It's not easy being green: Comparing typical skin colouration among amphibians with colour abnormalities associated with chromatophore deficits

Abstract Amphibians can obtain their colour from a combination of several different pigment and light reflecting cell types called chromatophores, with defects in one or several of the cells leading to colour abnormalities. There is a need for better recording of colour abnormalities within wild amphibian populations, as this may provide baseline data that can be used to determine changes in environmental conditions and population dynamics, such as inbreeding. In this study, we provide records of several types of chromatophore deficiencies, including those involving iridophores, xanthophores and melanophores, among two Australian tree frog species; the green and golden bell frog, Litoria aurea, and the eastern dwarf tree frog, L. fallax. We explore these colour abnormalities in terms of the chromatophores that have likely been affected and associated with their expression, in combination with typical colour phenotypes, colour variations and colour changes for these species. We intend for our photographs to be used as a visual guide that addresses the need for more accessible information regarding the physical manifestation of different chromatophore defects among amphibians.

pressures, leading to a range of typical phenotypes for each species, including colour variations within populations (colour polymorphism; Hoffman & Blouin, 2000) and between populations experiencing different environmental conditions (Wilkens, 1988).Amphibians obtain their colour via light interacting with a layered arrangement of three main types of specialised cells called chromatophores that exist within the dermis skin layer; two of which contain pigments that absorb light and one that contains reflective platelets that reflect or scatter light (Bechtel, 1995;Duellman & Trueb, 1994).
Each chromatophore unit consists of a xanthophore (containing yellow pigments that absorb short wavelengths such as blue-violet), which lies above an iridophore (containing reflective platelets that reflect or scatter the remaining short wavelengths yellow-green), and a melanophore (containing dark melanin pigment that absorbs long wavelengths such as red-orange) below (Bagnara et al., 1968;Bagnara & Hadley, 1973); layering differences have been detected in some species (e.g., Kobelt & Linsenmair, 1986).A visual representation of this unit can be found in Vitt and Caldwell (2013).It is the combination of all three chromatophores that give many frogs their stereotypical green colouration, as intermediate wavelengths (green) pass back through the skin (Bagnara & Hadley, 1973;Taylor & Bagnara, 1972).Beyond the chromatophore unit, melanocytes may also be present more superficially within the epidermis (Smith-Gill et al., 1972), and structural materials such as collagen within the skin may directly interact with light and contribute to skin colouration (Bagnara et al., 2007).In the absence of chromatophores, species with translucent skin may also attain colour from extracellular pigments (Franco-Belussi et al., 2016;Taboada et al., 2020).
The colouration of many amphibians is plastic, with stress, environmental background colour and temperature triggering pathways, notably hormonal, that allow individuals to adjust their phenotypic expression (Nilsson et al., 2013); an example is colour change that can occur rapidly via movement of pigments within chromatophore cells (Nielsen, 1978).The effects of chromatophore combinations and plasticity in their contribution to skin appearance highlight the complexity of colouration among amphibians.
Given its role in how individuals visually interact with their environment, colouration is under strong selecting forces that should lead to the rapid elimination overtime of deleterious colours or patterns (Andren & Nilson, 1981).Aberrant colourations are rare in natural populations (Henle & Dubois, 2017;Hoffman & Blouin, 2000), usually the result of genetic or environmental factors that affect the development, pigmentation, density and distribution of one or several of these cells (Duellman & Trueb, 1994).They may occur as part of the natural gene pool (Mitchell & Church, 2002), or they can be caused by environmental pollution (Henle & Dubois, 2017).Human disturbances such as habitat fragmentation can increase the visibility of rare alleles in affected populations via population isolation and inbreeding depression (Bensch et al., 2000;Vershinin, 2004).
No matter what their cause, these colour variations may be harmful for individuals if it causes them to be more susceptible to visually oriented predators-affecting cryptic species if it increases their conspicuousness (Childs Jr, 1953), or aposematic species if it decreases the strength of warning signals; for amphibian species that are primarily nocturnal the impact of abnormal colouring may be lessened due to the poor colour vision of most predator animals at night (Sillman et al., 1997).Variation in colour may also reduce reproductive potential if it reduces an individual's detectability by or their visual appeal to the opposite sex, especially since many amphibians show excellent colour vision in both dark and light environments (Aho et al., 1993;Cummings et al., 2008).Yet, colour mutations can sometimes be advantageous for animals, such as by increasing the inconspicuousness of individuals within human-altered environments (Askew et al., 1971), or by causing predators to be reluctant to attack visually novel prey (neophobia; Exnerová et al., 2006), or have neutral impacts on fitness, particularly beyond early life stages (Bensch et al., 2000).Further studies are warranted showing the occurrence rate and potential impacts of abnormal colouring among amphibians.
Understanding the ecological implications of colour mutations relies in part on measuring their occurrence in wild populations.
Potentially, changes in the occurrence rates of such abnormalities can be used as a rapid, visual means of monitoring populations for threats, such as inbreeding depression or environmental pollution.Indeed, colour aberrations may be associated with other deleterious mutations that may not be so easily detected at a distance (Browder, 1972;Sanabria et al., 2010).Unusual colouring or colour changes may also be an indication of the condition of individuals, such as changes in skin iridescence related to hydration (Kobelt & Linsenmair, 1992).Colour variants can also reveal information about the distribution of chromatophores of target species, which may not be visually apparent in individuals with typical colouration (Turner, 2017).While notes regarding observations of abnormal colouration are widespread among amphibians, occurrence rates within populations are not always provided, preventing the detection of changes in their occurrence from base rates that may provide valuable insights into the functioning of amphibian populations.
In this study, we present several different types of abnormal colouration that are the result of chromatophore deficiencies, detected during extensive field surveys of sympatric populations of two Australian tree frog species.Our primary objectives were to provide occurrence rates of each colour aberration and to compare these aberrations with typical colour phenotypes, adjustments and variability found within these species.We have inferred the chromatophore cell types that are likely affected for each aberration detected based on skin colouration and compared them with similar aberrations in other amphibians as part of a mini review.We have provided high quality photographs of wild individuals with both typical and abnormal colourations to be used as visual guides for understanding the physical manifestation of different chromophore defects among amphibians.

