This paper presents histological data on the long bones of different size (age) individuals of the basal cryptobranchid salamander Eoscapherpeton asiaticum from the Upper Cretaceous (Turonian) of Uzbekistan. E. asiaticum is similar to modern members of Cryptobranchidae in being relatively large (estimated body length up to 50–60 cm), aquatic, and neotenic. The analysis of growth series of femora demonstrates a significant histological maturation during ontogeny, expressed by the progressive appearance of highly organized parallel-fibred bone in the peripheral part of the periosteal cortex, appearance and increasing number of bone remodeling features, progressive resorption of calcified cartilage in the diaphyseal areas and formation of endochondral bone lining the erosion cavities in the calcified cartilage, progressive thickening of endosteal inner circumferential layer and increasing of vascularity and appearance of vascular network of longitudinal and oblique canals in the cortex. These ontogenetic changes in the long-bone histology of E. asiaticum generally correspond to those of other salamanders, except the appearance of the vascular network in the periosteal cortex — the feature that is characteristic for cryptobranchids and connected with their large body size. According to new data, the large Cenozoic cryptobranchids appear to have attained their larger size by extending the skeletal growth period.
Cet article présente des données histologiques sur les os longs d’individus différents en taille (âge) de la salamandre cryptobranchidée Eoscapherpeton asiaticum basale du Crétacé supérieur (Turonien) d’Ouzbékistan. E. asiaticum ressemble à des membres modernes de Cryptobranchidae par sa taille relativement grande (longueur estimée de son corps jusqu’à 50–60 cm, sa vie aquatique, ses caractères néoténiques). L’analyse de séries de croissance de fémurs démontre une maturation histologique significative pendant l’ontogénie, exprimée par l’apparition progressive d’os à fibres parallèles très organisées dans la partie périphérique du cortex périostique, l’apparition en nombre croissant de traits de remodelage osseux, la résorption progressive du cartilage calcifié dans les zones diaphysales et la formation d’os endochondral soulignant les cavités d’érosion dans le cartilage calcifié, l’épaississement progressif d’un feuillet circonférentiel interne endostéal et une croissance de la vascularité, ainsi que l’apparition d’un réseau vasculaire sous forme de canaux longitudinaux et obliques dans le cortex. Ces changements ontogénétiques dans l’histologie des os longs de E. asiaticum correspondent généralement à ceux observés chez d’autres salamandres, mise à part l’apparition du réseau vasculaire dans le cortex périostique, trait caractéristique des cryptobranchidés, en relation avec la grande taille de leur corps. Selon des données nouvelles, les grands cryptobranchidés du Cénozoïque semblent avoir atteint leur taille plus grande grâce à l’allongement de la période de croissance du squelette.
Caudata, Cryptobranchidae, Paleohistology, GigantismCaudata, Cryptobranchidae, Paléohistologie, GigantismepresentedHandled by Hans-Dieter SuesIntroduction
Giant salamanders (the family Cryptobranchidae Fitzinger, 1826) are a group of large (up to 2 m), strictly aquatic neotenic salamanders known in the fossil record since the Middle Jurassic (Gao and Shubin, 2003; see Vasilyan et al., 2013 for alternative opinion about taxonomic composition and temporal distribution of the Cryptobranchidae) and are represented in the modern fauna by two genera (Andrias and Cryptobranchus) and three species (namely, the Chinese (A. davidianus), Japanese (A. japonicas) and the North American (C. alleganiensis) giant salamanders) (Browne et al., 2012).
The Late Mesozoic–Paleogene cryptobranchid fossil record is relatively poor and the following fossil taxa from this time interval have been referred to this family: Chunerpeton tianyiensis Gao, Shubin, 2003 (Middle Jurassic, China), Eoscapherpeton asiaticumNesov, 1981, and Eoscapherpeton gracilisNesov, 1981 (both Late Cretaceous, Uzbekistan), Aviturus exsecratus Gubin, 1991, Ulanurus fractus Gubin, 1991, (both Paleocene, Mongolia), ‘Cryptobranchus’ (= Andrias?) saskatchewanensis Naylor, 1981 (Paleocene, Canada), Zaissanurus beliajevae Chernov, 1959 (Oligocene, Kazakhstan) (e.g., Browne et al., 2012, Holman, 2006 and Milner, 2000). All post-Paleogene cryptobranchids, except Ukrainurus hypsognathusVasilyan et al., 2013 (Miocene, Ukraine), were referred to two modern genera (e.g., Browne et al., 2012 and Vasilyan et al., 2013). The Mesozoic C. tianyiensis and E. asiaticum were considered stem cryptobranchid members, while Paleogene-Recent taxa were considered crown-group cryptobranchids (e.g., Browne et al., 2012, Holman, 2006 and Milner, 2000).
