Fur glowing under UV: a widespread consequence of porphyrin accumulation in mammals

Spectacular photoluminescence (PL) phenomena have been increasingly reported in various organisms from diverse ecosystems. However, the chemical basis of this PL remains poorly dened, and its potential ecological function is still blurry, especially in mammals. Here we used state-of-the-art spectroscopy and multispectral imaging techniques to document new cases of mammalian ultraviolet-induced PL (UV-PL) and to identify free-base porphyrins and natural derivatives as the organic compounds responsible for the reddish luminescence observed in the hairs and spines of distantly related species. We argue that pink to red UV-PL is predominantly observed in crepuscular and nocturnal mammals because porphyrins are photodegradable, and that this phenomenon might not have a specic function in intra- or interspecic communication but consists of a byproduct of a widespread physiological condition, overlooked in mammals.

Interestingly, this UV-PL can occur in different tissues, and the observed emitted colors vary a lot within the visible spectrum depending on the species. Reported observations include shades of dark blue from bone [10][11][12][13] , of light blue and green from skin and hairs 3,4,6,8,13 , of yellow from birds' feathers [14][15][16] , and shades of pink to red from feathers as well as from the pelage of most of the photoluminescent mammals [5][6][7][8][9]17 . This high variability implies that it is not the same UV-reactive molecular or structural compounds that are at the origin of this phenomenon depending on the species, suggesting probable different mechanisms at play. For instance, biological uids like urine are known to be photoluminescent 18, 19 , resulting in blue to yellow UV-PL observed. Recent studies found iridophores as responsible for greenish UV-PL in the dermis of a gecko 3 . The reddish UV-PL observed on the plumage of some birds was identi ed as caused by coproporphyrin III and protoporphyrin IX [20][21][22][23][24][25] , which are common molecules playing a crucial role in various biological processes and notably in heme biosynthesis 26 . A recent analysis on springhare pelage 9 identi ed a mixture of other porphyrins, usually found in urine or feces of mammals 27 , along with an unidenti ed molecule. However, the question remains whether these compounds are also responsible for the reddish UV-PL observed in other mammals. Moreover, the precise spatial and spectral repartitions of these compounds within the pelage are still unknown, as well as their inter-and intraspeci c variability. Answering these questions would greatly help improving our knowledge on the biological origin and potential function of such photoluminescent mechanism in animals.
In addition to molecular identi cation, it remains unclear whether PL is actually seen and used by the organisms in their natural environment 1,28 . So far, two cases of sexual selection using PL have been experimentally demonstrated in budgerigars 14 and in jumping spiders 29 . In mammals, based on the fact that UV-PL was predominantly observed in crepuscular or nocturnal animals, it was recently hypothesized that this may serve ecological functions in light-deprived or snow-covered environments such as intraspeci c communication or antipredator behavior 4,8,9 . These hypotheses have not been tested experimentally. Moreover, as the associated species are adapted to different ecosystems and lifestyles, from the semi-aquatic platypus to the terrestrial springhares and the arboreal ying squirrels, it remains unclear whether UV-PL has indeed a speci c ecological signi cance in nocturnal animals, or is simply a byproduct of a physiological condition such as pigmentation 1,28 .
Here we document a new selection of mammals exhibiting luminescence under UV, including hedgehogs (Erinaceus spp.) and ermine (Mustela erminea), substantiating the widespread occurrence of this phenomenon across this group. We focused on the reddish UV-PL observed in most of the species' pelage and used customized spectroscopy techniques to precisely identify in-situ the organic compounds at play as metal-free porphyrins. Furthermore, we used multispectral luminescence imaging to describe the spatial and spectral distributions of these porphyrinic compounds, demonstrating their speci c localization within hairs and spines along with their variable concentration across species. These results suggest a more pragmatic scenario in which this erratic accumulation of porphyrins into mammalian pelage, resulting in an observable photoluminescence under UV illumination, may not result from a speci c selection for nocturnal communication as previously argued, but from the non-pathological degradation process of porphyrins.

