Hidden colour signals as key drivers in the evolution of anti-predator coloration and defensive behaviours in snakes

Domain: biology
Published: 2025-12-26
Editorial outcome: minor_revision

Abstract
The initial evolution of warning coloration (“aposematism”) within a cryptic population of defended prey presents an evolutionary paradox. A recent phylogenetic analysis of amphibia suggests a new solution: prey that combine cryptic colours with conspicuous patches on concealed body parts (“hidden signallers”), may have mediated the transition of species from camouflage to aposematism. Here, we focus on the colour-diverse snake family Elapidae and test whether species with hidden colours could also serve as an intermediate stage in the evolution of aposematism in this group. Phylogenetic comparative analysis reveals several key patterns in their anti-predator colour evolution: (i) a few major transitions influenced the overall distribution of hidden-colours, camouflage, and aposematism in the group, and (ii) aposematism evolved multiple times, with hidden coloration a common precursor state, while direct transitions from camouflage to aposematism are also observed. We also quantify associations between colour patterns and defensive behaviours that reveal ventral surfaces (i.e. hidden signals). We find that venter-revealing defensive behaviours frequently co-occur with hidden colour signals. Our results suggest that aposematism can evolve through multiple routes and highlight the prevalence of co-evolution between venter-revealing defensive behaviour and anti-predator coloration in snakes.

Introduction
Most animals face predators at some stage in their life cycle. This has led to the evolution of a range of morphological and behavioural adaptations to avoid detection and/or deter their attack post-encounter1. In particular, many animal species have evolved camouflage, reducing their probability of detection or recognition by predators2. Conversely, some species have evolved active defences against predators such as stings or toxins and signal their unprofitability to their would-be predators through conspicuous warning signals, an association known as “aposematism”3. These warning signals can be mimicked by harmless species (Batesian mimicry)4 or copied by other defended species, thereby reducing the costs of predator education (Müllerian mimicry)5.

While conspicuous signals may serve to warn would-be predators, the initial spread of aposematic mutants from rarity is challenging to understand. The problem, first recognised by Fisher6, arises because the first conspicuous mutants would not only be more detectable than their cryptic conspecifics, but also not as readily recognised as defended. To learn the association, predators must sample the conspicuous mutant, yet in so doing, the rare mutant(s) may be driven to extinction. In spite of these challenges, aposematism has successfully evolved many times across the animal kingdom1. Several explanations as to how aposematic mutants overcome their two-fold disadvantage have been proposed. For example, some species may be able to deploy their noxious defences and survive capture by predators7. There are a variety of ways that this could occur; some insects, for example, have distasteful secretions or stings, which, combined with protective integuments, may allow them to survive taste rejection8. While resistance to handling may explain how aposematism has evolved in some taxa, it cannot provide a complete explanation because many poisonous aposematic species are soft-bodied and appear unlikely to be able to survive sampling9. Alternatively, some defences can be deployed or detected from a distance10, such as skunk spray or urticating hairs of New World theraphosid spiders11,12 but once again, not all defences are apparent before capture.

Fisher’s celebrated explanation for how aposematism evolves in these taxa was that while the individuals that are sampled die, aposematism can still be selected for if the prey have close relatives with the same anti-predator traits that benefit from any learned aversion. This kin selection13 hypothesis will be more likely to hold when relatives form aggregations, such that a predator may sample one individual but then ignore the rest of the group14. In support, many aposematic lepidoptera larvae do form aggregations15,16. However, phylogenetic analyses indicate that, in all lineages considered, conspicuous coloration likely preceded the evolution of gregariousness, suggesting that kin selection is unlikely to have contributed to the initial evolution of aposematism in these caterpillars15,17,18. Additionally, aposematism is observed in many non-gregarious taxa19, including coral snakes (Micrurus and Calliophis genera) that are highly cannibalistic outside of the breeding season and therefore unlikely to have exhibited gregarious behaviour at any point in their evolutionary history. Finally, an alternative explanation is that aposematism may evolve incrementally, with species gradually acquiring conspicuous coloration such that they may educate predators without fully abandoning crypsis, at least at first20. For this to work, predators must generalise learned avoidance of semi-conspicuous phenotypes to more readily detectable forms. A behavioural experiment using blue tits (Cyanistes caeruleus) as model predators found that prior exposure to semi-conspicuous distasteful targets did not reduce the birds’ probability of attacking more conspicuous similar-looking targets21. This led the authors to suggest that it is unlikely that aposematism evolves by gradual change.

Studies of the evolution of aposematism have typically only taken into account animal coloration that is always visible. However, many cryptic species exhibit patches of conspicuous colour on surfaces that are usually hidden and only transiently exposed during locomotion or as part of a behavioural display22–24. Unlike consistently visible aposematic signals, these hidden colour defences do not impose the costs of full conspicuousness during the initial stages of evolution. Their normally camouflaged appearance reduces the risk of detection, while still potentially deterring predators through post-attack displays that startle, warn, and/or facilitate avoidance learning25–27. This is an important distinction because, unlike rare aposematic forms, mutations causing hidden signals may provide a fitness benefit at the outset, for example, by startling a predator27. Once established, it is conceivable that species with hidden signals may function as a stepping stone, facilitating the evolution of aposematism if predators are able to generalise their learned avoidance of hidden signals to prey with permanently displayed signals.

In support of the hypothesis that species with hidden signals serve as stepping stones to species with full aposematism, a recent large-scale macroevolutionary study of transitions between different types of signaller in amphibians found that the acquisition of aposematic coloration in species was disproportionately preceded by species with hidden signals28. This evolutionary route to aposematism is likely not restricted to amphibians and may be widespread in the animal kingdom. Indeed, many aposematic species across various taxonomic groups, including fish, arthropods, and other tetrapods, have close relatives that are cryptic but possess conspicuous hidden signals27–29. Unfortunately, parallel analyses comparable to what was done in amphibians in many of these groups presents substantial challenges. This is because the analysis requires a few conditions to be met. First, relatively complete time-calibrated molecular phylogenies are required. Gaps in the phylogeny may result in intermediate forms being missed, preventing the detection of key transitions30. In many arthropods, for example, only a small proportion of the total described species have known phylogenetic information, and many more species remain undescribed31. Second, aposematism must have evolved several times independently. If all aposematic species in a taxon form a monophyletic clade, then the transition between colour states effectively happened once, not providing sufficient statistical power to estimate transition rates32.

One taxon that appears to be an excellent model to explore the evolutionary history of anti-predator coloration and test the hidden signal stepping stone hypothesis is elapid snakes. Aposematism is widespread in this group, and chemical defence (or offence) is the ancestral condition, with only a few congeneric species having secondarily lost this trait19,33. Additionally, the phylogenetic position of most extant described species is known except for the recently described species34. Intriguingly, elapids exhibit a variety of defensive behaviours (Fig. 1). For example, hooding describes a form of defensive display in which the anterior portion of the body is raised off the ground, exposing the ventral surface and the ribs are spread, potentially increasing the perceived size of the snake. While this behaviour is best known in cobras of the genus Naja, such as the Spectacled Cobra (Naja naja), it is widespread across other elapids. In addition to hooding, there exist other venter-revealing defensive behaviours, including rolling over, defensive coiling, tail displaying, and exposing the ventral surface in response to provocation by a predator27. These defensive behaviours involve revealing the usually hidden ventral surface, which raises the question of whether there are evolutionary associations between these defensive behaviours and anti-predator coloration.

Here, we have classified the anti-predator coloration of snakes in the family Elapidae (comprising approximately 400 known species from 55 genera) using a schema that recognises aposematism, crypsis, species with camouflaged dorsal surfaces and conspicuous ventral coloration that is usually hidden, as well as polymorphism in which individuals with different colour states are observed within a species. We then perform ancestral state reconstruction analysis of elapid anti-predator coloration and use discrete character evolution models to test the hidden signal stepping stone hypothesis in this taxon. We also evaluate the presence/absence of defensive behaviours in elapids using both video and pictorial documentation of their responses to human provocation. Using these data, we elucidate how this behavioural defence has coevolved with anti-predator coloration.

Results
The history of colour evolution in elapid snakes
We analysed 251 species (~62% of extant species) of elapid snakes with known phylogenetic relationships and colour information. Three human classifiers experienced with snakes categorised each species based on its anti-predator coloration: either cryptic (colours and patterns that match natural backgrounds), conspicuous (high contrast coloration typically associated with aposematism such as black and white, bright blue, bright yellow, red, or orange), or hidden with cryptic coloration on the dorsal surface and conspicuous coloration on the ventral surface (see Methods for details). The three classifiers all agreed on the colour classification for 236 elapid species (94% of analysed species). For the remaining 15 species, two classifiers agreed, and we used the most common classification for each species in the main analysis. Substantial variation existed in the type of defensive coloration in elapid snakes; 34% were classified as cryptic (Cry), 40% as conspicuous (Con), 21% as hidden-coloured (Hidden), and 5% as polymorphic (here, defined narrowly when a species exhibits two or more different defensive coloration strategies) (Fig. 2B, Table S1).

We generated six hypothetical models with different constraints on transition pathways or transition rate estimation (see “Methods” for details). We initially considered seven states in total, comprising three major colour states and four polymorphic states. However, our hypothetical models were generated using only the three monomorphic states because the small number of species in each polymorphic state limited reliable estimation of transition rates (see “Methods” for details). Among the competing six models (Fig. 2A), the Equal rates (ER) model, in which a single rate governs all transitional pathways, was the most strongly supported (64% support; Table 1; full transition rates among seven different states, including those for polymorphic states, can be found in Table S2), demonstrating that transitions of species between the three colour states occurred at similar rates. Stochastic character mapping results aligned well with the transition rate results in that the transitions among different strategies have occurred in similar frequencies (Fig. 3C). However, although the estimated rates were similar, the historical frequency of transitions from conspicuous to cryptic colour was considerably lower than that of other transitions, which is consistent with our analysis of the most likely transitional history for each species (Fig. 3D). This indicates that such transitions have rarely occurred in the history of elapid snake anti-predator coloration evolution, yet the comparable rate estimates suggest that whenever they do occur, they proceed just as quickly as other types of transitions. We mainly discuss the results of the ER model and the ancestral states it estimated, but we also include results from the second-best model, the Symmetrical (SYM) model (15% support) in Fig. S1.

Marginal and joint ancestral character reconstruction results matched 96% of cases in the most likely ancestral state for each node. Both the joint and marginal results suggest that the hidden colour state is the most likely ancestral state of elapid snakes (30% probability in the marginal ancestral character reconstruction when all seven states were considered), but there is also some support for the conspicuous (19%) and conspicuous/hidden polymorphic (19%) states (Fig. S2). The ancestral character reconstruction results (Fig. 3A) provide insights into several major patterns of colour evolution history, which we now discuss.

