For many aquatic creatures, the red blood cells that rush through their bodies are created in organs such as the liver or the kidney. In most land vertebrates however, blood-cell production occurs in the bone marrow. There, the process is shielded from the ultraviolet light or starker temperature changes experienced out of the water.

It is possible that this difference evolved long before the first animal with a backbone crawled out of the aquatic environment and faced new, harsher conditions: yet very little fossil evidence exists to support this idea. A definitive answer demands a close examination of fossils from the water-to-land transition including lobe-finned fish and early limbed vertebrates. To support the production of red blood cells, their fin and limb bones would have needed an internal cavity that can house a specific niche that opens onto a complex network of blood vessels.

To investigate this question, Estefa et al. harnessed the powerful x-ray beam produced by the European Synchrotron Radiation Facility and imaged the fin and limb bones from fossil lobe-finned fish and early limbed vertebrates. The resulting three-dimensional structures revealed spongy long bones with closed internal cavities where the bone marrow cells were probably entrapped. These could not have housed the blood vessels needed to create an environment that produces red blood cells.

In fact, the earliest four-legged land animals Estefa et al. found with an open marrow cavity lived 60 million years after vertebrates had first emerged from the aquatic environment, suggesting that blood cells only began to be created in bone marrow after the water-to-land transition. Future work could help to pinpoint exactly when the change in blood cell production occurred, helping researchers to identify the environmental and biological factors that drove this change.

Tetrapod long bones are among the most studied skeletal elements in the field of bone biology as they constitute a unit of reference for understanding the development and biomechanics of the appendicular skeleton (e.g. Duboule, 1994; Fröbisch, 2008; Hall, 2008; Shubin et al., 1997). The recent discovery of fossil tetrapod trackways (Ahlberg, 2018; Niedźwiedzki et al., 2010) suggested that limbs supported weight and engaged substrate locomotion earlier than previously thought in early tetrapod evolution. Not only crucial for their biomechanical properties, long bones also host bone marrow including stem-cell niches for the production of blood cells, that is haematopoiesis (Orkin and Zon, 2008). After birth, bone marrow is the definitive haematopoietic system in mostly terrestrial mammals and many other tetrapods (Akiyoshi and Inoue, 2012; Kapp et al., 2018; Orkin and Zon, 2008) but not in fish or some aquatic tetrapods (Akiyoshi and Inoue, 2012; Avagyan and Zon, 2016; Kapp et al., 2018). Indeed, red blood cells are produced in the supraspinal organ in the lamprey, the kidney and liver in actinopterygians (ray-finned fish) and some amphibians (tadpoles and aquatic adults, Akiyoshi and Inoue, 2012), and the kidney in lungfish (Amemiya et al., 2007; Kapp et al., 2018). Several studies proposed that the skeleton would have played a major role in hosting blood-cell production over the water-to-land transition and (1) protecting it against temperature changes (Weiss and Wislocki, 1956), (2) protecting it against potential DNA mutations induced by UV exposure on land (Horton, 1980; Kapp et al., 2018) or (3) providing a better efficiency in red-blood-cell production necessary for metabolically-demanding terrestrial locomotion and aerial respiration (Tanaka, 1976). Our study focusses on characterising the early evolution of the bone marrow and long-bone growth in fossils to contextualise these hypotheses.

Tetrapod long bones are regionalised in three parts mirrored from midshaft (Figure 1): (1) the middle of the shaft is called diaphysis; (2) the metaphyses are located at each extremity of the shaft and (3) the epiphyses start above the ossification notch (i.e. where the cortical bone stops forming), extend beyond the metaphyses, and comprise one or more condyles in the case of concave articulations (Francillon-Vieillot et al., 1990). Long bones elongate from the growth plate, which is located in the metaphysis (Figure 1). In this region, the cartilage is progressively substituted with bone: this process is called endochondral ossification (Francillon-Vieillot et al., 1990; Hall, 2005).

Figure 1

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In extant amniotes, long-bone elongation results from the proliferation of longitudinal columns of hypertrophic cartilage cells, called hypertrophic chondrocytes (Francillon-Vieillot et al., 1990; Haines, 1942; Xie et al., 2020; Figure 1A). The latter express collagen type X which facilitates the calcification of the surrounding matrix (Gudmann and Karsdal, 2016; Lüllmann-Rauch, 2015). To do so, the hypertrophic chondrocytes secrete matrix vesicles containing calcium phosphate crystals (Amizuka, 2012; Anderson and Shapiro, 2010). The vesicles align longitudinally along the septa. The crystals penetrate the vesicle membranes to form stellate clusters of needle-shaped apatite in the extra cellular matrix (Amizuka, 2012). The mineralisation thus propagates following the longitudinal organisation of the septa (Amizuka, 2012; Anderson and Shapiro, 2010; Figure 1A). Blood vessels and marrow processes invade the growth plate along these columns of hypertrophic cartilage (Lüllmann-Rauch, 2015; Figure 1A). Lytic enzymes secreted by the bone-marrow cells degrade the cartilage matrix, which is progressively substituted by bone deposition (Lüllmann-Rauch, 2015; Suzuki et al., 1981). Growth factors, such as the vascular endothelial growth factor (VEGF), trigger cartilage calcification and regulate endochondral ossification through stimulation of blood-vessel ingrowth into the diaphysis (Gerber et al., 1999). The lines of calcifying stellate clusters of crystals therefore form a scaffold for future trabecular bone deposition (Amizuka, 2012). This results in the formation of a bony mesh of longitudinal trabeculae (Figure 1A), which is progressively incorporated into the metaphysis where haematopoietic stem cell (HSC) niches (Figure 1A) are located (Calvi et al., 2003; Zhang et al., 2003) in the close vicinity of trabecular/endothelial surfaces (Gong, 1978; Nilsson et al., 2001; Wilson and Trumpp, 2006). HSC form localised niches whose environment is greatly controlled and regulated (Orkin and Zon, 2008; Sipkins et al., 2005; Zhang et al., 2003). Often in mature animals the growth plate disappears, causing the senescence of long-bone elongation (Kilborn et al., 2002). In most amniotes, the trabecular mesh in the metaphysis can be vastly remodelled (Haines, 1975). HSC can thereafter be observed adjacent to epiphyseal trabeculae (Askenasy and Farkas, 2002).

