Armored with skin and bone: A combined histological and μCT‐study of the exceptional integument of the Antsingy leaf chameleon Brookesia perarmata (Angel, 1933)

Madagascar's endemic ground‐dwelling leaf chameleons (Brookesiinae: Brookesia Gray, 1865 + Palleon Glaw, et al., Salamandra, 2013, 49, pp. 237–238) form the sister taxon to all other chameleons (i.e., the Chamaeleoninae). They possess a limited ability of color change, a rather dull coloration, and a nonprehensile tail assisting locomotion in the leaf litter on the forest floor. Most Brookesia species can readily be recognized by peculiar spiky dorsolateral projections (“Rückensäge”), which are caused by an aberrant vertebral structure and might function as body armor to prevent predation. In addition to a pronounced Rückensäge, the Antsingy leaf chameleon Brookesia perarmata (Angel, 1933) exhibits conspicuous, acuminate tubercle scales on the lateral flanks and extremities, thereby considerably enhancing the overall armored appearance. Such structures are exceptional within the Chamaeleonidae and despite an appreciable interest in the integument of chameleons in general, the morphology of these integumentary elements remains shrouded in mystery. Using various conventional and petrographic histological approaches combined with μCT‐imaging, we reveal that the tubercle scales consist of osseous, multicusped cores that are embedded within the dermis. Based on this, they consequently can be interpreted as osteoderms, which to the best of our knowledge is the first record of such for the entire Chamaeleonidae and only the second one for the entire clade Iguania. The combination of certain aspects of tissue composition (especially the presence of large, interconnected, and marrow‐filled cavities) together with the precise location within the dermis (being completely enveloped by the stratum superficiale), however, discriminate the osteoderms of B. perarmata from those known for all other lepidosaurs.

A striking morphological feature of this group concerns a more-orless developed row of spiky projections running along their back. This Brookesia-specific "Rückensäge" ("spinal saw"; Boettger, 1878Boettger, , 1893) and its underlying peculiar vertebral structure has been subject to several osteological studies, particularly in Brookesia superciliaris (Kuhl, 1820) (Parker & Taylor, 1942;Siebenrock, 1893). One of the larger species of the genus, the Antsingy leaf chameleon Brookesia perarmata (Angel, 1933), does not only exhibit a well-pronounced Rückensäge, but stands out among its congeners because also the lateral flanks and extremities exhibit additional and unique thorny elements or large tubercle scales, suggestive of some kind of veritable integumentary armor. Comparable structures are not known for any other member of Brookesia, and so far, it is unknown what these integumentary appendages truly are. The principle aim of the present study was to identify the histological structure and three-dimensional (3D) tissue composition of the different elements of the integumentary armor of this intriguing leaf chameleon. the present study. The specimens were kept preserved in ethanol according to standard museum procedures prior to this study.

| MATERIALS AND METHODS
One complete specimen (ZSM 17/2006) was scanned submersed in ethanol using a phoenix nanotom m (GE Measurement and Control) μCT-system with the following settings: tube voltage = 110 kV; tube current = 70 μA; target = tungsten, no filter; total sample rotation = 360 ; angular step size = 0.24 ; exposure time = 750 ms; binning = 1; averaging = 4; voxel size = 37.8 μm. The tomographic reconstruction was performed with the phoenix datos|x 2.2 software and converted to 8 bit in VG Studio 2.2. Digital rotation and cropping of the resulting image stack was performed in ImageJ (Schindelin et al., 2012) and textured mesh objects were extracted in Drishti 2.6.4 (Limaye, 2012). Final renderings were created in Blender 2.79 (blender.org). Using Daz Studio 4.10 (Daz Productions, Inc, Salt Lake City, UT), a reduced version of the digital hard tissue model was converted into a *.u3d file and embedded into an interactive 3D PDF by a custom LaTeX script.