First, ancestral hidden colours have been retained in several lineages, including those in the Naja, Sinomicrurus, and Calliophis genera. Second, a few major transitions largely determined the overall distribution of crypsis and conspicuousness (Fig. 3D): (i) a single transition from hidden colours to conspicuousness affected the prevalence of aposematism in the species-rich Micrurus genus, and (ii) a transition from hidden colours to cryptic colours at the basal node (the common ancestors from the Laticauda to the Hydrophis genera in Fig. 3A) influenced the prevalence of crypsis across descendant lineages. As a consequence, the majority of elapid species obtained their current anti-predator colour type by either no or a single transition event from the common ancestor (Fig. 3D). Third, conspicuous coloration evolved at least 18 times independently. The transition to conspicuous colour occurred in various lineages through one of two pathways: (i) ancestral hidden colours evolved to conspicuous colours or (ii) ancestral hidden colours evolved to cryptic colours, then conspicuous colour subsequently evolved (Fig. 3D). Fourth, crypsis evolved mostly from hidden colour states, mostly governed by a single transition at the basal node as explained above, with only a few exceptions that evolved a more complex route involving conspicuous colours (Fig. 3D). Once crypsis had evolved, multiple subsequent pathways emerged: most remained cryptic, while the rest evolved to either hidden or conspicuous colours. Fifth, the polymorphic states evolved multiple times independently in the phylogeny.

Smaller-scale transitional patterns in recently diverged lineages are lineage-specific, indicating no consistent trends among the lineages, as suggested by the relatively strong support for the equal-rate model. While a previous study on amphibians revealed that hidden colours served primarily as an intermediary stage in the transition from camouflage to aposematism28, when viewed within elapid snakes alone, the same pattern did not emerge; when the transition from crypsis to hidden colours occurred, the hidden colours remained in the extant lineages without further evolving to aposematism. However, this pattern could arise because the hidden colour state is the most likely ancestral state of elapid snakes, unlike amphibians, in which camouflage is the most likely ancestral state. We therefore explored the possibility that ancestors of the elapid snakes, such as common ancestors of Elapidae and sister clades (a multi-family clade including Pseudoxyrhophiidae, Psammophiidae, Lamprophiidae, Atractaspidadae, Cyclocoridae, and Buhoma sp), exhibited camouflage. Ancestral reconstruction incorporating 151 species from sister clades showed that elapid snakes likely diverged from cryptic ancestors (86% support; Figs. 3B, S3 for marginal reconstruction results; images of the sister clades were classified by KLH using the same criterion described in the “methods”). This suggests that stepwise evolution from crypsis to conspicuousness via hidden coloured states also occurred in snakes. However, unlike amphibians, where these transitional patterns appeared multiple times in various lineages, this stepwise evolution in elapid snakes (and relatives) is explained by a single ancestral transition from camouflage to the hidden colour state at a deep node. A more comprehensive examination of other snake families is warranted to confirm the prevalence and generality of this stepwise-evolution route and to assess its role in the evolution of aposematism in snakes.

The direct transitions of camouflaged species to species with conspicuous signals may be explained in part by the prevalence of Batesian and Müllerian mimicry in elapid snakes; thus, while it is challenging to understand how the novel conspicuous mutants of cryptic prey initially spread from rarity35, if one taxon first acquires aposematism through the hidden signal route, then new conspicuous mutants of other species who resemble this well-established warning signal may be recognised as unprofitable to attack at the outset. Indeed, the conspicuous coloration of non-venomous Emydocephalus species, which are Batesian mimics of venomous sea snakes like Aipysurus, Hydrophis, and Laticauda spp.36, likely evolved from cryptic ancestors, suggesting that direct transitions from crypsis to conspicuous forms can occur through mimicry. Additionally, unlike amphibians, in which no direct transition from crypsis to aposematism has been observed28, snakes possess hard keratinous scales compared to the relatively delicate skin of amphibians, which may allow them to better survive handling by predators. Likewise, the chemical defence of elapids involves venom that can be injected, rather than poison that must be ingested to take effect, further suggesting that elapids may be better able to survive sampling attempts than amphibians.

While these results provide insight into the evolutionary pathways through which lineages acquire aposematism, they do not explain why some lineages evolve this trait while others retain ancestral hidden signals. Indeed, in certain ecological contexts, hidden signalling may be an optimal strategy—allowing a snake to remain concealed from its prey while still being capable of signalling to predators when necessary. A recent phylogenetic study found that aposematism is more likely to evolve in elapid species that feed on other snakes37. Given that many snakes exhibit limited colour discrimination abilities, this may reduce the need to remain cryptic38. Under such conditions, there may be an advantage to signal their defences constantly to reduce the risk of initial attacks by their own predators. For example, if a surprise predatory attack leaves no chance for the snake to reveal its hidden signals, it may be costly to have cryptic dorsal coloration. This may happen in sea snakes such as Laticauda sp., where surprise strikes of potential predators, such as ospreys or sharks, often leave no chance for the snake to show hidden signals.

We conducted additional analyses to ensure robustness of our conclusions against (i) uncertainty in the phylogeny’s topology and node age, and (ii) uncertainty in colour classification. To examine how this uncertainty affects our results, we repeated the analysis using 100 randomly selected trees sampled from the posterior distribution of the supertree. The ER model remained the best-supported model in 63 trees, while the SYM model, the second-best, was supported in 31 trees. We also re-analysed our data using a dataset that removed 15 species with classification disagreements; the ER model still remained the best-supported model (Table S3). Additionally, we excluded all polymorphic species and re-analysed the data to mitigate the effects of species-poor states on major colour transition patterns. The ER model continued to be the best-supported model (Table S4). The ancestral character reconstruction results suggest that all supplementary analyses exhibit transitional patterns consistent with the main findings (Figs. S4, 5). These supplementary analyses confirm the robustness of the observed transitional patterns.

The evolutionary patterns of venter-revealing defensive behaviours
We analysed behaviours of 180 species for which both colour and behavioural data were available. Venter-revealing defensive behaviours were classified into one of three categories for each species: no behavioural display evident, presence of hooding behaviour, or other venter-revealing defensive behaviours (such as rolling over onto the back or lifting the tail to reveal the ventral surface) that are not hooding. A simple frequency comparison revealed strong evidence of an association between the colour group and the frequency of defensive behaviours (Chi-square test of independence; χ2 = 60, P < 0.001; Fig. 4B). Venter-revealing defensive displays (i.e. summed frequency of hooding and non-hooding defensive behaviours) are most common in the Hidden group (80%), followed by the Con (43%) and Cry (20%) groups. Specifically, hooding behaviour occurred more frequently in species with hidden colours than in other colour groups, and non-hooding defensive displays were similarly common in species with hidden and conspicuous colours, but none was found in cryptic species (Fig. 4B). While a previous study suggests that conspicuous colours are not associated with aggressive behaviours in snakes39, our results imply that defensive behaviours, particularly those that reveal the ventral surfaces, are more frequently found in species with both hidden and conspicuous colours in snakes. Ancestral character states for the colour dataset are described above; thus, our discussion here primarily focuses on defensive behaviour results.

Both the discrete character evolution model and the stochastic character mapping suggest that the hooding and non-hooding displays have evolved independently, each arising multiple times in different clades, with no transitions between the two display types (Table 2, Figs. 4, S6 for the model description, Fig. S7 for marginal ancestral character results, Fig. S8 for stochastic character mapping results); we found no evidence of any transitions between these two defensive behaviours. The type of defensive behaviours was conserved within each genus; most species in the Naja, Pseudonaja, Pseudechis, and Austrelaps genera exhibit only the hooding behaviour, if any. In contrast, species in Micrurus, Sinomicrurus, Calliophis, and some species in Suta, Vermicella, and Hydrophis exhibit the non-hooding defensive behaviours.

Both types of defensive behaviours have multiple independent origins (see Fig. 4C). First, hooding behaviour evolved at least six times. Among the six identified instances where hooding behaviour was acquired, two transitions occurred in ancestors with hidden colours, and four in ancestors with cryptic colours. After acquiring hooding behaviour, this behaviour was lost in some cryptic lineages, such as those in the genera Pseudechis and Pseudonaja, but was rarely lost in lineages with hidden colours, such as in the Naja genus. Once hooding behaviour evolved in cryptic lineages, many descendants subsequently developed hidden colours, retaining the hooding behaviour. This suggests two major evolutionary pathways for the correlated evolution of hooding behaviour and hidden colours: (i) hooding behaviour initially evolved in some cryptic lineages, followed by the subsequent evolution of hidden colours, and (ii) hooding behaviour evolved in species that already possessed hidden colours (the ancestral state of elapid snakes) and was retained within the lineage.

Non-hooding defensive displays seem to be the most likely ancestral trait of elapid snakes (Fig. 4C, 97% support). This ancestral state has been retained in many genera, such as Micrurus, Sinomicrurus, and Calliophis, with some species losing the defensive behaviour, primarily in aposematic species. Apart from this single ancestral effect, defensive behaviours have evolved independently multiple times, mostly in recently diverged lineages. Notably, these non-hooding defensive behaviours have evolved in either hidden-coloured or conspicuously-coloured lineages, but not in cryptically-coloured lineages.

Our study demonstrates general patterns of defensive colour evolution in elapid snakes and highlights the prevalence and key role of hidden colour states in the evolution of aposematism. As previously demonstrated in amphibians28, our phylogenetic analysis provides evidence that hidden warning signals can serve as an intermediary stage in the transition from camouflage to aposematism in elapid snakes, although there have also been direct transitions of species with camouflage to species with conspicuous signals. This finding offers a potential resolution to the paradox of the initial evolution of aposematism, suggesting that hidden colour states could facilitate this transition. That said, we stress that hidden-coloured states were likely present early in the Elapidae lineage, following a single transition from a cryptic ancestor. This early origin allowed hidden-coloured species to serve as a common precursor in all subsequent transitions. While our analysis is limited to the family Elapidae, where defensive coloration is more diverse than in other snake groups, most of which are predominantly camouflaged, it may not be a coincidence that aposematic species are rare in groups where hidden-coloured species are also rare. The paradox of the initial evolution of aposematism may persist in these groups, but hidden-coloured states could mitigate its associated costs and may facilitate the evolution of aposematism. This, in turn, may influence higher taxonomic patterns in the distribution of aposematic species.

In addition, our behavioural analyses highlight a close co-evolutionary relationship between hidden colours and the defensive behaviours. Hidden signals and associated defensive behaviours like flash displays and deimatism are widespread in the animal kingdom and are often observed in clades that contain aposematic species29. Therefore, it is possible that the hidden signal stepping stone route to aposematism is not restricted to herpetofauna. However, as our results in elapids demonstrate, other evolutionary routes can bypass the need for hidden signals in the transition from camouflage to aposematism, and this may extend beyond this taxon. For example, nudibranchs often mimic the toxic cnidarians from which they sequester their chemical defences, while many aposematic mammals utilise defences that do not require sampling or close contact by predators, such as the noxious sprays of skunks and African polecats12,40. Such defences may reduce the costs associated with the initial evolution of aposematism.

