Foss Rec 21 159ndash169 2018httpsdoiorg105194fr-21-159-2018copy Author(s) 2018 This work is distributed underthe Creative Commons Attribution 40 License

Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum(Amphibia Caudata) from the Late Cretaceous of UzbekistanPavel Skutschas Veniamin Kolchanov Elizaveta Boitsova and Ivan KuzminFaculty of Biology Department of Vertebrate Zoology Saint Petersburg State UniversityUniversitetskaya nab 79 199034 Saint Petersburg Russia

Correspondence Pavel Skutschas (skutchasmailru pskutschasspburu)

Received 25 December 2017 ndash Revised 20 March 2018 ndash Accepted 9 April 2018 ndash Published 27 April 2018

Abstract Osseous anomaliespathologies in the Late Creta-ceous cryptobranchid salamander Eoscapherpeton asiaticumfrom the Turonian Bissekty Formation of Uzbekistan are an-alyzed using gross morphological description microCT andhistologic analysis These pathologies result from trauma(fractured and subsequently healed dentary and femorahematoma on femur) possible infection due to trauma(prearticular with exostosis and necrotic cavities anteriortrunk vertebra and fused vertebra with pathological enlarge-ments formed during reactive periosteal growth) and congen-ital disorders (hemivertebra and fused vertebrae with short-ened asymmetrical centra and abnormal arrangements oftransverse processes) The origin of the pathologies of twoatlantal specimens (enlargement of transverse processes) isunclear Our report of hemivertebra in Eoscapherpeton isthe first occurrence of this congenital pathology in a fossillissamphibian The occurrence of several traumatic femoralpathologies in Eoscapherpeton could be a result of intraspe-cific aggressive behavior Bone pathologies are described forthe first time in fossil salamanders

1 Introduction

The study of paleopathology offers an effective tool for un-derstanding some aspects of vertebrate paleobiology includ-ing lifestyle and behavior (eg Hanna 2002 Martinelli etal 2015) as well as reaction of the skeletal tissues to dis-eases and injuries (traumas) through the time (Rothschildand Tanke 1991 Rothschild and Martin 2006 Waldron2009) Despite numerous paleopathological studies of dif-ferent tetrapods (eg dinosaurs crocodiles) paleopatholo-gies among fossil members of modern groups of amphibians

(ie lissamphibians frogs salamanders and caecilians) arepoorly documented (Rothschild 2012 Rothschild and Laub2013) Reported skeletal anomalies among fossil salaman-ders are limited to polydactyly abnormal phalangeal countsand supernumerary hind limbs in the Jurassic cryptobranchidChunerpeton tianyiensis from China (Wang et al 2016)Other pathologies in fossil salamanders have not been de-scribed and analyzed for etiology (Rothschild et al 2012)

Expeditions to the Kyzylkum Desert (Uzbekistan) in1977ndash1994 by Lev A Nesov and in 1997ndash2006 bythe international UzbekRussianBritishAmericanCanadianJoint Paleontological Expeditions (URBAC) yielded sev-eral thousand bones of the fossil cryptobranchid salamanderEoscapherpeton asiaticum from the Upper Cretaceous (Tur-onian) Bissekty Formation (Nesov 1981 1997 Skutschas2009 2013) This abundant material contains several ab-normal specimens of Eoscapherpeton asiaticum includingfused vertebrae fractured bones and some other osseouspathologies The main aims of this study are to describe os-seous anomalies in Eoscapherpeton asiaticum (using grossmorphological description microCT and histologic analy-sis) and to discuss their possible etiology

2 Materials and methods

A total of 17 specimens representing isolated elements fromthe Dzharakuduk locality of the Turonian Bissekty Forma-tion (Redman and Leighton 2009) that belong to the cryp-tobranchid salamander Eoscapherpeton asiaticum were ex-amined All these specimens are stored at the Paleoherpeto-logical collection (ZIN PH) of the Zoological Institute ofthe Russian Academy of Sciences Saint Petersburg Rus-sia The abnormal specimens include two bones of the lower

Published by Copernicus Publications on behalf of the Museum fuumlr Naturkunde Berlin

160 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticumTable

1Listofspecim

ensused

forthisstudy

Specimen

Skeletalelement

Sizeclass

Externalpathologicaldescription

Details

ofinternalstructureE

tiologyFigure

inthis

paper

ZIN

PH1243

prearticularm

ediumfocalbony

protrusion(ieexostosis)

erodedbone

surfacenecrotic

cavitiesconnected

with

vascularcanalstraum

atic-infectious(osteom

yelitis)Fig1

ZIN

PH2243

fragmentary

dentarym

ediumcoarse

callusporous

tissueofcallus

dueto

thepresence

ofradialcanalstraum

atic(healed

fracture)Fig2

ZIN

PH3243

atlantalcentrumsm

allenlarged

transverseprocess

nospecific

internaldetailsunknow

nFig3

ZIN

PH1830242

atlantalcentrumm

ediumasym

metry

(inanteriorand

posteriorviews)and

presenceofw

ell-developedtransverse

processesno

specificinternaldetails

unknown

Fig4

ZIN

PH13243

anteriortrunkvertebra

largeenlarged

(both)andelongated

(right)transverse

processesporous

transverseprocesses

with

largeirregularcavities

traumatic-infectious

(osteomyelitis)

Fig5

ZIN

PH1829242

co-ossifiedatlas

andhem

ivertebralarge

fusionof

vertebraeasym

metrical

atlasw

iththe

posteriorcotyle

orientedposterolaterally

narrowand

wedge-like

hemivertebralcentrum

with

alarge

transverseprocess

differencein

thedensities

ofatlantal

andthe

hemivertebral

centraenlargedtransverse

processofhem

ivertebraw

ithlarge

cavity

congenitalFig6

ZIN

PH1832242

fusedatlas

andthe

firsttrunk

vertebralarge

fusionofvertebraeenlargem

entson

thelateral

surfacesofthe

centraand

thehypapophyses

highlyporous

boneenlargem

entstraum

atic-infectious(osteom

yelitis)Fig7

ZIN

PH1831242

fusedatlas

andthe

firsttrunk

vertebralarge

fusionofvertebraeno

boneenlargem

entsshortening

ofthecentra

asymm

etryofthe

centracongenital

Fig8

ZIN

PH4243

fusedtw

oanteriortrunk

vertebraelarge

fusionofvertebraeenlargem

entson

thelateral

surfacesofthe

centraand

thehypapophyses

displacementofvertebra

porousstructure

ofenlargem

ents

traumatic-infectious

(osteomyelitis)

Fig9

ZIN

PH6243

fusedtw

oanteriortrunk

vertebraelarge

fusionofvertebraeno

boneenlargem

entsshortening

ofthe

centraasym

metric

arrangement

ofthetransverse

processes

asymm

etryofthe

centracongenital

Fig10

ZIN

PH7243

fusedtw

oposterior

trunkvertebrae

largefusion

ofvertebraenobone

enlargements

shorteningofthe

centraandasym

metric

arrangementofthe

transverseprocesses

asymm

etryofthe

centracongenital

Fig11

ZIN

PH5243

fusedthree

anteriortrunk

vertebraelarge

fusionofvertebraeno

boneenlargem

entsshortening

ofthecentra

nospecific

internaldetailscongenital

Fig12

ZIN

PH8243

fusedneuralarches

oftw

otrunk

vertebraelarge

fusionofvertebrae

nospecific

internaldetailscongenital

Fig13

ZIN

PH9243

distalpartofthefem

urm

ediumlarge

smooth

focalboneenlargem

entporous

structureofbone

enlargementdue

tothe

presenceofnum

erouscavities

enlargementis

composed

ofprim

arybone

traumatic

(hematom

a)Fig14andashh

ZIN

PH10243

distalpartofthefem

urm

ediumsm

oothfocalbone

enlargement(callus)

displacementofthe

distalendporous

structureofcallus

traumatic

(healedfracture)

Fig14indasho

ZIN

PH11243

distalpartofthefem

urm

ediumsm

oothfocalbone

enlargement(callus)

displacementofthe

distalendporous

structureofcallus

traumatic

(healedfracture)

Fig15andashf

ZIN

PH12243

distalpartofthefem

urlarge

smooth

focalboneenlargem

ent(callus)displacem

entofthedistalend

porousstructureofcallusdue

tothe

presenceofnum

erouslongitudinal

andobliquely

ori-ented

vascularcanals

traumatic

(healedfracture)

Fig15gndashp

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 161

Figure 1 The prearticular ZIN PH 1243 of Eoscapherpeton asi-aticum (a) Medial view (b) lateral view (c) medial view (digi-tal restoration) with the locations of the microCT digital sections(d) digital transverse section and (endashf) digital longitudinal sectionsAbbreviations E ndash exostosis NC ndash necrotic cavity

jaw (prearticular ZIN PH 1243 fragmentary dentary ZINPH 2243) two atlases (ZIN PH 3243 ZIN PH 1830242)trunk vertebra (ZIN PH 13243) fused vertebrae (ZINPH 1829242 ZIN PH 1831242 ZIN PH 1832242 ZINPH 4243 ZIN PH 5243 ZIN PH 6243 ZIN PH 7243 ZINPH 8243) and distal fragments of femora (ZIN PH 9243ZIN PH 10243 ZIN PH 11243 ZIN PH 12243) (Table 1)All specimens were CT-scanned (at 100 kV and 01 mA gen-erating a resolution of 29 microm of pixel size and an output of4000times 4000 pixels per slice) at the Saint Petersburg StateUniversity Research Centre for X-ray Diffraction Studies(Saint Petersburg Russia) using a Skyscan 1172 CT scan-ner CT scan data were imported to the software Amira 630(FEI-VSG Company) where the model was reconstructedand segmented

The CT data are deposited in the Department of VertebrateZoology of the Saint Petersburg State University Saint Pe-tersburg Russia and can be made available by the presentauthors for the purpose of scientific study

For the histologic analysis standard petrographic thin sec-tions (transverse) of two abnormal femora (distal fragmentsZIN PH 9243 ZIN PH 12243) were prepared The sectionswere observed under polarized light using an optical micro-scope (Leica 4500 Leica Microsystems Wetzlar Germany)in the Saint Petersburg State University Research Centre forX-ray Diffraction Studies Russia Histological terminologyfollows Francillon-Vieillot et al (1990) The thin sectionsused in study are housed in the histological collection of

Figure 2 The dentary ZIN PH 2243 of Eoscapherpeton asiaticum(a) Medial view (b) lateral view (c) ventral view (digital restora-tion) with the location of the transverse microCT digital section(d) dorsal view (digital restoration) with the location of the longi-tudinal microCT digital section (e) digital transverse section and(f) digital longitudinal section Abbreviations C ndash callus RC ndash ra-dial canals

the Department of Vertebrate Zoology Saint Petersburg StateUniversity Saint Petersburg Russia

3 Results

31 Pathologies of bones of the lower jaw

Osseous anomalies were found in the medium-sized preartic-ular ZIN PH 1243 and the fragmentary dentary ZINPH 2243 (Table 1) The prearticular ZIN PH 1243 (Fig 1)exhibits a focal bony protrusion (ie exostosis) on the medialsurface The bone surface around the exostosis is eroded dueto the presence of relatively deep notches of irregular shapeInternal examination reveals the presence of large necroticcavities which were connected with vascular canals (Fig 1dndashf) and with erosion notches on the bone surface The possi-ble diagnosis for this pathology is osteomyelitis ndash inflamma-tion of bone and bone marrow (usually posttraumatic) causedby bacteria (more rarely by fungi) resulting in the periostealreaction and irregular bone surface (Rothschild and Martin2006 Peterson and Vittore 2012 Marais et al 2014 Garciacuteaet al 2016 Ramiacuterez-Velasco et al 2016 )

The medium-sized fragmentary dentary ZIN PH 2243(Fig 2) displays a focal bone enlargement (ie callus) In-ternally the tissue of the callus is porous (in comparison tothat of normal tissue of the bone) and contains several radialcanals (Fig 2endashf) The callus was formed during the heal-ing of traumatic fracture of the dentary (traumatic nature of

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162 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 3 The atlantal centrum ZIN PH 3243 of Eoscapherpetonasiaticum (a) Ventral view (b) dorsal view (c) right lateral view(d) ventral view (digital restoration) (e) right lateral view (digi-tal restoration) with the locations of the microCT digital sections(f) digital transverse section and (g) digital longitudinal section(horizontal plane) in ventral view Abbreviations TP ndash transverseprocess

pathology) (Rothschild and Martin 2006 Pardo-Peacuterez et al2017)