| FIELD ME THODS
All case studies used in this study are derived from observations made on Kooragang Island, NSW, Australia (32.85837°S and 151.72480°E).This island is situated at the mouth of the Hunter River and has undergone widespread modification from agricultural and industrial activities, as well as recent remediation involving pond construction, resulting in the presence of semi-natural and artificial freshwater ponds interspersed by mangrove creeks and saltmarsh (Beranek et al., 2020;Gould et al., 2024).
Across four consecutive years from 2020 to 2024, we surveyed 80-90 freshwater ponds on the island between September and April to monitor one of the largest extant populations of the threatened green and golden bell frog (Litoria aurea).Each pond was surveyed at night by two or more researchers with headtorches for a maximum of 30 min by wading around the waterline and throughout emergent aquatic vegetation.During these surveys, we also recorded the presence of a sympatric species, the eastern dwarf tree frog (L.fallax) and any instances of colour abnormalities in both species.

| Amphibians with naturally blue skin
Few amphibians have blue skin colours or patterns that are considered typical.Exceptions include some morphs of the poison dart frog (Dendrobates tinctorius), where both sexes are almost entirely blue (d 'Orgeix et al., 2019), the moor frog (Rana arvalis), which shows dynamic sexual dichromatism as males turn blue during breeding periods (Rojas, 2017), and the Indian bullfrog (Hoplobatrachus tigerinus) in which breeding males exhibit blue vocal sacs (AmphibiaWeb, 2010;Thongproh et al., 2022).For these species, the blue colouring may be used as a visual cue for sex recognition (Sztatecsny et al., 2012), or as a warning sign (aposematism) to prevent predation (Caro & Ruxton, 2019;Rojas & Endler, 2013).Blue skin colours are caused by the reflectance of light from iridophores in the presence of melanophores below, but in the absence of overlying xanthophores (Bagnara et al., 2007;Rodríguez et al., 2020).