Methods
An extensive list of elapid species was obtained from the Reptile Database (http://www.reptile-database.org)41. We performed a phylogenetic analysis of the anti-predator coloration adopted by elapids based on classifying their images, as well as their defensive behaviours when approached by predators, based on both video and pictorial data.

Colour data acquisition and classification
All species on the list were categorised into one of three anti-predator coloration groups based on the images obtained from online sources (Data S1). In making the classification, conspicuous colours were defined as high contrast or bright colours that are uncommon in natural backgrounds and are commonly associated with aposematic signals37,42. These include bright yellows, reds, oranges, blues, and high contrast black and white markings. Cryptic coloration was defined as colours that resemble the substrates that are common in natural backgrounds, such as leaf litter, foliage, tree bark, rocks, and sand. The cryptic coloration usually includes brown, tan, grey, and green colours. A species was deemed conspicuous (Con) when its dorsal colours were conspicuous, regardless of ventral colours. A species was classified as cryptic (Cry) when its dorsal colours were cryptic and lacked any hidden conspicuous colour patches on the body. Species with hidden colour signals (Hidden) were defined as having a cryptic dorsal colour but possessing conspicuous colour patches, usually on the whole or part of the venter (e.g., underside of tails) that are typically hidden when resting. Additionally, some species, such as Naja naja, exhibit conspicuous coloration on the skin in between the scales that is only visible during hooding displays when the skin is stretched, and the scales are separated. These species were also categorised as having hidden signals.

Elapid snakes encompass considerable ecological diversity, so what constitutes a hidden signal may differ based on their ecological contexts, snake behaviours, and observer orientations, which are factors that we were not able to incorporate into our analysis. Generally, coloration on the ventral surface is clearly only exposed transiently and therefore qualifies as a hidden signal. However, a notable exception to this is in sea snakes (subfamilies Hydrophiinae and Laticaudinae), which swim suspended in the water column where their ventral surfaces are readily visible most of the time. Because of this, no sea snake species were classed as having hidden signals. However, we note that the colour categorisation of some sea snake species, such as Hydrophis platurus, may depend on the viewer. This species has a yellow belly and a dark brown dorsum, appearing aposematic underwater but camouflaged to aerial predators such as ospreys and sea eagles, which approach exclusively from above. In our list, H. platurus is the only species that exhibits such a bi-colour pattern.

There were some standard challenges to defining whether a species exhibits polymorphism or not. While the classical definition of colour polymorphism is based on the presence of multiple discrete forms within a single population, we used a restricted definition of the term, deeming a species as polymorphic when we observed two or more of the above colour classes exhibited by different individuals of the same species. This was because polymorphic forms with the same anti-predator signals would fall into the same category in our analysis. We appreciate that some morphs may occur at very low frequencies within a population or be restricted to a very limited geographical range, and as such, may not be represented in the data source we sampled.

To obtain images for categorisation, all species' scientific names were searched on Google® Images. Snake images were only used if they had been uploaded by reliable sources, usually a biodiversity database or naturalist community (Data S1). We restricted our image sampling to naturally occurring individuals and excluded those resulting from selective breeding or colour abnormalities (i.e., albinism). All species were categorised following the classification above by 3 independent observers: KLH and two independent observers (JB & RH) recruited from the Canadian Herpetological Society. The two external observers were trained to use the same categorisation scheme as KLH by being shown examples of clearly cryptic and aposematic species in non-elapid taxa to define what sorts of colours fall into the respective categories. Wherever possible, different images of each species were categorised by KLH and by the two external observers (Data S1). This was done to demonstrate the generality of the categorisation across separate individuals of the same species.

In most species (62%), the prepared images consisted of two images, one showing the dorsum and another showing the venter. For the rest of the species, both the dorsal and ventral coloration were visible from one image. Since any snake that exhibited conspicuous dorsal coloration would be ranked as conspicuous regardless of ventral coloration, some species for which images of the ventral surface were unavailable were provisionally included in the categorisation. This was done when KLH considered the dorsal coloration to be unambiguously conspicuous. When this occurred, the species was categorised as conspicuous by KLH and images of the dorsal surface were presented to the other categorisers. If the other categorisers had ranked any of these species as anything other than conspicuous, they would have been omitted from the analysis. However, this never occurred, and all 54 of these species were ranked with 100% agreement across all categorisers. When polymorphic forms were detected during image collection, each morph was categorised separately. All the images were presented to the external observers through a Zoom® meeting. For all species characterised by KLH as polymorphic, each morph was presented on a separate slide and categorised separately as if they were independent species. We classified 269 species, but ultimately analysed 251 species with known phylogenetic relationships.

Naturally, photo-based colour classification has drawbacks, as it does not account for snakes’ micro-habitat choices or predator (notably bird) vision—both crucial for assessing conspicuousness against natural backgrounds43,44. However, in global-scale comparative studies like ours, incorporating these factors is challenging, yet significant evolutionary patterns have still been successfully inferred from photos28,45. Public photos also lack ultraviolet (UV) colour, which can be seen by some natural predators such as birds and other snakes37. Recent evidence shows widespread evolution of UV colours in both camouflaged and aposematic snakes, especially in their venter46. In our classification, UV colours in visually conspicuous species would still be classified as conspicuous. For camouflaged species, UV might compromise camouflage, yet it may also help conceal against UV-reflecting substrates or serve as a predator deterrent via UV on the venter46. Thus, our classification may misidentify UV-hued species as cryptic if UV serves as a predator warning signal, although how frequently this occurs is unclear.

Defensive behaviour data acquisition and classification
KLH categorised the defensive behaviours of snakes using a scheme based on the descriptions provided by the California Herps webpage (www.californiaherps.com) and published literature47. Although snakes exhibit a variety of defensive behaviours, including acoustic signals such as hissing or tail rattling48,49, for the purposes of this study, we focused on visual defensive behaviours of snakes that involve showing their ventral surface. This includes (i) hooding, defined as lifting the anterior portion of the body off the ground and spreading the ribs to expose the ventral surface (Fig. 1D), and (ii) non-hooding defensive behaviours that encompass either flipping over entirely, lifting the underside of the tail, or lifting sections of the body to show the ventral surface (Fig. 1E for example). There were practical challenges in categorising defensive behaviours due to their diversity and the lack of clear distinctions between different behaviours. For analytical purposes, we classified each elapid species into one of three behavioural categories: (i) no evidence of the presence of venter-revealing defensive behaviours, (ii) showing hooding behaviours, (iii) showing a defensive behaviour that involves exposing the ventral surface that is not hooding. Naturally, how salient a behaviour depends on habitat and context. However, this generalised categorisation schema is useful in enabling broad multispecies comparisons.

There may be functional distinctions between hooding and non-hooding displays, with hooding displays in venomous snakes likely signalling imminent defence and dissuading predators through startle or by accentuating the size and ferocity of the snake, while non-hooding hidden signals could be aposematism or thanatosis50. As such, hooding may be more closely associated with an impending potent reaction, while non-hooding displays may be less dependent on the defence itself and more susceptible to being exploited by undefended Batesian mimics50. Naturally, acquiring behavioural data from over a hundred species of snakes distributed around the world is challenging. To overcome this, we used two independent methods to survey elapid snake defensive behaviour. First, we observed videos of snakes being confronted by humans; then, we surveyed images of snakes available on the internet (see Data S2, 3 for a list of sources). This resulted in the formation of two independently derived behavioural datasets whose collection is described below.

Video-based behavioural survey. Hooding behaviour is a characteristic display of elapid snakes that may function to increase the perceived size of the snake. Although best known from cobras of the genus Naja, hooding behaviour is also exhibited by many other species in this family. To assess whether a species exhibits hooding behaviour or not, the scientific names of all extant described elapid snakes were searched on YouTube® and Google® Video (Data S2, N=455 videos from 180 species). To be considered evidence that a species does not exhibit hooding behaviour, a video had to show a wild specimen not exhibiting hooding behaviour while being handled or manipulated in the field, either by hand or with tools such as snake hooks, snake tongs, sticks, etc. Because this effectively simulates a predation event, we assume that species that utilise hooding behaviour as part of their defensive behaviour will do so under these conditions. A video was considered evidence of the presence of hooding behaviour if a snake being handled as described above exhibited hooding behaviour. Additionally, some snakes exhibit hooding behaviour simply by being approached by the filmer, even without being touched. These videos were also considered evidence of hooding behaviour. Likewise, some videos of captive snakes exhibiting hooding behaviour were used as evidence of hooding behaviour. However, videos in which no physical contact was made with the snake were never considered evidence of a lack of hooding behaviour. Because snakes often habituate to humans when in captivity, videos of captive snakes not exhibiting hooding behaviour were never considered evidence that a species does not have hooding behaviour.

Timestamps are provided for the relevant times in each video (see uploaded data file), except for videos that are less than two minutes long or videos in which the entire duration is relevant. The timestamps correspond to the duration of handling time in videos with snakes not exhibiting hooding behaviour. In videos where snakes exhibit hooding behaviour, the timestamps correspond to the duration of the hooding behaviour. One to five videos were used for each species, depending on availability. All video analyses were done by KLH.

In addition to hooding behaviour, many elapids also engage in other defensive behaviours that involve revealing the ventral surface. However, these behaviours were generally not exhibited in the videos that we selected because they require the snake to be on a flat surface, and most videos of snakes being handled involved them being lifted off the ground. Because of this, we additionally utilised an image-based approach to evaluate the presence of snake defensive behaviour (see below).

Photo-based behavioural survey. In order to confirm and broaden our video-based categorisation of elapid snakes’ defensive behaviour, we collected an additional defensive behaviour dataset based on their images. This was accomplished by searching the names of all extant elapids in iNaturalist (http://www.inaturalist.org)51 and surveying all available images of each species. In order to be photographed, snakes must necessarily be approached by a human photographer. This is likely to be perceived by snakes as a predation cue. As such, it is expected that snakes will exhibit defensive behaviours in response. Therefore, if numerous photographs of a species are available yet none show evidence of a given defensive behaviour, it is likely that the species does not exhibit the defensive behaviour in question. If any images of snakes engaging in defensive displays that involved showing the venter were found, then the species was categorised as having such defensive behaviour. For the absence of defensive behaviours, we used an arbitrary criterion based on the availability of the images for each species; if more than 10 images of a species in the field were available and none showed evidence of defensive behaviour, it was considered that this species does not likely utilise such defensive behaviours (the mean and median number of surveyed images for each species were 227 and 66 respectively per species).