Terminological clari cations
Photoluminescence (PL) is a physical phenomenon in which a material reemits photons after a photonic excitation. It gathers two distinct phenomena, namely uorescence, or phosphorescence that can be differentiated by the difference in the electronic states involved in the radiative de-excitation pathway. Fluorescence and phosphorescence occur when the molecule in an excited state returns to the electronic ground state (essentially S 0 for most organic molecules), from the excited singlet or triplet state, respectively, by emission of a photon. The uorescence process occurs within a few tens of picoseconds to a few hundreds of nanoseconds, whereas phosphorescence, which corresponds to the forbidden transition between the excited and ground states, takes place on a timescale up to seconds, even minutes or more. UV-induced photoluminescence (UV-PL) is the process whereby absorbed UV photons trigger a radiative deexcitation at longer wavelengths. In contrast, bioluminescence (BL) corresponds to an emission of light generated by a living organism via chemical reactions.
Multiple cases of UV-PL and BL have been observed in various organisms either in their natural habitat or from preserved specimens. However, UV-PL is often referred to as "bio uorescence" in the literature, which might confuse either the scienti c community and the general public. Therefore, UV-PL is the appropriate technical term to use in this context, because it is neither proven whether it is uorescence or phosphorescence, and nor does it relate to bioluminescence. Moreover, although both phenomena result in spectacular luminous or glowing organisms, they consist in quite different mechanisms and are not observable under the same conditions. In practice, the UV-PL phenomenon is only observable to the human eye by using an arti cial source of ultraviolet light, associated with an optical lter to ensure error-free visual interpretation. Importantly, another noticeable difference is that the energetically demanding BL phenomenon can be correlated to identi able functions, while PL and UV-PL phenomena are more di cult to decipher 28 .

UV-induced photoluminescence is widespread in mammals
Our investigation reveals that multiple species of mammals, representing the three major mammalian clades (monotremes, marsupials, and placentals), indeed show conspicuous photoluminescence when observed under long-wave UV illumination (365-395 nm; Fig. 1, Table 1). Also, we hereby document cases of UV-PL observed in two eutherian genera hitherto overlooked: the hedgehog (Erinaceus spp., Erinaceomorpha) which appears reddish, and the common ermine (Mustela erminea, Carnivora) which appears lavender to the human eye. Emission spectroscopy reveals that all analyzed mammal specimens emit broadly in the blue region with varying intensities (see the broad band at 450-480 nm in Fig. 1b). This blue UV-PL is particularly visible to the human eye on the poorly pigmented pelage in hedgehogs, ermines, and ying squirrels (Fig. 1a). We also con rm a visible blueish to greenish UV-PL on the pelage of the platypus, as previously reported 4 . As low pigmented human hair is also known to be visibly photoluminescent under UV in these speci c ranges due to their keratin bers 30 , this phenomenon appears broadly shared in mammals.
Interestingly, a pink to red UV-PL is exhibited on the skin appendages (here hairs and spines) of phylogenetically distant species, such as marsupials (opossums) and placental mammals, the latter including ying squirrels (Euarchontoglires) and hedgehogs (Laurasiatheria) (Table 1, Fig. 1). Emission spectroscopy reveals in these mammals a series of peaks (often three) in the red region, between 600 and 690 nm, with variable intensity and position depending on the specimens (Fig. 1b), suggesting potential differences in the compounds at play or differences of the binding sites of the same compound 31 . We additionally observed that the associated specimens exhibited variable intensity of observable reddish UV-PL. Notably, the older preserved specimens investigated appeared less intense, suggesting that the responsible compounds are degradable probably by light exposure and might be sensitive to storage condition. Furthermore, we observed that the localization of this reddish UV-PL on the pelage seems to vary within species. In our specimens, the belly was the most luminescent body region of opossum marsupials and ying squirrels, while it was the dorsal spines in hedgehogs (Fig. 1a). Lastly, the red UV-PL is not homogeneously distributed within individuals, as also reported in ying squirrels and springhares 8,9 , suggesting potential biological differences within individuals.

