions necessary to\nfacilitate the evolution and diversification of biofluorescence in marine teleosts.”\nI think that this manuscript overall highlights how little we ultimately know about\nfluorescence in fishes. While I appreciate the effort to further document this\nphenomenon across many species, there is still a long way to go before we can identify\ncausal patterns and attribute function to this. A broader sampling and strong effort to\nexamine fluorescence in non-reef habitats and taxa would certainly be welcome.\nWhile we agree that greater sampling efforts are needed for biofluorescence across\nTeleostei, our methods reduce the effects of sampling disparities and provide useful\ninformation regarding the evolution of biofluorescence in fishes. This study does not\nattribute function to biofluorescence, but simply discusses current hypotheses\npreviously stated in the literature. The goal of this study is stated at the end of the\nintroduction:\n“We herein investigate the early evolution of biofluorescence in Teleostei, determine\nwhen the phenomenon first evolved in this group, reconstruct the evolutionary patterns\nof fluorescence emission colors in various lineages, and report the number of times the\nphenomenon is known to have independently evolve across this incredibly diverse\nvertebrate assemblage. We also investigate whether the rise and global expansion of\nmodern coral reefs contributed to the incredible diversity of biofluorescence we observe\nacross modern teleost lineages.”\nReviewer #2 (Remarks to the Author):\nI recommend that this manuscript be accepted, pending minor revisions. This is an\ninformative update to Sparks et al. 2014 and gives valuable new information on the\npresence and type of biofluorescence in a number of taxa previously not reported. Most\nof my comments are minor. The only serious issue I have is that the claim that their\nresults demonstrate a significant rise in reef associated biofluorescent species in\nparallel with a rise in coral species. This increase coincided with an increase in reef\nassociated fishes of all types after the K-T extinction, so the proliferation of\nbiofluorescent species may just be the result of a general burst of diversification and not\nspecifically tied to biofluorescence. The authors need to show that this rise is\nstatistically distinct from the overall increase of speciation on reefs that occurred since\nthe start of the Tertiary. Without this, it would be premature to say that biofluorescence is\na key innovation driving this rapid diversification.\nThank you for your comment. To investigate the relationship between biofluorescence\nand reef association, we added a new analysis using a MuHisse model. See new\nmethods under “Diversification Analysis of Biofluorescence and Reef Association”. This\nanalysis concluded that another factor besides biofluorescence or reef association is\ndriving diversification in our teleost phylogeny. The text stating that biofluorescence\ncould serve as a key innovation has been removed and replaced with the following\nbased on transition rates from the diversification analysis: “We show that reef-\nassociated species evolve fluorescence at 10x the rate of non-reef species and,\ntherefore, that the rise of modern coral reefs in the early Eocene could have provided\nthe abiotic and biotic conditions necessary to facilitate the evolution and diversification\nof biofluorescence in marine teleosts.”\nMethods\nIn addition to the percentages given for how many families/species of biofluorescent\ntaxa were represented in the dataset, it would be helpful to list raw numbers of both\nwhat was sampled and how many are known to be biofluorescent. Although this\ninformation is in Table S1 and some appears later in the Results, a summary at this\npoint in the manuscript would be useful. Also, state how many orders show\nbiofluorescence and provide a citation.\nTo provide an estimate for the number of new species sampled for biofluorescence, we\nadd a sample size to the following text “Live and frozen specimens used for examination\nand imaging (n=107)…” However, it is difficult to provide sample size for species\npreviously reported as biofluorescent in the literature because many studies do not\ninclude this information.\nThe number of biofluorescent species, families, and orders were added to this section\nand the text now reads: “This tree includes 32 of the 34 orders of known biofluorescent\nteleosts (Table S1). To avoid gaps in fluorescent data, we trimmed this dated phylogeny\nto only include species that have been investigated for biofluorescence (Table S1). The\nresulting trimmed tree contained 613 species, including 85% of the 87 known\nbiofluorescent teleost families and 58% of the 461 currently known biofluorescent\nspecies (Tables S1 and S4; Fig. S5).”\nWhen 89% is given for how complete the matrix is, please elaborate on how that was\ncalculated. Was it by aligned base pairs, by gene, etc? For example if only 200 bp of\ncytb was available for a species, was cytb considered “complete” for that species?\nWe no longer use this phylogeny for the analyses in this study. We provide the following\ninformation about the Rabosky et al. tree: “To assess the number of times\nbiofluorescence has evolved across ray-finned fishes, we used the dated Rabosky et al.\nphylogeny which included 31,517 species and was informed by 139 fossil calibrations.”\nFurther details can be found in the original publication (Rabosky et al. 2018).\nIf the journal follows the formatting of the software makers, then IQ-TREE is all caps\nwith a hyphen. Also please be more specific about which version of the software was\nused (e.g. the version as of June 26 is 2.3.5). The citation (33) for iqtree2 should appear\nwith its first mention, after talking about ModelFinder.\nThese methods have been removed.\nResults\nWhen listing the number of species with different kinds of biofluorescence, some more\ndetails are necessary. Based on the numbers, it seems like the 45 species that have\nboth red and green are each also included in the red biofluorescence category and\nagain in the green category (316+198=514 and 514-45=469). That makes sense but is\nnot immediately clear. It might be better to enumerate those that are red only, green\nonly, and both, so that each category is mutually exclusive.