Journal of

Anatomy

J. Anat. (2015) 226, pp334--347

doi: 10.1111/joa.12281

Long bone histology of the stem salamander Kokartus honorarius (Amphibia: Caudata) from the Middle Jurassic of Kyrgyzstan Pavel Skutschas1 and Koen Stein2,a 1

Vertebrate Zoology Department, Biological Faculty, Saint Petersburg State University, Saint Petersburg, Russian Federation € r Naturkunde Berlin, Berlin, Germany Museum fu

2

Abstract Kokartus honorarius from the Middle Jurassic (Bathonian) of Kyrgyzstan is one of the oldest salamanders in the fossil record, characterized by a mixture of plesiomorphic morphological features and characters shared with crown-group salamanders. Here we present a detailed histological analysis of its long bones. The analysis of a growth series demonstrates a significant histological maturation during ontogeny, expressed by the progressive appearance of longitudinally oriented primary vascular canals, primary osteons, growth marks, remodelling features in primary bone tissues, as well as progressive resorption of the calcified cartilage, formation of endochondral bone and development of cartilaginous to bony trabeculae in the epiphyses. Apart from the presence of secondary osteons, the long bone histology of Kokartus is very similar to that of miniaturized temnospondyls, other Jurassic stem salamanders, miniaturized seymouriamorphs and modern crown-group salamanders. We propose that the presence of secondary osteons in Kokartus honorarius is a plesiomorphic feature, and the loss of secondary osteons in the long bones of crown-group salamanders as well as in those of miniaturized temnospondyls is the result of miniaturization processes. Hitherto, all stem salamander long bong histology (Kokartus, Marmorerpeton and ‘salamander A’) has been generally described as having paedomorphic features (i.e. the presence of Katschenko’s Line and a layer of calcified cartilage), these taxa were thus most likely neotenic forms. The absence of clear lines of arrested growth and annuli in long bones of Kokartus honorarius suggests that the animals lived in an environment with stable local conditions. Key words: bone histology; Caudata; Jurassic salamander; Karauridae; Kokartus.

Introduction Salamanders (Caudata) are one of the three modern groups of amphibians. Salamanders are unique in many aspects, including neoteny as a common life strategy (Duellman & Trueb, 1986), high regeneration ability (salamanders can regenerate limbs, tail, eyes and even their spinal cord; see Monaghan et al. 2007 and references therein) and an enormously large genome (Gregory, 2001; Smith et al. 2009; Organ et al. 2011). Salamanders first appear in the fossil record in the Middle Jurassic (Bathonian). Remains of the oldest salamanders are known from geographically distant areas such as Western Siberia (Russia), Great Britain and Kyrgyzstan (Evans et al. Correspondence Pavel Skutschas, Vertebrate Zoology Department, Biological Faculty, Universitetskaya nab. 7/9, Saint Petersburg 199034, Russian Federation. E: [email protected] a Current address: Earth System Sciences – AMGC, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium

Accepted for publication 5 January 2015 Article published online 12 February 2015

1988; Nesov, 1988; Evans & Milner, 1994; Evans & Waldman, 1996; Nesov et al. 1996; Milner, 2000; Averianov et al. 2008; Skutschas & Krasnolutskii, 2011; Skutschas & Martin, 2011; Skutschas, 2013). Notably, the first salamanders in the Bathonian vertebrate assemblages of Western Siberia (Russia) and Great Britain were diverse, and are represented by primitive stem-group taxa and probably also crown-group taxa (Milner, 2000; Skutschas, 2013). All stem salamanders are usually referred to the family Karauridae, which is considered to be paraphyletic (Evans et al. 2005; Averianov et al. 2008). According to another opinion, only two taxa – the Middle Jurassic (Bathonian) Kokartus honorarius from Kyrgyzstan and the Late Jurassic (Kimmeridgian) Karaurus sharovi from Kazakhstan, constitute the monophyletic Karauridae, and the attribution of any other stem-group salamanders (e.g. Marmorerpeton) to the Karauridae is not supported by any synapomorphic features (Skutschas & Martin, 2011). Kokartus honorarius from the Middle Jurassic (Bathonian) of Kyrgyzstan is among the oldest salamanders in the fossil record (Nesov, 1988; Milner, 2000; Averianov et al. 2008; Skutschas & Martin, 2011; Skutschas, 2013; Marjanovi
c & © 2015 Anatomical Society

Long bone histology of a stem salamander, P. Skutschas and K. Stein 335

Laurin, 2014). It is known from isolated complete and fragmentary cranial and postcranial bones of individuals of different sizes, as well as a disarticulated partial skull and a partial skull roof (Averianov et al. 2008; Skutschas & Martin, 2011). Kokartus honorarius is characterized by a mixture of plesiomorphic morphological features shared with miniaturized temnospondyls and some lepospondyls and more derived features commonly associated with crown-group salamanders (Skutschas & Martin, 2011). Based on skeletal morphology, it has been shown that this salamander was relatively large (skull length about 40–50 mm) and, probably, neotenic (Skutschas & Martin, 2011). To obtain further insight into the lifestyle of this stem salamander, and how this corresponds to the environmental setting it was found in, we here present a detailed analysis of its bone histology. Institutional abbreviations: ZIN PH, Paleoherpetological collection Zoological Institute of Russian Academy of Sciences, Saint Petersburg, Russia.

