# The phylogenetic origin and evolution of acellular bone in teleost fishes: insights into osteocyte function in bone metabolism

Donald Davesne, François Meunier, Armin Schmitt, Matt Friedman, Olga

Otero, Roger Benson

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Donald Davesne, François Meunier, Armin Schmitt, Matt Friedman, Olga Otero, et al.. The phylogenetic origin and evolution of acellular bone in teleost fishes: insights into osteocyte function in bone metabolism. Biological Reviews, 2019, 94 (4), pp.1338-1363. 10.1111/brv.12505. hal-02167990

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The phylogenetic origin and evolution of acellular
bone in teleost fishes: insights into osteocyte function
in bone metabolism

Donald Davesne<fnref n="1" />,*, François J. Meunier<fnref n="2" />, Armin D. Schmitt<fnref n="1" />, Matt Friedman<fnref n="3" />, Olga Otero<fnref n="4" /> and Roger B. J. Benson<fnref n="1" />

<footnote n="1">¹ Department of Earth Sciences, University of Oxford, OX1 3AN Oxford, U.K.</footnote>

<footnote n="2">² BOREA (UMR 7208 CNRS, IRD, MNHN, Sorbonne Université), Muséum national d'Histoire naturelle, 75005 Paris, France</footnote>

<footnote n="3">³ Museum of Paleontology and Department of Earth and Environmental Sciences, University of Michigan, Ann Arbor, MI 48109-1079, U.S.A.</footnote>

<footnote n="4">⁴ PalEvoPrim (UMR 7262 CNRS), Université de Poitiers, 86000 Poitiers, France</footnote>

<footnote>* Corresponding author. ORCID: 0000-0002-4775-2360
donald.davesne@earth.ox.ac.uk</footnote>
ABSTRACT

Vertebrate bone is composed of three main cell types: osteoblasts, osteoclasts and osteocytes, the latter being by far the most numerous. Osteocytes are thought to play a fundamental role in bone physiology and homeostasis, however they are entirely absent in most extant species of teleosts, a group that comprises the vast majority of bony ‘fishes’, and approximately half of vertebrates. Understanding how this acellular (anosteocytic) bone appeared and was maintained in such an important vertebrate group has important implications for our understanding of the function and evolution of osteocytes. Nevertheless, although it is clear that cellular bone is ancestral for teleosts, it has not been clear in which specific subgroup the osteocytes were lost. This review aims at clarifying the phylogenetic distribution of cellular and acellular bone in teleosts, to identify its precise origin, reversals to cellularity, and their implications. We surveyed the bone type for more than 600 fossil and extant ray-finned fish species and optimised the results on recent large-scale molecular phylogenetic trees, estimating ancestral states. We find that acellular bone is a probable synapomorphy of Euteleostei, a group uniting approximately two-thirds of teleost species. We also confirm homoplasy in these traits: acellular bone occurs in some non-euteleosts (although rarely), and cellular bone was reacquired several times independently within euteleosts, in salmons and relatives, tunas and the opah (*Lampris* sp.). The occurrence of peculiar ecological (e.g. anadromous migration) and physiological (e.g. red-muscle endothermy) strategies in these lineages might explain the reacquisition of osteocytes. Our review supports that the main contribution of osteocytes in teleost bone is to mineral homeostasis (via osteocytic osteolysis) and not to strain detection or bone remodelling, helping to clarify their role in bone physiology.

*Keywords:* osteocyte, acellular bone, anosteocytic bone, Actinopterygii, Teleostei, Salmoniformes, Scombridae, ancestral state reconstruction, bone remodelling, endothermy
# I. INTRODUCTION

## (1) General introduction

Vertebrate bone is a living tissue that, besides its mineralised extracellular component, comprises cells of three different types. Surface-based osteoblasts and osteoclasts synthesise and resorb bone, respectively, and osteocytes are more versatile cells that fulfil various functions (Francillon-Vieillot et al., 1990; Ricqlès et al., 1991; Bonewald, 2011; Shahar & Dean, 2013; Hall, 2015). Osteocytes are by far the dominant cellular component, constituting up to 95% of bone cells in mammals. They derive from osteoblasts of the bone surfaces that become embedded into the bone matrix in cavities called osteocyte lacunae (Franz-Odendaal, Hall & Witten, 2006) and communicate with each other through a network of canaliculi (Cao et al., 2011).

Osteocytes play a key role in bone physiology: (1) they act as mechanical sensors detecting changes in bone strain; (2) they guide bone remodelling by activating or deactivating the osteoclasts they communicate with; (3) and they are involved in calcium and phosphorus metabolic regulation through direct resorption of the bone around their lacunae (Witten & Huysseune, 2009; Rochefort, Pallu & Benhamou, 2010; Bonewald, 2011; Wysolmerski, 2012; Shahar & Dean, 2013). This double role in mineral and mechanical homeostasis would suggest that osteocytes are indispensable for bone to function normally (Moss, 1961b; Shahar & Dean, 2013). However, bone is entirely devoid of osteocytes in most teleosts, (Kölliker, 1859; Stéphan, 1900; Enlow & Brown, 1956; Moss, 1963; Meunier, 1987, 1989; Meunier & Huysseune, 1992; Huysseune, 2000; Witten et al., 2004; Shahar & Dean, 2013) a group of ray-finned fishes that comprises more than half of modern vertebrate species.

Nineteenth century histologists noted the absence of 'bone corpuscles' (i.e. osteocyte lacunae) in the bone of some teleosts (Williamson, 1851; Gegenbaur, Kölliker, & Müller, 1853; Mettenheimer, 1854; Quekett, 1855). This inspired Kölliker (1859) to undertake a remarkable survey of more than 250 ray-finned fish species, distinguishing those with acellular bone (improperly named 'osteoid' at the time) from those with cellular bone. Moss and colleagues later described the structure, mineral composition and development of teleost acellular bone, confirming its nature as true bone (Moss & Posner, 1960; Moss, 1961a,b, 1962, 1963, 1965; Moss & Freilich, 1963). Later, Weiss & Watabe (1979) proposed the term 'anosteocytic bone', which is more precise because this tissue still bears other cell types (osteoblasts and osteoclasts) on its surface. Nevertheless, the term 'acellular bone' remains widely used in modern literature, and we apply that term here.

That bone is acellular in such a large and ecologically important group as teleosts raises numerous questions pertaining to: (1) the distribution of bone type within teleosts (does it follow ecological, physiological or phylogenetic patterns?), (2) the origin of acellular bone (does it have a unique origin, or multiple convergent appearances?), and (3) the function of such a bone type (does the absence of osteocytes impact bone structure, function and homeostasis?). Addressing these questions has critical implications to understanding the evolution of bone within vertebrates as a whole, and the role of osteocytes in bone physiology (Huysseune, 2000; Witten et al., 2004; Shahar & Dean, 2013; Currey, Dean & Shahar, 2017).
(2) The evolution of acellular bone: state of the art

Following the surveys of Kölliker (1859) and Moss (1961b), researchers attempted to explain the distributions of cellular and acellular bone among teleost species. For example, an early hypothesis proposed that acellular bone occurs because marine environments are richer in dissolved calcium, decreasing the need to use bone as an additional source of metabolic minerals (Moss, 1961b, 1963). However, acellular bone is also present in freshwater teleost taxa such as esocids (pikes), centrarchids (sunfishes), percids ('true' perches), and cichlids (Moss, 1965). In virtually all species, the entire skeleton seems to be composed exclusively of either cellular or acellular bone, and closely related species mostly seem to share the same bone type (Kölliker, 1859). Following these observations, cellularity was quickly recognised as a potentially significant phylogenetic character (e.g. Kölliker, 1859; Berg, 1947). Indeed, at least two studies have used the presence or absence of osteocytes to discuss the systematic position of enigmatic fossil taxa (Gaudant & Meunier, 2004; Mayrinck et al., 2017).

Deep divergences in teleost phylogeny have been poorly resolved until recently, meaning that the phylogenetic distribution of cellularity has not been clear. Nevertheless, there is broad consensus on two statements: (1) that cellular bone is the plesiomorphic condition for teleosts, actinopterygians and osteichthyans in general (Ørvig, 1951, 1967; Moss, 1961b, 1963); and (2) that acellular bone is found in 'advanced' or 'higher' teleost groups (Moss, 1961b, 1963; Meunier, 1987, 1989; Ricqlès et al., 1991; Meunier & Huysseune, 1992; Witten et al., 2004). As noted by the past authors themselves, these propositions are imprecise and potentially misleading. Indeed, the pattern appears to be much more complex: for example, acellular bone is found in certain 'lower' teleosts such as pikes and cellular bone is found in some 'higher' taxa such as tunas (Amprino & Godina, 1956; Moss, 1963; Meunier, 1989; Meunier & Huysseune, 1992). Moreover, the systematic distributions of both bone types have been described using subjective and poorly defined systematic categories (e.g. 'advanced teleosts'), not on an explicit phylogenetic framework based on character analysis.

Several authors used cellularity as a phylogenetic character: acellular bone is proposed as a synapomorphy uniting (1) Osmeriformes (true smelts) and Neoteleostei (the clade including spiny-rayed fishes, amongst others) by Rosen (1985); (2) Esociformes (pikes and mudminnows), Osmeriformes and Neoteleostei by Parenti (1986); (3) Esociformes and Neoteleostei by Johnson & Patterson (1996), the latter being the only phylogeny based on the analysis of a character matrix. However, the usefulness of this previous work is limited because the underlying phylogenetic frameworks have been superseded by more recent classifications based on molecular phylogenies that extensively sample both taxa and loci (e.g. Near et al., 2012; Betancur-R. et al., 2013, 2017). The most relevant changes relative to anatomical hypotheses include: (1) Esociformes do not form an exclusive clade with Neoteleostei, but instead consistently appear to be sister to Salmoniformes (Ramsden et al., 2003; Wilson & Williams, 2010; Near et al., 2012; Betancur-R. et al., 2013; Campbell et al., 2013); (2) Neoteleostei sensu Rosen (1973, 1985) is not a monophyletic group, with Stomiiformes (viperfishes and relatives) now considered sister to Osmeriformes (Li et al., 2010; Near et al., 2012; Betancur-R. et al., 2013, 2017).
### (3) Aim of this review

While most research on acellular teleost bone has been focused on its structure, development and function (Moss, 1961a; Meunier, 1989; Meunier & Huysseune, 1992; Cohen et al., 2012; Dean & Shahar, 2012; Shahar & Dean, 2013), the evolutionary origin and phylogenetic distribution of this bone type has not been studied in detail.

Explaining the evolutionary origins of acellular bone requires an explicitly phylogenetic approach that can distinguish the role of adaptation from that of phylogenetic history in the distribution of bone types among species. This review aims to clarify the distribution of cellular and acellular bone in teleosts within a phylogenetic context that is now available thanks to an array of recently published large-scale molecular analyses (e.g. Near et al., 2012; Betancur-R. et al., 2013; Hughes et al., 2018). We also review the structure of acellular bone, emphasising its functional similarity to cellular bone (Witten & Huysseune, 2009; Cohen et al., 2012; Shahar & Dean, 2013; Currey et al., 2017). Our review of the existing literature, complemented by our own observations, brings together most of the data published to date on actinopterygian bone to constitute a data set covering the whole diversity of the group. Including this data into an explicit phylogenetic framework for the first time, finally allows us to draw a possible historical scenario for the loss of osteocytes in teleosts.

## II. TELEOST ACELLULAR BONE: STRUCTURE AND FUNCTION

### (1) Structure and development

In teleosts, bone is found in the endoskeleton (the skeleton proper, i.e., cranial, axial and appendicular skeleton; Fig. 1A, C-F) and in the dermal exoskeleton, including scales. In teleosts, bone is found in the cranial, axial and appendicular skeleton (Fig. 1A, C-F) and in scales (Fig. 1B), lepidotrichia (fin rays) and the tissues that derive from them (Patterson, 1977; Schaeffer, 1977; Francillon-Vieillot et al., 1990). Despite its structural peculiarities that led historical authors to improperly designate it under other names (e.g. 'osteoid', Kölliker, 1859), teleost acellular bone is considered true bone because it shares its developmental origin and main characteristics with every other vertebrate bone tissue (Moss, 1961b; Witten & Huysseune, 2009; Dean & Shahar, 2012): (1) it is composed of hydroxyapatite crystals in a mesh of type I collagen fibres; (2) it has the same functional properties as other bone tissues (muscle insertion and organ support); (3) its extracellular matrix is secreted by osteoblasts and resorbed by osteoclasts; (4) it can be submitted to active remodelling.

Typical cellular bone contains numerous mature osteocytes that, despite being completely surrounded by mineralised tissue, communicate with each other and with the bone surface via a network of canaliculi containing cytoplasmic processes (Fig. 1C, D). This lacunocanalicular system permeates bone and gives osteocytes their characteristic star-shaped appearance (Meunier, 1987; Cao et al., 2011). It is however not clear whether osteocytes form a proper lacunocanalicular network in all teleosts with cellular bone (Fiaz, van Leeuwen & Kranenbarg, 2010; Totland et al., 2011). In acellular bone, on the other hand, there are no osteocytes or lacunae within the bone mineral matrix (Fig. 1E, F), but it is sometimes penetrated by osteoblastic canaliculi from the bone surface (Francillon-Vieillot et al., 1990; Sire & Meunier, 1994, 2017). In the 'tubular acellular bone' of a few taxa, tubules containing a bundle of collagen fibres and numerous osteoblastic cytoplasmic processes permeate acellular primary bone (Hughes, Bassett, & Moffat, 1994; Sire & Meunier, 2017; Meunier & Béarez, 2019).
These tubules are superficially similar, but structurally distinct from the canals of Williamson (Fig. 1D) that are known only from the cellular bone of holosteans and fossil stem teleosts (Williamson, 1849; Ørvig, 1951; Sire & Meunier, 1994; Meunier & Brito, 2004). Acellular bone can be vascular or avascular, osteoblastic canaliculi being more numerous in avascular acellular bone than in vascular acellular bone (Francillon-Vieillot et al., 1990).

In cellular bone, osteocytes originate from osteoblasts that become surrounded by the mineral matrix they secreted (Franz-Odendaal et al., 2006). Conversely, in acellular bone osteoblasts remain on the outer surface and secrete extracellular matrix exclusively towards the interior of bone, never ending up surrounded by bone to turn into osteocytes (Weiss & Watabe, 1979; Ekanayake & Hall, 1987, 1988). The hypothesis that acellular bone could form through intracellular mineralisation of osteocytes that are already entrapped in bone (Moss, 1961a) has been rejected since a study on the medaka *Oryzias latipes* (Ekanayake & Hall, 1987).

## (2) Functional properties of acellular bone

### (a) Mechanical properties

The mineral fraction in acellular bone is proportionally slightly higher than in cellular bone (Meunier, 1984a; Cohen et al., 2012). This higher mineral content, along with the reduction in porosity associated with the absence of osteocytes have been hypothesised to increase the stiffness of acellular bone (Horton & Summers, 2009). However, comparative studies of structural stress have suggested that acellular and cellular bone have equivalent stiffness (Horton & Summers, 2009; Cohen et al., 2012; Dean & Shahar, 2012; Currey & Shahar, 2013). On the other hand, the collagen fibre ultrastructure in acellular teleost bone gives it an increased toughness compared to tetrapod (e.g. human) cellular bone (Atkins et al., 2015b).

### (b) Resorption

Osteoclasts, the cells primarily responsible for bone resorption, were long thought to be absent from acellular teleost bone, although resorption was still observed (Blanc, 1953; Moss, 1963; Weiss & Watabe, 1979; Glowacki et al., 1986). Indeed, osteoclasts in acellular bone are structurally different from the 'typical' osteoclasts found in cellular bone, explaining why they long went undetected: they are generally mononucleated instead of multinucleated as in cellular bone (Sire, Huysseune, & Meunier, 1990; Witten, 1997; Witten & Villwock, 1997; Witten & Huysseune, 2009). This structural difference may be explained by the absence of osteocytes, which promote the growth of multinucleated osteoclasts (Witten & Huysseune, 2009, 2010).

### (c) Mineral metabolism

Bone plays a crucial role in calcium metabolism in vertebrates, both as a consumer and as a source of calcium. However, this role seems less critical in teleosts than in terrestrial vertebrates since, as aquatic animals, teleosts can mobilise calcium and other elements directly from the ambient water via their gills and/or digestive system (Takagi & Yamada, 1992; Witten & Huysseune, 2009; Shahar & Dean, 2013). Phosphorus availability appears to be more critical than that of calcium for healthy growth in both marine and freshwater teleosts (Witten & Huysseune, 2009; Shahar & Dean, 2013), and bone does not seem to mineralise when phosphorus is absent from the diet
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Examples of cellular (A–D) and acellular (E, F) bone in teleosts and close relatives. (A) Ground section through cellular bone in the jaw of the Devonian actinopterygian *†Cheirolepis canadensis* (MHNM 05-340), observed in transmitted natural light. Osteocyte lacunae are marked with black arrowheads. Modified from Meunier *et al.* (2018c). (B) Thin section through a scale of an osteoglossomorph, the arowana *Osteoglossum bicirrhosum*, observed in transmitted natural light. Osteocyte lacunae are visible in the superficial bony layer, and marked with black arrowheads. Photograph by F. J. Meunier. (C) Thin section through cellular bone in the rib of an ostariophysan, the barbel *Barbus barbus*, observed in transmitted natural light. Osteocyte lacunae and their associated lacunocanalicular network are clearly visible. Modified from Meunier & Herbin (2014). (D) ‘Virtual thin section’ obtained by stacking synchrotron</figcaption></figure>
tomographic slices of the dentary of the Jurassic stem teleost †*Dorsetichthys bechei* (OUMNH J.3369).
Star-shaped osteocyte lacunae and their canaliculi are visible (black arrowheads), as well as canals of
Williamson in cross-section (white arrowheads). Image produced by D. Davesne and A. D. Schmitt. (E)
Thin section through acellular bone in the rib of an acanthomorph, the sea bass *Dicentrarchus labrax*,
showing numerous radially arranged osteoblastic canaliculi. Photograph by D. Davesne. (F) Thin section
through acellular bone in the vertebra of an acanthomorph, the anglerfish *Lophius* sp. Bone is relatively
featureless, apart from visible successive growth marks (black arrowheads). Photograph by F. J. Meunier.

(Witten et al., 2016, 2018). Nevertheless, a specific type of bone resorption (osteocytic osteolysis) is undertaken by the osteocytes themselves and may be linked to periods of increased metabolic calcium and/or phosphorus requirement, as it occurs conspicuously in certain diadromous teleost species [e.g. European eel (*Anguilla anguilla*), salmoniforms] before and during migration (Kacem & Meunier, 2000, 2003; Sbaihi et al., 2007). In teleosts with acellular bone, osteocytic osteolysis is impossible, potentially making calcium and phosphorus more difficult to mobilise from and into the skeleton than in those with cellular bone (Moss, 1962; Simmons, Simmons & Marshall, 1970; Witten, 1997; Witten & Huysseune, 2009).

### (d) Remodelling

Teleost bony tissues consist mainly of primary bone in most species (Meunier, 1987) and bone remodelling appears to be less abundant in teleosts than in tetrapods – it was even long thought to be absent (Moss, 1961a). Nevertheless, bone remodelling occurs in teleosts, in taxa with both cellular (Witten, Hansen, & Hall, 2001; Witten & Hall, 2003; Nemoto et al., 2007; Witten & Huysseune, 2009) and acellular bone (Castanet & Ricqlès, 1986; Witten & Huysseune, 2009; Dean & Shahar, 2012; Shahar & Dean, 2013; Atkins et al., 2014, 2015a; Currey et al., 2017). For instance, hyperostoses are widespread in teleosts with acellular bone, and their formation requires an important remodelling activity (Meunier & Desse, 1986; Smith-Vaniz, Kaufman & Glowacki, 1995). In billfishes (Istiophoriformes), that lack osteocytes, bone in the rostrum is riddled with secondary osteons overlapping primary osteons, akin to what is found in the haversian bone of tetrapods and suggesting very intense remodelling activity as a response to fracture and load (Amprino & Godina, 1956; Poplin, Poplin & Ricqlès, 1976; Castanet & Ricqlès, 1986; Atkins et al., 2014). These examples suggest that, in the absence of osteocytes as sensors, acellular bone is nevertheless capable of detecting strain and damage by some mechanism that is yet not fully understood (Kranenbarg et al., 2005; Witten & Huysseune, 2009; Fiaz et al., 2010; Dean & Shahar, 2012; Shahar & Dean, 2013; Atkins et al., 2014, 2015a).