32 Atlantal pathologies

Two atlantal centra in the sample show the osseous anoma-lies the small centrum ZIN PH 3243 and one medium-sizedcentrum ZIN PH 1830242 (Table 1) The small atlas ZINPH 3243 (Fig 3) is characterized by an enlarged transverseprocess (ie rib bearer) on the right lateral surface The in-ner structure of the enlarged transverse process demonstratesthe presence of a large cavity (ie channel) that in life waslikely filled by cartilage (Fig 3fndashg) There are no differencesin porosity in the different parts of the cortical regions andno clear evidence of fractures or inflammation process Theorigin of the pathology is unknown and it could be traumatic-infectious developmental or neoplasm (Rothschild and Mar-tin 2006 Waldron 2009)

The centrum of the larger atlas ZIN PH 1830242 (Fig 4)is asymmetric (in anterior and posterior views) and bearswell-developed transverse processes on the both lateral sur-faces The inner structure of the enlarged transverse pro-cesses is similar to that in ZIN PH 3243 (the presence ofthe large cavity Fig 4g) Like in ZIN PH 3243 the originof the pathology is unclear

Figure 4 The atlantal centrum ZIN PH 1830242 of Eoscapher-peton asiaticum (a) Anterior view (b) posterior view (c) anteriorview (digital restoration) (d) posterior view (digital restoration)(e) right lateral view (digital restoration) with the locations of themicroCT digital sections (f) left lateral view (digital restoration)(g) digital longitudinal section (horizontal plane) in dorsal view and(h) digital transverse section in posterior view Abbreviations TP ndashtransverse process

33 Abnormal trunk vertebra

The large anterior trunk vertebra ZIN PH 13243 (Fig 5) ex-hibits an abnormal structure of the transverse processes onboth lateral surfaces of the centrum right transverse pro-cesses are slightly enlarged while left transverse processesare significantly enlarged and elongated (probably due to thefusion with a rib) The internal structure of the enlarged trans-verse processes is highly porous and contains large irregularcavities Reactive bone growth is likely the result of compli-cation of healing after trauma and the subsequent enlarge-ment of transverse processes could be caused by osteomyeli-tis (traumatic-infectious origin of pathology) (Rothschild andMartin 2006 Waldron 2009 Garciacutea et al 2016)

34 Hemivertebra

The specimen ZIN PH 1829242 is an asymmetrical (in dor-sal and ventral view) fragmentary large atlas that is co-ossified with a hemivertebra (Fig 6) The posterior cotyle

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 163

Figure 5 The anterior trunk vertebra ZIN PH 13243 of Eoscapher-peton asiaticum (a) Anterior view (b) posterior view (c) dor-sal view (d) left lateral view (e) anterior view (digital restora-tion) (f) posterior view (digital restoration) (g) dorsal view (digitalrestoration) (h) left lateral view (digital restoration) with the loca-tions of the microCT digital sections (i) digital transverse section inanterior view and (k) digital longitudinal section (horizontal plane)in dorsal view Abbreviations TP ndash transverse process R ndash rib

of the atlantal centrum is oriented posterolaterally Thehemivertebral centrum is narrow wedge-like and located atthe posterior left portion of the specimen The hemivertebralcentrum bears a large transverse process The bases of neu-ral arches of the atlas and hemivertebra are not fused Exter-nally the boundary between atlantal and the hemivertebralcentra is clearly traceable as a groove on the lateral surfaceof the hypapophysis Internally the boundary is not clearlyevident but some differences in the densities of atlantal andthe hemivertebral centra are visible in some virtual slices(Fig 6i) The inner structure of the enlarged transverse pro-cess demonstrates the presence of a large cavity (ie chan-nel)

35 Fusion of vertebrae

The sample of fused vertebrae (all vertebrae are relativelylarge in size) includes two specimens of fused atlas andthe first trunk vertebrae (ZIN PH 1831-1832242) twospecimens of the fused two anterior trunk vertebrae (ZINPH 4243 6243) one specimen of the fused three anteriortrunk vertebrae (ZIN PH 5243) one specimen of the fusedtwo posterior trunk vertebrae (ZIN PH 7243) and one spec-

Figure 6 The co-ossified atlas and hemivertebra ZIN PH 1829242of Eoscapherpeton asiaticum (a) Anterior view (b) posterior view(c) ventral view (d) dorsal view (e) dorsal view (digital restora-tion) (f) ventral view (digital restoration) (g) left lateral view (dig-ital restoration) with the locations of the microCT digital sectionsand (hndashi) digital longitudinal sections (horizontal plane) in ventralview Broken lines and an arrow show the border between atlas andhemivertebra Abbreviations HV ndash hemivertebra PC ndash posterioratlantal cotyle

imen of the fused neural arches of two trunk vertebrae (ZINPH 8243) (total number of specimens ndash 7 Table 1)

The specimen ZIN PH 1832242 (Fig 7) exhibits en-largements on the lateral surfaces of the centra and thehypapophyses along the suture between the atlas and thefirst trunk vertebrae that were formed during reactive bonegrowth The internal structure of these enlarged areasis highly porous The enlargements could be caused byosteomyelitis after trauma (traumatic-infectious nature ofpathology)

The specimen ZIN PH 1831242 (Fig 8) displays the fu-sion of the atlas and the first trunk vertebrae without notablepathological enlargements on the lateral surfaces of the cen-tra and the hypapophyses (only low ridge is present along thesuture between atlas and the first trunk vertebrae) This sug-gests the absence of the periosteal reactive bone growth Bothvertebrae are shortened in comparison with normal vertebraeor that in the specimen (ZIN PH 1832242) The absence ofany traces of trauma (eg fracture displacement callus) andinflammation of bone (eg enlargements formed during re-active bone growth necrotic cavities) suggests the congenitalorigin of the fusion

The fused two anterior trunk vertebrae ZIN PH 4243(Fig 9) are characterized by the enlargements on the lateralsurfaces of the centra and the hypapophyses along the suturebetween vertebrae and by deformation of the hypapophyses

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164 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 7 The fused atlas and the first trunk vertebra ZINPH 1832242 of Eoscapherpeton asiaticum (a) Dorsal view(b) ventral view (c) anterior view (d) dorsal view (digital restora-tion) (e) ventral view (digital restoration) (f) anterior view (digitalrestoration) with the location of longitudinal the microCT digitalsection (g) left lateral view (digital restoration) with the locationsof the microCT digital sections (h) digital longitudinal section (ver-tical plane) (i) digital longitudinal section (horizontal plane) in dor-sal view and (k) digital transverse section in posterior view Bro-ken lines show the border between atlas and trunk vertebra Arrowsshow bone enlargements Abbreviations AT ndash atlas TV ndash trunkvertebra

CT data show that the more posterior vertebra is slightly dis-placed in relation to the more anterior vertebrae (Fig 9l) sothe trauma took place before the forming of pathological en-largements The internal structure of enlarged areas is highlyporous and contains large necrotic cavities

The fused two anterior trunk vertebrae ZIN PH 6243(Fig 10) are characterized by shortening of the centra asym-metry of the centra (due to the unequal development of theanterior and posterior portions) and asymmetric arrangementof the transverse processes (widely separated on the left sideand closely spaced on the right side of the fused centra) Thebases of neural arches are fused and a large spinal nerve fora-men is present

Similarly to the specimen ZIN PH 6243 the fused twoposterior trunk vertebrae ZIN PH 7243 (Fig 11) exhibit thevertebral shortening with asymmetry of the centra and asym-metric arrangement of the transverse processes The absenceof any traces of trauma and inflammation of bone suggeststhe congenital origin of the fusion The asymmetric arrange-ment of the transverse processes and vertebral shortening

Figure 8 The fused atlas and the first trunk vertebra ZINPH 1831242 of Eoscapherpeton asiaticum (a) anterior view(b) posterior view (c) dorsal view (d) ventral view (e) left lat-eral view (f) anterior view (digital restoration) with the locationof longitudinal the microCT digital section (g) ventral view (digi-tal restoration) with the location of longitudinal the microCT digi-tal section (h) right lateral view (digital restoration) (i) left lateralview (digital restoration) (k) digital longitudinal section (horizon-tal plane) in dorsal view and (l) digital longitudinal section (verticalplane) Broken lines show the border between atlas and trunk verte-bra Abbreviations AT ndash atlas TV ndash trunk vertebra

could be the result of abnormal segmentation during embryo-genesis

The specimen ZIN PH 5243 consists of three fused ante-rior trunk vertebrae (Fig 12) The vertebrae exhibit the verte-bral shortening and do not display any signs of trauma andorinflammation of bone We suggest the congenital origin ofthe fusion in ZIN PH 5243

The neural arches of two trunk vertebrae ZIN PH 8243(Fig 13) are fused without any trace of suture The more an-terior neural arch is lower than the more posterior one Thereare no pathological enlargements (no signs of periosteal re-active growth) and the fusion of these neural arches is mostlikely of congenital origin

36 Femoral pathologies

The distal fragment of the medium-sized femur ZINPH 9243 (Fig 14andashh) displays the presence of large smoothfocal bone enlargement (ie periosteal elevation) in themetaphyseal region The internal structure of this bone en-largement is porous due to the presence of large cavities ofirregular shape (ie erosion bays) (Fig 14fndashh) The tissue

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 165

Figure 9 The fused two anterior trunk vertebrae ZIN PH 4243 ofEoscapherpeton asiaticum (a) Dorsal view (b) ventral view (c) an-terior view (d) right lateral view (e) left lateral view (f) anteriorview (digital restoration) (g) dorsal view (digital restoration) withthe location of longitudinal the microCT digital section (h) rightlateral view (digital restoration) with the location of longitudinalthe microCT digital section (i) left lateral view (digital restoration)(k) digital longitudinal section (vertical plane) and (l) digital lon-gitudinal section (horizontal plane) in dorsal view Blue lines showthe original median line of the centra prior to the trauma Arrowsshow bone enlargements Abbreviations NC ndash necrotic cavity

of the enlargement is entirely composed of a parallel-fiberedprimary bone of periosteal origin The parallel-fibered pri-mary bone in the outer part of the enlargement is more or-ganized than in the inner porous part and is similar to thatof the normal cortical bone of this femur (Fig 14h) No evi-dence of fracture was revealed by CT scanning and histolog-ical analysis Similar bone enlargement in modern reptileswas identified as hematoma (Rothschild et al 2010 2012)We also interpret the femoral pathology on ZIN PH 9243 ashematoma that could have been caused by trauma (traumaticorigin)

All other femoral specimens in the sample (two distal frag-ments ZIN PH 10-11243 of medium-sized femora and onedistal fragment ZIN PH 12243 of large femora Table 1Figs 14indasho 15) display the signs of healed fractures thatare traceable in CT images Externally all the specimensdemonstrate displacements of the distal end of the bone andhave large smooth focal bone enlargements (ie calluses) inthe metaphyseal region that were formed during the healingof traumatic fractures The internal structure of the callusesis porous and contains numerous longitudinal and obliquely

Figure 10 The fused two anterior trunk vertebrae ZIN PH 6243of Eoscapherpeton asiaticum (a) Dorsal view (b) ventral view(c) anterior view (d) left lateral view (e) right lateral view (f) ven-tral view (digital restoration) with the location of longitudinalthe microCT digital sections (g) anterior view (digital restora-tion) with the location of longitudinal the microCT digital section(h) left lateral view (digital restoration) (i) right lateral view (digitalrestoration) (kndashl) digital longitudinal sections (vertical plane) and(m) digital longitudinal section (horizontal plane) in ventral viewBroken lines show the border between trunk vertebrae Abbrevia-tions NA ndash neural arch TP1 ndash transverse process of more anteriorvertebra TP2 ndash transverse process of more posterior vertebra

oriented vascular canals that are typical for these structures(Fig 15nndashp)

4 Discussion

41 General discussion

Eoscapherpeton asiaticum demonstrates a wide spectrum ofosseous anomaliespathologies resulting from trauma pos-sible infection due to trauma and congenital disorders Trau-matic origins of pathologies are suggested for the dentaryZIN PH 2243 (the presence of the fracture and the callus)and for femoral fragments ZIN PH 9-12243 (hematoma andhealed fractures with the calluses on the distal portions of thebones) (Table 1) The possible traumatic-infectious patholo-gies are associated with bone enlargements formed duringreactive bone growth andor the presence of necrotic cavitiesare suggested for the prearticular ZIN PH 1243 the anteriortrunk vertebra ZIN PH 13243 the fused atlas and the firsttrunk vertebrae ZIN PH 1832242 and the fused two ante-