In extant urodeles (e.g. Pleurodeles waltl, De Ricqlès, 1964; De Ricqlès, 1965), the elongation of limb bones differs from the process in amniotes (Francillon-Vieillot et al., 1990; Haines, 1938; Figure 1B). Unlike mammals, endochondral ossification starts at a later stage in urodeles (De Ricqlès, 1964). The diaphyseal cartilaginous matrix is first hollowed by the formation of lacunae that are subsequently filled in with bone marrow, far before endochondral ossification starts (De Ricqlès, 1964). In mammals and birds (e.g. mouse, Zelzer et al., 2002; chicken, Carlevaro et al., 2000), VEGF initiates vascular ingrowth before endochondral ossification starts. In the amphibian Bufo gargarizan, a peak of VEGF expression is present in the hindlimb at metamorphic climax (Gao et al., 2018) paralleling an increase of endochondral ossification activity (Bo et al., 2018). VEGF would therefore seem to play a major role in amphibian long-bone endochondral ossification as well, but this role still needs to be characterised. The growth plate in the metaphysis of urodeles exhibits no aligned columns of hypertrophic cartilage cells or drastically reduced alignment of a few cells at most (De Ricqlès, 1965; Dickson, 1982; Felisbino and Carvalho, 1999; Felisbino and Carvalho, 2001; Figure 1B). Contrary to amniotes, when present, these aligned columns of hypertrophic cartilage do not constitute the location where the ossification takes place (Figure 1B). Instead, the mineralisation front is located in the underlying areas of the growth plate (i.e. in a layer of non-oriented hypertrophic cartilage or stratified non-oriented hypertrophic cartilage, De Ricqlès, 1965). There, after erosion of the cartilage, mineralisation occurs in urodeles via the formation of globular structures called globuli ossei (Figure 1B; De Ricqlès, 1965; Quilhac et al., 2014) and spherical mineralisation around them (forming Liesegang’s rings, Francillon-Vieillot et al., 1990). Globuli ossei are either (1) opened hypertrophic cartilaginous cells which died and were subsequently invaded by a cell from the blood/marrow system to initiate mineralisation or (2) uneroded hypertrophic cartilaginous cells modified into active cells which synthesise bone-like collagen fibrils (of intermediate size between type II of the cartilage and type I of the bone, Quilhac et al., 2014). The endochondral ossification therefore does not produce a longitudinally-oriented trabecular network (Figure 1B), but forms instead a light reticular mesh (De Ricqlès, 1965; Quilhac et al., 2014; Sanchez et al., 2008), rich in globuli ossei (De Ricqlès, 1964; Haines, 1938; Quilhac et al., 2014; Sanchez et al., 2010a). The epiphyses of urodeles remain cartilaginous while they often ossify in anurans (Castanet et al., 2003; Francillon, 1981; Sanchez et al., 2008). This is probably an adaptation to a demanding jumping locomotion and/or heterochronic mechanisms relevant to this clade (Francillon, 1981). In the medullary cavity of their long bones however, the cartilaginous cells hypertrophy with no preferential orientation as in urodeles (Dickson, 1982; Felisbino and Carvalho, 1999; Felisbino and Carvalho, 2001; Miura et al., 2008; Rozenblut and Ogielska, 2005). The resulting spongiosa is largely reduced (even quite often absent, Francillon, 1981). The epiphyseal cartilage hangs over the ossification notch and the shaft (Francillon, 1981) to ossify straight after the metamorphosis (Miura et al., 2008; Rozenblut and Ogielska, 2005). The function of bone marrow in amphibian long bones also differs from the function in extant amniotes. Indeed, in amphibians, the sites for haematopoiesis almost exclusively comprise the thymus, spleen and liver (Akiyoshi and Inoue, 2012; Hightower and Pierre, 1971). Bone marrow only plays a role of haematopoiesis in a few amphibian species (e.g. Xenopus laevis, Rana catesbeiana, Tanaka, 1976; Phillobates terribilis, Dendrobates tinctorius, Kapp et al., 2018). In these cases, haematopoiesis occurs in endosteal regions of the diaphysis between sinusoids (i.e. fenestrated capillaries; Figure 1B2) and endosteum (Tanaka, 1976). No HSC has been observed so far in the epiphysis of frogs.

Is the urodele model the plesiomorphic or the derived condition for tetrapod long-bone elongation and bone-marrow function? Very little attention has been given to these aspects of limb-bone evolution. On the one hand, some authors suggest that the amniote-like elongation process may have been the primitive state (Haines, 1942) but no fossil evidence was provided. On the other hand, early tetrapods had cartilaginous epiphyses like extant urodeles. Could that be an indication for a urodele-like primitive condition (e.g. Sanchez et al., 2008; Sanchez et al., 2010a)? This debate relied on the absence of evidence from stem-tetrapod data. Recently, palaeohistological studies revealed a fan-like longitudinal trabecular arrangement in the long-bone metaphysis of the 380-million-year-old lobe-finned fish Eusthenopteron (Sanchez et al., 2014), and in the 365-million-year-old limbed stem-tetrapod Acanthostega (Sanchez et al., 2016). These patterns would result from the same elongation process as in amniotes and would rather suggest that amphibians exhibit a derived condition. Using three-dimensional (3D) virtual histology based on propagation phase-contrast X-ray synchrotron radiation micro-computed tomography (PPC-SRµCT), as well as classical thin-section histology, we herein investigate several stem amphibians and stem amniotes to provide the first glimpses for characterising the early evolution of long-bone elongation and bone-marrow roles.

The diaphyseal and metaphyseal microarchitectures of the stem tetrapods Eusthenopteron and Hyneria were described by Sanchez et al., 2014 and Kamska et al., 2018, respectively. The diaphysis of Discosauriscus (Sanchez et al., 2008), Apateon (Sanchez et al., 2010a; Sanchez et al., 2010b), Metoposaurus (Konietzko-Meier and Sander, 2013) and Seymouria (Estefa et al., 2020) were thoroughly described, but the metaphyseal organisation of their humeri was only succinctly mentioned in the cited articles. Here, we provide a detailed description of them (Table 1) in 3D when possible.

Apateon caducus, juvenile specimen GPIM-N 1297, humerus

As the humerus was crushed (Figure 2A), only a small region of the metaphysis could be sectioned and visualised (Figure 2B). Nevertheless, a relatively complete sequence of calcification (extending over 600 µm) can be described here. The upper part of the section reflects the irregular surface of the calcification front (separating the unpreserved eroded non-calcified cartilage from the preserved calcified cartilage) (Figure 2B). Under this region, obvious figures of globuli ossei are entrapped in Liesegang’s rings (g.o. and l.r., Figure 2Bb1-2). They are numerous and unevenly arranged. Their sizes (ranging from 9 to 15 µm in diameter) seem as well unevenly distributed. The trabeculae are very few in this thin section (t., Figure 2Bb2).

Figure 2

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A. caducus, adult specimen GPIM-N 1572, radius and ulna

The epiphysis and metaphysis of the radius and ulna of this individual (Figure 3A) are more hollowed than those of the specimen GPIM-N 1297, with less cartilaginous matrix between the mineralised trabeculae (Figure 3B). Fewer globuli ossei are visible (g.o., Figure 3Bb1). Instead, large empty lacunae can be observed (75 µm) (e.l., Figure 3Bb1). Large bays of erosion open as well between these lacunae (e.b., Figure 3Bb2). The process of mineralisation therefore seems more advanced but no obvious trabecular organisation can be observed.

Figure 3

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Apateon pedestris, adult specimen SMNS 54981, humerus

In the humerus of SMNS 54981 (Figure 4A), the process of mineralisation seems relatively advanced as the globuli ossei only remain along a few mineralised trabeculae (g.o., Figure 4B). They are 16 µm large. In the metaphysis, the cartilage has been removed by erosional process (e.b., Figure 4B). The uncalcified cartilage in the epiphysis has not been preserved during the fossilisation (at least in this slide).

Figure 4

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A. pedestris, adult specimen SMNS 54988, humerus

This thin section in the humerus of SMNS 54988 (Figure 5A) shows a very remodelled bone with large bays of erosion in the cartilaginous matrix (e.b., Figure 5Bb) and only a few remaining globuli ossei at the surface of the bone trabeculae (g.o., Figure 5Bb). Most of the cartilaginous matrix has been eroded. There is no preserved cartilage in the epiphysis. The bony trabeculae have no preferential orientation.

Figure 5

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Metoposaurus sp., (sub-)adult specimen MUZ PGI OS-220/171, humerus

Transverse thin sections were made in the metaphysis of the femur (Konietzko-Meier and Sander, 2013) of Metoposaurus diagnosticus krasiejowensis (Sulej, 2002) recently re-diagnosed as Metoposaurus krasiejowensis (Brusatte et al., 2015). They revealed a dense trabecular mesh. The longitudinal virtual thin sections, made with PPC-SRµCT and presented here, were made in the proximal and distal metaphyses of a humerus of Metoposaurus sp. and confirm the presence of a dense trabecular mesh in the overall humerus (Figure 6Aa1-2). Additionally, a directional coloured light effect (cf. Materials and method section, Sanchez et al., 2014) shows that this mesh is oriented longitudinally and exhibits a fan-like shape in the metaphyses (purple trabeculae, Figure 6Aa2). The trabecular mesh covers the entire volume of the metaphysis and spreads into the diaphysis (Figure 6Aa1-2). The mineralisation front (m.f., Figure 6Aa1) contacts the sediment in which the bone is embedded (Figure 6Ba). The surface of the mineralisation front is irregular. No ossified epiphysis was found, thereby suggesting that a cartilaginous cap was probably covering the bone. This cap did not preserve over the fossilisation. In the metaphysis, the trabeculae are homogeneously distributed (t., Figure 6Bb). Some remnants of calcified cartilage are visible through Liesegang’s rings forming within the cartilage remaining between the metaphyseal trabeculae (Figure 6Bc). The mean thickness of the trabeculae is 117 µm (Table 2). Tubular structures can be observed (m.p., Figure 6Ba). They end blindly at the location of the mineralisation front. They are well defined tubes (248 µm in diameter, Table 2), although anastomosed. They ossified through endochondral ossification. These tubes are locally slightly eroded (Figures 6Ba and 10A). The size of these tubes, their intimate connection to each other and their location strongly support their identification as marrow processes (Haines, 1938).