The genus Eoscapherpeton (type species E. asiaticum from the Turonian Bissekty Formation of Uzbekistan) together with the genus Horezmia (type species H. gracile from the Cenomanian Khodzhakul Formation of Uzbekistan) were originally assigned by Nesov (1981) to the Scapherpetidae (a clade of extinct salamandroids; see Edwards, 1976 and Estes, 1981). Later, “Horezmia” gracile was considered a species of Eoscapherpeton and the genus Eoscapherpeton was referred to the Cryptobranchidae on the basis of the following characters in common with cryptobranchids: midline contact of the dorsal (= alary) processes of the premaxillae, frontal-maxillary contact, parietals strongly overlapped by the frontals, absence of a distinct medial process of the pterygoid, and presence of pterygoid-parasphenoid contact (Skutschas, 2009 and Skutschas, 2013). Apart from the Bissekty and Khodzhakul formations, Eoscapherpeton is also known from other geological units in Middle Asia (the Cenomanian Dzharakuduk Formation; the Coniacian–Santonian Aitym Formation, both in Uzbekistan; the Santonian Yalovach Formation in Tajikistan) and Kazakhstan (the Santonian–lower Campanian Bostobe Formation, Campanian Darbasa Formation). Generally, Cenomanian–Campanian vertebrate assemblages of those regions are characterized by the dominance of Eoscapherpeton (Skutschas, 2013).
Despite its wide geographical and temporal distributions and abundant available material, many aspects of the biology of Eoscapherpeton remain poorly known. However, it is clear that Eoscapherpeton was a relatively large salamander. The length of the holotype maxilla of E. asiaticum is about 1.5 cm (see Nesov, 1981 and Skutschas, 2013), the estimated length of the largest femur of E. asiaticum is about 3–3.5 cm (Table 1), and the estimated body length of the largest individuals is about 50–60 cm. Additionally, Eoscapherpeton, like modern cryptobranchids, was aquatic and neotenic (some large specimens of E. asiaticum retain a well-ossified hyobranchial apparatus; pers. obs.).
The abundance of E. asiaticum specimens collected to date (several thousand isolated bones and partly articulated specimens collected during expeditions to the Kyzylkum Desert in 1977–1994 by L.A. Nesov and in 1997–2006 by the international Uzbek/Russian/British/American/Canadian Joint Paleontological Expeditions (URBAC); Nesov, 1981, Skutschas, 2009 and Skutschas, 2013) provides an excellent opportunity to document the long-bone histology of this basal cryptobranchid and discuss evolutionary mechanisms of achievement of the giant body size in Cryptobranchidae.
Material and methods
A total of 13 specimens representing isolated complete and fragmentary femora of cryptobranchid salamander E. asiaticum from the Dzharakuduk locality of the Turonian Bissekty Formation (Redman and Leighton, 2009) were histologically studied to assess ontogenetic changes in long-bone histology.
For the present study, thin-sections were prepared based on the methodology outlined in Chinsamy and Raath (1992) and Lamm (2013).
The specimens were divided into three conventional size classes: small, medium, and large (see Table 1 for a list of material sampled). The small size class includes the two smallest femora, the medium size class comprises larger femora with a smooth bone surface (only a few vascular foramina could be present), and the large size class includes the largest femora that are characterized by the presence of numerous vascular foramina that open into vascular grooves (Fig. S1). We took thin-sections from or close to a standard level (midshaft region). Eight individual transverse sections (two small, three medium, and three large femora) and five individual longitudinal sections (three medium and two large femora) were prepared (Table 1).
The sections were observed under polarized light using an optical microscope (Leica 4500, Leica Microsystems, Wetzlar, Germany) in the Saint Petersburg State University Research Centre for X-ray Diffraction Studies, Russia. Histological terminology follows Francillon-Vieillot et al. (1990) and Prondvai et al. (2014).
We estimated the total length of the fragmentary femora by using maximal length/length from the proximal end to the base of trochanter ratio (measured on complete femora) that varies in E. asiaticum between 3.2 and 3.7. For the body length estimates of the largest individuals of E. asiaticum we used proportions of a skeleton of modern Cryptobranchus (femur/total skeleton length ratio is about 1: 16.5–17).