\nThe numbers for biofluorescent taxa listed here are now mutually exclusive and the text\nnow reads “Combined with our new records, this results in a total of 468 biofluorescent\nteleost species spanning 96 families and 26 orders (Table S1). Of these, fluorescent\nemissions are only red in 261 species, only green for 150 species, and both red and\ngreen for 48 species.”\nRelated to the above, the numbers don’t add up for the number of biofluorescent\nfamilies in the Ancestral State Reconstruction section, where 58 families show\nbiofluorescence, of which 35 are red, 48 are green and 27 are both. Using the same\nmath as above, 35+48-27=56, not 58. If this is accurate, please explain the difference of\ntwo families.\nThank you for pointing out this typo. This text has been replaced with the results of the\nnew analysis on the Rabosky et al. phylogeny:\n“Of the 267 biofluorescent teleost species in the Rabosky et al. trimmed phylogeny,\nfluorescent color was only red in 142 species, only green in 99 species, and both red\nand green in 26 species (Table S4, Fig. 2).”\nThe Phylogenetic Analysis section only discusses the monophyly of one family\n(Aulopidae), two orders (Anguilliformes and Aulopiformes), and two supraordinal\ngroups, Acanthomorpha and Percomorpha. This seems arbitrary and too brief. The\nauthors should mention the monophyly or non-monophyly of other major groups.\nBased on the feedback from all reviewers, we now use a trimmed Rabosky et al.\nphylogeny for the main analyses of this study. The structure and monophyly of this tree\nis discussed in detail in Rabosky et al. 2018. The “Phylogenetic Comparative Methods”\nsection now reads:\n“To assess the number of times biofluorescence has evolved across teleosts, we used\nthe time-calibrated Rabosky et al.32 phylogeny. This tree includes 32 of the 34 orders of\nknown biofluorescent teleosts (Table S1). To avoid gaps in fluorescent data, we trimmed\nthis dated phylogeny to only include species that have been investigated for\nbiofluorescence (Table S1). The resulting trimmed tree contained 613 species, including\n85% of the 87 known biofluorescent teleost families and 58% of the 461 currently known\nbiofluorescent species (Tables S1 and S4; Fig. S5).”\nWhen discussing orders and other higher level groups, especially Perciformes, it is\nnecessary to cite which classification is being followed. There are different fish\nclassifications in the last few years that have significantly altered the limits of many\ntraditional taxon names like Perciformes.\nWe used the classification of Rabosky et al. 2018. The citation is added when\ndiscussing orders and after each mention of Perciformes.\nDiscussion\nIt would be better to reword the number of biofluorescent taxa as “occurring in 26\norders, 96 families, and over 460 species” so that “over” unambiguously applies to just\nspecies.\nText edited as requested\nExplain what is meant by assemblage in “underestimate the true phylogenetic diversity\nwithin the assemblage” (biofluorescent reef fishes?)\nAssemblage refers to biofluorescent teleosts. The text now reads “However, to date we\nhave only sampled a small percentage of the total diversity of teleosts, and these\nnumbers almost certainly severely underestimate the true phylogenetic diversity of\nbiofluorescent taxa”\nExplain how “highly prevalent and exceptionally phenotypically variable in four diverse\nassemblages” was determined. Was there a percentage or threshold that warranted this\ndesignation?\nYes, we used an ancestral node likelihood of >60% for the presence of biofluorescence\nto define these groups. This is stated in the methods.\nCyclopteridae is said to be in Fig. 1E but I could not find it in the figure and no\nrepresentative of the family is listed in Table S3.\nThank you for this comment. In the previous figure, only Liparidae was present in Fig.\n1E. Cyclopteridae has been removed to avoid confusion.\nWhile some species occur in intertidal pools, Liparidae is not exactly a family that\ncomes to mind as an example of shallow water fishes.\nThe species “Liparis dennyi” was added to specify the example of intertidal liparids we\nare referring to. We also added a reference to Supplementary file S1, where two\nspecies of intertidal liparids are included (Liparis dennyi and Liparis florae). The text\nnow reads: “…from intertidal zones (e.g., Liparis dennyi, Perciformes; Fig. 1C, Table\nS1)…”\nBlenniiformes are said to be in Fig. 1C “Gobiiformes, Blenniiformes, and\nSyngnathiformes (Fig. 1C).” but that would contradict an earlier statement: “The third\nmajor clade includes Pseudochromidae, Gobiescocidae, and Blenniformes (Fig. 1D).”\nAlso, that order appears to be in 1D.\nThe reviewer is correct. This text has been removed and replaced by the results of the\nnew analysis of the Rabosky et al. phylogeny.\nPlural fishes should be used for “Thus, many reef fishes inhabit areas”\nText edited as requested\nAs adjectives, scorpaenids and nemipterids should not be capitalized\nText edited as requested\nPlease cite some examples to support the statement “Whereas Synodontidae is\nrecovered as nonmonophyletic in our phylogenetic reconstruction, this topology is\ncommon in studies of Aulopiformes” and explain what “this topology” means. Does that\nmean the non-monophyly of Synodontidae is common in other studies?\nWe agree that this sentence needed clarification. However, this section has since been\nremoved as the structure of Rabosky et al. phylogeny is discussed in the original\npublication (Rabosky et al. 2018).\nRefer to them as the Rabosky et al. dataset and Rabosky et al. phylogeny, not just\nRabosky dataset or Rabosky phylogeny.\nText edited as requested\nWhen discussing the distribution of red vs. green vs. both “Green fluorescence occurs\nindependent of red in 16 groups, whereas red fluorescence is only independent of green\nin five groups”, it would help the reader keep track if you explained what these groups\nare (from the figures, they seem to vary in taxonomic rank with some being single\nfamilies, others being multiple families, others being orders, etc.) and state how many\nbiofluorescent groups there are of each. From the manuscript, there are 16 green only\ngroups, 5 red only groups, but how many have both and how many total?