To verify more rigorously the absence of defensive behaviour, if more than 10 images were available on iNaturalist, and no evidence of defensive behaviours was found, then the species name was additionally searched in Google Images, first on its own and then with the phrases “defensive display,” “defensive behaviour,” and “defensive posture” successively. If an image of a ventral surface-exposing defensive display of a snake was found (Data S3), then that was considered evidence that the species has such defensive behaviour. However, if no such evidence was found, then the species was categorised as not having evidence of venter-revealing defensive behaviours. Whenever a defensive behaviour was found in a species, we deemed a species to have either hooding or non-hooding defensive behaviours. The classification of hooding behaviour followed the video-based definition. The non-hooding defensive behaviours include lifting the tail such that the ventral coloration is revealed, or rolling over, displaying the entire ventral surface.

Using the two behavioural datasets described above, we classified a species as exhibiting hooding behaviour if this behaviour was observed in either video or photo sources. Similarly, if evidence indicated that a species displays non-hooding defensive behaviour in either source, it was classified accordingly. Hooding behaviours were identifiable in both video and image sources, whereas information on non-hooding defensive behaviours was available only from images. Overall, video- and image-based data were largely consistent in detecting hooding behaviour. However, image sources provided more comprehensive information, as all species classified as exhibiting hooding behaviour based on video evidence were also classified as such from image data. Additionally, we identified five additional species displaying hooding behaviour in cases where no video data were available. Information on non-hooding defensive behaviours was exclusively derived from image sources.

Phylogenetic analysis
All analyses were conducted in the R programming environment52, version 4.4.3. We obtained Elapidae phylogenetic trees using Vertlife (https://vertlife.org). The Vertlife uses the phylogenetic tree of 9754 squamate species by Tonini et al.53 as a backbone and produces distributions of trees with subsets of taxa. We generated phylogenetic trees for 251 species (~62% of extant elapid snakes) for which images were available. We first extracted 1000 trees randomly drawn from the posterior distribution. Then, we generated the maximum clade credibility (MCC) tree using the ‘maxCladeCred’ function in the ‘phangorn’ package54. The main phylogenetic analyses were performed using the MCC tree, while the rest of the trees were also used for supplementary analyses to validate the robustness of the main results.

We employed discrete character evolution models implemented in the ‘fitMk’ function (but also used equivalent alternatives ‘fitHRM’ or ‘fitMk.parallel’ using the same model structure when ‘fitMk’ performed unstable or to speed up the process) in the ‘phytools’ package to estimate the transition rates between each colour state55. Treating polymorphic states needed special attention due to the low number of extant species in each state (Table S1). First, despite the low number of species for each group, we considered all polymorphic combinations (Cry/Hidden, Cry/Con, Hidden/Con, Cry/Hidden/Con) as separate states without merging them into one combined polymorphic state; this makes sense because polymorphic states were comprised of different combinations of colour groups which, in consequence, influence which colour state to evolve from or to. Next, we assumed that the transition rates between all polymorphic states were zero. This was based on the fact that all polymorphic states were located independently from each other in the phylogeny; thus, the direct transitions between different polymorphic states were unlikely. Additionally, we considered that each polymorphic state could evolve from, or to, one of the monomorphic states already included in the polymorphic forms (i.e., Cry/Hidden polymorphic states cannot evolve to or from Con states). Finally, we let the transitions from or to any polymorphic states to a monomorphic state free to vary (i.e., parameters were not set to zero and estimated by the model). Consequently, while we tested specific hypotheses for the transitions among the major monomorphic states, we did not test specific hypotheses regarding polymorphic states. This was (i) because the number of species in each polymorphic state is too low (Table S1; all less than six), thus the estimated transition rates were difficult to be generalised, and (ii) to still account for any contributions of polymorphic states to transition dynamics.

After determining how to handle the polymorphic states, we generated six hypothetical models for the transitions among the monomorphic states. These were All-rates-different (ARD), Symmetric (SYM), Equal-rates (ER), Intermediate, Cost-offset, and Secondary models (Fig. 2A). The ARD model allows all the transition rates between each monomorphic state to vary freely. The SYM model assumes that forward and reverse transitions share the same parameters. In the ER model, a single parameter governs all transition rates. The Intermediate model assumes that Hidden states serve as an intermediate stage between Cry and Con states by not allowing direct transitions between Cry and Con states. Cost-offset models do not allow direct transitions between Hidden and Cry states, making Hidden states evolve only from or to Con states. This assumes that Hidden states evolve from Con states to alleviate the high detectability costs of conspicuous colours27. The Secondary model assumes that the hidden colours in elapid snakes mainly evolve as a secondary defence of cryptic species; thus, the Secondary model only allows the Hidden states to evolve from or to Cry but not Con states. Then, we determined the best-supported model using corrected Akaike Information Criterion (AICc) and associated AIC weights.

We used the ‘ancr’ function in the ‘phytools’ package for ancestral character reconstruction55. The ancestral characters were reconstructed using the parameters of the best-supported model. We tried both marginal (finding the state at the current node that maximises the likelihood, integrating over all other states at all nodes; gives the results as proportional support for each state for each node) and joint (finding the set of character states at all nodes that jointly maximise the likelihood; gives the results as a single most likely state for each node) methods56. We primarily interpret the transitional patterns using the results from joint methods due to their ease of interpretation. However, we also compared the results of joint and marginal methods to confirm the consistency between the two methods and included the marginal results in the Supplementary materials. Additionally, we performed stochastic character mapping using the best-supported model to estimate a posterior distribution on the number of transitions that occurred among different states57. We sampled 100 stochastic character maps using ‘make.simmap’ function in ‘phytools’ package55.

For the analysis including behavioural data, we were able to analyse 180 species. For this dataset, we first excluded all polymorphic species from the analyses, because the number of species in each group is either zero or very few, making transition analysis impossible to conduct. Next, we first tried generating a nine-state dataset (three colour categories × three behaviour categories) and examining the transitions among the nine states. However, the estimated transition rates had low reliability (i.e., different functions of the discrete state evolution model, such as ‘fitMk’, ‘fitMk.parallel’, ‘corHMM’, yielded disparate results) in states with only a few species. Therefore, instead of analyzing the nine-state models, we deliberately decided to analyse colour and behaviour separately.