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166 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 11 The fused two posterior trunk vertebrae ZIN PH 7243 ofEoscapherpeton asiaticum (a) Ventral view (b) dorsal view (c) an-terior view (d) right lateral view (e) left lateral view (f) ventralview (digital restoration) with the location of the microCT digitalsections (g) anterior view (digital restoration) with the location oflongitudinal the microCT digital section (h) right lateral view (dig-ital restoration) (i) left lateral view (digital restoration) (k) digitallongitudinal section (vertical plane) (l) digital transverse section inanterior view and (m) digital longitudinal section (horizontal plane)in dorsal view Broken lines show the border between trunk verte-brae Abbreviations NA ndash neural arch TP1 ndash transverse process ofmore anterior vertebra TP2 ndash transverse process of more posteriorvertebra

rior trunk vertebrae ZIN PH 4243 (Table 1) The congenitalpathologies of E asiaticum are represented by the hemiver-tebra (specimen ZIN PH 182924 see discussion below) andby fusion of two (fused atlas and anterior trunk vertebra ZINPH 1831242 fused anterior trunk vertebrae ZIN PH 6243fused posterior trunk vertebrae ZIN PH 7243 and for fusedneural arches 8243) or three (fused anterior trunk vertebraeZIN PH 5243) vertebrae (Table 1) The total number of thesevertebral congenital pathologies in E asiaticum is very low(n= 5) in comparison with thousands of collected vertebraeof this salamander (only collection of atlases ZIN PH 242contains 1909 specimens while there are only two speci-mens of congenital pathologies with atlas ndash ZIN PH 182924and ZIN PH 1831242 This frequency of vertebral congen-ital pathologies in E asiaticum (for congenital pathologieswith atlas lt 01 ) is in accordance with the previous estima-tions for PaleozoicndashMesozoic temnospondyls and Holocenefrogs (Rothschild and Laub 2013 Witzmann et al 2013)

Figure 12 The fused three anterior trunk vertebrae ZIN PH 5243of Eoscapherpeton asiaticum (a) Ventral view (b) dorsal view(c) left lateral view (d) right lateral view (e) ventral view (digi-tal restoration) with the location of longitudinal the microCT digitalsection (f) left lateral view (digital restoration) with the locationof longitudinal the microCT digital section (g) right lateral view(digital restoration) (h) digital longitudinal section (vertical plane)and (i) digital longitudinal section (horizontal plane) in dorsal viewBroken lines show the border between trunk vertebrae

42 Hemivertebra in Eoscapherpeton asiaticum

A hemivertebra is a congenital malformation in which oneor more lateral halves of vertebrae are intercalated betweenneighboring vertebrae Formation of hemivertebrae is a resultof abnormal segmentation during embryogenesis (ie fail-ure of somitogenesis before chondrification and ossificationof the vertebral anlagen eg Witzmann et al 2008 2013Burnham et al 2013) In amniots single vertebrae are de-rived from the sclerotome of two adjacent somites and thisprocess is widely known as resegmentation (Piekarski andOlsson 2014 and references therein) Resegmentation havebeen proposed to be present in all modern lissamphibians butembryological and molecular evidence for this process wasprovided only for one salamander species ndash ambystomatidAmbystoma mexicanum (Mexican axolotl) (Piekarski andOlsson 2014) The development of the described hemiver-tebra in more basal cryptobranchid salamander Eoscapher-peton asiaticum can also be explained by a failure of reseg-mentation If this is correct then it is significant for the fol-

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 167

Figure 13 The fused neural arches of two trunk vertebrae ZINPH 8243 of Eoscapherpeton asiaticum (a) Dorsal view (b) rightlateral view (c) left lateral view (d) left lateral view (digital restora-tion) (e) right lateral view (digital restoration) (f) dorsal view (digi-tal restoration) with the location of longitudinal the microCT digitalsection and (g) digital longitudinal section (vertical plane)

lowing reasons (1) it is the oldest occurrence of resegmen-tation in salamanders and (2) it is evidence that resegmen-tation is a common process for salamanders (including rela-tively basal cryptobranchids and advanced ambystomatids)

A hemivertebra in E asiaticum was found in a relativelylarge (according to the large size of the atlas that was co-ossified with hemivertebra) and presumably adult individual(Fig 6) The presence of a hemivertebra in a large individualsuggests that this pathology did not cause its death

The hemivertebrae have been reported earlier for somePaleozoic and Mesozoic temnospondyls (Witzmann et al2013) The finding of hemivertebra in the Late Cretaceouscryptobranchid E asiaticum is the oldest (and the first de-scribed) occurrence of this pathology among modern groupsof amphibians (ie lissamphibians salamanders frogs andcaecilians)

43 Femoral pathologies in Eoscapherpeton possibleinsight into behavior

The traumatic origin of pathologies on the distal parts of thefemora (Figs 14ndash15) could be evidence of frequent occur-rence of hind limb injuries in Eoscapherpeton asiaticum Inmodern cryptobranchids such as Cryptobranchus limb in-juries are the most common physical abnormalities in wildpopulations (Miller and Miller 2005) The proposed maincause of limb injuries in Cryptobranchus is intraspecific ag-

Figure 14 The distal parts of the femora ZIN PH 9243 (andashh) andZIN PH 10243 (indasho) of Eoscapherpeton asiaticum Specimen ZINPH 9243 (a) ventral view (b) dorsal view (c) ventral view (dig-ital restoration) (d) dorsal view (digital restoration) (e) anterioror posterior view (digital restoration) with the locations of trans-verse histological sections and longitudinal the microCT digital sec-tion (f) digital longitudinal section and (gndashh) histological trans-verse section The arrow shows the border between cortical boneand pathological tissue Specimen ZIN PH 10243 (i) ventral view(k) dorsal view (l) ventral view (digital restoration) with the loca-tions of the microCT digital sections (m) anterior or posterior view(digital restoration) (n) digital longitudinal section and (o) digitaltransverse section Blue lines show the original bone position priorto the break Abbreviations BE ndash bone enlargement C ndash callus CBndash cortical bone

gression during the reproductive season (eg defending ofnesting sites) because fresh injuries were found only dur-ing the reproductive season (namely in August and Septem-ber) (Hiler et al 2005 Miller and Miller 2005) Takinginto account the phylogenetic position of Eoscapherpetonas a member of the cryptobranchid clade and their aquaticlifestyle (see Skutschas 2009 2013) the reason for the fre-quent occurrence of hind limb injuries in E asiaticum couldbe the same (intraspecific aggression during reproductiveseason) and this behavioral element could have appearedearly in the evolution of cryptobranchids ie at least in theLate Cretaceous

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168 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 15 The distal parts of the femora ZIN PH 11243 (andashf) andZIN PH 12243 (gndashp) of Eoscapherpeton asiaticum Specimen ZINPH 11243 (a) ventral view (b) dorsal view (c) dorsal view (dig-ital restoration) with the locations of the microCT digital sections(d) anterior or posterior view (digital restoration) (e) digital lon-gitudinal section and (f) digital transverse section Specimen ZINPH 12243 (g) ventral view (h) dorsal view (i) ventral view (dig-ital restoration) (k) dorsal view (digital restoration) (l) anterior orposterior view (digital restoration) with the locations of transversehistological sections and longitudinal the microCT digital section(m) digital longitudinal section and (nndashp) transverse histologicalsections Blue lines show the original bone position prior to thebreak Abbreviations C ndash callus

5 Conclusions

The study revealed diverse osseous anomaliespathologies inEoscapherpeton asiaticum which were interpreted as trau-matic possible traumatic-infectious and congenital in originpertaining to bones of the lower jaw vertebrae and femoraThe hemivertebra and fused vertebrae (without any traces oftrauma and inflammation) document the earliest occurrenceof vertebral congenital pathologies in crown-group salaman-ders The frequency of vertebral congenital pathologies in Easiaticum is low and in accordance with the previous estima-tions for temnospondyls and frogs The numerous traumaticpathologies of femora (hematoma and healed fractures) sug-gest the survival of accidents and possibly the presence of in-

traspecific aggression during the reproductive season in thissalamander (like in modern cryptobranchids)

Data availability Micro-CT data are archived at the Department ofVertebrate Zoology of the Saint Petersburg State University (SaintPetersburg Russia) and can be accessed through the present authorsfor the purpose of scientific study

Author contributions PS designed the project PS and VK per-formed the research VK and IK prepared 3-D reconstructions VKand EB prepared figures and PS wrote the manuscript All authorscommented on the manuscript

Competing interests The authors declare that they have no conflictof interest

Acknowledgements We are grateful to the staff of the SaintPetersburg State University Research Centre for X-ray DiffractionStudies (Saint Petersburg Russia) for their help with using theLeica 2500P microscope and for CT scanning of specimens Theauthors thank Judith Pardo-Peacuterez and Nadia Froumlbisch for providinghelpful comments that improved the quality of the manuscript Thisstudy was fulfilled under support of the Russian Scientific Fundproject 14-14-00015

Edited by Constanze BickelmannReviewed by Nadia Froumlbisch and Judith Pardo Perez

References

Burnham D A Rothschild B M Babiarz J P and Martin LD Hemivertebrae as pathology and as a window to behavior inthe fossil record Palarchrsquos Journal of Vertebrate Palaeontology10 1ndash6 2013

Francillon-Vieillot H de Buffreacutenil V Castanet J Geraudie JMeunier F Sire J Y Zylenberberg L and de Ricqles A DMicrostructure and mineralization of vertebrate skeletal tissuesin Skeletal biomineralization Patterns Processes and Evolu-tionary Trends Vol 1 edited by Carter J G Van NostrandReinhold New York 471ndash530 1990

Garciacutea R A Cerda I A Heller M Rothschild B M and Zur-riaguz V The first evidence of osteomyelitis in a sauropod di-nosaur Lethaia 50 227ndash236 httpsdoiorg101111let121892016

Hanna R R Multiple injury and infection in a sub adult thero-pod dinosaur Allosaurus fragilis with comparisons to allosaurpathology in Cleveland-Lloyd Dinosaur Quarry Collection JVertebr Paleontol 22 76ndash90 httpsdoiorg1016710272-4634(2002)022[0076MIAIIA]20CO2 2002

Hiler W R Wheeler B A and Trauth S E Abnormalities inthe Ozark Hellbender (Cryptobranchus alleganiensis bishopi) inArkansas A Comparison between Two Rivers with a Historical

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

160 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticumTable

1Listofspecim

ensused

forthisstudy

Specimen

Skeletalelement

Sizeclass

Externalpathologicaldescription

Details

ofinternalstructureE

tiologyFigure

inthis

paper

ZIN

PH1243

prearticularm

ediumfocalbony

protrusion(ieexostosis)

erodedbone

surfacenecrotic

cavitiesconnected

with

vascularcanalstraum

atic-infectious(osteom

yelitis)Fig1

ZIN

PH2243

fragmentary

dentarym

ediumcoarse

callusporous

tissueofcallus

dueto

thepresence

ofradialcanalstraum

atic(healed

fracture)Fig2

ZIN

PH3243

atlantalcentrumsm

allenlarged

transverseprocess

nospecific

internaldetailsunknow

nFig3

ZIN

PH1830242

atlantalcentrumm

ediumasym

metry

(inanteriorand

posteriorviews)and

presenceofw

ell-developedtransverse

processesno

specificinternaldetails

unknown

Fig4

ZIN

PH13243

anteriortrunkvertebra

largeenlarged

(both)andelongated

(right)transverse

processesporous

transverseprocesses

with

largeirregularcavities

traumatic-infectious

(osteomyelitis)

Fig5

ZIN

PH1829242

co-ossifiedatlas

andhem

ivertebralarge

fusionof

vertebraeasym

metrical

atlasw

iththe

posteriorcotyle

orientedposterolaterally

narrowand

wedge-like

hemivertebralcentrum

with

alarge

transverseprocess

differencein

thedensities

ofatlantal

andthe

hemivertebral

centraenlargedtransverse

processofhem

ivertebraw

ithlarge

cavity

congenitalFig6

ZIN

PH1832242

fusedatlas

andthe

firsttrunk

vertebralarge

fusionofvertebraeenlargem

entson

thelateral

surfacesofthe

centraand

thehypapophyses

highlyporous

boneenlargem

entstraum

atic-infectious(osteom

yelitis)Fig7

ZIN

PH1831242

fusedatlas

andthe

firsttrunk

vertebralarge

fusionofvertebraeno

boneenlargem

entsshortening

ofthecentra

asymm

etryofthe

centracongenital

Fig8

ZIN

PH4243

fusedtw

oanteriortrunk

vertebraelarge

fusionofvertebraeenlargem

entson

thelateral

surfacesofthe

centraand

thehypapophyses

displacementofvertebra

porousstructure

ofenlargem

ents

traumatic-infectious

(osteomyelitis)