\nThe quoted text has been removed and replaced by the results of the new analysis of\nbiofluorescent color. To avoid confusion with what “groups” refers to, we cite each group\nafter they are mentioned. The text now reads:\n“There are also many groups exhibiting only a single fluorescent emission color. We find\nthat green fluorescence evolves first in Anguilliformes (Fig. 2A) and is present in three\nlarger clades: the sculpins (Rhamphocottidae + Agonidae + Psychrolutidae; Fig. 2I),\nNemipteridae (Spariformes; Fig. 2F) and Cheilinus (Labriformes: Labridae; Fig. 2D).\nWhile red fluorescence evolves later in Syngnathiformes (Fig. 2J), it is far more\nwidespread than green fluorescence in the phylogeny (Fig. 2C, G, J, L, N, O), notably\nevolving in the ancestor of all Perciformes32 (Fig. 2G).”\nDelete “of” in “Many of coral reef fishes that possess opsins” or add “the” after “Many of”\nThe extra “of” was deleted.\nWith the number of authors, please specify who is responsible for a pers. obs.\nThis personal observation has been replaced with the citation Anthes et al. 2016 per\nReviewer #3’s comment.\nProvide a reference for the first evolution of bioluminescence in the following: “predating\nthe evolution of bioluminescence in marine fishes by over 30 million years.”\nThis text has been removed.\nWhen making the following statement (We have shown that a significant increase in\nbiofluorescent reef-associated families and species parallels the rise and diversification\nof modern coral reefs beginning in the early Eocene), it implies that there is statistical\nevidence showing the increase is significant, but no statistical tests were run to\ndemonstrate that the increase in biofluorescence is more than the general background\nincrease in actinoptergyian diversity that coincided with the start of the Cenozoic, where\nthere was an explosion of reef associated fishes in general.\nThis text is now removed and replaced by the results of the new diversification analysis,\nwhich shows that reef-associated species evolve biofluorescence at 10x the rate of non-\nreef species.\nReferences\nCapitalization of article titles is inconsistent, for example (1) Sparks et al. 2014 vs.\n(2) Kurup et al. 2013.\nAll articles titles have been changed to sentence case.\nDOIs are only given for (10) and (57)\nDOIs have been removed.\nMany scientific names in titles should be italicized: (7) (26) (29) (46) (47) (48) (49) (53)\n(65) (67) (68) (69)\nCitations fixed\n(9) Citation looks like it’s incomplete\nCitation fixed\n(35) Citation looks incomplete or copy and pasted from Google\nCitation fixed\n(58) Article title is in all caps\nSimpson, C., Kiessling, W., Mewis, H., Baron‐Szabo, R. C. & Müller, J.\nEVOLUTIONARY DIVERSIFICATION OF REEF CORALS: A COMPARISON OF THE\nMOLECULAR AND FOSSIL RECORDS. Evolution 65, 3274–3284 (2011).\nCitation fixed.\nOther\nAdd Table before S1, S2, S9\nText edited as requested\nAdd Figure before S5, S8, S10\nText edited as requested\ntime calibrated should be hyphenated in “time-calibrated phylogeny”\nText edited as requested\nreef associated should be hyphenated in “reef-associated acanthomorph families”\nHyphens were added to every instance of “reef associated” when used as an adjective.\nBlenniiformes is misspelled as “Blenniformes” twice\nText edited as requested\nFigure 1\nWhy is there a large gap in fish photos between Chlorophthalmidae and Aulostomidae?\nThere are two biofluorescent families (Zeidae and Holocentridae) in that part of the tree\nthat could have been included.\nAn image for Zeidae (Zeus faber) has been added to fill the gap in photos. There is no\nphoto available for the three biofluorescent Holocentridae species described in Anthes\net al. 2016.\nFigure 1 legend\nI do not know if the journal style requires it, but the statement starting “Major clades” is\nnot a complete sentence.\nSentence now reads “Major clades where biofluorescence is widespread (B-F), and the\noldest reconstructed node where biofluorescence is present (A) are shown.”\nFigure 5 legend\nAs a proper noun with an adjective form, Scleractinia should not be used as an\nadjective, instead it should be “scleractinian coral”. As an adjective, “acanthomorph”\nshould not be capitalized.\nText edited as requested\nTable S1\nFistulariidae is misspelled as “Fistulariiidae”\nTable edited as requested\nGobiesocidae is misspelled as “Gobiescocidae”\nTable edited as requested\nEuprymna berryi (Sepiolidae) is a cephalopod\nThis species has been removed\nTable S3\nTypeface is inconsistent, especially COI column\nTable has been removed.\nSeveral taxa (Lateolabrax_japonicus, Lepisosteus_oculatus, Mugil_cephalus,\nOncorhynchus_mykiss) are not italicized. If using italics, remove underscore between\ngenus and species names.\nItalics were removed for all species.\nFigure S10\nThe typeface is inconsistent between the x-axis and y-axis. The right edge of the parts A\nand B, representing the present (0 mya) are not aligned.\nIn part A, the x-axis extends beyond the y-axis and the right edge of the graph.\nIn part B, the shaded gray area representing cumulated species is separated from the x-\naxis by a small gap and the dotted line representing the K-T boundary extends past the\nx-axis. The shaded area does not start at the origin of the y-axis. The tick marks for the\ny-axis increments are misaligned and extend into the figure space.\nThank you for these detailed comments. This figure has since been removed.\nReviewer #3 (Remarks to the Author):\nEmily Carr and co-authors present a highly intriguing and well-founded study on the\nevolutionary history and phylogenetic distribution of 'green' and 'red' biofluorescence\namong ray-finned fish. This study achieves unprecedented coverage across fish\nphylogeny, establishing a critical reference point for future research to better understand\nthe evolution and adaptive functions of biofluorescence within a phylogenetic\nframework. Notably, it is the first to estimate the timing of biofluorescence origins in\nvarious fish lineages and connects these with a compelling hypothesis: that the greatest\ndiversification of biofluorescence may have coincided with the emergence and\ndiversification of shallow water reefs.