The femur of Cryptobranchus (used for comparisons) was CT-scanned (at 100 kV and 0.1 mA, generating a resolution of 12.79 μm of pixel size and an output of 2000 × 2000 pixels per slice) at the Saint Petersburg State University Research Centre for X-ray Diffraction Studies (Saint Petersburg, Russia) using a Skyscan 1172 CT scanner. CT scan data were imported to the software Amira 6.3.0 (FEI-VSG Company), where the model was reconstructed and segmented.
All studied specimens of E. asiaticum are stored at the Paleoherpetological collection (ZIN PH) of the Zoological Institute of the Russian Academy of Sciences, Saint Petersburg, Russia. The thin sections and a skeleton and a femur of subadult Cryptobranchus (specimen DVZ M 2/12; total length of skeleton is about 32–33 cm) used in study (for comparisons and for the body length estimations of E. asiaticum) are housed, respectively, in the histological and morphological collections of the Department of Vertebrate Zoology, Saint Petersburg State University, Saint Petersburg, Russia. The CT data are deposited in the Department of Vertebrate Zoology of the Saint Petersburg State University, Saint Petersburg, Russia, and can be made available by the present authors for the purpose of scientific study.
Histological descriptionFemora of small individuals (total length less than 7 mm)
Two femora of the smallest individuals (ZIN PH 20/85; 37/85 sectioned transversally; Fig. 1) contain a well-differentiated empty medullary cavity (maximum diameter of the medullary cavity — 0.1–0.2 mm). The medullary region is surrounded by a relatively thick cortex (maximum cortical thickness — 0.1–0.3 mm) that is composed of primary bone tissues in the periosteal part and endosteal bone tissue in the innermost part. The cortico-diaphyseal index (CDI, cortical thickness/local bone radius) is approximately 0.5–0.75. The periosteal cortex is compact and consists of almost avascular parallel-fibred bone. The periosteal cortex of ZIN PH 20/85 (Fig. 1C) contains one relatively large primary vascular canal. The endosteal lamellar bone formed a relatively thick inner circumferential layer (ICL) in the smaller specimen ZIN PH 20/85 (Fig. 1C and D). In this specimen, the border between periosteal and endosteal tissues is represented by undulating resorption line that suggests the erosion of periosteal bone along the margin of the medullary cavity and subsequent endosteal bone deposition. The larger specimen ZIN PH 37/85 lacks endosteal tissues.
The cortex of ZIN PH 37/85 contains a sequence of growth marks (Fig. 1B and D), but no well-defined lines of arrested growth. There are no signs of bone remodeling (erosion bays, secondary osteons) in the cortex.
Osteocyte lacunae in the periosteal cortex are rare and have a rounded appearance in the inner and an ellipsoid appearance in the peripheral part (in the area of the trochanteric crest). Osteocyte lacunae in the ICL have an ellipsoid appearance.
Femora of medium-sized individuals (total length between 7–12 mm)
The histology of the medium-sized bones is documented by six femora (specimens ZIN PH 22-23/85; 25/85, 32-34/85; Fig. 2 and Fig. 3), sectioned transversely (n = 3) and longitudinally (n = 3) (Table 1). The diaphyseal cross-sections have a rather thick cortex (maximum cortical thicknesses — 0.3–0.4 mm) relative to the diameter of the medullary cavity (maximum diameters of the medullary cavity — 0.1–0.4 mm). The CDI varies between 0.7 and 0.9. The structure of the periosteal cortex of the medium-sized femora differs from that of smaller individuals by the presence of:
highly organized (almost lamellar) parallel-fibred bone in the peripheral part;
cortical growth marks (the periosteal cortex contains growth marks, including one or two well-defined lines of arrested growth (LAG's) (Fig. 3A, C, E));
and several large erosion bays in the middle and inner parts of the cortex (Fig. 3).
The cortex retains a low degree of vascularization. One medium-sized femur (specimen ZIN PH 22/85) has a medullary cavity that is infilled by bone tissue (Fig. 3A). The presence of an infilled medullary cavity at the mid-diaphysis level is unusual for E. asiaticum and its appearance in specimen ZIN PH 22/85 could be a result of pathology (or a rare case of individual variation).
As in the small femur ZIN PH 20/85, the innermost part of the cortex of the medium-sized individuals is lined with endosteal bone (ICL).
In longitudinal sections of femora (specimens ZIN PH 32-34/85; Fig. 4), the epiphyseal and metaphyseal regions contain dense calcified cartilage with globular appearance. In the metaphyseal region, the calcified cartilage in the medullary cavity is marked by large erosion bays. Endochondral lamellar bone is only present in small restricted areas and do not form secondary bone trabeculae.
