Palaeoart is considered a discipline that can provide valuable information, combining graphic arts and science in order to create images of the reconstruction of extinct animals and their environments with scientific rigour. The methodology currently used for the reconstruction of fossil vertebrates is based on the sequential anatomical reconstruction proposed by Antón & Sánchez (Reference Antón, Sánchez, Baquedano and Rubio2004). It can be divided into different stages: the identification of the osteological correlations imprinted by soft tissue; the anatomically accurate reconstruction of the bones using comparative anatomy; the inference of muscle insertions – and other soft tissues such as fat, cartilage and tendons – from the basis of the previous osteological reconstruction; and the proposal of the external appearance, such as the skin, colour pattern and, in the case of extinct mammals, hair. Therefore, comparative anatomy becomes an extremely useful tool that allows to infer the live appearance of a fossil taxon. As could be expected, certain characters that are rarely preserved, and leave little to no evidence of how they would appear in the living organism, present some constraints for their reconstruction. Such as is the case for the trait analysed in this study: the colour pattern in extinct mammals.

Colour pattern is a character that is influenced by an extensive array of variables, such as natural or sexual selection (sometimes both), climate, habitat, behaviour, phylogenetic relationships, body size of the animals or pigment economy, among other factors (Bortolotti Reference Bortolotti, Hill and McGraw2006; Pérez González et al. Reference Pérez González, López Cantalapiedra, María Alcalde and Hernández Fernández2009; Brown et al. Reference Brown, Henderson, Vinther, Fletcher, Sistiaga, Herrera and Summons2017; Negro et al. Reference Negro, Finlayson and Galván2018), and therefore, whose analysis is complex. In the vast majority of fossils, physiological characters, for example, melanosome and carotenoid concentrations, structural colour, and molecular signals (Vinther et al. Reference Vinther, Briggs, Clarke, Mayr and Prum2010; McNamara et al. Reference McNamara, Birggs, Orr, Wedmann, Noh and Cao2011, Reference McNamara, Briggs and Orr2012, Reference McNamara, Kaye, Benton, Orr, Rossi, Ito and Wakamatsu2018; Li et al. Reference Li, Gao, Meng, Clarke, Shawkey, D'Alba, Pei, Ellison, Norell and Vinther2012; Brown et al. Reference Brown, Henderson, Vinther, Fletcher, Sistiaga, Herrera and Summons2017; Negro et al. Reference Negro, Finlayson and Galván2018; Pan et al. Reference Pan, Li, Wang, Zhao, Wang and Zheng2022), that might indicate the colouration of the taxon, are not preserved. This is further the case in fossil mammals, in which environmental and organic degradation make it very difficult for structures such as hair or skin to be well preserved (Tridico Reference Tridico2015). In particular, the colouration of the latter characters provides information on the behaviour of the animal, how it related intraspecifically and interspecifically, or how it interacted with its environment. As mentioned in Pérez González et al. (Reference Pérez González, López Cantalapiedra, María Alcalde and Hernández Fernández2009, and references therein), although climate and habitat have certain influence in the colouration of mammals, another determining factor is the phylogeny of the studied taxon, where the phylogenetic signal responsible for observed patterns of colouration might serve as a reliable source that allows to reconstruct the external appearance of select extinct taxa.

Therefore, phylogenetic information, from an actualist approach, when applied to palaeoartistic reconstructions, can provide to be a very useful frame of reference for making well supported inferences. Specifically, phylogenetically close taxa – current and extinct – give valuable information on the appearance or morphology of anatomical features that would otherwise not be evident nor preserved in the fossil record (Byrant & Russell Reference Byrant and Russell1992; Witmer Reference Witmer and Thomason1995).

Previous works have shown that phylogenetic analyses performed to study coloration patterns are a successful tool, often leading to either palaeoartistic reconstructions, or to understanding the coat pattern evolution within the studied groups. Some examples of such works, which focused on the colour pattern of mammals, include carnivorans (Ortolani & Caro Reference Ortolani, Caro and Gittleman1996; Werdelin & Olsson Reference Werdelin and Olsson1997; Ortolani Reference Ortolani1999; Mattern & McLennan Reference Mattern and McLennan2000; Werdelin et al. Reference Werdelin, Yamaguchi, Johnson, O'Brien, Macdonald and Loveridge2010), lagomorphs (Stoner et al. Reference Stoner, Bininda-Emonds and Caro2003a), rodents (Lai et al. Reference Lai, Shiroishi, Moriwaki, Motokawa and Yu2008; Ancillotto & Mori Reference Ancillotto and Mori2017) and artiodactyls (Stoner et al. Reference Stoner, Caro and Graham2003b; Caro et al. Reference Caro, Graham, Stoner and Vargas2004; Pérez González et al. Reference Pérez González, López Cantalapiedra, María Alcalde and Hernández Fernández2009).