\nGeneral Comments\n(a) Origins and Timing of Fluorescence\nThe reconstructed phylogenetic origins for red and green fluorescence are largely\nconvincing, but I have some concerns regarding the earliest estimated origins\n(Synodontidae at 184 mya—origin B in Figure 1, followed by Albulidae and Labridae). I\nwas surprised by the high confidence in placing the origin of biofluorescence at the base\nof these branches rather than somewhere along the branch leading to the single\ndescendant ‘biofluorescent clade’, each containing only a single OTU (family). Can we\nconfidently exclude the possibility that the origin of biofluorescence occurred (much)\nlater along these terminal branches in such cases? Moreover, what ages would be\nestimated for the earliest origin of biofluorescence using the Rabosky phylogeny?\nThank you for your comment. We now use a trimmed Rabosky phylogeny for our main\nanalysis to provide a more accurate age for the origin of biofluorescence. Based on an\nancestral node likelihood of 67% for the presence of biofluorescence, we conclude that\nbiofluorescence likely first evolved in the ancestor of Anguilliformes ~112 mya (Fig. 1).\nThe origin of biofluorescence is no longer placed on a terminal branch and placing the\norigin at the node is likely an underestimate of the actual age (i.e, minimum age).\n(b) Frequency of Independent Evolution Events\nThe study estimates 35 independent evolutions, with an even higher count using the\nRabosky dataset. Could the authors evaluate / discuss whether these estimates may be\nbiased upwards due to sampling gaps? For instance, in the large clade containing\nAcanthuridae, Nemipteridae, Antennariidae and others, with many independent\nevolutionary transitions, how might the picture change if more extensive sampling in\nfamilies like Leiognathiidae, Zanclidae, Lophidae or others would reveal new instances\nof biofluorescence?\nWe now use much more comprehensively sampled trimmed Rabosky phylogeny for our\nmain analysis and no longer include the original phylogeny addressed in this comment.\n(c) Methods for Detecting Biofluorescence\nThe criteria for distinguishing fluorescence from faint residual reflection during flash\nphotography could benefit from further clarification. For example, did the authors use\nlambda-max emission peaks to identify cases with peak fluorescence within the defined\nred or green spectral ranges? Where emission spectra standardized relative to a\nreflection standard?\nYes, the peak emissions (lambda-max) recorded by the spectrophotometer were used\nto determine the color of biofluorescence for most specimens. To clarify, the following\nsentence was added: “Peak emissions (lambda-max) were used to confirm fluorescent\nemission colors (Table S2).” For specimens where it was not possible to take\nspectrophotometer measurements, a scientific grade long-pass filter was used to\nensure reflection of excitation light did not provide a false positive for biofluorescence.\nAs a suggestion, I would find it useful if the supplement could contain spectra of the light\nsources and filters for future comparison.\nGraphs of the spectra for the Sola Nightsea and SeaDragon blue excitation lights were\nadded to the supplement (S3). Spectral ranges for excitation and long-pass emission\nfilters can be found in the methods.\nMoreover, would it be possible (and useful?) to publish lambda-max values for each\nmeasured species in the online supplement?\nPer request, we have added lambda-max values for all available newly described\nfluorescent species Table S2. Lambda-max values for species previously reported as\nfluorescent in the literature can be found in the original publications (e.g. Sparks et al.\n2014, Anthes et al. 2016).\n(d) Fluorescence in Shallow Waters\nI agree with the discussion's assertion that shallow waters offer particularly favorable\nconditions for biofluorescence compared to deep waters. However, to avoid\nmisinterpretation by readers, I suggest noting that fluorescence is likely overwhelmed by\nfull-spectrum sunlight in the upper few meters, likely limiting its contribution compared to\nreflection. Fluorescence may primarily offer advantages under specific conditions such\nas for short-distance communication, in shaded habitats, or around dusk and dawn.\nEarlier visual models for just noticeable differences (JND) in achromatic contrast\nsuggested that red fluorescent signals for intraspecific communication only become\ndetectable at depths below approximately 10 m depth in a triplefin species (ref. 69).\nThank you for the suggestion. The following text was added: “Biofluorescence is most\nconspicuous in bright, monochromatic blue water (~5-100 m), as it is likely overwhelmed\nby full-spectrum sunlight in the upper few meters of the water column, limiting its\ncontribution as a visual signal22,54… Further, complex structures like reef ledges and\ncoral heads cast shadows, altering the light regime under and around themselves56.\nWhen fish occupy these shadowed spaces, their fluorescence may be accentuated14,\nsuch that it can be observed by humans without a filter in clear blue water (Sparks pers.\nobs.).”\n(e) Biofluorescence as a Driver of Diversification\nWhile biofluorescence is a strong candidate as a driver of fish diversification, I suggest a\nword of caution when comparing it to bioluminescence. Bioluminescence can generate\nquite strong contrasts in deep-sea environments or shallow waters at night, whereas the\nuse of fluorescence is inherently restricted to diurnal conditions within the photic zone\n(see comment (d) above).\nThank you for your suggestion. We have removed the comparison between\nbiofluorescence and bioluminescence as a result of the new diversification analysis,\nwhich suggests neither biofluorescence or reef association is driving diversification in\nthe trimmed Rabosky et al. phylogeny. However, we do find that reef-associated species\nevolve biofluorescence at 10x the rate of non-reef species.\nMinor Comments\n- Introduction (earlier work): The study context and the current understanding of\nbiofluorescence functions are well framed. One section touches on earlier work\nexamining the phylogenetic diversity and distribution of biofluorescence among fish. For\nmy taste, this part could integrate refs. 42 and Michiels et al. 2008\n(https://link.springer.com/article/10.1186/1472-6785-8-16), which, along with refs. 1 and\n13, are the few studies to date to examine fish biofluorescence and its potential\nfunctions on a broader phylogenetic scale.