Osteocyte lacunae in the periosteal cortex are more numerous in the inner part. They have a rounded appearance in the transverse sections and slightly ellipsoid appearance in the longitudinal sections. Osteocyte lacunae in the ICL have an ellipsoid appearance.
Femora of large individuals (total length more than 12 mm)
The histology of the large individuals is documented by five femora (specimens ZIN PH 26-28/85; 35-36/85; Fig. 5 and Fig. 6), sectioned transversely (n = 3) and longitudinally (n = 2) (Table 1). The diaphyseal cross-sections reveal a thick cortex (maximum cortical thicknesses — 0.4–0.9 mm) relative to the diameter of the small medullary cavity (maximum diameters of the medullary cavity — 0.1–0.3 mm). The CDI varies between 0.7 and 0.9. The periosteal cortex is formed by less and highly organized parallel-fibred bone (Fig. 5D and F) and contains a sequence of growth marks, including two to three well-defined lines of arrested growth (LAG's) (Fig. 5A and C). The endosteal cortex is formed by a thick inner circumferential layer.
The large femora display fundamental differences from that of small and medium-sized individuals in having a significantly higher degree of vascularization of the cortex and, accordingly, in the presence of a vascular network of longitudinal and oblique canals (Fig. 5). The high degree of vascularization of the cortex is also visible externally: the bone surface contains numerous vascular foramina that could open into vascular grooves (at the proximal and distal ends) (Fig. S1).
In longitudinal sections of femoral proximal ends (specimens ZIN PH 35-36/85; Fig. 6), the structure of the epiphyseal, metaphyseal and (the ends of) diaphyseal regions is similar to that of medium-sized individuals in the presence of dense calcified cartilage and by large erosion bays, but it differs by the presence of more endochondral bone. The specimen ZIN PH 35/85 (Fig. 6A) retains a proximal end (= epiphysis) and its outer portion is formed by dense calcified cartilage (the structure of the inner portion is unclear due to preservation of the specimen).
The shape and distribution of osteocyte lacunae is similar to that of medium-sized individuals.
Femoral microanatomy of <italic>Cryptobranchus</italic>
The femur of subadult Cryptobranchus sp. (total length of the skeleton is about 32–33 cm; length of the femur is about 2 cm) is characterized by a thick cortex at the mid-diaphysis and thinner cortex in the proximal and distal metaphysis (Fig. 7C–E). The bone at the mid-diaphysis contains a small medullary cavity and few canals that could correspond to vascular canals and/or erosion cavities. The bone at the metaphyseal levels has a vascular network of longitudinal and oblique canals (Fig. 7B). Some of these canals opened up to the surface of the bone (Fig. 7A).
Discussion
The microanatomical structure and composition of the primary bone tissue in the long bones of E. asiaticum is similar to that of some modern crown and fossil stem salamanders, which have a narrow medullary cavity and a thick cortex (in the central part of diaphysis) that is formed by a highly organized (= lamellar sensuProndvai et al., 2014) or less organized (= non-lamellar sensuProndvai et al., 2014) parallel-fibred bone (e.g., De Buffrénil et al., 2015, Canoville and Laurin, 2009, Canoville et al., 2018, Castanet et al., 2003 and Skutschas and Stein, 2015). E. asiaticum is considered neotenic on the basis of a well-ossified hyobranchial apparatus (pers. obs.); the presence of calcified cartilage in the metaphyseal region (a feature that is characteristic for extant neotenic salamanders; Castanet et al., 2003) in large individuals further supports the neotenic hypothesis.
The analysis of a growth series of femora revealed the following main changes in the histology of E. asiaticum during ontogeny:
appearance of highly organized parallel-fibred bone in the peripheral part of the periosteal cortex (in medium-sized individuals);
appearance of growth marks (LAGs) in the cortex (in medium-sized individuals);
appearance (in medium-sized individuals) of erosion cavities in the cortex;
progressive resorption of calcified cartilage in the diaphyseal areas and formation of endochondral bone lining the erosion cavities in the calcified cartilage;
progressive thickening of ICL;
increase of vascularity and appearance of vascular network of longitudinal and oblique canals in the cortex (in large individuals).
Most of these changes reflect a significant histological maturation as individuals of E. asiaticum become larger and they are common for salamanders, except the appearance of vascular network of longitudinal and oblique canals in the cortex.
