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bioRxiv preprint doi: https://doi.org/10.1101/2020.07.27.222737; this version posted July 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Mineralisation of the Callorhinchus vertebral column

2 (Holocephali; Chondrichthyes)

3 4 Running title: Mineralisation of the Callorhinchus vertebral column 5 6 Jacob Pears1, Zerina Johanson2*, Kate Trinajstic1, Mason Dean3, Catherine 7 Boisvert1 8 1School of Molecular and Life Sciences, Curtin University, Perth, Australia. Email: 9 [email protected]; [email protected]; 10 [email protected]

11 2Department of Earth Sciences, Natural History Museum, London, UK. Email: 12 [email protected] 13 14 3Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Am 15 Muehlenberg 1, 14476 Potsdam, Germany. Email: [email protected]

16 17 *Correspondence: 18 Zerina Johanson: [email protected] 19 20 Keywords: Holocephali, Callorhinchus, tesserae, mineralisation, evolution, stem 21 group Holocephali 22 23 Abstract 24 Chondrichthyes (Elasmobranchii and Holocephali) are distinguished by their largely 25 cartilaginous endoskeleton that comprises an uncalcified core overlain by a mineralised layer; 26 in the Elasmobranchii (sharks, skates, rays) this mineralisation takes the form of calcified 27 polygonal tiles known as tesserae. In recent years, these skeletal tissues have been described 28 in ever increasing detail in sharks and rays but those of Holocephali (chimaeroids) have been 29 less well-described, with conflicting accounts as to whether or not tesserae are present. 30 During embryonic ontogeny in holocephalans, cervical vertebrae fuse to form a structure 31 called the synarcual. The synarcual mineralises early and progressively, anteroposteriorly and 32 dorsoventrally, and therefore presents a good skeletal structure in which to observe

33 mineralised tissues in this group. Here we describe the development and mineralisation of the 34 synarcual in an adult and stage 36 elephant shark embryo (Callorhinchus milii). Small, 35 discrete, but irregular blocks of cortical mineralisation are present in stage 36, similar to what 36 has been described recently in embryos of other chimaeroid taxa such as Hydrolagus, while 37 in Callorhinchus adults, the blocks of mineralisation have become more irregular, but remain 38 small. This differs from fossil members of the holocephalan crown group (Edaphodon), as 39 well as from stem group holocephalans (e.g., Symmorida, Helodus, Iniopterygiformes), 40 where tessellated cartilage is present, with tesserae being notably larger than in Callorhinchus 41 and showing similarities to elasmobranch tesserae, for example with respect to polygonal 42 shape.

44 Introduction

45 During ontogeny most vertebrate skeletons are initially composed predominantly of 46 hyaline cartilage and largely replaced by bone via endochondral ossification (Hall, 47 1975, 2005). In contrast, chondrichthyans, including elasmobranchs (sharks, skates, rays 48 and relatives) and holocephalans (chimaeroids) do not develop osseous skeletons, 49 having secondarily lost the ability to produce endoskeletal bone (Coates et al., 1998; 50 Dean and Summers, 2006; Ryll et al., 2014; Debiais-Thibaud, 2019). Instead, the 51 chondrichthyan endoskeleton remains primarily composed of hyaline-like cartilage, 52 with elasmobranchs developing a comparatively thin outer layer of cortical 53 mineralisation during ontogeny (Hall, 2005; Egerbacher et al., 2006; Dean et al., 2009, 54 2015; Seidel et al., 2016, 2019; Debiais-Thibaud, 2019). This mineralised tissue begins 55 as small separated islets near the cartilage surface, which gradually grow via mineral 56 accretion to fill the intervening spaces, eventually forming a thin cortex of abutting 57 polygonal tiles called tesserae (Dean and Summers, 2006; Dean et al., 2009, 2015; 58 Seidel et al., 2016, 2019; Dean, 2017). These tiles cover the uncalcified cartilage core 59 and are themselves overlain by a distal fibrous perichondrium (Dean and Summers, 60 2006; Dean et al., 2009, 2015). This mosaic of uncalcified cartilage, tesserae and 61 perichondrium is called tessellated cartilage and comprises most of the cranial and 62 postcranial skeleton (Kemp and Westrin, 1979; Dean and Summers, 2006; Seidel et al., 63 2016, 2017a).

64 Tessellated cartilage is therefore a major component of the skeleton and is 65 currently believed to be a synapomorphy for the entire chondrichthyan group (e.g., 66 Maisey et al. 2019, but see comments therein regarding morphological and histological 67 disparity in stem-chondrichthyans). Contemporary examination of extant 68 chondrichthyan mineralised skeletons and their tissues, however, have almost 69 exclusively focused on sharks (Kemp and Westrin, 1979; Peignoux-Deville et al., 1982; 70 Clement, 1986, 1992; Bordat, 1987, 1988; Egerbacher et al., 2006; Eames et al., 2007; 71 Enault et al., 2016) and rays (Dean et al., 2009, 2015; Claeson, 2011; Seidel et al., 2016, 72 2017a, b; Criswell et al., 2017a, b). In contrast, mineralised skeletal tissues of extant 73 chimaeroids (Holocephali) have been largely ignored, despite available descriptions of 74 vertebral development and morphology in the late nineteenth to mid-twentieth centuries 75 (Hasse, 1879; Schauinsland, 1903; Dean, 1906); fossil holocephalans have faced similar 76 neglect (e.g., Moy-Thomas, 1936; Patterson, 1965; Maisey, 2013). This has led to 77 contradictory descriptions of chimaeroid tissues (Lund and Grogan, 1997, 2004; Pradel 78 et al., 2009; Dean et al., 2015), prompting calls for more research (Eames et al., 2007; 79 Dean et al., 2015; Enault et al., 2016). Notably, recent examination of chimaeroid 80 mineralised skeletal tissues identified tesseral structures in the vertebral column 81 (synarcual) and Meckel’s cartilage of Chimaera and Hydrolagus (both Family 82 Chimaeridae; Finarelli and Coates, 2014; Debiais-Thibaud, 2019; Seidel et al., 2019a), 83 seemingly refuting the view that extant chimaeroids lack tessellated cartilage. 84 In order to address this controversy, and determine whether tessellated cartilage is 85 a shared character among cartilaginous fishes, we examine mineralisation in the skeletal 86 tissue of representatives of a second family of extant holocephalans, the 87 Callorhinchidae, focusing on the synarcual of the elephant shark (Callorhinchus milii). 88 The synarcual is a fused element in the anterior vertebral column (Claeson, 2011; 89 Johanson et al., 2013, 2015, 2019; VanBuren and Evans, 2017) and is one of the better 90 anatomical structures for mineralised tissue characterization, being formed early in 91 development and also mineralising early (Johanson et al., 2015, 2019). Synarcual 92 mineralisation progresses from anterior to posterior, and dorsal to ventral, allowing the 93 observation of different mineralisation patterns and stages within a single anatomical 94 structure (Johanson et al., 2015). We report the presence of a layer of mineralisation in 95 the Callorhinchus embryo, comprising small, irregularly-shaped units, maintained in 96 adults, and lacking many of the characteristics of tesserae in the elasmobranchs. To 97 provide further phylogenetic context we also examined mineralised tissues in fossil