Samples from the vertebrae, the skin of the lateral flanks, and anterior extremities containing both larger and smaller elements of the putative integumentary armor of the three other specimens were removed for histological analysis using a scalpel and forceps while keeping the entire specimens intact. The removed samples were transferred to 5% nitric acid. They were kept in this solution for about 48-60 hr to decalcify the tissue. Afterwards the samples were washed under running tap water for about 2 hr and returned to 70% ethanol. In addition, we took a sample of the skin of the lateral body wall devoid of any conspicuous armored elements for comparison.
Samples were dehydrated in ethanol and embedded in glycol methacrylate (Technovit 7,100, Heraeus Kulzer GmbH). The methacrylate blocks were sectioned at a thickness of 2-5 μm using a HM 350 rotary microtome (Microm International GmbH). The sections were stretched on a water bath and transferred to regular glass slides.
Staining was done with a solution of 0.1% toluidine blue in 0.1% borax and the slides were cover-slipped using Roti Histokitt II (Carl Roth GmbH + Co. KG). Staining time varied according to section thickness and we furthermore produced, with regard to the soft tissues, overstained preparations as those yielded better results for the osseous parts contained in several of the sections. All analyses and imaging was done using a Zeiss Axio Lab.A1 light microscope equipped with a Canon EOS 60D digital camera. The resulting images were processed using RawTherapee 5.4 and GIMP 2.8 (adjustments of white balance, contrast, slight color adjustments and the removal of the background).
One additional isolated armored element of the lateral flanks was removed from each ZSM 862/2000 and ZSM 914/2006 as described above, dehydrated in a graded series of ethanol and finally transferred to and immersed in hexamethyldisilazane (HMDS) for 10 min (Nation, 1983). The samples were air-dried overnight and placed in separate sealed containers filled with silica gel the next morning and kept there until further analysis for about a week.
The dry sample from ZSM 914/2006 was embedded in Araldite ® 2020 (Huntsman), cut with an IsoMet™ Low Speed Precision Cutter (Buehler), and ground with silicon carbide powder to produce petrographic ground sections. These sections were analyzed using a Leica DM LP polarizing microscope equipped with a Leica DFC 420 camera and further digitally processed as described for the conventional histological sections above.
Following the approach introduced by Rühr and Lambertz (2019), the dry sample from ZSM 862/2000 was gold-coated with a 108 auto sputter coater (Cressington Scientific Instruments) prior to μCT-scanning using a Skyscan 1272 device (Bruker microCT). The sputter-coating was required because the absorption indices of the sample's substructures differed so strongly that it was impossible to visualize the skin in the subsequent analysis steps. At high tube energies (>60 kV), the skin did not absorb enough photons to be visualized, while at lower energies (30-60 kV), the absorption of the underlying structure was so strong that its blurred outlines overlaid the weak skin signal in the digital slice reconstructions. The final μCT scan of the gold-coated sample was carried out with the following settings: tube voltage = 70 kV; tube current = 142 μA; target = tungsten; total sample rotation = 180 ; angular step size = 0.19 ; exposure time = 1925 ms; binning = 2 × 2; filter = Al 0.5 mm; averaging = 8; random movement = 15; voxel size = 4.4 μm. Thermal drift correction and digital section reconstruction was done in NRecon 1.7 (Bruker microCT). Textured mesh creation of the skin and the osteoderm, final rendering and 3D PDF creation procedures were carried out as described above for the whole body scan. Additionally, a digital endocast of the observed cavernous structure was generated with the region competition algorithm of ITK-SNAP (Yushkevich et al., 2006).

| Normal skin of the lateral flanks
The skin is divided into a dermis and an epidermis. The dermis is divided into a basal stratum compactum of more regularly arranged collagen fibers and a stratum superficiale of irregular connective tissue.
Numerous pigment cells are present in the apical regions of the stratum superficiale. The overlying, multilayered epidermis is covered by a micro-ornamented Oberhäutchen.