As was recently shown by Canoville et al. (2018), modern salamanders, with the exception of the cryptobranchid Andrias, usually have an avascular cortex in the limb bones. The presence of few vascular canals in the largest extant salamander Andrias is a likely result of its large body size and a significant thickness of the periosteal cortex; the absolute cortical thickness is positively correlated with bone vascular density in lissamphibians (Canoville et al., 2018), lepidosaurs, and birds (Cubo et al., 2014). Additionally to Andrias, we found a vascularity of the cortex in the modern Cryptobranchus (Fig. 7) and the Late Cretaceous E. asiaticum (this study). Thus, the relatively high vascular density of the periosteal cortex in adult individuals Andrias, Cryptobranchus and Eoscapherpeton is connected with their large body size of all those salamanders (Canoville et al., 2018; this study) — a key feature of cryptobranchids.
The potentially oldest cryptobranchid C. tianyiensis from the Middle Jurassic of China demonstrates several morphological characters of Cryptobranchidae (see Gao and Shubin, 2003), but it was relatively small (the total body length of the holotype is approximately 180 mm; Wang and Evans, 2006: 68) in comparison with modern taxa (Andrias up to 180 cm; Cryptobranchus up to 75 cm; Browne et al., 2014) and E. asiaticum (the estimated length of the largest femur ZIN PH 28/85 is about 3.0–3.5 cm, and, correspondingly, the total body length (using femur/total skeleton length ratio of Cryptobranchus) — up to 50–60 cm). The bone histology of Chunerpeton is unknown. Accordingly, at the current state of knowledge, Eoscapherpeton is the geologically oldest member of Cryptobranchidae that acquired histological cryptobranchid features (vascular network of canals in the cortex) related to large body size, and this taxon represents the first stage of evolution of gigantism of cryptobranchids that finally led to the appearance of really giant forms (up to 2 m).
The comparisons of bone histology of Eoscapherpeton and large cryptobranchids (Andrias) provide new information about possible evolutionary mechanisms of achievement of giant body size in the Cenozoic Cryptobranchidae. Both Eoscapherpeton (all size classes) and Andrias (a large individual, see Canoville et al., 2018: fig. 2A, C–F) have parallel-fibred bone in the periosteal cortex and there are no differences in primary bone tissue composition of these taxa. So, there is no evidence of increasing growth rates (e.g., presence of a fast growing fibrolamellar bone) in Andrias in comparison with a more basal and smaller Eoscapherpeton. We suggest that bone formation was not accelerated in Andrias, and that large Cenozoic cryptobranchids appear to have attained their larger size by extending the skeletal growth period (prolongation of lifespan or at least prolongation of the time to reach adult size). This suggestion is indirectly supported by the presence of fewer LAG's (cyclical growth marks that are usually formed annually) in large individuals of Eoscapherpeton in comparison with Andrias (see Canoville et al., 2018: fig. 2) and Aviturus (Skutschas et al., in press).
The significant increase of body size in crown salamanders only started in the Late Cretaceous, after the extinction of relict stem salamanders (Skutschas, 2013 and Skutschas et al., in press). The relatively large body size (at least up to 40–50 cm) was achieved by crown-group salamanders during the Late Cretaceous independently in two isolated clades — Cryptobranchidae in Asia (Eoscapherpeton) and Scapherpetidae in North America (Scapherpeton, Lisserpeton, Piceoerpeton; e.g., Gardner, 2012 and Holman, 2006), but really giant body size (up 2 m) was achieved only in the Paleocene (cryptobranchid Aviturus; Vasilyan and Böhme, 2012).
Conclusions
E. asiaticum is a crucial taxon for understanding the evolution of cryptobranchids. It provides important information on character evolution along the lineage leading to the advanced crown Cryptobranchidae. It also represents one of the earliest known cryptobranchids to achieve large body sizes (up to 50–60 cm) and to acquire a size-related histological cryptobranchid feature (vascular network of canals in the cortex). Like Cenozoic cryptobranchids (and the basalmost cryptobranchid C. tianyiensis from the Middle Jurassic), E. asiaticum was a neotenic salamander and neoteny is the only life history strategy of Cryptobranchidae. Histological analysis of a growth series of femora of E. asiaticum allows new insights into the possible mechanism of the evolution of the gigantic body size of the cryptobranchids. E. asiaticum shows the same long-bone histology (parallel-fibred bone in the periosteal cortex) as the modern cryptobranchids and other salamanders. This similarity in bone histology suggests that Cenozoic cryptobranchids reached a giant body size without increasing growth rate compared to the ancestor (acceleration), but, possibly, by extending ontogeny. More histological studies of fossil and recent Cenozoic cryptobranchids are needed to test this hypothesis.