In its general structure, biomechanics, and mechanisms of bone resorption and remodelling, acellular teleost bone then appears to be functionally very similar to cellular teleost bone. This suggests that the presence of osteocytes is not strictly necessary to achieve these functions. This leaves osteocytic osteolysis, a potentially important mechanism involved in calcium and/or phosphorus metabolism (Witten & Huysseune, 2009; Cohen et al., 2012; Shahar & Dean, 2013; Doherty, Ghalambor & Donahue, 2015), as the main function entirely lacking in acellular bone.
### III. PHYLOGENETIC DISTRIBUTION OF ACELLULAR BONE

#### (1) Acellular bone outside of actinopterygians

##### (a) Palaeozoic jawless vertebrates

246 A peculiar bone-like tissue devoid of osteocytes, called aspidin, has long been known in the dermal skeleton of heterostracans, a group of Palaeozoic jawless vertebrates (Gross, 1930; Halstead, 1969). Similar tissues were later described in other early jawless stem gnathostome lineages, such as tanaspids, thelodonts and tgaleaspids (Stensiö, 1958; Sire, Donoghue & Vickaryous, 2009; Keating & Donoghue, 2016). Aspidin appears to be structurally very similar to teleost acellular bone, with probable collagen bundles (akin to the 'tubules' of teleosts) penetrating the mineralised tissue (Keating et al., 2018). The occurrence of either cellular or acellular bone in various early vertebrate lineages (Fig. 2) led to a debate over which one was phylogenetically older (Ørvig, 1951; Denison, 1963; Halstead, 1963; Smith & Hall, 1990). The earliest vertebrates with cellular bone are the jawless osteostracans that appear in the Silurian (Stensiö, 1958; Smith & Hall, 1990; Donoghue & Sansom, 2002), although osteocytes have also been described in the dermal bone of a late Ordovician tarandaspid (Sansom et al., 2013). Abundant evidence supports the placement of osteostracans as the sister group to gnathostomes (jawed vertebrates): it seems likely that cellular bone would then be a synapomorphy of the clade uniting osteostracans and gnathostomes (Donoghue & Sansom, 2002; Brazeau & Friedman, 2014), with a potential convergent appearance in tarandaspids (Fig. 2). This would imply that bone in tanaspids, thelodonts, heterostracans and tgaleaspids is primitively devoid of osteocytes, making acellular bone the plesiomorphic state for skeletonising vertebrates (Denison, 1963; Halstead, 1963, 1969; Donoghue & Sansom, 2002; Keating et al., 2018).

270
271 (b) Jawed vertebrates
272 As a plesiomorphic character for gnathostomes (Fig. 2), cellular bone is found in Palaeozoic jawed stem gnathostomes such as ‘placoderms’ (Ørvig, 1951; Downs & Donoghue, 2009; Sire et al., 2009; Giles, Rücklin & Donoghue, 2013) and in fossils interpreted as stem osteichthyans, such as Andreolepis, Lophosteus and Psarolepis (Jerve et al., 2016; Qu et al., 2017). Bone is cellular in sarcopterygians, the sister group to actinopterygians, including modern coelacanths, modern lungfishes, modern tetrapods (lissamphibians, mammals, diapsids) and fossil taxa falling on their respective stem groups (Sire et al., 2009; Zylberberg, Meunier & Laurin, 2010; Schultze, 2016; Meunier, Cupello & Clément, 2019).

281 On the other hand, acellular bone also occurs in different gnathostome lineages. A prominent example is the basal bone layer in the odontodes of various chondrichthyans (cartilaginous fishes) and their close relatives, including Palaeozoic ‘acanthodians’ (Sire et al., 2009; Chevrinais, Sire & Cloutier, 2017). Acellular perichondral bone is also found in the modified dorsal fin of the Palaeozoic stem holocephalan Akmonistion (Coates et al., 1998), while the fin rays of the African lungfish Protopterus are composed of acellular dermal bone (Géraudie & Meunier, 1984). Finally, acellular bone is found in very localised zones of specialised tissues in a few tetrapods, for example in cranial bones and sutures of pachycephalosaurid and ceratopsian dinosaurs (Goodwin & Horner, 2004; Bailleul & Horner, 2016). In all these taxa, acellular bone is found exclusively in dermal bone, leaving teleosts as the only known vertebrates with occurrence of acellular endochondral bone.
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Distribution of cellular and acellular bone in the phylogeny of vertebrates (modified from Keating *et al.*, 2018). The coloured circles at the tip of branches reflect bone type in the clade: acellular (yellow), cellular (dark blue), or bone absent (white). Taxon pictures from N. Tamura, and Iglésias (2014a, 2014b).</figcaption></figure>

(2) Phylogenetic distribution of acellular bone in teleosts and other actinopterygians

(a) *Material of study*

To evaluate the phylogenetic distribution of cellular and acellular bone in actinopterygians, we reviewed more than 150 years of literature on ray-finned fish bone. The most comprehensive sources of information were the extensive surveys by Kölliker (1859) and Moss (1961b, 1965), to which we added data from various fossil and extant species where required to better resolve the phylogenetic and temporal distribution (see online Supporting information, Table S1, for details on these sources). In total, our database includes 677 fossil and extant taxa. In addition, we obtained propagation phase contrast synchrotron microtomography (PPC-SRµCT) data from museum specimens of 108 extant and fossil species (Table 1, Table S1), bringing new information or corroborating our knowledge on the presence or absence of osteocytes in their bones. The SRµCT scans were carried out at the ID-19 (microtomography) beamline of the European Synchrotron Radiation Facility (ESRF), using a white beam with energy levels between 35 and 105 keV, obtaining a voxel size of 0.72 µm.
For all extant and fossil taxa, we used the dentary as a bone of study (and in some cases, a rib). This bone appears to be cellular, even when both bone types coexist in the skeleton (Weigele & Franz-Odendaal, 2016). We then consider that the lack of osteocytes in the dentary is likely to reflect genuine acellularity in a given taxon.

**(b) Non-teleost actinopterygians**

Cellular bone is present in the earliest actinopterygians from the Devonian (Table S1): for example, in the bones and scales of †*Cheirolepis* (Zylberberg, Meunier & Laurin, 2016) and †*Moythomasia* (Sire *et al.*, 2009; Schultze, 2016) and in the scales of †*Mimipiscis* (Richter & Smith, 1995). Bone and scales are always cellular in modern non-teleost actinopterygians, for example in bichirs (Polypteriformes), bowfins and gars (Holostei) (Kölliker, 1859; Moss, 1961b; Sire & Meunier, 1994; Daget *et al.*, 2001; Sire *et al.*, 2009) and in their Mesozoic fossil relatives (Goodrich, 1907; Ørvig, 1978; Gayet & Meunier, 1992; Meunier & Brito, 2004; Meunier *et al.*, 2016). Sturgeons and paddlefishes (Acipenseriformes) have a poorly mineralised skeleton, but it is nonetheless composed of cellular bone (e.g. Kölliker, 1859; Stéphan, 1900; Buffrénil *et al.*, 2016; Leprévost *et al.*, 2017). Finally, many clades of extinct Mesozoic actinopterygians have been surveyed histologically and show cellular bone, for example: †saurichthyids (Scheyer *et al.*, 2014), †aspidorhynchids (Brito & Meunier, 2000), †pachycormids (Meunier & Brito, 2004; Liston *et al.*, 2013), †pholidophorids (Meunier & Brito, 2004). Our SRµCT data provide additional information on a series of fossil non-teleost actinopterygians, revealing the presence of cellular bone in the Jurassic stem chondrostean †*Chondrosteus acipenseroides*, the Jurassic †pycnodontiform †*Proscinetes elegans*, the Jurassic †dapediid †*Dapedium* sp., the Triassic holosteans †*Heterolepidotus dorsalis* and †*Eoeugnathus megalepis* and the Jurassic stem bowfin †*Caturus furcatus*. These data also confirm the presence of cellular bone in 17 Jurassic and Cretaceous taxa (Tables 1, S1) interpreted as stem-group teleosts (e.g. Arratia, 2015).

**(c) Elopomorpha**

Within Elopomorpha, cellular bone is found in tarpons and their relatives (Elopiformes), including in scales (Kölliker, 1859; Meunier & Brito, 2004). Several eels (Anguilliformes) are described as having acellular bone by Kölliker (1859). However, they all seem to pertain to an outdated taxonomy that treated leptocephalus larvae as separate taxa (Table S1). For example, Kölliker (1859) reports cellular bone in the sorcerer eel *Nettastoma melanurum* and acellular bone in *'Hyoprorus messanensis'*, corresponding to the larva of *N. melanurum* (Eschmeyer, Fricke & van der Laan, 2018). Although Moss (1961b) reports acellular bone in the moray eel *Gymnothorax moringa*, we confirm the presence of osteocytes in this species, as well as in the adults of every other anguilliform surveyed, including the freshwater eels *Anguilla anguilla* and *A. rostrata* (Stéphan, 1900; Moss, 1965; Lopez, 1970), the conger eel *Conger conger* and the pike conger *Muraenesox cinereus* (Table 1). The bonefish *Albula vulpes* was described as having a mix of cellular and acellular bone (Moss, 1961b), but this is contradicted by our observations (see Section III.3a). Finally, our SRµCT data reveal cellular bone in several fossil albuliforms (e.g. †*Istieus*, †*Lebonichthys*), elopiforms (e.g. †*Ichthyemidion*, †*Anaethalion*, †*Flindersichthys*) and anguilliforms (†*Urenchelys*). In conclusion, we find that cellular bone is present in post-larval individuals of all elopomorphs surveyed so far.
(d) *Osteoglossomorpha*

Fossil and extant bony-tongue fishes (Osteoglossomorpha) have cellular bone in their skeleton, including scales (Kölliker, 1859; Meunier & Brito, 2004; Meunier, Brito & Leal, 2013a; Meunier, Dutheil & Brito, 2013b). Moss (1965) reported acellular bone in the two modern mooneye (Hiodontidae) species, *Hiodon alosoides* and *H. tergisus*. However, Kölliker (1859) described cellular bone in ‘*Hyodon claudulus*’ that could be synonymised with *H. alosoides* (Eschmeyer *et al.*, 2018). We resolved this uncertainty using unambiguous observations of osteocyte lacunae in SRµCT images of dentaries and/or ribs from *H. alosoides*, *H. tergisus* and their Eocene close relative †*Eohiodon falcatus*, confirming the presence of cellular bone in hiodontids. We also find cellular bone in *Arapaima gigas*, in the arowana *Osteoglossum bicirrhosum* and its extinct Eocene relatives †*Brychaetus muelleri* and †*Phareodus encaustus*, as well as in the featherback *Chitala chitala* (Table 1). In conclusion, it is likely that cellular bone is present in all osteoglossomorphs (Table S1).

(e) *Clupeomorpha*

Herrings and their relatives (Clupeomorpha) appear to have cellular bone (Kölliker, 1859; Moss, 1961b, 1965). Although Moss (1961b) reported acellular bone in the anchovy *Anchoviella* sp. and the American shad *Alosa sapidissima*, he later updated this observation by reporting cellular bone in *A. sapidissima* and three other *Alosa* species (Moss, 1965). Our SRµCT data reveal cellular bone in all clupeomorphs surveyed (Table 1), including the Cretaceous †*Armigatus namourensis* and †*Ellimmichthys longicostatus* and the Eocene †*Knightia* sp., as well as the extant wolf-herring *Chirocentrus dorab*, the Pacific sardine *Sardinops sagax* and the alewife *Alosa pseudoharengus*. In conclusion, it is likely that cellular bone is present in all clupeomorphs (Table S1), with the possible exception of *Anchoviella* that needs further appraisal.

(f) *Ostariophysi*

Kölliker (1859) and Moss (1961b, 1965), extensively sampled the considerable diversity of the mostly freshwater ostariophysans, including milkfishes (Gonorhynchiformes), carps and relatives (Cypriniformes), characins and relatives (Characiformes), catfishes (Siluriformes) and electric ‘eels’ (Gymnotiformes). Their surveys totalled 115 species, virtually all of which appear to have cellular bone (Table S1). We also observed cellular bone in our SRµCT images of the carp *Cyprinus carpio*, the tench *Tinca tinca*, the bream *Abramis brama* (Cypriniformes), the trahira *Hoplias malabaricus*, the payara *Hydrolycus scomberoides*, the piranha *Serrasalmus spilopleura* (Characiformes), the catfishes *Ariopsis felis*, *Galeichthys feliceps* and *Pimelodella gracilis* (Siluriformes), and the banded knifefish *Gymnotus carapo* (Gymnotiformes), as well as in the Early Cretaceous gonorhynchiform †*Tharrias araripes* (Table 1). Acellular bone is only described in two ostariophysan species (Table S1): in the diminutive pencil catfish *Trichomycterus punctulatus* (Kölliker, 1859), which is confirmed by our SRµCT data from the dentary of another *Trichomycterus* species, and in some cranial dermal bones of the zebrafish *Danio rerio* (Weigele & Franz-Odendaal, 2016). In conclusion, cellular bone is present in all ostariophysans surveyed so far, with the notable exceptions of *Trichomycterus*. In addition, slickheads (Alepocephaliformes) are consistently recovered as sister to ostariophysans in molecular phylogenies (Lavoué *et al.*, 2008; Near *et al.*, 2012;
408 Betancur-R. et al., 2013; Straube et al., 2018; Hughes et al., 2018). The only species
409 surveyed from the group, Alepocephalus rostratus, has cellular bone (Kölliker, 1859).

410
411 (g) *Non-neoteleost Euteleostei*

412 Bone type is variable amongst Euteleostei, but generally homogeneous within a given
413 lineage (Table S1). Acellular bone is found in galaxiids (but only two species of *Galaxias*
414 have been surveyed), pikes and mudminnows (Esociformes; Kölliker, 1859; Moss,
415 1961b, 1965), smelts (Osmeridae; Moss, 1961b, 1965) and viperfishes and their
416 relatives (Stomiiformes; Kölliker, 1859; Germain, Schnell & Meunier, in press).
417 Conversely, cellular bone is found in *Argentina silus* (the only member of
418 Argentiniformes that was sampled) and we observe it in the Late Cretaceous
419 †*Spaniodon elongatus*, a taxon whose phylogenetic position within euteleosts is
420 uncertain (e.g. Taverne & Filleul, 2003). Salmons, trouts and their relatives
421 (Salmoniformes) are generally described as having cellular bone (Kölliker, 1859; Moss,
422 1961b, 1965; Hughes et al., 1994; Witten & Hall, 2002; Totland et al., 2011), but our
423 extensive SRµCT sampling within the group complicates this pattern (Table 1). Bone
424 appears always to be cellular in the 'typical' trouts and salmons (Salmoninae). We
425 confirm this for extant and fossil representatives of *Oncorhynchus*, *Salmo*, *Salvelinus*
426 and *Parahucho*. The whitefishes *Coregonus reighardi*, *Prosopium williamsoni* and
427 *Stenodus leucichthys* (Coregoninae) also seem to have osteocytes, but they are much
428 scarcer than in salmonines, and irregularly distributed inside of bone. This is consistent
429 with the observation of Moss (1965), who described variation in osteocyte abundance
430 within the skeleton in some salmoniforms. Finally, in the grayling *Thymallus thymallus*
431 (Thymallinae), bone seems to be acellular.

432
433
434 (h) *Neoteleostei*, including *Acanthomorpha*

435 Within the euteleost subclade Neoteleostei (sensu Betancur-R. et al., 2017), acellular
436 bone is found in various lizardfishes (Aulopiformes), including the Late Cretaceous
437 †*Eurypholis* sp., in the lanternfish (Myctophiformes) *Notoscopelus elongatus* and in the
438 Cretaceous genus of uncertain placement †*Ctenothrissa vexillifer* (Kölliker, 1859; Moss,
439 1961b; Davesne et al., 2018). Spiny-rayed fishes (Acanthomorpha) contribute the
440 greatest fraction of neoteleost species diversity. Amongst the approximately 17,000
441 acanthomorph species (more than 300 being surveyed in the present study), acellular
442 bone is virtually universal (Kölliker, 1859; Moss, 1961b, 1965) and is found throughout
443 taxa displaying a broad range of morphologies and ecologies (Table S1), from marine
444 benthic taxa such as toadfishes and sculpins (Simmons et al., 1970; Horton & Summers,
445 2009), to pelagic fast-swimming taxa like jacks and billfishes (Smith-Vaniz et al., 1995;
446 Atkins et al., 2014), deep-sea eelpouts (Meunier & Arnulf, 2018), or freshwater ricefishes
447 and tilapias (Ekanayake & Hall, 1987; Cohen et al., 2012). Within acanthomorphs,
448 cellular bone is only known conclusively in two relatively species-poor lineages: the 'true'
449 tunas *Auxis*, *Euthynnus*, *Katsuwonus* and *Thunnus* (Kölliker, 1859; Stéphan, 1900;
450 Amprino & Godina, 1956; Moss, 1961b; Meunier & Huysseune, 1992; Santamaria et al.,
451 2018) and the opah *Lampris* (Davesne et al., 2018). At least in tunas, osteocytes are
452 present not only in bones, but also in scales, fin rays and spines (Meunier et al., 2008a;
453 Wainwright, Ingersoll & Lauder, 2018; Santamaria et al., 2018).
(3) Intra-specific and intra-individual variation

(a) Occurrence of mixed bone types

Comparative literature generally states that when cellular or acellular bone is found, it
occurs throughout the whole skeleton, including dermal and endochondral bone, fin rays
and spines (Kölliker, 1859; Moss, 1961b, 1963; Meunier & Huysseune, 1992). The
incompletely mineralised elasmoid scales of most modern teleosts are an exception:
they are often acellular when the rest of the skeleton is cellular (see Section III.4). Moss
(1961b) reported that the bonefish *Albula vulpes* displayed a mix of cellular and acellular
bone, with the latter being found in the operculum and gill arches. However, our SRµCT
data including the operculum and gill arches show osteocytes in all of these elements.
These observations suggest that the whole skeleton of *A. vulpes* is cellular, contradicting
Moss' (1961b) statement.

Weigele & Franz-Odendaal (2016) showed that in the zebrafish *Danio rerio*, bones with and without osteocytes coexist within the cranial skeleton of a given individual. Both dermal and endochondral bones can be cellular or acellular, but dermal intramembranous bones of the neurocranium seem more likely to be acellular, while endochondral bones of the splanchnocranium (i.e. palatoquadrate, hyoid and branchial arches) are all cellular. These results imply that using only the dermal neurocranium to describe bone type in a teleost species can potentially be misleading. Conversely, jaw bones (such as the dentary) and the postcranium (vertebrae excepted) are all cellular in *D. rerio* (Weigele & Franz-Odendaal, 2016). This suggests that our SRµCT data (Table 1), which rely on dentaries and/or ribs, accurately reflect cellularity: if acellular bone is found in these elements it is most likely to reflect the rest of the skeleton.

A possibility is that this pattern of mixed bone types stems from the very small adult
body size of *D. rerio*. In very thin bones, there might not be enough bone matrix for
osteoblasts to become entrapped and turn into osteocytes. For instance, some of the
acellular bones observed in *D. rerio* are approximately 10 µm thick (Weigele & Franz-
Odendaal, 2016), in an animal which rarely exceeds 40 mm in total adult length (Spence
et al., 2008). While the frontal bone is described as acellular in *D. rerio* (Weigele &
Franz-Odendaal, 2016), we observe with SRµCT osteocytes in the frontal bone of the
carp *Cyprinus carpio*, a closely related cypriniform. Since this observation comes from a
carp of 452 mm in total length, it would corroborate our hypothesis of a size-related
acellularity in *D. rerio*, and potentially other teleosts. Surveying various cranial bones in
other teleost taxa and on specimens of various sizes would help clarify whether this
pattern of mixed bone types is widespread in teleosts, or specific to *D. rerio*.
Observations based on ontogenetic series of other taxa also corroborate that the
absence of osteocytes might be explained by the size of the bone. For example,
Huysseune (2000) reports that very young individuals of teleosts with cellular bone often
lack osteocytes, which appear once bone becomes thicker. This would also explain
Kölliker's (1859) observations of acellular bone in larval anguilliforms (see Section
III.2c).