Figure 6

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Table 2

Species	Thickness of the trabeculae (µm)		Diameter of the marrow processes (µm)
	Diaphysis	Metaphysis	Metaphysis
Metoposaurus sp. (Subadult or Adult, MUZ PGI OS-220/171)	131	117	248
Seymouria sanjuanensis (Juvenile, MNG 7747)	94	25	100
S. sanjuanensis (Adult, CM 28597)	79	30	100
Discosauriscus austriacus (Subadult, SNM Z 15568)	80	54	111

Seymouria sanjuanensis, juvenile specimen MNG 7747, humerus

This specimen was investigated using PPC-SRµCT. The spongiosa occupies the entire bone area (Figure 7A). The metaphyseal trabeculae are about four times thinner (25 µm on average) than the diaphyseal trabeculae (94 µm on average, Table 2 and Estefa et al., 2020). A longitudinal section reveals that the trabecular mesh becomes denser towards the distal and proximal ends of the bone (Figure 7Aa1-2). As the shape of the bone widens and flattens from midshaft towards the metaphyseal surfaces, the longitudinal trabeculae tilt, thereby forming a fan-like configuration (Figure 7Aa2). In the metaphysis, the trabecular mesh is mostly arranged longitudinally (obviously appearing purple and progressively shifting to green as the deltopectoral crest tilts to 90 degrees, Figure 7Aa2) although a few anastomoses (highlighted in green in most of the metaphysis apart from the tilted region of the deltopectoral crest, Figure 7Aa2) run radially. A few remnants of calcified cartilage (Francillon-Vieillot et al., 1990) are very rarely visible (Estefa et al., 2020). Marrow processes form an intricate network, while anastomosing to each other, and connecting to cavities of irregular shapes and sizes (Figure 7Ba,c and 10B). These tubular structures are around 100 µm in diameter under the mineralisation front (Table 2). They contact each other when they reach the mineralisation front (Figure 10B). No ossified epiphysis was found. The humeral epiphysis was probably not preserved due to being unmineralised cartilage.

Figure 7

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S. sanjuanensis, adult specimen CM 28597, humerus

A longitudinal virtual thin section from the PPC-SRµCT data shows that the trabecular network remains relatively dense in the metaphyses (Figure 8A) at the adult stage. The trabecular mesh is longitudinally and radially oriented like a fan although slightly less organised than in the juvenile specimen (purple trabeculae, Figure 8Aa2). The trabeculae appear to be more remodelled, leaving large cavities resulting from an intense erosional process (Figure 8Aa2). The cortex is almost inexistent in the metaphysis (Figure 8Bb). The thickness of the trabeculae averages 30 µm (Table 2, Estefa et al., 2020), which is equivalent to the thickness of the trabeculae in the juvenile metaphysis (MNG 7747). There is no endosteal bone on the surface of the medullary cavity. Very few remnants of calcified cartilage were found in the metaphysis of the adult humerus, that is in much lower frequency than in the juvenile specimen (Estefa et al., 2020). The spongiosa contains a few longitudinal interconnected marrow processes (100 µm in diameter, Figure 8Bb-c and 10C, Table 2). When present, these processes exhibit the same distribution as in the juvenile humerus. The epiphyses were not ossified. A resting surface (yellow arrow, Figure 8Aa2), red arrows, (Figure 8—figure supplement 1A), can be observed 2-to-4 mm under the mineralisation front. It is observed as well in the distal metaphysis (Figure 8—figure supplement 1A). This resembles Harris lines identified in mammals (Garn et al., 1968; Harris, 1933) and birds (Wegner, 1874). Although these lines are very common in mammals (including extant and extinct taxa, Duckler and Van Valkenburgh, 1998), they have not been comprehensively studied in other groups. We find that they can also be encountered in groups with no secondary ossification centre such as chelonians (e.g. Centrochelys sulcata, Figure 8—figure supplement 1B) and crocodilians (e.g. Crocodylus niloticus, P.T. pers. obs.). Harris lines seem to result from both short- and long-term pressures (e.g. starvation – Park, 1964; disease and deficiencies – Duckler and Van Valkenburgh, 1998).

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Discosauriscus austriacus, subadult specimen SNM Z 15568, humerus

Thin sections were made and described in the distal metaphysis of the femur of the specimen KO224 of D. austriacus (Sanchez et al., 2008). They revealed a dense trabecular mesh and the absence of calcified cartilage. Virtual thin sections from three-dimensional PPC-SRµCT scans in the humerus SNM Z 15568 complete these observations despite the fact that the metaphyseal spongiosa is partly crushed (Figure 9Aa2). As the femur KO224 (Sanchez et al., 2008), the humerus SNM Z 15568 exhibits a dense trabecular mesh in both the proximal and distal metaphyses with a fan-like trabecular orientation (purple trabeculae, Figure 9Aa2). The trabeculae are homogeneously distributed in the metaphysis. They are 54 µm thick on average (Table 2). They are eroded at the base of the metaphysis (Figure 9Aa1-2). Some tubular marrow processes (average diameter: 111 µm, Table 2) open up directly towards the diaphysis into the metaphyseal space of the medullary cavity left vacant after erosion (Figures 9B and 10D). The surface of the mineralisation front is irregular (Figure 9Ba). The latter probably was covered by uncalcified cartilage.

Figure 9

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Figure 10 with 1 supplement see all

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The trabecular bone tissues observed in these long bones exhibit characteristics of endochondral ossification (remnants of calcified cartilage, globuli ossei and/or columnar trabecular mesh) as seen in stem- (Sanchez et al., 2014; Sanchez et al., 2016) and crown-tetrapods (Estefa et al., 2020; Francillon-Vieillot et al., 1990; Sanchez et al., 2008; Sanchez et al., 2010a).

Early evolution of tetrapod limb-bone elongation

Although all limb bones studied here have cartilaginous epiphyses, their metaphyseal organisation, and the underlying long-bone elongation processes, can greatly differ between taxa.

The long-bone elongation in Apateon probably results from the hypertrophying action of scattered cartilaginous cells in the upper part of the metaphysis (Figure 11). The fossils revealed a mineralisation front characterised by a large number of globuli ossei (Figures 2–5), which progressively replaced these hypertrophic chondrocytes, as in urodeles (De Ricqlès, 1964; De Ricqlès, 1965; Haines, 1938; Quilhac et al., 2014). As the uncalcified cartilage is not preserved in Apateon, it is not possible to check whether the growth zone and calcifying zone are distant as in amphibians (De Ricqlès, 1965; Felisbino and Carvalho, 1999; Felisbino and Carvalho, 2001). However, the presence of very few bony trabeculae, with no preferential orientation in the metaphyseal region of Apateon’s long bones (Figures 2–5) strongly support the idea that the ossification did not occur within a zone of columnar hypertrophic cartilage. Bone elongation was probably followed by mineralisation through globuli ossei as previously observed in the temnospondyl dissorophoid Doleserpeton (De Ricqlès, 1963; Figure 11).

Figure 11

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On the contrary, the metaphyseal trabecular meshes in the limb bones of Metoposaurus, Seymouria and Discosauriscus all exhibit the same fan-like pattern of longitudinal trabeculae. This implies that the growth plate comprised longitudinal columns of hypertrophic cells where endochondral ossification occurred through columnar cartilage-to-bone substitution (Francillon-Vieillot et al., 1990; Kronenberg, 2003) as seen in turtles and crocodiles (e.g. Haines, 1938; Haines, 1942), lepidosaurs (e.g. Haines, 1969), dinosaurs (De Ricqlès, 1968; Horner et al., 2000; Horner et al., 2001), birds (e.g. Horner et al., 2001) and mammals (e.g. Jacenko et al., 1993; Figure 11). Although this fan-like trabecular configuration can be greatly remodelled in extant amniotes, it is only slightly remodelled here in the adult Seymouria and subadult Discosauriscus (Figures 8 and 9).

