Fluorescence of bony tubercles in chameleons

We discovered that the crests and tubercles on the heads of many chameleon species emit blue fluorescence when excited with UV light. Focusing on the genus Calumma we investigated the osteological and histological basis of this phenomenon and the sex/species specificity of its patterns, and its phylogenetic distribution across all chameleon genera.

Living and ethanol-preserved Calumma chameleons exhibit characteristic tubercle patterns of blue fluorescence on their heads (Fig. 1A). The optimal excitation wavelength is in the UV-A spectrum at 353 nm, inducing an emission spectrum from 360 nm to 500 nm, with a maximum at 433 nm (Fig. 1B) measured from C. globifer and C. crypticum without notable variation between the species. The fluorescent elements are the centres of raised scales (Fig. 1C) in most cases. Micro-CT scans reveal these raised scales to be caused by tubercular outgrowths of the underlying bones (Fig. 1A,D, Supplementary Figs S1–4). Histological sections of a fluorescent tubercle (FT, Fig. 2) demonstrate that the top of the bony tubercle is only covered by a thin layer (20–25 µm) of epidermis that functions as a ‘window’ through which the bone is directly visible (Fig. 2B,D,F, Supplementary Fig. S5A). The bony tubercle displaces the dermis containing melanophores and chromatophores24, which are present around the protuberance and identical to that of a non-tubercle scale (Fig. 2A,C,E), rendering the layer covering the tubercle thin and transparent. A comparison of fluorescence spectra of a FT with the underlying bone showed that the emission peak of the bone covers the same range and is broadened towards longer wavelengths with a maximum at about 445 nm (Supplementary Fig. S5F,G). This indicates that the FT spectrum is included in that of the bone. Obviously the thin layer of epidermis acts as an optical filter and shifts the fluorescence towards a ‘bluer’ emission spectrum.

Figure 1 Chameleons of the genus Calumma with fluorescent tubercles of bony origin. (A) Male C. globifer (ZSM 141/2016) showing congruent tubercle/fluorescent patterns (from left to right); top row: alive in the field under sunlight, micro-CT scan of head surface (probable edge artefact in cheek region), micro-CT scan of the skull; bottom row: alive in the field under UV light, ethanol-preserved under UV light. (B) Excitation-emission matrix (intensity in arbitrary units) of fluorescent tubercles on right temporal region of C. globifer (ZSM 221/2002). (C) Fluorescent tubercles (FTs) on temporal region (right body side) of a male C. crypticum (ZSM 503/2014) under visible (above) and additional UV light (below); framed area of the skin, including a FT, was histologically sectioned (Fig. 2). (D) Distribution of FTs on the head surface (left) and micro-CT scan (right) of the head of C. guibei (ZSM 2855/2010) shows the bony origin of the FTs. Full size image

Figure 2 Histological sections of skin of a male Calumma crypticum (ZSM 503/2014) from the temporal region (framed in Fig. 1C). (A,B) 3D-reconstruction (volume rendering) of 279 histological sections of skin (without tubercle) (A) and of tubercle (B), i.e. upper margin of frame in Fig. 1C). (C) Section of skin and underlying bone in adjacent skin, stained (Richardson). (D) Section of FT (centre, stained). (E) Detail of skin near FT (stained). (F) Detail of FT (stained). For detailed views of the chromatophores, see Supplementary Fig. S5. Full size image

Distribution patterns of FTs

This externally visible bone-based fluorescence is not restricted to Calumma but also occurs in at least 8 of the 12 chameleon genera currently recognized (Fig. 3A). We focused on the species specificity and sexual dimorphism of Calumma. A quantitative analysis of FTs in 24 of the 34 species25,26 (126 adult specimens in total) shows that male individuals have on average more tubercles than females in almost all species (Fig. 3B). As we were restricted to using only relatively fresh material, despite having one of the largest collections of Calumma specimens outside Madagascar at our disposal, sample sizes per species were low, and the statistical significance of sexual dimorphism is therefore limited; although ANOVA results provide significantly more tubercles in the C. brevicorne group, with p = 0.016, and C. nasutum group, with p = 0.003 (Supplementary Table S1). The trend appears to be strong across all species (Figs 3B and 4A–D), and we expect that greater sampling will only strengthen it further.

Figure 3 Analysis of distribution of FTs on adult individuals of Calumma species groups. (A) Schematic chameleon phylogeny of Tolley et al.27 updated with genus Palleon, light blue lines indicate genera where fluorescence occurs (* indicates genus where no data was available). (B) Mean value of fluorescent tubercles (FTs) per head side and species, males plotted against females of 126 individuals/24 species assigned to four species groups; dashed diagonal line shows 1:1 ratio, samples below it show more tubercles in males than females. (C,D) PCA scatter plots assigned to four species groups, factor loadings are given in Supplementary Tables S2, 3; (C) Distribution of FTs per head side based on 12 characters (Supplementary Table S4) of 140 individuals/29 species. (D) Number of FTs per cranial bone from lateral view of 25 adult males of different Calumma species based on 6 characters (Supplementary Table S5). (E) Bar charts of number of FTs per cranial bone (M, maxilla; PRF, prefrontal; Flat, frontal seen laterally; POF, postorbitofrontal; JU, jugal; SQ, squamosal; see Fig. 1D) from lateral view of 25 adult males of different Calumma species. Full size image