For the scope of this study, we focus on the order Artiodactyla, specifically the family Cervidae, a group of small to large herbivore mammals that includes 54 extant species (Burgin et al. Reference Burgin, Wilson, Mittermeier, Rylands, Lacher and Sechrest2020). In particular, Cervidae present a complicated case study for the determination of coat pattern, since there are extensive factors that contribute to the observed phenotypes. One of the influencing factors to consider is intraspecific variation which is present in many other mammalian groups. Additionally, the ontogeny of the individual is involved, as in other artiodactyls, although it is highly noticeable in this group (Fig. 1a). Furthermore, the colour arrangement in specimens varies considerably between different populations of the same species (Fig. 1b). Finally, the colour variations occurring between winter and summer coats are extreme in many cervids (Fig. 1c), unlike in other artiodactyls, although not all genera experience a significantly noticeable change between these two seasons. Conversely, differences in colour pattern as a result of sexual dimorphism are not as marked in this group as they are in, for example, some genera of bovids.

Figure 1. Different colour pattern variations in Cervidae. (a) Ontogeny (Capreolus capreolus, fawn, left, and adult, right). (b) Different populations and/or subspecies of the same species (winter coat of Rangifer tarandus, from left to right, Rangifer tarandus caribou, Rangifer tarandus groenlandicus, Rangifer tarandus tarandus and Rangifer tarandus platyrhynchus). (c) Seasons of the year (Cervus canadensis, summer coat, left, and winter coat, right). Not to scale. Illustrations by Jesús Gamarra.

However, despite these limitations, the large number of species and the relatively well resolved phylogenetic relationships within Cervidae (Heckeberg Reference Heckeberg2020), makes this family an ideal group for the aim of this work, offering a good framework to test the evolution and phylogenetic signal of their colour patterns.

The taxon used to test the inference of the colour pattern in an extinct cervid is Heteroprox moralesi Azanza Reference Azanza1989, a basal deer from the Middle Miocene of Central Spain. Its phylogenetic position close to the base of the family Cervidae makes it ideal to be the subject of an analysis of the basal coloration of this group (Heckeberg Reference Heckeberg2020).

Heteroprox comprises four Eurasian species of medium-sized cervids characterised by the presence of small proto-antlers with 2–3 ramifications, elongated extracranial, supraorbital pedicles, with a relatively smooth surface, and with an ornamentation consisting of fine striae. Males had well-developed canines (Azanza Reference Azanza1989; Rössner Reference Rössner2010; Heckeberg Reference Heckeberg2017), as occurs in other species of Cervidae, in either extinct (such as Procervulus) or extant groups (such as Muntiacus or Elaphodus).

Further classification of the genus Heteroprox placed it within the subfamily Procervulinae, together with the cervid Procervulus, and considered it as basalmost in regards to its phylogenetic relationships within family Cervidae, as is suggested by various authors (Vislobokova Reference Vislobokova1983; Azanza Reference Azanza1989, Reference Azanza1993; Rössner Reference Rössner1995; Gentry et al. Reference Gentry, Rössner, Heizmann, Rössner and Heissig1999; Mennecart et al. Reference Mennecart, Rössner, Métais, De Miguel, Schulz, Muller and Costeur2016, Reference Mennecart, De Miguel, Bibi, Rössner, Métais, Neenan, Wang, Schulz, Müller and Costeur2017). However, other works classify Heteroprox as belonging to the Dicrocerini tribe, as they find it to have a greater resemblance to the genus Dicrocerus (Bubenik & Bubenik Reference Bubenik and Bubenik1990; Grubb Reference Grubb2000). The phylogenetic analysis done by Heckeberg (Reference Heckeberg2020) also places Heteroprox close to both Procervulus and Dicrocerus, in a position close to the Muntiacini tribe, which would place them close to the crown group of extant cervids.

In this study, for the purpose of estimating the phylogenetic position of Heteroprox, we choose to follow the criteria of considering Heteroprox a member of Procervulinae to estimate its phylogenetic position since, of the two, this proposal is the most agreed upon classification and has been extensively supported. In this context, we perform a character trace analysis using the well understood phylogeny of the family Cervidae, relating extinct species with extant ones; the latter for which colouration pattern information is available. From this analysis, the objective of the present work is to produce a tentative reconstruction of the coat colour pattern of basal cervids, such as Heteroprox and its closely related taxa.

