Marine fishes exhibit exceptional variation in biofluorescent emission spectra

Abstract

Biofluorescence is a phylogenetically widespread phenomenon among marine fishes, yet the phenotypic diversity in fluorescent emission wavelengths (e.g., green, red) remains poorly studied across the broad diversity of marine teleosts. In this study we investigate the fluorescent emission spectra from a diverse array of 18 teleost families and record fluorescent emission peaks over multiple body regions. Our results show that fluorescent emission spectra are remarkably diverse among teleost families, as well as within genera. Fluorescent emissions also vary across different body regions within some individuals. We show that members of the families Gobiidae, Oxudercidae, and Bothidae exhibit at least six distinct, non-overlapping fluorescent emission peaks. Nine of the 18 families examined in this study were found to have at least four distinct and non-overlapping fluorescent emission peaks. Further, we find that several families exhibit multiple discrete emission peaks for a single fluorescent color (i.e., wavelength range), including multiple distinct peaks within the green and red portions of the spectrum. The interplay between different fluorescent emission wavelengths and notable variation in the distribution of fluorescence on the body could allow for a wide array of fluorescent patterns to be produced by an individual or among closely related species. Our results reveal far more diversity in both fluorescent emission wavelengths (colors) and in the distribution of fluorescent molecules across the body than had previously been reported in the literature. We characterize this novel variation in biofluorescent emissions across an array of teleost families and discuss the potential implications of this exceptional phenotypic variability.

Introduction

Biofluorescence results from the absorption of high energy, shorter wavelength light by an organism and its reemission at longer, lower energy wavelengths [1]. Although present in numerous invertebrate and vertebrate lineages [1–7], fluorescence is particularly phylogenetically widespread and phenotypically variable in marine fishes, where fluorescent emissions are generally reported within the red and green portions of the visible spectrum [1,8,9]. Several recent studies have documented this widespread presence of biofluorescence across ray-finned fishes [1,8,10–12], but relatively few of these report fluorescent emission spectra [1,8,12–14]. Anthes et al. [14] documented variation in red fluorescent emissions within several teleost families, finding three general groups of emission peaks: near red, deep red, and far red. However, their analysis excluded all emission peaks below 580 nm (i.e., green-yellow) [14]. No study to date has focused on investigating variation in fluorescent emission peaks across the full range of fluorescent wavelengths observed in teleosts [1,13]. This highlights a large gap in knowledge, especially considering prior studies have reported the presence of multiple fluorescent colors (e.g., green and red) even within an individual [1,9,13].

Fishes have been hypothesized to use biofluorescence for functions such as intraspecific signaling, visual enhancement, and camouflage [1,10,11,15]. However, these potential functions require that signal receivers can visualize fluorescent emissions, either as a color signal or as enhanced contrast (e.g., against a background or substrate) [1]. Most fluorescent teleosts are cryptically patterned reef fishes [1] whose eyes are generally most sensitive to shorter wavelengths of blue, green, and yellow [16,17]. Blue and green wavelengths are the most common at depth, as the attenuation of sunlight through water rapidly removes longer (orange-red) wavelengths. This creates a monochromatic blue environment of 470–480 nm by around 150 m depth in clear oceanic waters [18]. One of the potential benefits of fluorescence in marine environments is that it restores longer wavelengths (green-red) in these habitats where only shorter blue wavelengths can penetrate [1,11]. Interestingly, many reef fishes (e.g., Pomacentridae, Gobiidae, Labridae) possess long wavelength sensitivity (LWS) opsins in their eyes, that may allow them to visualize orange and red wavelengths [16,19,20].

Although the potential function of biofluorescence remains unknown in most lineages of marine fishes, recent studies hypothesize that it could serve as a visual aid. Bright green fluorescence was shown to significantly increase contrast at depth in catsharks, making it easier for conspecifics to see each other in these dimly lit environments [11]. Red fluorescence in the eyes of certain reef fishes or on the fins in cryptically-patterned species is hypothesized to provide a visual aid and may function in intraspecific signaling [12,14]. In addition, many reef lineages possess yellow intraocular filters in their lenses or corneas [21]. These filters can function as long-pass filters, which may enhance the perception of longer wavelength fluorescent emissions within a primarily blue ambient environment [1]. Although further studies are needed to determine and compare the diversity of visual spectral ranges broadly among teleosts, in general, many reef fishes are capable of visualizing the green through red portions of the spectrum common in fluorescent emissions [16,19,20].

In this study, we compare emission spectra within 18 biofluorescent families across the phylogeny of Teleostei [1,9]. We document significant variation among families and genera, and over body anatomy within individual species. The objectives of this study are to: 1) record detailed fluorescence emission spectra across a wide array of teleost families that have been shown to exhibit biofluorescence; 2) determine and characterize how fluorescence emission spectra vary within a family, genus, and species; 3) analyze variation in fluorescence emission wavelengths by anatomical region within an individual; and 4) discuss the implications of variation in fluorescent emission spectra (i.e., the presence of several distinct emission peaks within a lineage or species). This study expands our understanding of the variation of biofluorescence in teleosts and highlights the importance of measuring fluorescent emission spectra as it relates to taxonomy, fluorescent proteins, and potential visual functions.

Methods

Specimens and imaging

Specimens used in this study are housed at the American Museum of Natural History (AMNH), New York, and are from collections made in the Solomons Islands (2012, 2013, and 2019), Greenland (2019), and Thailand (2024) (S1 Table). Research, collecting, and export permits were obtained from the Ministry of Fisheries and Ministry of Environment (Honiara, Solomon Islands), local fisheries authorities in Greenland, and the Department of Fisheries and Chulalonghorn University (Bangkok, Thailand). 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).