| Case study-Litoria aurea
Among Australian amphibians, there are no species that are naturally blue across a majority of their body surfaces (Anstis, 2007), yet some do show blue markings in select body regions.The most notable include those within the bell frog species group, such as L. aurea.Both sexes of this species have vivid turquoise blue along the thighs, running into the lower sides, as well as the armpits (Figure 1).The adaptive purpose of this blue marking, if any, remains undetermined.Its presence in both sexes suggests that its occurrence is unlikely to be for intraspecific communication but it may be form of hidden aposematism (flash colouration) exhibited against visual orientated predators during escape or posturing (Ferreira et al., 2019).Consistent with that hypothesis, the blue coloured skin is almost entirely hidden when these frogs are resting in a neutral position (Figure 1).

| Axanthism among amphibians
Blue skin colour aberrations have been recorded globally among amphibians (Henle & Dubois, 2017), particularly species in the family Randiae and more often in frogs from North America (Berns & Uhler, 1966;Jablonski et al., 2014).Yet, it has been recorded in only a few Australian species and rarely photographed.Exceptions include a completely blue green tree frog (L.caerulea) and a partially blue Barrington Tops tree frog (L.barringtonensis; Robinson, 2022).
These aberrations are most likely the result of mutations causing a lack of the carotenoid-bearing xanthophores or their pigment and leading to axanthism (Bagnara et al., 1978;Berns & Narayan, 1970;Browder, 1968).This prevents the filtering of short wavelengths of light that are scattered from the iridophore cells within the chromatophore unit, which results in the expression of blue skin across affected surfaces where green skin would have been expressed, typically the dorsum (Berns & Narayan, 1970;Miller et al., 2018).et al., 2007).However, we are not aware of visual comparisons of blue colouration caused by these two different processes.
Axanthism is a rare aberration, particularly among adults, which may be related to the reduced fitness of individuals if the abnormal colouring results in increased predation or is associated with other harmful mutations (Dubois, 1979;Jablonski et al., 2014).For example, the rate of axanthism in the green frog (Lithobates clamitans), has been recorded at 0.2-0.3%(Berns & Uhler, 1966).Disparities in the frequency of its occurrence between species and regions may be an artefact of research intensity (Jablonski et al., 2014), yet there may be a higher predisposition for its occurrence which is species specific.

| Case study-Litoria aurea
We recently made the first observation of partial axanthism in L. aurea.In December 2023, an adult L. aurea female showing blue skin patches was captured in a constructed wetland pond within a Typha stand on Kooragang Island (32.86976° S, 151.73119°E).An opaque, aquamarine colouration was confined to small patches on the face, including the nose, upper eyelids and upper lip, as well as the dorsum and forelimbs (Figure 2).Normal colouration was found across remaining areas of dorsum (light green), as well as the vent (opaque white), irises (metallic gold with black venation) and thighs (opaque turquoise).The individual was partially gravid and showed good condition (as noted by the large size of the body relative to the head).This axanthism differs from the typical blue flash thigh colouration that also occurs in this species, with the relatively greenish hue of the abnormal blue skin suggesting that there was only a deficit in xanthophores or their pigment as opposed to a complete absence.Across four consecutive years (2020-2023), we detected only one partially axanthic blue L. aurea out of 7193 observations (0.01%).

| Case study-Litoria fallax
We recently made the first observation of near complete axanthism in L. fallax.In December 2023, we observed a blue adult of unknown sex within a constructed pond on Kooragang Island (32.86481° S, 151.73507°E), resting on a Baumea plant 60 cm above water.The frog was almost entirely opaque, cerulean blue, including the face, dorsum, flanks and limbs (Figure 3 noticeable example of the latter occurs in male eastern stony creek frogs (Litoria wilcoxii), which undergo rapid physiological colour change from brown to opaque yellow that occurs after mate selection and amplexus has been initiated (Kindermann & Hero, 2016b); a similarly rapid colour change has been exhibited in adult male Bufo luetkenii (Doucet & Mennill, 2010).This rapid dichromatism in L.
wilcoxii may be used as a visual cue to deter conspecific males and is under neuro-hormonal control.Epinephrine induced pathways cause melanosomes within melanophores to become aggregated within the centre of these cells, thus allowing for the exposure of the yellow xanthophores on top that are usually covered by the melanophore pigment when it is dispersed (Kindermann et al. 2014;Kindermann & Hero, 2016a).In this species, iridophores are also absent from the yellow skin (Kindermann & Hero, 2016a) but in other species yellow skin contain both xanthophores and iridophores (e.g., Ambystoma maculatum; Taylor & Bagnara, 1972).We are unsure how the presence of iridophores influences the expression of yellow skin colouration except to brighten the xanthophore pigment and/or increase the opaqueness of the skin (Bagnara, 1966).