Therefore, our study shows that the amniote-like long-bone elongation is more commonly distributed than previously thought. It is not restrained to the appendicular skeleton of amniotes – including Discosauriscus and Seymouria (as demonstrated here), Ophiacodon, Dicynodon and some kannemeyeriids (De Ricqlès, 1972; Haines, 1938), marine reptiles, Plesiosaurus and Nothosaurus (Haines, 1938) – neither to that of stem tetrapods (Kamska et al., 2018; Sanchez et al., 2014; Sanchez et al., 2016; Figure 11). Indeed, our study demonstrates for the first time that certain temnospondyls like Metoposaurus also elongated their appendicular skeleton like amniotes (Figure 11). We can confidently conclude that (1) endochondral ossification based on the mineralisation of longitudinal columns of hypertrophic cartilage is a primitive process for the elongation of the appendicular skeleton in tetrapods and that (2) ossification through globuli ossei is restricted to a limited group of stem- and crown-batrachians (Figure 11).

The mineralisation processes in extant amphibians and amniotes largely differ in many points: (1) their timing and microstructural relationships (i.e. ossification dependant on a calcified scaffold in amniotes, Amizuka, 2012, but not in amphibians, Felisbino and Carvalho, 2001), (2) their initiation (i.e. stacks of hypertrophic cells in amniotes, Lüllmann-Rauch, 2015, versus isolated hypertrophic cells in amphibians, De Ricqlès, 1965; Quilhac et al., 2014) and (3) their molecular mechanisms (i.e. collagen type X secreted in amniotes, Gudmann and Karsdal, 2016; versus fibrillar collagens in amphibians, Quilhac et al., 2014). How could such distinct mineralisation processes play the same functional role in long-bone elongation in different vertebrates? In addition to a columnar pattern of trabeculae, several remnants of Liesegang’s rings could be observed in the fossil limb bones of Metoposaurus (Figure 6Bc and Konietzko-Meier and Sander, 2013) and, to a lesser extent, Seymouria (Estefa et al., 2020) and stem-tetrapods (i.e. Hyneria, Eusthenopteron and Acanthostega, Kamska et al., 2018; Sanchez et al., 2014; Sanchez et al., 2016). The most parsimonious evolutionary scenario therefore suggests that stem tetrapods were able to produce globular calcified cartilage although they were elongating their bone through a columnar configuration (Figure 11). Long-bone elongation exclusively based on the intensive production of globuli ossei would have been a derived feature emerging within temnospondyls and restricted to extant batrachians and their close dissorophoid temnospondyl relatives (e.g. Apateon and Doleserpeton). Within amniotes – including stem amniotes – the globular calcification of the cartilage would have drastically reduced to be fully abandoned to the benefit of an exclusive columnar elongation (Figure 11). Two exceptions persist in amniotes: (1) large aquatic animals usually produce a large amount of globular calcified cartilage to balance their buoyancy or in extremely retarded developmental conditions, such as paedomorphosis (e.g. pachy-osteosclerotic amniotes, De Buffrénil et al., 2008; De Ricqlès and De Buffrénil, 2001; crocodiles, Haines, 1938), (2) diseased amniotes can have osteosclerotic problems which result in the production and retention of globuli ossei (Gussen, 1967). In such cases, the production of globuli ossei is not solely located in the epiphysis and does not play any role in the elongation process of limb bones. Even though these cases reflect derived and/or rare conditions, they show that amniotes keep the ability to produce globuli ossei although they do not allocate them to the limb-bone elongation process.

The regions of interest of the reconstructed data are available on http://paleo.esrf.eu/. The raw data have been deposited to the European Synchrotron Radiation Facility. All the fossils and thin sections observed are available in the museum/university collections cited in the Materials and Methods section.

1. 1. Sanchez S
   2. Estefa J
   3. Tafforeau P

   (2021) European Synchrotron Radiation Facility

   Radiographic data of the complete scan of Discosauriscus humerus SNMZ 15568 and associated partial 3D reconstructions.

   https://doi.org/10.15151/ESRF-DC-385889323
2. 1. Sanchez S
   2. Estefa J
   3. Tafforeau P

   (2021) European Synchrotron Radiation Facility

   Radiographic data of the complete scan of Seymouria juvenile humerus MNG7747 and associated partial 3D reconstructions.

   https://doi.org/10.15151/ESRF-DC-386797910
3. 1. Sanchez S
   2. Estefa J
   3. Tafforeau P

   (2021) European Synchrotron Radiation Facility

   Radiographic data of the complete scan of Seymouria adult humerus CM-28597 and associated partial 3D reconstructions.

   https://doi.org/10.15151/ESRF-DC-386801611
4. 1. Sanchez S
   2. Estefa J
   3. Tafforeau P

   (2021) European Synchrotron Radiation Facility

   Radiographic data of the complete scan of Metoposaurus humerus MUZ-PGI-OS-220171 and associated partial 3D reconstructions.

   https://doi.org/10.15151/ESRF-DC-386788297

1. 1. Ahlberg PE

   (2018) Follow the footprints and mind the gaps: a new look at the origin of tetrapods

   Earth and Environmental Science Transactions of the Royal Society of Edinburgh 109:115–137.

   https://doi.org/10.1017/S1755691018000695

   • Google Scholar
2. 1. Akiyoshi H
   2. Inoue AM

   (2012) Comparative histological study of hepatic architecture in the three orders amphibian livers

   Comparative Hepatology 11:2.

   https://doi.org/10.1186/1476-5926-11-2

   • PubMed
   • Google Scholar
3. 1. Amemiya CT
   2. Saha NR
   3. Zapata A

   (2007) Evolution and development of immunological structures in the lamprey

   Current Opinion in Immunology 19:535–541.

   https://doi.org/10.1016/j.coi.2007.08.003

   • PubMed
   • Google Scholar
4. 1. Amizuka N

   (2012) Histology of epiphyseal cartilage calcification and endochondral ossification

   Frontiers in Bioscience E4:2085–2100.

   https://doi.org/10.2741/e526

   • Google Scholar
5. Book

   1. Anderson JS

   (2007)

   Incorporating ontogeny into the matrix: a phylogenetic evaluation of developmental evidence for the origin of modern amphibians

   In: Anderson J. S, Sues H. -D, editors. Major Transitions in Vertebrate Evolution. Bloomington: Indiana University Press. pp. 182–227.

   • Google Scholar
6. 1. Anderson JS

   (2008) Focal review: the origin(s) of modern amphibians

   Evolutionary Biology 35:231–247.

   https://doi.org/10.1007/s11692-008-9044-5

   • Google Scholar
7. Book

   1. Anderson HC
   2. Shapiro IM

   (2010)

   The Epiphyseal growth plate

   In: Bronner F, Farach-Carson M, Roach H, editors. Bone and Development, Topics in Bone Biology, 6. London: Springer-Verag London Limited. pp. 39–64.

   • Google Scholar
8. 1. Askenasy N
   2. Farkas DL

   (2002) Optical imaging of PKH-labeled hematopoietic cells in recipient bone marrow in vivo

   Stem Cells 20:501–513.

   https://doi.org/10.1634/stemcells.20-6-501

   • PubMed
   • Google Scholar
9. 1. Avagyan S
   2. Zon LI

   (2016) Fish to learn: insights into blood development and blood disorders from zebrafish hematopoiesis

   Human Gene Therapy 27:287–294.

   https://doi.org/10.1089/hum.2016.024

   • PubMed
   • Google Scholar
10. 1. Bazzini MD
    2. Reynolds, III JE
    3. Essman RA

    (1986) Erythropoiesis and granulopoiesis in the west indian Manatee, Trichechus manatus (Mammalia: sirenia)

    Cells Tissues Organs 126:150–152.

    https://doi.org/10.1159/000146205

    • Google Scholar
11. 1. Berman DS
    2. Reisz RR
    3. Eberth DA

    (1987a) A new genus and species of trematopid amphibian from the Late Pennsylvanian of North-Central New Mexico

    Journal of Vertebrate Paleontology 7:252–269.

    https://doi.org/10.1080/02724634.1987.10011659

    • Google Scholar
12. 1. Berman DS
    2. Reisz RR
    3. Eberth DA

    (1987b) Seymouria sanjuanensis (Amphibia, Batrachosauria) from the Lower Permian Cutler Formation of North-Central New Mexico and the occurrence of sexual dimorphism in that genus questioned

    Canadian Journal of Earth Sciences 24:1769–1784.

    https://doi.org/10.1139/e87-169

    • Google Scholar
13. 1. Berman DS
    2. Henrici AC
    3. Sumida SS
    4. Martens T

    (2000) Redescription of Seymouria sanjuanensis (Seymouriamorpha) from the Lower Permian of Germany based on complete, mature specimens with a discussion of paleoecology of the Bromacker locality assemblage

    Journal of Vertebrate Paleontology 20:253–268.

    https://doi.org/10.1671/0272-4634(2000)020[0253:ROSSSF]2.0.CO;2

    • Google Scholar
14. 1. Berman DS
    2. Martens T

    (1993)

    First occurence of seymouria (Amphibia: Batrachosauria) in the Lower Permian rotliegend of central germany

    Annals of Carnegie Museum 62:63–79.