98 members of the Callorhinchidae (Edaphodon; Nelson et al. 2006), as well as stem-group 99 holocephalan taxa (e.g., Cladoselache, Cobelodus, Helodus, Iniopterygiformes; Coates 100 et al., 2017, 2018; Frey et al., 2019). The tesserae in these stem-group holocephalans are 101 larger than in Callorhinchus, and more similar in shape to polygonal elasmobranch 102 tesserae. Thus, the evolution of skeletal mineralisation in Chondrichthyes may have 103 involved a progressive reduction of mineralisation in the Holocephali, relative to the 104 elasmobranchs. 105 106 Materials and Methods 107 2.1 Histological sections of Callorhinchus milii synarcual 108 To gain insight into the development of mineralised tissues, slides of the synarcual from 109 a sectioned embryo of an elephant shark (Callorhinchus millii; section thickness 110 ~30µm; Life Sciences Department, Natural History Museum, London) were examined 111 by light microscopy using an Olympus BX51 compound microscope and Olympus 112 DP70 camera and management software. These slides were prepared sometime during 113 the 1980s and the animal is estimated to represent stage 36 (near hatching, based on the 114 calculated size of the individual (110–135 mm; Didier et al., 1998). This developmental 115 stage is ideal to study mineralisation as it is small enough to section but mature enough 116 to show mineralisation, including more mineralisation anterodorsally, progressing 117 posteroventrally. This in effect provides ontogenetic information on how mineralisation 118 develops, in one individual. 119 120 2.2 Adult Callorhinchus milii 121 Two adult female of C. milii were captured by rod and reel from Western Port Bay, 122 Victoria, Australia (Permits: RP1000, RP 1003 and RP1112) with the authorisation and 123 direction of the Monash University Animal Ethics Committee (Permit: MAS-ARMI- 124 2010-01) and kept according to established husbandry methods (Boisvert et al., 2015). 125 These specimens died in captivity and were frozen. 126 127 2.3 Scanning Electron Microscopy 128 The synarcual of one of these adult C. milii specimens was dissected out and either 129 small layers of mineralised tissue or cross sections of the vertebrae were collected. 130 Samples were macerated in a trypsin solution (0.25g Trypsin Sigma T-7409 Type II-S 131 from porcine pancreas in 100mL 10%PBS) and warmed in a 38oC water bath. Samples

132 were extracted from the solution every hour to remove macerated flesh and fascia using 133 scalpels, needles and forceps. This was repeated until sufficient flesh had been removed 134 to observe the mineralised surface. To prevent distortion, samples were placed between 135 Teflon blocks before being air-dried until firm. Cross sections were embedded in a 136 Struers CitoVac using Struers EpoFix Resin and EpoFix Hardener mixed in a 50:6 137 weight ratio and polished using a Struers Tegramin-30. All samples were given a 3nm 138 conductive coating of pure platinum using a Cressington 208HR sputter coater. Samples 139 were imaged using a TESCAN MIRA3 XMU variable pressure field emission scanning 140 electron microscope (VP-FESEM) using backscatter mode (voltage: 15 kv; working 141 distance: 6–15 mm; Tescan Mira3 VP-FESEM instrumentation, John de Laeter Centre, 142 Curtin University). The remaining adult synarcual (Johanson et al. 2015: fig. 7) was 143 dissected out, air dried and imaged using a FEI Quanta 650 FEG SEM in secondary 144 electron mode (voltage: 10 kv; working distance: 14.7 mm). 145 146 2.4 Macrophotography 147 Five fossil holocephalans from the Earth Sciences Department, NHM (NHMUK PV P) 148 were chosen to represent extinct taxa, phylogenetically important with respect to the 149 Callorhinchidae and crown-group holocephalans (Coates et al., 2017, 2018; Frey et al. 150 2019). These comprised: Cladoselache (NHMUK PV P.9285), Cobelodus (NHMUK 151 PV P.62281a), Sibirhynchus (NHMUK PV P.62316b), Edaphodon (NHMUK PV 152 P.10343), and Helodus (NHMUK PV P.8212). One specimen preserving mineralised 153 cartilage was chosen from each taxon, and photographed using a Canon EOS 600D 154 camera, EOS Utility. Five to ten images of each specimen were taken at different focal 155 depths and the resultant image stack imported into Helicon Focus (v. 6.8.0) to create 156 images with high depth of focus. These specimens were also photographed using a Zeiss 157 Axio Zoom microscope with camera to provide closeup images; as well, tesseral width 158 was determined using the measurement function in the Zen Pro 2 software 159 accompanying the Axio Zoom microscope (Table 1). 160 The second adult Callorhinichus milii synarcual (Johanson et al. 2015) was air- 161 dried and photographed using Zeiss Axio Zoom microscope to illustrate tesseral shape. 162 163 3 RESULTS 164 3.1 Histology 165 3.1.1 General Morphology