For fluorescence imaging, live and freshly frozen specimens were placed in a narrow photographic tank and gently held flat against a thin glass front. Nearly all specimens were imaged for fluorescence prior to freezing. We note that fluorescence does not degrade over time if fish are frozen promptly after capture. For example, frozen specimens that were collected in 2012 are still brightly fluorescent and spectra readings of the same specimens align with the original emission spectra taken over 10 years ago. Fluorescent emissions were imaged while 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 or A7RV camera outfitted with a Sony 90 mm macro lens. Flashes (Nikon SB910 Speedlights) were covered with blue interference bandpass excitation filters (490 nm + /- 5 nm; Omega Optical, Inc., Brattleboro, VT; Semrock, Inc., Rochester, NY) to elicit fluorescence. Long-pass (LP) emission filters (Semrock, Inc.) were attached to the front of the camera lens to block any blue excitation light and record only emitted fluorescence. The two Nikon SB910 Speedlights were mounted on light stands and placed approximately two feet from the tank at 45-degree angles to the specimen. To capture fluorescent images of all specimens, multiple LP filter pairs were used. A 514 nm LP filter was initially used to image all specimens, allowing longer (green-red) wavelengths of fluorescence to be imaged. However, in specimens that exhibited multiple fluorescent colors (i.e., overlapping fluorescent wavelengths), a 561 nm LP filter was additionally used to block any emitted green fluorescence and restrict imaging to longer-wavelength fluorescence (yellow through red). Detailed imaging specifications for each fluorescent specimen shown in the Figures and supplementary materials (S2 Fig) are available in the supplementary materials (S3 Fig).

Fluorescent spectra measurements

Emission spectra were recorded from fresh specimens prior to freezing while in a dark room by using an Ocean Optics USB2000 + portable spectrophotometer (Dunedin, FL) equipped with a hand-held fiber optic probe (Ocean Optics ZFQ-12135). To provide excitation light to elicit fluorescence, specimens collected in the Solomon Islands and Greenland were illuminated with Royal Blue LED lights (Pierce Lab, Yale University, New Haven, CT), collimated to ensure perpendicular incidence on the scientific grade 490 nm (+/- 5 nm) interference filter surface (Omega Optical, Inc., Brattleboro, VT), thereby minimizing the transmission of out-of-band energy. Two Sola NightSea (Marina, CA, USA) lights set on full power in flood mode were used to excite fluorescence in specimens collected in Thailand. Fluorescence excitation lights were placed ~15–20 cm from the specimen, located rostrally and caudally, and at 45-degree angles to the specimen. Emission spectra were then recorded by placing the fiber optic probe proximate to specific anatomical parts of the individual fish specimen exhibiting biofluorescence. This process was repeated several times for each specimen and each anatomical region to ensure the accuracy and repeatability of the spectrophotometer readings. To visualize and record longer (i.e., yellow-orange) wavelength emissions in members of Chlopsidae, which exhibit bright green fluorescence over their entire body, it was necessary to use a LP emission filter (561 nm) to block these green fluorescent emissions (see [1]; S2 Fig). Additional technical specifications for all excitation light sources, excitation and emission filters, and spectrophotometer specifications and settings are available in the supplementary materials (S3 Fig).

Fluorescent emission peaks (lambda-max) are defined as the wavelengths that correspond to the highest intensity value (S4 Table). Due to variation in the strength of excitation light source and distances between the spectrophotometer probe and specimens, fluorescence intensity values are relative and do not signify overall brightness. When a single spectrum reading exhibited multiple distinct emission peaks (e.g., one green peak and another red peak), or multiple peaks within a single wavelength/color category), peak values were reported for each distinct emission wavelength by determining each local maxima. As a result, taxa may exhibit multiple emission peaks within a distinct color range (Table 1). All resulting emission spectra were graphed in R [22] using the package “ggplot2” [23] and lines were smoothed in Adobe Illustrator to decrease unnecessary noise without altering data. For some spectra plots, only the emission readings with the highest intensity value were included when redundant lower intensity peaks were present. Plots for all spectra readings taken can be found in supplemental materials (S2 Fig). The scatter plot of peak values for each family (Fig 1A) was plotted in R [22] using the package “ggplot2” [23]. Visual pigment data (S5 Table) were collected from the literature [24,25] and plotted in R [22] using the package “ggplot2” [23]. We compare the total range of visual pigments within a family due to a lack of visual data for the individual species measured in this study. Families are listed throughout such that families within specific orders appear sequentially [26].

[Figure omitted. See PDF.]

[Figure omitted. See PDF.]

A) Emission peaks recorded from species representing 18 teleost families. White dots for emission spectra of Scorpaenidae, which exhibits a continuous range of fluorescent emission peaks from 524-583 nm, indicate that this family is an exception to the distinct fluorescent wavelength ranges reported for other families. B) Corresponding spectra recorded for all nine teleost orders investigated: Acanthuriformes (Cepolidae, Nemipteridae), Anguilliformes (Chlopsidae, Muraenidae), Aulopiformes (Synodontidae), Blenniiformes (Blenniidae, Tripterygiidae), Carangiformes (Bothidae, Cynoglossidae, Soleidae), Gobiiformes (Gobiidae, Oxudercidae), Lophiiformes (Antennariidae), Perciformes (Labridae, Liparidae, Scorpaenidae), and Syngnathidae (Aulostomidae, Mullidae). Dashed lines indicate fluorescent emission peaks. Intensity values are relative and do not signify overall brightness. White brackets correspond to the four main wavelength/color ranges.