    • Google Scholar
15. 1. Bo X
    2. Wu M
    3. Xiao H
    4. Wang H

    (2018) Transcriptome analyses reveal molecular mechanisms that regulate endochondral ossification in amphibian Bufo gargarizans during metamorphosis

    Biochimica et Biophysica Acta (BBA) - General Subjects 1862:2632–2644.

    https://doi.org/10.1016/j.bbagen.2018.07.032

    • Google Scholar
16. 1. Boy JA

    (2003) Paläoökologische Rekonstruktion von Wirbeltieren: Möglichkeiten und Grenzen

    Paläontologische Zeitschrift 77:123–152.

    https://doi.org/10.1007/BF03004564

    • Google Scholar
17. 1. Brusatte SL
    2. Butler RJ
    3. Mateus O
    4. Steyer JS

    (2015) A new species of Metoposaurus from the Late Triassic of Portugal and comments on the systematics and biogeography of metoposaurid temnospondyls

    Journal of Vertebrate Paleontology 35:e912988.

    https://doi.org/10.1080/02724634.2014.912988

    • Google Scholar
18. 1. Calvi LM
    2. Adams GB
    3. Weibrecht KW
    4. Weber JM
    5. Olson DP
    6. Knight MC
    7. Martin RP
    8. Schipani E
    9. Divieti P
    10. Bringhurst FR
    11. Milner LA
    12. Kronenberg HM
    13. Scadden DT

    (2003) Osteoblastic cells regulate the haematopoietic stem cell niche

    Nature 425:841–846.

    https://doi.org/10.1038/nature02040

    • Google Scholar
19. 1. Carlevaro MF
    2. Cermelli S
    3. Cancedda R
    4. Descalzi Cancedda F

    (2000)

    Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation

    Journal of Cell Science 113:59–69.

    • PubMed
    • Google Scholar
20. Book

    1. Castanet J
    2. Francillon-Vieillot H
    3. De Ricqlès A

    (2003)

    The Skeletal histology of the Amphibia

    In: Heatwole H, Davies M, editors. Amphibian Biology. Chipping Norton: Surrey Beatty & Sons. pp. 1598–1683.

    • Google Scholar
21. 1. Cau A
    2. Beyrand V
    3. Voeten D
    4. Fernandez V
    5. Tafforeau P
    6. Stein K
    7. Barsbold R
    8. Tsogtbaatar K
    9. Currie PJ
    10. Godefroit P

    (2017) Synchrotron scanning reveals amphibious ecomorphology in a new clade of bird-like dinosaurs

    Nature 552:395–399.

    https://doi.org/10.1038/nature24679

    • PubMed
    • Google Scholar
22. 1. De Buffrénil V
    2. Houssaye A
    3. Böhme W

    (2008) Bone vascular supply in monitor lizards (Squamata: Varanidae): Influence of size, growth, and phylogeny

    Journal of Morphology 269:533–543.

    https://doi.org/10.1002/jmor.10604

    • Google Scholar
23. Thesis

    1. De Ricqlès A

    (1963)

    Recherches paléohistologiques sur les os longs des tétrapodes

    PhD thesis.

    • Google Scholar
24. 1. De Ricqlès A

    (1964)

    La formation des os longs des membres de Pleurodeles watlii (Michahelles) - Première partie

    Bulletin De La Société Zoologique De France 89:797–808.

    • Google Scholar
25. 1. De Ricqlès A

    (1965)

    La formation des os longs des membres de Pleurodeles waltlii (Michahelles) - Deuxième partie

    Bulletin De La Société Zoologique De France 90:267–286.

    • Google Scholar
26. 1. De Ricqlès A

    (1968)

    Recherches paléohistologiques sur les os longs des tétrapodes: origine Du tissu osseux plexiforme des dinosauriens sauropodes

    Annales De Paléontologie 54:133–145.

    • Google Scholar
27. 1. De Ricqlès A

    (1972)

    Nature et signification des "surfaces épiphysaires" chez les tétrapodes fossiles

    Compte-Rendu De l'Académie Des Sciences De Paris 274:3527–3530.

    • Google Scholar
28. 1. De Ricqlès A

    (1979)

    Quelques remarques sur l'histoire évolutive des tissus squelettiques chez les vertébrés et plus particulièrement chez les tétrapodes

    L'Année Biologique 18:1–35.

    • Google Scholar
29. Book

    1. De Ricqlès A
    2. De Buffrénil V

    (2001)

    Bone histology, heterochronies and the return of tetrapods to life in water: where are we?

    In: Mazin J. M, De Buffrénil V, editors. Secondary Adaptation of Tetrapods to Life in Water. München: Verlag: Dr. Friedrich Pfeil. pp. 289–310.

    • Google Scholar
30. 1. Dickson GR

    (1982)

    Ultrastructure of growth cartilage in the proximal femur of the frog, Rana temporaria

    Journal of Anatomy 135:549–564.

    • PubMed
    • Google Scholar
31. 1. Duboule D

    (1994) How to make a limb?

    Science 266:575–576.

    https://doi.org/10.1126/science.7939709

    • Google Scholar
32. 1. Duckler GL
    2. Van Valkenburgh B

    (1998) Exploring the health of late Pleistocene mammals: the use of Harris lines

    Journal of Vertebrate Paleontology 18:180–188.

    https://doi.org/10.1080/02724634.1998.10011042

    • Google Scholar
33. 1. Estefa J
    2. Klembara J
    3. Tafforeau P
    4. Sanchez S

    (2020) Limb-Bone development of seymouriamorphs: implications for the evolution of growth strategy in stem amniotes

    Frontiers in Earth Science 8:97.

    https://doi.org/10.3389/feart.2020.00097

    • Google Scholar
34. 1. Felisbino SL
    2. Carvalho HF

    (1999) The epiphyseal cartilage and growth of long bones in Rana catesbeiana

    Tissue and Cell 31:301–307.

    https://doi.org/10.1054/tice.1999.0036

    • PubMed
    • Google Scholar
35. 1. Felisbino SL
    2. Carvalho HF

    (2001) Growth cartilage calcification and formation of bone trabeculae are late and dissociated events in the endochondral ossification of Rana catesbeiana

    Cell and Tissue Research 306:319–323.

    https://doi.org/10.1007/s004410100446

    • PubMed
    • Google Scholar
36. 1. Francillon H

    (1981)

    Contribution à l'étude des structures et du fonctionnement des épiphyses fémorales chez les amphibiens anoures

    Archives De Zoologie Expérimentale & Générale pp. 289–310.

    • Google Scholar
37. Book

    1. Francillon-Vieillot H
    2. De Buffrénil V
    3. Castanet J
    4. Géraudie J
    5. Meunier F-J
    6. Sire J-Y
    7. Zylberberg L
    8. De Ricqlès A

    (1990)

    Microstructure and mineralization of vetebrate skeletal tissues

    In: Carter J. G, editors. Skeletal Biomineralization: Patterns, Processes and Evolutionary Trends. New York: Van Nostrand Reinhold. pp. 471–530.