166 The axial skeleton of chondrichthyans typically includes a series of dorsal and ventral 167 cartilages, and in the elasmobranchs, centra associated with the notochord (e.g., Dean, 168 1895, Gadow and Abbott, 1895, Goodrich, 1930; Compagno, 1977; Criswell et al., 169 2017b). Mineralisation of the axial skeleton takes a variety of forms, recently 170 summarized by Debiais-Thibaud (2019), with the dorsal and ventral cartilages of most 171 species, as well as the outer centrum, composed of tessellated cartilage (Dean and 172 Summers, 2006; Dean et al., 2009; Criswell et al., 2017b; Johanson et al., 2019). Most 173 of the spool-shaped vertebral centrum comprises areolar mineralisation, with substantial 174 variation in patterns of mineralisation between elasmobranch species (Ridewood, 1921; 175 Dean and Summers, 2006; Porter et al., 2007). Holocephalans also possess dorsal and 176 ventral cartilages (e.g., Dean, 1895; Johanson et al., 2012, 2015), but centra do not 177 develop. Instead, the notochord is surrounded by a fibrous chordal sheath, which 178 contains many calcified rings, except in the Callorhinchidae (Patterson, 1965; Didier, 179 1995). Holocephalans possess a synarcual, absent in elasmobranchs, which is the focus 180 of the following description. 181 In the Callorhinchus embryo examined (stage 36), several tissue layers 182 concentrically surround the notochord. Most proximal is a thin basophilic membrane, 183 the elastic interna, adherent to the outside of the notochord (Figure 1A–D, nc, el.int). 184 Distal to this membrane is a thick (~665µm) fibrous sheath (Figure 1A, B, fb.sh), which 185 is largely composed of spindle shaped cells (Figure 1C, D). Abutting the sheath dorsally 186 and ventrally are separate bilateral pairs of cartilages, the basidorsals and basiventrals, 187 respectively (Figure 1B, D, bv, bd). Immediately dorsal to the sheath is the spinal 188 cavity, containing the spinal cord, which is surrounded ventrolaterally by the basidorsal 189 cartilages and dorsally by the neural arch cartilage (Figure 1B, sp.c, sp.cd, bd, na). 190 Spinal nerves are also visible in section, with the dorsal root exiting the neural tube 191 towards the dorsal root ganglion situated lateral to the vertebral column (Figure 1A, B, 192 d.rt, d.rt.g). The hyaline cartilages associated with the vertebral column—the neural 193 arch, basidorsals and basiventrals— fuse anteriorly to form the synarcual, which 194 surrounds the majority of the fibrous sheath and spinal cavity, while maintaining 195 foramina for the dorsal root (Figure 1A). 196 In these histological slides, areas of mineralisation are limited to the distal 197 peripheries of the vertebral column-associated cartilages (Figures 1A, B, 2, min). These 198 mineralised tissues are bordered externally by a fibrous perichondrium and a thin, cell-

199 rich layer of cartilage (Figures 2, 3, FP, SC), similar to the supratesseral cartilage in the 200 stingray Urobatis halleri (Seidel et al., 2017). 201 202 3.1.2 Cellular Aspects 203 Cells within the neural arch, basidorsal and basiventral cartilages can be categorised 204 with respect to morphology, and distribution within the cartilage. Chondrocytes located 205 deeper in the cartilage interior (Figures 2A, 3B, IN, ch) are approximately similar in 206 terms of cell morphology and density: ≥200μm from the periphery, cells are sparsely 207 distributed within the intracellular matrix, with most being ovoid in shape and located in 208 open circular spaces identified as lacunae (diameter: ~15 µm). Chondrocytes often 209 occur in pairs, which may indicate recent mitotic activity (Figures 2A, B, 3A, ch; Kheir 210 and Shaw, 2009). This deeper (≥200μm from the periphery), interior cartilage also 211 contains relatively greater quantities of empty lacunae compared to the more peripheral 212 cartilage (Figures 2A, B, 3A, el), which may indicate chondroptosis (chondrocyte 213 apoptosis; Roach et al., 2004), although this is normally associated with chondrocyte 214 hypertrophy, which has not been observed in chondrichthyans (Dean et al., 2015; Seidel 215 et al., 2017b; but see Debiais-Thibaud [2019] for a summary of contrary opinions). 216 Closer to the periphery, within ≥100 μm of the outer edge, and immediately proximal to 217 mineralised tissue, chondrocytes are clustered within a distinct layer (Figures 2, 3B, 218 CPL) and appear uniformly ovoid. This area displays a greater variation in cell size as it 219 contains many smaller chondrocytes (diameter: 5–10 µm), and fewer empty lacunae 220 compared to the interior. In addition, this area contains notably more isogenous groups 221 relative to the interior, which may indicate higher rates of chondrocyte proliferation 222 (Figures 2A, B, 3B, E, iso; Kheir and Shaw, 2009). In some regions, mineralisation is 223 absent at the periphery (i.e., there is no tesseral layer); in these areas, cell distribution 224 and morphology are more similar to the interior (Figure 3A). 225 There appears to be a gradient of cell morphology from the perichondrium, 226 through the supratesseral cartilage, potentially relating to a cellular transition from 227 fibroblasts cells in the perichondrium, differentiating to chondroblasts in the 228 supratesseral cartilage, and then to chondrocytes in the main body of the cartilage as 229 described in Genten et al. (2009; Figures 1C, D, 2A, B, CP, FP, fb, cb, ch). These 230 cartilages seem to display interstitial and appositional growth, with the former involving 231 the mitotic division of single chondrocytes into a cluster of cells (the isogenous groups), 232 and matrix deposition between these to increase the size of the element (e.g., Figures