(b) Alleged osteocytes in tubular and hyperostotic bone

The presence of few osteocytes in very localised zones of otherwise acellular bone has
been suggested for some species, relying upon two specific cases. In the first case,
osteocytes were detected in tubules containing collagen bundles and osteoblastic
canaliculi in three species of sparids (sea breams), an acanthomorph family otherwise
501 characterised by acellular bone (Hughes et al., 1994). However these results are
502 seemingly contradicted by more recent data (Sire & Meunier, 2017): at least in the case
503 of *Sparus aurata* these tubules do not appear to contain osteocyte nuclei. In the second
504 case, osteocytes were described within areas of hyperostosis in the cleithrum of the jack
505 *Caranx latus* (Smith-Vaniz et al., 1995) and in dorsal pterygiophores of the oarfish
506 *Regalecus russellii* (Paig-Tran, Barrios & Ferry, 2016), two acanthomorphs that
507 otherwise have acellular bone. However, such osteocytes do not appear to be present
508 systematically in acanthomorph hyperostotic bone: they are absent from the
509 hyperostoses of the scabbardfish *Trichiurus lepturus*, the jack mackerel *Trachurus*
510 *trachurus*, the sicklefish *Drepane africana*, the grunt *Pomadasys kaakan* and the
511 searobin *Prionotus stephanophrys* (Desse et al., 1981; Meunier & Desse, 1994;
512 Meunier, Béarez & Francillon-Vieillot, 1999; Meunier, Gaudant & Bonelli, 2010). The
513 black skipjack tuna *Euthynnus lineatus* has cellular bone in its hyperostotic vertebrae
514 (Béarez, Meunier & Kacem, 2005), however this is consistent with the presence of
515 cellular bone throughout the rest of its skeleton. The occurrence of osteocytes in
516 hyperostotic regions of an otherwise acellular skeleton then appears to be the exception
517 rather than the rule; it nevertheless requires explanation.

518 A possibility is that these localised osteocytes could form via an accidental incorporation
519 of osteoblasts during the exceptionally rapid growth of hyperostotic bone. This
520 arrangement may be temporary and accidental, and would differ from 'true' cellular
521 bone. Determining whether these osteocytes are present in all hyperostotic individuals of
522 a given species, for example, would help to assess the nature of this phenomenon.

523
524 (4) Phylogenetic distribution of acellular bone in actinopterygian scales

525 The phylogenetic distribution of osteocytes in actinopterygian scales (Table S1) has
526 been less studied than in the rest of the skeleton (Parenti, 1986). Scales in
527 actinopterygians primitively consist of a bony basal plate covered by dentine and
528 ganoine (an enamel-like tissue). The bony component remains as a thin external layer in
529 the elasmoid scales of most teleosts (Francillon-Vieillot et al., 1990; Meunier &
530 Huysseune, 1992; Sire et al., 2009). In ganoid scales, bone is always cellular, as shown
531 in early actinopterygians (Richter & Smith, 1995; Sire et al., 2009; Zylberberg et al.,
532 2016), bichirs (Daget et al., 2001; Sire et al., 2009), holosteans (Meunier, François &
533 Castanet, 1978; Meunier et al., 2016; Brito, Meunier & Gayet, 2000) and stem teleosts
534 (Brito & Meunier, 2000; Meunier & Brito, 2004). In elasmoid scales, found in all teleosts
535 but also in amiids and the extant coelacanth *Latimeria* (Smith, Hobdell & Miller, 1972;
536 Meunier, 1984b; Meunier et al., 2008b; Sire et al., 2009), the situation is more complex.
537 In this type of scales, the basal layer develops into an incompletely mineralised plywood-
538 like structure called elasmodine (previously described as isopedine). The basal layer in
539 the scales in amiids and some teleosts (e.g. *Megalops*, *Hiodon*, *Arapaima*, *Chanos*)
540 incorporates cells superficially similar to osteocytes, called elasmocytes (Meunier,
541 1984b, 1987; Meunier & Brito, 2004). The bony layer is cellular in the elasmoid scales of
542 amiids (Meunier & Poplin, 1995), elopomorphs (e.g. *Megalops*, *Elops*, *Albula*) and at
543 least some osteoglossomorphs (Meunier, 1984b; Meunier & Brito, 2004). It is, however,
544 acellular in other taxa with cellular bone including clupeomorphs, ostariophysans and
545 salmoniforms (Meunier, 1987; Meunier & Brito, 2004; Meunier, Sorba & Béarez, 2004;
546 Sire et al., 2009). Taxa with acellular bone always seem to have acellular scales as well
547 (Kölliker, 1859). In the tunas *Thunnus alalunga* and *T. obesus* scales are composed of
cellular bone (Meunier & Sire, 1981; Wainwright et al., 2018), in agreement with the rest of the skeleton. Since many teleosts with cellular bone lack osteocytes in their scales, it then seems that acellularisation in scales phylogenetically precedes that of the rest of the skeleton (Kölliker, 1859; Meunier, 1987; Meunier & Huysseune, 1992).

## IV. PHYLOGENETIC ORIGIN AND EVOLUTION OF ACELLULAR BONE
### (1) Ancestral character state reconstruction

For our entire data set of 677 fossil and extant actinopterygians, we scored the presence of cellular or acellular bone (Table S1; scales scored separately). When bone lacks osteocytes only in certain skeletal elements (e.g. teleosts with cellular bone but acellular scales) or ontogenetic stages (e.g. in larval anguilliforms) we scored its status as 'cellular'.

This data set was mapped onto three time-calibrated trees stemming from three recent multi-locus or phylogenomic studies of actinopterygian intra-relationships. Topology #1 (T1) was obtained in an analysis of nine nuclear protein-coding loci including 232 taxa, all extant (Near et al., 2012); Topology #2 (T2) is based on an analysis (Betancur-R. et al., 2013) of 20 nuclear and one mitochondrial loci including 1582 extant taxa, to which 240 fossil taxa were added based on previously argued phylogenetic placements (Betancur-R., Ortí & Pyron, 2015); Topology #3 (T3) was obtained from a transcriptomic analysis of 1721 exons (Hughes et al., 2018). In order to achieve consistency in clade names, we relied on the phylogenetic classification proposed by Betancur-R. et al. (2017), itself based on the molecular phylogeny that yielded T2.

All three topologies mostly differ at the level of the first dichotomies within Euteleostei. They all recover an Osmeriformes + Stomiiformes clade (Stomiati) and a Salmoniformes + Esociformes clade, but Galaxiiformes are sister to Neoteleostei *sensu stricto* in T1, to Salmoniformes + Esociformes in T2, and to Stomiati in T3. Similarly, Argentiniformes are sister to Salmoniformes + Esociformes in T1, to this clade + Galaxiiformes in T2, and to Galaxiiformes + Stomiati in T3.

We used a sub-sample of taxa that are included in both our cellularity data set and at least one of the topologies. When two different species of the same genus were used in two different data sets, we considered the genus as a whole, since no case of variability of cell type between species of the same genus is known. This sub-sample retains 100 extant taxa for T1, 292 taxa including 26 fossils for T2, and 121 extant taxa for T3. Every major actinopterygian lineage is present in the resulting trees with a few exceptions for which osteohistological data are lacking entirely, such as the salamanderfish (Lepidogalaxiiformes) and the jellynose fishes (Ateleopodiformes). Ancestral states at the nodes were reconstructed with the *ace* function of the APE package in R (Paradis, Claude & Strimmer, 2004). Two models of ancestral character state estimations were tested: an 'all rates different' (ARD) model (that allows transitions from cellular to acellular and from acellular to cellular to have different frequencies) and a 'symmetrical' model (that constrains transition frequencies to be equal). The difference between the transition frequencies was very low even with the ARD model, but the Akaike information criterion (AIC) very slightly favoured the symmetrical model, leading us to apply the latter to our analyses.
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Time-calibrated multilocus tree of actinopterygians (ray-finned fishes), obtained from the optimisation of the character states ‘cellular bone’ (in dark blue) and ‘acellular bone’ in (yellow) on the topology T2 (Betancur-R. *et al*., 2015). Character states for coded species are at the tips, and the reconstructed ancestral states at the nodes. A few key taxa, discussed in the text, are signalled in bold case. Taxon pictures are from Iglésias (2014b).</figcaption></figure>

**(2) Reconstructed origin of acellular bone**

Results from all topologies recover cellular bone as the plesiomorphic state for actinopterygians, teleosts and every other node outside of Euteleostei, with a very high likelihood of 0.99 (Figs 3, S1–S3). T2 includes fossil taxa but they did not affect the ancestral state reconstructions, since those that were sampled all possess cellular bone in a region of the tree where it is also found in extant taxa (Fig. 3).

The reconstructed ancestral state for Euteleostei is ambiguous and varies from one topology to the other. With T1, the ancestral state for Euteleostei is equivocal. The likelihoods of the ancestral state being ‘cellular’ or ‘acellular’ are between 0.45 and 0.55
609 for three clades: Euteleostei, Argentiniformes + (Esociformes + Salmoniformes) and
610 Esociformes + Salmoniformes (Figs 4, S1). In this scenario, whether cellular bone in
611 argentiniforms and salmoniforms is a secondary reacquisition or the retention of an
612 ancestral state is unclear.

613 With T2, the ancestral state for Euteleostei is acellular bone with a very high likelihood of
614 0.95 (Figs 3, 4, S2), implying that argentiniforms and salmoniforms both reacquired
615 cellular bone secondarily and separately. T3 also implies an ancestral acellular bone for
616 Euteleostei (and a secondary reacquisition of cellular bone in argentiniforms and
617 salmoniforms), albeit with a slightly lower likelihood of 0.89 (Figs. 4, S3).

618 T1 and T2 were both produced with similar methods involving multi-locus molecular data
619 sets adequately covering actinopterygian diversity, and it is difficult to establish whether
620 one is more credible than the other. Phylogenetic resolution at the base of the euteleost
621 tree is poor due to conflict between molecular markers and sparse taxon sampling, and
622 remains a point of contention in the literature (Campbell et al., 2017; Straube et al.,
623 2018; Hughes et al., 2018).

624 Bone histology of the salamanderfish *Lepidogalaxias* has never been studied, but could
625 be critical to accurately reconstruct the ancestral euteleostean state, since it is
626 consistently recovered by molecular studies as the sister group to all other euteleosts (Li
627 et al., 2010; Near et al., 2012; Betancur-R. et al., 2013; Campbell et al., 2017; Straube et
628 al., 2018; Hughes et al., 2018). As long as the phylogeny of euteleosts is not stabilised,
629 and the osteohistology of more taxa not sampled (e.g. other argentiniforms and
630 galaxiids, *Lepidogalaxias*), ambiguity concerning the exact phylogenetic origin of
631 acellular bone will remain. Certain early fossil euteleosts, such as the Late Cretaceous
632 †*Spaniodon* (that has cellular bone) could also potentially play a key role in elucidating
633 this character's evolution. However, their usefulness is hampered by even greater
634 phylogenetic uncertainty than that for living lineages. For example †*Spaniodon* was
635 included in a clade grouping esociforms, salmoniforms and osmeriforms in a
636 phylogenetic analysis (Taverne & Filleul, 2003), but this topology is rejected by modern
637 molecular phylogenies, leaving the position of this fossil taxon unknown.
638 The megadiverse Neoteleostei (more than 18,000 extant species) are reconstructed as
639 having acellular bone ancestrally with all three topologies (likelihood = 0.99; Figs 3, 4,
640 S1-S3). Two distinct neoteleost lineages are reconstructed as having reacquired cellular
641 bone independently: (1) the 'true' tunas *Auxis*, *Katsuwonus*, *Euthynnus* and *Thunnus*,
642 forming the probably monophyletic tribe Thunnini within Scombridae; (2) the opah
643 *Lampris* in the monotypic Lamprididae (Fig. 3).

644 In conclusion, the clade in which acellular bone appears is equivocal with our ancestral
645 state reconstructions. T2 and T3 clearly support that acellular bone appears in
646 Euteleostei, while the ancestral state for Euteleostei is equivocal with T1, leaving open
647 the possibility of an independent appearance of acellular bone in Esociformes and in the
648 clade that unites Stomiati, Galaxiiformes and Neoteleostei (Figs 4, S1-S3).

649 In any case, acellular bone is almost entirely absent outside of Euteleostei, being
650 notably described in: (1) some larval anguilliforms, (2) the clupeiform *Anchoviella* sp., (3)
651 certain cranial dermal bones of the cypriniform *Danio rerio*, and (4) the siluriform
652 *Trichomycterus* sp. (see Section III.2; Fig. 3). It is noteworthy that all these occurrences
653 correspond to either larvae or to taxa with characteristically small adult body sizes. A
654 size-related explanation for the absence of osteocytes cannot be excluded in this
655 context (see Section III.3a for an exploration in the case of *D. rerio*).
Acellular bone seems to appear phylogenetically earlier in scales than in the rest of the skeleton (Meunier, 1987; Meunier & Huysseune, 1992). Since acellular scales are described in clupeomorphs, ostariophysans and every euteleost with the exception of tunas (Table S1), we hypothesise that acellular scales are a character state of the clade Clupeocephala (i.e. all modern teleosts but elopomorphs and osteoglossomorphs). A systematic review of the histology of teleost scales is needed to test this hypothesis suitably. The nature of the external layer of teleost scales is controversial, and some authors have proposed that it has a different evolutionary origin to bone (e.g. Sire et al., 2009), potentially explaining why cellularity is lost earlier in this tissue than in 'true' bone.

**(3) Secondary reacquisition of cellular bone**

*(a) Probable occurrence in salmoniforms*

Our ancestral state reconstructions suggest that cellular bone was secondarily reacquired in salmons, trouts and their relatives (Salmoniformes), but this is equivocal due to topological uncertainty at the base of the euteleost tree (Fig. 4). The same reconstructions also equivocally support a separate secondary reacquisition of osteocytes in argentiniforms. However, since our data only rely on one species (*Argentina silus*) and the phylogenetic position of argentiniformes is highly uncertain, we refrain from commenting until more observations are available.

As described above, cellular bone does not seem to be distributed uniformly within salmoniforms, according to our SRµCT data (Tables 1, S1): (1) in the grayling *Thymallus thymallus* (Thymallinae), we did not observe osteocytes conclusively; (2) in the shortnose cisco *Coregonus reighardi* (Coregoninae), osteocytes are present, but sparsely distributed within bone; (3) in *Stenodus leucichthys* (Coregoninae) and all observed Salmoninae, osteocytes are present, and uniformly distributed inside bone.

Moss (1965) already noted that osteocyte abundance varies within bone elements in at least some salmoniforms, which has been interpreted by Parenti (1986) as a possible 'intermediate' stage between cellular and acellular bone.

Salmoniform phylogeny is currently disputed, particularly in the relationships between thymallines, coregonines and salmonines. Recent molecular studies have recovered three different topologies: Coregoninae + Salmoninae (Alexandrou et al., 2013; Horreo, 2017), Thymallinae + Salmoninae (Near et al., 2012; Crête-Lafrenière, Weir & Bernatchez, 2012; Betancur-R. et al., 2013), and Coregoninae + Thymallinae (Campbell et al., 2013; Macqueen & Johnston, 2014; Hughes et al., 2018), also affecting the three topologies we used in our analyses. These competing phylogenies mean that the pattern of evolution of cellular bone in salmoniforms as a whole is uncertain.

Many salmoniforms are anadromous, meaning that sexually mature individuals migrate upstream over sometimes long distances. This behaviour involves intense and sustained swimming activity, which is likely to affect physiology and metabolism. How it influences bone growth and structure is not fully understood, but it appears that bone responds adaptively to the anadromous lifestyle. In the Atlantic salmon (*Salmo salar*), bones undergo halastasis (a diffuse demineralisation without degradation of the organic matrix) during spawning migration (Kacem & Meunier, 2003, 2009). In addition, *S. salar* shows a prominent increase in the volume of osteocyte lacunae in adult specimens compared to juveniles, which is probably explained by osteocytic osteolysis (Kacem & Meunier, 2000). Moreover, bone in salmons exposed to sustained swimming shows increases in
T1

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

T2

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

T3

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>
Figure 4. Sections of the time-calibrated multilocus trees obtained from the optimisation of the character states ‘cellular bone’ (in dark blue) and ‘acellular bone’ in (yellow) on topologies T1 (Near et al., 2012), T2 (Betancur-R. et al., 2015) and T3 (Hughes et al., 2018), highlighting divergences at the level of the euteleost clade. Character states for coded species are at the tips, and the reconstructed ancestral states at the nodes. Taxon pictures are from Iglésias (2014b).

osteocyte abundance (Totland et al., 2011). These observations support the hypothesis that osteocytes play an important role in resorbing salmon bone during anadromous migration.

Anadromy is likely to be a trait that evolved multiple times in various lineages within salmoniforms from strictly freshwater ancestors (McDowall, 1997, 2001; Alexandrou et al., 2013). Anadromy is widespread in salmonines (especially in the clade formed by Salmo, Oncorhynchus and Salvelinus), and in most species of Coregonus (Alexandrou et al., 2013). Osteocytes are also observed in all of these taxa, while they seem to be absent in the non-migrating freshwater Thymallus and in esociforms, the probable sister group to salmoniforms. The occurrence of cellular bone then roughly follows that of anadromy in this particular teleost clade. A notable exception occurs in the genus Prosopium, a non-migrating taxon that possesses cellular bone. Nevertheless, it is possible that the hypothesised reacquisition of cellular bone in at least some salmoniforms would have allowed or facilitated the evolution of anadromy in these animals, using a combination of halastasis and osteocytic osteolysis to function as a source of calcium and/or phosphorus for metabolism and muscle activity. A more extensive survey of bone histology in salmoniforms, especially for taxa that have not been studied so far (such as the non-migrating salmonines Hucho and Brachymystax), and in anadromous euteleosts outside of salmoniforms, is necessary to investigate the potential coevolution of bone cellularity with anadromous habits.

(b) Convergent occurrences in red-muscle endotherms

Unlike salmoniforms, there is no ambiguity that osteocytes were reacquired secondarily in two acanthomorph lineages (Figs 3, 5A): tunas and the opah (Davesne et al., 2018). Tunas are scombrids, a family that molecular analyses place reliably into the clade Pelagiaria, itself included in the ultradiverse acanthomorph clade Percomorpha (Betancur-R. et al., 2013, 2017; Near et al., 2013; Miya et al., 2013; Alfaro et al., 2018). The ‘true’ tunas (Thunnini) consist of five genera (Allothunnus, Auxis, Euthynnus, Katsuwonus and Thunnus), and their monophyly is supported by morphological (Collette et al., 1984; Carpenter, Collette & Russo, 1995) and most molecular phylogenies (Block et al., 1993; Betancur-R. et al., 2013; Miya et al., 2013). The opah (Lampris sp.) is a lampridiform, a clade whose phylogenetic position within acanthomorphs is uncertain, but that branches outside of Percomorpha in any case (Betancur-R. et al., 2013; Near et al., 2013; Davesne et al., 2014, 2016; Alfaro et al., 2018). There is then clear evidence that the secondary reacquisition of osteocytes occurred independently in both lineages (Davesne et al., 2018).

While cellular bone has long been known in tunas (Kölliker, 1859; Stéphan, 1900; Amprino & Godina, 1956; Moss, 1961b), fewer data were available on other scombrid taxa and acellular bone was known only from the Atlantic mackerel Scomber scombrus and the Spanish mackerel Scomberomorus maculatus (Kölliker, 1859; Amprino & Godina, 1956; Moss, 1961b). Our SRµCT data allow us to confirm the absence of
osteocytes from the ribs of a larger sample of scombrids: the butterfly kingfish *Gasterochisma melampus*, the blue mackerel *Scomber australasicus*, the wahoo *Acanthocybium solandri*, the bonito *Sarda orientalis* and the dogtooth 'tuna' *Gymnosarda unicolor* (Table 1, Fig. 5C, D). *Sarda* and *Gymnosarda* are particularly relevant because they probably constitute the sister group to Thunnini (Collette *et al.*, 1984; Block *et al.*, 1993; Miya *et al.*, 2013). All of these taxa are outside of Thunnini, supporting that 'true' tunas are the only scombrids with cellular bone (Fig. 5A, E).