Materials and methods

A

© 2015 Anatomical Society

The sections were observed under polarized light using an optical microscope (Leica DM 4500 LP microscope, Leica Microsystems, Wetzlar, Germany). Images were obtained using a Leica firecam (DFC 450) and processed and measured using Imagic ImageAccess software. Histological terminology follows Francillon-Vieillot et al. (1990), Stein & Prondvai (2014) and Prondvai et al. (2014).

Results

For this study, we sampled nine fragments of long bones (humeri, femora) of Kokartus honorarius from the Kugart 1 locality (site KUG-3) in the Kugart River Basin of the Fergana Depression in Kyrgyzstan. This locality is confined to the lower part of the Balabansai Svita, which is dated as Bathonian (Burakova & Fedorov, 1989; Nesov & Fedorov, 1989). Because isolated diaphyseal fragments

Fig. 1 Anatomical sketch of the femur (A) and humerus (B) of Kokartus honorarius, indicating plane of specimen sectioning for histological examination.

from the Kugart 1 locality could not be referred with certainty to Kokartus honorarius, we used only proximal and distal fragments of humeri and femora (Fig. 1), which demonstrate a typical morphology of this species (e.g. no dorsal crest on the humerus, the ventral crest is confluent with the humerus head; high, massive trochanteric crest extending distolaterally from a blunt trochanter; Averianov et al. 2008, p. 10). The specimens for histological study were divided into three size classes: smallest, medium-sized and large (all specimens are listed in Table 1). In total, four longitudinal sections (one medium-sized humeral distal fragment, one medium-sized femoral distal fragment, one large humeral proximal fragment, and one large femoral proximal fragment) and five crosssections (one of the smallest femoral proximal fragment, one medium-sized humeral distal fragment, one medium-sized femoral distal fragment, one large humeral proximal fragment and one of the large femoral proximal fragment) were made.

General microanatomical features The humeral and femoral thin sections of Kokartus honorarius demonstrate a microanatomical organization that is generally similar to that of modern salamanders (Castanet B

336 Long bone histology of a stem salamander, P. Skutschas and K. Stein

Table 1 List of specimens sampled for this study. Specimen no.

Element

Size class

Sectioning plane

ZIN ZIN ZIN ZIN ZIN ZIN ZIN ZIN ZIN

Femur prox. Humerus dist. Humerus dist. Femur dist. Femur dist. Humerus prox. Humerus prox. Femur prox. Femur prox.

Small Medium Medium Medium Medium Large Large Large Large

Transv. Transv. Longit. Longit. Tranv. Longit. Transv. Transv. Longit.

PH PH PH PH PH PH PH PH PH

65/47 66/47 67/47 68/47 69/47 22/47 23/47 43/47 44/47

et al. 2003; Figs 2–6). Most specimens contain a well-differentiated empty medullary cavity (Figs 2–3 and 5–6), with remains of calcified cartilage in various degrees of resorption and preservation, but an exceptionally large amount of calcified cartilage is present in large proximal femoral fragment ZIN PH 44/47 (Fig. 4). The medullary region of the humeri and femora contains cartilaginous and endochondral bone trabeculae in the proximal and distal parts, but closer to the midshaft such trabeculae are absent. The medullary region is surrounded by a relatively thick cortex; both are separated from each other by a clearly defined Katschenko’s Line. This discontinuity between the periosteal bone and calcified cartilage in the medullary cavity is present in all specimens, and only partly eroded in the largest individuals. The periphery of the periosteal cortex is composed of avascular or poorly vascular lamellar bone and contains growth marks, but no well-defined lines of arrested growth (LAGs). The inner part of the periosteal cortex (i.e. excluding the calcified cartilage) in medium-sized and the largest individuals (presumably sub-adults and adults) is characterized by the presence of active bone remodelling processes (erosion bays, secondary osteons). The rare primary vascular canals in the cortex are longitudinally oriented.

Histology General histological features In the smallest femur, the periosteal cortex is avascular (without primary vascular canals or primary osteons) and

contains no growth marks. A perimedullary layer of calcified cartilage lines the internal surface of the cortex (demarcated by Katschenko’s Line) and the medullary region contains no trabeculae. In medium-sized femoral (as well as humeral) specimens, vascular canals are present in the central part of the periosteal cortex, and initial signs of active remodelling processes in the deepest periosteal cortex and in the calcified cartilage of the medullary cavity are present. In the largest individuals, the number of vascular canals, as well as primary and secondary osteons and erosion bays increases. In the medium-sized and largest specimens, growth marks are present in the periosteal part of the cortex. The number of growth marks varies between one and two in the medium-sized individuals, to up to five in the largest individuals. The medullary cavity of the largest individuals contains a smaller amount of calcified cartilage and shows erosion of Katschenko’s Line. Buildups of endochondral bone line the remaining calcified cartilage, and form trabeculae in the metaphyseal and epiphyseal regions. Except for the smallest femur specimen, the osteocyte lacunae of the innermost cortex of most specimens (humeral as well as femoral) appear larger than the lacunae in the outermost cortex. These enlarged lacunae are mostly situated in the same regions as the large erosion bays. Femur of a small individual (< 1.8 mm small diameter) Only one femur specimen of the smallest known individuals (which were presumably juveniles) was sampled for bone histology, and it is documented by a shaft cross-section (specimen ZIN PH 65/47; Figs 2 and 7). The cortex of this femur is relatively thin (540 lm maximally) in comparison with a large medullary cavity (1240 lm in maximal diameter), and the cortico-diaphyseal index (CDI, cortical thickness/local bone radius) varies between 0.26 and 0.39. The cortical periosteal tissue is compact and formed by avascular parallel-fibred to lamellar bone (Fig. 7A). We did not observe any growth marks (e.g. annuli, LAGs) in the cortex, and the only sign of remodelling in the periosteal bone is a single resorption space. Osteocyte lacunae in the periosteal cortex are numerous and interconnected by thin numerous canaliculi. The osteocyte lacunae generally have a rounded appearance