| Armor-like elements of the lateral flanks
The histological structure of the integument immediately surrounding the armor-like element of the lateral flanks agrees with the condition for the normal skin described above. The tubercle scale itself consists of an osseous core that is embedded within the stratum superficiale of the dermis and fully enveloped by it (Figures 3a and 4a). As on the rest of the body, the stratum superficiale covering the osseous core is F I G U R E 2 Brookesia perarmata, vertebral histology. Cross section of the last dorsal vertebra in a posterior plane (a) and hemisection at about its midpoint (b). Note the cartilaginous zygapophyseal joint with the adjacent vertebra (c). The externally visible dorsal projection (d) shows a small island of cartilage at its base, just dorsal to the neural spine (arrow). The entire accessory, nontypical vertebral structures bulge out the dermis (e), which causes the external visibility. Scale bars equal 500 μm in a and b, 50 μm in c and e, and 100 μm in d F I G U R E 3 Brookesia perarmata, histological comparison of the integumentary armor. Both the armored elements of the lateral flanks (a, resting on the lateral body wall immediately above a rib) and those of the extremities (b) show the same principal architecture, most notably characterized by the osseous element (osteoderm) containing numerous large internal cavities. Note the larger blood vessels penetrating the bone at its basal (medial) side (asterisk) as well as the smaller ones at its superficial (lateral) one (arrow). All scale bars equal 500 μm F I G U R E 4 Brookesia perarmata, histological details of the lateral flank integumentary armor. Note that the osteoderm is fully enveloped by the stratum superficiale of the dermis (a), whereas the superficial dermal and epidermal layers resemble the ordinary squamate condition (b). Large blood vessels on the basal (medial) side (c) are, just as smaller ones on the superficial (lateral) side (not shown, but see arrow in Figure 3), connected to a marrow-like, potentially haematopoetic tissue within the osteoderm's cavities (d). All scale bars equal 50 μm F I G U R E 5 Brookesia perarmata, polarized microscopy of the lateral flank osteoderm. Note the remarkably complex architecture of the bone that is composed of parallel-fibered (plus sign), metaplastic (asterisk), and secondary lamellar bone (arrow). Fibers (black arrowhead) anchor the osteoderm within the dermis. At least one growth mark (white arrowhead) is visible in the parallel-fibered portion of the cortex. All scale bars equal 100 μm

| Armor-like elements of the anterior extremities
The histological structure of the integument surrounding the armor-like element of the extremities again agrees with the condition for the normal skin of the flanks described above. In addition, also the histological composition of the armor-like element itself agrees with those of the flanks: a stratum superficiale-embedded, multicusped, osseous element containing numerous cavities filled with marrow-like tissue. The above-mentioned shape reminiscent of a half-open bracelet is clearly evident (Figure 3b).

| 3D-morphology of the lateral flank integumentary armor
The central bony element of the lateral flank armor can clearly be separated from the surrounding soft tissue of the skin in the μCT scans ( Figure 6a). The osseous core directly resembles that of the externally visible, cone-shaped structure with its several minor cusps of the "tubercle scale" itself (Figure 6a (Figure 6d), which became evident due to the fact that the semiautomatic reconstruction employed produced a complete and continuous "endocast" after several starting points were set in the central portion of the osteoderm.

| DISCUSSION
The macroscopic external appearance of the conspicuous putative body armor of Brookesia perarmata has been known since the original description of this species by Angel (1933). We were able to corroborate the osteological findings on the vertebral projections in B. superciliaris by Siebenrock (1893) and Parker and Taylor (1942) also for the Antsingy leaf chameleon. A bridge-like arch extends between the pre-and postzygapophyses, connects both of them, projects laterally, and extends into the dermis.
These also externally visible projections constitute unique structural elements not known for any other lepidosaur outside the genus Brookesia and contribute to generating the appearance of body armor.