| Case study-Litoria aurea
We have observed dynamic sexual dichromatism in L. aurea during the breeding season, with male individuals exhibiting relatively more yellowing of green dorsal skin and white banding, as well as discoloured yellowish throats when compared to females that possess the typical green dorsal skin colouration with white banding and white throats (Figure 5).This yellowing in L. aurea appears to be extended for the duration of the breeding season (several weeks or more) and may be under steroid-hormonal control (e.g., Richards, 1982), particularly elevated testosterone levels that causes either a (i) slow, temporary morphological colour change via the increase in xanthophore pigment synthesis (Kindermann  (Tang et al., 2014).As yellowing in males occurs over areas of white skin for extended periods with relatively little difference in brown dorsal patterning between the sexes, we hypothesise that increased testosterone leading to xanthophore pigment synthesis is the primary cause for dorsal yellowing in L. aurea as opposed to a rapid change in the aggregation of melanosomes within melanophores as seen in L. wilcoxii, Seasonal changes in testicular structure and consequently hormones have also been associated with the cyclic hypertrophy/darkening and regression/lightening of male nuptial pads (Lofts, 1964;Lynch & Blackburn, 1995), a secondary sex characteristic used to assist with grasping females during amplexus.It remains to be determined whether this extended sexual dichromatism seen in L. aurea has an adaptive function in terms of intraspecific visual communication (mate choice or malemale competition) or is simply an artefact of hormonal changes that occur with reproductive cycles.

| Hypomelanism among amphibians
Conditions that lead to deficiencies or the absence in melanin pigment are referred to as hypomelanism.This includes albinism (total amelanism), which is an autosomal recessive gene inheritance that results in the complete or near complete absence in biosynthesis of melanin pigment across all pigmented tissues including the eyes (both the iris and retina) (Lunghi et al., 2017;Sazima, 1974), and leucism which affects the skin and irises but not the retinas.In the absence of melanin (hypomelanism), amphibians may still express colouring due to the presence of non-melanin pigment cell types in the skin that are not affected (Smith-Gill et al., 1972).For example, fire salamanders (Salamandra salamandra) have black skin that is melanin rich with likely few other chromatophore cell types present, with yellow patches that are low in melanin but contain high abundances of xanthophores (and possibly iridophores).Albino individuals of this species express flesh/pink skin and eyes due to the absence of melanin in areas that are typically black but continue to express yellow skin patches that are the result of the unaffected presence of xanthophores (Lunghi et al., 2017).

| Case study-Litoria aurea
Across four consecutive breeding seasons, we detected three adult L. aurea individuals out of 7193 observations (0.04%) exhibiting partial hypomelanism to varying extents across Kooragang Island (Figure 6).These individuals exhibited abnormal, opaque yellowing of dorsal skin that is typically opaque green, suggesting the loss of melanin but the maintenance of other chromatophores, namely iridophores and xanthophores.Depigmentation occurred (i) only on the extremities, including completely on the forelimbs and partially on the hindlimbs, with no other skin sections affected, (ii) the front of the face, flanks and extremities, with the central dorsum of the back and head retaining normal skin colouration and (iii) almost all F I G U R E 5 Dichromatism in adult green and golden bell frogs, Litoria aurea, during the breeding season.Male (left) shows yellowing of dorsal and throat surfaces while female (right) has retained green dorsal surfaces and white throat.The yellowing of the male's skin compared with that of the female can be seen in the mating pair in amplexus.skin surfaces except for partial, mottled green colouration of the dorsum (Figure 6).Individuals with their head dorsum affected showed a noticeable lack of black banding running between the nose and eyes, further evidence of melanin deficiency.The affected skin was also opaque yellow in appearance when compared to ventral skin that remained opaque white, the latter of which is typical for this species.All individuals possessed normal pigmentation of the iris, which was metallic gold with black venation and surrounded by a rim of black, and black retinas, indicating no other chromatophores besides melanin pigment cells in the skin were affected.The yellow colouration caused by hypomelanism differs from the typical yellowing that is also present among adult males of this species during the breeding season.The rate of hypomelanism we detected in L. aurea is much lower than for other species such as Zeus' robber frog (Eleutherodactylus (Syrrophus) zeus) where 26% of detected individuals showed abnormal white patches suggestive of partial leucism (García-Padrón & Bosch, 2019).