cb eb

ZIN PH 65/47

cc

KL

Fig. 2 Microanatomy of small femur specimen ZIN PH 65/47. Histological composite image under crossed plane polarized light with lambda waveplate and interpretative sketch. Note the thin layer of parallel-fibred bone immediately above Katschenko’s line, showing different orientation of osteocyte lacunae compared with the main cortex. Abbreviations: cb, cortical bone; cc, calcified cartilage; eb, endochondral bone; KL, Katschenko’s line. © 2015 Anatomical Society

Long bone histology of a stem salamander, P. Skutschas and K. Stein 337

ZIN PH 69/47

KL

cc

cb

H ZIN P 68/47

Fig. 3 Microanatomy of medium-sized distal femur specimens ZIN PH 69/47 and ZIN PH 68/47, sectioned transversely and longitudinally, respectively. Crossed plane polarized light with lambda waveplate composite images and interpretative sketch. Abbreviations: cb, cortical bone; cc, calcified cartilage; KL, Katschenko’s line.

cc

cc

ZIN PH

44/47

er

Fig. 4 Microanatomy of large femoral proximal end ZIN PH 44/47 and large femur shaft ZIN PH 43/47 with interpretative drawings. Note the large amount of calcified cartilage and absence of endochondral bone in the medullary cavity of specimen ZIN PH 44/47 compared with ZIN PH 43/47. Abbreviations: cb, cortical bone; cc, calcified cartilage; er, erosion room. © 2015 Anatomical Society

cc

cb

ZIN PH 43/47

338 Long bone histology of a stem salamander, P. Skutschas and K. Stein

ZIN PH 66/47

cc? cb

/47 67 PH ZIN

Fig. 5 Microanatomy of medium-sized humeri ZIN PH 66/47 and ZIN PH 67/47. Please note the poor preservation of ZIN PH 66/47. Note that the cortex of ZIN PH 66/47 is relatively thick, but the poor preservation precludes more detailed description of the tissues. Humerus ZIN PH 67/47 also shows a thick cortex, and significant amounts of calcified cartilage. The calcified cartilage shows various stages of preservation (indicated by different shades of grey). Abbreviations: cb, cortical bone; cc, calcified cartilage.

cc cc

ZIN PH 22/ 47

eb

cc

cb cc ZIN PH 23/47 throughout the cortex, and are arranged in rows parallel to the surface of the cortex. However, the innermost very thin (only a few cells thick) layer of cortical bone has osteocyte lacunae with an ellipsoid appearance, indicating a change in osteocyte lacuna orientation from this layer of cortical bone to the rest of the cortex (Fig. 7A–C). Osteocytic lacunae are large (the largest ellipsoid lacunae have diameters about 30 lm and 10 lm). Katschenko’s Line is well preserved, and separates the cortex from a layer of calcified cartilage with a thickness varying between 70 and 230 lm (Fig. 7B,C). The calcified cartilage is well identifiable by the presence of Liesegang waves and rings, and has a typical globular appearance (cf. Quilhac et al. 2014). Endochondral lamellar bone is only present in restricted areas on the sur-

Fig. 6 Microanatomy of large humeri ZIN PH 22/47 and ZIN PH 23/47. Most of the calcified cartilage has been resorbed and, locally, endochondral bone has been deposited on the resorption surfaces in the calcified cartilage. Also in the cross-section, a large patch of endochondral bone is present. Abbreviations: cb, cortical bone; cc, calcified cartilage; eb, endochondral bone.

face of the erosion rooms in the calcified cartilage (Fig. 7C). Trabeculae are absent. Femora of medium-sized individuals (small diameter between 1.8 and 2.2 mm) Histology of the femora of the medium-sized, and presumably sub-adult individuals, is documented by two femoral distal fragments (specimen ZIN PH 69/47 and ZIN PH 68/47), sectioned transversely and longitudinally, respectively (Figs 3 and 8A–E). The shaft cross-section has a relatively thick cortex (up to 685 lm) in comparison with the diameter of the medullary cavity (maximally 1330 lm), and the CDI varies between 0.38 and 0.46. The structure of the periosteal cortex of the medium-sized femora differs from that © 2015 Anatomical Society