233 1D, 2A, B, iso), while the latter occurs at the cartilage margin through chondrocyte 234 differentiation and matrix deposition (Figure 1B–D; Hall, 2005; Kheir and Shaw, 2009). 235 For example, in several sections, the basidorsal and basiventral cartilages are still 236 separate (e.g., Figure 1B), but appear to show a region of appositional and interstitial 237 growth or matrix deposition at their margins (Figure 1B–D, black arrows showing 238 direction of growth). 239 240 3.1.3 Mineralisation 241 In the neural arches, the distribution of mineralised tissue is more complete, 242 extending along almost the entire periphery excluding only the ventro-mesial concave 243 part of the arch (Figures 2C, 4B). Within the basiventrals, mineralised tissue is also 244 found near the periphery, but by comparison to the neural arches, is only patchily 245 distributed (Figures 3, 4), with individual units more variable and irregular in shape 246 (Figures 3B, E, 4, min). In the neural arches, these units are more rectangular and flatter 247 (Figures 2A, B, 5A, B). Nevertheless, mineralised tissues in all vertebral elements lack a 248 regular geometry and any differentiation into inner and outer regions. Additionally, 249 beyond being limited to the cartilage periphery beneath the fibrous perichondrium, these 250 tissues lack any organisation, reflecting the lack of a regular geometric shape to the 251 individual units. 252 Tissue mineralisation appears to be preceded by the clustering of chondrocytes 253 within cartilage below the perichondrium (Figures 3A, D, 5B, cc, FP), because amongst 254 these clustered chondrocytes, the inception of mineralisation can be observed via the 255 formation of small islands of calcification (≤25 µm). Initially, these partially encircle 256 these cells (Figures 3B, E, 5B, gc, ic), but come to surround the chondrocytes, forming a 257 thin (≤50 µm) layer of irregular units of mineralised tissue (Figures 4C, D, 5A, B, dm, 258 min, ic). The anteroposterior order of this sequence suggests that the initial islands 259 expand, through the calcification of the surrounding tissue. Alternatively, these islands 260 may grow by fusing together to form larger units, as indicated by the presence of small 261 mineralisation foci between units (Figure 6). More anteriorly, the irregular units (~50– 262 150 µm wide, in cross section, variable in shape), are acellular, suggesting the 263 mineralisation eventually completely engulfs the chondrocytes, resulting in their death. 264 We identify these units as tesserae, with comparison of these mineralised units to the 265 tesserae of sharks and rays discussed further below.

266 The presence of less mineralised tissue in posterior sections (Figures 3, 5), 267 compared to anterior (Figures 2, 4), indicates that mineralisation progresses 268 anteroposteriorly along the vertebral column. Thus, tesserae are smaller in more 269 posterior sections (50–100 µm) and more regularly separated by regions of 270 unmineralised cartilage (Figures 4F, 5A, B, min, uc), reflecting their earlier 271 developmental stage. 272 273 3.2 Scanning Electron Mircoscopy (SEM) 274 In planar views of the external surface of the synarcual of adult Callorhinchus milii, the 275 mineralised layer appears to comprise a tessellated surface of irregular tiles that are 276 separated by (~5um) thin strips of uncalcified cartilage (Figure 7A, B min, uc). These 277 tesserae do not have a uniform shape or size, ranging from 50–150µm in width (Figures 278 7B; Supplementary Info Figure 1). From this perspective, these tesserae do not appear 279 to possess features that are currently considered common among elasmobranch tesserae 280 (e.g., mineralised ‘spokes’ at the contact points between tesserae, intertesseral joints, 281 vital chondrocytes; reviewed in Seidel et al., 2016, 2019b). 282 In transverse view, the mineralised tissue forms a single layer of tesserae, tightly 283 arranged units of irregular blocks separated by very thin (<5 µm) strips of uncalcified 284 cartilage (Figure 7C, min, SC, IN, uc). In this perspective, the tesserae are 30–50 µm 285 thick and 30–150 µm wide (Figure 7C). Some cracking of the mineralisation during 286 sample preparation is visible, but individual tesserae can be identified by comparing and 287 matching Liesegang lines between adjacent fragments (Figure 7C, ll, f). Liesegang lines 288 are concentric, wave-like patterns of varying mineral density visible in the mineralised 289 tissue, and are particularly prominent near the lateral margins of the mineralised units 290 (Figure 7C, D, ll). 291 Spheroidal mineralised regions, surrounded by Liesegang lines and approximately 292 the size and shape of chondrocytes also permeate the tesserae (Figure 7D, ch). These are 293 likely calcified (micropetrotic) cells, are variously sized (~1–5 µm), and appear to be 294 organised in clusters (isogenous groups), suggesting some have been calcified during 295 mitosis (Figure 7D, ch, iso). 296 297 3.3 Mineralisation in stem Holocephali and fossil Callorhinchidae 298 Following phylogenetic review Coates et al. (2017, 2018; Dearden et al., 2019; 299 Frey et al., 2019), several taxa that were previously resolved as stem group

300 chondrichthyans (basal to the clade Elasmobranchii + Holocephali; Pradel et al., 2011), 301 are now resolved as stem holocephalans, joining more crownward stem holocephalans 302 including the Iniopterygiformes (Zangerl and Case, 1973), Helodus (Moy-Thomas, 303 1936), Kawichthys, Debeerius and Chondrenchelys, the latter being the sister taxon to 304 the crown group Holocephali (chimaeroids) (Figure 8). Tessellated calcified cartilage 305 has been variously identified among these stem-group Holocephali: this includes taxa 306 assigned to the Symmorida, such as Dwykaselachus (Coates et al., 2017: extended data 307 figure 1d), Ozarcus (Pradel et al., 2014), Cladoselache (“minute granular 308 calcifications”; Dean, 1894; Figure 9A), Akmonistion (“prismatic calcified cartilage” 309 Coates and Sequiera, 2001), Damocles and Falcatus (Lund and Grogan, 1997), also 310 present in Cobelodus (Figure 9B). In all of these taxa, the tesselated layer is comprised 311 of recognizable polygonal units, although in Cladoselache, the edges of the units appear 312 less regular. This may represent the presence of mineralised ‘spokes’ extending between 313 the tesserae: spokes are hypermineralised tissue regions associated with points of 314 contact between elasmobranch tesserae, often represented externally by lobulated 315 extensions along tesseral margins (Seidel et al., 2016; Jayasankar et al. 2020). Such 316 structural extensions, suggestive of mineralised spokes, are even more clearly present in 317 Cobelodus (Figure 9C). 318 In the more crownward stem holocephalans, comparable polygonal tesserae are 319 also present, including in Kawichthys (“tesserate prismatic calcified cartilage”, Pradel et 320 al., 2011) and the Iniopterygiformes (“calcified cartilage prisms”, Zangerl and Case, 321 1973), represented by Sibirhynchus in Figure 9D. Particularly small tesserae (Table 1) 322 are present in Helodus (“minute tesserae”, Moy-Thomas, 1936; Figure 9G), and 323 Chondrenchelys (“tessellated calcified cartilage”, Finarelli and Coates, 2014: fig. 7B). 324 There appears to be more variation in the shape of these polygons, and signs of 325 mineralised spokes are less apparent in these taxa, but this may be due to postmortem 326 distortion. With respect to the fossil taxa assigned to the Callorhinchidae (crown group 327 Holocephali), mineralised tissue units in Edaphodon appear to maintain a polygonal 328 shape, compared to the stem holocephalans just described (Figure 9E, F). The width of 329 tesserae in these fossil taxa was measured (Table 1) for comparison to the size of 330 mineralised units in adult Callorhinchus; (50–150µm, as noted above) the tesserae of all 331 fossil taxa were notably larger than in Callorhinchus, discussed further below. 332 333 4 Discussion