Results

Variation in fluorescence among families

In general, four distinct, non-overlapping wavelength (i.e., color) ranges of fluorescent emission peaks are observed among the teleost families we investigated. These peaks correspond to the green (~500–565 nm), yellow-orange (~565–625 nm), near red (~625–700 nm), and far red (~700–750 nm) portions of the visible spectrum (Figs 1 and 2; Table 1). Fluorescent emission peaks ranging from 514–560 nm (green) are present in Chlopsidae, Muraenidae, Synodontidae, Cepolidae, Labridae, Liparidae, Mullidae, Nemipteridae, Scorpaenidae, Gobiidae, Oxudercidae, Tripterygiidae, Blenniidae, Cynoglossidae, Bothidae, and Soleidae. Emission peaks ranging from 573–613 nm (yellow-orange) are present in Chlopsidae, Synodontidae, Cepolidae, Nemipteridae, Scorpaenidae, Gobiidae, Oxudercidae, Tripterygiidae, Antennariidae, Bothidae, and Soleidae. Near red emission peaks ranging from 633–692 nm are observed in Synodontidae, Cepolidae, Aulostomidae, Labridae, Liparidae, Scorpaenidae, Gobiidae, Oxudercidae, Bothidae, and Soleidae. We also find additional far-red emission peaks ranging from 705–751 nm in Muraenidae, Synodontidae, Cepolidae, Labridae, Liparidae, Scorpaenidae, Gobiidae, Oxudercidae, Tripterygiidae, Blenniidae, Bothidae, and Soleidae.

[Figure omitted. See PDF.]

A) Eviota prasites (Gobiidae), B) Trimma fangi (Gobiidae), C) Enneapterygius niger (Tripterygiidae), D) Helcogramma striata (Tripterygiidae), E) Ecsenius axelrodi (Blenniidae), F) Engyprosopon mozambiqense (Bothidae), G) Japonolaeops dentatus* (Bothidae), H) Cynoglossus microlepis (Cynoglossidae), I) Gymnothorax zonipectis (Muraenidae), J) Cheilinus oxycephalus (Labridae), K) Pseudocheilinus evanidus (Labridae), L) Saurida micropectoralis*^,‡ (Synodontidae), M) Upeneus sundaicus* (Mullidae), N) Taenianotus triacanthus (Scorpaenidae), O) Sebastapistes fowleri (Scorpaenidae), P) Aulostomus chinensis (Aulostomidae), Q) Kaupichthys diodontus (Chlopsidae). *Images of the same specimen without (top) and with (bottom) a 561 nm long-pass filter to block emitted green fluorescence. ^‡Saurida micropectoralis (L) is shown in dorsal and lateral views for green fluorescence. Scale bars (white lines) shown in each image are 1 cm.

Members of Synodontidae, Cepolidae, Scorpaenidae, Gobiidae, Oxudercidae, Bothidae, and Soleidae exhibit fluorescent emission peaks in all four of these wavelength ranges (Fig 1; Table 1). However, the scorpaenid genus Sebastapistes is an exception to the distinct wavelength ranges identified above and exhibits a continuous range of fluorescent emission peaks from 524–583 nm, spanning wavelengths that correspond to green and yellow-orange (Figs 1 and 3C; Table 1). Nine of the 18 families examined were found to exhibit at least four distinct fluorescent emission peaks, including members of Synodontidae, Cepolidae, Labridae, Liparidae, Scorpaenidae, Gobiidae, Oxudercidae, Bothidae, and Soleidae (Figs 1 and 3; S2 Fig).

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Variation among genera is shown for members of: A) Labridae; B) Gobiidae; C) Scorpaenidae; D) Oxudercidae; E) Bothidae; and F) Synodontidae. Dashed lines represent fluorescent emission peaks. Images of Japonolaeops (Bothidae; E) are of the same specimen without (top) and with (bottom) a 561 nm long-pass filter to block emitted green fluorescence. Intensity values are relative and do not signify overall brightness.

Dual fluorescent emission peaks

Members of some biofluorescent families exhibit dual emission peaks, where a spectral reading shows two distinct emission peaks within a small wavelength (i.e., single color) range (e.g., Fig 1B Pleuronectiformes and Fig 3B Myersina). Dual green emission peaks are present in members of Chlopsidae (520–528 and 551–554 nm), Cepolidae (525 and 550 nm), Mullidae (517–523 and 547–550 nm), Nemipteridae (517–536 and 549–558 nm), Gobiidae (523–527 and 546–555 nm; Fig 3B), Cynoglossidae (526–529 and 548–552 nm), and Bothidae (523–526 and 551–552 nm) (S2 Fig). Dual emission peaks within the near red portion of the spectrum are only present in Liparidae (643–645 and 676 nm) and Bothidae (635 and 674 nm) (S2 Fig).

Variation within families

Large variation is present in fluorescent emission peaks among genera within Synodontidae, Labridae, Scorpaenidae, Gobiidae, Oxudercidae, and Bothidae. In Synodontidae, green fluorescent emissions are present as a single peak in Synodus (516–529 nm) and as a dual peak in Saurida (517–525 and 542–550 nm) (Figs 1 and 3F; Table 1). Yellow-orange fluorescent emissions produce a peak in Synodus (594 nm) and Saurida (590 nm). Only Synodus has fluorescent emissions corresponding to near red (674–678 nm) and far red (738–670 nm) wavelengths; red fluorescent emissions are absent in Saurida (Fig 3F). Within Labridae, fluorescent emission peaks in Cheilinus are recorded at 517–524 nm (green), 675–692 nm (near red), and 729–737 nm (far red), whereas Cirrhilabrus and Pseudocheilinus both exhibit near red peaks at 652–662 nm (Figs 1 and 3A; Table 1) and lack green fluorescent emissions.

In Scorpaenidae, all genera examined exhibit green fluorescent emission peaks (Figs 1 and 3C; Table 1). These peaks range from 526–530 nm in Caracanthus and 524 nm in Taenianotus. Sebastapistes exhibits a wide range of emission peaks spanning wavelengths from green to yellow-orange (526–584 nm). However, a distinct yellow emission peak is present in Taenianotus at 573 nm. Near red emission peaks are present in Caracanthus (679 nm), Sebastapistes (670 nm), and Taenianotus (673–677 nm), whereas far red emission peaks are present in Sebastapistes (717–744 nm) and Taenianotus (732–740 nm) (Fig 3C).