The question remains though how they are formed. Romer (1956), without further elaborating, which species he examined nor how he came to this conclusion, considered them as "[s]uperficial dermal ossification[s]" (p. 539). Our histological analyses revealed continuous growth lines and the absence of discrete sutures for the lateral F I G U R E 6 Brookesia perarmata, 3D-reconstruction of the lateral flank integumentary armor of Brookesia perarmata. Note that the shape of the multicusped osteoderm directly reflects the external morphology of the tubercle scale (a, b) and that there are numerous smaller superficial (lateral) and larger basal (medial) vascular canals within the bone (b, c). All the internal cavities are connected to each other (d). Scale bar equals 1 mm. To view an interactive 3D model (PDF version only), click on the Figure. Standard views available in toolbar at top. Individual meshes of skin, bone and endocast of osteoderm can be toggled on/off when "Model Tree" is activated in tool bar. Additional mouse controls: Left click: rotate scene; right click/ mouse wheel: zoom; both mouse buttons: pan. Figure best viewed with Adobe Acrobat Reader Version 9 or later projections with the vertebral body, suggesting that the vertebrae and these accessory structures in fact form a developmental unity. Dorsally, however, the externally visible projection appears to be somewhat separable from the dorsal tip of the vertebral neural spine in terms of its bone architecture, and the presence of cartilaginous remnants may be suggestive of a fusion of two discrete structures. Based on the material available to us, we can neither rule out that the accessory structures are of dermal origin fusing with the vertebrae, nor that they form as endochondral ossification and as outgrowths of the vertebrae themselves, or even that it is a combination of both processes.
In order to unambiguously answer the question of developmental origin, ontogenetic studies based on an appropriate series of specimens at different ages seem ultimately needed.
While the normal skin in the Antsingy leaf chameleon follows the general morphology of the squamate integument, the osteoderms themselves are rather unusual. Osteoderm morphology differs greatly within squamates, not only in relation to shape, size and distribution on the body (Paluh et al., 2017;Vickaryous & Sire, 2009), but also with respect to tissue composition (Vickaryous et al., 2015;Vickaryous & Sire, 2009). Most frequently, osteoderms are limited to the dorsal surface of the head and trunk (Gadow, 1909), whereas in other taxa (e.g., anguids, some gekkotans, and scincids) they enclose the entire body (Vickaryous et al., 2015;Vickaryous & Sire, 2009). In many squamate taxa, osteoderms are relatively small or thin (Otto, 1909;Paluh et al., 2017) and do not change or influence the outer silhouette of the animal. Even though their distribution is much more localized in B. perarmata, the osteoderms are conspicuous and large compared with the animal's size and, especially in combination with the Rückensäge, dramatically alter its body contour. Osteoderm shape ranges from vermicular in varanids (Erickson et al., 2003), imbricating and flat in anguids and scincids Otto, 1909;Schmidt, 1910Schmidt, , 1914a, to robust and bead-like in helodermatids (Mead et al., 2012;Vickaryous & Sire, 2009), or elongated with branching processes in anniellids (Bhullar & Bell, 2008).
However, large, conical, and multicusped osteoderms such as those of B. perarmata seem to be exceptional. Vickaryous and Sire (2009) found that in all lepidosaurians they investigated, osteoderms were embedded into the dermis directly at the juncture of the stratum superficiale and stratum compactum. The osteoderms of B. perarmata, however, are completely enveloped by the stratum superficiale, so that there is no contact with the stratum compactum. Though fundamentally different with regard to the osteoderm structure itself, this is reminiscent of the condition found in Geckolepis maculata Peters, 1880(Paluh et al., 2017. For the gecko Tarentola mauritanica (Linnaeus, 1758), Levrat-Calviac and Zylberberg (1986) described bundles of collagen fibers comparable to Shapey's fibers anchoring the osteoderms within the dermis, and Vickaryous et al. (2015) confirmed this interpretation.
Corresponding fibers are also present in B. perarmata and appear to secure the osteoderm within the surrounding superficial dermis.
Tissue composition of squamate osteoderms also varies greatly and is by no means restricted to osseous components, comprising a diverse spectrum of other mineralized and unmineralized tissues (Moss, 1969;Vickaryous & Sire, 2009). As a rough generalization, two types of osteoderms can be distinguished: (a) those that (at least primarily) consist of bone (e.g., in Anguis fragilis Linnaeus, 1758 and some gekkonids) (Vickaryous et al., 2015;Zylberberg & Castanet, 1985), and (b) those that additionally contain a mostly avascular and acellular, hypermineralized dental-like tissue (de Buffrénil et al., 2010;Iacoviello et al., 2020;Moss, 1969;Vickaryous et al., 2015), recently termed osteodermine (de Buffrénil, Dauphin, Rage, & Sire, 2011). The osteoderms of B. perarmata clearly fall into the former category. But in contrast to, for instance, A. fragilis in which the osteoderms are divided into a basal layer of lamellar bone and a superficial layer of woven-fibered bone (Zylberberg & Castanet, 1985), the osteoderms of the Antsingy leaf chameleon do not show such a two-part organization and lamellar bone is only found around the inner cavity walls.