Within lampridiforms, acellular bone has been described in the ribbonfishes *Trachipterus trachypterus* and *Zu cristatus* (Kölliker, 1859), in the oarfish *Regalecus russelii* (Paig-Tran *et al.*, 2016) and in the veliferid *Velifer hypselopterus* (Davesne *et al.*, 2018). Our SRµCT data show that the veliferid *Metavelifer multiradiatus* also lacks osteocytes (Table 1), and veliferids are probably sister to all other lampridiforms (Olney, Johnson & Baldwin, 1993; Wiley, Johnson & Dimmick, 1998; Davesne *et al.*, 2014). The absence of osteocytes in veliferids, and in the Cretaceous stem lampridiform †*Aipichthys* velifer supports that acellular bone is plesiomorphic for lampridiforms (Davesne *et al.*, 2018). Thus, the opah is secondarily cellular within lampridiforms, akin to 'true' tunas within scombrids (Fig. 5A).

Tunas and the opah share many life-history traits, to which the reappearance of osteocytes could potentially be imputed. However, a closer examination of these traits across acanthomorph diversity reveals that most do not correlate with the presence of osteocytes. (1) Sustained, active swimming is also found in other large-bodied pelagic predators with acellular bone, such as carangids (Smith-Vaniz *et al.*, 1995), the dolphinfish *Coryphaena hippurus* (Moss, 1961b), billfishes (Kölliker, 1859; Amprino & Godina, 1956; Moss, 1961b; Atkins *et al.*, 2014) and several scombrids outside of 'true' tunas (Fig. 5A, C, D). (2) A large body size does not seem to be a factor either: within scombrids, the osteocytic bullet tuna *Auxis rochei* rarely exceeds 350 mm in total length as an adult (Collette & Nauen, 1983), while the dogtooth 'tuna' *Gymnosarda unicolor* and wahoo *Acanthocybium solandri* both commonly exceed 1000 mm in total length (Collette & Nauen, 1983) and are anosteocytic (Fig. 5D). Other very large pelagic acanthomorphs such as the oarfish *Regalecus* sp., billfishes, or the oceanic sunfish *Mola mola* (Kölliker, 1859) all have acellular bone as well. (3) Finally, the reacquisition of osteocytes does not seem to be linked with structural homeostasis: bone in tunas, opah and billfishes appears to have active, intense and sustained resorption and remodelling activities (Fig. 5B, D, E) evidenced by the extensive presence of secondary bone (Amprino & Godina, 1956; Poplin *et al.*, 1976; Castanet & Ricqlès, 1986; Atkins *et al.*, 2014; Davesne *et al.*, 2018). However, bone in billfishes is acellular (Fig. 5B), confirming that this intense remodelling activity does not require the presence of osteocytes (Atkins *et al.*, 2014; Currey *et al.*, 2017).

Conversely, a correlation between cellular bone and endothermy in acanthomorphs appears to be more substantiated (Meunier, 1987; Ricqlès *et al.*, 1991; Meunier & Huysseune, 1992; Davesne *et al.*, 2018). Our new SRµCT data confirm that cellular bone co-occurs with a modification in the distribution and position of the lateral aerobic red muscles (Fig. 5A), that concentrate in the anterior portion of the body and become internalised within myotomes, coming closer to the axial skeleton; this configuration is unique to 'true' tunas amongst scombrids (Graham, Koehrn, & Dickson, 1983; Block *et al.*, 1993; Graham & Dickson, 2000, 2004).

796
<figure><img src="image_11.png" /><figcaption>**Figure 5.** (A) Phylogenetic distribution of bone type in endothermic acanthomorph teleosts and their close relatives (modified from Davesne *et al.*, 2018). The squares represent bone type (acellular in yellow, cellular in dark blue) and thermal physiology (ectothermy in white, cranial endothermy in salmon pink, red-muscle endothermy in red). Taxon pictures from Iglésias (2014b) and R. N. Cada (www.fishbase.org). (B) Thin section through the rostrum of the marlin *Makaira nigricans*, a billfish. Bone is acellular, but shows secondary osteons delimited by resorption lines (white arrowheads). Photograph courtesy of A. Atkins. (C) Synchrotron tomographic slice in a rib of the butterfly kingfish *Gasterochisma melampus*, a scombrid (AMNH I-93480 SD). Bone is acellular. (D) Synchrotron tomographic slice in a rib of the dogtooth 'tuna' *Gymnosarda unicolor*, a scombrid (MNHN.ICOS.00492). Bone is acellular. Note secondary bone deposition around the blood vessels, delimited by resorption lines (white arrowheads). (E) Synchrotron tomographic slice in a rib of the 'true' tuna *Euthynnus affinis* (AMNH I-56274 SD). Bone is cellular (osteocytes marked with black arrowheads), with extensive deposition of secondary bone delimited by resorption lines (white arrowheads). (C-E) Images produced by D. Davesne.</figcaption></figure>

This configuration is thought to be associated with heat production and retention (i.e. endothermy): the heat that is produced by muscle activity during swimming is insulated from the exterior and retained within the body due to a network of specialised blood vessels, named retia (Graham *et al.*, 1983; Graham & Dickson, 2001; Katz, 2002). This peculiar configuration has been called 'red-muscle endothermy' by various authors

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Block et al., 1993; Dickson & Graham, 2004; Watanabe et al., 2015). The opah developed a distinct form of red-muscle endothermy in which the red pectoral-fin muscles produce most of the heat, are insulated from the outside by a thick fatty layer, and the heat is kept and redistributed *via* retia located within the gills (Wegner et al., 2015). A form of endothermy is also found in two other acanthomorph lineages: billfishes and the butterfly kingfish *Gasterochisma melampus*, a non-Thunnini scombrid (Fig. 5A). In these cases, heat is produced by specialised modified ocular muscles (the superior rectus in billfishes and the lateral rectus in *G. melampus*) that lost their contractile activity and cycle calcium ions between the cytoplasm and sarcoplasmic reticulum (Carey, 1982; Block, 1986, 1994; Dickson & Graham, 2004). Since it only warms the brain and the eyes, this configuration is often called 'cranial endothermy' (Dickson & Graham, 2004). Osteocytes are absent in the bill and ribs of billfishes (Atkins et al., 2014), and our SRµCT data failed to find them in a rib of *G. melampus* (Table 1, Fig. 5B,C), implying that cranial endotherms, unlike red-muscle endotherms, have acellular bone. We also observe acellular bone in the sclerotic ossicles of *G. melampus* and of the billfishes *Kajikia albida* and *Xiphias gladius* (Table 1), confirming that the cellularity of a bone is not affected by its proximity to the heat-generating muscles. In the opah, the sclerotic ossicles have cellular bone like the rest of the skeleton (Table 1).

Heat production by red muscles involved in swimming (rather than modified ocular muscles) and redistribution in a large proportion of the body (rather than in the brain region only) is the key distinction between red-muscle and cranial endothermy. Given that both acanthomorph lineages that developed red-muscle endothermy are also the only ones that reacquired osteocytes, a correlation between these characters is likely (Davesne et al., 2018). As for salmoniforms, we can hypothesise that the correlation stems from an intense muscular activity associated with sustained swimming. The latter is necessary both to hunt prey and to produce heat *via* the myotomal or pectoral red muscles. Since muscles are important consumers of calcium, an element primarily found in bony tissues, osteolytic osteolysis potentially played an important role in the appearance of red-muscle endothermic strategies. Whether the reacquisition of osteocytes facilitated the evolution of red-muscle endothermy, or both characters coevolved under a common selective pressure is unclear.

(c) *Structural evidence for a re-acquisition in salmoniforms, tunas and opahs*

While osteocyte morphology is very diverse in vertebrate bone in general, two main morphologies seem to occur in teleost cellular bone (Fig. 6). In the first type, osteocytes have a rounded or irregular cell body, and show numerous, thin cytoplasmic processes that branch into canaliculi in all directions. This gives these osteocytes a typically 'star-shaped' morphology (Fig. 6A). In the second type, osteocytes are much more elongate ('spindle-shaped') and orientate in a preferential direction, presumably following the collagen lamellae of the extracellular matrix (Kerschnitzki et al., 2011). Their cell bodies are more regular in shape, and they have only two cytoplasmic processes that are located at the extremities of the cell body, aligning with its long axis. They also have very few, non-branching canaliculi, that tend to orientate in preferential directions (Fig. 6B, C). Both osteocyte types seem to coexist within teleost cellular bone, for example in *D. rerio* (Weigele & Franz-Odendaal, 2016). Conversely, in the bone of salmoniforms and 'true' tunas (Stéphan, 1900; Meunier & Huysseune, 1992; Totland et al., 2011; Davesne et al., 2018), the spindle-shaped osteocytes seem to be the only type that is present (Fig.
6B,C). In the opah, osteocytes are close to the 'spindle-shaped' morphology, since they have very few cytoplasmic processes and canaliculi that all orientate in a preferential direction, but they are not located at the extremities of the cell body like in tunas and salmoniforms (Fig. 6D). It is not clear whether this second type of osteocytes forms a connected canalicular system; at least in salmons they might not be connected to each other at all (Totland et al., 2011). Moreover, their morphology does not seem to change significantly between primary and remodelled bone, for example in the opah (Davesne et al., 2018).

It appears that the three lineages that have in common an inferred or likely reacquisition of cellular bone share these structural similarities in osteocyte morphology. This suggests that their peculiar morphology might be linked with the evolutionary reacquisition of osteocytes from an ancestral acellular bone. Weigele & Franz-Odendaal (2016) proposed that these types of osteocytes have different developmental origins, and that the elongate, spindle-shaped osteocytes are derived from the elongate 'osteoblast-like' cells that line the bone. It is possible that all secondarily reacquired osteocytes share this unique developmental origin, and that the other, 'typical' osteocytes derive from a mode of formation that does not occur in salmoniforms, tunas and opahs and was possibly lost at the euteleost node. Structural similarities in osteocyte morphology appear further to support that their reacquisition is underlined by shared, and not fully understood, mechanisms.

<figure><img src="image_12.png" /><figcaption>**Figure 6.** Osteocyte morphology in taxa that retain the ancestral cellular bone (A) or that secondarily reacquired it from acellular ancestors (B–D). (A) 'Star-shaped' osteocytes in the dorsal-fin spine of the carp *Cyprinus carpio*, an ostariophysan. Note the irregular shape of the lacunae, and the numerous cytoplasmic processes ending in canaliculi branching in all directions (arrows). Modified from Meunier & Huysseune (1992). (B) 'Spindle-shaped' osteocytes in the coracoid of the salmon *Salmo salar* (NHMUK, uncatalogued), a salmoniform. Note the two cytoplasmic processes located at both extremities of the cell axis (arrows). Photograph by D. Davesne and A. D. Schmitt. (C) 'Spindle-shaped' osteocytes in the dorsal-fin spine of the tuna *Katsuwonus pelamis*, an acanthomorph. Note the two cytoplasmic processes located at both extremities of the cell axis (arrows). Photograph by F. J. Meunier. (D) 'Spindle-shaped'</figcaption></figure>
osteocytes in the rib of the opah *Lampris* sp. (MNHN-ZA-AC-A-7506), an acanthomorph. Note the few
canaliculi, all pointing in the same direction (arrows). Modified from Davesne *et al.* (2018).

898
899
V. THE ROLE OF MINERAL HOMEOSTASIS IN THE LOSS AND
REACQUISITION OF OSTEOCYTES

900 Of the main functions of bone, those related to mechanical homeostasis (e.g. strain
901 detection and bone remodelling) function in the absence of osteocytes (see Section II.2).
902 Mineral homeostasis, on the other hand, relies on a variety of mechanisms including
903 halastasis, i.e. a diffuse demineralisation of the bone without affecting its organic matrix
904 (Lopez, 1976; Kacem & Meunier, 2003; Sbaihi et al., 2007), osteoblast-mediated bone
905 resorption (Francillon-Vieillot et al., 1990; Ricqlès et al., 1991), and osteocyte-mediated
906 bone resorption (osteocytic osteolysis). Halastasis has only been observed so far in taxa
907 with cellular bone, and evidently osteocytic osteolysis is lacking in acellular bone. This
908 suggests that acellular bone is less efficient than cellular bone in regulating mineral
909 content in the body. In aquatic animals like teleosts, however, it is likely that enough
910 calcium and phosphorus is available from the diet and ambient water to compensate the
911 less-efficient mineral homeostasis (Witten & Huysseune, 2009; Cohen et al., 2012;
912 Shahar & Dean, 2013; Doherty et al., 2015). Therefore, it is possible that osteocytes are
913 not required either for mechanical or mineral homeostasis in teleosts because both
914 functions can be achieved by other means (Dean & Shahar, 2012). In that context, the
915 disappearance of osteocytes in at least some euteleosts could hypothesised to be due
916 to a relaxed selective pressure that does not compensate the cost of maintaining them
917 (Shahar & Dean, 2013; Doherty et al., 2015). However, this hypothesis alone clearly
918 does not explain the phylogenetic distribution of acellular bone: if a low selective
919 pressure was not preventing the loss of osteocytes, we would expect this phenomenon
920 to be widespread in teleosts and other aquatic vertebrates. Our data support the
921 contrary: probably just a single main disappearance of cellular bone, potentially in
922 euteleosts, along with other, extremely rare losses in species-poor lineages (at least in
923 *Trichomycterus* sp. and some bones of *Danio rerio*) that could be size-related (see
924 Section III.3a). Other mechanisms may have been involved, such as developmental
925 heterochrony (e.g. Parenti, 1986).
926

927 Tunas, opahs and potentially salmoniforms all reacquired osteocytes secondarily (see
928 Section IV.3). They also share specific adaptations that lead to increased and sustained
929 muscular activity: an anadromous migrating behaviour in salmoniforms, and specialised
930 red muscles involved in heat production in tunas and opahs. At least in these taxa, the
931 main function of osteocytes could be that of osteocytic osteolysis, as has been proposed
932 for teleosts as a whole by previous authors (e.g. Cohen et al., 2012). Reacquiring
933 osteocytes would allow the use of bone as a major source of calcium and phosphorus,
934 which would constitute a key adaptive advantage in an organism experiencing increased
935 pressure on maintaining efficient muscle activity. Mineral homeostasis is then proposed
936 to have played a major role in the evolution of acellular bone in teleost fishes.
937

938
939
VI. CONCLUSION

(1) According to our ancestral state reconstructions (Figs. 3,4), acellular bone is a
940 synapomorphy of either Euteleostei (as supported by two out of three tree topologies), or
941 of a smaller clade consisting of Stomiati, Galaxiiformes and Neoteleostei (as supported
942 by one tree topology). New analyses incorporating histological information on more
943 euteleost taxa (e.g. *Lepidogalaxias*, more argentiniforms and galaxiiforms) including
944 early fossil representatives, and a stabilisation of the euteleost phylogeny, are both
945 necessary to clarify the ambiguity on the exact clade in which acellular bone evolved.
946 Given the equivocal support for the euteleost ancestral state in T1 (Fig. 4), and
947 considering other lines of evidence (such as structural similarities between osteocytes in
948 salmoniforms and tunas; Fig. 6), we consider it to be more likely that osteocytes were
949 lost in Euteleostei, with a secondary reacquisition in salmoniforms.

950 (2) Scales became acellular earlier than the rest of the skeleton in teleost phylogeny,
951 probably in the clade Clupeocephala, which includes clupeomorphs, ostariophysans and
952 euteleosts. More comparative data are needed to confirm this hypothesis.

953 (3) Scales aside, acellular bone appears to be almost absent outside of Euteleostei (Fig.
954 3). We reject its occurrence in the bonefish *Albula vulpes* and the mooneyes *Hiodon* sp.
955 The catfish *Trichomycterus* sp. appears to be acellular and the zebrafish *Danio rerio* has
956 both cellular and acellular bone in its cranial skeleton, but the occurrence of acellular
957 bone in both may be explained by their small adult body sizes. More comparative data
958 encompassing multiple bones in multiple teleost species will be necessary to support
959 whether these are isolated or more widespread occurrences.

960 (4) Within spiny-rayed teleosts (Acanthomorpha), osteocytes have been secondarily
961 reacquired in tunas (Thunnini) and in the opah *Lampris* sp. The exact co-occurrence of
962 osteocytes with that of an endothermic physiology based on red muscle activity (Fig. 5)
963 strongly suggests that these traits are correlated in acanthomorph teleosts. Other traits
964 shared by tunas and the opah are also present in some acanthomorphs with acellular
965 bone (e.g. large body size, cranial endothermy, intense bone remodelling), and so are
966 less plausible as explanations of the evolutionary reacquisition of osteocytes.

967 (5) Acellular teleost bone can perform every structural and mechanical function of
968 cellular bone (e.g. detection of strains and constraints, adaptive remodelling) and both
969 have very similar mechanical properties. However, acellular bone seems to be less
970 efficient in terms of mineral homeostasis, probably because it lacks the possibility to
971 perform osteocytic osteolysis. Osteocytes are secondarily reacquired in lineages that
972 may have increased requirements for minerals, mostly to support an intense and
973 sustained muscular activity: the red-muscle endotherms and (potentially) the
974 anadromous salmoniforms. This pattern seems to support the hypothesis that the most
975 fundamental role of osteocytes in teleost bone physiology is that of mineral, rather than
976 mechanical homeostasis.

977 (6) Our review of the available evidence with the addition of new data allowed us to
978 establish for the first time a detailed phylogenetic hypothesis for the evolution of
979 osteocytes in teleosts. Acellular bone is a fundamental model to understand bone
980 function, because it lacks a cell type that is classically thought to play a major role in the
981 structure and maintenance of bony tissues. This review highlights the need to use large-
982 scale comparative histological data, backed by a rigorous phylogenetic framework, to
983 address fundamental questions on the interplay of bone structure, function and
984 physiology.

