Abstract The discovery of fluorescent proteins has revolutionized experimental biology. Whereas the majority of fluorescent proteins have been identified from cnidarians, recently several fluorescent proteins have been isolated across the animal tree of life. Here we show that biofluorescence is not only phylogenetically widespread, but is also phenotypically variable across both cartilaginous and bony fishes, highlighting its evolutionary history and the possibility for discovery of numerous novel fluorescent proteins. Fish biofluorescence is especially common and morphologically variable in cryptically patterned coral-reef lineages. We identified 16 orders, 50 families, 105 genera, and more than 180 species of biofluorescent fishes. We have also reconstructed our current understanding of the phylogenetic distribution of biofluorescence for ray-finned fishes. The presence of yellow long-pass intraocular filters in many biofluorescent fish lineages and the substantive color vision capabilities of coral-reef fishes suggest that they are capable of detecting fluoresced light. We present species-specific emission patterns among closely related species, indicating that biofluorescence potentially functions in intraspecific communication and evidence that fluorescence can be used for camouflage. This research provides insight into the distribution, evolution, and phenotypic variability of biofluorescence in marine lineages and examines the role this variation may play.

Citation: Sparks JS, Schelly RC, Smith WL, Davis MP, Tchernov D, Pieribone VA, et al. (2014) The Covert World of Fish Biofluorescence: A Phylogenetically Widespread and Phenotypically Variable Phenomenon. PLoS ONE 9(1): e83259. https://doi.org/10.1371/journal.pone.0083259 Editor: Diego Fontaneto, Consiglio Nazionale delle Ricerche (CNR), Italy Received: September 28, 2013; Accepted: October 31, 2013; Published: January 8, 2014 Copyright: © 2014 Sparks et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the American Museum of Natural History, City University of New York, National Science Foundation grants DEB-0444842, IOS-0749943, and DEB-1258141 to JSS, MCB-0920572 and DRL-1007747 to DFG, DEB-0732642 and DEB-1060869 to WLS, DEB-1257555 and DEB-1258141 to MPD, WLS, and JSS, National Institutes of Health (NIH) grants U24NS057631 and R01NS083875 to VAP and National Geographic Waitt Grants #W101-10 to DFG and #W214-12 to JSS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction The primarily monochromatic blue spectrum that characterizes large areas of the photic ocean provides a unique filtered-light environment for visual organisms. Compared to the terrestrial environment, marine organisms reside in a spectrally restricted visual domain. The red, orange, yellow, and green components of sunlight are selectively removed with depth resulting in a narrow, near-monochromatic, band of blue light between 470 and 480 nm [1]. Spectrally restricted illumination in the ocean provides unique lighting conditions for organisms to exploit fluorescence to produce visual contrast and patterns. In the marine environment, biofluorescence is highly prevalent in cnidarians (particularly Anthozoans) [2], and also in a ctenophore [3], copepods [4], mantis shrimp [5], amphioxus [6] and some fishes [7]. In addition, the photosynthetic apparatus associated with chlorophyll fluoresces red and provides a background of biofluorescence in areas of high algal growth on coral reefs. Biofluorescence results from the absorption of electromagnetic radiation at one wavelength by an organism, followed by its reemission at a longer and lower energy wavelength, visually resulting in green, orange, and red emission coloration in marine organisms. Biofluorescence signaling has previously been reported in butterflies [7], parrots [9], spiders [10], and flowers [11], as well as a deep-sea siphonophore [12]. In scleractinian corals, biofluorescence has been suggested to function in photoprotection [13], antioxidation [14], regulation of symbiotic dinoflagellates [15], photoacclimation [16], visual contrast [2], and coral health [17]. Whereas insight into the evolution and function of biofluorescence has greatly enhanced our knowledge of coral biology, little to nothing is known regarding the impact of biofluorescence on other organisms that thrive in coral-reef habitats, particularly those with advanced visual systems that could readily exploit fluorescent coloration and contrast. Investigating the evolution of biofluorescence across marine fishes is particularly appealing because they are visual animals, many of which possess yellow intraocular (lenses or cornea) filters [18], which function as long-pass filters and could enable enhanced perception of biofluorescence in the ocean. Worldwide, there are more than 8,000 species of fishes that inhabit coral reefs. Many reef fish species are known for their striking color patterns, whereas many others are cryptically patterned and appear well camouflaged. However, nearly nothing is known regarding the evolution or function of fluorescence in fishes. Only recently has a fluorescent protein, a novel fatty-acid-binding protein, been isolated from a vertebrate, a Japanese eel [19]. Here we report, for the first time, that biofluorescence is widespread throughout the tree of life for fishes, and it appears particularly common and phenotypically variable in marine lineages, especially cryptically patterned, well camouflaged coral-reef lineages. Our findings identify a widespread and previously unrecognized evolutionary phenomenon that provides new insights into the evolution of marine fishes and the function of light and visual systems in a marine environment, as well as providing a framework for the discovery of additional novel fluorescent proteins.