Acknowledgments
This article is dedicated to the great paleoherpetologist Jean-Claude Rage who recently passed away. Fieldwork in Uzbekistan was facilitated by and conducted in cooperation with the Zoological Institute of the National Academy of Sciences of Uzbekistan, particularly D.A. Azimov and Y.A. Chikin. For their efforts in the field, scientific expertise, and camaraderie, we thank A.V. Abramov, J.D. Archibald, G.O. Cherepanov, I.G. Danilov, S. Dominguez, C. King, N. Morris, C.M. Redman, A.S. Resvyi, C. Skrabec, E.V. Syromyatnikova, and D.J. Ward. The authors thank anonymous reviewer for providing helpful comments that improved the quality of the manuscript. We are grateful to the staff of the Saint Petersburg State University Research Centre for X-ray Diffraction Studies (Saint Petersburg, Russia) for their help with using the Leica 2500P microscope and for CT scanning of specimens. This study was fulfilled under support of the Russian Scientific Fund project 14-14-00015 and the Russian Foundation for Basic Research project 19-04-00060.
Supplementary data
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Transverse histological sections of femora of smallest individuals of Eoscapherpeton asiaticum ZIN PH 20/85, ZIN PH 37/85 under polarized light with a Lamb wave plate (A–D). A and B. Microanatomical overview of the femora of smallest individuals. Note empty medullary cavity surrounded by a relatively thick cortex. C. Close-up of the cortex (ZIN PH 20/85) showing compact periosteal part and the endosteal bone composed by the lamellar bone tissue in the innermost part. D. Details of the cortex composition. Periosteal cortex (ZIN PH 37/85) is compact and consists of parallel-fibred bone with relatively large primary vascular canal. cx: cortex; eb: endosteal bone; gm: growth mark; mc: medullary cavity; oc: osteocyte lacunae; pb: periosteal bone; trcr: trochanteric crest; vc: vascular canal.
Sections histologiques transversales de fémurs des plus petits individus de Eoscapherpeton asiaticum ZIN PH 20/85, ZIN PH 37/85 sous lumière polarisée avec une plaque d'onde de Lamb (A–D). A et B. Vue d’ensemble microanatomique de fémur des plus petits individus. À noter la cavité médullaire vide et entourée d’un cortex relativement épais. C. Fermeture du cortex (ZIN PH 20/85) montrant la partie compacte du périoste et l’os endostéal, composé d’un tissu osseux lamellaire dans sa partie la plus profonde. D. Détail de la composition du cortex. Le cortex du périoste (ZIN PH 37/85) est compact et consiste en un os à fibres parallèles, avec un canal vasculaire primaire relativement grand. cx : cortex ; eb : os endostéal ; gm : marque de croissance ; mc : cavité médullaire ; oc : lacunes d’ostéocytes ; pb : os périostique ; trcr : crête du trochanter ; vc : canal vasculaire.
Transverse histological sections of femora of medium-sized individuals of Eoscapherpeton asiaticum ZIN PH 22/85, ZIN PH 23/85, ZIN PH 25/85 under polarized light with a Lamb wave plate (A and C) and without lambda waveplate (B). A–C. Microanatomical overview of the femora of three medium-sized individuals. Note an almost avascular thick cortex. Additionally, note that the specimen ZIN PH 22/85 (A) has a medullary cavity that is infilled by bone tissue. The line of arrested growth is marked by the yellow arrow. cx: cortex; hpfb: highly organized parallel-fibred bone; mc: medullary cavity; trcr: trochanteric crest.
Coupes histologiques transversales de fémurs d’individus de taille moyenne d’Eoscapherpeton asiaticum ZIN PH 22/85, ZIN PH 23/85, ZIN PH 25/85 sous lumière polarisée avec plaque d'onde de Lamb (A et C) et sans plaque d'onde de Lamb (B). A–C. Vue d’ensemble microanatomique des fémurs de trois individus de taille moyenne. À noter un épais cortex non vascularisé. À noter, en outre, que le spécimen ZIN PH 22/85 (A) a une cavité médullaire remplie de tissu osseux. La ligne d’arrêt de croissance est marquée en jaune. cx : cortex ; hpfb : os à fibres parallèles très bien organisées ; mc : cavité médullaire ; trcr : crête du trochanter.