Long bone histology of a stem salamander, P. Skutschas and K. Stein 339

A

cb KL cc

cb

B

KL cc

C

KL

eb cc Fig. 7 Histology of small femur ZIN PH 65/47. (A) Overview. (B) Detail of the calcified cartilage in the medullary cavity, Katschenko’s line and the innermost cortical bone. (C) Detail of the perimedullary (endochondral) bone lining the calcified cartilage in the medullary cavity. Abbreviations: cb, cortical bone; cc, calcified cartilage; eb, endochondral bone; KL, Katschenko’s line.

of smaller individuals by the presence of the following features: (i) longitudinally oriented primary vascular canals in the peripheral part of the cortex (Fig. 8E); (ii) cortical growth marks; (iii) several primary osteons in the central part of the cortex; (iv) several large erosion bays and secondary osteons in the innermost part of the cortex (Fig. 8B). A well-preserved Katschenko’s Line separates the cortex, as © 2015 Anatomical Society

in smaller individuals, from a layer of calcified cartilage (thickness between 70 and 230 lm; Fig. 8A,C). Endochondral lamellar bone lines the internal surface of the calcified cartilage in restricted areas, but trabeculae are absent. In a longitudinal section of a femoral distal end of a medium-sized individual, the epiphyseal region contains dense calcified cartilage that fills the medullary region. In the metaphysis, cartilaginous and endochondral bone trabeculae are present, but the perimedullary region is mostly composed of calcified cartilage (Fig. 8A,B). The periosteal cortex is composed of dynamic osteogenesis (DO)-derived nonlamellar and lamellar parallel-fibred bone (sensu Ferretti et al. 2002; Marotti, 2010; Prondvai et al. 2014; Stein & Prondvai, 2014; Fig. 8C). Osteocytic lacunae are more numerous in the innermost part of the periosteal cortex (Fig. 8C). Katschenko’s Line is well preserved in both epiphyseal and metaphyseal regions (Fig. 8A–C). Femora of large individuals (small diameter > 2.2 mm) Histology of the femora of large and presumably, adult individuals is documented from two femoral proximal fragments (Fig. 4). The shaft cross-section (specimen ZIN PH 43/ 47) reveals a thick cortex (maximal cortical thickness – 1570 lm; maximal diameter of the medullary cavity – 1140 lm) with CDI varying between 0.47 and 0.72. The periosteal cortex is formed by non-lamellar and lamellar parallel-fibred bone and contains a sequence of growth marks (Fig. 9A,C), but no well-defined lines of arrested growth. The peripheral part of the cortex is poorly vascularized by very few primary vascular canals. As in the cross-section of the femur of the medium-sized individuals, the periosteal cortex contains primary osteons (with deposition of lamellar bone in the vascular space) in its central part, and large erosion bays and secondary osteons (Fig. 9B) in the innermost part of the cortex. Katschenko’s Line is well preserved and only partly eroded by secondary osteons. The calcified cartilage in the medullary cavity is mostly resorbed, leaving only a thin layer in contact with Katschenko’s Line. The remaining cartilage in the perimedullar region is coated with a nearly continuous layer of endochondral lamellar bone. Cartilaginous and endochondral bone trabeculae are present in the epiphyses. In a longitudinal section of a femoral proximal end (specimen ZIN PH 44/47), the epiphyseal region contains dense calcified cartilage with globular appearance. In the metaphyseal region, the calcified cartilage in the medullary cavity is marked by numerous erosion bays, and cartilaginous and endochondral bone trabeculae are present (Fig. 9D,E). Katschenko’s Line is well preserved in both epiphyseal and metaphyseal regions. Humeri of medium-sized individuals (small diameter between 1.8 and 2.0 mm) The humeri are similar to the femora in their histological features. In the humeral cross-section (Fig. 5) of the

340 Long bone histology of a stem salamander, P. Skutschas and K. Stein

A

B

cc cb er cc tr

D

cb

C

KL

cb

E

cc

medium-sized (and presumably sub-adult) individuals (specimens ZIN PH 66/47 and 67/47), the cortex is relatively thick (up to 580 lm), and a distinct medullary cavity (maximal diameter of medullary cavity is 1370 lm) is present. The CDI varies between 0.45 and 0.54. The preserved regions of the outer part of the periosteal cortex are composed of a parallel-fibred to lamellar bone matrix (Fig. 10A), and contain primary longitudinal vascular canals (Fig. 10B) as well as some growth marks. Clear annuli or lines of arrested growth could not be identified. The innermost part of the periosteal cortex contains numerous round osteocyte lacunae and several primary as well as secondary osteons (Fig. 10B,C). A thin layer of endochondral lamellar bone lines the remaining calcified cartilage (Fig. 10A). A longitudinal thin section of a humeral distal end (Fig. 5) shows that the epiphyseal region contains dense calcified cartilage. The metaphyseal region contains cartilaginous to endochondral bone trabeculae, and the perimedullary wall is mostly composed of calcified cartilage (Fig. 10D,E). The periosteal bone cortex is very thin and composed of a parallel-fibred to lamellar bone matrix (Fig. 10D). Katschenko’s

Fig. 8 Histology of medium-sized distal femur specimens ZIN PH 68/47 (A, B) and ZIN PH 69/47 (C–E).(A) ZIN PH 68/47 longitudinal section showing histology of the medullary cavity, calcified cartilage and transition to cortical bone (Katschenko’s line). Note that the struts in the MC are composed of calcified cartilage. (B) Close-up of calcified cartilage in the MC and the lamellar and nonlamellar parallel-fibred periosteally deposited cortical bone, as well as large erosion cavities transecting Katschenko’s line. (C) ZIN PH 69/ 47 transverse section showing calcified cartilage and cortical bone with erosion spaces with secondary bone deposits. (D) Detail of calcified cartilage in (C) with typical globular appearance. (E) Detail of incipient secondary osteons from (C). Abbreviations: cb, cortical bone; cc, calcified cartilage; er, erosion room; KL, Katschenko’s line; tr, trabeculae consisting of cartilage and endochondral bone.