1. Materials and methods

The methodology used in this work largely follows the one proposed by Pérez González et al. (Reference Pérez González, López Cantalapiedra, María Alcalde and Hernández Fernández2009) for the colour pattern reconstruction of the Middle Miocene bovid Tethytragus, which is recorded in association in some fossil sites to Heteroprox. The analysis employed to infer the ancestral states was the maximum likelihood (ML) (Maddison & Maddison Reference Maddison and Maddison1989), using software Mesquite version 3.61 (Maddison & Maddison Reference Maddison and Maddison2021), an analysis which assumes the least number of changes in the character (in this case, the colours) to explain the distribution of states of that character in the taxa that can be observed at the extreme points of the phylogenetic tree (Pérez González et al. Reference Pérez González, López Cantalapiedra, María Alcalde and Hernández Fernández2009). ML analysis, combined with Markov algorithms, takes into account the length of the phylogenetic tree branches, in order to infer which character or characters of the studied taxa emerged from a specific ancestor. The most probable percentages of each character in a node between two phylogenetic branches are shown.

The ancestor of all the extant cervids, referenced in this study as the hypothetical ancestor, was used as the most closely related taxon to Heteroprox and to other Miocene cervids. These basal cervids were more closely related between them than with the extant cervids, as the work of Heckeberg (Reference Heckeberg2020) shows. This makes it necessary to have an accurate phylogenetic tree of the extant cervids. We followed Heckeberg (Reference Heckeberg2020) as the main source of information; Meijaard & Groves (Reference Meijaard and Groves2004) for the genus Axis; Świsłocka et al. (Reference Świsłocka, Matosiuk and Ratkiewicz2020) for the genus Alces; and Zhang et al. (Reference Zhang, Lwin, Li, Maung, Li and Quan2021) for the genus Muntiacus.

A total of 51 species of cervids belonging to the two extant subfamilies, Cervinae and Capreolinae, were analysed in order to create a thorough coat colouration database. To follow a same criterion, only images of fully grown adult males were used, to treat the data homogeneously. Also, only the colour pattern for the summer coat of the taxa was considered, since H. moralesi is associated with warm environments (López-Martínez et al. Reference López-Martínez, Élez, Hernando, Luis, Mazo, Mínguez Gandú, Morales, Polonio, Salesa and Sánchez2000), and most likely experienced little to no discernible changes of coat during the winter. Moreover, the colouration patterns of the summer coat were selectively used because they are present during the season in which male individuals are at their maximum sexual maturation stage and where the expression of characters (e.g., antlers, colourful coats, etc.) is most conspicuous in preparation for the breeding season.

Several images were retrieved for each of the different species, obtained from Google Images. Quality profile photographs were selected with comparable light conditions, except for those of Mazama chunyi, Muntiacus malabaricus, Muntiacus putaonensis and Muntiacus truongsonensis, for which case the illustrations as shown in Burgin et al. (Reference Burgin, Wilson, Mittermeier, Rylands, Lacher and Sechrest2020) were consulted, since quality images with discernible colour pattern for these taxa were not available.

Some species were discarded in this analysis: Mazama bricenii as it is possibly a junior synonym of Mazama rufina (Heckeberg Reference Heckeberg2020, and references therein); Muntiacus rooseveltorum as it is considered a junior synonym of Muntiacus feae (Heckeberg Reference Heckeberg2020, and references therein); Muntiacus puhoatensis since no studies have been found that clarify its systematics; and Rucervus schomburgki as it is extinct and no adequate images of its fur could be found, since taxidermy specimens lose their original colouration over time.

To study the colour variations occurring in the same specimen, a schematic template of a general cervid was made (Fig. 2) based on the bovid template created by Pérez González et al. (Reference Pérez González, López Cantalapiedra, María Alcalde and Hernández Fernández2009) with some modifications. This template sectors the different parts of the body of a cervid where the most common colour changes observed in cervid colour pattern are placed, 26 in total. This template was coloured for each species studied, painting it in flat colours without considering the volume (i.e., lights and shadows) so that the colour pattern was studied uniformly and without alterations.

Figure 2. Body sectorisation of a generic cervid used in this work. Based on Pérez González et al. (Reference Pérez González, López Cantalapiedra, María Alcalde and Hernández Fernández2009).