334 4.1 Mineralised Tissue Development 335 Currently, the ultrastructure and ontogeny of mineralised endoskeletal tissues of 336 chimaeroids is poorly described, with previous work only providing a broad overview of 337 developmental trajectories, showing that mineralisation in the vertebral skeleton (in the 338 synarcual) progresses from anterior to posterior and dorsal to ventral, as indicated by a micro- 339 CT scan of a Callorhinchus milii adult (Johanson et al., 2015; Figure 10A). The series 340 described above in the stage 36 embryo goes beyond this to capture fine histological detail 341 related to the progression of this mineralisation. 342 The development of mineralised tissue described here for C. milii shares some 343 similarities with the development of elasmobranch tesserae (Seidel et al., 2016; Debiais- 344 Thibaud, 2019). Tesserae in elasmobranchs such as the batoid ray Urobatis halleri 345 initially develop as patches of globular mineralisation interposing within clusters of 346 flattened, subperichondral chondrocytes (at a distance from the perichondrium). These 347 chondrocytes become entombed by the growth of these mineralised intra-chondrocyte 348 septa, by mineral accretion (Dean et al., 2009; Seidel et al., 2016). This accretion and 349 entombment process is similar to the inception of mineralisation observed in C. milii 350 and elasmobranch mineralised septae bear resemblance in shape and size to the 351 developing mineralisation of C. milii (Figures 4C, D, 5A, B). Through development of 352 U. halleri, these mineralised septae continue to grow and engulf chondrocytes, 353 eventually forming discrete, but abutting tesserae, which contain vital chondrocytes and 354 closely border the perichondrium (Seidel et al., 2016; also in the batoid Raja clavata, 355 Debiais-Thibaud, 2019). In a general sense, elasmobranch tesserae are not dissimilar to 356 some of the developing units of mineralisation in C. milii (Figure 4F, dm, ic), which 357 also border the perichondrium, grow via calcification of surrounding cartilage matrix 358 and, at least early in development, contain chondrocytes which appear to be vital. 359 Additionally, mineralised tissues in C. milii appear to be overlain by a distinct layer of 360 uncalcified cartilage, beneath the perichondrium (Figure 2A, SC). This resembles the 361 thin layer of ‘supratesseral uncalcified cartilage’ intervening between tesserae and 362 perichondrium in elasmobranchs such as U. halleri and Scyliorhinus canicula (Bordat, 363 1988; Egerbacher et al., 2006; Enault et al., 2015; Seidel et al., 2016, 2017a, Debiais- 364 Thibaud, 2019, contra Kemp and Westrin, 1979). 365 Despite these similarities, mineralisation in adult C. milii appears to be a distinct 366 form of tessellated calcified cartilage. From a planar perspective, the mineralised tissue 367 seems to comprise a more irregular mosaic of tesserae than typically seen in

368 elasmobranchs, with near-abutting calcified tiles separated by uncalcified cartilage 369 (Figure 7A, B, min, uc; Supplementary Info Figure 1). From a transverse perspective, 370 these tissues are similar to that of embryo, arranged as a single layer of tightly arranged 371 units separated by very thin strips of uncalcified cartilage and sandwiched between a 372 supratesseral layer and internal uncalcified cartilage (Figure 7C, D, min, uc, SC, IN). 373 From this perspective, the tesserae of C. milii most closely resemble the tesserae of the 374 sevengill shark (Notorynchus cepedianus) in terms of their arrangement, being very 375 tightly organised and separated by minimal uncalcified cartilage, while also lacking 376 vital chondrocytes; however, they are not comparable in size, being ~19–57% of the 377 size (Seidel et al., 2016). This is notable, as the Hexanchiformes, to which N. 378 cepedianus belongs, are considered one of the most primitive of modern selachian 379 groups (Barnett et al., 2012; Tanaka et al., 2013; da Cunha et al., 2017). However, 380 despite this resemblance, it is likely that this tissue organisation does not represent a 381 plesiomorphic trait given the morphology of the skeletal tissues of stem holocephalans 382 such as Cobelodus (see section 4.2). 383 Despite similarities in the early stages of development between elasmobranch and 384 C. milii tesserae (e.g., with early growth surrounding vital chondrocytes; Figures 3B, 385 4E, 5B), the tesserae of C. milii differ significantly from those elasmobranchs, and 386 particularly batoids, in terms of size and ultrastructure. The tesserae observed in the 387 stage 36 embryo and adult C. milii (Figures 2, 4, Supplementary Info Figure 1, min) are 388 much smaller compared to most elasmobranch tesserae that have been examined (Seidel 389 et al., 2016; Figure 10, t), generally being less than 50 µm thick and ranging from 50– 390 150 µm in width (Figures 2A, 7C), comparable in size to the ~100 µm tesserae of the 391 catshark Scyliorhinus (Egerbacher et al., 2006; Seidel et al, 2016; Debiais-Thibaud, 392 2019). Additionally, C. milii tesserae display no internal regionalisation into the cap and 393 body zones (regions in elasmobranch tesserae delineated by cell shape and collagen 394 type, Figure 10, bz, cz; Kemp & Westrin, 1979; Seidel et al, 2016; Chaumel et al, 395 2020), with no differences observed between the surfaces closer to the fibrous 396 perichondrium, and surfaces surrounded by hyaline cartilage (e.g., Figures 6A, 7C). 397 Chimaeroid tesserae also apparently lack the intertesseral joints and mineralised spokes 398 characteristic of elasmobranch tesserae (Figure 10, itj, sp; Seidel et al, 2016), as well as 399 the Sharpey’s fibres that extend from the perichondrium into the tesserae cap zone in 400 elasmobranchs ( e.g. Kemp and Westrin, 1979; Peignoux-Deville et al., 1982; Clement, 401 1992; Summers, 2000; Seidel et al., 2017). With respect to growth, the presence of