Fig9

ZIN

PH6243

fusedtw

oanteriortrunk

vertebraelarge

fusionofvertebraeno

boneenlargem

entsshortening

ofthe

centraasym

metric

arrangement

ofthetransverse

processes

asymm

etryofthe

centracongenital

Fig10

ZIN

PH7243

fusedtw

oposterior

trunkvertebrae

largefusion

ofvertebraenobone

enlargements

shorteningofthe

centraandasym

metric

arrangementofthe

transverseprocesses

asymm

etryofthe

centracongenital

Fig11

ZIN

PH5243

fusedthree

anteriortrunk

vertebraelarge

fusionofvertebraeno

boneenlargem

entsshortening

ofthecentra

nospecific

internaldetailscongenital

Fig12

ZIN

PH8243

fusedneuralarches

oftw

otrunk

vertebraelarge

fusionofvertebrae

nospecific

internaldetailscongenital

Fig13

ZIN

PH9243

distalpartofthefem

urm

ediumlarge

smooth

focalboneenlargem

entporous

structureofbone

enlargementdue

tothe

presenceofnum

erouscavities

enlargementis

composed

ofprim

arybone

traumatic

(hematom

a)Fig14andashh

ZIN

PH10243

distalpartofthefem

urm

ediumsm

oothfocalbone

enlargement(callus)

displacementofthe

distalendporous

structureofcallus

traumatic

(healedfracture)

Fig14indasho

ZIN

PH11243

distalpartofthefem

urm

ediumsm

oothfocalbone

enlargement(callus)

displacementofthe

distalendporous

structureofcallus

traumatic

(healedfracture)

Fig15andashf

ZIN

PH12243

distalpartofthefem

urlarge

smooth

focalboneenlargem

ent(callus)displacem

entofthedistalend

porousstructureofcallusdue

tothe

presenceofnum

erouslongitudinal

andobliquely

ori-ented

vascularcanals

traumatic

(healedfracture)

Fig15gndashp

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 161

Figure 1 The prearticular ZIN PH 1243 of Eoscapherpeton asi-aticum (a) Medial view (b) lateral view (c) medial view (digi-tal restoration) with the locations of the microCT digital sections(d) digital transverse section and (endashf) digital longitudinal sectionsAbbreviations E ndash exostosis NC ndash necrotic cavity

jaw (prearticular ZIN PH 1243 fragmentary dentary ZINPH 2243) two atlases (ZIN PH 3243 ZIN PH 1830242)trunk vertebra (ZIN PH 13243) fused vertebrae (ZINPH 1829242 ZIN PH 1831242 ZIN PH 1832242 ZINPH 4243 ZIN PH 5243 ZIN PH 6243 ZIN PH 7243 ZINPH 8243) and distal fragments of femora (ZIN PH 9243ZIN PH 10243 ZIN PH 11243 ZIN PH 12243) (Table 1)All specimens were CT-scanned (at 100 kV and 01 mA gen-erating a resolution of 29 microm of pixel size and an output of4000times 4000 pixels per slice) at the Saint Petersburg StateUniversity Research Centre for X-ray Diffraction Studies(Saint Petersburg Russia) using a Skyscan 1172 CT scan-ner CT scan data were imported to the software Amira 630(FEI-VSG Company) where the model was reconstructedand segmented

The CT data are deposited in the Department of VertebrateZoology of the Saint Petersburg State University Saint Pe-tersburg Russia and can be made available by the presentauthors for the purpose of scientific study

For the histologic analysis standard petrographic thin sec-tions (transverse) of two abnormal femora (distal fragmentsZIN PH 9243 ZIN PH 12243) were prepared The sectionswere observed under polarized light using an optical micro-scope (Leica 4500 Leica Microsystems Wetzlar Germany)in the Saint Petersburg State University Research Centre forX-ray Diffraction Studies Russia Histological terminologyfollows Francillon-Vieillot et al (1990) The thin sectionsused in study are housed in the histological collection of

Figure 2 The dentary ZIN PH 2243 of Eoscapherpeton asiaticum(a) Medial view (b) lateral view (c) ventral view (digital restora-tion) with the location of the transverse microCT digital section(d) dorsal view (digital restoration) with the location of the longi-tudinal microCT digital section (e) digital transverse section and(f) digital longitudinal section Abbreviations C ndash callus RC ndash ra-dial canals

the Department of Vertebrate Zoology Saint Petersburg StateUniversity Saint Petersburg Russia

3 Results

31 Pathologies of bones of the lower jaw

Osseous anomalies were found in the medium-sized preartic-ular ZIN PH 1243 and the fragmentary dentary ZINPH 2243 (Table 1) The prearticular ZIN PH 1243 (Fig 1)exhibits a focal bony protrusion (ie exostosis) on the medialsurface The bone surface around the exostosis is eroded dueto the presence of relatively deep notches of irregular shapeInternal examination reveals the presence of large necroticcavities which were connected with vascular canals (Fig 1dndashf) and with erosion notches on the bone surface The possi-ble diagnosis for this pathology is osteomyelitis ndash inflamma-tion of bone and bone marrow (usually posttraumatic) causedby bacteria (more rarely by fungi) resulting in the periostealreaction and irregular bone surface (Rothschild and Martin2006 Peterson and Vittore 2012 Marais et al 2014 Garciacuteaet al 2016 Ramiacuterez-Velasco et al 2016 )

The medium-sized fragmentary dentary ZIN PH 2243(Fig 2) displays a focal bone enlargement (ie callus) In-ternally the tissue of the callus is porous (in comparison tothat of normal tissue of the bone) and contains several radialcanals (Fig 2endashf) The callus was formed during the heal-ing of traumatic fracture of the dentary (traumatic nature of

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162 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 3 The atlantal centrum ZIN PH 3243 of Eoscapherpetonasiaticum (a) Ventral view (b) dorsal view (c) right lateral view(d) ventral view (digital restoration) (e) right lateral view (digi-tal restoration) with the locations of the microCT digital sections(f) digital transverse section and (g) digital longitudinal section(horizontal plane) in ventral view Abbreviations TP ndash transverseprocess

pathology) (Rothschild and Martin 2006 Pardo-Peacuterez et al2017)

32 Atlantal pathologies

Two atlantal centra in the sample show the osseous anoma-lies the small centrum ZIN PH 3243 and one medium-sizedcentrum ZIN PH 1830242 (Table 1) The small atlas ZINPH 3243 (Fig 3) is characterized by an enlarged transverseprocess (ie rib bearer) on the right lateral surface The in-ner structure of the enlarged transverse process demonstratesthe presence of a large cavity (ie channel) that in life waslikely filled by cartilage (Fig 3fndashg) There are no differencesin porosity in the different parts of the cortical regions andno clear evidence of fractures or inflammation process Theorigin of the pathology is unknown and it could be traumatic-infectious developmental or neoplasm (Rothschild and Mar-tin 2006 Waldron 2009)

The centrum of the larger atlas ZIN PH 1830242 (Fig 4)is asymmetric (in anterior and posterior views) and bearswell-developed transverse processes on the both lateral sur-faces The inner structure of the enlarged transverse pro-cesses is similar to that in ZIN PH 3243 (the presence ofthe large cavity Fig 4g) Like in ZIN PH 3243 the originof the pathology is unclear

Figure 4 The atlantal centrum ZIN PH 1830242 of Eoscapher-peton asiaticum (a) Anterior view (b) posterior view (c) anteriorview (digital restoration) (d) posterior view (digital restoration)(e) right lateral view (digital restoration) with the locations of themicroCT digital sections (f) left lateral view (digital restoration)(g) digital longitudinal section (horizontal plane) in dorsal view and(h) digital transverse section in posterior view Abbreviations TP ndashtransverse process

33 Abnormal trunk vertebra

The large anterior trunk vertebra ZIN PH 13243 (Fig 5) ex-hibits an abnormal structure of the transverse processes onboth lateral surfaces of the centrum right transverse pro-cesses are slightly enlarged while left transverse processesare significantly enlarged and elongated (probably due to thefusion with a rib) The internal structure of the enlarged trans-verse processes is highly porous and contains large irregularcavities Reactive bone growth is likely the result of compli-cation of healing after trauma and the subsequent enlarge-ment of transverse processes could be caused by osteomyeli-tis (traumatic-infectious origin of pathology) (Rothschild andMartin 2006 Waldron 2009 Garciacutea et al 2016)

34 Hemivertebra

The specimen ZIN PH 1829242 is an asymmetrical (in dor-sal and ventral view) fragmentary large atlas that is co-ossified with a hemivertebra (Fig 6) The posterior cotyle

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 163

Figure 5 The anterior trunk vertebra ZIN PH 13243 of Eoscapher-peton asiaticum (a) Anterior view (b) posterior view (c) dor-sal view (d) left lateral view (e) anterior view (digital restora-tion) (f) posterior view (digital restoration) (g) dorsal view (digitalrestoration) (h) left lateral view (digital restoration) with the loca-tions of the microCT digital sections (i) digital transverse section inanterior view and (k) digital longitudinal section (horizontal plane)in dorsal view Abbreviations TP ndash transverse process R ndash rib

of the atlantal centrum is oriented posterolaterally Thehemivertebral centrum is narrow wedge-like and located atthe posterior left portion of the specimen The hemivertebralcentrum bears a large transverse process The bases of neu-ral arches of the atlas and hemivertebra are not fused Exter-nally the boundary between atlantal and the hemivertebralcentra is clearly traceable as a groove on the lateral surfaceof the hypapophysis Internally the boundary is not clearlyevident but some differences in the densities of atlantal andthe hemivertebral centra are visible in some virtual slices(Fig 6i) The inner structure of the enlarged transverse pro-cess demonstrates the presence of a large cavity (ie chan-nel)

35 Fusion of vertebrae

The sample of fused vertebrae (all vertebrae are relativelylarge in size) includes two specimens of fused atlas andthe first trunk vertebrae (ZIN PH 1831-1832242) twospecimens of the fused two anterior trunk vertebrae (ZINPH 4243 6243) one specimen of the fused three anteriortrunk vertebrae (ZIN PH 5243) one specimen of the fusedtwo posterior trunk vertebrae (ZIN PH 7243) and one spec-

Figure 6 The co-ossified atlas and hemivertebra ZIN PH 1829242of Eoscapherpeton asiaticum (a) Anterior view (b) posterior view(c) ventral view (d) dorsal view (e) dorsal view (digital restora-tion) (f) ventral view (digital restoration) (g) left lateral view (dig-ital restoration) with the locations of the microCT digital sectionsand (hndashi) digital longitudinal sections (horizontal plane) in ventralview Broken lines and an arrow show the border between atlas andhemivertebra Abbreviations HV ndash hemivertebra PC ndash posterioratlantal cotyle

imen of the fused neural arches of two trunk vertebrae (ZINPH 8243) (total number of specimens ndash 7 Table 1)

The specimen ZIN PH 1832242 (Fig 7) exhibits en-largements on the lateral surfaces of the centra and thehypapophyses along the suture between the atlas and thefirst trunk vertebrae that were formed during reactive bonegrowth The internal structure of these enlarged areasis highly porous The enlargements could be caused byosteomyelitis after trauma (traumatic-infectious nature ofpathology)

The specimen ZIN PH 1831242 (Fig 8) displays the fu-sion of the atlas and the first trunk vertebrae without notablepathological enlargements on the lateral surfaces of the cen-tra and the hypapophyses (only low ridge is present along thesuture between atlas and the first trunk vertebrae) This sug-gests the absence of the periosteal reactive bone growth Bothvertebrae are shortened in comparison with normal vertebraeor that in the specimen (ZIN PH 1832242) The absence ofany traces of trauma (eg fracture displacement callus) andinflammation of bone (eg enlargements formed during re-active bone growth necrotic cavities) suggests the congenitalorigin of the fusion

The fused two anterior trunk vertebrae ZIN PH 4243(Fig 9) are characterized by the enlargements on the lateralsurfaces of the centra and the hypapophyses along the suturebetween vertebrae and by deformation of the hypapophyses