    • Google Scholar
38. 1. Fröbisch NB

    (2008) Ossification patterns in the tetrapod limb - conservation and divergence from morphogenetic events

    Biological Reviews 83:571–600.

    https://doi.org/10.1111/j.1469-185X.2008.00055.x

    • Google Scholar
39. 1. Gao J
    2. Li X
    3. Zhang Y
    4. Wang H

    (2018) Endochondral ossification in hindlimbs during Bufo gargarizans metamorphosis: A model of studying skeletal development in vertebrates

    Developmental Dynamics 247:1121–1134.

    https://doi.org/10.1002/dvdy.24669

    • Google Scholar
40. 1. Garn SM
    2. Silverman FN
    3. Hertzog KP
    4. Rohmann CG

    (1968)

    Lines and bands of increased density

    American Journal of Physical Anthropology 31:89–92.

    • Google Scholar
41. 1. Gerber H-P
    2. Vu TH
    3. Ryan AM
    4. Kowalski J
    5. Werb Z
    6. Ferrara N

    (1999) VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation

    Nature Medicine 5:623–628.

    https://doi.org/10.1038/9467

    • Google Scholar
42. 1. Gong J

    (1978) Endosteal marrow: a rich source of hematopoietic stem cells

    Science 199:1443–1445.

    https://doi.org/10.1126/science.75570

    • Google Scholar
43. Book

    1. Gudmann NS
    2. Karsdal MA

    (2016) Type X Collagen

    In: Karsdal M. A, editors. Biochemistry of Collagens, Laminins and Elastin: Structure, Function and Biomarkers. Academic Press. pp. 73–76.

    https://doi.org/10.1016/C2015-0-05547-2

    • Google Scholar
44. 1. Gussen R

    (1967) Globuli interossei as a manifestation of bone resorption

    Acta Oto-Laryngologica 63:411–422.

    https://doi.org/10.3109/00016486709128775

    • PubMed
    • Google Scholar
45. 1. Haines RW

    (1938)

    The primitive form of epiphysis in the long bones of tetrapods

    Journal of Anatomy 72:323–343.

    • PubMed
    • Google Scholar
46. 1. Haines RW

    (1942) The evolution of epiphyses and of endochondral bone

    Biological Reviews 17:267–292.

    https://doi.org/10.1111/j.1469-185X.1942.tb00440.x

    • Google Scholar
47. Book

    1. Haines RW

    (1969) Epiphyses and sesamoids

    In: Gans C, AdA B. ellairs, editors. Biology of the Reptilia. New York: Academic Press. pp. 81–115.

    https://doi.org/10.1093/icb/icp063

    • Google Scholar
48. 1. Haines RW

    (1975)

    The histology of epiphyseal union in mammals

    Journal of Anatomy 120:1–25.

    • Google Scholar
49. Book

    1. Hall BK

    (2005)

    Bones and Cartilage: Developmental and Evolutionary Skeletal Biology

    London: Elsevier Academic Press.

    • Google Scholar
50. Book

    1. Hall BK

    (2008) Fins Into Limbs: Evolution, Development and Transformation

    Chicago: University of Chicago Press.

    https://doi.org/10.1111/j.1469-7580.2008.00863.x

    • Google Scholar
51. Book

    1. Harris HA

    (1933) Bone Growth in Health and Disease: The Biological Principles Underlying the Clinical, Radiological, and Histological Diagnosis of Perversions of Growth and Disease in the Skeleton

    London: Oxford University Press.

    https://doi.org/10.1001/jama.1933.02740520053040

    • Google Scholar
52. 1. Hightower JA
    2. Pierre RLS

    (1971) Hemopoietic tissue in the adult newt, Notopthalmus viridescens

    Journal of Morphology 135:299–307.

    https://doi.org/10.1002/jmor.1051350304

    • PubMed
    • Google Scholar
53. 1. Horner JR
    2. De Ricqlès A
    3. Padian K

    (2000) Long bone histology of the hadrosaurid dinosaur Maiasaura peeblesorum: growth dynamics and physiology based on an ontogenetic series of skeletal elements

    Journal of Vertebrate Paleontology 20:115–129.

    https://doi.org/10.1671/0272-4634(2000)020[0115:LBHOTH]2.0.CO;2

    • Google Scholar
54. 1. Horner JR
    2. Padian K
    3. de Ricqlès A

    (2001) Comparative osteohistology of some embryonic and perinatal archosaurs: developmental and behavioral implications for dinosaurs

    Paleobiology 27:39–58.

    https://doi.org/10.1666/0094-8373(2001)027<0039:COOSEA>2.0.CO;2

    • Google Scholar
55. 1. Horton JD

    (1980) Development and differentiation of vertebrate lymphocytes: review of the Durham symposium — September 1979

    Developmental & Comparative Immunology 4:177–181.

    https://doi.org/10.1016/S0145-305X(80)80019-X

    • Google Scholar
56. 1. Jacenko O
    2. LuValle PA
    3. Olsen BR

    (1993) Spondylometaphyseal dysplasia in mice carrying a dominant negative mutation in a matrix protein specific for cartilage-to-bone transition

    Nature 365:56–61.

    https://doi.org/10.1038/365056a0

    • Google Scholar
57. 1. Kamska V
    2. Daeschler EB
    3. Downs JP
    4. Ahlberg PE
    5. Tafforeau P
    6. Sanchez S

    (2018) Long-bone development and life-history traits of the Devonian tristichopterid Hyneria lindae

    Earth and Environmental Science Transactions of the Royal Society of Edinburgh 109:75–86.

    https://doi.org/10.1017/S175569101800083X

    • Google Scholar
58. 1. Kapp FG
    2. Perlin JR
    3. Hagedorn EJ
    4. Gansner JM
    5. Schwarz DE
    6. O'Connell LA
    7. Johnson NS
    8. Amemiya C
    9. Fisher DE
    10. Wölfle U
    11. Trompouki E
    12. Niemeyer CM
    13. Driever W
    14. Zon LI

    (2018) Protection from UV light is an evolutionarily conserved feature of the haematopoietic niche

    Nature 558:445–448.

    https://doi.org/10.1038/s41586-018-0213-0

    • PubMed
    • Google Scholar
59. 1. Kilborn SH
    2. Trudel G
    3. Uhthoff H

    (2002)

    Review of growth plate closure compared with age at sexual maturity and lifespan in laboratory animals

    Contemporary Topics in Laboratory Animal Science 41:21–26.

    • PubMed
    • Google Scholar
60. 1. Klembara J

    (1995) The external gills and ornementation of skull roof bones of the Lower Permian tetrapod Discosauriscus (Kuhn 1933) with remarks to its ontogeny

    Paläontologische Zeitschrift 69:265–281.

    https://doi.org/10.1007/BF02985990

    • Google Scholar
61. 1. Klembara J
    2. Martens T
    3. Bartík I

    (2001) The postcranial remains of a juvenile seymouriamorph tetrapod from the Lower Permian Rotliegend of the Tambach Formation of Central Germany

    Journal of Vertebrate Paleontology 21:521–527.

    https://doi.org/10.1671/0272-4634(2001)021[0521:TPROAJ]2.0.CO;2

    • Google Scholar
62. 1. Klembara J
    2. Berman DS
    3. Henrici AC
    4. Ĉerňanský A
    5. Werneburg R

    (2006) Comparison of cranial anatomy and proportions of similarly sized Seymouria sanjuanensis and Discosauriscus austriacus

    Annals of Carnegie Museum 75:37–49.

    https://doi.org/10.2992/0097-4463(2006)75[37:COCAAP]2.0.CO;2

    • Google Scholar
63. 1. Klembara J

    (2009) New cranial and dental features of Discosauriscus austriacus (Seymouriamorpha, Discosauriscidae) and the ontogenetic conditions of Discosauriscus

    Special Papers in Palaeontology 81:61–69.

    https://doi.org/10.1111/j.1475-4983.2009.00864.x

    • Google Scholar
64. 1. Klembara J
    2. Clack JA
    3. Milner AR
    4. Ruta M

    (2014) Cranial anatomy, ontogeny, and relationships of the Late Carboniferous tetrapod Gephyrostegus bohemicus Jaekel, 1902

    Journal of Vertebrate Paleontology 34:774–792.