402 Liesegang lines parallel to tesseral edges (Figure 7C, D, ll) suggests calcification in C. 403 milii accretes at the margins of tesserae (Figures 2–5, min, dm, gc) in the same manner 404 as elasmobranch tesserae. Additionally and/or alternatively, C. milii tesserae may grow 405 through the development and fusion of new, smaller mineralisation foci between 406 existing tesserae (e.g., Figure 6A). Indeed, the irregular and less concentric arrangement 407 of Liesegang lines in C. milii tesserae relative to those in elasmobranchs may be 408 indicative of a more multimodal and/or haphazard form of growth, perhaps explaining 409 the varied shape of the observed tesserae (Figure 7). 410 As noted, chondrocytes appear to be engulfed during mineralisation in C. milii 411 (Figures 3B, E, 4E) and may be vital in early stages (Figures 4C, D, F, 5, dm, ic). 412 However, more developed tesserae in embryos and adults appear to be acellular 413 (Figures 2, 4A, 6, min) as any previously entombed chondrocytes appear to have 414 calcified (Figure 7D, cc), a major difference when compared to most elasmobranch 415 tesserae (Seidel et al., 2017; Debiais-Thibaud, 2019; Figure 10). The absence of vital 416 chondrocytes in the tesserae of C. milii may have important implications for their 417 maintenance. In batoids, chondrocytes entombed in tesserae (Figure 10) remain vital in 418 uncalcified lacunar spaces and form passages not unlike the canalaculi found in bone 419 (Dean et al., 2010; Seidel et al., 2016; Chaumel et al., 2020). These chondrocytes and 420 the networks they form are thought to have important functions with regard to 421 maintaining the endoskeleton by communicating information about the mechanical 422 environment in a manner similar to osteocytes in bone (Dean et al., 2010; Seidel et al., 423 2016; Chaumel et al., 2020). Thus, vital chondrocytes are absent in the tissues of the 424 adult and the anterior older (anterior) regions of the synarcual, suggesting these are lost 425 during ontogeny, along with their associated putative mechanosensory networks, and 426 that these functions are either absent or achieved through alternative means. 427 428 4.2 Chimaeroid Endoskeleton: Form Across Phylogeny 429 In contrast with elasmobranchs, the mineralised components of the chimaeroid 430 endoskeleton have been subjected to little study. The limited literature available 431 proposes conflicting forms of endoskeletal mineralisation: tesselated calcified cartilage 432 akin to that of elasmobranchs (Hasse, 1879; Seidel et al., 2019b), smooth superficial 433 sheets of continuous calcified cartilage formed from the fusion of tesserae during 434 ontogeny (Lund and Grogan, 1997; Grogan and Lund, 2004; Pradel et al., 2009; Grogan 435 et al., 2015), or a granular texture (Hydrolagus, Finarelli and Coates, 2014). Recent

436 histological data from the synarcual of a sub-adult (20 cm) Hydrolagus illustrates two 437 forms of mineralised tissue (Debiais-Thibaud, 2019: 116). This includes small (≤50 µm) 438 subperichondral tissues “reminiscent of globular mineralisation” at the periphery of the 439 vertebral body and neural arch that appear to follow a tessellated pattern, and a more 440 irregular form of globular mineralisation deep within the vertebral body surrounding the 441 fibrous chordal sheath. 442 Based on these few recent reported data on chimaeroid mineralisation, tesserae in C. 443 milii seem to share similarities with those of Hydrolagus. In both taxa, mineralisation is 444 tessellated and limited to the periphery of structures composed of hyaline cartilage, including 445 the neural arches, basidorsals and basiventrals (vertebral body), though C. milii lacks the 446 second deeper layer of globular mineralisation (Debiais-Thibaud, 2019: fig. 6.1). The 447 mineralised tissues of Hydrolagus also take the form of small, irregular acellular units, 448 lacking clear separation into upper cap and lower body zones (Seidel et al., 2016; Debiais- 449 Thibaud, 2019). Likewise, in Chimaera, mineralisation more clearly takes the form of 450 tesserae, although differences with respect to the more developed batoid tesserae have been 451 described (Seidel et al., 2016). 452 These few recent descriptions of mineralisation in modern chimaeroids, including 453 that provided here for C. millii, indicate that these taxa do not possess sheets of 454 continuous calcified cartilage, nor a granular texture (Lund and Grogan, 1997; Grogan 455 and Lund, 2004; Pradel et al., 2009; Finarelli and Coates, 2014). Instead they appear to 456 support more historical claims (Hasse, 1879) that these organisms possess tesselated 457 skeletal tissues, though contrary to these sources, these are distinctly different from 458 most elasmobranch tesserae. These discrepant accounts may arise from the tissue 459 arrangements; in taxa such as C. milii the tesserae are very tightly arranged, being 460 separated by very thin portions of uncalcified cartilage, which may give the impression 461 that the surface comprises of a sheet. The tesserae themselves are covered in a type of 462 fascia (see Materials and Methods, above), which could account for the observations of 463 a granular texture. 464 By comparison, in a series of stem holocephalans, cartilage mineralisation in what 465 are presumed to be adults occurs as small polygonal units that are very similar among 466 disparate taxa (Figure 9). The polygonal shape is more comparable to tesserae in the 467 Elasmobranchii, including the suggested presence of mineralised spokes at tesseral 468 joints in taxa such as Cobelodus. In contrast, polygonal tesserae are more irregular in 469 shape, and spokes appear absent, in more crownward taxa, including Edaphodon