Transverse histological sections of femora of medium-sized individuals of Eoscapherpeton asiaticum ZIN PH 22/85, ZIN PH 23/85, ZIN PH 25/85 under polarized light with a Lamb wave plate (A, C and D) and without lambda waveplate (B). A. Details of the cortex composition (ZIN PH 22/85) showing a highly organized parallel-fibred bone in the peripheral part, an endosteal bone in the innermost part and infilled medullary cavity. Note the presence of two lines of arrested growth (yellow arrows). B–D. Close-up of the cortex (ZIN PH 23/85) with highly organized parallel-fibred bone (B), lines of arrested growth (yellow arrows) (C) and erosion bays (D). E. Periosteal and the endosteal bone of the cortex (ZIN PH 25/85) with highly organized parallel-fibred bone and one line of arrested growth (yellow arrow). eb: endosteal bone; erb: erosion bays; hpfb: highly organized parallel-fibred bone; mc: medullary cavity; oc: osteocyte lacunae; vc: vascular canals.
Coupes biologiques transversales de fémurs d’individus de taille moyenne d’Eoscapherpeton asiaticum 22/85, ZIN PH 23/85, ZIN PH 25/85, sous lumière polarisée avec plaque d’onde de Lamb (A, C et D) et sans plaque d’onde de Lamb (B). A. Détails de la composition du cortex (IN PH 22/85) montrant un os à fibres parallèles très bien organisées à la partie périphérique, un os endostéal dans la partie la plus interne et une cavité médullaire remplie. À noter la présence de deux lignes d’arrêt de croissance (flèches jaunes). B–D. Fermeture du cortex (ZIN PH 23/85) avec de l’os à fibres parallèles très bien organisées (B), des lignes d’arrêt de croissance (flèches jaunes) (C) et des baies d’érosion (D). E. Os périostique et endostéal du cortex (ZIN PH 25/85) avec de l’os à fibres parallèles très bien organisées et une ligne d’arrêt de croissance (flèche jaune). eb : os endostéal ; erb : baies d’érosion ; hpfb : os à fibres parallèles très bien organisées ; cmc : cavité médullaire ; oc : lacunes d’ostéocytes ; vc : canaux vasculaires.
Longitudinal histological sections of femora of medium-sized individuals of Eoscapherpeton asiaticum ZIN PH 33/85, ZIN PH 34/85 under polarized light with a Lamb wave plate (A–C, E and F) under normal light (D). A. Microanatomical overview of the femur of medium-sized individual (ZIN PH 33/85). B and C. The epiphyseal regions. Note the presence of dense calcified cartilage with a globular appearance and an endochondral lamellar bone in small restricted areas. D. Microanatomical overview of the femora of medium-sized individual (ZIN PH 34/85). D and E. Details of the diaphyseal (D) and epiphyseal (E) regions showing highly organized parallel-fibred bone with slightly ellipsoid osteocyte lacunae and large erosion bays. cc: calcified cartilage; ehb: endochondral bone; erb: erosion bays; hpfb: highly organized parallel-fibred bone; oc, osteocyte lacunae; vc, vascular canals.
Coupes biologiques longitudinales de fémurs d’individus de taille moyenne d’Eoscapherpeton asiaticum ZN PH 33/85, ZIN PH 34/85 sous lumière polarisée avec une plaque d’onde de Lamb (A–C, E et F) et sous lumière normale (D). A. Vue d’ensemble du fémur d’un individu de taille moyenne (ZN PH 33/85). B et C. Zones de l’épiphyse. À noter la présence de cartilage calcifié dense à l’apparence globulaire et d’os lamellaire endochondral en petites zones restreintes. D. Vue d’ensemble microanatomique de fémur d’un individu de taille moyenne (ZIN PH 35/85). D et E. Détails des zones diaphysaire (D) et épiphysaire (E) montrant de l’os à fibres parallèles très bien organisées avec des lacunes d’ostéocytes légèrement ellipsoïdales et de grandes baies d’érosion. cc : cartilage calcifié ; ehb : os endochondral ; erb : baies d’érosion ; hpfb : os à fibres parallèles très bien organisées ; os : lacunes d’ostéocytes ; vc : canaux vasculaires.
Transverse histological sections of femora of large individuals of Eoscapherpeton asiaticum ZIN PH 26/85, ZIN PH 27/85, ZIN PH 28/85 under polarized light with a Lamb wave plate. A–C. Microanatomical overview of the femora of large-sized individuals. Note significantly high degree of vascularization of the cortex and a sequence of well-defined lines of arrested growth (yellow arrows). D–F. Details of the femoral cortex of large individuals showing highly organized parallel-fibred bone and high degree of vascularization. cx: cortex; eb: endosteal bone; mc: medullary cavity; trcr: trochanteric crest; vc: vascular canals.