Porphyrin accumulation into mammalian pelage is ubiquitous
We used excitation spectroscopy at 700 nm to probe, directly on solid samples, the absorption properties of the compounds responsible for the pink to red UV-PL observed in mammals' pelages. Spectra collected on hairs or spines show an intense band between 390 and 430 nm (so-called Soret band), followed by a series of bands, ten to a hundred times less intense, between 480 and 660 nm (so-called Q-bands) characteristic of free-base (nonmetalated) porphyrins (Fig. 2a). on the affected step of the heme biosynthetic pathway 35,36 . In addition to humans, this condition has been described in a few other mammals such as ruminants, horses, and cats 27,37 . Interestingly, several cases of protoporphyrin accumulation not causing any disease were also reported in mammals. Notably, all members of the fox squirrel Sciurus niger are known to accumulate the free-base uroporphyrin I in their internal organs and skin without showing detrimental symptoms, as a result of the low activity of the enzyme uroporphyrinogen III synthase 34 . Other free-base porphyrins were recently identi ed as causing a non-pathological conspicuous reddish UV-PL on the pelage of several springhare species 9 . Non-pathological porphyrin accumulation has also been described in a pet hedgehog 38 .
As our analyses were conducted on preserved collection specimens, we could not determine whether they exhibited porphyria symptoms before their death, and the causes of porphyrin accumulation in the mammals that we have sampled remain to be identi ed. Nonetheless, previous observations on living individuals suggest that this condition is predominantly not harmful for most mammal species. Also, as dermal lesions in humans are found in the skin where hairs are sparse 35 , it is possible that porphyrin accumulation occurring in the inert hair or spine tissues is naturally less problematic than the one occurring in living tissues such as those of the skin. Therefore, we suppose that most species may exhibit physiological solutions to adapt to the potential toxicity of porphyrin overproduction 34 . This suggests that porphyrin accumulation in the pelage of mammals, resulting in a reddish UV-PL phenomenon, is likely a much more common trait than previously assumed.
The platypus and ermine likely have other photoluminescent compounds in their pelage that remain to be identi ed. The analysis for these speci c UV-PL colors (blue-green and lavender) would require further investigation, because contrary to porphyrins they are not associated with characteristic spectra.