For the defensive behaviour data, we compared the model fits of ARD, SYM, and ER models. We also generated three hypothetical models that have the equivalent model structure of Intermediate, Cost-offset, and Secondary models in colour analysis and compared their model fits. These models included scenarios where (i) non-hooding defensive behaviours can only evolve from/to non-display states via the hooding behaviour state (Hooding Intermediate), the hooding behaviour can only evolve from/to non-display states via non-hooding defensive behaviour states (Other Displays Intermediate), and (ii) (iii) hooding behaviour and non-hooding defensive behaviours evolve independently without direct transitions between the two (Independent) (see Fig. S1 for visual description of the models). We then estimated ancestral character states using the best-fitted model based on AICc.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "GENERAL\nBeautifully written introduction. There is a general problem set up in the Introduction as to how aposematism evolves. The\nhypothesis to be tested is that hidden signals enable conspicuousness to evolve within the context of aposematism. This\nrequires venom to precede hidden signals which I don’t think was shown. I think we are being asked to accept that all the\nspecies here are venomous – may be this needs better documentation?\nSo now we have cryptic venomous snakes (venom used for capturing food) but this offense can be used in defense to deter\nwould-be predators and hidden signals allow conspicuous signals to evolve. So why be fully conspicuous at all – why don’t\nall aposematic species have hidden signals only? The costs of fully conspicuousness might need discussion.\nThe analyses show that hidden signals are an evolutionary route, but conspicuous dorsa can also evolve from crypticity –\nthis isn’t really discussed much – and it still leaves us searching for a solution to the paradox in these species. Can this be\naddressed more explicitly?\nI think there is a conceptual difference between hidden hooded reveals and non-hooded reveals. The former in venomous\nsnakes is also a signal of imminent defense (such that the signal and the defense are closely associated), whereas the non-\nhooded hidden signal could be deimatism or thanatosis and hence open to cheating at least in non-venomous snakes.\nSome discussion of this in Caro & Ruxton TREE.\nCould we broaden the discussion at the end. Is the hidden signals route something restricted to herps or can we expect to\nsee it in arthropods, nudibranchs, mammals and so on. If so why/or why not? I understand there are methodologicsal issues\nin examining this but some speculation would be useful.\nAnd could deimatism and flash coloration be related/additional routes to aposematism along with hidden signals?\nSPECIFIC\n74 and many species are not gregarious but solitary\n88-89 fitness benefit at the outset – you still have to make the connection between a conspicuous venter and distastefulness\n– presumably by sampling unless you are suggesting deimatism?\n182 doesn’t the mimicry route beg the question of how the model evolved aposematism or are you assuming models used\nthe hidden signal route?\n262 hooding and non-hooding defensive behaviours are found in venomous species only – all species here are venomous, I\ntake it? I don’t think that the genus Emydocephalus is venomous?? Perhaps this needs more discussion?\n325 ‘Since any snake that exhibited conspicuous dorsal coloration would be ranked as conspicuous regardless of ventral\ncoloration, some species for which images of the ventral surface were unavailable were provisionally included in the\ncategorization. This was done when considering the dorsal coloration to be unambiguously conspicuous.’ Is this OK? I\nwould think it rather incautious as assumes hidden will end up as aposematic.\n414 – this is a lot of images – is this correct?", "reviewer_2": "This is an interesting and well conducted study examining the role of ‘hidden signals’ in the evolution of conspicuous\naposematic patterns in elapid snakes (the majority of which are defended with potent venoms). The initial evolution of\nconspicuous aposematism can be difficult to explain, as predators will be naïve to the original conspicuous mutant and may\ndrive it to extinction before avoidance behaviour can become established. In this manuscript, the authors postulate that\nhidden signals, which do not attract unnecessary attention but which can still be learnt by predators, may act as a stepping-\nstone between cryptic and conspicuous phenotypes. By categorising the colour of 251 species of elapid snake and\nexamining evolutionary transitions between different anti-predator strategies, the authors conclude that hidden signals may\nbe an intermediate step in the evolution of conspicuous colouration.\nOverall, I found this to be a very interesting study, that would make an excellent contribution to the field. Before\nrecommending acceptance, I would however like to ask for come clarification on the authors’ interpretation of the data, and I\nhave some comments and suggestion on the manuscript which I hope the authors will find useful. I outline each of these in\nturn below.\nMain comments\n1. The conclusions appear to be much more strongly worded than the Results indicate. The Abstract and Conclusion both\nstate that hidden signals are a transition between camouflage and aposematism, but the data does not appear to strongly\nsupport this conclusion. I believe the data is of interest whether it supports the hypothesised model or not. I therefore\nrecommend some careful consideration of how the results are interpreted and discussed to ensure clarity and consistency\nthroughout the manuscript.\n2. It is not clear to me how hidden signals escape the costs of naïve predators as it seems they may still suffer from a lack of\navoidance behaviour in naïve predators. How can hidden signals increase the rate of non-lethal avoidance learning? Does\nthis rely on startle (as mentioned in the introduction (lines 86-87)? I think this justification needs some further elaboration /\ndiscussion throughout the introduction.\n3. Some important ecological information is not discussed in detail. I think it is important to acknowledge how other\nbehavioural / ecological traits may have influenced colour evolution in these animals. For example, the potential effect of\ndifferences in visual perception among predator species is acknowledged in the Methods, but there are several other factors\nI think are worthy of discussion. This include: 1) colour is discussed as if it is necessarily defensive, but many of these\nsnakes also need to hide from their own prey – a constraint which may restrict conspicuous colours to hidden body regions\neven if being fully conspicuous would be advantageous for deterring predators, 2) this clade includes terrestrial, fossorial,\narboreal, and aquatic species, as well as both nocturnal and diurnal species, and so what constitutes a hidden signal or\nconspicuous behaviour may well differ according to observer orientation or snake activity – how may this affect the evolution\nof colour or your analyses? I recognise that it would not be possible to add all these factors to the analysis, but I think the\nmanuscript would benefit from including some discussion of their potential role (especially the role of needed to hide form\nprey). Moreover, it would be nice to consider whether these factors may explain some of differences between your findings\nfor amphibians and for these snakes.\n4. The journal format requires the methods to be presented at the end of manuscript. I therefore think it would be beneficial to\ninclude some (brief) additional methodological information at the beginning of the Results & Discussion section. I think this\nhelp the reader understand what the data means when it is first introduced. For example, stating what the ‘three classifiers’\nand the ‘competing six models’ refer to when first used.\nLine-by-line comments\nLines 24-26: Can you be more specific about your findings here please?\nLines 29-31: Can you clarify the results here please? As I understand the results (and most of the discussion) there is only\nweak evidence to support the assertion that hidden signals are an important transition.\nLine 42: I’m not sure the statement “Most animal species have evolved camouflage…” is supported by the cited work. I agree\nwith the underlying point of this sentence, but I think it is worth revising this into a more general statement.\nLine 46-47: The terms “Batesian” and “Mullerian” mimicry are used later in the manuscript (i.e., lines 181-188) but have not\nbeen defined here. I think adding these terms in here would be beneficial.\nLine 60: Reference to Wallace (1867) does not follow the superscript number convention.\nLine 68-70: I think this statement is too strongly worded. Kin selection may well have played some role in the evolution or\nelaboration of aposematic signals, despite caterpillars not forming aggregations prior to the initial evolution of bright colours.\nPerhaps adding the word ‘initial’ would be more accurate, e.g., “… kin selection is unlikely to have contributed to the initial\nevolution of aposematism in these caterpillars.”.\nLine 120: typo – missing ‘the’ before ‘Elapidae family’.\nLines 133-141: Given the journal’s format (with methods at the end). I think care should be taken to introduce key\nmethodological details when introducing the data. For example, here you could quickly mention that three human classifiers\nwere used to classify the main defensive strategy of each snake.\nLine 138: Does your definition of polymorphic only include to species where colour variation crosses between CON, CRY, &\nHID categories? For example, would a species with two distinct and brightly coloured morphs (e.g., red and yellow) be\ncategorised as polymorphic?\nLines 138-147: Can you explain in more detail, how the statement on line 141 (“… transitions of species between the three\ncolour states occurred at similar rates” is compatible with the following statement on lines 143-145 (“…the transition from\nconspicuous to cryptic color occurred considerably less than other transitions”.\nLines 175-188: This paragraph appears to be contradictory. The paragraphs before and after this one seem to suggest that\nthere is weak (or no) evidence to support the hidden signal intermediate model, but this paragraph seems to argue that it is\nkey to the evolution of aposematism.\nLine 178: typo – missing ‘a’ before ‘cryptic ancestor’.\nLines 181-183: This argument appears to be incomplete as there may be reasons, other than mimicry, why conspicuous\ncolouring would evolve directly from camouflage. As discussed in the Introduction (lines 55-62), conspicuous aposematism\nmay be more likely to evolve where defences can be deployed before an animal is captured or consumed. Bites from elapid\nsnakes would appear to fall into this category (also see spitting cobras), so may not require mimicry. I think this should be\ndiscussed in more detail.\nI think it is also worth discussing whether snakes need colour to identify themselves as dangerous when many mammal and\nbird species will instinctively avoid snakes. This may again shift the balance away from conspicuous colouring as\nspecialised snake predators can often avoid/withstand bites from venomous snakes (i.e., secretary birds or mongoose).\nLine 185-188: How might your result change if known mimics were removed from the data set?\nLines 196-199: The classification methods and the sample size for these taxa are missing (from here and from the Methods).\nLines 196-205: This analysis effectively reduces your sample size to one (as explained in the introduction – lines 104-106). I\nthink extra caution should be taken here to explain this result, not as a confirmation of the stepwise model, but rather as a\nsuggestion for additional work across a broader range of snake taxa.\nLine 214-215: Does this support the hidden signals as a transition to aposematism or not? It is unclear to me.\nLine 265-270: I think this needs to be clarified throughout. How confident can we be in this statement given the data /\ndiscussion presented earlier in the manuscript? If this conclusion is relying on the ancestor of all elapids having hidden\nsignals, then I would recommend stating this caveat and being more cautious with the interpretation throughout.\nLine 288: Does this classification method also apply to the sister clades mentioned on lines 189-205?\nLine 305-312: Did you restrict this to naturally occurring colour morphs, for example excluding colour abnormalities and\ncolour forms selectively bred in captivity (i.e., albinism). Similarly, how did you categorise species with recorded melanism?\nLine 433: typo – unnecessary “(“ at the end of the sentence.\nLine 440 onwards: Missing mention that the analysis was conducted in R.\nSupplementary Data: Can you include a more detailed README file along with your data and R code please? Some\nabbreviations seem to differ between the data and the main manuscript (e.g., “FV” in data vs “Hid” in the manuscript)."}, "author_response": "RESPONSE TO REVIEWERS’ COMMENTS\nReviewer #1 (Remarks to the Author):\nGENERAL\nBeautifully written introduction. There is a general problem set up in the Introduction as to how\naposematism evolves. The hypothesis to be tested is that hidden signals enable conspicuousness\nto evolve within the context of aposematism. This requires venom to precede hidden signals\nwhich I don’t think was shown. I think we are being asked to accept that all the species here are\nvenomous – may be this needs better documentation?\nThank you very much for the positive feedback. The presence of venom is indeed the\nancestral state and nearly ubiquitous in elapid snakes (it is known to have been lost only\nonce in a recently diverged lineage containing only three extant species with two of these\nspecies included in our analysis). We now explicitly state this on lines 111 – 113, along with\nsupporting references: “Aposematism is widespread in this group, and chemical defense (or\noffence) is the ancestral condition, with only a few congeneric species having secondarily\nlost this trait”\nSo now we have cryptic venomous snakes (venom used for capturing food) but this offense can\nbe used in defense to deter would-be predators and hidden signals allow conspicuous signals to\nevolve. So why be fully conspicuous at all – why don’t all aposematic species have hidden\nsignals only? The costs of fully conspicuousness might need discussion.