Figure 4 Fluorescent tubercles showing sexual dimorphism under UV light at 365 nm (A–D) and fluorescence in further chameleon genera (E–G). (A) Male Calumma crypticum ZSM 32/2016. (B) Female C. crypticum ZSM 67/2005. (C) Male C. cucullatum ZSM 655/2014. (D) Female C. cucullatum ZSM 654/2014. (E) Brookesia superciliaris, male (only UV light at 365 nm). (F) Bradypodion transvaalense, male (dim light and additional UV light at 395 nm). (G) Furcifer pardalis, male (daylight and additional UV light at 365 nm). For details see ‘fluorescent photography’ in Materials and Methods. Full size image

FTs are concentrated around the eye and the temporal region (Supplementary Figs S1–4), which are important areas for colour signalling among chameleons. Their distribution differs however among species, and even more so among species groups (Fig. 3C). A quantitative comparison of the FTs per cranial bone (Fig. 1D) in males of different Calumma species is systematically and phylogenetically informative (Fig. 3D,E). Members of the C. parsonii group for instance differ clearly from the other species by the presence of a high number of FTs in the temporal region, caused by the broadened postorbitofrontal and squamosal bone which are fused and are densely covered with tubercles (Supplementary Fig. S4). The differences in FT patterns among closely related species are inconsistent, with some sister-species pairs (e.g. C. brevicorne and C. crypticum27) having strongly overlapping values, while others (e.g. C. tsaratananaense and C. hafahafa) are distinctly different (Supplementary Table S4). FT patterns strongly reflect species groups, and are therefore of significant systematic value at the supraspecific level within Calumma (Fig. 3E), but their role within species groups requires further study.

Hypothesis of ecological relevance of fluorescence in chameleons

Phylogenetically, it seems FTs are plesiomorphic to Chamaeleonidae, but are most widespread in Calumma being found in almost all known species (Fig. 3A, Supplementary Table S4). In their sister genus Furcifer, by contrast, we found only six of 20 studied species to have FTs (Fig. 4G, Supplementary Table S6). Furcifer is typically found in more open and dryer habitats in Western Madagascar whereas Calumma are usually found in shady, humid forest habitats28. Also in Brookesia, which are terrestrial and forest dwelling chameleons from Madagascar, bone-based fluorescence even across the whole body is common (Fig. 4E). As shorter (UV, blue) wavelengths are scattered more strongly than longer wavelengths29 the UV component under the diffuse irradiation in the forest shade is relatively higher compared to the direct irradiation by the sunlight. Consequently, using UV reflections for communication is apparently more common in closed habitats than in open habitats, as has been shown in chameleons of the genus Bradypodion30. Members of the latter genus also show fluorescence in the head region (Fig. 4F). A function of FTs as UV reflectors could therefore be hypothesised. This can be ruled out however, as these structures do not reflect UV light at 365 or 390 nm (Fig. 1B, Supplementary Fig. S6A) and at least some chameleon lenses are only transmissive above 350 nm31. On the other hand, the emission spectrum of FTs with a maximum at 433 nm is deep blue, a colour that is reasonably rare in a tropical forest and appears to be a conspicuous signal against the background reflectance of grey brown leaf litter or green vegetation as shown in Fig. 1 of Andersson et al.32. The glowing blue of the FTs is near to the maximum absorption of the pigments of the SWS (short-wave-sensitive) cones with 440–450 nm of the examined chameleons31. Additionally, wavelengths of around 433 nm might appear brighter to chameleons as their visual spectrum is shifted towards shorter wavelengths (from about 350 nm to 650 nm) compared to the human visual perception. Assuming that in the shade of a closed forest canopy the relative intensity of the diffuse UV-light is even higher the fluorescent part of the total reflectance might increase considerably. A quantum yield calculation revealed 0.29% of absorbed photons being emitted as fluorescence. This is a typical fluorescence quantum yield for a dye that is immobilised in a matrix (here the bone) and not in a solution (Schwager unpubl.).

Constant fluorescent patterns potentially give chameleons a secondary, stable signalling system that is not influenced by their well-known communication by colour change, and does not compromise their camouflage. Moreover, the systematic relevance of the FTs suggests that they at least correlate with trends in skull evolution, and they may have provided substrate for sexual selection. On this basis, the distribution of FTs could also be used by taxonomists as an additional character to delimitate between taxa. Sexual dichromatism based on fluorescence has been shown in birds33 which are also highly visual animals. The use of chameleons as model organisms in the understanding of the role of visual ecology and behaviour in sexual selection34,35 will require some adjustment to account for this newly discovered phenomenon. Important future steps will be behavioural trials as well as the creation of colour visual models (accounting for the spatial acuity of chameleon vision) in order to explore the biological relevance of this phenomenon.

Given the recent discoveries of fluorescence, especially among terrestrial vertebrates11,36, it is clear that fluorescence evolved by several independent and unique mechanisms. Apparently this tends to occur by the evolution of new mechanisms, rather than implementing features that already have fluorescent properties. Use of such features would be expected to be the most obvious route to fluorescence. We suspect, however, that chameleons are not unique in this regard, and that bone-based fluorescence is in fact widespread among taxa that use bones in their ornamentation, especially other squamates. Fluorescence in terrestrial vertebrates has been underestimated until now, and its role in the evolution of ornamentation remains largely unexplored, but this is a promising avenue for future research.