Methods 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. This study was carried out in strict accordance with the recommendations in the Guidelines for the Use of Fishes in Research of the American Fisheries Society and the American Museum of Natural History's Institutional Animal Care and Use Committee (IACUC). Fishes were collected via SCUBA, using both standard open circuit systems and closed circuit rebreathers, via the application of rotenone and quinaldine to a targeted variety of shallow to deep (mesophotic) habitats in each sampling location where collecting was permitted. Taxonomic field surveys of biofluorescence in marine fishes were conducted during the following expeditions: Little Cayman Island, January 2011, working out of the Central Caribbean Marine Institute; the Exumas, Bahamas, May 2011 and December 2011, at the Perry Institute for Marine Science on Lee Stocking Island; and a taxonomically comprehensive survey conducted at numerous localities in the Solomon Islands (June–July, 2012 and September 2013). In addition, we have supplemented these field studies with specimens available in the aquarium trade and by imaging specimens at aquariums after hours (e.g., Mystic Aquarium and Institute for Exploration, Mystic, CT; Birch Aquarium, Scripps Institution of Oceanography, La Jolla, CA). All collected specimens were placed on ice to preserve coloration and digitally imaged upon return to shore using Nikon D300s, D7000, or D800 DSLR cameras affixed with either a 60 or 105 mm Nikkor macro lens under white light. Fishes were subsequently scanned for fluorescence using bright LED light sources equipped with excitation filters and observed using emission filter glasses/goggles. All fluorescent fishes were then imaged (Fig. 1) using the “Fluorescent Macro Photography” protocol outlined below. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Diversity of fluorescent patterns and colors in marine fishes. A, swell shark (Cephaloscyllium ventriosum); B, ray (Urobatis jamaicensis); C, sole (Soleichthys heterorhinos); D, flathead (Cociella hutchinsi); E, lizardfish (Synodus dermatogenys); F, frogfish (Antennarius maculatus); G, false stonefish (Scorpaenopsis diabolus); H, false moray eel (Kaupichthys brachychirus); I, false moray eel (Kaupichthys nuchalis); J, pipefish (Corythoichthys haematopterus); K, sand stargazer (Gillellus uranidea); L, goby (Eviota sp.); M, goby (Eviota atriventris); N, surgeonfish (Acanthurus coeruleus, larval); O, threadfin bream (Scolopsis bilineata). https://doi.org/10.1371/journal.pone.0083259.g001 The list and phylogenetic distribution of biofluorescence across cartilaginous and bony fishes presented in Figure 2 and Table S1 are the result of this survey work, and they also include data from [7] that specifically examined red fluorescence in some shallow, reef-associated fishes. In addition, we have summarized other accounts of biofluorescence in fishes from the popular literature (underwater photography magazines and websites) and available on the internet. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Observed occurrences of green and red fluorescent emissions indicate the evolution of biofluorescence is widespread across the evolutionary history of ray-finned fishes (Actinopterygii). Family-level tree showing evolutionary relationships of ray-finned fishes inferred from maximum likelihood analysis of 221 species and six (one mitochondrial, five nuclear) genes. Note: Not all biofluorescent lineages are shown due to sampling limitations (see Table S1, Fig. S1). https://doi.org/10.1371/journal.pone.0083259.g002 Emission Spectra Emission spectra were collected using an Ocean Optics USB2000+ miniature spectrometer (Dunedin, FL) equipped with a hand-held fiber optic probe (Ocean