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164 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 7 The fused atlas and the first trunk vertebra ZINPH 1832242 of Eoscapherpeton asiaticum (a) Dorsal view(b) ventral view (c) anterior view (d) dorsal view (digital restora-tion) (e) ventral view (digital restoration) (f) anterior view (digitalrestoration) with the location of longitudinal the microCT digitalsection (g) left lateral view (digital restoration) with the locationsof the microCT digital sections (h) digital longitudinal section (ver-tical plane) (i) digital longitudinal section (horizontal plane) in dor-sal view and (k) digital transverse section in posterior view Bro-ken lines show the border between atlas and trunk vertebra Arrowsshow bone enlargements Abbreviations AT ndash atlas TV ndash trunkvertebra

CT data show that the more posterior vertebra is slightly dis-placed in relation to the more anterior vertebrae (Fig 9l) sothe trauma took place before the forming of pathological en-largements The internal structure of enlarged areas is highlyporous and contains large necrotic cavities

The fused two anterior trunk vertebrae ZIN PH 6243(Fig 10) are characterized by shortening of the centra asym-metry of the centra (due to the unequal development of theanterior and posterior portions) and asymmetric arrangementof the transverse processes (widely separated on the left sideand closely spaced on the right side of the fused centra) Thebases of neural arches are fused and a large spinal nerve fora-men is present

Similarly to the specimen ZIN PH 6243 the fused twoposterior trunk vertebrae ZIN PH 7243 (Fig 11) exhibit thevertebral shortening with asymmetry of the centra and asym-metric arrangement of the transverse processes The absenceof any traces of trauma and inflammation of bone suggeststhe congenital origin of the fusion The asymmetric arrange-ment of the transverse processes and vertebral shortening

Figure 8 The fused atlas and the first trunk vertebra ZINPH 1831242 of Eoscapherpeton asiaticum (a) anterior view(b) posterior view (c) dorsal view (d) ventral view (e) left lat-eral view (f) anterior view (digital restoration) with the locationof longitudinal the microCT digital section (g) ventral view (digi-tal restoration) with the location of longitudinal the microCT digi-tal section (h) right lateral view (digital restoration) (i) left lateralview (digital restoration) (k) digital longitudinal section (horizon-tal plane) in dorsal view and (l) digital longitudinal section (verticalplane) Broken lines show the border between atlas and trunk verte-bra Abbreviations AT ndash atlas TV ndash trunk vertebra

could be the result of abnormal segmentation during embryo-genesis

The specimen ZIN PH 5243 consists of three fused ante-rior trunk vertebrae (Fig 12) The vertebrae exhibit the verte-bral shortening and do not display any signs of trauma andorinflammation of bone We suggest the congenital origin ofthe fusion in ZIN PH 5243

The neural arches of two trunk vertebrae ZIN PH 8243(Fig 13) are fused without any trace of suture The more an-terior neural arch is lower than the more posterior one Thereare no pathological enlargements (no signs of periosteal re-active growth) and the fusion of these neural arches is mostlikely of congenital origin

36 Femoral pathologies

The distal fragment of the medium-sized femur ZINPH 9243 (Fig 14andashh) displays the presence of large smoothfocal bone enlargement (ie periosteal elevation) in themetaphyseal region The internal structure of this bone en-largement is porous due to the presence of large cavities ofirregular shape (ie erosion bays) (Fig 14fndashh) The tissue

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 165

Figure 9 The fused two anterior trunk vertebrae ZIN PH 4243 ofEoscapherpeton asiaticum (a) Dorsal view (b) ventral view (c) an-terior view (d) right lateral view (e) left lateral view (f) anteriorview (digital restoration) (g) dorsal view (digital restoration) withthe location of longitudinal the microCT digital section (h) rightlateral view (digital restoration) with the location of longitudinalthe microCT digital section (i) left lateral view (digital restoration)(k) digital longitudinal section (vertical plane) and (l) digital lon-gitudinal section (horizontal plane) in dorsal view Blue lines showthe original median line of the centra prior to the trauma Arrowsshow bone enlargements Abbreviations NC ndash necrotic cavity

of the enlargement is entirely composed of a parallel-fiberedprimary bone of periosteal origin The parallel-fibered pri-mary bone in the outer part of the enlargement is more or-ganized than in the inner porous part and is similar to thatof the normal cortical bone of this femur (Fig 14h) No evi-dence of fracture was revealed by CT scanning and histolog-ical analysis Similar bone enlargement in modern reptileswas identified as hematoma (Rothschild et al 2010 2012)We also interpret the femoral pathology on ZIN PH 9243 ashematoma that could have been caused by trauma (traumaticorigin)

All other femoral specimens in the sample (two distal frag-ments ZIN PH 10-11243 of medium-sized femora and onedistal fragment ZIN PH 12243 of large femora Table 1Figs 14indasho 15) display the signs of healed fractures thatare traceable in CT images Externally all the specimensdemonstrate displacements of the distal end of the bone andhave large smooth focal bone enlargements (ie calluses) inthe metaphyseal region that were formed during the healingof traumatic fractures The internal structure of the callusesis porous and contains numerous longitudinal and obliquely

Figure 10 The fused two anterior trunk vertebrae ZIN PH 6243of Eoscapherpeton asiaticum (a) Dorsal view (b) ventral view(c) anterior view (d) left lateral view (e) right lateral view (f) ven-tral view (digital restoration) with the location of longitudinalthe microCT digital sections (g) anterior view (digital restora-tion) with the location of longitudinal the microCT digital section(h) left lateral view (digital restoration) (i) right lateral view (digitalrestoration) (kndashl) digital longitudinal sections (vertical plane) and(m) digital longitudinal section (horizontal plane) in ventral viewBroken lines show the border between trunk vertebrae Abbrevia-tions NA ndash neural arch TP1 ndash transverse process of more anteriorvertebra TP2 ndash transverse process of more posterior vertebra

oriented vascular canals that are typical for these structures(Fig 15nndashp)

4 Discussion

41 General discussion

Eoscapherpeton asiaticum demonstrates a wide spectrum ofosseous anomaliespathologies resulting from trauma pos-sible infection due to trauma and congenital disorders Trau-matic origins of pathologies are suggested for the dentaryZIN PH 2243 (the presence of the fracture and the callus)and for femoral fragments ZIN PH 9-12243 (hematoma andhealed fractures with the calluses on the distal portions of thebones) (Table 1) The possible traumatic-infectious patholo-gies are associated with bone enlargements formed duringreactive bone growth andor the presence of necrotic cavitiesare suggested for the prearticular ZIN PH 1243 the anteriortrunk vertebra ZIN PH 13243 the fused atlas and the firsttrunk vertebrae ZIN PH 1832242 and the fused two ante-

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166 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 11 The fused two posterior trunk vertebrae ZIN PH 7243 ofEoscapherpeton asiaticum (a) Ventral view (b) dorsal view (c) an-terior view (d) right lateral view (e) left lateral view (f) ventralview (digital restoration) with the location of the microCT digitalsections (g) anterior view (digital restoration) with the location oflongitudinal the microCT digital section (h) right lateral view (dig-ital restoration) (i) left lateral view (digital restoration) (k) digitallongitudinal section (vertical plane) (l) digital transverse section inanterior view and (m) digital longitudinal section (horizontal plane)in dorsal view Broken lines show the border between trunk verte-brae Abbreviations NA ndash neural arch TP1 ndash transverse process ofmore anterior vertebra TP2 ndash transverse process of more posteriorvertebra

rior trunk vertebrae ZIN PH 4243 (Table 1) The congenitalpathologies of E asiaticum are represented by the hemiver-tebra (specimen ZIN PH 182924 see discussion below) andby fusion of two (fused atlas and anterior trunk vertebra ZINPH 1831242 fused anterior trunk vertebrae ZIN PH 6243fused posterior trunk vertebrae ZIN PH 7243 and for fusedneural arches 8243) or three (fused anterior trunk vertebraeZIN PH 5243) vertebrae (Table 1) The total number of thesevertebral congenital pathologies in E asiaticum is very low(n= 5) in comparison with thousands of collected vertebraeof this salamander (only collection of atlases ZIN PH 242contains 1909 specimens while there are only two speci-mens of congenital pathologies with atlas ndash ZIN PH 182924and ZIN PH 1831242 This frequency of vertebral congen-ital pathologies in E asiaticum (for congenital pathologieswith atlas lt 01 ) is in accordance with the previous estima-tions for PaleozoicndashMesozoic temnospondyls and Holocenefrogs (Rothschild and Laub 2013 Witzmann et al 2013)

Figure 12 The fused three anterior trunk vertebrae ZIN PH 5243of Eoscapherpeton asiaticum (a) Ventral view (b) dorsal view(c) left lateral view (d) right lateral view (e) ventral view (digi-tal restoration) with the location of longitudinal the microCT digitalsection (f) left lateral view (digital restoration) with the locationof longitudinal the microCT digital section (g) right lateral view(digital restoration) (h) digital longitudinal section (vertical plane)and (i) digital longitudinal section (horizontal plane) in dorsal viewBroken lines show the border between trunk vertebrae

42 Hemivertebra in Eoscapherpeton asiaticum

A hemivertebra is a congenital malformation in which oneor more lateral halves of vertebrae are intercalated betweenneighboring vertebrae Formation of hemivertebrae is a resultof abnormal segmentation during embryogenesis (ie fail-ure of somitogenesis before chondrification and ossificationof the vertebral anlagen eg Witzmann et al 2008 2013Burnham et al 2013) In amniots single vertebrae are de-rived from the sclerotome of two adjacent somites and thisprocess is widely known as resegmentation (Piekarski andOlsson 2014 and references therein) Resegmentation havebeen proposed to be present in all modern lissamphibians butembryological and molecular evidence for this process wasprovided only for one salamander species ndash ambystomatidAmbystoma mexicanum (Mexican axolotl) (Piekarski andOlsson 2014) The development of the described hemiver-tebra in more basal cryptobranchid salamander Eoscapher-peton asiaticum can also be explained by a failure of reseg-mentation If this is correct then it is significant for the fol-

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 167

Figure 13 The fused neural arches of two trunk vertebrae ZINPH 8243 of Eoscapherpeton asiaticum (a) Dorsal view (b) rightlateral view (c) left lateral view (d) left lateral view (digital restora-tion) (e) right lateral view (digital restoration) (f) dorsal view (digi-tal restoration) with the location of longitudinal the microCT digitalsection and (g) digital longitudinal section (vertical plane)

lowing reasons (1) it is the oldest occurrence of resegmen-tation in salamanders and (2) it is evidence that resegmen-tation is a common process for salamanders (including rela-tively basal cryptobranchids and advanced ambystomatids)

A hemivertebra in E asiaticum was found in a relativelylarge (according to the large size of the atlas that was co-ossified with hemivertebra) and presumably adult individual(Fig 6) The presence of a hemivertebra in a large individualsuggests that this pathology did not cause its death

The hemivertebrae have been reported earlier for somePaleozoic and Mesozoic temnospondyls (Witzmann et al2013) The finding of hemivertebra in the Late Cretaceouscryptobranchid E asiaticum is the oldest (and the first de-scribed) occurrence of this pathology among modern groupsof amphibians (ie lissamphibians salamanders frogs andcaecilians)

43 Femoral pathologies in Eoscapherpeton possibleinsight into behavior

The traumatic origin of pathologies on the distal parts of thefemora (Figs 14ndash15) could be evidence of frequent occur-rence of hind limb injuries in Eoscapherpeton asiaticum Inmodern cryptobranchids such as Cryptobranchus limb in-juries are the most common physical abnormalities in wildpopulations (Miller and Miller 2005) The proposed maincause of limb injuries in Cryptobranchus is intraspecific ag-

Figure 14 The distal parts of the femora ZIN PH 9243 (andashh) andZIN PH 10243 (indasho) of Eoscapherpeton asiaticum Specimen ZINPH 9243 (a) ventral view (b) dorsal view (c) ventral view (dig-ital restoration) (d) dorsal view (digital restoration) (e) anterioror posterior view (digital restoration) with the locations of trans-verse histological sections and longitudinal the microCT digital sec-tion (f) digital longitudinal section and (gndashh) histological trans-verse section The arrow shows the border between cortical boneand pathological tissue Specimen ZIN PH 10243 (i) ventral view(k) dorsal view (l) ventral view (digital restoration) with the loca-tions of the microCT digital sections (m) anterior or posterior view(digital restoration) (n) digital longitudinal section and (o) digitaltransverse section Blue lines show the original bone position priorto the break Abbreviations BE ndash bone enlargement C ndash callus CBndash cortical bone

gression during the reproductive season (eg defending ofnesting sites) because fresh injuries were found only dur-ing the reproductive season (namely in August and Septem-ber) (Hiler et al 2005 Miller and Miller 2005) Takinginto account the phylogenetic position of Eoscapherpetonas a member of the cryptobranchid clade and their aquaticlifestyle (see Skutschas 2009 2013) the reason for the fre-quent occurrence of hind limb injuries in E asiaticum couldbe the same (intraspecific aggression during reproductiveseason) and this behavioral element could have appearedearly in the evolution of cryptobranchids ie at least in theLate Cretaceous