Considerably more emission peak variation is recorded among genera in Gobiidae (Figs 1 and 3B; Table 1). Green fluorescence is present in Callogobius (526–531 nm), Eviota (524 nm), and Trimma (517–526 nm) as a single peak, and as a dual peak in Acentrogobius (523 and 555 nm) and Myersina (517–527 and 546–555 nm). Yellow-orange fluorescent emissions are present as a single peak in Eviota (594–611 nm), Myersina (590 nm), and Pleurosicya (606–611 nm). Near red emission peaks are present in Eviota (672–678 nm), Myersina (635 nm), Pleurosicya (674 nm), and Trimma (670–674 nm). Far red fluorescent emissions are present in Eviota (728–731 nm) and Trimma (731–745 nm). In the gobiiform family Oxudercidae, green fluorescence is present as a single peak in both Gnatholepis (524–527 nm) and Trypauchen (517 nm), and as a dual peak in Oxyurichthys (514–519 and 553 nm) (Figs 1 and 3D; Table 1). Yellow-orange fluorescence is present as a single peak in Oxyurichthys (592–594 nm). Near red fluorescent emissions are present in both Gnatholepis (672 nm) and Oxyurichthys (635 nm), however, far red fluorescence is only present in Gnatholepis at (739–744 nm) (Fig 3D).

In Bothidae, green fluorescent emissions are present as a single peak in Japonolaeops (515 nm) and as a dual peak in Engyprosopon (523–526 and 551–552 nm) (Figs 1 and 3E; Table 1). Yellow-orange, near red, and far red fluorescent emissions are only present in Japonolaeops at 594 nm, 635 and 674 nm (dual emission), and 705 nm, respectively (Fig 3E).

We find little to no variation in fluorescent emission wavelength peaks among the two genera of Tripterygiidae (Enneapterygius and Helcogramma), Nemipteridae (Nemipterus and Scolopsis), or Soleidae (Heteromycteris and Zebrias) that we examined in this study (S2 Fig). In the remaining families investigated, we were only able to analyze a single genus (Chlopsidae, Muraenidae, Cepolidae, Aulostomidae, Liparidae, Mullidae, Blenniidae, Antennariidae, and Cynoglossidae) so intrafamilial variation could not be assessed (S2 Fig).

Eye fluorescent emissions

Fluorescence from the eye (iris and lens), is commonly observed in marine teleosts (Fig 2). Fluorescent emission spectra were recorded from the eyes in members of eleven teleost families (Fig 4; Table 1). We recorded a similar double green emission peak from the eyes of members of Chlopsidae (520–522 and 554 nm), Synodontidae (518–529 and 542–543 nm), Cepolidae (525 and 551 nm), Mullidae (520–523 and 546–550 nm), Nemipteridae (518–521 and 552–555 nm), and Gobiidae (Myersina, 525–527 and 546–551 nm). Green emissions from the eyes are also present as a single peak in Scorpaenidae (524–544 nm) and Bothidae (Japonolaeops, 560 nm). In the scorpaenid genus Sebastapistes, we recorded a yellow-orange emission peak from the eyes at 584 nm, whereas in the tripterygiid genus Helcogramma, a yellow-orange emission peak was recorded at 605–608 nm (Fig 4). Aulostomidae, Liparidae, and Scorpaenidae (Taenionotus) have eyes that emit near red peaks at 680 nm, 675 nm, and 677 nm, respectively. The eyes of members of Scorpaenidae also emit a far red emission peak at 732–742 nm.

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Eleven teleost families were measured: Chlopsidae, Synodontidae, Cepolidae, Aulostomidae, Liparidae, Mullidae, Nemipteridae, Scorpaenidae, Gobiidae, Tripterygiidae, and Bothidae. Dashed lines represent fluorescent emission peaks. Intensity values are relative and do not signify overall brightness.

Fluorescent emission variation by anatomical region

In some individuals, fluorescent emissions vary by body region (Fig 5; Table 1). In Helcogramma striata (Tripterygiidae), the upper and lower flank exhibit a green emission peak ranging from 530–532 nm, whereas only the eye and portion of the upper flank share a similar yellow-orange emission peak of 594–608 nm (Fig 5A). The lower flank of H. striata also has far red emission peaks ranging from 733–751 nm. In Scorpaenidae, Taenionotus triacanthus exhibits four distinct emission peaks over different body regions, including the eye, mouth, and upper flank (Fig 5B; Table 1). Emission peaks are green (524 nm) for the mouth and eye, yellow-orange (573 nm) for the mouth, eye, and entire flank, near red (673–677 nm) for the mouth and eye, and far red (732–740 nm) for the upper flank and eye. In Sebastapistes strongia (Scorpaenidae), the eye exhibits a green emission peak at 544 nm and a single broad yellow-orange peak at 584 nm, spanning ~566–596 nm (Fig 5C). The upper flank exhibits a green emission peak at 560–564 nm and an additional far red peak in the same spectra reading at 737–743 nm (Fig 5C). The anterior and posterior body of one Gymnothorax zonipectis (Muraenidae) specimen exhibits a similar green emission peak from 529–532 nm, however, the posterior body has an additional far red peak from the same spectra reading at 732–736 nm (Fig 5D). In Cepola schlegelii (Cepolidae), green emission peaks are present in the eyes (double peak, 525 and 551 nm) and posterior region of the body (516 nm) but are absent in the anterior section of the body (S2 Fig); both the anterior and posterior regions of the body have a yellow-orange emission peak at 594 nm and a near red emission peak at 635 nm. However, only the anterior region of the body of C. schlegelii exhibits a far red emission peak at 706 nm (S2 Fig). All other specimens examined show little to no variation over the various body regions that were scanned for fluorescence.