Coupes biologiques transversales de fémurs d’individus de grande taille d’Eoscarphermeton asiaticum ZIN PH 26/85, ZIN PH 27/85, ZIN PH 28/85 sous lumière polarisée avec une plaque d’onde de Lamb. A–C. Vue d’ensemble microanatomique de fémurs d’individus de grande taille. À noter le degré significativement élevé de la vascularisation du cortex et une séquence de lignes bien définies d’arrêt de croissance (flèches jaunes). D–F. Détails du cortex fémoral d’individus de grande taille, montrant de l’os à fibres parallèles très bien organisées et un haut degré de vascularisation. cx : cortex ; eb : os endostéal ; mc : cavité médullaire ; trcr : crête du trochanter ; vc : canaux vasculaires.
Longitudinal histological sections of femora of large individuals of Eoscapherpeton asiaticum ZIN PH 35/85, ZIN PH 36/85 under polarized light with a Lamb wave plate. A. Microanatomical overview of the femur of a large individual (ZIN PH 35/85). B and C. Close-up of the cortex (ZIN PH 35/85) with dense calcified cartilage with globular appearance and endochondral lamellar bone. D. Microanatomical overview of the femur of a large individual (ZIN PH 36/85). E Close-up of the cortex (ZIN PH 36/85) with dense calcified cartilage with globular appearance and endochondral lamellar bone. cc: calcified cartilage; ehb: endochondral bone; erb: erosion bays; vc: vascular canals.
Coupes histologiques longitudinales de fémurs d’individus de grande taille d’Eoscarpherpeton asiaticum ZIN PH 35/85, ZIN 36/85 sous lumière polarisée avec plaque d’onde de Lamb. A. Vue d’ensemble microanatomique du fémur d’un individu de grande taille (ZIN PH 35/85). B et C. Fermeture du cortex (ZIN PH 35/85) avec du cartilage calcifié dense d’apparence globulaire et de l’os lamellaire endochondral. D. Vue d’ensemble du fémur d’un individu de grande taille (ZIN PH 36/85). E Fermeture du cortex (ZIN PH 36/85) avec du cartilage calcifié dense d’apparence globulaire et de l’os lamellaire endochondral. cc : cartilage calcifié ; ehb : os endochondral ; erb : baies d’érosion ; vc : canaux vasculaires.
Digital restoration of the right femur of subadult Cryptobranchus sp. (specimen DVZ M 2/12) in lateral view (A) and in dorsal view (B) with reconstructed vascular network (in red) and with the locations of the microCT transverse digital sections (C–E). C. Digital section at the level of proximal metaphysis. D. Digital section at the mid-diaphysis. E. Digital section at the level of distal metaphysis. Note the presence of vascular foramina, which open into vascular grooves on the bone surface (A). Scales: A and B = 5 mm; C–E = 2 mm.
Restauration numérique du fémur droit d’un subadulte Cryptobranchus sp. (spécimen DVZ M 2/12) en vues latérale (A) et dorsale (B) avec le réseau vasculaire reconstitué (en rouge) et les localisations des coupes numériques transversales micro-CT (C–E). C. Coupe numérique au niveau de la métaphyse proximale. D. Coupe numérique à mi-diaphyse. E. Coupe numérique au niveau de la métaphyse distale. À noter la présence de foramens vasculaires qui s’ouvrent en rainures vasculaires à la surface de l’os (A). Barres d’échelle : A et B = 5 mm ; C–E = 2 mm.
List of specimens sampled for this study
Liste des spécimens échantillonnés pour cette étude.
Table 1No.Femur length (mm)Width of the proximal end (mm)Width of the distal end (mm)Diameter of the central part of diaphysis (mm)Maximal cortex thickness (mm)Maximal medullary cavity diameter (mm)Size classSectioning planeLeft/RightZIN PH 20/855.61.61.90.40.10.2SmallTransverseRZIN PH 37/855.81.71.90.80.30.1SmallTransverseLZIN PH 32/85Estimated length 7.4–8.52.6––––MediumLongitudinalRZIN PH 22/858.32.12.30.70.30.1MediumTransverseLZIN PH 23/859.22.63.00.90.30.4MediumTransverseLZIN PH 33/859.83.03.4–––MediumLongitudinalRZIN PH 34/8511.23.53.4–––MediumLongitudinalRZIN PH 25/8511.43.33.61.20.40.2MediumTransverseLZIN PH 26/8514.03.64.41.00.40.1LargeTransverseRZIN PH 27/85Estimated length 14.7–17.04.05.91.90.70.2LargeTransverseRZIN PH 35/85Estimated length 16.0–18.54.0––––LargeLongitudinalLZIN PH 28/85Estimated length 24.6–28.56.9–2.00.90.3LargeTransverseLZIN PH 36/85Estimated length 30.1–34.87.3––––LargeLongitudinalR