470 (Callorhinchidae), a member of the crown group Holocephali. In these features, the 471 tissues of crownward taxa bear the closest resemblance to those of C. milii 472 (Callorhinchidae), however, tesserae in C. milii are more irregular in shape and much 473 smaller than the irregular polygonal tesserae of Edaphodon (Table 1). The presence of 474 shape and structural features in these fossil taxa that echo those in modern 475 elasmobranch tesserae suggests that substantial changes have occurred in mineralisation 476 in living chimaeroids, with a loss of many characteristics of tesserae in other 477 chondrichthyans. 478 479 5.0 Conclusion 480 Whilst tessellated cartilage has been suggested to be a shared characteristic of the 481 chondrichthyan endoskeletons, the data presented here indicate that this type of 482 mineralisation has been significantly modified within the holocephalans. The 483 mineralised components of the endoskeleton of Callorhinchus milii (Family 484 Callorhinchidae) consist of small units that form a layer of tightly arranged, irregularly 485 shaped tesserae, also present in Hydrolagus (Family Chimaeridae). These tesserae in 486 Callorhinchus and Hydrolagus differ in many respects from most shark and ray 487 tesserae, being smaller and simpler, lacking features such as distinct cap and body 488 zones, mineralised spokes between the tesserae and retention of lacunae housing vital 489 chondrocytes. Nevertheless some similarities in development are present, such as the 490 intra-chondrocyte septa that surround the chondrocytes early in the development of the 491 tesserae, described above in Callorhinchus and the ray Urobatis (Dean et al. 2009; 492 Seidel et al. 2016). Tesserae in sharks such as Scyliorhinus and Notorynchus may also 493 lack some features seen in the other elasmobranchs (Debiais-Thibaud, 2019: fig. 6.3; 494 Seidel et al. 2016: fig. 11A). Tesserae in stem group holocephalans, as well as in fossil 495 relatives of Callorhinchus such as Edaphodon, within the Family Callorhinchidae (Fig. 496 9F), also appear to possess the polygonal shape more characteristic of ray tesserae with 497 these being larger and better developed than the mineralisation in the adult of 498 Callorhinchus. Thus it appears that these smaller units may be the characteristic 499 mineralised structure in extant holocephalans, representing a reduction of mineralisation 500 occurring separately within the Callorhinchidae and Chimaeridae, and within the 501 Elasmobranchii. 502 503 6 Conflict of Interest

504 The authors declare that the research was conducted in the absence of any commercial 505 or financial relationships that could be constructed as a potential conflict of interest. 506 507 7 Author Contributions 508 ZJ, CB, JP conceived this project, JP, CB, ZJ contributed data to the project from fossil 509 and extant holocephalans; all authors contributed to interpretation of the data and 510 writing of the manuscript. 511 512 8 Funding 513 Curtin University Faculty of Science and Engineering Research and Development 514 Committee Small Grant: JP & CB; Curtin Research fellowship: CB, Australian 515 Government Research Training Program Scholarship (AGRTP): JP 516 517 9 Acknowledgements: We would like to thank Ollie Crimmen and James MacLaine 518 (NHM Life Sciences Department) for providing access to the slides of the 519 Callorhinchus embryo and Innes Claxworthy (NHM Core Research Labs) for SEM 520 imaging of the Callorhinchus adult. We would also like to acknowledge Elaine Miller 521 and the John de Laeter Centre at Curtin University for expert advice, assistance, and 522 service in the imaging of Callorhinchus embryo tissues, and the Curtin Faculty of 523 Science and Engineering, School of Molecular and Life Sciences and Centre of Health 524 and Innovation Research for support. The John de Laeter Centre is funded by the 525 Australian Research Council (ARC LE130100053). JP is supported by the Australian 526 Government through the AGRTP Scholarship. We would also like to thank Alan Pradel 527 and John Maisey for discussion of tesserae in fossil chondrichthyans. 528 529 References 530 Barnett, A., Braccini, J. M., Awruch, C. A., Ebert, D. A. (2012). An overview on the role of 531 Hexanchiformes in marine ecosystems: biology, ecology and conservation status of a 532 primitive order of modern sharks. J. Fish Biol. 80(5), 966–990. 533 Boisvert, C. A., Martins, C. L., Edmunds, A. G., Cocks, J., Currie, P. 2015. Capture, 534 transport, and husbandry of elephant sharks (Callorhinchus milii) adults, eggs, and 535 hatchlings for research and display. Zoo. Biol. 34, 94–98. doi:10.1002/zoo.21183

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727 728 729 FIGURE 1. Histological sections through the synarcual (anterior fused vertebrae) of a 730 stage 36 embryo of Callorhinchus milii (Holocephali; Callorhinchidae). A, B, section 731 showing neural arch surrounding the spinal cord and basidorsal and basiventral arches 732 surrounding the notochord. C, closeup of region indicated in A; D, closeup of region 733 indicated in B. Black s in C, D indicate direction of appositional growth of the 734 basiventral cartilage. Abbreviations: bd, basidorals, bv, basiventral; cb, chondroblasts; 735 ch, chondrocyte; CP, perichondrium including chondroblast cells; d.rt, dorsal root of the 736 spinal nerve; d.rt.g, dorsal root ganglion of the spinal nerve; el. ex, elastica externa; 737 el.int, elastic interna; fb, fibroblasts; fb.sh, fibrous sheath surrounding the notochord; fc, 738 fused cartilage; FP, perichondrium including fibroblast cells; iso, isogenous group of 739 chondrocytes; min, mineralisation; msc, musculature; msch, mesenchymal cells; na, 740 neural arch, nc, notochord; sp.c, spinal cavity; sp.cd, spinal cord. Black silhouette of C. 741 millii indicates approximate region shown in the figure. 742 743

744 745 FIGURE 2. Histological sections through the anterior synarcual (anterior fused 746 vertebrae) of a stage 36 embryo of Callorhinchus milii (Holocephali; Callorhinchidae). 747 A, B, closeups showing perichondrium, cartilage and mineralisation in the neural arch; 748 C, overview of section with locations of closeup views indicated by white squares. 749 Abbreviations: As in Figure 1, also cc, clustered chondrocytes CPL, chondrocyte 750 proliferative layer; el, empty chondrocyte lacunae; gc, calcification globule; ic, 751 chondrocyte that is being engulfed or has been incorporated; uc, uncalcified cartilage; 752 IN, internal cartilage; SC, supratesseral/mineral cartilage. Black silhouette of C. millii 753 indicates approximate region shown in the figure. 754

755 756 757 FIGURE 3. Histological sections through the posterior synarcual (anterior fused 758 vertebrae) of a stage 36 embryo of Callorhinchus milii (Holocephali; Callorhinchidae). 759 A, B, closeups showing initial mineralisation in a basiventral and clustered 760 chondrocytes in the same location in the preceding section; C, overview with location of 761 closeup view of initial mineralisation indicated by a black square; D, E, close ups of A, 762 B, locations indicated by white squares. Abbreviations: As in previous Figures, also dm, 763 developing mineralisation. Black silhouette of C. millii indicates approximate region 764 shown in the figure. 765 766