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A) Helcogramma striata (Tripterygiidae, n = 1); B) Taenianotus triacanthus (Scorpaenidae, n = 1); C) Sebastapistes strongia (Scorpaenidae, n = 1); D) Gymnothorax zonipectis (Muraenidae, n = 2). Note: For each panel the top image shows the species imaged under white light and all others show the species fluorescing. Dashed lines represent fluorescent emission peaks. Intensity values are relative and do not signify overall brightness.

Visual pigments

Visual pigment data is available for species representing nine of the 18 families examined in this study (Fig 6; S5 Table) [24,25]. All families have visual pigments with peak absorbances (lambda max) within the 366–500 nm range [24,25]. Visual pigments with peak absorbances in green wavelengths are present in Muraenidae (510 nm), Synodontidae (503 nm), Labridae (505–555 nm), Liparidae (522–527 nm), Mullidae (515–533 nm), Scorpaenidae (501–530 nm), Gobiidae (508–553 nm), and Blenniidae (500–561 nm). Visual pigments with peak absorbances in yellow-orange wavelengths are present in Gobiidae (565 nm) and Blenniidae (570 nm).

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Peak absorbance (lambda max) is reported for each photoreceptor type: rod, single cone, double cone, and twin cone. Intensity values are relative and do not signify overall brightness.

Discussion

In this study we compare the emission spectra of numerous species representing 18 diverse teleost families that exhibit biofluorescence. Recent studies have shown that biofluorescence is phylogenetically widespread and phenotypically variable in marine fishes, particularly across ray-finned lineages, where fluorescent emissions have generally been reported simply as either red or green [1,9]. Our results, however, show that fluorescence is far more variable in emitted wavelength and corresponding color, both among and within lineages at the family, genus, and species scale (Figs 1–5; Table 1). Further, in many species, fluorescent emissions vary within an individual (Fig 5; Table 1). In general, we recover a pattern of four distinct emission peak ranges spanning green (514–560 nm), yellow-orange (573–613 nm), near red (633–692 nm), and far red (705–751 nm) wavelengths (Fig 1; Table 1). Interestingly, fluorescent emissions in yellow wavelengths (~565–590 nm) are quite rare, only occurring in two scorpaenid genera, Sebastapistes (584 nm) and Taenianotus (573 nm) (Fig 3C; Table 1; S4 Table). However, due to sampling constraints, the results reported herein may represent a minimum of variation in fluorescent emissions in teleosts, setting a baseline for additional, more taxonomically comprehensive studies of lineages that exhibit a high degree of variability.

Overall, we find that fluorescent emissions are highly variable in marine teleosts. For example, three families— Gobiidae, Oxudercidae, and Bothidae – exhibit at least six distinct, non-overlapping fluorescent emission peaks (Figs 1 and 3; Table 1). A lack of variation in fluorescent emissions was rare, and only two of the 18 families examined exhibited a single fluorescent peak: yellow-orange in Antennariidae and near red in Aulostomidae (Fig 1; Table 1). All other families examined had at least two distinct fluorescent emission peaks, with some falling within the same emission wavelength range (e.g., green) (Fig 1; Table 1). We note that previous studies that include species not examined here have reported fluorescence in other wavelength ranges (colors) in members of both Antennariidae, and Mullidae [1,9,14], in addition to the spectra reported herein.

Fluorescent molecules

To date, proteins that produce observable fluorescence in fishes have only been identified and isolated from catsharks and true eels (Anguilliformes), all of which only produce green fluorescence [27–31]. While one red fluorescent protein, sandercyanin, has been identified in the North American walleye (Sander vitreus), this species is not naturally fluorescent (i.e., fluorescence is only observed from isolated proteins) [32]. No other molecules or fluorescent proteins that emit yellow through red fluorescent wavelengths have been identified or isolated from fluorescent fishes, despite the remarkable variation in yellow-orange, near red, and far red fluorescent emission spectra reported in this study (Fig 1; Table 1). Based on the results from prior studies focused on isolating and characterizing fluorescent proteins from false moray eels, moray eels, and catsharks, fluorescent proteins and other fluorescent molecules in fishes tend to have very narrow emission peaks of around 3 nm maximum [27,29–31]. Our results also show that it is quite common for members of several families to emit multiple distinct fluorescent emission peaks within either the green or red (near and far red) portions of the visible spectrum (Fig 1; Table 1). This ability to produce distinct emission signals within a single-color bandwidth greatly increases the variability of potential fluorescent signals that could be produced by an individual.

Our results suggest that either 1) numerous fluorescent proteins or molecules capable of producing fluorescence are present in marine fishes (e.g., multiple green or red fluorescent proteins that result in the dual peaks reported in several families), or 2) that certain lineages can alter emitted wavelengths produced by a single fluorescent protein or molecule. These hypothesized wavelength shifts could be produced via interactions with other pigment producing cells like chromatophores in combination with some type of filter or lens [33], or via some other mechanism leading to a shift in emitted wavelength [34]. Regardless, our results point to a fascinating array of fluorescent colors that can be emitted by marine fishes, frequently within a single individual, and highlight the need for further investigation of the fluorescent proteins or other molecules responsible for producing these fluorescent emissions.