Generally, reptilian dermal bone rarely possesses any cavities and is rather poorly vascularized (Moss, 1969). Schmidt (1910, 1912a, 1912b, 1912c, 1914a) noted the presence of small vascular canals for gekkotans, gerrhosaurids, scincids, helodermatids, and anguids. For Gerrhosaurus Wiegman, 1828and Zonosaurus Boulenger, 1887, Schmidt (1912b also described small cavities ("Markräume"), probably resulting from resorption, but did not provide further information about the soft tissue occupying these spaces. As a generalized remark, Moss (1969) stated that, if present, cavities were filled by fat cells and hematopoietic tissue, but he did not mention differences between the taxa he investigated. Otto (1909) reported small cavities in the osteoderms of Chalcides chalcides (Linnaeus, 1758) and C. ocellatus (Forskål, 1775). He interpreted the internal tissue as vascularized adipose cells, possibly combined with connective tissue and pigment cells. More recently, Broeckhoven, du Plessis, and Hui (2017)  The medullary region of B. perarmata's osteoderms shows considerable areas of resorption and redeposition with secondary infillings of lamellar bone bordered by a cement line along the trabeculae and inner walls of the cavities. In reptiles, the formation of lamellar bone is considered to require the presence of a periosteum (Moss, 1969;or endosteum), which would indicate a formation by intramembranous ossification. Except for secondary infillings, collagen arrangement within the osteoderms of B. perarmata is rather irregular and directly continuous with that of the surrounding superficial dermis, which, on the other hand, suggests a formation by metaplasia (Haines & Mohuiddin, 1968;Moss, 1969). These findings may indicate that osteoderm formation in B. perarmata is achieved by a combination of intramembranous ossification and metaplasia, which would be in congruence with the current knowledge for Lepidosauria in general (Vickaryous & Sire, 2009).
The general structure of the Antsingy leaf chameleon's osteoderms can be characterized as somewhat "spongy" and thus at least superficially resembles that of crocodylian osteoderms (see also those of the South American horned frogs, Quinzio & Fabrezi, 2012).
Osteoderm coverage in crocodylians is extensive, and depending on the species is not restricted to the dorsolateral surface but also extends to the ventral abdomen (Schmidt, 1914b;Vickaryous & Hall, 2008). Crocodylian osteoderms are more or less disc like and often possess a central protuberance or keel and apical ornamentation (Vickaryous & Hall, 2008). Microstructurally, crocodylian osteoderms exhibit a distinct diploe structure consisting of a compact cortex and a cancellous central portion (de Buffrénil et al., 2015;Vickaryous & Sire, 2009) similar to that found in B. perarmata. In contrast to most lepidosaurians, crocodylian osteoderms lie within the stratum superficiale and are anchored by Sharpey's fibers (de Buffrénil et al., 2015;Vickaryous & Sire, 2009), again reminiscent of the situation in B. perarmata. Apical and basal sides of crocodylian osteoderms are penetrated by small neurovascular foramina (Schmidt, 1914b;Vickaryous & Hall, 2008), but cavities are not limited to vascular spaces alone (Schmidt, 1914b). Schmidt (1914b) found that, at least in Crocodylus niloticus Laurenti, 1768, cavities were filled by a combination of connective tissue, blood vessels, nerves, and pigment cells. In contrast to that, the cavities of the osteoderms in B. perarmata appear to lack pigment cells, but are filled by potential hematopoietic tissue.