Optics ZFQ-12135). Excitation spectra were achieved during illumination with a band-pass filter (450–500 nm, Omega Optical, Inc., Brattleboro, VT, or Semrock, Inc., Rochester, NY). Emission spectra were recorded by applying the fiber optic probe to specific anatomical parts of the individual fish specimen exhibiting biofluorescence. This was repeated several times for each specimen to ensure the accuracy of measurements. Fluorescent Macro Photography Individual fish specimens were placed in a narrow photographic tank and held flat against a thin plate glass front. Fluorescent macro images [7360×4912 (Nikon D800); 4928×3264 (Nikon D7000); 2180×1800 pixel (Nikon D300S)] were produced in a dark room by covering the flash (Nikon SB 600, SB 800, or SB910) with interference bandpass excitation filters (Omega Optical, Inc., Brattleboro, VT; Semrock, Inc., Rochester, NY). Longpass (LP) and bandpass (BP) emission filters (Semrock) were attached to the front of the camera lens. A variety of excitation/emission filter pairs were tested on each sample to elicit the strongest fluorescence emission: excitation 450–500 nm, emission 514 LP; excitation 500–550 nm, emission 561 LP. Phylogeny reconstruction A majority of the DNA sequence data used in this study is from [20], but additional sequences were obtained from many studies [21]–[84]; the GenBank accession numbers for these sequences as well as our added GenBank accession numbers (KF768155-KF768177) can be found in Table S2. Mitochondrial and nuclear genes were aligned using the program MAFFT v6.0 with default parameters [85]. The phylogenetic analysis presented herein had a total of 5,238 base pairs including: one mitochondrial gene (cytochrome oxidase I, 812 bps), and five protein-coding genes (glycosyltransferase gene, 732 bps; myosin heavy chain 6 alpha gene, 737 bps; pleiomorphic adenoma protein-like 2-like gene, 659 bps; recombination activating gene 1, 1403 bps; zic family member protein, 890 bps). For each maximum likelihood analysis, the dataset was partitioned by individual gene fragments. A model of molecular evolution was chosen by the program jMODELTEST v.2.1 [86] with the best fitting model under the Akaike information criteria (AIC) for each individual gene partition assigned, including: cytochrome oxidase I (GTR+I+Γ), glycosyltransferase (GTR+ Γ), myosin heavy chain 6 alpha (GTR+I+Γ), pleiomorphic adenoma protein-like 2-like gene (GTR+I+Γ), recombination activating gene 1 (SYM+I+Γ), and zic family member protein (GTR+I+Γ). Maximum likelihood analyses were performed in GARLI v2.0 [87]. Ten separate analyses were conducted, and the tree having the best likelihood score is presented here (Fig. S1, Fig. 2) to evaluate evolutionary relationships.

Acknowledgments We are grateful to Ray and Barbara Dalio and the Dalio Family Foundation, Fabio Amador and Dominique Rissolo of the National Geographic Society/Waitt Program, Zipolo Habu Resort and Dive Gizo, Solomon Islands, Perry Institute for Marine Science, Lee Stocking Island, and Central Caribbean Marine Institute, Little Cayman Island, for providing facilities, boats, submersibles, and logistical support. 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. Thanks also to D. Harrington and T. Romano at Mystic Aquarium and N. Hillgarth and R. Elkus at Birch Aquarium (UCSD) for access to their collections; to M. Lombardi and J. Godfrey for deep diving assistance; and to the J.B. Pierce Lab machine shop for equipment design.

Author Contributions Conceived and designed the experiments: JSS RCS WLS MPD DT VAP DFG. Performed the experiments: JSS RCS WLS MPD DT VAP DFG. Analyzed the data: JSS RCS WLS MPD VAP DFG. Contributed reagents/materials/analysis tools: JSS RCS WLS MPD VAP DFG. Wrote the paper: JSS RCS WLS MPD VAP DFG.