\nThank you for the suggestion. The following text has been edited to incorporate all the\nsuggested citations: “Previous investigations of the phylogenetic distribution of\nbiofluorescence in ray-finned fishes found that the phenomenon is both phylogenetically\nwidespread and phenotypically variable across the assemblage1,30,31.”\nIntroduction (last paragraph): Review the use of the word 'incredible'—it appears\nredundant and could be removed.\nThe word “huge” has been removed to avoid redundancy. The sentence now reads\n“…and report the number of times the phenomenon is known to independently evolve\nacross this incredibly diverse vertebrate assemblage.”\n- Discussion: Regarding the variation in red emission wavelengths within lineages, this\nhas been documented extensively in ref. 42, which shows evidence for up to three\ndistinct emission peaks within single species and significant variation among individuals.\nThank you for bringing this to our attention. We now cite Anthes et al. 2016 instead of\nthe personal observation.\nResponse to Reviewers – Revision 2\nThe authors would like to thank all reviewers for their helpful comments, which have\ngreatly improved this manuscript. In this revision, we’ve added further explanations of\nthe diversification methods and a description of the resulting parameters (transition rate\nvs diversification rate). We have also re-run the diversification analysis using a more\naccurate sampling fraction that better accounts for the uneven sampling of reef vs non-\nreef fishes. This resulted in a change in one new transition rate, suggesting that reef-\nassociated fluorescent fishes are 2.6x more likely to transition to living off reefs. Thus,\nwhile fluorescence is more likely to evolve in fishes on reefs, it is not restricted to reef\nenvironments. We’ve also added further discussion regarding the loss of fluorescence\nand numerous character state changes throughout the phylogeny, concluding that\nfluorescence may be a labile trait.\nReviewer #1 (Remarks to the Author):\nThe manuscript by Carr and colleagues investigates the evolution and drivers of\nfluorescence in fishes and is a resubmission of a previous draft. I believe that the\nauthors have addressed many of the concerns of the prior three reviewers, and overall\npresents a more cohesive, precise study. By changing to a MuHiSSE model, the\nconclusions of the manuscript changed to find that fluorescence, nor reef dwelling,\ndrove diversification dynamics in their study. This is a major shift in the findings of the\nstudy, but I believe it is more likely realistic, and I do believe that this was the correct\nanalytical approach to take.\nHowever, my only critique of this version is that the Discussion still reads like the former\nversion in parts, where the importance of coral reefs for fluorescent evolution is\nstressed. For example:\nLine 322: \"Thus, biofluorescence may be particularly advantageous for reef fishes given\nits numerous potential functions in camouflage, mate identification, and intraspecific\ncommunication.\" <- I agree that camouflage is certainly an advantage for fluorescence\nin reef environments (although fluorescence exists in macroalgae and other substrates\na fish could hide on in addition to reefs). However, the latter two points (identification\nand communication) could occur in any environment, and may even be more important\nin an environment where the water isn't as clear as on a reef.\nWe agree with the reviewer that the latter two points (identification and communication)\nmay be even more important in turbid/low visibility conditions and have added text to\nclarify that these functions could also be important in non-reef habitats (“We note,\nhowever, that intraspecific communication, including mate recognition, could also be\nfacilitated by fluorescence in non-reef habitats with lower visibility, such as turbid\nwater.\").\nAlthough these functions can occur in both reef and non-reef environments, this\nparagraph focuses on discussing how coral reefs likely exhibit the ideal lighting\nconditions for functional biofluorescence in fishes. The studies referred to in this\nparagraph that hypothesize the use of biofluorescence in mate identification and\nintraspecific communication focus on reef fishes. We’ve clarified this in the previous\nsentence, which now reads: “Further, biofluorescent pigments are prevalent in the fins\nof reef fishes, such as Eviota (Gobiidae), Enneapterygius (Tripterygiidae), and\nCorythoichthys (Syngnathidae), structures that these groups use for intraspecific\ncommunication.”\nWe’ve also removed the word “particularly” in Line 322 and acknowledge these\nfunctions may also be useful in non-reef environments under certain conditions.\n“Thus, biofluorescence may be advantageous for reef fishes, given its numerous\npotential functions in camouflage, mate identification, and intraspecific communication.\nWe note, however, that intraspecific communication, including mate recognition,\ncould also be facilitated by fluorescence in non-reef habitats with lower visibility,\nsuch as turbid water.”\nLine 333 states your finding that reef fishes are more likely to evolve fluorescence,\nsupporting your argument and the message of this paragraph. However, you also found\nthat reef fishes are more likely to LOSE fluorescence as well, which may suggest that\nit's not that advantageous on reefs or that it may come at a cost.\nThank you for this comment. We agree that the loss of fluorescence in reef-associated\nspecies provides an interesting perspective and should also be mentioned in the\ndiscussion. However, we offer an alternative interpretation of these results:\n“Through a diversification analysis, we find that reef-associated teleosts evolve\nfluorescence at a much faster rate (10x transition rate) than non-reef species (Figs. 3A\nand 4). This indicates that a lineage already occupying a reef environment is much\nmore likely to evolve fluorescence than a lineage that is not reef-associated. However,\nwhile fluorescence in teleosts is more likely to evolve on reefs, it is not restricted\nto reef environments, as fluorescent fish are 2.6x more likely to transition to living\noff reefs (Fig. 3A). We also find that reef-associated lineages have similar\ntransition rates for the loss (q = 0.008) and gain (q = 0.01) of fluorescence (Fig.