    https://doi.org/10.1080/02724634.2014.837055

    • Google Scholar
65. 1. Klembara J
    2. Bartík I

    (1999) The postcranial skeleton of Discosauriscus Kuhn, a seymouriamorph tetrapod from the Lower Permian of the Boskovice Furrow (Czech Republic)

    Transactions of the Royal Society of Edinburgh: Earth Sciences 90:287–316.

    https://doi.org/10.1017/S0263593300002649

    • Google Scholar
66. 1. Klembara J
    2. Meszároš S

    (1992) New finds of Discosauriscus austriacus (Makowsky 1876) from the Lower Permian of Boskovice Furrow (Czecho-Slovakia)

    Geologica Carpathica 43:305–312.

    https://doi.org/10.1111/j.1096-3642.1993.tb00298.x

    • Google Scholar
67. 1. Konietzko-Meier D
    2. Sander PM

    (2013) Long bone histology of Metoposaurus diagnosticus (Temnospondyli) from the Late Triassic of Krasiejów (Poland) and its paleobiological implications

    Journal of Vertebrate Paleontology 33:1003–1018.

    https://doi.org/10.1080/02724634.2013.765886

    • Google Scholar
68. 1. Kronenberg HM

    (2003) Developmental regulation of the growth plate

    Nature 423:332–336.

    https://doi.org/10.1038/nature01657

    • PubMed
    • Google Scholar
69. Book

    1. Lamm E-T

    (2013)

    Preparation and sectioning of specimens

    In: Padian K, Lamm E. -T, editors. Bone Histology of Fossil Tetrapods: Advancing Methods, Analysis, and Interpretation. University of California Press. pp. 55–160.

    • Google Scholar
70. Book

    1. Lüllmann-Rauch R

    (2015)

    Taschenlehrbuch Histologie. 5. Ausgabe

    New York: Georg Thieme Verlag.

    • Google Scholar
71. 1. Lyckegaard A
    2. Johnson G
    3. Tafforeau P

    (2011)

    Correction of ring artifacts in X-ray tomographic images

    International Journal of Tomography & Statistics 18:1–9.

    • Google Scholar
72. 1. Marjanović D
    2. Laurin M

    (2013) The origin(s) of extant amphibians: a review with emphasis on the “lepospondyl hypothesis”

    Geodiversitas 35:207–272.

    https://doi.org/10.5252/g2013n1a8

    • Google Scholar
73. Book

    1. Milner AR

    (1988) The relationships and origin of living amphibians

    In: Benton M. J, editors. The Phylogeny and Classification of the Tetrapods. Oxford: Clarendon Press. pp. 59–102.

    https://doi.org/10.1002/ajpa.1330820116

    • Google Scholar
74. 1. Mirone A
    2. Brun E
    3. Gouillart E
    4. Tafforeau P
    5. Kieffer J

    (2014) The PyHST2 hybrid distributed code for high speed tomographic reconstruction with iterative reconstruction and a priori knowledge capabilities

    Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms 324:41–48.

    https://doi.org/10.1016/j.nimb.2013.09.030

    • Google Scholar
75. 1. Miura S
    2. Hanaoka K
    3. Togashi S

    (2008) Skeletogenesis in Xenopus tropicalis: characteristic bone development in an anuran amphibian

    Bone 43:901–909.

    https://doi.org/10.1016/j.bone.2008.07.005

    • PubMed
    • Google Scholar
76. 1. Niedźwiedzki G
    2. Szrek P
    3. Narkiewicz K
    4. Narkiewicz M
    5. Ahlberg PE

    (2010) Tetrapod trackways from the early Middle Devonian period of Poland

    Nature 463:43–48.

    https://doi.org/10.1038/nature08623

    • Google Scholar
77. 1. Nilsson SK
    2. Johnston HM
    3. Coverdale JA

    (2001) Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches

    Blood 97:2293–2299.

    https://doi.org/10.1182/blood.V97.8.2293

    • Google Scholar
78. 1. Orkin SH
    2. Zon LI

    (2008) Hematopoiesis: an evolving paradigm for stem cell biology

    Cell 132:631–644.

    https://doi.org/10.1016/j.cell.2008.01.025

    • PubMed
    • Google Scholar
79. 1. Paganin D
    2. Mayo SC
    3. Gureyev TE
    4. Miller PR
    5. Wilkins SW

    (2002) Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object

    Journal of Microscopy 206:33–40.

    https://doi.org/10.1046/j.1365-2818.2002.01010.x

    • PubMed
    • Google Scholar
80. 1. Pardo JD
    2. Small BJ
    3. Huttenlocker AK

    (2017) Stem caecilian from the Triassic of Colorado sheds light on the origins of Lissamphibia

    PNAS 114:E5389–E5395.

    https://doi.org/10.1073/pnas.1706752114

    • PubMed
    • Google Scholar
81. 1. Park EA

    (1964)

    The imprinting of nutritional disturbances on the growing bone

    Pediatrics 33:815–861.

    • Google Scholar
82. 1. Quilhac A
    2. de Ricqlès A
    3. Lamrous H
    4. Zylberberg L

    (2014) Globuli ossei in the long limb bones of Pleurodeles waltl (Amphibia, Urodela, Salamandridae)

    Journal of Morphology 275:1226–1237.

    https://doi.org/10.1002/jmor.20296

    • Google Scholar
83. 1. Rozenblut B
    2. Ogielska M

    (2005) Development and growth of long bones in European water frogs (Amphibia: Anura: Ranidae), with remarks on age determination

    Journal of Morphology 265:304–317.

    https://doi.org/10.1002/jmor.10344

    • Google Scholar
84. 1. Ruta M
    2. Coates MI

    (2007) Dates, nodes and character conflict: addressing the lissamphibian origin problem

    Journal of Systematic Palaeontology 5:69–122.

    https://doi.org/10.1017/S1477201906002008

    • Google Scholar
85. 1. Sanchez S
    2. Klembara J
    3. Castanet J
    4. Steyer JS

    (2008) Salamander-like development in a seymouriamorph revealed by palaeohistology

    Biology Letters 4:411–414.

    https://doi.org/10.1098/rsbl.2008.0159

    • Google Scholar
86. 1. Sanchez S
    2. de Ricqlès A
    3. Schoch R
    4. Steyer JS

    (2010a) Developmental plasticity of limb bone microstructural organization in Apateon: histological evidence of paedomorphic conditions in branchiosaurs

    Evolution & Development 12:315–328.

    https://doi.org/10.1111/j.1525-142X.2010.00417.x

    • PubMed
    • Google Scholar
87. 1. Sanchez S
    2. Steyer JS
    3. Schoch RR
    4. De Ricqlès A

    (2010b) Palaeoecological and palaeoenvironmental influences revealed by long-bone palaeohistology: the example of the Permian branchiosaurid Apateon

    Geological Society, London, Special Publications 339:139–149.

    https://doi.org/10.1144/SP339.12

    • Google Scholar
88. 1. Sanchez S
    2. Ahlberg PE
    3. Trinajstic KM
    4. Mirone A
    5. Tafforeau P

    (2012) Three-dimensional synchrotron virtual paleohistology: a new insight into the world of fossil bone microstructures

    Microscopy and Microanalysis 18:1095–1105.

    https://doi.org/10.1017/S1431927612001079

    • PubMed
    • Google Scholar
89. 1. Sanchez S
    2. Tafforeau P
    3. Ahlberg PE

    (2014) The humerus of Eusthenopteron: a puzzling organization presaging the establishment of tetrapod limb bone marrow

    Proceedings of the Royal Society B: Biological Sciences 281:20140299.

    https://doi.org/10.1098/rspb.2014.0299

    • Google Scholar
90. 1. Sanchez S
    2. Tafforeau P
    3. Clack JA
    4. Ahlberg PE

    (2016) Life history of the stem tetrapod Acanthostega revealed by synchrotron microtomography

    Nature 537:408–411.

    https://doi.org/10.1038/nature19354

    • PubMed
    • Google Scholar
91. 1. Schoch RR

    (2019) The putative lissamphibian stem-group: phylogeny and evolution of the dissorophoid temnospondyls

    Journal of Paleontology 93:137–156.

    https://doi.org/10.1017/jpa.2018.67

    • Google Scholar
92. Book

    1. Schoch RR
    2. Milner AR

    (2000)

    Stereospondyli: Stem-Stereospondyli, Rhinesuchidae, Rhytidostea, Trematosauroidea, Capitosauroidea

    In: Wellnhofer P, editors. Handbuch Der Paläoherpetologie. Munich: F. Pfeil. pp. 1–219.