Line is clearly distinguishable in the metaphyseal (Fig. 10D) but not in the epiphyseal region, and the cortical osteocyte lacunae are large (between 30 and 50 lm in length; Fig. 10D,F). Humeri of large individuals (small diameter > 2.0 mm) In cross-section (Fig. 6), the cortex of a large (and presumed adult) individual (specimen ZIN PH 23/47) is relatively thick (maximal cortical thickness – 1080 lm; maximal diameter of the medullary cavity – 1890 lm), and CDI varies between 0.44 and 0.58. The periosteal cortex is composed of a parallel-fibred to lamellar bone matrix with few primary vascular canals and four ill-defined growth marks in the peripheral part (Fig. 11A,C). The innermost cortex contains several primary and secondary osteons and large erosion bays (Fig. 11A–C). Katschenko’s Line is preserved, but partly obscured by secondary osteons, and islands of calcified cartilage with an endochondral lamellar bone lining can be observed in the perimedullar region adjacent to Katschenko’s Line (Fig. 11B). Trabecular structures are absent from the medullary cavity. © 2015 Anatomical Society

Long bone histology of a stem salamander, P. Skutschas and K. Stein 341

A Fig. 9 Histological detail of large distal femur specimens ZIN PH 43/47 (A–C) and ZIN PH 44/47 (D, E). (A) Overview showing the medullary cavity with remnants of calcified cartilage and endochondral bone, the cortex with remodelling features in the innermost part and growth marks in the outermost part. Note the osteocyte lacunae affected by resorption, associated with remodelling features in the innermost cortex. (B) Secondary osteon in the innermost cortex. (C) Detail of the growth marks in the outermost cortex. (D) Longitudinal section in the shaft region, showing thick cortical bone and large erosion spaces, but little to no calcified cartilage. (E) Longitudinal section in the epiphyseal region, showing thinner cortex, with significant amounts of calcified cartilage and large erosion spaces. Abbreviations: cb, cortical bone; cc, calcified cartilage; er, erosion room; gm, growth marks; hl, howship lacunae; KL, Katschenko’s line; ROc, zone of enlarged osteocyte lacunae associated with remodelling features.

B

cb

ROc

cc

gm

eb

D

E

cb

cc

er

cb

B

cb ol

C

cl

ol

D

cb cc E

F

eb

cc

A longitudinal section (Fig. 6) of a humeral proximal end of a large individual (specimen ZIN PH 22/47) is generally similar to that of the humeral distal end of the © 2015 Anatomical Society

hl

C

A

Fig. 10 Histology of medium-sized humeri ZIN PH 66/47 (A–C) and ZIN PH 67/47 (D–F). (A) Overview. (B) Detail of primary vascular canal in the outermost cortex. (C) Detail of secondary osteon in the innermost cortex. Despite poor preservation, calcified cartilage and endochondral bone can be identified. (D) Overview of cortex in longitudinal section. Note the presence of a layer of endochondral bone lining the calcified cartilage. (E) Detail of the calcified cartilage lined with endochondral bone. (F) Osteocyte lacunae. Note the large size of the lacunae. This may be the original size of the osteocyte lacunae, but they may have also been enlarged by osteocytic osteolysis, see text for details. Abbreviations: cb, cortical bone; cc, calcified cartilage; cl, cementing line; eb, endochondral bone; ol, osteocyte lacuna.

KL

medium-sized individual, except that it contains a higher number of erosion bays in the innermost periosteal cortex (Fig. 11D,E).

342 Long bone histology of a stem salamander, P. Skutschas and K. Stein

A

B

KL eb

gm ROc cc er C

D

er

er

E

cb

cc eb

er cb cb

Fig. 11 Histology of large humeri ZIN PH 23/47 (A–C) and ZIN PH 22/47 (D, E). (A) Overview of the cortex. Note the advanced features of resorption, including enlarged osteocyte lacunae associated with large resorption cavities in the innermost cortex. Endochondrally formed osteon in the layer of calcified cartilage lining the medullary cavity. (C) Primary vascular canal surrounded by osteocyte lacunae and canaliculi in the outermost cortex. Note the generally rounded appearance and small size of the transversely sectioned osteocyte lacunae. (D) Overview of the shaft region in longitudinal section, showing a transition of calcified cartilage to the periosteal cortical bone perturbed by large erosion cavities. (E) Close-up of the medullary cavity–cortex interface. Note the globular appearance of the calcified cartilage, but also the rounded appearance of the osteocyte lacunae in the innermost layer of cortical bone directly on the calcified cartilage. These lacunae have a different orientation than those in the subsequentially deposited bone. Abbreviations: cb, cortical bone; cc, calcified cartilage; er, erosion room; gm, growth marks; KL, Katschenko’s line; ROc, zone of enlarged osteocyte lacunae associated with remodelling features.