\n3A). This could imply that fluorescence is a labile trait on reefs.”\nWe reiterate this conclusion in the following paragraph as well after discussing Figure 1:\n“However, we find that multiple transitions between absence and presence of\nbiofluorescence even occur within teleost orders (e.g. Labriformes; Fig. 1B), which\ncould indicate that fluorescence is more evolutionarily labile in some clades.”\nThese are nit-picky examples, I agree, but highlight how I do feel the Discussion may be\na tad misleading at times to the actual findings of the study.\nWe thank you for your comments, which have improved our manuscript. We have\nchanged wording accordingly throughout the discussion to highlight that our conclusions\non the importance of coral reefs is a hypothesis based on the results of our analyses.\nWe have also added further discussion regarding the loss of fluorescence and\nnumerous character state changes through the tree, concluding that fluorescence may\nbe a labile trait.\nOverall, though, I think this is a much improved revision, and I commend the authors on\ntheir work. I only have a few other minor comments:\nFirst word line 226: Biofluorescence misspelled 'Bioluorescence'\nThank you for pointing out this error, which has now been resolved.\nLine 393: The term 'ancient' can be a loaded term, and is relative. Many would argue\nthat 112 million years isn't that ancient.\nWe agree that this term is relative to the clade being discussed and have revised our\nwording accordingly. The text now reads “Biofluorescence is a phylogenetically\nwidespread and phenotypically diverse phenomenon that is found at deep ancestral\nnodes in teleost fishes.”\nFigure 4: It would be really interested to see trend lines for Reef-associated non-\nfluorescent lineages and for Non-reef non-fluorescent lineages as well. This could help\nreinforce some of what you're saying in your Discussion.\nWe agree and a lineage over time plot with all character states has now been added to\nthe supplement (S6). The results reflect the same trends found in the diversification\nanalysis. By highlighting fluorescence only in Figure 4, we allow readers to better\nvisualize the trends of the diversification analysis, especially for readers who are not\nfamiliar with these methods and may have difficulty interpreting the results in Fig. 3A.\nReviewer #3 (Remarks to the Author):\nThe authors have carefully addressed the issues raised by the three referees, and I can\nconfirm that the manuscript has greatly benefited from the revision. I found particularly\nhelpful the revised data analysis based on the more fine-grained Rabosky et al.\nphylogeny and the addition of explicit diversification rate models along the suggestions\nof ref. 1.\nMy only remaining major comment relates to the way that this diversification rate\nanalysis is presented (L. 183 ff). (note that I am not familiar with this technique in any\ndetail, so consider myself a naïve reader who loves to roughly understand what is\ngoing on.)\n(i) A first aspect that left me wondering is the disbalanced sample of species from reef\nvs. non-reef-habitats (479 vs. 134 species). Could this lead to a situation where\ndiversification rate estimates for non-reef inhabitants are biased downwards – perhaps\nalso because of sampling bias towards non-fluorescent taxa? Or put differently: Is the\napproach mentioned in l. 199-203 sufficient to take this into account?\nWe thank you for this comment, which has led to a more robust result. As referenced\nhere, we mitigate the effects of sampling bias by incorporating a sampling fraction into\nour analysis. However, our previous sampling fractions did not account for the uneven\nsampling of reef and non-reef fishes. To correct this, we ran the same analyses with a\nsampling fraction that was based on the number of reef-associated (1501) and non-reef\n(1838) fish in the full Larouche et al. dataset of 3339 marine fishes. This provides a\nmore accurate proportion of reef association in marine teleosts (~45% of species are\nreef associated). We then used the dataset of fluorescent and nonfluorescent fish\nreported in this study (Table S1) to estimate the expected number of species in each\nfluorescence habitat state (e.g. fluorescent and non-reef). The sampling fractions for\neach state were then calculated as the proportion of the actual number of species\nsampled in each state divided by the expected number of species in each state. Thus,\nthe new sampling fraction better accounts for the uneven sampling of reef vs non-reef\nfishes compared to the original values. The text describing the sampling fraction in the\nmethods now reads:\n“To account for incomplete phylogenetic sampling, we estimated sampling fractions for\neach fluorescence-habitat state (non-fluorescent/non-reef, fluorescent/non-reef, non-\nfluorescent/reef-associated, fluorescent/reef-associated). We first used the proportion\nof fluorescent fishes in the dataset reported in this study (Table S1) to estimate\nthe number of fluorescent and nonfluorescent species in the Larouche et al.41\ndataset, which assigns a character state of reef-associated or non-reef for 3339\nmarine species. We then estimated sampling fractions based on the proportion of\nspecies sampled in the trimmed Rabosky et al.33 phylogeny with each\nfluorescence-habitat state out of the full Larouche et al.41 dataset.”\nHowever, the results of the diversification analysis using this more accurate sampling\nfraction were almost identical to the results of the original analysis. Net diversification\nrates (Fig. 3B) were slightly affected, but as the reviewer states in a later comment, the\ndiversification rates (the rate that new species evolve) of all character combinations are\nlikely not statistically different due to highly overlapping standard deviations. The only\nnotable change in results is in the transition rate (rate of change between character\nstates; Fig. 3A) from reef-associated + fluorescent to non-reef + fluorescent. In the\noriginal analysis, fluorescent fish transitioned 1.3x faster from non-reef environments to\nreefs. However, the new analysis suggests that reef-associated fluorescent fishes are\n2.6x more likely to leave reefs. These new values have been added to the\n“Diversification Analysis of Biofluorescence and Reef Association” section of the results,\nwhich now reads,\n“The best fitting SSE models were character-independent models, indicating that factors\nbesides fluorescence and reef-association influence lineage diversification. The best\nfitting models were the MuCID model with seven and eight hidden states. Average\nnet diversification rates, informed by the ancestral state reconstructions, were similar (r\n= 0.029-0.031) with overlapping standard deviations across all character state\ncombinations (Fig. 3B). However, based on transition rates, the rate of change\nbetween character states, teleost fishes on reefs evolved fluorescence at a rate 10x\nfaster than species off reefs (Fig. 3A, bolded arrows; Fig. 4; Fig. S6). However, reef\nfishes also lost fluorescence at a rate 1.38x faster than non-reef fishes. Additionally,\nfishes with fluorescence transitioned to living off reefs at a rate 2.6x faster than\nthey transitioned on reefs (Fig. 3A).”