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168 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 15 The distal parts of the femora ZIN PH 11243 (andashf) andZIN PH 12243 (gndashp) of Eoscapherpeton asiaticum Specimen ZINPH 11243 (a) ventral view (b) dorsal view (c) dorsal view (dig-ital restoration) with the locations of the microCT digital sections(d) anterior or posterior view (digital restoration) (e) digital lon-gitudinal section and (f) digital transverse section Specimen ZINPH 12243 (g) ventral view (h) dorsal view (i) ventral view (dig-ital restoration) (k) dorsal view (digital restoration) (l) anterior orposterior view (digital restoration) with the locations of transversehistological sections and longitudinal the microCT digital section(m) digital longitudinal section and (nndashp) transverse histologicalsections Blue lines show the original bone position prior to thebreak Abbreviations C ndash callus

5 Conclusions

The study revealed diverse osseous anomaliespathologies inEoscapherpeton asiaticum which were interpreted as trau-matic possible traumatic-infectious and congenital in originpertaining to bones of the lower jaw vertebrae and femoraThe hemivertebra and fused vertebrae (without any traces oftrauma and inflammation) document the earliest occurrenceof vertebral congenital pathologies in crown-group salaman-ders The frequency of vertebral congenital pathologies in Easiaticum is low and in accordance with the previous estima-tions for temnospondyls and frogs The numerous traumaticpathologies of femora (hematoma and healed fractures) sug-gest the survival of accidents and possibly the presence of in-

traspecific aggression during the reproductive season in thissalamander (like in modern cryptobranchids)

Data availability Micro-CT data are archived at the Department ofVertebrate Zoology of the Saint Petersburg State University (SaintPetersburg Russia) and can be accessed through the present authorsfor the purpose of scientific study

Author contributions PS designed the project PS and VK per-formed the research VK and IK prepared 3-D reconstructions VKand EB prepared figures and PS wrote the manuscript All authorscommented on the manuscript

Competing interests The authors declare that they have no conflictof interest

Acknowledgements We are grateful to the staff of the SaintPetersburg State University Research Centre for X-ray DiffractionStudies (Saint Petersburg Russia) for their help with using theLeica 2500P microscope and for CT scanning of specimens Theauthors thank Judith Pardo-Peacuterez and Nadia Froumlbisch for providinghelpful comments that improved the quality of the manuscript Thisstudy was fulfilled under support of the Russian Scientific Fundproject 14-14-00015

Edited by Constanze BickelmannReviewed by Nadia Froumlbisch and Judith Pardo Perez

References

Burnham D A Rothschild B M Babiarz J P and Martin LD Hemivertebrae as pathology and as a window to behavior inthe fossil record Palarchrsquos Journal of Vertebrate Palaeontology10 1ndash6 2013

Francillon-Vieillot H de Buffreacutenil V Castanet J Geraudie JMeunier F Sire J Y Zylenberberg L and de Ricqles A DMicrostructure and mineralization of vertebrate skeletal tissuesin Skeletal biomineralization Patterns Processes and Evolu-tionary Trends Vol 1 edited by Carter J G Van NostrandReinhold New York 471ndash530 1990

Garciacutea R A Cerda I A Heller M Rothschild B M and Zur-riaguz V The first evidence of osteomyelitis in a sauropod di-nosaur Lethaia 50 227ndash236 httpsdoiorg101111let121892016

Hanna R R Multiple injury and infection in a sub adult thero-pod dinosaur Allosaurus fragilis with comparisons to allosaurpathology in Cleveland-Lloyd Dinosaur Quarry Collection JVertebr Paleontol 22 76ndash90 httpsdoiorg1016710272-4634(2002)022[0076MIAIIA]20CO2 2002

Hiler W R Wheeler B A and Trauth S E Abnormalities inthe Ozark Hellbender (Cryptobranchus alleganiensis bishopi) inArkansas A Comparison between Two Rivers with a Historical

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 161

Figure 1 The prearticular ZIN PH 1243 of Eoscapherpeton asi-aticum (a) Medial view (b) lateral view (c) medial view (digi-tal restoration) with the locations of the microCT digital sections(d) digital transverse section and (endashf) digital longitudinal sectionsAbbreviations E ndash exostosis NC ndash necrotic cavity

jaw (prearticular ZIN PH 1243 fragmentary dentary ZINPH 2243) two atlases (ZIN PH 3243 ZIN PH 1830242)trunk vertebra (ZIN PH 13243) fused vertebrae (ZINPH 1829242 ZIN PH 1831242 ZIN PH 1832242 ZINPH 4243 ZIN PH 5243 ZIN PH 6243 ZIN PH 7243 ZINPH 8243) and distal fragments of femora (ZIN PH 9243ZIN PH 10243 ZIN PH 11243 ZIN PH 12243) (Table 1)All specimens were CT-scanned (at 100 kV and 01 mA gen-erating a resolution of 29 microm of pixel size and an output of4000times 4000 pixels per slice) at the Saint Petersburg StateUniversity Research Centre for X-ray Diffraction Studies(Saint Petersburg Russia) using a Skyscan 1172 CT scan-ner CT scan data were imported to the software Amira 630(FEI-VSG Company) where the model was reconstructedand segmented

The CT data are deposited in the Department of VertebrateZoology of the Saint Petersburg State University Saint Pe-tersburg Russia and can be made available by the presentauthors for the purpose of scientific study

For the histologic analysis standard petrographic thin sec-tions (transverse) of two abnormal femora (distal fragmentsZIN PH 9243 ZIN PH 12243) were prepared The sectionswere observed under polarized light using an optical micro-scope (Leica 4500 Leica Microsystems Wetzlar Germany)in the Saint Petersburg State University Research Centre forX-ray Diffraction Studies Russia Histological terminologyfollows Francillon-Vieillot et al (1990) The thin sectionsused in study are housed in the histological collection of

Figure 2 The dentary ZIN PH 2243 of Eoscapherpeton asiaticum(a) Medial view (b) lateral view (c) ventral view (digital restora-tion) with the location of the transverse microCT digital section(d) dorsal view (digital restoration) with the location of the longi-tudinal microCT digital section (e) digital transverse section and(f) digital longitudinal section Abbreviations C ndash callus RC ndash ra-dial canals

the Department of Vertebrate Zoology Saint Petersburg StateUniversity Saint Petersburg Russia

3 Results

31 Pathologies of bones of the lower jaw

Osseous anomalies were found in the medium-sized preartic-ular ZIN PH 1243 and the fragmentary dentary ZINPH 2243 (Table 1) The prearticular ZIN PH 1243 (Fig 1)exhibits a focal bony protrusion (ie exostosis) on the medialsurface The bone surface around the exostosis is eroded dueto the presence of relatively deep notches of irregular shapeInternal examination reveals the presence of large necroticcavities which were connected with vascular canals (Fig 1dndashf) and with erosion notches on the bone surface The possi-ble diagnosis for this pathology is osteomyelitis ndash inflamma-tion of bone and bone marrow (usually posttraumatic) causedby bacteria (more rarely by fungi) resulting in the periostealreaction and irregular bone surface (Rothschild and Martin2006 Peterson and Vittore 2012 Marais et al 2014 Garciacuteaet al 2016 Ramiacuterez-Velasco et al 2016 )

The medium-sized fragmentary dentary ZIN PH 2243(Fig 2) displays a focal bone enlargement (ie callus) In-ternally the tissue of the callus is porous (in comparison tothat of normal tissue of the bone) and contains several radialcanals (Fig 2endashf) The callus was formed during the heal-ing of traumatic fracture of the dentary (traumatic nature of

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162 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 3 The atlantal centrum ZIN PH 3243 of Eoscapherpetonasiaticum (a) Ventral view (b) dorsal view (c) right lateral view(d) ventral view (digital restoration) (e) right lateral view (digi-tal restoration) with the locations of the microCT digital sections(f) digital transverse section and (g) digital longitudinal section(horizontal plane) in ventral view Abbreviations TP ndash transverseprocess

pathology) (Rothschild and Martin 2006 Pardo-Peacuterez et al2017)

32 Atlantal pathologies

Two atlantal centra in the sample show the osseous anoma-lies the small centrum ZIN PH 3243 and one medium-sizedcentrum ZIN PH 1830242 (Table 1) The small atlas ZINPH 3243 (Fig 3) is characterized by an enlarged transverseprocess (ie rib bearer) on the right lateral surface The in-ner structure of the enlarged transverse process demonstratesthe presence of a large cavity (ie channel) that in life waslikely filled by cartilage (Fig 3fndashg) There are no differencesin porosity in the different parts of the cortical regions andno clear evidence of fractures or inflammation process Theorigin of the pathology is unknown and it could be traumatic-infectious developmental or neoplasm (Rothschild and Mar-tin 2006 Waldron 2009)

The centrum of the larger atlas ZIN PH 1830242 (Fig 4)is asymmetric (in anterior and posterior views) and bearswell-developed transverse processes on the both lateral sur-faces The inner structure of the enlarged transverse pro-cesses is similar to that in ZIN PH 3243 (the presence ofthe large cavity Fig 4g) Like in ZIN PH 3243 the originof the pathology is unclear

Figure 4 The atlantal centrum ZIN PH 1830242 of Eoscapher-peton asiaticum (a) Anterior view (b) posterior view (c) anteriorview (digital restoration) (d) posterior view (digital restoration)(e) right lateral view (digital restoration) with the locations of themicroCT digital sections (f) left lateral view (digital restoration)(g) digital longitudinal section (horizontal plane) in dorsal view and(h) digital transverse section in posterior view Abbreviations TP ndashtransverse process

33 Abnormal trunk vertebra

The large anterior trunk vertebra ZIN PH 13243 (Fig 5) ex-hibits an abnormal structure of the transverse processes onboth lateral surfaces of the centrum right transverse pro-cesses are slightly enlarged while left transverse processesare significantly enlarged and elongated (probably due to thefusion with a rib) The internal structure of the enlarged trans-verse processes is highly porous and contains large irregularcavities Reactive bone growth is likely the result of compli-cation of healing after trauma and the subsequent enlarge-ment of transverse processes could be caused by osteomyeli-tis (traumatic-infectious origin of pathology) (Rothschild andMartin 2006 Waldron 2009 Garciacutea et al 2016)

34 Hemivertebra

The specimen ZIN PH 1829242 is an asymmetrical (in dor-sal and ventral view) fragmentary large atlas that is co-ossified with a hemivertebra (Fig 6) The posterior cotyle

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 163

Figure 5 The anterior trunk vertebra ZIN PH 13243 of Eoscapher-peton asiaticum (a) Anterior view (b) posterior view (c) dor-sal view (d) left lateral view (e) anterior view (digital restora-tion) (f) posterior view (digital restoration) (g) dorsal view (digitalrestoration) (h) left lateral view (digital restoration) with the loca-tions of the microCT digital sections (i) digital transverse section inanterior view and (k) digital longitudinal section (horizontal plane)in dorsal view Abbreviations TP ndash transverse process R ndash rib

of the atlantal centrum is oriented posterolaterally Thehemivertebral centrum is narrow wedge-like and located atthe posterior left portion of the specimen The hemivertebralcentrum bears a large transverse process The bases of neu-ral arches of the atlas and hemivertebra are not fused Exter-nally the boundary between atlantal and the hemivertebralcentra is clearly traceable as a groove on the lateral surfaceof the hypapophysis Internally the boundary is not clearlyevident but some differences in the densities of atlantal andthe hemivertebral centra are visible in some virtual slices(Fig 6i) The inner structure of the enlarged transverse pro-cess demonstrates the presence of a large cavity (ie chan-nel)

35 Fusion of vertebrae

The sample of fused vertebrae (all vertebrae are relativelylarge in size) includes two specimens of fused atlas andthe first trunk vertebrae (ZIN PH 1831-1832242) twospecimens of the fused two anterior trunk vertebrae (ZINPH 4243 6243) one specimen of the fused three anteriortrunk vertebrae (ZIN PH 5243) one specimen of the fusedtwo posterior trunk vertebrae (ZIN PH 7243) and one spec-