985
986 VII. ACKNOWLEDGEMENTS
987 We thank the colleagues, curators and collection managers that granted or facilitated
988 access to specimens: R. Arrindell and B. Brown (AMNH), A. López-Arbarello (LMU
989 Munich), G. Clément, D. Germain and P. Béarez (MNHN), E. Bernard and Z. Johanson
990 (NHMUK), H. Ketchum, J. Hay, M. Carnall and E. Westwig (OUMNH), H. Furrer and C.
991 Klug (PIMUZ), D. Nelson (UMMZ). R. Betancur-R. (University of Oklahoma) provided the
992 tree files critical for the T2 ancestral state reconstruction analysis. A. Bailleul (Chinese
993 Academy of Sciences) is thanked for discussion on acellular bone in tetrapods. H.
994 Middleton and C. Nicklin are thanked for providing fossil specimens for the study, and A.
995 Atkins (BINA), S. Iglésias (MNHN) and N. Tamura for providing illustrations. J. Wells and
996 O. Green (University of Oxford) are thanked for preparing histological sections and for
997 help with their imaging. ESRF beam time was obtained via two proposals (LS2614,
998 LS2758). The authors thank S. Sanchez (Uppsala University), V. Fernandez (NHMUK)
999 and P. Tafforeau (ESRF) for their crucial help during the synchrotron experiments,
1000 image treatment and reconstruction, as well as for scientific discussion. Two anonymous
1001 reviewers provided valuable comment that improved a previous version of the
1002 manuscript. This work was supported by the Leverhulme Trust (RPG-2016-168) and by
1003 a Junior Research Fellowship at Wolfson College, University of Oxford (D.D.).
1004
1005 REFERENCES
1006 *References marked with an asterisk are cited only within the supporting information.*
1007
1008 ALEXANDROU, M.A., SWARTZ, B.A., MATZKE, N.J. & OAKLEY, T.H. (2013). Genome
duplication and multiple evolutionary origins of complex migratory behavior in
1009 Salmonidae. *Molecular Phylogenetics and Evolution* **69**, 514–523.
1010
1011 ALFARO, M.E., FAIRCLOTH, B.C., HARRINGTON, R.C., SORENSON, L., FRIEDMAN, M.,
1012 THACKER, C.E., OLIVEROS, C.H., ČERNÝ, D. & NEAR, T.J. (2018). Explosive
diversification of marine fishes at the Cretaceous–Palaeogene boundary. *Nature*
1013 *Ecology & Evolution* **2**, 688–696.
1014
1015 AMPRINO, R. & GODINA, G. (1956). Osservazioni sul rinnovamento strutturale dell’osso in
1016 Pesci Teleostei. *Pubblicazioni della Stazione Zoologica di Napoli* **28**, 62–71.
1017
1018 ARRATIA, G. (2015). Complexities of early Teleostei and the evolution of particular
morphological structures through time. *Copeia* **103**, 999–1025.
1019
1020 ATKINS, A., DEAN, M.N., HABEGGER, M.L., MOTTA, P.J., OFER, L., REPP, F., SHIPOV, A.,
WEINER, S., CURREY, J.D. & SHAHAR, R. (2014). Remodeling in bone without
1021 osteocytes: billfish challenge bone structure–function paradigms. *Proceedings of
the National Academy of Sciences* **111**, 16047–16052.
1022
1023 ATKINS, A., MILGRAM, J., WEINER, S. & SHAHAR, R. (2015a). The response of anosteocytic
1024 bone to controlled loading. *Journal of Experimental Biology* **218**, 3559–3569.
1025
1026 ATKINS, A., REZNIKOV, N., OFER, L., MASIC, A., WEINER, S. & SHAHAR, R. (2015b). The
three-dimensional structure of anosteocytic lamellated bone of fish. *Acta
Biomaterialia* **13**, 311–323.
1027
1028 BAILLEUL, A.M. & HORNER, J.R. (2016). Comparative histology of some craniofacial
sutures and skull-base synchondroses in non-avian dinosaurs and their extant
1029 phylogenetic bracket. *Journal of Anatomy* **229**, 252–285.
1030
1031 BEAREZ, P., MEUNIER, F.J. & KACEM, A. (2005). Description morphologique et
histologique de l'hyperostose vertébrale chez la thonine noire, *Euthynnus lineatus*
1032 (Teleostei: Perciformes: Scombridae). *Cahiers de Biologie Marine* **46**, 21–28.
1033
1034 BERG, L.S. (1947). *Classification of fishes both Recent and fossil*. J. W. Edwards, Ann
1035 Arbor.
1036
BETANCUR-R., R., BROUGHTON, R.E., WILEY, E.O., CARPENTER, K., LOPEZ, J.A., LI, C.,
1037 HOLCROFT, N.I., ARCILA, D., SANCIANGCO, M., CURETON, J.C., ZHANG, F., BUSER, T., CAMPBELL, M.A., BALLESTEROS, J.A., ROA-VARON, A., *ET AL*. (2013). The tree of life and a new classification of bony fishes. *PLoS Currents Tree of Life* **2013 Apr 1**, DOI: 10.1371/currents.tol.53ba26640df0ccaee75bb165c8c26288.

1041 BETANCUR-R., R., ORTI, G. & PYRON, R.A. (2015). Fossil-based comparative analyses reveal ancient marine ancestry erased by extinction in ray-finned fishes. *Ecology Letters* **18**, 441–450.

1044 BETANCUR-R., R., WILEY, E.O., ARRATIA, G., ACERO, A., BAILLY, N., MIYA, M., LECOINTRE, G. & ORTÍ, G. (2017). Phylogenetic classification of bony fishes. *BMC Evolutionary Biology* **17**, 162.

1047 BLANC, M. (1953). Contribution à l'étude de l'ostéogénèse chez les Poissons Téléostéens. *Mémoires du Muséum national d'Histoire naturelle, Série A* **7**, 1–146.

1049 BLOCK, B.A. (1986). Structure of the brain and eye heater tissue in marlins, sailfish, and spearfishes. *Journal of Morphology* **190**, 169–189.

1051 BLOCK, B.A. (1994). Thermogenesis in muscle. *Annual Review of Physiology* **56**, 535–577.

1053 BLOCK, B.A., FINNERTY, J.R., STEWART, A.F.R. & KIDD, J. (1993). Evolution of endothermy in fish: mapping physiological traits on a molecular phylogeny. *Science* **260**, 210–214.

1056 BONEWALD, L.F. (2011). The amazing osteocyte. *Journal of Bone and Mineral Research* **26**, 229–238.

1058 BRAZEAU, M.D. & FRIEDMAN, M. (2014). The characters of Palaeozoic jawed vertebrates. *Zoological Journal of the Linnean Society* **170**, 779–821.

1060 *BRITO, P.M., ALVARADO-ORTEGA, J. & MEUNIER, F.J. (2017). Earliest known lepisosteoid extends the range of anatomically modern gars to the Late Jurassic. *Scientific Reports* **7**, 17830.

1063 BRITO, P.M. & MEUNIER, F.J. (2000). The morphology and histology of the scales of Aspidorhynchidae (Actinopterygii, Halecostomi). *Geobios* **33**, 105–111.

1065 BRITO, P.M., MEUNIER, F.J. & GAYET, M. (2000). The morphology and histology of the scales of the Cretaceous gar *Obaichthys* (Actinopterygii, Lepisosteidae): phylogenetic implications. *Comptes Rendus de l'Académie des Sciences, Paris, Sciences de la Terre et des Planètes* **331**, 823–829.

1069 *BRUNEEL, B. & WITTEN, P.E. (2015). Power and challenges of using zebrafish as a model for skeletal tissue imaging. *Connective Tissue Research* **56**, 161–173.

1071 BUFFRÉNIL, V. DE, CLARAC, F., CANOVILLE, A. & LAURIN, M. (2016). Comparative data on the differentiation and growth of bone ornamentation in Gnathostomes (Chordata: Vertebrata). *Journal of Morphology* **277**, 634–670.

1074 CAMPBELL, M.A., ALFARO, M.E., BELASCO, M. & LOPEZ, J.A. (2017). Early-branching euteleost relationships: areas of congruence between concatenation and coalescent model inferences. *PeerJ* **5**, e3548.

1077 CAMPBELL, M.A., LÓPEZ, J.A., SADO, T. & MIYA, M. (2013). Pike and salmon as sister taxa: detailed intraclade resolution and divergence time estimation of Esociformes + Salmoniformes based on whole mitochondrial genome sequences. *Gene* **530**, 57–65.

1081 CAO, L., MORIISHI, T., MIYAZAKI, T., IIMURA, T., HAMAGAKI, M., NAKANE, A., TAMAMURA, Y., KOMORI, T. & YAMAGUCHI, A. (2011). Comparative morphology of the osteocyte lacunocanalicular system in various vertebrates. *Journal of Bone and Mineral*
1084 *Metabolism* **29**, 662–670.

1085 CAREY, F.G. (1982). A brain heater in the swordfish. *Science* **216**, 1327–1329.

1086 CARPENTER, K.E., COLLETTE, B.B. & RUSSO, J.L. (1995). Unstable and stable classifications of scombroid fishes. *Bulletin of Marine Science* **56**, 379–405.

1088 CASTANET, J. & RICQLES, A. DE (1986). Sur la relativité de la notion d'ostéones primaires et secondaires et de tissus osseux primaire et secondaire en général. *Annales des Sciences naturelles, Zoologie, Paris* **8**, 103–109.

1091 CHEVRINAIS, M., SIRE, J. & CLOUTIER, R. (2017). From body scale ontogeny to species ontogeny: Histological and morphological assessment of the Late Devonian acanthodian *Triazeugacanthus affinis* from Miguasha, Canada. *PLoS ONE* **12**, e0174655.

1095 COATES, M.I., SEQUEIRA, S.E.K., SANSOM, I.J. & SMITH, M.M. (1998). Spine and tissues of ancient sharks. *Nature* **396**, 729–730.

1097 COHEN, L., DEAN, M.N., SHIPOV, A., ATKINS, A., MONSONEGO-ORNAN, E. & SHAHAR, R. (2012). Comparison of structural, architectural and mechanical aspects of cellular and acellular bone in two teleost fish. *The Journal of Experimental Biology* **215**, 1983–1993.

1101 COLLETTE, B.B. & NAUEN, C.E. (1983). *FAO Species Catalogue, Vol. 2: Scombrids of the World. An Annotated and Illustrated Catalogue of Tunas, Mackerels, Bonitos and Related Species Known to Date*. Food and Agriculture Organization of the United Nations, Rome.

1105 COLLETTE, B.B., POTTHOFF, T., RICHARDS, W.J., UEYANAGI, S., RUSSO, J.L. & NISHIKAWA, Y. (1984). Scombroidei: development and relationships. In *Ontogeny and Systematics of Fishes, Special Publication Number 1, American Society of Ichthyologists and Herpetologists* (eds H.G. MOSER, W.J. RICHARDS, D.M. COHEN, M.P. FAHAY, A.W. KENDALL & S.L. RICHARDSON), pp. 591–620. Allen Press, Lawrence, Kansas.

1111 CRÊTE-LAFRENIÈRE, A., WEIR, L.K. & BERNATCHEZ, L. (2012). Framing the Salmonidae family phylogenetic portrait: a more complete picture from increased taxon sampling. *PLoS ONE* **7**, e46662.

1114 CURREY, J.D., DEAN, M.N. & SHAHAR, R. (2017) Revisiting the links between bone remodelling and osteocytes: insights from across phyla. *Biological Reviews* **92**, 1702–1719.

1117 CURREY, J.D. & SHAHAR, R. (2013). Cavities in the compact bone in tetrapods and fish and their effect on mechanical properties. *Journal of Structural Biology* **183**, 107–122.

1120 DAGET, J., GAYET, M., MEUNIER, F.J. & SIRE, J.Y. (2001). Major discoveries on the dermal skeleton of fossil and recent polypteriforms: a review. *Fish and Fisheries* **2**, 113–124.

1123 DAVESNE, D., FRIEDMAN, M., BARRIEL, V., LECOINTRE, G., JANVIER, P., GALLUT, C. & OTERO, O. (2014). Early fossils illuminate character evolution and interrelationships of Lampridiformes (Teleostei, Acanthomorpha). *Zoological Journal of the Linnean Society* **172**, 475–498.

1127 DAVESNE, D., GALLUT, C., BARRIEL, V., JANVIER, P., LECOINTRE, G. & OTERO, O. (2016). The phylogenetic intrarelationships of spiny-rayed fishes (Acanthomorpha, Teleostei, Actinopterygii): fossil taxa increase the congruence of morphology with molecular data. *Frontiers in Ecology and Evolution* **4**, 129.
1131 DAVESNE, D., MEUNIER, F.J., FRIEDMAN, M., BENSON, R.B.J. & OTERO, O. (2018).

1132 Histology of the endothermic opah (*Lampris* sp.) suggests a new structure–function

1133 relationship in teleost fish bone. *Biology Letters* **14**, 20180270.

1134 DEAN, M.N. & SHAHAR, R. (2012). The structure-mechanics relationship and the
1135 response to load of the acellular bone of neoteleost fish: a review. *Journal of
1136 Applied Ichthyology* **28**, 320–329.

1137 DENISON, R.H. (1963). The early history of the vertebrate calcified skeleton. *Clinical*

1138 *Orthopaedics and Related Research* **31**, 141–152.

1139 DESSE, G., MEUNIER, F.J., PERON, M. & LAROCHE, J. (1981). Hyperostose vertébrale chez l’animal. *Rhumatologie* **33**, 105–119.

1141 DICKSON, K.A. & GRAHAM, J.B. (2004). Evolution and consequences of endothermy in fishes. *Physiological and Biochemical Zoology* **77**, 998–1018.

1143 DOHERTY, A.H., GHALAMBOR, C.K. & DONAHUE, S.W. (2015). Evolutionary physiology of bone: bone metabolism in changing environments. *Physiology* **30**, 17–29.

1145 DONOGHUE, P.C.J. & SANSOM, I.J. (2002). Origin and early evolution of vertebrate skeletonization. *Microscopy Research and Technique* **59**, 352–372.

1147 DOWNS, J.P. & DONOGHUE, P.C.J. (2009). Skeletal histology of *Bothriolepis canadensis* (Placodermi, Antiarchi) and evolution of the skeleton at the origin of jawed vertebrates. *Journal of Morphology* **270**, 1364–1380.

1150 EKANAYAKE, S. & HALL, B.K. (1987). The development of acellularity of the vertebral bone of the Japanese medaka, *Oryzias latipes* (Teleostei; Cyprinidontidae). *Journal of Morphology* **193**, 253–261.

1153 EKANAYAKE, S. & HALL, B.K. (1988). Ultrastructure of the osteogenesis of acellular vertebral bone in the Japanese medaka, *Oryzias latipes* (Teleostei, Cyprinodontidae). *The American Journal of Anatomy* **182**, 241–249.

1156 ENLOW, D.H. & BROWN, S.O. (1956). A comparative histological study of fossil and recent bone tissues. Part I. *The Texas Journal of Science* **8**, 405–443.

1158 ESCHMEYER, W.N., FRICKE, R. & VAN DER LAAN, R. (2018). Catalog of Fishes: genera, species, references. http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp [accessed 19 March 2018].

1162 *ESTÊVÃO, M.D., SILVA, N., REDRUELLO, B., COSTA, R., GREGÓRIO, S., CANÁRIO, A.V.M. & POWER, D.M. (2011). Cellular morphology and markers of cartilage and bone in the marine teleost *Sparus auratus*. *Cell and Tissue Research* **343**, 619–635.

1165 FIAZ, A.W., VAN LEEUWEN, J.L. & KRANENBARG, S. (2010). Phenotypic plasticity and mechano-transduction in the teleost skeleton. *Journal of Applied Ichthyology* **26**, 289–293.

1168 FRANCILLON-VIEILLOT, H., BUFFRENIL, V. DE, CASTANET, J., GERAUDIE, J., MEUNIER, F.J., SIRE, J.-Y., ZYLBERBERG, L. & RICQLES, A. DE (1990). Microstructure and mineralization of vertebrate skeletal tissues. In *Skeletal Biomineralization: Patterns, Processes and Evolutionary Trends. Volume I* (ed J.G. CARTER), pp. 471–530. Van Nostrand Reinhold, New York.

1173 FRANZ-ODENDAAL, T.A., HALL, B.K. & WITTEN, P.E. (2006). Buried alive: how osteoblasts become osteocytes. *Developmental Dynamics* **235**, 176–190.

1175 *FROESE, R. & PAULY, D. (2019). FishBase. www.fishbase.org [accessed 29 January 2019].

1177 *GAUDANT, J. & MEUNIER, F.J. (1996). Etude d'un cas de pachyostose chez un Clupeidae
1178 fossile du Miocène terminal de l'ouest algérien, *Sardina? crassa* (Sauvage). *Cybium* 20, 169-183.

1179

1180 GAUDANT, J. & MEUNIER, F.J. (2004). Un test pour déterminer la position systématique du

1181 genre *Thaumaturus* Reuss 1844 (poisson téléostéen) : l'approche

1182 paléohistologique. *Courier Forschung-Institut Senckenberg* **252**, 79-93.

1183 GAYET, M. & MEUNIER, F.J. (1992). Polyptériformes (Pisces, Cladistia) du Maastrichtien

1184 et du Paléocène de Bolivie. *Geobios* **14**, 159-168.

1185 GEGENBAUR, C., KÖLLIKER, A. & MÜLLER, H. (1853). Bericht über einige im Herbste 1852

1186 in Messina angestellte vergleichend-anatomische Untersuchungen. *Zeitschrift für*

1187 wissenschaftliche Zoologie **4**, 299-373.

1188 GÉRAUDIE, J. & MEUNIER, F.J. (1984). Structure and comparative morphology of

1189 camptotrichia of lungfish fins. *Tissue and Cell* **16**, 217-236.

1190 GERMAIN, D., SCHNELL, N.K. & MEUNIER, F.J. (in press). Some histological data on bone

1191 and teeth in the barbeled dragonfishes *Borostomias panamensis* Regan &

1192 Trewavas, 1929 and *Stomias boa* Reinhardt 1842 (Stomiidae; Stomiiformes).

1193 *Cybium*.

1194 GILES, S., RÜCKLIN, M. & DONOGHUE, P.C.J. (2013). Histology of "placoderm" dermal

1195 skeletons: implications for the nature of the ancestral gnathostome. *Journal of*

1196 *Morphology* **274**, 627-644.

1197 GLOWACKI, J., COX, K.A., O'SULLIVAN, J., WILKIE, D. & DEFTOS, L.J. (1986). Osteoclasts

1198 can be induced in fish having an acellular bony skeleton. *Proceedings of the*

1199 *National Academy of Sciences* **83**, 4104-4107.

1200 GOODRICH, E.S. (1907). On the scales of fish, living and extinct, and their importance in

1201 classification. *Proceeding of the Zoological Society, London* **2**, 751-774.

1202 GOODWIN, M.B. & HORNER, J.R. (2004). Cranial histology of pachycephalosaurs

1203 (Ornithischia: Marginocephalia) reveals transitory structures inconsistent with head-

1204 butting behavior. *Paleobiology* **30**, 253-267.

1205 GRAHAM, J.B. & DICKSON, K.A. (2000). The evolution of thunniform locomotion and heat

1206 conservation in scombrid fishes: new insights based on the morphology of

1207 *Allothunnus fallai*. *Zoological Journal of the Linnean Society* **129**, 419-466.

1208 GRAHAM, J.B. & DICKSON, K.A. (2001). Anatomical and physiological specialization for

1209 endothermy. In *Fish Physiology. Volume 19: Tuna: Physiology, Ecology, and*

1210 *Evolution* pp. 121-165.

1211 GRAHAM, J.B. & DICKSON, K.A. (2004). Tuna comparative physiology. *Journal of*

1212 *Experimental Biology* **207**, 4015-4024.

1213 GRAHAM, J.B., KOEHRN, F.J. & DICKSON, K.A. (1983). Distribution and relative proportions

1214 of red muscle in scombrid fishes: consequences of body size and relationships to

1215 locomotion and endothermy. *Canadian Journal of Zoology* **61**, 2087-2096.

1216 GROSS, W. (1930). Die Fische des mittleren Old Red Südlivlands. *Geologische und*

1217 *Palaeontologische Abhandlungen* **18**, 123-156.

1218 HALL, B.K. (2015). *Bones and Cartilage, Developmental and Evolutionary Skeletal*

1219 *Biology - Second Edition*. Academic Press.

1220 HALSTEAD, L.B. (1963). Aspidin: The precursor of bone. *Nature* **199**, 46-48.

1221 HALSTEAD, L.B. (1969). Calcified tissues in the earliest vertebrates. *Calcified Tissue*

1222 *Research* **3**, 107-124.

1223 HORREO, J.L. (2017). Revisiting the mitogenomic phylogeny of Salmoninae: new insights

1224 thanks to recent sequencing advances. *PeerJ* **5**, e3828.
1225 HORTON, J.M. & SUMMERS, A.P. (2009). The material properties of acellular bone in a
teleost fish. *Journal of Experimental Biology* **212**, 1413–1420.

1226 HUGHES, D.R., BASSETT, J.R. & MOFFAT, L.A. (1994). Histological identification of
1227 osteocytes in the allegedly acellular bone of the sea breams *Acanthopagrus
1228 australis*, *Pagrus auratus* and *Rhabdosargus sarba* (Sparidae, Perciformes,
1229 Teleostei). *Anatomy and Embryology* **190**, 163–179.

1230 HUGHES, L.C., ORTÍ, G., HUANG, Y., SUN, Y., BALDWIN, C.C., THOMPSON, A.W., ARCILA, D.,
1231 BETANCUR-R., R., LI, C., BECKER, L., BELLORA, N., ZHAO, X., LI, X., WANG, M., FANG,
1232 C., ET AL. (2018). Comprehensive phylogeny of ray-finned fishes (Actinopterygii)
1233 based on transcriptomic and genomic data. *Proceedings of the National Academy
1234 of Sciences* **115**, 6249–6254.

1235 *HUYSSEUNE, A. (1986). Late skeletal development at the articulation between upper
1236 pharyngeal jaws and neurocranial base in the fish, *Astatotilapia elegans*, with the
1237 participation of a chondroid form of bone. *The American Journal of Anatomy* **177**,
1238 119–137.

1239 HUYSSEUNE, A. (2000). Skeletal system. In *The Laboratory Fish* (ed G. OSTRANDER), pp.
1240 307–317. Academic Press.