Use of fluorescence in taxonomy

While some variation in fluorescent emissions may not provide a visual function, fluorescence has the potential to aid in taxonomic studies. In closely related species where patterns under white light are almost identical, variations in fluorescent emissions may serve as a tool for species identification (e.g., reef lizardfishes, Synodontidae; see [1] Fig 3). In Synodontidae, Synodus species exhibit both near red and far red emission peaks that are absent in members of the closely related genus Saurida. These fluorescent wavelengths are outside of the visual range of Synodontidae, which only extends to ~500 nm (Fig 6). However, these differences in fluorescent emissions may aid in distinguishing these otherwise morphologically similar genera (Fig 3F) [1]. Fluorescence has already been used as a character in species descriptions [35–38]. For example, fluorescence has been used to discriminate two morphologically cryptic and sympatric bonefishes (Albulidae) [38]. Fluorescent patterns in fairy wrasse (Labridae: Cirrhilabrus) were also found to be species-specific and sexually dimorphic [35]. Thus, the use of fluorescent emission spectra may provide additional variation among species, and could be used similarly to hyperspectral data of white light coloration to identify cryptic diversity [39].

Vision

A number of recent studies [40–42] provide a useful set of criteria for attributing a visual function to biofluorescence to determine whether fluorescent emissions should be considered visually significant to the individuals expressing them, including: 1) light sufficiently excites biofluorescent molecules when in their natural habitat; 2) fluorescent emissions/signals are viewed against or next to contrasting backgrounds; 3) fluorescent emissions/signals are located on regions of the body that are utilized for signaling behaviors; 4) signal-receivers have optimal spectral sensitivities to the emitted fluorescent wavelengths; and 5) fluorescence is associated with a behavioral change. This creates a narrow set of circumstances where fluorescence may contribute to a visual task, and it is possible that fluorescence does not serve a visual function in some species or at certain emission wavelengths. It is also possible that fluorescence may serve a non-visual function in some groups, such as reducing oxidative stress or photoprotection [43,44]. Thus, studies that directly test each of the above criteria are needed before a function can be attributed to a certain fluorescent taxa.

It is currently unknown whether slight variations in fluorescent emission wavelengths could serve a functional role, such as aiding in either intra- or interspecific recognition, camouflage, or by providing visual cues. Red fluorescence has been tied to intraspecific signaling in some cryptic reef fishes [e.g., members of Syngnathidae, Gobiidae (Eviota), and Tripterygiidae] when present on certain body parts used in mating (fins) [8]. However, we find no variation in the wavelength of fluorescence emissions over the body in species of Tripterygiidae or Eviota that were investigated in this study (S2 Fig). Thus, the potential role of fluorescence in intraspecific signaling in these groups could be more dependent on the location of the fluorescent emission rather than emission wavelength itself. However, we did find variation in fluorescent emissions over the body of some individuals (Figs 4 and 5). For example, Helcogramma striata (Tripterygiidae) exhibits green and yellow-orange fluorescence over all body regions measured (eye, upper and lower flank), whereas far red emissions are only present on the lower flank (Fig 5A). Future studies are needed to determine if the behavior and distribution of fluorescent tissue over the body of individuals influences success in intraspecific signaling.

In the marine environment, fluorescence has two hypothesized functions, enhancing the color signal (i.e., specific emission wavelengths are visible) or increasing luminosity contrast with the surrounding background [11,12]. Longer wavelengths from sunlight or moonlight are rapidly absorbed in oceanic habitats and fluorescence can restore these lower-energy, longer wavelengths (green-red) at depths where only shorter blue wavelengths can penetrate [1,11]. Reef fishes generally have good color vision in blue and green wavelengths, including many of the families examined this study (e.g., Gobiidae, Labridae) [16,19,20]. Species representing nine of the families investigated in this study had available visual pigment data in the literature (Fig 6; S5 Table) [24,25]. Except for Aulostomidae, all families had visual pigments with peak absorbances (lambda max) at similar green wavelengths to their fluorescent emissions (500–565 nm; Figs 1 and 6A). However, three families we examined – Blenniidae, Gobiidae, and Labridae – had longer wavelength shifted vision, with some visual pigments exhibiting peak absorbances extending to 570 nm (Fig 6A). Gobiidae in particular had certain visual pigments with peak absorbances around the green fluorescent dual emission peaks found in Myersina and Acentrogobius (Fig 6B). Moreover, many lineages of reef associated fishes (e.g., Synodontidae, Labridae, Scorpaenidae, Pleuronectiformes) that exhibit fluorescence have been shown to possess yellow intraocular (lenses or cornea) filters [21] that absorb short wavelengths of light. These filters could enable enhanced perception of fluorescent emissions in a blue ambient environment.

While members of several reef families (e.g., Pomacentridae, Gobiidae, Labridae) possess long wavelength sensitivity (LWS) opsins in their eyes, this only allows them to visualize orange-yellow wavelengths up to ~600 nm [16,19,20]. For example, Eviota pellucida (Gobiidae), a species that emits yellow-orange fluorescence (~600 nm), has a spectral sensitivity that peaks at 450–550 nm, extending to ~600 nm at 50% absorptance [8]. Whereas the yellow-orange fluorescence of E. pellucida could potentially be detected, their visual pigments are not optimized for these wavelengths, one of the key criteria for functional fluorescence [40–42]. Thus, it is possible that long-wavelength fluorescence (>600 nm) may not provide a broadscale visual function in fishes, and further direct testing of the above criteria for functional fluorescence is needed.

Some fluorescent wavelengths may also function to create greater luminosity contrast with surrounding backgrounds in a monochromatic blue ambient environment at depth, as shown in catsharks [11]. In dimmer oceanic waters (e.g., turbid coastal habitats, deeper environments where only blue light penetrates), emitted fluorescent photons at longer wavelengths may function to enhance luminosity contrast in the individual [11]. The particular functionality of this enhancement depends largely on the spectral sensitivity and environment of the signal receiver (see Marshall and Johnsen [40] for specific criteria). In addition, color vision is rarely possible in these low light environments, and organisms would rely more on rod-mediated vision [41]. For example, the swell shark (Cephaloscyllium ventriosum) has only a single visual pigment and lacks pre-retinal filters. As a result, detecting a fluorescent pattern against a background must rely entirely on variation in luminosity (i.e., brightness). Although the visual pigment (maximum absorbance 484 nm + /-3 nm) in the swell shark is not spectrally situated to maximize contrast between the individual’s bright green fluorescence and the blue background light, the absorbance of their visual pigment overlaps the spectral bandwidth of their green fluorescence. As a result, the reticulated patterns of dark and light on the body can still be detected [11]. Luminosity contrast resulting from fluorescence in C. ventriosum can result in a conspecific being more apparent than a non-fluorescent shark [11].