\nThe greater transition rate for fluorescent fishes to migrate off reefs is an interesting\nresult. This may support our hypothesis regarding the importance of reefs in the\nproliferation of fluorescence throughout teleosts. While fluorescence is more likely to\nevolve in fishes on reefs, it is not restricted to reef environments. Accordingly, the\nfollowing statement has been added to the discussion:\n“However, while fluorescence in teleosts is more likely to evolve on reefs, it is not\nrestricted to reef environments, as fluorescent fish are 2.6x more likely to transition to\nliving off reefs (Fig. 3A).”\n(ii) Second, the analysis estimates not only diversification rates (= no significant\ndifference among groups), but also state transition rates (finding a 10x difference\nbetween non-reef vs. reef-associated species for the transition to biofluorescence –\nhere I would love to see some indicator of estimate precision).\nThe model does not report a confidence interval for transition rates, and no precision\nestimate is commonly reported (e.g., McCord et al. 2021, Román‐Palacios et al. 2022;\nplease see citations below). However, the transition rates reported in this study are\nbased on the best fitting model, the model with the highest AIC value.\nMcCord, Charlene L., et al. \"Phylogeny of the damselfishes (Pomacentridae) and\npatterns of asymmetrical diversification in body size and feeding ecology.\" PLoS\nOne 16.10 (2021): e0258889.\nRomán‐Palacios, Cristian, Daniela Moraga‐López, and John J. Wiens. \"The origins of\nglobal biodiversity on land, sea and freshwater.\" Ecology letters 25.6 (2022): 1376-1386.\nThe role that these two components have in the models, coupled with an explanation\nhow a model that assumes state-independence can generate a 10x difference in\nestimated transition rates, is needed to make the models more transparent to non-\nexperts in these techniques. Ultimately, I understand that both estimates target very\ndifferent aspects, and possibly even arise from separate models (?). Anyway, this does\nnot become apparent to me from the current Method description.\nThank you for pointing this out. The best fitting models estimate transition rates between\ncharacter states, which is then used to perform ancestral state reconstructions on the\nphylogeny. The ancestral state reconstructions are then used to calculate the model-\naveraged diversification rate of each tip in the phylogeny, which in our case equates to\neach species. To clarify for all readers, we have edited the methods explaining how\ntransition and diversification rates are calculated.\n“We identified the best-fitting models using weighted AIC values, which estimated the\ntransition rates between character states. We then used the transition rates to\nconduct marginal ancestral state reconstructions with the hisse R package. The\nresults of the ancestral state reconstructions were summarized by calculating the\ntip-associated net diversification rates by model-averaging parameters from all models\nthat contributed >5% of the AICc weight. Transition rates measure how frequently a\ntrait is gained or lost (i.e., the rate of change between character states). A higher\ntransition rate towards the loss of a character state, for example, indicates that a\nlineage is more likely to lose that character over evolutionary time. Diversification\nrates indicate the rate that new species evolve. If diversification rates are higher\nin lineages with a certain character state, that character state is likely\ncontributing to the proliferation of species over time.”\nThe possibility for a character-independent model to estimate different transition rates\nbetween states is explained by the incorporation of hidden states into the model. Hidden\nstates are used to account for unknown factors besides fluorescence and reef-\nassociation that may affect transition rates and lineage diversification. The finding that a\nCID model is the best fitting model suggests that the rates at which transitions occur are\nnot dependent on the ancestral states. For instance, the gain of fluorescence among\nreef fishes is not fast solely because they are on reefs. Instead, our models suggest that\nadditional unknown factors (represented by the hidden states) also promote faster\ntransition rates in reef fishes. It should also be noted that the inclusion of hidden states\nalso explains how different transition rates may result in similar diversification rates\nacross states. To explain the importance of including hidden states, the following\nstatement has been added to the methods:\n“Hidden states are used to account for unknown factors besides fluorescence and reef-\nassociation that may affect transition rates and lineage diversification.”\n(iii) My third issue is probably largely resolved once issue (ii) is clarified, but let me list it\nhere for completeness. So: At first reading, I was surprised by the description of the\nResults (l. 277 ff): The first sentence states that the best fitting model predicted a global\ndiversification rate that was independent of reef association or biofluorescence – to be\nthen followed by a description of strikingly different transition rates among habitat-\nfluorescence-combinations. I took me two times back-and-forth checking until I\nunderstood that we talk about two rather different aspects here. (See also formulations\nin Discussion: Line 331-332 (10x faster rate under reef-association) vs. Line 340-41\n(character-independent models having the best fit).