Once the template of a species was coloured, saturation was reduced to the minimum to obtain a grey scale drawing (Fig. 3b). Using grey scales allow to evaluate the same criteria in the study for each sector in the different species. As each shade in a grey scale can be attributed to a specific and unique colour code, the Adobe Photoshop dropper tool was used to identify the colour code (value) for each shade. The average colour of each sector was then selected and compared with a reference grey scale table (Fig. 3a), which allowed for a more objective determination, rather than just a visual assessment.

Figure 3. Grey scales used in this work. (a) Grey shades used in this work. Under the top five are their colour codes. Contrasted is where, in a same sector, abrupt changes from light to dark shades can be found. (b) Example of a grey coloured template (in this case, the species is Axis axis). Abbreviation: Spt. = spotted.

The reference grey scale table used is divided into five shades, which represent a range of values: white; light; neutral; dark; and black. The shade values obtained from the image sampling of individuals, were then assigned to one of the five shade categories within the reference greyscale table. The results were then added to the database, a value was assigned to each shade, obtaining a total of 10 values: white (1); light (2); neutral (3); dark (4); black (5); contrasted (6); spotted light (7); spotted neutral (8); spotted dark (9); and spotted black (10). The values of the first five shades were taken from the template of the species Axis axis (Fig. 3b), as it has all the grey shades in its colour pattern, from pure white to deep black.

Once the database was completed with all the species, a ML analysis of ancestral states was performed.

2. Results

In Table 1, the results are shown as the most likely percentage for each shade of each sector to appear in the basal node for all extant Cervidae, and further represent the probability of the hypothetical basal cervid ancestor having that colouration.

Table 1. Sectors with the probabilities of appearance of each grey shade in the basal node of the family Cervidae, where Heteroprox would be found.

Values and sectors in boldface type are those that are highly significant. To reduce the table size, values were rounded to the third decimal place. Abbreviations: Wh = white; Li = light; Ne = neutral; Da = dark; Bl = black; Co = contrasted; Sp. Li = spotted light; Sp. Ne = spotted neutral; Sp. Da = spotted dark; Sp. = black.

To follow a criterion, the sectors considered to have significant values are those with at least twice or more the value than the next highest value (which represents the next most likely shade), and were considered as the final results for that sector. These sectors are shown as in Fig. 4a. Sectors not showing significant values (as the cases aforementioned) were studied separately, and appear in white (see Fig. 4a). In the analysis, these sectors were: 7, forehead; 10, neck; 11, beard; 12, dewlap; 13, forearm; 14, metacarpals; 16, tibia; 17, metatarsals; 20, abdomen; 22, side stripe; 24, dorsal stripe; and 26, tail.

Figure 4. Results of the analysis shown in the template. (a) Sectors with the significant values (coloured) with the problematic sectors (in white). (b) Final result in the template. (c) Palaeoartistic reconstruction of Heteroprox moralesi with the results of this study. Illustration by Jesús Gamarra.

Therefore, to assess the shades of sectors with ambiguous results, and complement the results which did show significant values, further information was needed. Consequently, additional factors affecting colour pattern in H. moralesi, such as its inferred habitat, were evaluated.

Heteroprox, as most cervids, inhabited forested areas (López-Martínez et al. Reference López-Martínez, Élez, Hernando, Luis, Mazo, Mínguez Gandú, Morales, Polonio, Salesa and Sánchez2000). While the other Eurasian Heteroprox species were known to live in humid forests, the conditions during the Middle Miocene in much of the Central Iberian Peninsula were significantly arid or even semi-desertic (Fesharaki Reference Fesharaki2016; Menéndez et al. Reference Menéndez, Gómez Cano, García Yelo, Domingo, Domingo, Cantalapiedra, Blanco and Hernández Fernández2017). Therefore, the habitats of H. moralesi were most likely forest patches near savannas and arid environments. Some current biomes that resemble Heteroprox habitats are savannas, deciduous tropical forests and Mediterranean forests.