Discussion Ontogenetic changes in long bone histology The analysis of a growth series of femora and humeri revealed the following main changes in histology of Kokartus honorarius during ontogeny: (i) appearance and increase in number of longitudinally oriented primary vascular canals in the peripheral parts and primary osteons in the central part of the periosteal cortex; (ii) appearance and increase in number of growth marks in the outermost part of the cortex; (iii) appearance and increasing number of bone remodelling features in primary bone tissues; (iv) progressive resorption of calcified cartilage in the diaphyseal areas and formation of endochondral bone lining the erosion cavities in the calcified cartilage; (v) development of

trabecular structures in the epiphyseal regions of the medullary cavity. Thus, there appears to be a significant histological maturation as individuals are getting larger. The enlargement of the osteocyte lacunae in the region of the large erosion bays (Figs 8C, 9A and 11A) in the medium-sized and large individuals may have several possible explanations. Possibly, the osteocyte lacunae were enlarged in the living animals, by a process called osteocytic osteolysis (for a review, see Hall, 2005; Wysolmerski, 2012), where the osteocytes themselves contribute to bone resorption, thereby enlarging their own lacunae. This could have facilitated osteoclasts entering into the innermost cortex, and compensate the lack of vascular canals in the smallest individuals. Alternatively, the enlargement happened after death, by acidic enzymes resulting from autolytic processes or bacterial activity in the medullary cavity. The absence of © 2015 Anatomical Society

Long bone histology of a stem salamander, P. Skutschas and K. Stein 343

enlarged lacunae in the smallest specimen suggests that the former hypothesis is more likely; however, different tissues in the marrow cavity (i.e. higher amount of non-calcified cartilage) of the living animal may have resulted in different taphonomic processes in this specimen. Whatever may be the cause, this phenomenon could potentially affect the results of paleogenomic studies (see, e.g. Organ et al. 2007, 2009, 2011). Despite these lacunae having a DO origin, the eroded features of the enlarged lacunae make an estimation of volume difficult as they do not have an ellipsoid shape, nor do we have an immediate grasp on their three-dimensional structure. This is also the reason why DO-derived osteocytes and not static osteogenesis-derived osteocytes (also called woven bone osteocytes) should be used in paleogenomic studies (see Ferreti et al. 2002; Marotti, 2010; Stein & Prondvai, 2014; Prondvai et al. 2014 for more details). In the longitudinal sections of Kokartus, features of lacuna enlargement are not readily identifiable, or completely absent, most likely because the section was made through an area with lacunae with less to no resorption features.

Comparisons of long bone histology of Kokartus honorarius and modern salamanders Modern salamanders have a rather simple microanatomical organization of the long bones that are formed by very simple periosteal bone surrounding a large marrow cavity, the latter of which may contain bone tissues of endochondral origin. The innermost cortex usually shows evidence of resorption but not secondary osteons (Castanet et al. 2003). In contrast to modern (= crown-group) salamanders, larger individuals of Kokartus honorarius have secondary osteons in the cortex (like in some Paleozoic and Mesozoic temnospondyls and even in fossil sarcopterygians, e.g. Eusthenopteron; see Meunier & Laurin, 2012). We hypothesize here that the active bone remodelling process with the formation of secondary osteons in Kokartus honorarius is a plesiomorphic feature, and that long bones of modern salamanders show a derived simplification of the histological structure. This simplification could be the result of miniaturization during the transition from stem-group to crown-group salamanders. All stem-group taxa were rather large salamanders compared with Jurassic and Early Cretaceous crown-group members. Most stem-group salamanders had a skull length of about 40–50 mm and an estimated body length about
nil et al. 200–400 mm (Skutschas & Martin, 2011; de Buffre 2015), whereas early crown-group members had a skull length about 20 mm and an estimated body length about 120–180 mm (Wang & Evans, 2006). Crown-group salamanders, with body sizes comparable to large stemgroup taxa, appeared only in the Late Cretaceous (e.g. the cryptobranchid Eoscapherpeton in Asia, scapherpetontids in North America). © 2015 Anatomical Society

Moreover, the miniaturized dissorophoid temnospondyl Apateon does not show any secondary osteons in the long bones (Sanchez et al. 2010a,b), while large temnospondyls do (e.g. Metoposaurus; Konietzko-Meier & Sander, 2013). The loss of secondary osteons in long bones of different groups of amphibians may therefore be a result of miniaturization processes.