Figure 6 The co-ossified atlas and hemivertebra ZIN PH 1829242of Eoscapherpeton asiaticum (a) Anterior view (b) posterior view(c) ventral view (d) dorsal view (e) dorsal view (digital restora-tion) (f) ventral view (digital restoration) (g) left lateral view (dig-ital restoration) with the locations of the microCT digital sectionsand (hndashi) digital longitudinal sections (horizontal plane) in ventralview Broken lines and an arrow show the border between atlas andhemivertebra Abbreviations HV ndash hemivertebra PC ndash posterioratlantal cotyle

imen of the fused neural arches of two trunk vertebrae (ZINPH 8243) (total number of specimens ndash 7 Table 1)

The specimen ZIN PH 1832242 (Fig 7) exhibits en-largements on the lateral surfaces of the centra and thehypapophyses along the suture between the atlas and thefirst trunk vertebrae that were formed during reactive bonegrowth The internal structure of these enlarged areasis highly porous The enlargements could be caused byosteomyelitis after trauma (traumatic-infectious nature ofpathology)

The specimen ZIN PH 1831242 (Fig 8) displays the fu-sion of the atlas and the first trunk vertebrae without notablepathological enlargements on the lateral surfaces of the cen-tra and the hypapophyses (only low ridge is present along thesuture between atlas and the first trunk vertebrae) This sug-gests the absence of the periosteal reactive bone growth Bothvertebrae are shortened in comparison with normal vertebraeor that in the specimen (ZIN PH 1832242) The absence ofany traces of trauma (eg fracture displacement callus) andinflammation of bone (eg enlargements formed during re-active bone growth necrotic cavities) suggests the congenitalorigin of the fusion

The fused two anterior trunk vertebrae ZIN PH 4243(Fig 9) are characterized by the enlargements on the lateralsurfaces of the centra and the hypapophyses along the suturebetween vertebrae and by deformation of the hypapophyses

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164 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 7 The fused atlas and the first trunk vertebra ZINPH 1832242 of Eoscapherpeton asiaticum (a) Dorsal view(b) ventral view (c) anterior view (d) dorsal view (digital restora-tion) (e) ventral view (digital restoration) (f) anterior view (digitalrestoration) with the location of longitudinal the microCT digitalsection (g) left lateral view (digital restoration) with the locationsof the microCT digital sections (h) digital longitudinal section (ver-tical plane) (i) digital longitudinal section (horizontal plane) in dor-sal view and (k) digital transverse section in posterior view Bro-ken lines show the border between atlas and trunk vertebra Arrowsshow bone enlargements Abbreviations AT ndash atlas TV ndash trunkvertebra

CT data show that the more posterior vertebra is slightly dis-placed in relation to the more anterior vertebrae (Fig 9l) sothe trauma took place before the forming of pathological en-largements The internal structure of enlarged areas is highlyporous and contains large necrotic cavities

The fused two anterior trunk vertebrae ZIN PH 6243(Fig 10) are characterized by shortening of the centra asym-metry of the centra (due to the unequal development of theanterior and posterior portions) and asymmetric arrangementof the transverse processes (widely separated on the left sideand closely spaced on the right side of the fused centra) Thebases of neural arches are fused and a large spinal nerve fora-men is present

Similarly to the specimen ZIN PH 6243 the fused twoposterior trunk vertebrae ZIN PH 7243 (Fig 11) exhibit thevertebral shortening with asymmetry of the centra and asym-metric arrangement of the transverse processes The absenceof any traces of trauma and inflammation of bone suggeststhe congenital origin of the fusion The asymmetric arrange-ment of the transverse processes and vertebral shortening

Figure 8 The fused atlas and the first trunk vertebra ZINPH 1831242 of Eoscapherpeton asiaticum (a) anterior view(b) posterior view (c) dorsal view (d) ventral view (e) left lat-eral view (f) anterior view (digital restoration) with the locationof longitudinal the microCT digital section (g) ventral view (digi-tal restoration) with the location of longitudinal the microCT digi-tal section (h) right lateral view (digital restoration) (i) left lateralview (digital restoration) (k) digital longitudinal section (horizon-tal plane) in dorsal view and (l) digital longitudinal section (verticalplane) Broken lines show the border between atlas and trunk verte-bra Abbreviations AT ndash atlas TV ndash trunk vertebra

could be the result of abnormal segmentation during embryo-genesis

The specimen ZIN PH 5243 consists of three fused ante-rior trunk vertebrae (Fig 12) The vertebrae exhibit the verte-bral shortening and do not display any signs of trauma andorinflammation of bone We suggest the congenital origin ofthe fusion in ZIN PH 5243

The neural arches of two trunk vertebrae ZIN PH 8243(Fig 13) are fused without any trace of suture The more an-terior neural arch is lower than the more posterior one Thereare no pathological enlargements (no signs of periosteal re-active growth) and the fusion of these neural arches is mostlikely of congenital origin

36 Femoral pathologies

The distal fragment of the medium-sized femur ZINPH 9243 (Fig 14andashh) displays the presence of large smoothfocal bone enlargement (ie periosteal elevation) in themetaphyseal region The internal structure of this bone en-largement is porous due to the presence of large cavities ofirregular shape (ie erosion bays) (Fig 14fndashh) The tissue

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 165

Figure 9 The fused two anterior trunk vertebrae ZIN PH 4243 ofEoscapherpeton asiaticum (a) Dorsal view (b) ventral view (c) an-terior view (d) right lateral view (e) left lateral view (f) anteriorview (digital restoration) (g) dorsal view (digital restoration) withthe location of longitudinal the microCT digital section (h) rightlateral view (digital restoration) with the location of longitudinalthe microCT digital section (i) left lateral view (digital restoration)(k) digital longitudinal section (vertical plane) and (l) digital lon-gitudinal section (horizontal plane) in dorsal view Blue lines showthe original median line of the centra prior to the trauma Arrowsshow bone enlargements Abbreviations NC ndash necrotic cavity

of the enlargement is entirely composed of a parallel-fiberedprimary bone of periosteal origin The parallel-fibered pri-mary bone in the outer part of the enlargement is more or-ganized than in the inner porous part and is similar to thatof the normal cortical bone of this femur (Fig 14h) No evi-dence of fracture was revealed by CT scanning and histolog-ical analysis Similar bone enlargement in modern reptileswas identified as hematoma (Rothschild et al 2010 2012)We also interpret the femoral pathology on ZIN PH 9243 ashematoma that could have been caused by trauma (traumaticorigin)

All other femoral specimens in the sample (two distal frag-ments ZIN PH 10-11243 of medium-sized femora and onedistal fragment ZIN PH 12243 of large femora Table 1Figs 14indasho 15) display the signs of healed fractures thatare traceable in CT images Externally all the specimensdemonstrate displacements of the distal end of the bone andhave large smooth focal bone enlargements (ie calluses) inthe metaphyseal region that were formed during the healingof traumatic fractures The internal structure of the callusesis porous and contains numerous longitudinal and obliquely

Figure 10 The fused two anterior trunk vertebrae ZIN PH 6243of Eoscapherpeton asiaticum (a) Dorsal view (b) ventral view(c) anterior view (d) left lateral view (e) right lateral view (f) ven-tral view (digital restoration) with the location of longitudinalthe microCT digital sections (g) anterior view (digital restora-tion) with the location of longitudinal the microCT digital section(h) left lateral view (digital restoration) (i) right lateral view (digitalrestoration) (kndashl) digital longitudinal sections (vertical plane) and(m) digital longitudinal section (horizontal plane) in ventral viewBroken lines show the border between trunk vertebrae Abbrevia-tions NA ndash neural arch TP1 ndash transverse process of more anteriorvertebra TP2 ndash transverse process of more posterior vertebra

oriented vascular canals that are typical for these structures(Fig 15nndashp)

4 Discussion

41 General discussion

Eoscapherpeton asiaticum demonstrates a wide spectrum ofosseous anomaliespathologies resulting from trauma pos-sible infection due to trauma and congenital disorders Trau-matic origins of pathologies are suggested for the dentaryZIN PH 2243 (the presence of the fracture and the callus)and for femoral fragments ZIN PH 9-12243 (hematoma andhealed fractures with the calluses on the distal portions of thebones) (Table 1) The possible traumatic-infectious patholo-gies are associated with bone enlargements formed duringreactive bone growth andor the presence of necrotic cavitiesare suggested for the prearticular ZIN PH 1243 the anteriortrunk vertebra ZIN PH 13243 the fused atlas and the firsttrunk vertebrae ZIN PH 1832242 and the fused two ante-

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166 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 11 The fused two posterior trunk vertebrae ZIN PH 7243 ofEoscapherpeton asiaticum (a) Ventral view (b) dorsal view (c) an-terior view (d) right lateral view (e) left lateral view (f) ventralview (digital restoration) with the location of the microCT digitalsections (g) anterior view (digital restoration) with the location oflongitudinal the microCT digital section (h) right lateral view (dig-ital restoration) (i) left lateral view (digital restoration) (k) digitallongitudinal section (vertical plane) (l) digital transverse section inanterior view and (m) digital longitudinal section (horizontal plane)in dorsal view Broken lines show the border between trunk verte-brae Abbreviations NA ndash neural arch TP1 ndash transverse process ofmore anterior vertebra TP2 ndash transverse process of more posteriorvertebra

rior trunk vertebrae ZIN PH 4243 (Table 1) The congenitalpathologies of E asiaticum are represented by the hemiver-tebra (specimen ZIN PH 182924 see discussion below) andby fusion of two (fused atlas and anterior trunk vertebra ZINPH 1831242 fused anterior trunk vertebrae ZIN PH 6243fused posterior trunk vertebrae ZIN PH 7243 and for fusedneural arches 8243) or three (fused anterior trunk vertebraeZIN PH 5243) vertebrae (Table 1) The total number of thesevertebral congenital pathologies in E asiaticum is very low(n= 5) in comparison with thousands of collected vertebraeof this salamander (only collection of atlases ZIN PH 242contains 1909 specimens while there are only two speci-mens of congenital pathologies with atlas ndash ZIN PH 182924and ZIN PH 1831242 This frequency of vertebral congen-ital pathologies in E asiaticum (for congenital pathologieswith atlas lt 01 ) is in accordance with the previous estima-tions for PaleozoicndashMesozoic temnospondyls and Holocenefrogs (Rothschild and Laub 2013 Witzmann et al 2013)

Figure 12 The fused three anterior trunk vertebrae ZIN PH 5243of Eoscapherpeton asiaticum (a) Ventral view (b) dorsal view(c) left lateral view (d) right lateral view (e) ventral view (digi-tal restoration) with the location of longitudinal the microCT digitalsection (f) left lateral view (digital restoration) with the locationof longitudinal the microCT digital section (g) right lateral view(digital restoration) (h) digital longitudinal section (vertical plane)and (i) digital longitudinal section (horizontal plane) in dorsal viewBroken lines show the border between trunk vertebrae

42 Hemivertebra in Eoscapherpeton asiaticum

A hemivertebra is a congenital malformation in which oneor more lateral halves of vertebrae are intercalated betweenneighboring vertebrae Formation of hemivertebrae is a resultof abnormal segmentation during embryogenesis (ie fail-ure of somitogenesis before chondrification and ossificationof the vertebral anlagen eg Witzmann et al 2008 2013Burnham et al 2013) In amniots single vertebrae are de-rived from the sclerotome of two adjacent somites and thisprocess is widely known as resegmentation (Piekarski andOlsson 2014 and references therein) Resegmentation havebeen proposed to be present in all modern lissamphibians butembryological and molecular evidence for this process wasprovided only for one salamander species ndash ambystomatidAmbystoma mexicanum (Mexican axolotl) (Piekarski andOlsson 2014) The development of the described hemiver-tebra in more basal cryptobranchid salamander Eoscapher-peton asiaticum can also be explained by a failure of reseg-mentation If this is correct then it is significant for the fol-

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 167

Figure 13 The fused neural arches of two trunk vertebrae ZINPH 8243 of Eoscapherpeton asiaticum (a) Dorsal view (b) rightlateral view (c) left lateral view (d) left lateral view (digital restora-tion) (e) right lateral view (digital restoration) (f) dorsal view (digi-tal restoration) with the location of longitudinal the microCT digitalsection and (g) digital longitudinal section (vertical plane)

lowing reasons (1) it is the oldest occurrence of resegmen-tation in salamanders and (2) it is evidence that resegmen-tation is a common process for salamanders (including rela-tively basal cryptobranchids and advanced ambystomatids)