    • Google Scholar
93. Book

    1. Schoch RR
    2. Milner AR

    (2004)

    Structure and implications of theories on the origin of lissamphibians

    In: Arratia G, Wilson M. V. H, Cloutier R, editors. Recent Advances in the Origin and Early Radiation of Vertebrates. München: Verlag Dr. Friedrich Pfeil. pp. 345–377.

    • Google Scholar
94. 1. Shubin N
    2. Tabin C
    3. Carroll S

    (1997) Fossils, genes and the evolution of animal limbs

    Nature 388:639–648.

    https://doi.org/10.1038/41710

    • Google Scholar
95. 1. Sigurdsen T
    2. Green DM

    (2011) The origin of modern amphibians: a re-evaluation

    Zoological Journal of the Linnean Society 162:457–469.

    https://doi.org/10.1111/j.1096-3642.2010.00683.x

    • Google Scholar
96. 1. Sipkins DA
    2. Wei X
    3. Wu JW
    4. Runnels JM
    5. Côté D
    6. Means TK
    7. Luster AD
    8. Scadden DT
    9. Lin CP

    (2005) In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment

    Nature 435:969–973.

    https://doi.org/10.1038/nature03703

    • PubMed
    • Google Scholar
97. 1. Sulej T

    (2002)

    Species discrimination of the Late Triassic temnospondyl amphibian Metoposaurus diagnosticus

    Acta Palaeontologica Polonica 47:535–546.

    • Google Scholar
98. 1. Sulej T

    (2007)

    Osteology, variability, and evolution of Metoposaurus, a temnospondyl from the Late Triassic of Poland

    Palaeontologia Polonica 64:29–139.

    • Google Scholar
99. 1. Suzuki F
    2. Takase T
    3. Takigawa M
    4. Uchida A
    5. Shimomura Y

    (1981) Simulation of the initial stage of endochondral ossification: in vitro sequential culture of growth cartilage cells and bone marrow cells

    PNAS 78:2368–2372.

    https://doi.org/10.1073/pnas.78.4.2368

    • PubMed
    • Google Scholar
100. 1. Tafforeau P
     2. Boistel R
     3. Boller E
     4. Bravin A
     5. Brunet M
     6. Chaimanee Y
     7. Cloetens P
     8. Feist M
     9. Hoszowska J
     10. Jaeger J-J
     11. Kay RF
     12. Lazzari V
     13. Marivaux L
     14. Nel A
     15. Nemoz C
     16. Thibault X
     17. Vignaud P
     18. Zabler S

     (2006) Applications of X-ray synchrotron microtomography for non-destructive 3D studies of paleontological specimens

     Applied Physics A 83:195–202.

     https://doi.org/10.1007/s00339-006-3507-2

     • Google Scholar
101. 1. Tanaka Y

     (1976) Architecture of the marrow vasculature in three amphibian species and its significance in hematopoietic development

     American Journal of Anatomy 145:485–497.

     https://doi.org/10.1002/aja.1001450407

     • Google Scholar
102. Book

     1. Trueb L
     2. Cloutier R

     (1991)

     A phylogenetic investigation of the inter-and intrarelationships of the Lissamphibia (Amphibia: Temnospondyli)

     In: Schultze H. -P, Trueb L, editors. Origins of the Higher Groups of Tetrapods: Controversy and Consensus. New York: Cornell University Press. pp. 223–313.

     • Google Scholar
103. 1. Vallin G
     2. Laurin M

     (2004) Cranial morphology and affinities of Microbrachis, and a reappraisal of the phylogeny and lifestyle of the first amphibians

     Journal of Vertebrate Paleontology 24:56–72.

     https://doi.org/10.1671/5.1

     • Google Scholar
104. 1. Vaughn PP

     (1966)

     Seymouria from the Lower Permian of southeastern Utah, and possible sexual dimorphism in that genus

     Journal of Paleontology 40:603–612.

     • Google Scholar
105. 1. Wegner G

     (1874) Ueber das normale und pathologische wachsthum der röhrenknochen

     Archiv Für Pathologische Anatomie Und Physiologie Und Für Klinische Medicin 61:44–76.

     https://doi.org/10.1007/BF01960485

     • Google Scholar
106. 1. Weiss LP
     2. Wislocki GB

     (1956) Seasonal variations in hematopoiesis in the dermal bones of the nine-banded armadillo

     The Anatomical Record 126:143–163.

     https://doi.org/10.1002/ar.1091260203

     • Google Scholar
107. 1. Wilson A
     2. Trumpp A

     (2006) Bone-marrow haematopoietic-stem-cell niches

     Nature Reviews Immunology 6:93–106.

     https://doi.org/10.1038/nri1779

     • Google Scholar
108. 1. Xie M
     2. Gol'din P
     3. Herdina AN
     4. Estefa J
     5. Medvedeva EV
     6. Li L
     7. Newton PT
     8. Kotova S
     9. Shavkuta B
     10. Saxena A
     11. Shumate LT
     12. Metscher BD
     13. Großschmidt K
     14. Nishimori S
     15. Akovantseva A
     16. Usanova AP
     17. Kurenkova AD
     18. Kumar A
     19. Arregui IL
     20. Tafforeau P
     21. Fried K
     22. Carlström M
     23. Simon A
     24. Gasser C
     25. Kronenberg HM
     26. Bastepe M
     27. Cooper KL
     28. Timashev P
     29. Sanchez S
     30. Adameyko I
     31. Eriksson A
     32. Chagin AS

     (2020) Secondary ossification center induces and protects growth plate structure

     eLife 9:e55212.

     https://doi.org/10.7554/eLife.55212

     • PubMed
     • Google Scholar
109. 1. Zelzer E
     2. McLean W
     3. Ng YS
     4. Fukai N
     5. Reginato AM
     6. Lovejoy S
     7. D'Amore PA
     8. Olsen BR

     (2002)

     Skeletal defects in VEGF120/120 mice reveal multiple roles for VEGF in skeletogenesis

     Development 129:1893–1904.

     • PubMed
     • Google Scholar
110. 1. Zhang J
     2. Niu C
     3. Ye L
     4. Huang H
     5. He X
     6. Tong W-G
     7. Ross J
     8. Haug J
     9. Johnson T
     10. Feng JQ
     11. Harris S
     12. Wiedemann LM
     13. Mishina Y
     14. Li L

     (2003) Identification of the haematopoietic stem cell niche and control of the niche size

     Nature 425:836–841.

     https://doi.org/10.1038/nature02041

     • Google Scholar

Author details

1. Jordi Estefa

   Department of Organismal Biology, Evolution and Development, Uppsala University, Uppsala, Sweden

   Contribution

   Formal analysis, Investigation, Visualization, Methodology

   For correspondence

   jordi.estefa@gmail.com

   Competing interests

   No competing interests declared

2. Paul Tafforeau

   European Synchrotron Radiation Facility, Grenoble, France

   Contribution

   Resources, Software, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5962-1683

3. Alice M Clement

   Flinders University, College of Science and Engineering, Adelaide, Australia

   Contribution

   Funding acquisition, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0380-7347

4. Jozef Klembara

   Comenius University in Bratislava, Faculty of Natural Sciences, Department of Ecology, Bratislava, Slovakia

   Contribution

   Funding acquisition, Investigation

   Competing interests

   No competing interests declared

5. Grzegorz Niedźwiedzki

   Department of Organismal Biology, Evolution and Development, Uppsala University, Uppsala, Sweden

   Contribution

   Funding acquisition, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4775-5254

6. Camille Berruyer

   European Synchrotron Radiation Facility, Grenoble, France

   Contribution

   Investigation, Data acquisition

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0046-2972

7. Sophie Sanchez

   1. Department of Organismal Biology, Evolution and Development, Uppsala University, Uppsala, Sweden
   2. European Synchrotron Radiation Facility, Grenoble, France

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology

   For correspondence

   sophie.sanchez@ebc.uu.se

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3611-6836

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