\nThis is a very good point, as the adoption of full aposematism appears counterintuitive\nwhen hidden signals seem to offer the “best of both worlds”—facilitating both signaling to\npredators and hiding from prey. One potential drawback to the hidden signaling strategy is\nthat, unlike full aposematism, it requires the snake to be aware of the predator before the\nattack in order to deploy a behavioural display. If the snake is caught by surprise, it may\nnever have the chance to signal.\nA recent phylogenetic study of elapid and colubrid snakes by Kojima et al. (2024) found\nthat species which feed on other snakes (which generally have rudimentary colour vision)\nare more likely to evolve aposematism(https://www.pnas.org/doi/10.1073/pnas.2318857121),\nwhich may be possible because snakes have limited colour discrimination. The same study\nalso revealed that non-conspicuous coloration is associated with a sit-and-wait strategy.\nThis suggests that when visual detection by prey is not a factor, aposematism becomes a\nmore adaptive strategy.\nWe are currently exploring the relative costs and benefits of aposematism versus hidden\nsignaling in some unpublished theoretical work. We have added the following passage at\nlines 227 – 238 to address this point:\n“While these results provide insight into the evolutionary pathways through which lineages\nacquire aposematism, they do not explain why some lineages evolve this trait while others\nretain ancestral hidden signals. Indeed, in certain ecological contexts, hidden signaling may\nbe an optimal strategy—allowing a snake to remain concealed from its prey while still\nbeing capable of signaling to predators when necessary. A recent phylogenetic study found\nthat aposematism is more likely to evolve in elapid species that feed on other snakes\n(Kojima et al. 2024). Given that many snakes exhibit limited colour discrimination abilities,\nthis may reduce the need to remain cryptic (Simões et al. 2016). Under such conditions,\nthere may be an advantage to signal their defences constantly to reduce the risk of initial\nattacks—for example, if an initial predatory attack leaves no chance for the snake to reveal\nits hidden signals, it may be costly to have cryptic dorsal coloration. This may happen in\nsea snakes such as Laticauda sp. where initial strikes of potential predators, such as ospreys\nor sharks, often leave no chance for the snake to show hidden signals.”\nThe analyses show that hidden signals are an evolutionary route, but conspicuous dorsa can also\nevolve from crypticity – this isn’t really discussed much – and it still leaves us searching for a\nsolution to the paradox in these species. Can this be addressed more explicitly?\nThis is an interesting point of difference between our findings with amphibians and the\nfindings presented here with respect to elapid snakes. Our previous work found that direct\ntransition from crypsis to aposematism occur at negligible rates in amphibians (Loeffler-\nHenry et al. 2023). Our results suggest a rather different pattern in elapids, with direct\ntransitions of camouflaged species to species with conspicuous signals being observed at\nnotably higher frequencies. While it is challenging to understand how novel conspicuous\nmutants of cryptic prey spread from rarity, elapids appear to possess a range of defensive\nand ecological characteristics not seen in amphibians, that may partially mitigate costs\nfaced by early/novel conspicuous phenotypes. For instance, compared to the relatively\ndelicate skin of amphibians, the hard keratinous scales of snakes may enable them to better\nsurvive handling by a predator. Likewise, the chemical defense of elapids consists of venom\nthat can be injected rather than poison that must be ingested to take effect, further suggests\nthat elapids may be better able to survive sampling attempts than amphibians. In addition,\nthe prevalence of Batesian and Müllerian mimicry in elapid snakes may explain direct\ntransitions from camouflage to conspicuousness in some lineages. This is because if one\ntaxon first acquires aposematism through the stepping-stone route, then new conspicuous\nmutants of other species who resemble this well-established warning signal may be\nrecognized as unprofitable to attack at the outset. Indeed, the conspicuous coloration of\nnon-venomous Emydocephalus species, which are Batesian mimics of venomous sea snakes\nlike Aipysurus, Hydrophis, and Laticauda spp., likely evolved from cryptic ancestors,\nsuggesting that direct transitions from crypsis to conspicuous forms can occur through\nmimicry.\nSome of the above points have already been discussed in lines 213 – 221. We have also\nadded the following passage to lines 221 – 226.\n“Additionally, unlike amphibians in which no direct transition from crypsis to\naposematism has been observed (Loeffler et al. 2023), snakes possess hard keratinous scales\ncompared to the relatively delicate skin of amphibians, which may allow them to better\nsurvive handling by predators. Likewise, the chemical defence of elapids involves venom\nthat can be injected, rather than poison that must be ingested to take effect, further\nsuggesting that elapids may be better able to survive sampling attempts than amphibians.”\nI think there is a conceptual difference between hidden hooded reveals and non-hooded reveals.\nThe former in venomous snakes is also a signal of imminent defense (such that the signal and the\ndefense are closely associated), whereas the non-hooded hidden signal could be deimatism or\nthanatosis and hence open to cheating at least in non-venomous snakes. Some discussion of this\nin Caro & Ruxton TREE.\nThank you for suggesting this. We have added the following passage to the discussion\nhighlighting this functional difference and the non-hooding displays that may be exploited\nby Batesian mimics.\n“There may be functional distinctions between hooding and non-hooding displays, with\nhooding displays in venomous snakes likely signal imminent defence and dissuade\npredators through startle or by accentuating the size and ferocity of the snake, while non-\nhooding hidden signals could be aposematism or thanatosis (Caro & Ruxton, 2009). As\nsuch, hooding may be more closely associated with an impending potent reaction, while\nnon-hooding displays may be less dependent on the defense itself and more susceptible to\nbeing exploited by undefended Batesian mimics (Caro & Ruxton, 2009).”\nCould we broaden the discussion at the end. Is the hidden signals route something restricted to\nherps or can we expect to see it in arthropods, nudibranchs, mammals and so on. If so why/or\nwhy not? I understand there are methodologicsal issues in examining this but some speculation\nwould be useful.\nIn the revised conclusion section, we highlight the above point in lines 320 - 329: “Hidden\nsignals and associated defensive behaviours like flash displays and deimatism are\nwidespread in the animal kingdom and are often observed in clades that contain\naposematic species (Loeffler-Henry et al. 2019). Therefore, it is possible that the hidden\nsignal stepping-stone route to aposematism is not restricted to herpetofauna. However, as\nour results in elapids demonstrate, other evolutionary routes can bypass the need for\nhidden signals in the transition from camouflage to aposematism, and this may extend\nbeyond this taxon. For example, nudibranchs often mimic the toxic cnidarians that they\nsequester their chemical defenses from, while many aposematic mammals utilise defenses\nthat do not require sampling or close contact by predators, such as the noxious sprays of\nskunks and African polecats (Howell et al. 2021, Avila et al. 2018). Such defences may\nreduce the costs associated with the initial evolution of aposematism.”\nAnd could deimatism and flash coloration be related/additional routes to aposematism along with\nhidden signals?\nThank you for this suggestion. We have now incorporated this feedback into the last\nparagraph of the discussion (see above).\nSPECIFIC\n74 and many species are not gregarious but solitary\nFixed thank you. Now it reads as “Additionally, aposematism is observed in many non-\ngregarious taxa, including coral snakes (Micrurus and Calliophis genera) that are highly\ncannibalistic outside of the breeding season and therefore unlikely to have exhibited\ngregarious behaviour at any point in their evolutionary history” in lines 72 – 75.\n88-89 fitness benefit at the outset – you still have to make the connection between a conspicuous\nventer and distastefulness – presumably by sampling unless you are suggesting deimatism?\nThe sentence now reads in line 88 – 91: “This is an important distinction because it\nsuggests that, unlike rare aposematic forms, mutations that cause hidden signals may\nprovide a fitness benefit at the outset, by either startling a predator or generating confusion\n(Drinkwater et al. 2021).”\n182 doesn’t the mimicry route beg the question of how the model evolved aposematism or are\nyou assuming models used the hidden signal route?\nThis sentence now reads in lines 216 – 218: “…, if one taxon first acquires aposematism\nthrough the hidden signal route, then new conspicuous mutants of other species that\nresemble this well-established warning signal may be recognised as unprofitable to attack\nat the outset.”\n262 hooding and non-hooding defensive behaviours are found in venomous species only – all\nspecies here are venomous, I take it? I don’t think that the genus Emydocephalus is venomous??\nPerhaps this needs more discussion?\nThat is correct—all species with behavioral displays are venomous. Given that this is now\nclearly stated in the Introduction, we have not reiterated it here (see response to the first\ncomment of this revision). We also noted elsewhere that Emydocephalus are non-venomous\nvia: “Indeed, the conspicuous coloration of non-venomous Emydocephalus species, which\nare Batesian mimics of venomous sea snakes …” in lines 218 – 220.\n325 ‘Since any snake that exhibited conspicuous dorsal coloration would be ranked as\nconspicuous regardless of ventral coloration, some species for which images of the ventral\nsurface were unavailable were provisionally included in the categorization. This was done when\nconsidering the dorsal coloration to be unambiguously conspicuous.’ Is this OK? I would think it\nrather incautious as assumes hidden will end up as aposematic.\nThis was only done for species that were ranked as unambiguously conspicuous by KLH\nduring image collecting. Because any species that has conspicuous coloration on the dorsal\nsurface will be classed as CON regardless of the coloration on the ventral surface, the\ninformation about the ventral coloration would not affect the classification. However, if the\nother categorizers had ranked any of these species as CRY, they would have been removed\nfrom the data. Since a species with cryptic dorsal colour could be ranked as hidden\ndepending on the ventral colour, this never happened, however, due to the generally high\nrate of agreement among categorizers.\n414 – this is a lot of images – is this correct?\nThat is correct! All images on iNaturalist were surveyed to determine the presence/absence\nof defensive behaviour for every species.\nTim Caro\nReviewer #2 (Remarks to the Author):\nThis is an interesting and well conducted study examining the role of ‘hidden signals’ in the\nevolution of conspicuous aposematic patterns in elapid snakes (the majority of which are\ndefended with potent venoms). The initial evolution of conspicuous aposematism can be difficult\nto explain, as predators will be naïve to the original conspicuous mutant and may drive it to\nextinction before avoidance behaviour can become established. In this manuscript, the authors\npostulate that hidden signals, which do not attract unnecessary attention but which can still be\nlearnt by predators, may act as a stepping-stone between cryptic and conspicuous phenotypes. By\ncategorising the colour of 251 species of elapid snake and examining evolutionary transitions\nbetween different anti-predator strategies, the authors conclude that hidden signals may be an\nintermediate step in the evolution of conspicuous colouration.\nOverall, I found this to be a very interesting study, that would make an excellent contribution to\nthe field. Before recommending acceptance, I would however like to ask for come clarification\non the authors’ interpretation of the data, and I have some comments and suggestion on the\nmanuscript which I hope the authors will find useful. I outline each of these in turn below.\nThank you very much for this encouraging feedback.\nMain comments\n1. The conclusions appear to be much more strongly worded than the Results indicate. The\nAbstract and Conclusion both state that hidden signals are a transition between camouflage and\naposematism, but the data does not appear to strongly support this conclusion. I believe the data\nis of interest whether it supports the hypothesised model or not. I therefore recommend some\ncareful consideration of how the results are interpreted and discussed to ensure clarity and\nconsistency throughout the manuscript.\nWe agree with the reviewer and have modified both Abstract and Conclusion sections to\naccount for the presence of multiple routes to aposematism in the results. Please, see the\nrevised abstract and conclusions below. The modified texts are highlighted in bold.\nAbstract\nThe initial evolution of warning coloration (“aposematism”) within a cryptic population of\ndefended prey presents an evolutionary paradox. A recent phylogenetic analysis of\namphibia suggested a new solution: prey that combine cryptic colours with conspicuous\npatches on concealed body parts (“hidden signallers”), may have mediated the transition of\nspecies from camouflage to aposematism. Here, we focus on the colour-diverse snake family\nElapidae and test whether species with hidden colours could also serve as an intermediate\nstage in the evolution of aposematism in this group. Phylogenetic comparative analysis\nrevealed several key patterns in their anti-predator colour evolution: (i) a few major\ntransitions influenced the overall distribution of hidden-colours, camouflage, and\naposematism in the group, and (ii) aposematism evolved multiple times, with hidden\ncoloration a common precursory state, while direct transitions from camouflage to\naposematism were also observed. We also quantified associations between colour patterns\nand defensive behaviours that reveal ventral surfaces (i.e. hidden signals). We found that\nventer-revealing defensive behaviours frequently co-occur with hidden colour signals. Our\nresults suggest that aposematism can evolve through multiple routes and highlight the\nprevalence of co-evolution between venter-revealing defensive behaviour and anti-predator\ncoloration in snakes.