| Amphibians with naturally white or translucent skin
Skin transparency, where light can easily pass through, or translucency, where light can partially pass through, are known to be the natural condition of several amphibians.This includes glass frogs, which possess almost totally transparent ventral surfaces that may be used for camouflage (Barnett et al., 2020;Rudh & Qvarnström, 2013).'Seethrough' skin results from the low concentration or lack of all chromatophores types within the skin.This may lead to the expression of skin translucency to varying extents, or true transparency where internal blood vessels, bones and organs are visible (Bruni et al., 2020;Sumida et al., 2016).Increased skin transparency can also occur when melanosomes are aggregated within melanophores in sections of skin where it is the primary chromatophore present (Nilsson et al., 2013).
In contrast, opaque white or silver skin among amphibians occurs when iridophores are the only chromatophore type present (Bagnara et al., 2007;Fernandez & Bagnara, 1993;Rudh & Qvarnström, 2013), or when melanophores are also present but their pigment is aggregated to prevent the concealment of the iridophores (Fernandez & Bagnara, 1993), or when there is an increase in the abundance of iridophores that creates a highly reflective layer above other chromatophores also present (Kobelt & Linsenmair, 1986).High skin reflectance leading to white skin colouration is dictated by the abundance of iridophores in the skin, their thickness and the arrangement of light reflecting platelets within these cells (Kobelt & Linsenmair, 1992).For many amphibians, lighter skin colours, including opaque white, are commonly expressed over ventral surfaces while darker and colourful skin is expressed across the dorsum; possibly as a form of crypsis in land animals (Rowland et al., 2008).This spatial separation of skin colouration in amphibians is at least partly caused by the presence of a melanisation inhibiting factor (MIF) specifically in ventral skin that leads to a lack of melanophore presence and promotes iridophore localisation (Bagnara & Fukuzawa, 1990;Fukuzawa et al., 1995).
In addition to producing opaque white skin, iridophores can also result in iridescent colours, producing 'glittery' or metallic silver and copper/gold to green (Kobelt & Linsenmair, 1992); these are commonly expressed in the iris of many amphibians above a background of melanophores (Duellman & Trueb, 1994;Glaw & Vences, 1997).
In the skin, the presence of iridophores has also been suggested to brighten the xanthophore pigment in the absence of melanophores (Bagnara, 1966).

| Lack of iridophores
Skin transparency or translucency can be a chromatic aberration, resulting from genetic mutations that cause a deficiency in all skin chromatophores within affected areas (Bruni et al., 2020;Sumida et al., 2016).If multiple chromophores are present in the skin, then  (Turner, 2017), leading to transparent skin, while partial deficiency in chromatophores leading to transparent skin patches has been detected in Po's tree frog (Hyla perrini; Bruni et al., 2020).

| Case study-Litoria aurea
In February 2021, we observed an adult male L. aurea with relatively translucent skin across most of its ventral surface in a constructed pond of Kooragang Island (32.87179° S, 151.74016°E, Figure 7).This suggests a near total lack of chromatophores; the vent of this species is typically opaque white, indicating that iridophores should typically be present and xanthophores and melanophores typically absent.Internal tissues and blood vessels were apparent but not well defined through this skin, potentially due to other structures within the skin leading to some light scattering or the presence of a small number of iridophores.Three opaque, white patches of skin consistent with typical ventral colouration caused by the presence of iridophores were located in the middle of its vent and over smaller sections of its throat, indicating piebaldlike deficiency in chromatophores.The L. aurea dorsum showed normal skin colouration, consisting of a background of green skin with gold markings, indicating the presence of all chromophores at typical levels in this region (Figure 7).We have only detected translucency in one out of 7193 observations (0.01%) across four consecutive breeding seasons.