1241 IGLÉSIAS, S.P. (2014a). Handbook of the marine fishes of Europe and adjacent waters (a
1242 natural classification based on collection specimens, with DNA barcodes and
1243 standardized photographs) - Volume I (Chondrichthyans and Cyclostomata) -
1244 Provisional version 08. http://iccanam.mnhn.fr

1245 IGLÉSIAS, S.P. (2014b). Handbook of the marine fishes of Europe and adjacent waters (a
1246 natural classification based on collection specimens, with DNA barcodes and
1247 standardized photographs) - Volume II (Actinopterygians) - Provisional version 10.
1248 http://iccanam.mnhn.fr

1249 JERVE, A., QU, Q., SANCHEZ, S., BLOM, H. & AHLBERG, P.E. (2016). Three-dimensional
1250 paleohistology of the scale and median fin spine of *Lophosteus superbus* (Pander
1251 1856). *PeerJ* **4**, e2521.

1252 JOHNSON, G.D. & PATTERSON, C. (1996). Relationships of lower euteleostean fishes. In
*Interrelationships of Fishes* (eds M.L.J. STIASSNY, L.R. PARENTI & G.D. JOHNSON),
1253 pp. 251–317. Academic Press, San Diego.

1254 KACEM, A. & MEUNIER, F.J. (2000). Mise en évidence de l'ostéolyse périostéocytaire
1255 vertébrale chez le saumon Atlantique *Salmo salar* (Salmonidae, Teleostei), au
1256 cours de sa migration anadrome. *Cybium* **24**, 105–112.

1257 KACEM, A. & MEUNIER, F.J. (2003). Halastatic demineralization in the vertebrae of Atlantic
1260 salmon, during their spawning migration. *Journal of Fish Biology* **63**, 1122–1130.

1261 KACEM, A. & MEUNIER, F.J. (2009). Transformations of the texture and the mineralization
1262 of the dentary bone in the Atlantic salmon, *Salmo salar* L. (Salmonidae), during
1263 anadromous migration. *Cybium* **33**, 61–72.

1264 KATZ, S.L. (2002). Design of heterothermic muscle in fish. *The Journal of Experimental
1265 Biology* **205**, 2251–2266.

1266 KEATING, J.N. & DONOGHUE, P.C.J. (2016). Histology and affinity of anaspids, and the
1267 early evolution of the vertebrate dermal skeleton. *Proceedings of the Royal Society
1268 B: Biological Sciences* **283**, 20152917.

1269 KEATING, J.N., MARQUART, C.L., MARONE, F. & DONOGHUE, P.C.J. (2018). The nature of
1270 aspidin and the evolutionary origin of bone. *Nature Ecology & Evolution* **2**, 1501–
1271 1506.
1272 KERSCHNITZKI, M., WAGERMAIER, W., ROSCHGER, P., SETO, J., SHAHAR, R., DUDA, G.N., MUNDLOS, S. & FRATZL, P. (2011). The organization of the osteocyte network mirrors the extracellular matrix orientation in bone. *Journal of Structural Biology* **173**, 303–311.

1276 *KHEMIRI, S., MEUNIER, F.J., LAURIN, M. & ZYLBERBERG, L. (2001). Morphology and structure of the scales in the Gadiformes (Actinopterygii: Teleostei: Paracanthopterygii) and a comparison to the elasmoid scales of other Teleostei. *Cahiers de Biologie Marine* **42**, 345–362.

1280 KÖLLIKER, A. (1859). On the different types in the microscopic structure of the skeleton of osseous fishes. *Proceedings of the Royal Society of London* **9**, 656–668.

1282 KRANENBARG, S., VAN CLEYNENBREUGEL, T., SCHIPPER, H. & VAN LEEUWEN, J. (2005). Adaptive bone formation in acellular vertebrae of sea bass (*Dicentrarchus labrax* L.). *The Journal of Experimental Biology* **208**, 3493–3502.

1285 LAVOUÉ, S., MIYA, M., POULSEN, J.Y., MÖLLER, P.R. & NISHIDA, M. (2008). Monophyly, phylogenetic position and inter-familial relationships of the Alepocephaliformes (Teleostei) based on whole mitogenome sequences. *Molecular Phylogenetics and Evolution* **47**, 1111–1121.

1289 *LECOMTE, F., MEUNIER, F.J. & ROJAS-BELTRAN, R. (1989). Some data on the growth of *Arius proops* (Ariidae, Siluriforme) in the estuaries of French Guyana. *Aquatic Living Resources* **2**, 63–38.

1292 LEPRÉVOST, A., AZAÏS, T., TRICHET, M. & SIRE, J.-Y. (2017). Vertebral development and ossification in the Siberian sturgeon (*Acipenser baerii*), with new insights on bone histology and ultrastructure of vertebral elements and scutes. *The Anatomical Record* **300**, 437–449.

1296 LI, J., XIA, R., MCDOWALL, R.M., LÓPEZ, J.A., LEI, G. & FU, C. (2010). Phylogenetic position of the enigmatic *Lepidogalaxias salamandroides* with comment on the orders of lower euteleostean fishes. *Molecular Phylogenetics and Evolution* **57**, 932–936.

1300 LISTON, J., NEWBREY, M.G., CHALLANDS, T.J. & ADAMS, C.E. (2013). Growth, age and size of the Jurassic pachycormid *Leedsichthys problematicus* (Osteichthyes: Actinopterygii). In *Mesozoic Fishes 5 - Global Diversity and Evolution* (eds G. ARRATIA, H.-P. SCHULTZE & M.V.H. WILSON), pp. 145–175. Verlag Dr. Friedriech Pfeil, Munich.

1305 LOPEZ, E. (1970). L'os cellulaire d'un poisson téléostéen *Anguilla anguilla* L. I. Etude histocytologique et histophysique. *Zeitschrift für Zellforschung und Mikroskopische Anatomie* **109**, 552–565.

1308 LOPEZ, E. (1976). Effects of calcitonin and ultimobranchialectomy (UBX) on calcium and bone metabolism in the eel, *Anguilla anguilla* L. *Calcified Tissue Research* **20**, 173–186.

1311 MACQUEEN, D.J. & JOHNSTON, I.A. (2014). A well-constrained estimate for the timing of the salmonid whole genome duplication reveals major decoupling from species diversification. *Proceedings of the Royal Society B: Biological Sciences* **281**, 20132881.

1315 MAYRINCK, D., BRITO, P.M., MEUNIER, F.J., ALVARADO-ORTEGA, J. & OTERO, O. (2017). †*Sorbinicharax verraesi*: an unexpected case of a benthic fish outside Acanthomorpha in the Upper Cretaceous of the Tethyan Sea. *PLoS ONE* **12**, e0183879.
1319 McDOWALL, R.M. (1997). The evolution of diadromy in fishes (revisited) and its place in
1320 phylogenetic analysis. *Reviews in Fish Biology and Fisheries* **7**, 443-462.

1321 McDOWALL, R.M. (2001). The origin of salmonid fishes: Marine, freshwater... or neither?
1322 *Reviews in Fish Biology and Fisheries* **11**, 171-179.

1323 METTENHEIMER, C. (1854). Anatomisch-histologische Untersuchungen über den
1324 *Tetragonurus Cuvieri Risso. Abhandlungen der Senckenbergischen
1325 Naturforschenden Gesellschaft* **1**, 214-257.

1326 MEUNIER, F.J. (1984a). Etude de la minéralisation de l'os chez les téléostéens à l'aide de
1327 la microradiographie quantitative: résultats préliminaires. *Cybium* **8**, 43-49.

1328 MEUNIER, F.J. (1984b). Spatial organization and mineralization of the basal plate of
1329 elasmoid scales in osteichthyans. *American Zoologist* **24**, 953-964.

1330 MEUNIER, F.J. (1987). Os cellulaire, os acellulaire et tissus dérivés chez les
1331 Ostéichthyens: les phénomènes de l'acellularisation et de la perte de minéralisation.
1332 *L'Année Biologique* **26**, 201-233.

1333 MEUNIER, F.J. (1989). The acellularisation process in osteichthyan bone. In *Trends in
1334 Vertebrate Morphology: Proceedings of the 2nd International Symposium on
1335 Vertebrate Morphology*, Vienna, 1986 pp. 443-446.

1336 *MEUNIER, F.J. (2009). Structure and mineralization of the scales in the clown trigger-fish
1337 *Balistoides conspicillum* (Teleostei: Tetraodontiformes: Balistidae). *Cahiers de
1338 Biologie Marine* **50**, 47-56.

1339 *MEUNIER, F.J. (2011). The Osteichtyes, from the Paleozoic to the extant time, through
1340 histology and palaeohistology of bony tissues. *Comptes Rendus Palevol* **10**, 347-
1341 355.

1342 MEUNIER, F.J. & ARNULF, I. (2018). Some histological data of bone and teeth in the Rift
1343 Eelpout, *Thermarces cerberus* (Zoarcidae). *Cybium* **42**, 83-86.

1344 MEUNIER, F.J. & BÉAREZ, P. (2019). Histological study of the cutaneous bony scutes in
1345 the John dory, *Zeus faber* Linnaeus, 1758 (Teleostei; Zeiformes; Zeidae). *Cahiers
1346 de Biologie Marine* **60**. DOI: 10.21411/CBM.A.260F6487

1347 MEUNIER, F.J., BEAREZ, P. & FRANCILLON-VIEILLOT, H. (1999). Some morphological and
1348 histological aspects of hyperostosis in the Eastern Pacific marine fish *Prionotus
1349 stephanophrys* Lockington, 1880 (Triglidae). In *Proceedings of the 5th Indo-Pacific
1350 Fish Conference, Nouméa, 1997* (eds B. SÉRET & J.-Y. SIRE), pp. 125-133. Société
1351 Française d'Ichthyologie, Paris.

1352 MEUNIER, F.J. & BRITO, P.M. (2004). Histology and morphology of the scales in some
1353 extinct and extant teleosts. *Cybium* **28**, 225-235.

1354 MEUNIER, F.J., BRITO, P.M. & LEAL, M.-E.C. (2013a). Morphological and histological data
1355 on the structure of the lingual toothplate of *Arapaima gigas* (Osteoglossidae;
1356 Teleostei). *Cybium* **37**, 263-271.

1357 MEUNIER, F.J., CUPELLO, C.D. & CLÉMENT, G. (2019). The skeleton and the mineralized
1358 tissues of the living coelacanths. *Bulletin of Kitakyushu Museum of Natural History
1359 and Human History* **17**, 37-48.

1360 MEUNIER, F.J., DESCHAMPS, M.-H., LECOMTE, F. & KACEM, A. (2008a). Le squelette des
1361 poissons téléostéens : structure, développement, physiologie, pathologie. *Bulletin
1362 de la Société Zoologique de France* **133**, 9-32.

1363 MEUNIER, F.J. & DESSE, G. (1986). Les hyperostoses chez les Téléostéens: description,
1364 histologie et problemes étiologiques. *Ichthyophysiologica Acta* **10**, 130-141.

1365 MEUNIER, F.J. & DESSE, J. (1994). Histological structure of hyperostotic cranial remains
1366 of *Pomadasys hasta* (Osteichthyes, Perciformes, Haemulidae) from archaeological
1367 sites of the Arabian Gulf and the Indian Ocean. *Annalen - Koninklijk Museum voor*
1368 *Midden-Afrika - Zoologische Wetenschappen* **274**, 47-53.

1369 MEUNIER, F.J., DUTHEIL, D.B. & BRITO, P.M. (2013b). Histological study of the median
1370 lingual dental plate of the Cretaceous fish †*Palaeonotopterus greenwoodi*
1371 (Teleostei: Osteoglossomorpha) from the Kem-Kem beds, Morocco. *Cybium* **37**,
1372 121-125.

1373 MEUNIER, F.J., ERDMANN, M. V., FERMON, Y. & CALDWELL, R.L. (2008b). Can the
1374 comparative study of the morphology and histology of the scales of *Latimeria
1375 menadoensis* and *L. chalumnae* (Sarcopterygii: Actinistia, Coelacanthidae) bring
1376 new insight on the taxonomy and the biogeography of recent coelacanthids?
1377 *Geological Society, London, Special Publications* **295**, 351-360.

1378 MEUNIER, F.J., EUSTACHE, R.-P., DUTHEIL, D. & CAVIN, L. (2016). Histology of ganoid
1379 scales from the early Late Cretaceous of the Kem Kem beds, SE Morocco:
1380 systematic and evolutionary implications. *Cybium* **40**, 121-132.

1381 *MEUNIER, F.J. & FRANCILLON-VIEILLOT, H. (1999). Histological structure of the caudal
1382 spine of the surgeonfish *Ctenochaetus striatus* (Teleostei: Acanthuridae). In
1383 *Proceedings of the 5th Indo-Pacific Fish Conference, Nouméa, 1997* (eds B. SÉRET
1384 & J.-Y. SIRE), pp. 117-124. Société Française d'Ichtyologie, Paris.

1385 MEUNIER, F.J., FRANÇOIS, Y. & CASTANET, J. (1978). Etude histologique et
1386 microradiographique des écailles de quelques Actinoptérygiens primitifs actuels.
1387 *Bulletin de la Société Zoologique de France* **103**, 309-318.

1388 MEUNIER, F.J., GAUDANT, J. & BONELLI, E. (2010). Morphological and histological study of
1389 the hyperostoses of *Lepidopus albyi* (Sauvage, 1870), a fossil Trichiuridae from the
1390 Tortonian (Upper Miocene) of Piedmont (Italy). *Cybium* **34**, 293-301.

1391 *MEUNIER, F.J. & GAYET, M. (1992). Nouveau remaniement de la ganoïne chez un
1392 Semionotidae du Crétacé supérieur de Bolivie : intérêt paléobiologique. *Geobios* **25**,
1393 767-774.

1394 *MEUNIER, F.J. & GAYET, M. (1996). A new polypteriform from the Late Cretaceous and
1395 the middle Paleocene of South America. In *Mesozoic Fishes - Systematics and*
1396 *Paleoecology* (eds G. ARRATIA & G. VIOHL), pp. 95-103. Verlag Dr. Friedriech Pfeil,
1397 Munich.

1398 *MEUNIER, F.J. & GERMAIN, D. (2018). The histological structure of teeth in the northern
1399 wolffish *Anarhichas denticulatus* (Teleostei: Perciformes: Anarhichadidae). *Cahiers*
1400 *de Biologie Marine* **59**, 217-224.

1401 *MEUNIER, F.J., GERMAIN, D. & OTERO, O. (2018a). A histological study of the lingual
1402 molariform teeth in *Hyperopisus bebe* (Mormyridae; Osteoglossomorpha). *Cybium*
1403 **42**, 87-90.

1404 MEUNIER, F.J. & HERBIN, M. (2014). La collection de préparations histologiques
1405 effectuées par Paul Gervais (1816-1879) sur le squelette des 'poissons'. *Cybium*
1406 **38**, 23-42.

1407 MEUNIER, F.J. & HUYSSEUNE, A. (1992). The concept of bone tissue in Osteichthyes.
1408 *Netherlands Journal of Zoology* **42**, 445-458.

1409 *MEUNIER, F.J., JOURNIAC, N., LAVOUE, S. & RABET, N. (2002). Caractéristiques
1410 histologiques des marques de croissance squelettique chez l'atipa, *Hoplosternum*
1411 *littorale* (Hancock, 1828) (Teleostei, Siluriformes) dans le marais de Kaw (Guyane
1412 Française). *Bulletin Français de la Pêche et de la Pisciculture* **364**, 71-86.
1413 *MEUNIER, F.J., LECOMTE, F. & DUHAMEL, G. (2018b). Some histological data on bone and teeth in the grey notothen (*Lepidonotothen squamifrons*) and in the mackerel icefish (*Champsocephalus gunnari*) (Notothenioidei; Perciformes; Teleostei).
*Cybium* **42**, 91–97.

1417 MEUNIER, F.J., OTERO, O. & LAURIN, M. (2018c). Histological study of the jaw teeth in the Devonian actinopterygian †*Cheirolepis canadensis* (Whiteaves). *Cybium* **42**, 67–74.

1419 MEUNIER, F.J. & POPLIN, C. (1995). Paleohistological study of the scales of *Amia robusta* Priem, 1901, Amiidae from the Thanetian (Paleocene) of Cernay (France). *Geobios* **19**, 39–43.

1422 *MEUNIER, F.J. & SAUR, F. (2007). Étude morphologique et structurale des écailles de *Tetragonurus cuvieri* (Tetragonuridae) et de *Cleidopus gloriamaris* (Monocentridae).
*Cybium* **31**, 123–132.

1425 MEUNIER, F.J. & SIRE, J. (1981). Sur la structure et la minéralisation des écailles de germon, *Thunnus alalunga* (Téleostéen, Perciforme, Thunnidae). *Bulletin de la Société Zoologique de France* **106**, 327–336.

1428 MEUNIER, F.J., SORBA, L. & BEAREZ, P. (2004). Presence of vascularized acellular bone in the elasmoid scales of *Micropogonias altipinnis* (Osteichthyes, Perciformes, Sciaenidae). *Cybium* **28**, 25–31.

1431 MIYA, M., FRIEDMAN, M., SATOH, T.P., TAKESHIMA, H., SADO, T., IWASAKI, W., YAMANOUE, Y., NAKATANI, M., MABUCHI, K., INOUE, J.G., POULSEN, J.Y., FUKUNAGA, T., SATO, Y. & NISHIDA, M. (2013). Evolutionary origin of the Scombridae (tunas and mackerels): members of a Paleogene adaptive radiation with 14 other pelagic fish families.
*PLoS ONE* **8**, e73535.

1436 MOSS, M.L. (1961a). Osteogenesis of acellular teleost fish bone. *American Journal of Anatomy* **108**, 99–110.

1438 MOSS, M.L. (1961b). Studies of the acellular bone of teleost fish. I. Morphological and systematic variations. *Acta Anatomica* **46**, 343–462.

1440 MOSS, M.L. (1962). Studies of the acellular bone of teleost fish. II. Response to fracture under normal and acalcemic conditions. *Acta Anatomica* **48**, 46–60.

1442 MOSS, M.L. (1963). The biology of acellular teleost bone. *Annals of the New York Academy of Sciences* **109**, 337–350.

1444 MOSS, M.L. (1965). Studies of the acellular bone of teleost fish. V. Histology and mineral homeostasis of fresh-water species. *Acta Anatomica* **60**, 262–276.

1446 MOSS, M.L. & FREILICH, M. (1963). Studies of the acellular bone of teleost fish. IV. Inorganic content of calcified tissues. *Acta Anatomica* **55**, 1–8.

1448 MOSS, M.L. & POSNER, A.S. (1960). X-ray diffraction study of acellular teleost bone. *Nature* **188**, 1037–1038.

1450 NEAR, T.J., DORNBURG, A., EYTAN, R.I., KECK, B.P., SMITH, W.L., KUHN, K.L., MOORE, J.A., PRICE, S.A., BURBRINK, F.T., FRIEDMAN, M. & WAINWRIGHT, P.C. (2013). Phylogeny and tempo of diversification in the superradiation of spiny-rayed fishes. *Proceedings of the National Academy of Sciences* **110**, 12738–12743.

1454 NEAR, T.J., EYTAN, R.I., DORNBURG, A., KUHN, K.L., MOORE, J.A., DAVIS, M.P., WAINWRIGHT, P.C., FRIEDMAN, M. & SMITH, W.L. (2012). Resolution of ray-finned fish phylogeny and timing of diversification. *Proceedings of the National Academy of Sciences* **109**, 13698–13703.

1458 NEMOTO, Y., HIGUCHI, K., BABA, O., KUDO, A. & TAKANO, Y. (2007). Multinucleate osteoclasts in medaka as evidence of active bone remodeling. *Bone* **40**, 399–408.
1460 OLNEY, J.E., JOHNSON, G.D. & BALDWIN, C.C. (1993). Phylogeny of lampridiform fishes.

1461 *Bulletin of Marine Science* **52**, 137–169.

1462 ØRVIG, T. (1951). Histologic studies of placoderms and fossil elasmobranchs. 1- The endoskeleton, with remarks on the hard tissues of lower vertebrates in general.

1464 *Arkiv för Zoologi* **2**, 321–454.

1465 ØRVIG, T. (1967). Phylogeny of tooth tissues: evolution of some calcified tissues in early vertebrates. In *Structural and Chemical Organization of Teeth, Vol. I* pp. 45–105.