\nConclusion\nOur study demonstrates general patterns of defensive colour evolution in elapid\nsnakes and highlights the prevalence and key role of hidden colour states in the evolution of\naposematism. As previously demonstrated in amphibians28, our phylogenetic analysis\nprovides evidence that hidden warning signals can serve as an intermediary stage in the\ntransition from camouflage to aposematism in elapid snakes, although there have also been\ndirect transitions of species with camouflage to species with conspicuous signals. This\nfinding offers a potential resolution to the paradox of the initial evolution of aposematism,\nsuggesting that hidden colour states could facilitate this transition. That said, we stress that\nhidden-coloured states were likely present early in the Elapidae lineage, following a single\ntransition from a cryptic ancestor. This early origin allowed hidden-coloured species to\nserve as a common precursor in all subsequent transitions. While our analysis is limited to\nthe family Elapidae –where defensive coloration is more diverse than in other snake\ngroups, most of which are predominantly camouflaged, it may not be a coincidence that\naposematic species are rare in groups where hidden-coloured species are also rare; the\nparadox of initial evolution of aposematism may persist in these groups, but the hidden-\ncoloured states could mitigate its associated costs and may facilitate the evolution of\naposematism. This, in turn, may influence higher taxonomic patterns in the distribution of\naposematic species.\nIn addition, our behavioural analyses highlight a close co-evolutionary relationship\nbetween hidden colours and the defensive behaviours. Hidden signals and associated\ndefensive behaviours like flash displays and deimatism are widespread in the animal\nkingdom and are often observed in clades that contain aposematic species29. Therefore, it is\npossible that the hidden signal stepping-stone route to aposematism is not restricted to\nherpetofauna. However, as our results in elapids demonstrate, other evolutionary routes\ncan bypass the need for hidden signals in the transition from camouflage to aposematism,\nand this may extend beyond this taxon. For example, nudibranchs often mimic the toxic\ncnidarians that they sequester their chemical defenses from, while many aposematic\nmammals utilise defenses that do not require sampling or close contact by predators, such\nas the noxious sprays of skunks and African polecats40,41. Such defences may reduce the\ncosts associated with the initial evolution of aposematism.\n2. It is not clear to me how hidden signals escape the costs of naïve predators as it seems they\nmay still suffer from a lack of avoidance behaviour in naïve predators. How can hidden signals\nincrease the rate of non-lethal avoidance learning? Does this rely on startle (as mentioned in the\nintroduction (lines 86-87)? I think this justification needs some further elaboration / discussion\nthroughout the introduction.\nThis is a good point. We have added the following passage to clarify our reasoning on lines\n85 – 88.\n“Unlike consistently visible aposematic signals, these hidden colour defenses do not impose\nthe costs of full conspicuousness during the initial stages of evolution. Their normally\ncamouflaged appearance reduces the risk of detection, while still potentially deterring\npredators through post-attack displays that startle, warn, and/or facilitate avoidance\nlearning.”\n3. Some important ecological information is not discussed in detail. I think it is important to\nacknowledge how other behavioural / ecological traits may have influenced colour evolution in\nthese animals. For example, the potential effect of differences in visual perception among\npredator species is acknowledged in the Methods, but there are several other factors I think are\nworthy of discussion. This include: 1) colour is discussed as if it is necessarily defensive, but\nmany of these snakes also need to hide from their own prey – a constraint which may restrict\nconspicuous colours to hidden body regions even if being fully conspicuous would be\nadvantageous for deterring predators, 2) this clade includes terrestrial, fossorial, arboreal, and\naquatic species, as well as both nocturnal and diurnal species, and so what constitutes a hidden\nsignal or conspicuous behaviour may well differ according to observer orientation or snake\nactivity – how may this affect the evolution of colour or your analyses? I recognise that it would\nnot be possible to add all these factors to the analysis, but I think the manuscript would benefit\nfrom including some discussion of their potential role (especially the role of needed to hide form\nprey). Moreover, it would be nice to consider whether these factors may explain some of\ndifferences between your findings for amphibians and for these snakes.\nThank you for these suggestions. Please see how we have incorporated them into the\nmanuscript. We have included some considerations regarding (1) in lines 229 – 233 as\nfollows “Indeed, in certain ecological contexts, hidden signaling may be an optimal\nstrategy—allowing a snake to remain concealed from its prey while still being capable of\nsignaling to predators when necessary. A recent phylogenetic study found that\naposematism is more likely to evolve in elapid species that feed on other snakes (Kojima et\nal. 2024).”\nAlso, we have added the following passage to acknowledge how ecological variability would\naffect what constitutes a conspicuous colour in lines 355 - 365; this is particularly relevant\nto sea snakes:\n“Elapid snakes encompass considerable ecological diversity, so what constitutes a hidden\nsignal may differ based on their ecological contexts, snake behaviours, and observer\norientations which are factors that we were not able to incorporate into our analysis.\nGenerally, coloration on the ventral surface is clearly only exposed transiently and\ntherefore qualifies as a hidden signal. However, a notable exception to this is in sea snakes\n(subfamilies Hydrophiinae and Laticaudinae) who swim suspended in the water column\nwhere their ventral surfaces are readily visible most times. Because of this, no sea snake\nspecies were classed as having hidden signals. However, we note that the colour\ncategorisation of some sea snake species, such as Hydrophis platurus, may depend on the\nviewer. This species has a yellow belly and a dark brown dorsum, appearing aposematic\nunderwater but camouflaged to aerial predators such as ospreys and sea eagles, which\napproach exclusively from above. In our list, H. platurus is the only species that exhibits\nsuch a bicolour pattern.”\nFor behaviour in lines 426 – 428:\n“Naturally how salient a behaviour depends on habitat and context. However, this\ngeneralised categorisation schema is useful in enabling broad multispecies comparisons.”\nFor hiding from prey see lines 227 – 229 and above paragraph that starts with the sentence:\n“While these results provide insight into the evolutionary pathways through which lineages\nacquire aposematism, they do not explain why some lineages evolve this trait while others\nretain ancestral hidden signals.”\nAnd with respect to how this may have contributed to the disparate findings between\namphibians and elapids see lines 221 – 224 and above paragraph that starts with the\nsentence:\n“Additionally, unlike amphibians in which no direct transition from crypsis to\naposematism has been observed (Loeffler et al. 2023), snakes possess hard keratinous scales\ncompared to the relatively delicate skin of amphibians, which may allow them to better\nsurvive handling by predators.”\n4. The journal format requires the methods to be presented at the end of manuscript. I therefore\nthink it would be beneficial to include some (brief) additional methodological information at the\nbeginning of the Results & Discussion section. I think this help the reader understand what the\ndata means when it is first introduced. For example, stating what the ‘three classifiers’\nand the ‘competing six models’ refer to when first used.\nWe have revised the first two paragraphs of the results discussion to help frame what was\ndone for the reader in lines 135 – 143 with the changes made highlighted in bold; see below\n“We analysed 251 species (~62% of extant species) of elapid snakes with known\nphylogenetic relationship and colour information. Three human classifiers experienced\nwith snakes categorised each species based on its anti-predator coloration: either cryptic\n(colours and patterns that match natural backgrounds), conspicuous (high contrast\ncoloration typically associated with aposematism such as black and white, bright blue,\nbright yellow, red, or orange), or hidden with cryptic coloration on the dorsal surface and\nconspicuous coloration on the ventral surface (see Methods for details). The three\nclassifiers all agreed on the colour classification for 236 elapid species (94% of analysed\nspecies). For the remaining 15 species, two classifiers agreed, and we used the most\ncommon classification for each species in the main analysis.”\nAlso, in lines 147 – 153, “We generated six hypothetical models with different constraints\non transition pathways or transition rate estimation (see Methods for details). We\nconsidered seven states in total, comprising three major colour states and four polymorphic\nstates. Our hypothetical models were generated using only the three monomorphic states\nbecause the small number of species in each polymorphic state limited reliable estimation\nof transition rates (see Methods for details). … Among the competing six models (Fig. 2A),\nthe Equal rates (ER) model, in which a single rate governs all transitional pathways, was\nthe most strongly supported …”\nLine-by-line comments\nLines 24-26: Can you be more specific about your findings here please?\nThis part has been revised as “a few major transitions influenced the overall distribution of\nhidden-colours, camouflage, and aposematism in the group, and (ii) aposematism evolved\nmultiple times, with hidden coloration a common precursory state, while direct transitions\nfrom camouflage to aposematism were also observed.” in lines 25 – 27.\nLines 29-31: Can you clarify the results here please? As I understand the results (and most of the\ndiscussion) there is only weak evidence to support the assertion that hidden signals are an\nimportant transition.\nRevised to “Our results suggest that aposematism can evolve through multiple routes and\nhighlight the prevalence of co-evolution between venter-revealing defensive behaviour and\nanti-predator coloration in snakes.”\nLine 42: I’m not sure the statement “Most animal species have evolved camouflage…” is\nsupported by the cited work. I agree with the underlying point of this sentence, but I think it is\nworth revising this into a more general statement.\nThis now reads “many animal species have evolved camouflage, reducing their probability\nof detection or recognition by predators” in lines 43 – 44.\nLine 46-47: The terms “Batesian” and “Mullerian” mimicry are used later in the manuscript (i.e.,\nlines 181-188) but have not been defined here. I think adding these terms in here would be\nbeneficial.\nThat’s a good point. Revised to “These warning signals can be mimicked by harmless\nspecies (Batesian mimicry) or copied by other defended species thereby reducing the costs\nof predator education (Müllerian mimicry)” with citations in lines 46 – 68.\nLine 60: Reference to Wallace (1867) does not follow the superscript number convention.\nFixed, thank you for spotting this.\nLine 68-70: I think this statement is too strongly worded. Kin selection may well have played\nsome role in the evolution or elaboration of aposematic signals, despite caterpillars not forming\naggregations prior to the initial evolution of bright colours. Perhaps adding the word ‘initial’\nwould be more accurate, e.g., “… kin selection is unlikely to have contributed to the initial\nevolution of aposematism in these caterpillars.”.\nThank you for this suggestion; we have revised this sentence accordingly in line 71.\nLine 120: typo – missing ‘the’ before ‘Elapidae family’.\nFixed, thank you for spotting this as well.\nLines 133-141: Given the journal’s format (with methods at the end). I think care should be\ntaken to introduce key methodological details when introducing the data. For example, here you\ncould quickly mention that three human classifiers were used to classify the main defensive\nstrategy of each snake.\nThank you for this suggestion. As we describe in our response to your main comments (4),\nwe have rephrased the first two paragraphs of Results and Discussion section to help frame\nwhat was done for the reader.\nLine 138: Does your definition of polymorphic only include to species where colour variation\ncrosses between CON, CRY, & HID categories? For example, would a species with two distinct\nand brightly coloured morphs (e.g., red and yellow) be categorised as polymorphic?\nThat is correct. Our narrow definition of polymorphism only includes species that have\nmorphs in different coloration categories. For example, Bungarus fasciatus exhibits both a\nyellow and black form and a white and black form. But because both are highly\nconspicuous, the species is classified as CON, not polymorphic. We have revised the\nsentence a bit to “(here, defined narrowly when a species exhibits two or more different\nanti-predator strategies)” in lines 145 – 146.\nLines 138-147: Can you explain in more detail, how the statement on line 141 (“… transitions of\nspecies between the three colour states occurred at similar rates” is compatible with the\nfollowing statement on lines 143-145 (“…the transition from conspicuous to cryptic color\noccurred considerably less than other transitions”.\nIn the revised manuscript, we have clarified why there exists some discrepancy between the\ntwo sentences in lines 154 – 162 as below.