| CON CLUS ION
The occurrence of colour anomalies within wild populations are rare and while they can occur naturally by chance, they may also be an indication of inbreeding processes, exposure to pollution or the effects of disease.Long term population studies should thus record changes in the frequency of each type of colour anomaly, as an effective visual means of monitoring populations and detecting the consequence of changes that may not be immediately apparent using typical monitoring techniques.We hope that our records of colour abnormalities in wild amphibian populations when compared to normal colour phenotypes can be used as a guide to assist other projects in explaining similar colour mutations.

ACK N OWLED G EM ENTS
This work was conducted under ethics number A2023-204 approved by the University of Newcastle.All experimental procedures were performed in accordance with the Australian code for the F I G U R E 7 Ventral skin translucency detected in an adult green and golden bell frog, Litoria aurea, from Kooragang Island, NSW.Australia.The typical ventral skin colour for this species is opaque white caused by light scattering from a layer of iridophores, with skin translucency indicating a deficiency in this chromatophore.The individual displays typical green dorsal colouration with brown patterning and white banding.
Another potential cause of blue skin aberrations is the absence of both xanthophores and iridophores within the skin, with the presence of collagen leading to the scattering of blue light (Bagnara F I G U R E 1 Blue flash colouration exhibited by adult male and female green and golden bell frogs, Litoria aurea, from Kooragang Island, NSW, Australia.The blue skin is (a) and (b) expressed along the inner thighs and armpits and (c) hidden when individuals are in a resting position.

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), suggesting a deficit in xanthophores.Freckling of green skin colouration typical for this species was apparent on the dorsum, indicating normal functioning of all chromatophores in these specific areas, including all three chromatophore cell types.The ventral surface showed normal colouration (opaque white), as well as the irises (melic gold with black venation), indicating that all other chromatophores besides xanthophores were unaffected.Across four consecutive years (2021-2023), we detected only one axanthic blue L. fallax out of 12,350 observations (0.008%).The total blue colouration of all dorsal surfaces in this L. fallax individual differed from the typical colouration of this species.In particular, L. fallax typically show dorsal skin that ranges from bronze to dark green in both sexes (Figure 4), with no expression of blue colouration in any skin region.Amphibians with typical yellow colouration Yellow colouration is part of the typical phenotype of some amphibians and forms via the presence of xanthophores when melanophores are either absent or if the melanosome organelles storing the melanin pigment within melanophores are aggregated to prevent xanthophore concealment (Kindermann & Hero, 2016a).The most F I G U R E 2 Partial axanthism in an adult female green and golden bell frog, Litoria aurea, captured on Kooragang Island, NSW, Australia.The individual shows the typical green dorsal colouration with patches of light blue across the face, back and front limbs.

F
Near complete axanthism in an adult eastern dwarf tree frog, Litoria fallax, captured on Kooragang Island, NSW, Australia.The individual shows only a few patches of typical green dorsal colouration.F I G U R E 4 Variations in dorsal skin colouration in the eastern dwarf tree frog, Litoria fallax, that is considered part of its normal phenotype.Both adult males and females photographed in amplexus range from dark green to light brown.et al. 2014), or (ii) a slow, temporary physiological colour change via the dispersion of xanthophore pigment organelles simultaneous to the aggregation of melanosomes within the melanophores

F
Partial hypomelanism of dorsal skin surfaces in adult green and golden bell frogs, Litoria aurea, from a wild population on Kooragang Island, NSW, Australia.Individuals show melanin deficiency across (a) the limbs, (b) the limbs and flanks and (c) all dorsal surfaces.transparency or translucency can only occur if all chromatophore types are deficient.Skin transparency aberrations have been observed in several frogs.For example, total deficiency in iridophores across typically opaque white ventral surfaces has been recorded in an Australian tree frog, L. rothii Conceptualization (lead); investigation (equal); methodology (lead); visualization (lead); writing -original draft (lead); writing -review and editing (equal).Colin McHenry: Investigation (equal); supervision (lead); writing -original draft (supporting); writing -review and editing (equal).