1467 Academic Press, New York.

1468 ØRVIG, T. (1978). Microstructure and growth of the dermal skeleton in fossil actinopterygian fishes: *Birgeria* and *Scanilepis*. *Zoologica Scripta* **7**, 33–56.

1470 PAIG-TRAN, E.W.M., BARRIOS, A.S. & FERRY, L.A. (2016). Presence of repeating hyperostotic bones in dorsal pterygiophores of the oarfish, *Regalecus russellii*.

1472 *Journal of Anatomy* **229**, 560–567.

1473 PARADIS, E., CLAUDE, J. & STRIMMER, K. (2004). APE: Analyses of phylogenetics and evolution in R language. *Bioinformatics* **20**, 289–290.

1475 PARENTI, L.R. (1986). The phylogenetic significance of bone types in euteleost fishes. *Zoological Journal of the Linnean Society* **87**, 37–51.

1477 PATTERSON, C. (1977). Cartilage bones, dermal bones and membrane bones, or the exoskeleton versus the endoskeleton. In *Problems in Vertebrate Evolution. Linnean Society Symposium Series, No. 4* (eds S. MAHALA ANDREWS, R.S. MILES & A.D.

1479 WALKER), pp. 77–121. Academic Press, London.

1481 POPLIN, C., POPLIN, F. & RICQLÈS, A. DE (1976). Quelques particularités anatomiques et histologiques du rostre de l'espadon (*Xiphias gladius* L.). *Comptes Rendus de l'Académie des sciences, Paris, série D* **282**, 1105–1108.

1483 QU, Q., SANCHEZ, S., ZHU, M., BLOM, H. & AHLBERG, P.E. (2017). The origin of novel features by changes in developmental mechanisms: ontogeny and three-dimensional microanatomy of polyodontode scales of two early osteichthyans.

1487 *Biological Reviews* **92**, 1189–1212.

1488 QUEKETT, J. (1855). *Descriptive and Illustrated Catalogue of the Histological Series Contained in the Museum of The Royal College of Surgeons of England. Vol. II.*

1490 *Structure of the Skeleton of Vertebrate Animals*. Taylor & Francis, London.

1491 RAMSDEN, S.D., BRINKMANN, H., HAWRYSHYN, C.W. & TAYLOR, J.S. (2003). Mitogenomics and the sister of Salmonidae. *Trends in Ecology and Evolution* **18**, 605–607.

1493 RICHTER, M. & SMITH, M.M. (1995). A microstructural study of the ganoine tissue of selected lower vertebrates. *Zoological Journal of the Linnean Society* **114**, 173–212.

1495 RICQLÈS, A. DE, MEUNIER, F.J., CASTANET, J. & FRANCILLON-VIEILLOT, H. (1991). Comparative microstructure of bone. In *Bone: A Treatise, Volume III* (ed B.K. HALL), pp. 1–78. CRC Press.

1498 ROCHEFORT, G.Y., PALLU, S. & BENHAMOU, C.L. (2010). Osteocyte: the unrecognized side of bone tissue. *Osteoporosis International* **21**, 1457–1469.

1500 *ROLVIEN, T., NAGEL, F., MILOVANOVIC, P., WUERTZ, S., MARSHALL, R.P., JESCHKE, A., SCHMIDT, F.N., HAHN, M., WITTEN, P.E., AMLING, M. & BUSSE, B. (2016). How the European eel (*Anguilla anguilla*) loses its skeletal framework across lifetime.

1503 *Proceedings of the Royal Society B: Biological Sciences* **283**, 20161550.

1504 ROSEN, D.E. (1973). Interrelationships of higher euteleostean fishes. In *Interrelationships of Fishes* (eds P.H. GREENWOOD, R.S. MILES & C. PATTERSON), pp. 397–513.

1506 Academic Press, London.
1507 ROSEN, D.E. (1985). An essay on euteleostean classification. *American Museum
1508 Novitates **2827**, 1–57.

1509 SANSOM, I.J., HAINES, P.W., ANDREEV, P. & NICOLL, R.S. (2013). A new pteraspidomorph
1510 from the Nibil Formation (Katian, Late Ordovician) of the Canning Basin, Western
1511 Australia. *Journal of Vertebrate Paleontology* **33**, 764–769.

1512 SANTAMARIA, N., BELLO, G., PASSANTINO, L., DI COMITE, M., ZUPA, R., POUSIS, C.,
1513 VASSALLO-AGIUS, R., CICIRELLI, V., BASILONE, G., MANGANO, S. & CORRIERO, A.
1514 (2018). Micro-anatomical structure of the first spine of the dorsal fin of Atlantic
1515 bluefin tuna, *Thunnus thynnus* (Osteichthyes: Scombridae). *Annals of Anatomy* **219**,
1516 1–7.

1517 SBAIHI, M., KACEM, A., AROUA, S., BALOCHE, S., ROUSSEAU, K., LOPEZ, E., MEUNIER, F. &
1518 DUFOUR, S. (2007). Thyroid hormone-induced demineralisation of the vertebral
1519 skeleton of the eel, *Anguilla anguilla*. *General and Comparative Endocrinology* **151**,
1520 98–107.

1521 SCHAEFFER, B. (1977). The dermal skeleton in fishes. In *Problems in Vertebrate
1522 Evolution*. Linnean Society Symposium Series, No. 4 (eds S. MAHALA ANDREWS,
1523 R.S. MILES & A.D. WALKER), pp. 25–52. Academic Press, London.

1524 SCHEYER, T.M., SCHMID, L., FURRER, H. & SÁNCHEZ-VILLAGRA, M.R. (2014). An
1525 assessment of age determination in fossil fish: the case of the opercula in the
1526 Mesozoic actinopterygian *Saurichthys*. *Swiss Journal of Palaeontology* **133**, 243–
1527 257.

1528 SCHULTZE, H.-P. (2016). Scales, enamel, cosmine, ganoine, and early osteichthyans.
1529 *Comptes Rendus Palevol* **15**, 83–102.

1530 SHAHAR, R. & DEAN, M.N. (2013). The enigmas of bone without osteocytes. *BoneKEy
1531 Reports* **2**, 343.

1532 SIMMONS, D.J., SIMMONS, N.B. & MARSHALL, J.H. (1970). The uptake of calcium-45 in the
1533 acellular-boned toadfish. *Calcified Tissue Research* **5**, 206–221.

1534 SIRE, J.-Y., DONOGHUE, P.C.J. & VICKARYOUS, M.K. (2009). Origin and evolution of the
1535 integumentary skeleton in non-tetrapod vertebrates. *Journal of Anatomy* **214**, 409–
1536 440.

1537 *SIRE, J. & MEUNIER, F.J. (1993). Ornamentation superficielle et structure des plaques
1538 osseuses dermiques de quelques Siluriformes cuirassés (Loricariidae,
1539 Callichthyidae, Doradidae). *Annales des Sciences naturelles, Zoologie, Paris* **14**,
1540 101–123.

1541 SIRE, J.-Y. & MEUNIER, F.J. (1994). The canaliculi of Williamson in holostean bone
1542 (Osteichthyes, Actinopterygii): a structural and ultrastructural study. *Acta Zoologica
1543 75*, 235–247.

1544 SIRE, J.-Y. & MEUNIER, F.J. (2017). Typical tubules in the acellular bone of gilthead sea
1545 bream *Sparus aurata* (Teleostei: Perciformes: Sparidae). *Cahiers de Biologie
1546 Marine* **58**, 467–474.

1547 SIRE, J.Y., HUYSSEUNE, A. & MEUNIER, F.J. (1990). Osteoclasts in teleost fish: light-and
1548 electron-microscopical observations. *Cell and Tissue Research* **260**, 85–94.

1549 SMITH, M.M. & HALL, B.K. (1990). Development and evolutionary origins of vertebrate
1550 skeletogenic and odontogenic tissues. *Biological Reviews* **65**, 277–373.

1551 SMITH, M.M., HOBDELL, M.H. & MILLER, W.A. (1972). The structure of the scales of
1552 *Latimeria chalumnae*. *Journal of Zoology* **167**, 501–509.

1553 SMITH-VANIZ, W.F., KAUFMAN, L.S. & GLOWACKI, J. (1995). Species-specific patterns of
1554 hyperostosis in marine teleost fishes. *Marine Biology* **121**, 573–580.

1555 SPENCE, R., GERLACH, G., LAWRENCE, C. & SMITH, C. (2008). The behaviour and ecology of the zebrafish, *Danio rerio*. *Biological Reviews* **83**, 13–34.

1557 STENSIÖ, E. (1958). Les Cyclostomes fossiles ou Ostracodermes. In *Traité de Zoologie* *Tome XIII : Agnathes et Poissons vol. 3* pp. 173–425. Masson, Paris.

1559 STEPHAN, P. (1900). Recherches histologiques sur la structure du tissu osseux des poissons. *Bulletin des Sciences de France et de la Belgique* **33**, 281–429.

1561 STRAUBE, N., LI, C., MERTZEN, M., YUAN, H. & MORITZ, T. (2018). A phylogenomic approach to reconstruct interrelationships of main clupeocephalan lineages with a critical discussion of morphological apomorphies. *BMC Evolutionary Biology* **18**, 158.

1565 TAKAGI, Y. & YAMADA, J. (1992). Effects of calcium deprivation on the metabolism of acellular bone in tilapia, *Oreochromis niloticus*. *Comparative Biochemistry and Physiology A* **102**, 481–485.

1568 TAVERNE, L. & FILLEUL, A. (2003). Osteology and relationships of the genus *Spaniodon* (Teleostei, Salmoniformes) from the Santonian (Upper Cretaceous) of Lebanon. *Palaeontology* **46**, 927–944.

1571 TOTLAND, G.K., FJELLDAL, P.G., KRYVI, H., LØKKA, G., WARGELIUS, A., SAGSTAD, A., HANSEN, T. & GROTMOL, S. (2011). Sustained swimming increases the mineral content and osteocyte density of salmon vertebral bone. *Journal of Anatomy* **219**, 490–501.

1575 WAINWRIGHT, D.K., INGERSOLL, S. & LAUDER, G. V. (2018). Scale diversity in bigeye tuna (*Thunnus obesus*): Fat-filled trabecular scales made of cellular bone. *Journal of Morphology* **279**, 828–840.

1578 WATANABE, Y.Y., GOLDMAN, K.J., CASELLE, J.E., CHAPMAN, D.D. & PAPASTAMATIOU, Y.P. (2015). Comparative analyses of animal-tracking data reveal ecological significance of endothermy in fishes. *Proceedings of the National Academy of Sciences* **112**, 6104–6109.

1582 WEGNER, N.C., SNODGRASS, O.E., DEWAR, H. & HYDE, J.R. (2015). Whole-body endothermy in a mesopelagic fish, the opah, *Lampris guttatus*. *Science* **348**, 786–790.

1585 WEIGELE, J. & FRANZ-ODENDAAL, T.A. (2016). Functional bone histology of zebrafish reveals two types of endochondral ossification, different types of osteoblast clusters and a new bone type. *Journal of Anatomy* **229**, 92–103.

1588 WEISS, R.E. & WATABE, N. (1979). Studies on the biology of fish bone. III. Ultrastructure of osteogenesis and resorption in osteocytic (cellular) and anosteocytic (acellular) bones. *Calcified Tissue International* **28**, 43–56.

1591 WILEY, E.O., JOHNSON, G.D. & DIMMICK, W.W. (1998). The phylogenetic relationships of lampridiform fishes (Teleostei: Acanthomorpha), based on a total-evidence analysis of morphological and molecular data. *Molecular Phylogenetics and Evolution* **10**, 417–425.

1595 WILLIAMSON, W.C. (1849). On the microscopic structure of the scales and dermal teeth of some ganoid and placoid fish. *Philosophical Transactions of the Royal Society of London. Series B* **139**, 435–475.

1598 WILLIAMSON, W.C. (1851). Investigations into the structure and development of the scales and bones of fishes. *Philosophical Transactions of the Royal Society of London* **141**, 643–702.
1601 WILSON, M.V.H. & WILLIAMS, R.R.G. (2010). Salmoniform fishes: key fossils, supertree, and possible morphological synapomorphies. In *Origin and Phylogenetic Interrelationships of Teleosts* (eds J.S. NELSON, H.-P. SCHULTZE & M.V.H. WILSON), pp. 379–409. Verlag Dr. Friedrich Pfeil, Munich.

1605 WITTEN, P.E. (1997). Enzyme histochemical characteristics of osteoblasts and mononucleated osteoclasts in a teleost fish with acellular bone (*Oreochromis niloticus*, Cichlidae). *Cell and Tissue Research* **287**, 591–599.

1608 WITTEN, P.E., FJELLDAL, P.G., HUYSSEUNE, A., MCGURK, C., OBACH, A. & OWEN, M.A.G. (2018). Bone without minerals and its secondary mineralization in Atlantic salmon (*Salmo salar*): the recovery from phosphorus deficiency. *Journal of Experimental Biology*, jeb.188763.

1612 WITTEN, P.E. & HALL, B.K. (2002). Differentiation and growth of kype skeletal tissues in anadromous male Atlantic salmon (*Salmo salar*). *International Journal of Developmental Biology* **46**, 719–730.

1615 WITTEN, P.E. & HALL, B.K. (2003). Seasonal changes in the lower jaw skeleton in male Atlantic salmon (*Salmo salar* L.): remodelling and regression of the kype after spawning. *Journal of Anatomy* **203**, 435–450.

1618 WITTEN, P.E., HANSEN, A. & HALL, B.K. (2001). Features of mono- and multinucleated bone resorbing cells of the zebrafish *Danio rerio* and their contribution to skeletal development, remodeling, and growth. *Journal of Morphology* **250**, 197–207.

1621 WITTEN, P.E. & HUYSSEUNE, A. (2009). A comparative view on mechanisms and functions of skeletal remodelling in teleost fish, with special emphasis on osteoclasts and their function. *Biological Reviews* **84**, 315–346.

1624 WITTEN, P.E. & HUYSSEUNE, A. (2010). The unobtrusive majority: mononucleated bone resorbing cells in teleost fish and mammals. *Journal of Applied Ichthyology* **26**, 225–229.

1627 WITTEN, P.E., HUYSSEUNE, A., FRANZ-ODENDAAL, T.A., FEDAK, T., VICKARYOUS, M.K., COLE, A. & HALL, B.K. (2004). Acellular teleost bone: dead or alive, primitive or derived? *The Palaeontological Association Newsletter* **55**, 37–41.

1630 WITTEN, P.E., OWEN, M.A.G., FONTANILLAS, R., SOENENS, M., MCGURK, C. & OBACH, A. (2016). A primary phosphorus-deficient skeletal phenotype in juvenile Atlantic salmon *Salmo salar*: The uncoupling of bone formation and mineralization. *Journal of Fish Biology* **88**, 690–708.

1634 WITTEN, P.E. & VILLWOCK, W. (1997). Growth requires bone resorption at particular skeletal elements in a teleost fish with acellular bone (*Oreochromis niloticus*, Teleostei: Cichlidae). *Journal of Applied Ichthyology* **13**, 149–158.

1637 WYSOLMERSKI, J.J. (2012). Osteocytic osteolysis: time for a second look? *BoneKey Reports* **1**, 229.

1639 ZYLBERBERG, L., MEUNIER, F.J. & LAURIN, M. (2010). A microanatomical and histological study of the postcranial dermal skeleton in the Devonian sarcopterygian *Eusthenopteron foordi*. *Acta Palaeontologica Polonica* **55**, 459–470.

1642 ZYLBERBERG, L., MEUNIER, F.J. & LAURIN, M. (2016). A microanatomical and histological study of the postcranial dermal skeleton in the Devonian actinopterygian *Cheirolepis canadensis*. *Acta Palaeontologica Polonica* **61**, 363–376.

1645
1646
1647 IX. SUPPORTING INFORMATION

1648 Additional supporting information may be found in the online version of this article.

1649 **Table S1.** Complete list of actinopterygian (ray-finned fish) taxa surveyed by our literature review, including additional species obtained with our synchrotron microtomography (SRµCT) data.

1652 The supra-specific taxonomy follows Betancur-R. et al. (2017). In the case of older literature, species names were often outdated. We used FishBase (Froese & Pauly, 2019) and the Catalog of Fishes (Eschmeyer et al., 2019) to identify the corresponding valid names.

1656 Key for the ‘Notes’ column: ¹leptocephalus larva; ²possible error in identifying bone type; ³localised cellular bone in hyperostoses; ⁴acellular hyperostotic bone; ⁵alleged osteocytes in cytoplasmic tubules.

1659 **Fig. S1.** Time-calibrated multilocus tree of extant actinopterygians (ray-finned fishes), obtained from the optimisation of the character states ‘cellular bone’ (in dark blue) and ‘acellular bone’ (in yellow) on the topology T1 (Near *et al.*, 2012).

1662 **Fig. S2.** Time-calibrated multilocus tree of extant and fossil actinopterygians (ray-finned fishes), obtained from the optimisation of the character states ‘cellular bone’ (in dark blue) and ‘acellular bone’ (in yellow) on the topology T2 (Betancur-R. *et al.*, 2015).

1665 **Fig. S3.** Time-calibrated multilocus tree of extant actinopterygians (ray-finned fishes), obtained from the optimisation of the character states ‘cellular bone’ (in dark blue) and ‘acellular bone’ (in yellow) on the topology T3 (Hughes *et al.*, 2018).