767 768 FIGURE 4. Histological section through the anterior synarcual (anterior fused 769 vertebrae) of a stage 36 embryo of Callorhinchus milii (Holocephali; Callorhinchidae). 770 A, C, D, E, F, closeups showing perichondrium, cartilage and mineralisation in 771 basiventrals; B, overview of section through the neural arch with locations of closeup 772 views indicated by black squares. Abbreviations: As in previous Figures. Black 773 silhouette of C. millii indicates approximate region shown in the figure. 774 775

776 777 FIGURE 5. Histological section through the posterior synarcual (anterior fused 778 vertebrae) of a stage 36 embryo of Callorhinchus milii (Holocephali; Callorhinchidae). 779 A, B, closeups showing perichondrium, cartilage and mineralisation in the neural arch; 780 C, overview of section with locations of closeup views indicated by white squares. 781 Abbreviations: As in previous Figures. Black silhouette of C. millii indicates 782 approximate region shown in the figure. 783

784 785 786 FIGURE 6. Histological section through the synarcual (anterior fused vertebrae) of a 787 stage 36 embryo of Callorhinchus milii (Holocephali; Callorhinchidae). A, B, closeups 788 showing the potential formation of new mineralisation foci between already existing 789 units; C, overview of section through the neural arch with locations of closeup views 790 indicated by white squares. Abbreviations: as in previous Figures. Black silhouette of C. 791 millii indicates approximate region shown in the figure. 792 793

794 795 FIGURE 7. SEM images of mineralisation from the synarcual (anterior fused vertebrae) 796 of an adult Callorhinchus milii (Holocephali; Callorhinchidae). A, overview of 797 tesselated mineralisation from a planar perspective; B, close up of mineralisation from a 798 planar perspective; C, mineralisation from a transverse perspective; D, close up of 799 mineralisation surface in transverse perspective; Note brightness and contrast of C, D 800 have been altered to more clearly visualise morphology. Abbreviations: As in previous 801 Figures, also f, fragments ll, Liesegang lines. 802

803 804 805 FIGURE 8. Chondricthyan phylogeny (after Coates et al. 2018). 1, Crown–group 806 Chondrichthyes (Holocephali + Elasmobranchii); 2, Holocephali; 3, Elasmobranchii; 4, 807 Crown–group Holocephali; 5, Crown–group Elasmobranchii. 808 809

810 811 FIGURE 9. Mineralised cartilage in stem Holocephali and crown group Holocephali 812 (Fig. 8). A, NHMUK PV P.9285, Cladoselache, stem Holocephali, palatoquadrate and 813 Meckel’s cartilage; asterisk indicates area shown in B; B, polygonal mineralisation 814 (tesserae), with irregular margins; C, NHMUK PV P.62281a, Cobelodus, stem 815 Holocephali, more regular polygonal mineralisation (tesserae); D, NHMUK PV 816 P.62316b, Sibirhynchus, stem Holocephali, polygonal mineralisation (tesserae); E, F, 817 NHMUK PV P.10343, Edaphodon, Family Callorhinchidae, crown group Holocephali 818 (Fig. 8, 2), E, dorsal fin endoskeletal support (with dorsal fin spine, anterior to left),

819 asterisk indicates area shown in F; F, closeup showing polygonal mineralisation 820 (tesserae); G, NHMUK PV P.8212, Helodus, polygonal mineralisation. See Table 1 for 821 tessera sizes in these taxa. 822

823 824 FIGURE 10. Histological section of tessellated cartilage of a batoid ray (Raja). 825 Abbreviations: as in previous Figures also bz, body zone; CP, chondrogenic 826 perichondrium cz, cap zone; itj, intertesseral joint; FP, fibrous perichondrium; sp, 827 spoke; t, tesserae; vc, vital chondrocytes 828 829

830 831 832 FIGURE 11. A, B, micro–CT scan of a synarcual from an adult Callorhinchus milii 833 (Holocephali; Callorhinchidae). A,synarcual, lateral view; B, ocronal section (virtual) 834 through synarcual; C, macrophotograph of lateral synarcual surface showing 835 mineralisation with a granular appearance. 836

Table 1: Tesserae width taken from stem group Callorhinchidae (Edaphodon) and several stem group Holocephali (Sibirhynchus, Cobelodus, Helodus, Cladoselache). Measurements in microns.

Helodus Cobelodus Sibirhynchus Cladoselache Edaphodon NHMUK PV P8212 NHMUK PV P.62281a NHMUK PV P.62316b NHMUK PV P.9285 NHMUK PV P.10343

198.2 299.1 256.5 526.2 223.4 239.6 345.4 213.8 416.5 237.2 229.2 309.8 256.5 576.8 237.2 198.2 341.9 320.5 519.8 259.8 239.8 455.8 356.1 421.6 255.2 198.2 373.9 384.8 495.6 227.9 251.9 242.1 270.7 427.4 232.48 188.7 295.5 235 501.4 205.1 208.6 320.5 348.9 655.4 209.7 250 227.9 377.5 290.6 223.5 270.8 370.3 306.5 415.9 264.33 291.7 338.2 228 387.4 132.3 230.1 373.9 349 558.4 259.8 260.4 352.5 306.2 512.7 223.4 323.1 288.4 363.1 495.7 227.9 312.5 327.6 270.7 381.7 136.9 250.9 331.2 285.2 478.7 246.2 229.2 327.6 242.5 484.2 223.4 230.1 395.2 327.6 415.9 223.3 291.9 363.2 434.6 524.1 278 208.6 320.5 341.8 490 214.3 292.4 366.7 370.4 359.1 232.5 241.6 352.5 384.6 330.5 227.9 261.7 359.7 320.8 433.1 255.2 247.7 270.6 306.2 427.3 255.3 312.5 327.6 235.1 535.8 246.1 312.8 320.5 159.8 438.8 250.7 287.6 270.6 164.1 450.1 259.8 184.1 356.1 282.6 581.1 237 183.6 413.1 364.6 415.9 218.8 250 381 314.5 501.5 232.6 258.4 306.2 259.8 353.4 250.7 191.9 277.7 300.8 218.9 183.3 338.3 196.2 240.7 192.4 324 273.6 200.5 166.7 237 bioRxiv preprintMean doi: https://doi.org/10.1101/2020.07.27.222737240.788889 ; this 333.288571version posted July 28, 2020. The copyright298.591176 holder for this preprint 462.58125 230.694722 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.