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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 1 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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. 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. 43 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). 2 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. 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 3 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. 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