Comparisons of long bone histology of Kokartus honorarius and other stem-group salamanders Hitherto, the bone histology of stem-group salamanders is only known from the humerus of two taxa – Marmorerpeton and ‘salamander A’ from the Middle Jurassic of Eng
nil et al. 2015). Contrary to Kokartus, land (de Buffre humeral shafts of juvenile or sub-adult individuals of Marmorerpeton are almost amedullar (i.e. without medullary cavity); the periosteal cortex is thick and contains signs of growth marks and numerous randomly situated erosion bays. Calcified cartilage is present in the small medullary cavity. There is no trace of secondary osteons or endochondral ossification, unlike in Kokartus. In ‘salamander A’, the structure of the periosteal cortex of the humeral shaft is similar to that of Marmorerpeton, but the medullary cavity is larger and nearly completely filled by calcified
nil et al. 2015). Additionally, the prescartilage (de Buffre ence of cortical woven bone was reported for Marmorerp
nil et al. 2015), a feature that we could not eton (de Buffre confirm in the provided images of Marmorerpeton, or in our sample of Kokartus. The long bone histology of Kokartus honorarius shares similarities with that of Marmorerpeton and ‘salamander A’ because of the presence of erosion bays in the cortical bone and calcified cartilage in the medullary region. Kokartus honorarius differs from Marmorerpeton and ‘salamander A’ in having secondary osteons (in medium-sized and large individuals) and endochondral bone (even in the smallest individual). The bone histology of Kokartus honorarius also differs from Marmorerpeton in the location of erosion bays, which are randomly situated in the cortex of Marmorerpeton and mostly located in the innermost cortex of Kokartus. The absence of secondary osteons and endochondral bone in the humeri of juvenile or sub-adult Marmorerpeton
nil et al. 2015) is and sub-adult ‘salamander A’ (de Buffre most likely an ontogenetic feature. Therefore, they may still be present in the largest (and likely the oldest) individuals belonging to those taxa, which were unfortunately not
nil et al. (2015). sampled by de Buffre

Comparison of Kokartus honorarius and extinct amphibian long bone histology Without considering the basalmost tetrapods (e.g. baphetids, colosteids, anthracosaurs and various stem tetrapods), there are three major groups of extinct amphibians

344 Long bone histology of a stem salamander, P. Skutschas and K. Stein

(according to a paraphyletic concept of ‘Amphibia’): temnospondyls, seymouriamorphs and lepospondyls. Temnospondyls and lepospondyls are of particular interest for histological investigation because both groups have frequently been suggested as close relatives of some or all of the modern amphibian clades, including salamanders (Laurin & Reisz, 1997; Ruta et al. 2003; Anderson, 2007; Ruta & Coates, 2007; Anderson et al. 2008; Marjanovi
c & Laurin, 2013). The histology of the long bones of large temnosondyls (e.g. metoposaurids and trematosaurids; Steyer et al. 2004; Mukherjee et al. 2010; Konietzko-Meier & Sander, 2013) differs from Kokartus honorarius in having a significantly more vascularized cortex (sometimes with development of a woven-parallel fibred complex sensu Prondvai et al. 2014). Large temnospondyls furthermore possess a dense network of bony trabeculae in the medullary cavity and mostly lack calcified cartilage as well as Katschenko’s Line in adults. Miniaturized temnospondyls (e.g. dissorophoid Apateon; Sanchez et al. 2010a,b), which have been proposed to be closely related to salamanders (Ruta et al. 2003), had a relatively simple long bone histology, similar to that of Kokartus honorarius (thick lamellar cortex, presence of Katschenko’s Line and calcified cartilage, deposition of a thin layer of perimedullary bone coating in adult specimens). However, Apateon did not possess secondary osteons. Miniaturizied seymouriamorphs (Discosauriscus; Sanchez et al. 2008) have a long bone histology that is generally similar to extant crown-group salamanders of small body size (e.g. Euproctus asper; Montori, 1990; Desmognathus monticola, Castanet et al. 1996) and different from Kokartus honorarius because they do not possess calcified cartilage, Katschenko’s Line or secondary osteons. Unfortunately, a comparison with lepospondyl is not possible at this stage because detailed information about the bone histology in this group is largely unknown, and a detailed analysis of lepospondyl bone histology is beyond the scope of the current manuscript.

Paedomorphic honorarius

histological

features

in

Kokartus

Kokartus honorarius is considered to be a neotenic salamander on the basis of a number of skeletal morphological characters: the presence of ossified hypobranchials 1 and 2, tooth-bearing coronoid bones, non-pedicellate teeth, and transverse processes on the atlas (Skutschas & Martin, 2011). The presence of Katschenko’s Line in dia- and metaphyseal regions and the presence of a layer of calcified cartilage in the diaphyseal region furthermore support the neotenic nature of Kokartus honorarius. These features are also characteristic for extant neotenic salamanders (Castanet et al. 2003), the presumed neotenic stem-group salamanders
nil et al. Marmorerpeton and ‘salamander A’ (de Buffre 2015), as well as the neotenic miniaturized dissorophoid temnospondyl Apateon (Sanchez et al. 2010c).