Fluorescence in the eyes of some reef fishes may also enhance prey detection [12,14]. In Tripterygion delaisi (Tripterygiidae), fluorescence in the iris (~600 nm) may induce high contrast reflections in the eyes of cryptic prey, increasing conspicuousness and thus capture success [45]. Similar mechanisms are hypothesized to be used by deep-sea dragonfishes (Stomiiformes) and flashlight fishes (Anomalopidae) [46,47]. Whereas we find comparable orange wavelengths of fluorescence in the eyes of members of Tripterygiidae (605–608 nm), we also find longer wavelengths of near red fluorescence in the eyes of members of Aulostomidae, Liparidae, and Scorpaenidae (near red, 674–680 nm) (Fig 4; Table 1). These red fluorescent emissions are more similar in wavelength to those observed in dragonfishes (678 nm) and could possibly serve a similar visual function in prey capture [47].

Conclusion

Our results show that biofluorescence in teleosts is not only phylogenetically widespread, but phenotypically variable. We find an exceptional degree of variation in fluorescence emission spectra within both families and genera (Figs 1 and 3; Table 1), and even over the body within certain individuals (Figs 4 and 5). This remarkable variation found across a wide array of fluorescent teleost families could allow for an incredibly diverse and elaborate fluorescent emission signaling system that is highly variable in emitted wavelength, potentially resulting in unique, species-specific fluorescent patterns. The resulting fluorescent emission spectra could provide numerous uses in taxonomic studies, such as aiding in species identification and uncovering cryptic diversity [35–38]. Given that marine fishes can produce such a fascinating diversity of fluorescent emission wavelengths (colors), novel studies across a broader range of fluorescent lineages are needed to determine the visual systems/capabilities of additional fluorescent species, identify and isolate the fluorescent molecules capable of producing these stunning displays, and investigate the potential functions of this exceptionally variable phenomenon.

Supporting information

S1 Table. All specimens used in this study with collection locality data.

https://doi.org/10.1371/journal.pone.0316789.s001

(XLSX)

S2 Fig. Additional fluorescent emission spectra for all families and genera analyzed in this study.

https://doi.org/10.1371/journal.pone.0316789.s002

S3 Fig. Fluorescent imaging and emission spectra setup and technical specifications.

https://doi.org/10.1371/journal.pone.0316789.s003

(DOCX)

S4 Table. Fluorescent emission peaks (lambda max) for all species and body.

https://doi.org/10.1371/journal.pone.0316789.s004

(XLSX)

S5 Table. Visual pigment data or species representing nine of the families investigated in this study from the literature.

https://doi.org/10.1371/journal.pone.0316789.s005

(XLSX)

Acknowledgments

We are grateful to Somsak Panha and Piyoros (Pomme) Tongkerd (CU) for facilitating our collections and studies in Thailand. We thank Sven Gust of Northern Explorers Diving & Expeditions for SCUBA diving, boat, and logistical support in eastern Greenland. We are grateful to David Gruber (CUNY), Robert Schelly (USFWS), and Brennan Phillips (URI) for considerable assistance in the field and for help with imaging and recording of emission spectra. For assistance with the identification of specimens used in this study we thank Christine Thacker (UCSB), Gerald Allen (WAM), Thomas Monroe (NOAA), and Kunio Amaoka (Hokkaido University).

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Citation: Carr EM, Thurman MA, Martin RP, Sparks TS, Sparks JS (2025) Marine fishes exhibit exceptional variation in biofluorescent emission spectra. PLoS One 20(6): e0316789. https://doi.org/10.1371/journal.pone.0316789

About the Authors:

Emily M. Carr

Roles: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing

E-mail: [email protected]

¶These authors contributed equally to this work.

Affiliations: Division of Vertebrate Zoology, Department of Ichthyology, American Museum of Natural History, New York, New York, United States of America, Richard Gilder Graduate School, American Museum of Natural History, New York, New York, United States of America

ORICD: https://orcid.org/0000-0001-7985-863X

Mason A. Thurman

Roles: Conceptualization, Investigation, Validation, Writing – original draft, Writing – review & editing

¶MAT, RPM, TSS and JSS also contributed equally to this work.

Affiliation: Department of Biological Sciences, Clemson University, Clemson, South Carolina, United States of America

Rene P. Martin

Roles: Conceptualization, Data curation, Validation, Visualization, Writing – review & editing

¶MAT, RPM, TSS and JSS also contributed equally to this work.

Affiliations: Division of Vertebrate Zoology, Department of Ichthyology, American Museum of Natural History, New York, New York, United States of America, School of Natural Resources, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America

Tate S. Sparks

Roles: Conceptualization, Data curation

¶MAT, RPM, TSS and JSS also contributed equally to this work.

Affiliation: Rutgers, School of Engineering, The State University of New Jersey, New Brunswick, New Jersey, United States of America

John S. Sparks

Roles: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing – review & editing

¶MAT, RPM, TSS and JSS also contributed equally to this work.