\nTo clarify the difference between the two values we have edited the “Diversification\nAnalysis of Biofluorescence and Reef Association” results section. The text now reads:\n“The best fitting SSE models were character-independent models, indicating that\nfactors besides fluorescence and reef-association influence lineage\ndiversification. The best fitting models were the MuCID model with seven and eight\nhidden states. Average net diversification rates, informed by the ancestral state\nreconstructions, were similar (r = 0.029-0.031) with overlapping standard\ndeviations across all character state combinations (Fig. 3B). However, based on\ntransition rates, the rate of change between character states, teleost fishes on reefs\nevolved fluorescence at a rate 10x faster than species off reefs (Fig. 3A, bolded arrows;\nFig. 4; Fig. S6). However, reef fishes also lost fluorescence at a rate 1.38x faster than\nnon-reef fishes. Additionally, fishes with fluorescence transitioned to living off reefs at a\nrate 2.6x faster than they transitioned on reefs (Fig. 3A).”\nThe following statements in the discussion have also been edited to clarify this issue.\nLines (346-349): “Through a diversification analysis, we find that reef-associated\nteleosts evolve fluorescence at a much faster rate (10x transition rate) than non-reef\nspecies (Figs. 3A and 4). This indicates that a lineage already occupying a reef\nenvironment is much more likely to evolve fluorescence than a lineage that is not reef-\nassociated.”\nLines (359-362): “However, due to the character independent models having the best fit,\nour results imply that another factor besides reef-association or biofluorescence is likely\ndriving global diversification across the entire tree.”\nFigure 3A: The difference in arrow length is currently exclusive to the two bold arrows\n(to stress the 10x difference), but not used for the other (narrow) arrows. This could\ncreate some confusion among readers. I would favour a more display type for these\narrows. For example, use arrows of equal length throughout, but use arrow thickness to\nroughly scale with transition rate values. The two arrows that readers should focus\nattention on could then be additionally highlighted by different colour or line type.\nThank you for the feedback. The figure has been edited according to the reviewer’s\nsuggestions.\nFigure 3B: The graph shows means +/- SD. This is fine, but allows no direct “statistical”\ncomparison among the four groups. Does the model also provide means with their\nstandard errors, as to be statistically more informative? Does the underlying statistical\nmodel take into account the right-skewed data distributions – probably requiring a log-\nlink error term? Such information would also be needed to support the statement that\n“average diversification rate was highest in reef-associated fish” (Figure caption as well\nas line 279-81 in the Results). Strictly, the models imply that there is no difference in\ndiversification rate between groups, so the formulation should probably be even a bit\nmore careful.\nThe diversification model only provides a mean +/- SD, which is commonly used to\ncompare diversification rates (e.g. McCord et al. 2021, Conway et al. 2019; see citations\nbelow). Because we are not statistically comparing the diversification rates, it is not\nnecessary to conduct a log transformation. The right-skewed data distributions are likely\ncaused by outliers that have high diversification rates due to unrelated factors outside\nthe scope of this study (e.g. diet, morphology). We’ve edited the results to added the\nfollowing statement to the results (lines 291-293):\n“Average net diversification rates, informed by the ancestral state reconstructions, were\nsimilar (r = 0.029-0.031) with overlapping standard deviations across all character\nstate combinations (Fig. 3B).”\nMcCord, Charlene L., et al. \"Phylogeny of the damselfishes (Pomacentridae) and\npatterns of asymmetrical diversification in body size and feeding ecology.\" PLoS\nOne 16.10 (2021): e0258889.\nConway, Meaghan, and Brian J. Olsen. \"Contrasting drivers of diversification rates on\nislands and continents across three passerine families.\" Proceedings of the Royal\nSociety B 286.1915 (2019): 20191757.\nMinor comments:\nL. 181: „as a result, the latter method was used”: I cannot trace to which method exactly\nthe “latter” referent is referring to, please specify.\nThe sentence has been clarified to read “as a result, marginal ancestral state\nreconstructions were used.”\nL. 305: “73 % of biofluo species are reef-associated”: I think this number needs context\nwith respect to the fraction among the totally sampled species that were reef-associated\n(because there has been sampling bias towards reefs – see also previous referee\ncomments).\nThe text has been edited to reflect the total number of reef-associated species and now\nreads, “Of the 267 biofluorescent species within the tree, 73% are reef associated.\nThese reef-associated fluorescent species account for 196 of the total 479 reef-\nassociated species.”\nL. 350: replace ‘show’ by ‘imply’ or similar – these values still remain likelihood-based\nestimates (same in Line 399).\nText edited as requested.\nL. 374: evolves -> replace by ‘evolved’ (past tense; same in L. 381, L. 398, L. 400).\nText edited as requested.\nL. 377: which -> replaced by ‘who’?\nText edited as requested.\nFigure 2: Have the authors checked whether brightness contrasts between the chosen\ncolours remain discernible for people with red-green-issues?\nYes, this is a color blind friendly palette (the senior author is color blind)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I feel that the authors have done a good job addressing reviewer concerns on multiple resubmissions. I have no further major concerns or suggestions to add at this point. I commend the authors on a job well done.", "reviewer_3": "I wish to thank the authors for their careful and well-explained revision along the remaining suggestions by the two referees. I am satisfied with all responses and text editions, and see no need for further revision. I wish to congratulate the authors to this very nice piece of work!"}, "author_response": ""}]

Decision letter