Determination of age and developmental stages The observed growth marks most likely correspond to temporary cessation of osteogenesis, and mark the ending and start of regular growth cycles. We did not observe any clearly delineated LAGs. In modern amphibians, LAGs can be formed annually during a single lethargic period (‘simple-LAG pattern’; Castanet et al. 2003). In the case of the presence of two lethargic periods (hibernation and aestivation) during 1 year, two LAGs may be formed (‘double-LAG pattern’). The distances between LAGs depends on ontogenetic stage: large distances are typical of juveniles with fast somatic growth and high rate of bone deposition. A decrease of the distances between subsequent LAGs is usually indicative of individuals that attained sexual maturity and have protracted growth with a low bone deposition rate. Several factors may affect age determination via skeletochronology in extant amphibians (e.g. ‘double-LAG pattern’, rapprochement and endosteal resorption; Castanet & Smirina, 1990; Smirina, 1994; Eden et al. 2007). However, the general problem is that the number of LAGs does not always correspond to the absolute age of a specimen, and skeletochronological studies that do not use individuals of known age for calibration may underestimate ages (Eden et al. 2007). In the modern salamander Ambystoma tigrinum from Arizona, the number of LAGs in large individuals with known age (15 years) varied from 4 to 11, although most ranged from 4 to 6 (Eden et al. 2007). Similarly, Matsuki & Matsui (2009) found that Hynobius nebulosus from Japan does permit accurate age estimation using skeletochronology in most individuals. These authors suggested that variations in life history traits in A. tigrinum may be responsible for a poor skeletochronological signal. Kokartus was most likely a neotenic salamander with highly variable life history traits comparable to modern neotenic salamanders. Because we cannot confidently calibrate the growth mark record of extinct amphibians, attempting to reconstruct life history and growth curves of Kokartus is prone to a high degree of error and the results would be highly unreliable.

Palaeoenvironmental conditions at the Kugart 1 locality All Kokartus material in the Kugart 1 locality (site KUG-3) was found in red thin-layered clay that overlies red sandstones and underlies brownish-red sandy clays (Nesov et al. 1994, 1996). The depositional environment and the taphonomy of the red thin-layered clay with remains of Kokartus honorarius in the Kugart 1 locality are not well understood. According to Nesov et al. (1996), this clay was deposited in shallow water conditions. The composition of the vertebrate assemblage (see Skutschas & Martin, 2011) and the presence of conchostracans and gastropods Valvata sp. (see Nesov et al. 1996) in the Kugart 1 locality indicate a © 2015 Anatomical Society

Long bone histology of a stem salamander, P. Skutschas and K. Stein 345

freshwater (possibly endorheic lacustrine) rather than an estuarine or firth environment. The histology of the long bones of Kokartus honorarius shows no evidence (i.e. LAGs) of strong seasonality in the local climate where these salamanders lived. The observed growth marks in Kokartus honorarius indicate that there were periods of unfavourable conditions (possibly due to moderate seasonality), but generally the paleoecosystem of the Kugart 1 locality was stable. Additionally, the absence of LAGs and annuli could suggest that the local population of Kokartus honorarius did not hibernate or aestivate.

Concluding remarks Histological analysis of one of the oldest and most primitive stem salamanders (Kokartus honorarius) shows that the histology of long bones in this taxon shares similarities to that of miniaturized temnospondyls, Jurassic stem salamanders, miniaturized seymouriamorphs and modern crown-group salamanders, but differs from all above-mentioned taxa in having secondary osteons. The presence of secondary osteons in Kokartus honorarius could be a size-related plesiomorphic feature, and the loss of secondary osteons in the long bones in crown-group salamanders and miniaturized temnospondyls (e.g. dissorophoid Apateon) may be correlated with miniaturization processes. Until now, three stem salamanders with described long bong histology (Kokartus honorarius, Marmorerpeton and ‘salamander A’) demonstrate that larval (= paedomorphic) bone histological features are retained, suggesting that all of them were neotenic. Despite general similarities related to neoteny, Kokartus honorarius was probably less paedomorphic than other stem salamanders because it had secondary osteons (even in medium-sized individuals) and endochondral bone (even in the smallest individual). However, this could be due to the fact that no large and fully mature specimens of Marmorerpeton or other stem salamanders have been studied histologically thus far. The presence of enlarged osteocyte lacunae (potential remodelling feature) adjacent to the large erosion bays in the medium-sized and large individuals may be explained by osteocytic osteolysis. If correct, this interpretation would raise further awareness of potential errors in histomorphometric and paleogenomic studies aimed at estimating genome sizes of extinct vertebrates based on osteocyte lacuna size. Finally, the absence of clearly defined LAGs in the long bones of Kokartus honorarius suggests that seasonality was moderate and that the Kugart 1 locality had a relatively stable paleoclimate.

Acknowledgements The authors thank all the members of the expeditions in Kyrgyz€ lfer stan for their help. The authors are very grateful to Olaf Du © 2015 Anatomical Society

€ontologie, € r Geologie, Mineralogie und Pala (Steinmann-Institut fu €t Bonn, Germany) for helping with the preparation of Universita € let Dinosaur Research the thin sections; Edina Prondvai (Lendu € tvo € s Lora
nd University, Budapest, Hungary) and P. MarGroup, Eo € r Geologie, Mineralogie und tin Sander (Steinmann-Institut fu €ontologie, Universita €t Bonn, Germany) for fruitful discussions Pala and literature advice. The authors thank Michel Laurin and one anonymous reviewer for providing helpful comments that improved the quality of the manuscript. This work was supported by a Postdoctoral Research Fellowship and a Return Fellowship of the Alexander von Humboldt Foundation (Germany) and the Russian Foundation for Basic Research (project 14-04-01507) to P.S., K.S. was supported by DFG funding in the framework of the € bisEmmy Noether Programme FR 2657/5-1 awarded to Nadia Fro ch. K.S. is currently a Postdoctoral Fellow of the Research Foundation Flanders (FWO).

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