Does pelage photoluminescence have an ecological function?
Our multispectral imaging analysis reveals that the porphyrinic compounds responsible for the reddish UV-PL are located inside the spine and hair bers of mammals (Fig. 2b), as previously seen in springhares 9 . This is particularly visible in the hedgehog' spines, where the PPIX is located within the inner lumen (Fig. 2b). This suggests that these porphyrinic compounds are excreted within the skin appendages, and throughout the natural process of continuous pelage growth. Also, we found that the spatial and spectral repartition of these compounds is not homogeneously distributed along the hairs and spines, and neither between individuals nor species. Indeed, in the hedgehog's spines, PPIX is coating the walls of the inner lumen from base to apex (Fig. 2b). In the hairs of marsupials and rodents, the porphyrins are present either throughout the total hair length (marsupials) or restricted to a more basal region ( ying squirrels). Additionally, we found that the intensity of UV-PL is also lower in areas that are otherwise less pigmented and in hairs that are thinner (e.g., ying squirrels) compared to the thicker and more pigmented spines of hedgehogs (Fig. 2b). Additionally, we found that alcohol-preserved specimens (e.g., marsupial from JAGUARS collections) show higher intensity of UV-PL, compared to the older and dry-preserved specimens of hedgehog or ying squirrels that we analyzed. This variation in degrees of reddish UV-PL intensity were also previously noted in museum specimens 6,8,9 . Because porphyrins are photodegradable compounds 39 , it appears thus likely that the preservation of the reddish UV-PL on specimens' pelage may vary depending on both the preservation method employed and the amount of light they have been exposed to through time. This entails that one cannot draw the conclusion that a given species is not accumulating porphyrins in its pelage based on the apparent lack of UV-PL in museum specimens.
As this reddish UV-PL is predominantly found in crepuscular and nocturnal species, this phenomenon has been interpreted as potentially related to visual functions for intraspeci c communication or antipredator behavior in light-deprived environments 4,8,9,24 . However, in showing that the reddish UV-PL of mammals' pelage is induced by the accumulation of photodegradable protoporphyrins, it appears more likely that this phenomenon might simply be overrepresented in crepuscular and nocturnal specimens mainly because in their case the porphyrinic compounds are less degraded than in diurnal species. Furthermore, it should be kept in mind that it is far from evident that natural UV illuminating sources, even at dusk or dawn, are su cient for this UV-PL phenomenon to be perceived and contrasted from the re ected visible light by co-speci cs or predators 11 . Indeed, for the UV-PL to have a visual function, it would require that the animals are exposed, voluntarily or not, to su cient UV illumination so that the photoluminescence reemitted is seen by other individuals or species. Additionally, one should keep in mind that when it comes to UV-PL, the reemitted light is in the "visible spectrum" (400-700 nm), so whether the animals have an improved UV light vision (200-400 nm) does not provide them with any advantage to perceive such photoluminescence.
Given these results, we thus suggest an alternative hypothesis to interpret the accumulation of PPIX derivatives in the pelage of mammals, resulting in this observable UV-PL. We suggest that this phenomenon is a byproduct of the heme biosynthesis with no other function than to be excreted from the organism by being naturally degraded through the pelage 1,28 . Storing these residual molecules of the heme pathway -which might become toxic when produced in too high quantities 26,35,36 -in skin appendages that are composed of inert tissues such as hair or spines and that are likely to be exposed to light, could hence help excreting these compounds without further energetically demanding metabolic processes. Given its distribution in the phylogeny, this perhaps represents a mechanism that was acquired early in the evolution of mammals.  Table 1. To proceed with photoluminescent spectroscopy and multispectral imaging, we collected several samples of complete hairs and spines of known orientation and spatial localization on the pelage of several selected specimens (Table 1, Fig. 1a., Fig. 2b.). Spectroscopy and multispectral imaging measurements were also carried out on two complete specimens preserved in alcohol (JAGUARS-M535 and JAGUARS-M2838).
UV-induced photoluminescence macro-analysis and photography. Specimens were observed and photographed rst under natural or standard white light conditions. Then they were placed in the dark and illuminated using speci c UV light setups emitting at a distance of about 20 cm from the specimens. We initially used a 395 nm LED torch (100 LED ashlight, Youthink) following ref. 8 and associated with a yellow lter (K&F Concept) to annul the purple light emitted by this torch. Nevertheless, this setup may leave out some wavelengths that are not captured by the yellow lter and which can be misinterpreted as UV-PL. We therefore used a professional 365 nm compact lamp (4 W, UVL-21, UVP) associated with a strict UV black lter (Filter Band U-360 2IN SQ, Edmund Optics Ltd).
Photographs presented in Fig. 1.a. were obtained using the second setup, when the lter was placed directly on the UV emitting surface of the lamp. Photographs were taken using a Canon EOS 5D Mark III equipped with a 50mm macro lens and a UV blocking lter (UV390 Protect Filter, hama). White balance was systematically readjusted for each specimen.
Photoluminescence spectroscopy. Emission and excitation spectroscopy were performed using a spectro uorometer Fluorolog 3-22 from HORIBA Jobin Yvon. Spectra were collected using a bundle of optical ber equipped with a focusing optics allowing to perform measurements out of analysis compartment. Entrance and exit slits widths of the monochromators were set at 10 nm in order to ensure a balance between the collection of spectra with good signal-to-noise and a spectral resolution compatible with the detection of Q bands. The emission spectra were collected using an excitation at 400 nm. To record the excitation spectra, a 732 ± 34 nm bandpass lters (from Semrock) was placed at the entrance of the emission monochromator in order to collect the signal at 700 nm without any interference due to the second order diffraction of excitation light or due to stray light.
Photoluminescence multispectral imaging. False color luminescence images were assembled using black and white images collected with a home-made setup which consists of a low-noise 4-megapixel Si C-MOS camera (ORCA ash V4.0 V2 -Hamamatsu) with a sensitivity ranging from 200 to 1100 nm. The camera is tted with a UV-VIS-IR 60 mm 1:4 Apo Macro lens (CoastalOptics) in front of which is positioned a lter wheel holding 8 Interference band-pass lters (Semrock) to collect images in speci c spectral ranges. Illumination was provided by 16 LED lights ranging from 365 up to 700 nm (CoolLED pE-4000), coupled to a liquid light-guide ber tted with a ber-optic ring light-guide, allowing homogeneous illumination of the region of interest. False color images were generated by assigning red, green and blue color to speci c emission ranges using ImageJ 40 software.

Declarations
Data availability statement The dataset generated for this study, i.e., the raw multispectral images and emission and excitation spectra, are available on Figshare (https://doi.org/10.6084/m9. gshare.XXX).