\n“…transitions of species between the three colour states occurred at similar rates.\nStochastic character mapping results aligned well with the transition rate results in that the\ntransitions among different strategies have occurred in similar frequencies (Fig. 3C).\nHowever, although the estimated rates were similar, the historical frequency of transitions\nfrom conspicuous to cryptic colour was considerably lower than that of other transitions,\nwhich is consistent with our analysis of the most likely transitional history for each species\n(Fig. 3D). This indicates that such transitions have rarely occurred in the history of elapid\nsnake anti-predator coloration evolution, yet the comparable rate estimates suggest that\nwhenever they do occur, they proceed just as quickly as other types of transitions. ”\nLines 175-188: This paragraph appears to be contradictory. The paragraphs before and after this\none seem to suggest that there is weak (or no) evidence to support the hidden signal intermediate\nmodel, but this paragraph seems to argue that it is key to the evolution of aposematism.\nThis paragraph can be better framed when the ancestral character estimation of deeper\nnode comes first (which we presented in later paragraph in the original manuscript).\nHowever, we found that the contents of this paragraph somewhat overlap with the\nparagraphs before and after this one. After careful consideration, we have decided to\nremove the paragraph.\nLine 178: typo – missing ‘a’ before ‘cryptic ancestor’.\nThe paragraph has now been removed. Thank you for spotting this.\nLines 181-183: This argument appears to be incomplete as there may be reasons, other than\nmimicry, why conspicuous colouring would evolve directly from camouflage. As discussed in\nthe Introduction (lines 55-62), conspicuous aposematism may be more likely to evolve where\ndefences can be deployed before an animal is captured or consumed. Bites from elapid snakes\nwould appear to fall into this category (also see spitting cobras), so may not require mimicry. I\nthink this should be discussed in more detail. I think it is also worth discussing whether snakes\nneed colour to identify themselves as dangerous when many mammal and bird species will\ninstinctively avoid snakes. This may again shift the balance away from conspicuous colouring as\nspecialised snake predators can often avoid/withstand bites from venomous snakes (i.e., secretary\nbirds or mongoose).\nThis is another excellent point, which was also raised by Reviewer #1. Our answer can be\nfound above, but we repeat our revised paragraph below as well.\n“The direct transitions of camouflaged species to species with conspicuous signals may be\npartly explained by the prevalence of Batesian and Müllerian mimicry in elapid snakes;\nthus, while it is challenging to understand how the novel conspicuous mutants of cryptic\nprey initially spread from rarity35, if one taxon first acquires aposematism through the\nhidden signal route, then new conspicuous mutants of other species who resemble this well-\nestablished warning signal may be recognised as unprofitable to attack at the outset.\nIndeed, the conspicuous coloration of non-venomous Emydocephalus species, which are\nBatesian mimics of venomous sea snakes like Aipysurus, Hydrophis, and Laticauda spp.36,\nlikely evolved from cryptic ancestors, suggesting that direct transitions from crypsis to\nconspicuous forms can occur through mimicry. Additionally, unlike amphibians in which\nno direct transition from crypsis to aposematism has been observed28, snakes possess hard\nkeratinous scales compared to the relatively delicate skin of amphibians, which may allow\nthem to better survive handling by predators. Likewise, the chemical defence of elapids\ninvolves venom that can be injected, rather than poison that must be ingested to take effect,\nfurther suggesting that elapids may be better able to survive sampling attempts than\namphibians.”\nLine 185-188: How might your result change if known mimics were removed from the data set?\nThat is an excellent question and one we would also very much like to know the answer to.\nWe indeed attempted to investigate model–mimic relationships and to incorporate this into\nthe analysis. Unfortunately, it was not possible to fully quantify the extent of mimicry\nwithin this clade because we have colour information for only 62% of species. As a result,\nmany potential venomous (co-)models remain unknown, and the true model–mimic\nrelationships likely extend beyond this clade, primarily into the family Colubridae. While\nwe attempted to identify model–mimic relationships, it was difficult to confirm these\nrelationships with confidence for many species that are potentially involved in mimicry.\nWe predict that if known mimics were removed from the dataset, hidden-signal routes\nmight become a more prevalent pathway for transitions to aposematism. However, we have\nno data to confirm this. Our speculation regarding the potential role of mimicry in shaping\nthese transitional patterns was included in the original manuscript and can now be found\nin lines 213 – 226 (the same paragraph in the above comment).\nLines 196-199: The classification methods and the sample size for these taxa are missing (from\nhere and from the Methods).\nNow the text has been revised to “Ancestral reconstruction incorporating 151 species from\nsister clades showed that elapid snakes likely diverged from cryptic ancestors (86%\nsupport; Fig. 3B; Fig. S3 for marginal reconstruction results; images of the sister clades\nwere classified by KLH using the same criterion described in the methods).” in lines 203 –\n206.\nLines 196-205: This analysis effectively reduces your sample size to one (as explained in the\nintroduction – lines 104-106). I think extra caution should be taken here to explain this result, not\nas a confirmation of the stepwise model, but rather as a suggestion for additional work across a\nbroader range of snake taxa.\nThe transition from crypsis to hidden-coloured states occurred once. Then, the transition\nfrom hidden-coloured states to aposematism occurred multiple times. We agree that, while\nmany aposematic elapid species evolved following stepwise-evolution route, all of them are\nfrom a single ancestral transition. We already mention this in lines 206 – 210 as “This\nsuggests that stepwise evolution from crypsis to conspicuousness via hidden coloured states\nalso occurred in snakes. However, unlike amphibians, where these transitional patterns\nappeared multiple times in various lineages, this stepwise evolution in elapid (and relative)\nsnakes is explained by a single ancestral transition from camouflage to the hidden colour\nstate at a deep node.”. In addition to this, we have added a sentence highlighting the need\nfor additional work across broad range of snake taxa to examine the prevalence and\ngenerality of this route in shaping aposematism in snakes in lines 210 – 212 as “A more\ncomprehensive examination of other snake families is warranted to confirm the prevalence\nand generality of this stepwise-evolution route and to assess its role in the evolution of\naposematism in snakes.”.\nLine 214-215: Does this support the hidden signals as a transition to aposematism or not? It is\nunclear to me.\nOur main results show multiple transitional pathways to aposematism, involving both\nhidden signal routes and direct routes. The supplementary analyses reveal patterns\nconsistent with the main findings. We have added a sentence to highlight this in lines 247 –\n249 as follows. “The ancestral character reconstruction results suggest that all\nsupplementary analyses exhibit transitional patterns consistent with the main findings\n(Figs. S4, 5).”\nLine 265-270: I think this needs to be clarified throughout. How confident can we be in this\nstatement given the data / discussion presented earlier in the manuscript? If this conclusion is\nrelying on the ancestor of all elapids having hidden signals, then I would recommend stating this\ncaveat and being more cautious with the interpretation throughout.\nWe have modified this statement to express more caution about the interpretation of the\nfindings (see below). Also, as our reviewer remarked, we have emphasized the fact that the\nancestral species of Elapids was most likely a hidden signaller of all the possible signalling\nstates 303 - 312.\n“Our study demonstrates general patterns of defensive colour evolution in elapid snakes\nand highlights the prevalence and key role of hidden colour states in the evolution of\naposematism. As demonstrated in amphibians28, our phylogenetic analysis provides\nevidence that hidden warning signals can serve as an intermediary stage in the transition\nfrom camouflage to aposematism in elapid snakes, although there have also been direct\ntransitions of species with camouflage to species with conspicuous signals. This finding\noffers a potential resolution to the paradox of the initial evolution of aposematism,\nsuggesting that hidden colour states could facilitate this transition. That said, we stress that\nhidden-coloured states were likely present early in the Elapidae lineage, following a single\ntransition from a cryptic ancestor. This early origin allowed hidden-coloured species to\nserve as a common precursor in all subsequent transitions.”\nLine 288: Does this classification method also apply to the sister clades mentioned on lines 189-\n205?\nThat is correct with the exception that the sister clade was exclusively classified by KLH\nfor logistical reasons. As we noted above, we now explain this in our revised manuscript.\nLines 305-312: Did you restrict this to naturally occurring colour morphs, for example excluding\ncolour abnormalities and colour forms selectively bred in captivity (i.e., albinism). Similarly,\nhow did you categorise species with recorded melanism?\nThat is correct, only images of naturally occurring forms were utilized. Semi-domesticated\nselectively bred recessive mutations were never included in the classification. We highlight\nthis in the revised manuscript in lines 376 – 378 as “We restricted our image sampling to\nnaturally occurring individuals and excluded those resulting from selective breeding or\ncolour abnormalities (i.e. albinism).”\nLine 433: typo – unnecessary “(“ at the end of the sentence.\nThank you for spotting this. We have now removed it.\nLine 440 onwards: Missing mention that the analysis was conducted in R.\nThank for spotting this as well. We have added it.\nSupplementary Data: Can you include a more detailed README file along with your data and R\ncode please? Some abbreviations seem to differ between the data and the main manuscript (e.g.,\n“FV” in data vs “Hid” in the manuscript).\nReviewer #2 (Remarks on code availability):\nI am unfamiliar with the phylogenetic analysis and I am not able to fully examine the analysis\ncode.\nThe raw data appears to be present and correct.\nThe README file (\"Explanation.txt\"), explains what each file is for but is lacking information\nabout what is contained within each data file.\nWe have uploaded a new README file that provides details about our data and R code.\nWe also ensured that the manuscript, data, and R code use consistent terminology.\nDear Editor,\nWe wish to thank you and the two reviewers for the final comments. Below, we provide our\nresponses to each of the reviewers’ points.\nSincerely,\nChangku Kang (corresponding author) on behalf of all authors,"}, {"round": 2, "reviewer_comments": {"reviewer_1": "This is an excellent paper ready for publication.\nSome very minor points:\n118 give a well-known example here\n120 are there others apart from rolling over?\n239-250 could this go in SOM?\nFigure 3 D not clear what the colours of the bars refer to?", "reviewer_2": "I would like to thank the authors for their responses to my comments and for answering my questions. I believe this study addresses a very interesting question for the evolution of morphology and behaviour generally, as well as for the colour of elapid snakes specifically. I enjoyed reading the manuscript and I only have one (very) minor comment on the revised draft:\nLines 312-317: This is a very long and complex sentence (with an em-dash, multiple commas, and a semi-colon). I think it could be split into two."}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis is an excellent paper ready for publication. Some very minor points:\nOur response: We have removed this part from the manuscript. It now reads: ‘…because,\nunlike rare aposematic forms, mutations causing hidden signals may provide a fitness\nbenefit at the outset, for example, by startling a predator.\n118 give a well-known example here\nOur response: It now reads as “While this behaviour is best known in cobras of the genus\nNaja, such as the Spectacled Cobra (Naja naja),”\n120 are there others apart from rolling over?\nOur response: Now it reads as “In addition to hooding, there exists other venter-revealing\ndefensive behaviours including rolling over, defensive coiling, tail displaying, and exposing\nthe ventral surface in response to provocation by a predator.”\n239-250 could this go in SOM?\nOur response: We appreciate the suggestion. Although placing this paragraph in the SOM\nis one possibility, we believe it is helpful to retain it in the main text. The related analyses\nare already detailed in the SOM, and presenting their key implications here strengthens\nour main findings and offers readers clearer context.\nFigure 3 D not clear what the colours of the bars refer to?\nOur response: The bar colors did not carry any specific meaning. To avoid any confusion,\nwe have updated the bars to use a uniform color. Additionally, we have added a legend\nclarifying which strategies are represented by each colored circle.\nReviewer #2 (Remarks to the Author):\nI would like to thank the authors for their responses to my comments and for answering my\nquestions. I believe this study addresses a very interesting question for the evolution of\nmorphology and behaviour generally, as well as for the colour of elapid snakes specifically. I\nenjoyed reading the manuscript and I only have one (very) minor comment on the revised draft:\nLines 312-317: This is a very long and complex sentence (with an em-dash, multiple commas,\nand a semi-colon). I think it could be split into two.\nOur response: Thank you for the helpful suggestion. We have now split the sentence into\ntwo."}]

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