Crocodylian osteoderms are composed of a mixture of woven bone, parallel-fibered bone, lamellar bone, and mineralized and unmineralized connective tissue (de Buffrénil et al., 2015;Vickaryous & Hall, 2008). While Vickaryous and Hall (2008) did not find signs of intramembranous ossification in Alligator mississippiensis (Daudin, 1802) and consider bone metaplasia to be the only mode of osteoderm formation in crocodylians, de Buffrénil et al. (2015) investigated numerous extant and fossil Crocodylomorpha and found endosteal bone deposits in older (i.e., larger) individuals suggestive of osteoblast activity. As already stated, a similar combination of metaplasia and intramembranous ossification might also be present in B. perarmata. However, all of this taken together, renders the osteoderm morphology of the Antsingy leaf chameleon quite remarkable and unique among lepidosaurs, and thus expands our knowledge about the structural diversity of the amniote integument.
Concerning the functional significance of these structures for B. perarmata, the situation is even more confounded. Traditionally, reptilian osteoderms have been regarded solely as defensive structures, that is, dermal armor in the literal sense. However, even though this might be true for some taxa, it does not explain the occurrence of relatively thin and fragile osteoderms (Broeckhoven, Diedericks, & Mouton, 2015;Paluh et al., 2017).
More recent hypotheses widen the presumed function of osteoderms. Vickaryous et al. (2015) proposed a possible protection during aggressive intraspecific behavior as well as against well-fortified, large prey items in several species of geckos. Dacke et al. (2015) studied labile calcium sources in reproducing alligators and suggested that osteoderms serve as calcium deposits for eggshell production. A similar function as mineral reservoirs has also been discussed for sauropod dinosaur osteoderms (Curry Rogers, D'Emic, Rogers, Vickaryous, & Cagan, 2011). Furthermore, a possible role in thermoregulation in crocodilians and squamates has been discussed by several authors (Broeckhoven et al., 2017;Clarac, de Buffrénil, Cubo, & Quilhac, 2018;Clarac & Quilhac, 2019;Drane & Webb, 1980;Vickaryous & Hall, 2008;Vickaryous & Sire, 2009). For B. perarmata, no information on thermoregulation and calcium metabolism is available (although it is restricted to karstic limestone habitats), therefore a possible influence of such physiological processes on osteoderm advantageousness can hardly be discussed.
In his work on B. superciliaris, Siebenrock (1893) interpreted the accessory arches and zygapophyseal bridges of the vertebrae as strengthenings of the vertebral column, and considered the lateral spines as adornments ("Zierde"; possibly in the sense of display structures). The fact that both males and females of the species exhibit these armor-like elements appears to contradict the hypothesis of a display structure that would play a signaling role during mating behaviors. However, we cannot exclude a simultaneous visual and mechanical function, and especially the potential for bone-based fluorescence known for other chameleons warrants further examination (Prötzel et al., 2018).
Chameleons mainly rely on camouflage and crypsis to avoid predation, and leaf chameleons are no exception. The Rückensäge and osteoderms could actually facilitate such a strategy. In addition, Raxworthy (1991) documented that at least some Brookesia species not only rely on passive defense behavior when gripped, but switch to active vibrating or even thrusting of the dorsolateral-spines to deter predators. It is conceivable that B. peramata may use both, the pointed osteoderms and dorsolateral-spines, in such a spine thrusting response.
Birds and snakes have been identified as the main predators for Malagasy chameleons, and specifically several Brookesia serve as a substantial dietary component of for instance the short-legged ground roller (Jenkins, Rabearivony, & Rakotomanana, 2009). Against avian and ophidian predators, the exceptional osteoderms of B. perarmata in fact may contribute toward their general defense strategy. All species of Brookesia exhibit a somewhat sculptured skull presenting several prominent crests that may be harmful to potential predators trying to swallow them in one piece. The same applies to the vertebral projections of the Rückensäge that is characteristic for most species of the genus. The entire physiognomy of the Antsingy leaf chameleon with its pronounced "spikyness" over larger parts of the body, as a result of the numerous large osteoderms, may enhance such effects.

ACKNOWLEDGMENTS
P. Martin Sander and Olaf Dülfer are thanked for providing access to the bone histology laboratory, as well as for helpful discussions.
We are grateful to the Malagasy authorities for research, collection, and export permits, and to the German authorities for import permits.