1668
<table><caption>**Table 1.** Bone type (presence or absence of osteocytes) in the taxa surveyed by our synchrotron microtomography (SR-µCT) data. C = cellular bone; A = acellular bone. ¹Bones sampled for *Cyprinus carpio*: frontal, maxilla, dentary, pharyngobranchial, opercle, abdominal vertebra, rib, dorsal-fin spine, cleithrum, pelvic bone. Specimens were obtained from private collectors and from the following natural history collections: American Museum of Natural History, New York City, USA (AMNH); Muséum national d'Histoire naturelle, Paris, France (MNHN); Natural History Museum, London, UK (NHMUK); Oxford University Museum of Natural History, Oxford, UK (OUMNH); Paleontological Institute and Museum, Zurich, Switzerland (PIMUZ); University of Michigan Museum of Paleontology (UMMP) and of Zoology (UMMZ); Université de Poitiers, France.</caption><thead><tr><th>Taxon</th><th></th><th>Species name</th><th>Vernacular name</th><th>Type</th><th>Bone sampled</th><th>Specimen used</th></tr></thead><tbody><tr><td>†Cheirolepidiformes</td><td>†Cheirolepididae</td><td>†Cheirolepis canadensis</td><td>-</td><td>C</td><td>dentary</td><td>UMMP 3453</td></tr><tr><td rowspan="2">Polypteriformes</td><td rowspan="2">Polypteridae</td><td>Erpetoichthys calabaricus</td><td>reedfish</td><td>C</td><td>dentary</td><td>Université de Poitiers, uncat.</td></tr><tr><td>Polypterus delhezi</td><td>barred bichir</td><td>C</td><td>dentary</td><td>Université de Poitiers, uncat.</td></tr><tr><td>Incertae sedis</td><td>Incertae sedis</td><td>†Birgeria stensioei</td><td>-</td><td>C</td><td>dentary</td><td>PIMUZ T2188</td></tr><tr><td rowspan="2">Chondrostei</td><td>†Chondrosteidae</td><td>†Chondrosteus acipenseroides</td><td>-</td><td>C</td><td>dentary</td><td>NHMUK PV P 2261a</td></tr><tr><td>Aipenseridae</td><td>Acipenser gueldenstaedtii</td><td>Danube sturgeon</td><td>C</td><td>dentary</td><td>MNHN.ICOS.01529</td></tr><tr><td>†Pycnodontiformes</td><td>†Pycnodontidae</td><td>†Proscinetes elegans</td><td>-</td><td>C</td><td>dentary</td><td>NHMUK PV P 1626</td></tr><tr><td>†Dapediiformes</td><td>†Dapediidae</td><td>†Dapedium sp.</td><td>-</td><td>C</td><td>dentary</td><td>OUMNH J.3041</td></tr><tr><td rowspan="4">Holostei</td><td rowspan="4">Incertae sedis</td><td>†Eoeugnathus megalepis</td><td>-</td><td>C</td><td>dentary</td><td>PIMUZ T344</td></tr><tr><td>†Heterolepidotus dorsalis</td><td>-</td><td>C</td><td>dentary</td><td>NHMUK PV P 10290</td></tr><tr><td>†Hulettia americana</td><td>-</td><td>C</td><td>dentary</td><td>UMMP 11217</td></tr><tr><td>Amiidae</td><td>Amia calva</td><td>bowfin</td><td>C</td><td>dentary</td><td>OUMNH 21648</td></tr><tr><td>†Caturidae</td><td>†Caturus furcatus</td><td>-</td><td>C</td><td>dentary</td><td>private collection</td></tr><tr><td rowspan="2">Lepisosteidae</td><td rowspan="2">†Semionotidae</td><td>Atractosteus tropicus</td><td>tropical gar</td><td>C</td><td>dentary</td><td>MNHN.ICOS. PB-901</td></tr><tr><td>Lepisosteus oculatus</td><td>spotted gar</td><td>C</td><td>dentary</td><td>UMMZ 178806/S</td></tr><tr><td>†Aspidorhynchiformes</td><td>†Aspidorhynchidae</td><td>†Semionotus elegans</td><td>-</td><td>C</td><td>dentary</td><td>UMMP 13664</td></tr><tr><td rowspan="3">†Pachycormiformes</td><td rowspan="3">†Pachycormidae</td><td>†Aspidorhynchus cf.eodus</td><td>-</td><td>C</td><td>dentary</td><td>private collection</td></tr><tr><td>†Vinctifer comptoni</td><td>-</td><td>C</td><td>dentary</td><td>UMMP 101950</td></tr><tr><td>†Euthynotus incognitus</td><td>-</td><td>C</td><td>dentary, rib</td><td>NHMUK PV P 2044</td></tr><tr><td rowspan="3">†Pholidophoriformes</td><td rowspan="3">Incertae sedis</td><td>†Hypsocormus sp.</td><td>-</td><td>C</td><td>dentary</td><td>private collection</td></tr><tr><td>†Leedsichthys problematicus</td><td>-</td><td>C</td><td>gill raker</td><td>private collection</td></tr><tr><td>†Pachycormus macropterus</td><td>-</td><td>C</td><td>dentary</td><td>MNHN.F.JRE87</td></tr><tr><td>†Pholidophoriformes</td><td>Incertae sedis</td><td>†Pholidophoroides crenulata</td><td>-</td><td>C</td><td>dentary</td><td>NHMUK PV OR 36313</td></tr></tbody></table>
<table>
  <tbody>
    <tr>
      <td>†Dorsetichthyiformes</td>
      <td>†Dorsetichthyidae</td>
      <td>†Pholidophoropsis caudalis</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>OUMNH J.3363</td>
    </tr>
    <tr>
      <td>†Dorsetichthyiformes</td>
      <td>†Ascalaboidae</td>
      <td>†Dorsetichthys bechei</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>OUMNH J.3369</td>
    </tr>
    <tr>
      <td>†Leptolepidiformes</td>
      <td>†Ascalaboidae</td>
      <td>†Ascalabos voithii</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>NHMUK PV P 3673a</td>
    </tr>
    <tr>
      <td>†Leptolepidiformes</td>
      <td>†Ascalaboidae</td>
      <td>†Tharsis dubius</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>NHMUK PV OR 37852b</td>
    </tr>
    <tr>
      <td>†Leptolepididae</td>
      <td>†Leptolepis macropthalmus</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>private collection</td>
      <td></td>
    </tr>
    <tr>
      <td>†Ichthyodectiformes</td>
      <td>Incertae sedis</td>
      <td>†Allothrissops regleyi</td>
      <td>-</td>
      <td>C</td>
      <td>dentary, rib</td>
      <td>NHMUK PV P 921</td>
    </tr>
    <tr>
      <td>†Ichthyodectidae</td>
      <td>Incertae sedis</td>
      <td>†Pachythrissops laevis</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>NHMUK PV P 41859</td>
    </tr>
    <tr>
      <td>†Ichthyodectidae</td>
      <td>Incertae sedis</td>
      <td>†Thrissops formosus</td>
      <td>-</td>
      <td>C</td>
      <td>dentary, rib</td>
      <td>NHMUK PV OR 35013</td>
    </tr>
    <tr>
      <td>†Crossognathiformes</td>
      <td>†Crossognathidae</td>
      <td>†Ichthyodectes cf.ctenodon</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>UMMP V56318</td>
    </tr>
    <tr>
      <td>Elopomorpha</td>
      <td>Incertae sedis</td>
      <td>†Xiphactinus cf.audax</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>UMMP 11003</td>
    </tr>
    <tr>
      <td>†Crossognathiformes</td>
      <td>†Crossognathidae</td>
      <td>†Rhacolepis buccalis</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>UMMP 101952</td>
    </tr>
    <tr>
      <td>Elopomorpha</td>
      <td>Incertae sedis</td>
      <td>†Osmeroides sp.</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>OUMNH K.64151</td>
    </tr>
    <tr>
      <td>Elopomorpha</td>
      <td>Incertae sedis</td>
      <td>†Urenchelys germanus</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>NHMUK PV P 62726</td>
    </tr>
    <tr>
      <td>Albulidae</td>
      <td>Albula vulpes</td>
      <td>bonefish</td>
      <td>-</td>
      <td>C</td>
      <td>rib, opercle, ceratobranchial</td>
      <td>UMMZ 186965/S</td>
    </tr>
    <tr>
      <td>Elopidae</td>
      <td>Albula vulpes</td>
      <td>bonefish</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>NHMUK PV P 3886</td>
    </tr>
    <tr>
      <td>Elopidae</td>
      <td>†Anaethalion angustus</td>
      <td>-</td>
      <td>C</td>
      <td>dentary, rib</td>
      <td>NHMUK PV OR 37926</td>
      <td></td>
    </tr>
    <tr>
      <td>Elopidae</td>
      <td>†Anaethalion angustus</td>
      <td>-</td>
      <td>C</td>
      <td>dentary, rib</td>
      <td>NHMUK PV P 63231</td>
      <td></td>
    </tr>
    <tr>
      <td>Megalopidae</td>
      <td>Elops saurus</td>
      <td>ladyfish</td>
      <td>-</td>
      <td>C</td>
      <td>dentary, rib</td>
      <td>UMMZ 189366/S</td>
    </tr>
    <tr>
      <td>Megalopidae</td>
      <td>†Flindersichthys denmaedi</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>NHMUK PV P 59694</td>
      <td></td>
    </tr>
    <tr>
      <td>Anguillidae</td>
      <td>Megalops cyprinoides</td>
      <td>Indo-Pacific tarpon</td>
      <td>-</td>
      <td>C</td>
      <td>dentary, rib</td>
      <td>MNHN.ICOS.00987</td>
    </tr>
    <tr>
      <td>Congridae</td>
      <td>Anguilla anguilla</td>
      <td>European eel</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>MNHN.ICOS. D-35</td>
    </tr>
    <tr>
      <td>Muraenesocidae</td>
      <td>Conger conger</td>
      <td>European conger</td>
      <td>-</td>
      <td>C</td>
      <td>dentary, rib</td>
      <td>MNHN.ICOS.SP-24</td>
    </tr>
    <tr>
      <td>Muraenesocidae</td>
      <td>Muraenesox cinereus</td>
      <td>daggertooth pike-conger</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>MNHN.ICOS.00286</td>
    </tr>
    <tr>
      <td>Muraenesocidae</td>
      <td>Gymnothorax moringa</td>
      <td>spotted moray</td>
      <td>-</td>
      <td>C</td>
      <td>dentary, rib</td>
      <td>UMMZ 173403/S</td>
    </tr>
    <tr>
      <td>Muraenesocidae</td>
      <td>Muraena helena</td>
      <td>Mediterranean moray</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>MNHN.ICOS.01039</td>
    </tr>
    <tr>
      <td>Osteoglossomorpha</td>
      <td>Hiodontidae</td>
      <td>†Eohiodon falcatus</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>NHMUK PV P 61245</td>
    </tr>
    <tr>
      <td>Osteoglossomorpha</td>
      <td>Hiodontidae</td>
      <td>Hiodon alosoides</td>
      <td>goldeye</td>
      <td>C</td>
      <td>dentary</td>
      <td>UMMZ 189540/S</td>
    </tr>
    <tr>
      <td>Osteoglossomorpha</td>
      <td>Hiodontidae</td>
      <td>Hiodon tergisus</td>
      <td>mooneye</td>
      <td>C</td>
      <td>rib</td>
      <td>UMMZ 180315/S</td>
    </tr>
    <tr>
      <td>Osteoglossomorpha</td>
      <td>Hiodontidae</td>
      <td>Chitala chitala</td>
      <td>giant featherback</td>
      <td>C</td>
      <td>dentary, rib</td>
      <td>UMMZ 193675/S</td>
    </tr>
  </tbody>
</table>
<table><tbody><tr><td rowspan="10">Clupeomorpha</td><td rowspan="10">Incertae sedis</td><td>Osteoglossidae</td><td>Arapaima gigas</td><td>arapaima</td><td>C</td><td>dentary, rib</td><td>MNHN.ICOS.00557</td></tr><tr><td>†Brychaetus muelleri</td><td>-</td><td>C</td><td>dentary</td><td>NHMUK PV OR 28424</td></tr><tr><td>Osteoglossum bicirrhosum</td><td>silver arowana</td><td>C</td><td>dentary, rib</td><td>MNHN.ICOS.00630</td></tr><tr><td>†Phareodus encaustus</td><td>-</td><td>C</td><td>dentary</td><td>NHMUK PV P 64636I</td></tr><tr><td>†Knightia sp.</td><td>-</td><td>C</td><td>dentary</td><td>UMMP Tmp-1008</td></tr><tr><td>†Armigatidae</td><td>†Armigatus namourensis</td><td>-</td><td>C</td><td>dentary, rib</td><td>NHMUK PV P 63151a</td></tr><tr><td>†Ellimmichthyidae</td><td>†Ellimmichthys longicostatus</td><td>-</td><td>C</td><td>dentary, rib</td><td>NHMUK PV P 9855</td></tr><tr><td>Chirocentridae</td><td>Chirocentrus dorab</td><td>wolf-herring</td><td>C</td><td>dentary, rib</td><td>UMMZ 220543/S</td></tr><tr><td>Clupeidae</td><td>Alosa pseudoharengus</td><td>alewife</td><td>C</td><td>dentary, rib</td><td>UMMZ 187300/S</td></tr><tr><td></td><td>Sardinops sagax</td><td>Pacific sardine</td><td>C</td><td>dentary</td><td>MNHN.ICOS.5036</td></tr><tr><td rowspan="5">Gonorhynchiformes</td><td rowspan="5">Chanidae</td><td>†Tharrias araripes</td><td>-</td><td>C</td><td>dentary, rib</td><td>NHMUK PV P 54675b</td></tr><tr><td>Catostomidae</td><td>Catostomus commersonii</td><td>white sucker</td><td>C</td><td>dentary, rib</td><td>UMMZ 178869/S</td></tr><tr><td>Leuciscidae</td><td>Abramis brama</td><td>freshwater bream</td><td>C</td><td>dentary</td><td>MNHN.ICOS.00756</td></tr><tr><td>Cyprinidae</td><td>Cyprinus carpio</td><td>common carp</td><td>C</td><td>various bones<sup>1</sup></td><td>MNHN.ICOS.00610</td></tr><tr><td>Tincidae</td><td>Tinca tinca</td><td>tench</td><td>C</td><td>dentary</td><td>MNHN.ICOS.00585</td></tr><tr><td rowspan="4">Characiformes</td><td>Cynodontidae</td><td>Hydrolycus scomberoides</td><td>payara</td><td>C</td><td>dentary</td><td>MNHN.ICOS.01021</td></tr><tr><td>Erythrinidae</td><td>Hoplias malabaricus</td><td>trahira</td><td>C</td><td>dentary, rib</td><td>MNHN.ICOS.00631</td></tr><tr><td>Serrasalmidae</td><td>Serrasalmus spilopleura</td><td>speckled piranha</td><td>C</td><td>dentary, rib</td><td>MNHN.ICOS.01027</td></tr><tr><td>Ariidae</td><td>Ariopsis felis</td><td>hardhead sea catfish</td><td>C</td><td>dentary, rib</td><td>UMMZ 223241/S</td></tr><tr><td rowspan="5">Siluriformes</td><td></td><td>Galeichthys feliceps</td><td>white barbel</td><td>C</td><td>dentary</td><td>MNHN.ICOS.00875</td></tr><tr><td>Heptapteridae</td><td>Pimelodella gracilis</td><td>graceful pimelodella</td><td>C</td><td>dentary, rib</td><td>UMMZ 204550/S</td></tr><tr><td>Trichomycteridae</td><td>Trichomycterus sp.</td><td>pencil catfish</td><td>A</td><td>dentary</td><td>MNHN.ICOS.00887</td></tr><tr><td>Gymnotiformes</td><td>Gymnotidae</td><td>Gymnotus carapo</td><td>banded knifefish</td><td>C</td><td>dentary, rib</td><td>UMMZ 207893/S</td></tr><tr><td>Euteleostei</td><td>Incertae sedis</td><td>†Spaniodon elongatus</td><td>-</td><td>C</td><td>dentary, rib</td><td>NHMUK PV OR 44831</td></tr><tr><td rowspan="5">Salmoniformes</td><td rowspan="5">Salmonidae</td><td>Coregonus reighardi</td><td>shortnose cisco</td><td>C</td><td>dentary, rib</td><td>UMMZ 172476/S</td></tr><tr><td>Oncorhynchus clarkii</td><td>cutthroat trout</td><td>C</td><td>dentary</td><td>UMMZ 191615/S</td></tr><tr><td>†Oncorhynchus lacustris</td><td>-</td><td>C</td><td>dentary</td><td>UMMP 47839</td></tr><tr><td>Oncorhynchus mykiss</td><td>rainbow trout</td><td>C</td><td>dentary</td><td>UMMZ uncat.</td></tr><tr><td>Oncorhynchus tshawytscha</td><td>Chinook salmon</td><td>C</td><td>dentary</td><td>UMMZ uncat.</td></tr></tbody></table>
<table>
  <tbody>
    <tr>
      <td></td>
      <td>†Paleolox larsoni</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>UMMP 50352</td>
    </tr>
    <tr>
      <td></td>
      <td>Parahucho perryi</td>
      <td>Japanese huchen</td>
      <td>C</td>
      <td>dentary</td>
      <td>UMMZ 187612</td>
    </tr>
    <tr>
      <td></td>
      <td>†Prosopium prolixus</td>
      <td>-</td>
      <td>C</td>
      <td>dentary</td>
      <td>UMMP 21728</td>
    </tr>
    <tr>
      <td></td>
      <td>Prosopium williamsoni</td>
      <td>mountain whitefish</td>
      <td>C</td>
      <td>dentary</td>
      <td>UMMZ 182503/S</td>
    </tr>
    <tr>
      <td></td>
      <td>Salmo salar</td>
      <td>Atlantic salmon</td>
      <td>C</td>
      <td>dentary</td>
      <td>MNHN.ICOS.00619</td>
    </tr>
    <tr>
      <td></td>
      <td>Salmo trutta</td>
      <td>sea trout</td>
      <td>C</td>
      <td>dentary</td>
      <td>UMMZ uncat.</td>
    </tr>
    <tr>
      <td></td>
      <td>Salvelinus confluentus</td>
      <td>bull trout</td>
      <td>C</td>
      <td>dentary</td>
      <td>UMMZ uncat.</td>
    </tr>
    <tr>
      <td></td>
      <td>Salvelinus fontinalis</td>
      <td>brook trout</td>
      <td>C</td>
      <td>dentary</td>
      <td>UMMZ uncat.</td>
    </tr>
    <tr>
      <td></td>
      <td>Salvelinus namaycush</td>
      <td>lake trout</td>
      <td>C</td>
      <td>dentary</td>
      <td>UMMZ 177542</td>
    </tr>
    <tr>
      <td></td>
      <td>Stenodus leucichthys</td>
      <td>inconnu</td>
      <td>C</td>
      <td>dentary, rib</td>
      <td>UMMZ 187119/S</td>
    </tr>
    <tr>
      <td></td>
      <td>Thymallus thymallus</td>
      <td>grayling</td>
      <td>A</td>
      <td>rib</td>
      <td>MNHN.ICOS.00626</td>
    </tr>
    <tr>
      <td></td>
      <td>Metavelifer multiradiatus</td>
      <td>spinyfin velifer</td>
      <td>A</td>
      <td>rib</td>
      <td>AMNH I-91798 SD</td>
    </tr>
    <tr>
      <td rowspan="10">Acanthomorpha</td>
      <td>Veliferidae</td>
      <td>Lampris sp.</td>
      <td>opah</td>
      <td>C</td>
      <td>sclerotic ossicle</td>
      <td>AMNH I-21766 SD</td>
    </tr>
    <tr>
      <td>Lamprididae</td>
      <td>Polymixiidae</td>
      <td>stout beardfish</td>
      <td>A</td>
      <td>rib</td>
      <td>AMNH I-210677 SD</td>
    </tr>
    <tr>
      <td>Polymixiidae</td>
      <td>Coryphaenidae</td>
      <td>common dolphinfish</td>
      <td>A</td>
      <td>rib</td>
      <td>MNHN.ICOS.00189</td>
    </tr>
    <tr>
      <td>Coryphaenidae</td>
      <td>Trachurus trachurus</td>
      <td>horse mackerel</td>
      <td>A</td>
      <td>rib</td>
      <td>MNHN.ICOS.A-14</td>
    </tr>
    <tr>
      <td>Carangidae</td>
      <td>Xiphias gladius</td>
      <td>swordfish</td>
      <td>A</td>
      <td>rib, sclerotic<br>ossicle</td>
      <td>MNHN.ICOS.6988,<br>AMNH I-15658 SD</td>
    </tr>
    <tr>
      <td>Xiphidae</td>
      <td>Xiphias gladius</td>
      <td>Atlantic white marlin</td>
      <td>A</td>
      <td>rib, sclerotic<br>ossicle</td>
      <td>UMMZ 198674/S</td>
    </tr>
    <tr>
      <td>Istiophoridae</td>
      <td>Kajikia albida</td>
      <td>European seabass</td>
      <td>A</td>
      <td>rib</td>
      <td>private collection</td>
    </tr>
    <tr>
      <td>Moronidae</td>
      <td>Dicentrarchus labrax</td>
      <td>steephead parrotfish</td>
      <td>A</td>
      <td>rib</td>
      <td>MNHN.ICOS.00912</td>
    </tr>
    <tr>
      <td>Scaridae</td>
      <td>Chlorurus microrhinos</td>
      <td>wahoo</td>
      <td>A</td>
      <td>rib</td>
      <td>MNHN.ICOS.01010</td>
    </tr>
    <tr>
      <td>Scombridae</td>
      <td>Acanthocybium solandri</td>
      <td>little tunny</td>
      <td>C</td>
      <td>rib</td>
      <td>AMNH I-56274 SD</td>
    </tr>
    <tr>
      <td></td>
      <td>Euthynnus affinis</td>
      <td>butterfly kingfish</td>
      <td>A</td>
      <td>rib, sclerotic<br>ossicle</td>
      <td>AMNH I-93480 SD</td>
    </tr>
    <tr>
      <td></td>
      <td>Gasterochisma melampus</td>
      <td>dogtooth 'tuna'</td>
      <td>A</td>
      <td>rib</td>
      <td>MNHN.ICOS.00492</td>
    </tr>
    <tr>
      <td></td>
      <td>Gymnosarda unicolor</td>
      <td>striped bonito</td>
      <td>A</td>
      <td>rib</td>
      <td>MNHN.ICOS.00954</td>
    </tr>
    <tr>
      <td></td>
      <td>Sarda orientalis</td>
      <td>blue mackerel</td>
      <td>A</td>
      <td>rib</td>
      <td>MNHN.ICOS.00254</td>
    </tr>
    <tr>
      <td></td>
      <td>Scomber australasicus</td>
      <td>bigeye tuna</td>
      <td>C</td>
      <td>rib</td>
      <td>MNHN.ICOS.00374</td>
    </tr>
    <tr>
      <td></td>
      <td>Thunnus obesus</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
  </tbody>
</table>