Affiliations: Division of Vertebrate Zoology, Department of Ichthyology, American Museum of Natural History, New York, New York, United States of America, Richard Gilder Graduate School, American Museum of Natural History, New York, New York, United States of America

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References

1. Sparks JS, Schelly RC, Smith WL, Davis MP, Tchernov D, Pieribone VA, et al. The covert world of fish biofluorescence: a phylogenetically widespread and phenotypically variable phenomenon. PLoS One. 2014;9(1):e83259. pmid:24421880

2. Kurup R, Johnson AJ, Sankar S, Hussain AA, Sathish Kumar C, Sabulal B. Fluorescent prey traps in carnivorous plants. Plant Biol (Stuttg). 2013;15(3):611–5. pmid:23696970

3. Lim MLM, Land MF, Li D. Sex-specific UV and fluorescence signals in jumping spiders. Science. 2007;315(5811):481. pmid:17255504

4. Mazel CH, Fuchs E. Contribution of fluorescence to the spectral signature and perceived color of corals. Limnol Oceano. 2003;48(1part2):390–401.

5. Welch VL, Hooijdonk EV, Intrater N, Vigneron J-P. Fluorescence in insects. The nature of light: light in nature IV. SPIE; 2012. p. 25–39. https://doi.org/10.1117/12.929547

6. Cox JL, Fitzpatrick BM. Biofluorescent sexual dimorphism revealed in a southern Appalachian endemic salamander, Plethodon metcalfi. Sci Rep. 2023;13(1):3588. pmid:36869050

7. Travouillon KJ, Cooper C, Bouzin JT, Umbrello LS, Lewis SW. All-a-glow: spectral characteristics confirm widespread fluorescence for mammals. R Soc Open Sci. 2023;10(10):230325. pmid:37800154

8. Michiels NK, Anthes N, Hart NS, Herler J, Meixner AJ, Schleifenbaum F, et al. Red fluorescence in reef fish: a novel signalling mechanism? BMC Ecol. 2008;8:16. pmid:18796150

9. Carr E, Martin R, Thurman M, Cohen K, Huie J, Gruber D, et al. Repeated and widespread evolution of biofluorescence in marine fishes. Nat Commun. 2025;16:4826. https://doi.org/10.21203/rs.3.rs-4513221/v1

10. Cohen KE, Summers AP. Dimorphic fluorescence in the Pacific spiny lumpsucker. Ichthyol Herpetol. 2022;110(2):350–3.

11. Gruber DF, Loew ER, Deheyn DD, Akkaynak D, Gaffney JP, Smith WL, et al. Biofluorescence in catsharks (Scyliorhinidae): Fundamental description and relevance for elasmobranch visual ecology. Sci Rep. 2016;6:24751. pmid:27109385

12. Meadows MG, Anthes N, Dangelmayer S, Alwany MA, Gerlach T, Schulte G, et al. Red fluorescence increases with depth in reef fishes, supporting a visual function, not UV protection. Proc Biol Sci. 2014;281(1790):20141211. pmid:25030989

13. Gruber DF, Sparks JS. First report of biofluorescence in Arctic snailfishes and rare occurrence of multiple fluorescent colors in a single species. Am Mus Novit. 2021;2020(3967):1–12.

14. Anthes N, Theobald J, Gerlach T, Meadows MG, Michiels NK. Diversity and ecological correlates of red fluorescence in marine fishes. Front Ecol Evol. 2016;4.

15. Ludt WB, Clardy TR. First detection of biofluorescence in a deep-sea anglerfish. J Fish Biol. 2022;100(3):843–6. pmid:34982469

16. Marshall NJ, Cortesi F, de Busserolles F, Siebeck UE, Cheney KL. Colours and colour vision in reef fishes: Past, present and future research directions. J Fish Biol. 2019;95(1):5–38. pmid:30357835

17. Marshall J, Carleton KL, Cronin T. Colour vision in marine organisms. Curr Opin Neurobiol. 2015;34:86–94. pmid:25725325

18. Jerlov NG. Optical oceanography. 1st ed. Amsterdam: Elsevier Publishing Company; 1968.

19. Stieb SM, Cortesi F, Sueess L, Carleton KL, Salzburger W, Marshall NJ. Why UV vision and red vision are important for damselfish (Pomacentridae): structural and expression variation in opsin genes. Mol Ecol. 2017;26(5):1323–42. pmid:27997050

20. Cortesi F, Camacho DE, Luehrmann M, Sommer GM, Musilova Z. Multiple ancestral duplications of the red-sensitive opsin gene (LWS) in teleost fishes and convergent spectral shifts to green vision in gobies. bioRxiv. 2021:2021.05.08.443214.

21. Heinermann PH. Yellow intraocular filters in fishes. Exp Biol. 1984;43(2):127–47. pmid:6398222

22. R Core Team. R: A language and environment for statistical computing. 2023. Available from: https://www.R-project.org/

23. Wilkinson L. ggplot2: elegant graphics for data analysis by WICKHAM, H. Oxford University Press; 2011. Available from: https://academic.oup.com/biometrics/article-abstract/67/2/678/7381027

24. Schweikert LE, Fitak RR, Caves EM, Sutton TT, Johnsen S. Spectral sensitivity in ray-finned fishes: diversity, ecology and shared descent. J Exp Biol. 2018;221(Pt 23):jeb189761. pmid:30322978

25. Losey GS, McFarland WN, Loew ER, Zamzow JP, Nelson PA, Marshall NJ. Visual biology of Hawaiian coral reef fishes. I. Ocular transmission and visual pigments. Montgomery WL, editor. Copeia. 2003;2003(3):433–54.

26. Van der Laan R, Fricke R, Eschmeyer WN. Eschmeyer’s Catalog of Fishes: Classification. 2024.

27. Hayashi S, Toda Y. A novel fluorescent protein purified from eel muscle. Fish Sci. 2009;75(6):1461–9.

28. Gruber DF, Gaffney JP, Mehr S, DeSalle R, Sparks JS, Platisa J, et al. Adaptive evolution of eel fluorescent proteins from fatty acid binding proteins produces bright fluorescence in the marine environment. PLoS One. 2015;10(11):e0140972. pmid:26561348

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