Taking this into consideration, we should expect to see some similarity to H. moralesi in the assortment of shades in extant deer species inhabiting the three types of biomes. We then selected shade values (colour code) of several extant deer species that live within those biomes, in order to infer which colouration Heteroprox was most likely to have in these problematic sectors based on its habitat. Species habitat information was obtained from Myers et al. (Reference Myers, Espinosa, Parr, Jones, Hammond and Dewey2022): those living in savanna are Axis axis, Axis porcinus, Blastocerus dichotomus, Dama dama, Dama mesopotamica, Mazama americana, Mazama gouazoubira, Muntiacus muntjak, Rucervus duvaucelii, Odocoileus hemionus and Odocoileus virginianus; in deciduous tropical forests, A. axis, A. porcinus, Blastocerus dichotomus, Hydropotes inermis, M. americana, M. gouazoubira, Muntiacus muntjak, Muntiacus reevesi, Rucervus duvaucelii, Rucervus eldii, Rusa timorensis, Rusa unicolor and O. virginianus; and in Mediterranean forests, Capreolus capreolus, Cervus elaphus, D. dama, D. mesopotamica and Odocoileus hemionus.

For each one of the biomes, we selected the most common colouration for each ambiguous sector (refer to Table 2) most frequently observed among the different species. The results showed that the predominant shades for the sectors evaluated were similar across all selected biomes. Based on the shades of the compared extant cervids for these arid habitats, new supporting data was obtained which was compared to the previous results (the shades showing similar values, or no significant values; see Table 1), which allowed us to determine the final colour pattern proposed for the ambiguous sectors.

Table 2. Results of the problematic sectors analysis according to the habitat.

Colour abbreviations are the same as in Table 1. Values in boldface type are the chosen ones when certainly significant compared to the other values of the likelihood analysis.

The results for the ambiguous sectors were as follows: 7, forehead, dark to black; 10, neck, neutral; 11, beard, white; 12, dewlap, light; 13, forearm, dark; 14, metacarpals, dark to black; 16, tibia, dark; 17, metatarsals, dark to black; 20, abdomen, light; 22, side stripe, neutral; 24, dorsal stripe, black; and 26, tail, contrasted.

The result for Heteroprox general colouration shows an animal with a slight general countershading dorsoventrally, as is usual in most cervid species and appears in many other animals. This type of colouration is very common in the animal kingdom since, as mentioned by Cott (Reference Cott1940), it is the basis of camouflage in both predators and prey.

Nonetheless, the results of the countershading in this work indicate further variation, also varying anteroposteriorly, with the neck and the head presenting neutral tones – to the exception of the upper part of the neck – while the rest of the body would appear darker in tone. This may be due to the fact that, much like the countershading of the abdomen, a slightly lighter colouration on the anteroventral sector of the neck would have been more favourable for blending in, as it would counteract the effect of the shadow – usually produced in areas at an angle less exposed to the sunlight – making it less noticeable by reducing its contrast against the rest of the body (Stoner et al. Reference Stoner, Caro and Graham2003b; Rowland Reference Rowland2009). As seen from the results in Table 2, the beard (11) and dewlap (12) sectors are usually light in the more frequent phenotype of the species from the two most open environments, the savanna and Mediterranean forest, in comparison to those of the more closed and denser deciduous tropical forest. The shade values of the former (i.e., species from the savanna and Mediterranean forest) were then given preference as a source of reference and were, consequently, used to fill the gaps in the ambiguous-resulting sectors for the reconstruction of Heteroprox. The lighter shades in these sectors would suggest that the colouration patterns in Heteroprox were more transitional at the head and at the neck, and thus less conspicuous to potential predators, an advantageous morphological trait in the more exposed areas of open vegetation.

The results for Heteroprox also suggest that some sectors were darker in comparison to the rest of the general body colour, more distinctively the extremities, dorsal parts of the head and neck, and the dorsal stripe on the back. Darker limbs, which serve in extant cervids as a form of disruptive colouration, are associated with large social groups, open environments and desert habitats (Stoner et al. Reference Stoner, Caro and Graham2003b) and might have aided Heteroprox in intraspecific communication. The results for the perianal region and tail sectors further produce a more evident contrast, and result in a conspicuous colouration pattern. Many extant cervids have these strongly contrasted areas, or are directly white (either only on the inner part or on the entire tail). In some species such as O. virginianus, for example, this colouration serves for communication when a predator is detected (Blindstein Reference Blindstein1983). It may be that in Heteroprox, if it had any gregarious nature, that could have served to make other individuals aware of its presence, or to warn of a nearby danger.

Thus, the final colour pattern reconstruction is obtained from the results, as shown in the Fig. 4b template. Overall, Heteroprox had a pelage with darker tones and countershading yet some areas in which the colour patterns were more conspicuous. This darker colouration might have served Heteroprox and, therefore, basal cervids to go unnoticed among the foliage of the forests where they lived. The countershading could have aided with camouflage, blending its colouration among the warm forest patches near a semiarid savanna (Fig. 4c).