A hemivertebra in E asiaticum was found in a relativelylarge (according to the large size of the atlas that was co-ossified with hemivertebra) and presumably adult individual(Fig 6) The presence of a hemivertebra in a large individualsuggests that this pathology did not cause its death

The hemivertebrae have been reported earlier for somePaleozoic and Mesozoic temnospondyls (Witzmann et al2013) The finding of hemivertebra in the Late Cretaceouscryptobranchid E asiaticum is the oldest (and the first de-scribed) occurrence of this pathology among modern groupsof amphibians (ie lissamphibians salamanders frogs andcaecilians)

43 Femoral pathologies in Eoscapherpeton possibleinsight into behavior

The traumatic origin of pathologies on the distal parts of thefemora (Figs 14ndash15) could be evidence of frequent occur-rence of hind limb injuries in Eoscapherpeton asiaticum Inmodern cryptobranchids such as Cryptobranchus limb in-juries are the most common physical abnormalities in wildpopulations (Miller and Miller 2005) The proposed maincause of limb injuries in Cryptobranchus is intraspecific ag-

Figure 14 The distal parts of the femora ZIN PH 9243 (andashh) andZIN PH 10243 (indasho) of Eoscapherpeton asiaticum Specimen ZINPH 9243 (a) ventral view (b) dorsal view (c) ventral view (dig-ital restoration) (d) dorsal view (digital restoration) (e) anterioror posterior view (digital restoration) with the locations of trans-verse histological sections and longitudinal the microCT digital sec-tion (f) digital longitudinal section and (gndashh) histological trans-verse section The arrow shows the border between cortical boneand pathological tissue Specimen ZIN PH 10243 (i) ventral view(k) dorsal view (l) ventral view (digital restoration) with the loca-tions of the microCT digital sections (m) anterior or posterior view(digital restoration) (n) digital longitudinal section and (o) digitaltransverse section Blue lines show the original bone position priorto the break Abbreviations BE ndash bone enlargement C ndash callus CBndash cortical bone

gression during the reproductive season (eg defending ofnesting sites) because fresh injuries were found only dur-ing the reproductive season (namely in August and Septem-ber) (Hiler et al 2005 Miller and Miller 2005) Takinginto account the phylogenetic position of Eoscapherpetonas a member of the cryptobranchid clade and their aquaticlifestyle (see Skutschas 2009 2013) the reason for the fre-quent occurrence of hind limb injuries in E asiaticum couldbe the same (intraspecific aggression during reproductiveseason) and this behavioral element could have appearedearly in the evolution of cryptobranchids ie at least in theLate Cretaceous

wwwfoss-recnet211592018 Foss Rec 21 159ndash169 2018

168 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 15 The distal parts of the femora ZIN PH 11243 (andashf) andZIN PH 12243 (gndashp) of Eoscapherpeton asiaticum Specimen ZINPH 11243 (a) ventral view (b) dorsal view (c) dorsal view (dig-ital restoration) with the locations of the microCT digital sections(d) anterior or posterior view (digital restoration) (e) digital lon-gitudinal section and (f) digital transverse section Specimen ZINPH 12243 (g) ventral view (h) dorsal view (i) ventral view (dig-ital restoration) (k) dorsal view (digital restoration) (l) anterior orposterior view (digital restoration) with the locations of transversehistological sections and longitudinal the microCT digital section(m) digital longitudinal section and (nndashp) transverse histologicalsections Blue lines show the original bone position prior to thebreak Abbreviations C ndash callus

5 Conclusions

The study revealed diverse osseous anomaliespathologies inEoscapherpeton asiaticum which were interpreted as trau-matic possible traumatic-infectious and congenital in originpertaining to bones of the lower jaw vertebrae and femoraThe hemivertebra and fused vertebrae (without any traces oftrauma and inflammation) document the earliest occurrenceof vertebral congenital pathologies in crown-group salaman-ders The frequency of vertebral congenital pathologies in Easiaticum is low and in accordance with the previous estima-tions for temnospondyls and frogs The numerous traumaticpathologies of femora (hematoma and healed fractures) sug-gest the survival of accidents and possibly the presence of in-

traspecific aggression during the reproductive season in thissalamander (like in modern cryptobranchids)

Data availability Micro-CT data are archived at the Department ofVertebrate Zoology of the Saint Petersburg State University (SaintPetersburg Russia) and can be accessed through the present authorsfor the purpose of scientific study

Author contributions PS designed the project PS and VK per-formed the research VK and IK prepared 3-D reconstructions VKand EB prepared figures and PS wrote the manuscript All authorscommented on the manuscript

Competing interests The authors declare that they have no conflictof interest

Acknowledgements We are grateful to the staff of the SaintPetersburg State University Research Centre for X-ray DiffractionStudies (Saint Petersburg Russia) for their help with using theLeica 2500P microscope and for CT scanning of specimens Theauthors thank Judith Pardo-Peacuterez and Nadia Froumlbisch for providinghelpful comments that improved the quality of the manuscript Thisstudy was fulfilled under support of the Russian Scientific Fundproject 14-14-00015

Edited by Constanze BickelmannReviewed by Nadia Froumlbisch and Judith Pardo Perez

References

Burnham D A Rothschild B M Babiarz J P and Martin LD Hemivertebrae as pathology and as a window to behavior inthe fossil record Palarchrsquos Journal of Vertebrate Palaeontology10 1ndash6 2013

Francillon-Vieillot H de Buffreacutenil V Castanet J Geraudie JMeunier F Sire J Y Zylenberberg L and de Ricqles A DMicrostructure and mineralization of vertebrate skeletal tissuesin Skeletal biomineralization Patterns Processes and Evolu-tionary Trends Vol 1 edited by Carter J G Van NostrandReinhold New York 471ndash530 1990

Garciacutea R A Cerda I A Heller M Rothschild B M and Zur-riaguz V The first evidence of osteomyelitis in a sauropod di-nosaur Lethaia 50 227ndash236 httpsdoiorg101111let121892016

Hanna R R Multiple injury and infection in a sub adult thero-pod dinosaur Allosaurus fragilis with comparisons to allosaurpathology in Cleveland-Lloyd Dinosaur Quarry Collection JVertebr Paleontol 22 76ndash90 httpsdoiorg1016710272-4634(2002)022[0076MIAIIA]20CO2 2002

Hiler W R Wheeler B A and Trauth S E Abnormalities inthe Ozark Hellbender (Cryptobranchus alleganiensis bishopi) inArkansas A Comparison between Two Rivers with a Historical

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

162 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 3 The atlantal centrum ZIN PH 3243 of Eoscapherpetonasiaticum (a) Ventral view (b) dorsal view (c) right lateral view(d) ventral view (digital restoration) (e) right lateral view (digi-tal restoration) with the locations of the microCT digital sections(f) digital transverse section and (g) digital longitudinal section(horizontal plane) in ventral view Abbreviations TP ndash transverseprocess

pathology) (Rothschild and Martin 2006 Pardo-Peacuterez et al2017)

32 Atlantal pathologies

Two atlantal centra in the sample show the osseous anoma-lies the small centrum ZIN PH 3243 and one medium-sizedcentrum ZIN PH 1830242 (Table 1) The small atlas ZINPH 3243 (Fig 3) is characterized by an enlarged transverseprocess (ie rib bearer) on the right lateral surface The in-ner structure of the enlarged transverse process demonstratesthe presence of a large cavity (ie channel) that in life waslikely filled by cartilage (Fig 3fndashg) There are no differencesin porosity in the different parts of the cortical regions andno clear evidence of fractures or inflammation process Theorigin of the pathology is unknown and it could be traumatic-infectious developmental or neoplasm (Rothschild and Mar-tin 2006 Waldron 2009)

The centrum of the larger atlas ZIN PH 1830242 (Fig 4)is asymmetric (in anterior and posterior views) and bearswell-developed transverse processes on the both lateral sur-faces The inner structure of the enlarged transverse pro-cesses is similar to that in ZIN PH 3243 (the presence ofthe large cavity Fig 4g) Like in ZIN PH 3243 the originof the pathology is unclear

Figure 4 The atlantal centrum ZIN PH 1830242 of Eoscapher-peton asiaticum (a) Anterior view (b) posterior view (c) anteriorview (digital restoration) (d) posterior view (digital restoration)(e) right lateral view (digital restoration) with the locations of themicroCT digital sections (f) left lateral view (digital restoration)(g) digital longitudinal section (horizontal plane) in dorsal view and(h) digital transverse section in posterior view Abbreviations TP ndashtransverse process

33 Abnormal trunk vertebra

The large anterior trunk vertebra ZIN PH 13243 (Fig 5) ex-hibits an abnormal structure of the transverse processes onboth lateral surfaces of the centrum right transverse pro-cesses are slightly enlarged while left transverse processesare significantly enlarged and elongated (probably due to thefusion with a rib) The internal structure of the enlarged trans-verse processes is highly porous and contains large irregularcavities Reactive bone growth is likely the result of compli-cation of healing after trauma and the subsequent enlarge-ment of transverse processes could be caused by osteomyeli-tis (traumatic-infectious origin of pathology) (Rothschild andMartin 2006 Waldron 2009 Garciacutea et al 2016)

34 Hemivertebra

The specimen ZIN PH 1829242 is an asymmetrical (in dor-sal and ventral view) fragmentary large atlas that is co-ossified with a hemivertebra (Fig 6) The posterior cotyle

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 163

Figure 5 The anterior trunk vertebra ZIN PH 13243 of Eoscapher-peton asiaticum (a) Anterior view (b) posterior view (c) dor-sal view (d) left lateral view (e) anterior view (digital restora-tion) (f) posterior view (digital restoration) (g) dorsal view (digitalrestoration) (h) left lateral view (digital restoration) with the loca-tions of the microCT digital sections (i) digital transverse section inanterior view and (k) digital longitudinal section (horizontal plane)in dorsal view Abbreviations TP ndash transverse process R ndash rib

of the atlantal centrum is oriented posterolaterally Thehemivertebral centrum is narrow wedge-like and located atthe posterior left portion of the specimen The hemivertebralcentrum bears a large transverse process The bases of neu-ral arches of the atlas and hemivertebra are not fused Exter-nally the boundary between atlantal and the hemivertebralcentra is clearly traceable as a groove on the lateral surfaceof the hypapophysis Internally the boundary is not clearlyevident but some differences in the densities of atlantal andthe hemivertebral centra are visible in some virtual slices(Fig 6i) The inner structure of the enlarged transverse pro-cess demonstrates the presence of a large cavity (ie chan-nel)

35 Fusion of vertebrae

The sample of fused vertebrae (all vertebrae are relativelylarge in size) includes two specimens of fused atlas andthe first trunk vertebrae (ZIN PH 1831-1832242) twospecimens of the fused two anterior trunk vertebrae (ZINPH 4243 6243) one specimen of the fused three anteriortrunk vertebrae (ZIN PH 5243) one specimen of the fusedtwo posterior trunk vertebrae (ZIN PH 7243) and one spec-

Figure 6 The co-ossified atlas and hemivertebra ZIN PH 1829242of Eoscapherpeton asiaticum (a) Anterior view (b) posterior view(c) ventral view (d) dorsal view (e) dorsal view (digital restora-tion) (f) ventral view (digital restoration) (g) left lateral view (dig-ital restoration) with the locations of the microCT digital sectionsand (hndashi) digital longitudinal sections (horizontal plane) in ventralview Broken lines and an arrow show the border between atlas andhemivertebra Abbreviations HV ndash hemivertebra PC ndash posterioratlantal cotyle

imen of the fused neural arches of two trunk vertebrae (ZINPH 8243) (total number of specimens ndash 7 Table 1)

The specimen ZIN PH 1832242 (Fig 7) exhibits en-largements on the lateral surfaces of the centra and thehypapophyses along the suture between the atlas and thefirst trunk vertebrae that were formed during reactive bonegrowth The internal structure of these enlarged areasis highly porous The enlargements could be caused byosteomyelitis after trauma (traumatic-infectious nature ofpathology)

The specimen ZIN PH 1831242 (Fig 8) displays the fu-sion of the atlas and the first trunk vertebrae without notablepathological enlargements on the lateral surfaces of the cen-tra and the hypapophyses (only low ridge is present along thesuture between atlas and the first trunk vertebrae) This sug-gests the absence of the periosteal reactive bone growth Bothvertebrae are shortened in comparison with normal vertebraeor that in the specimen (ZIN PH 1832242) The absence ofany traces of trauma (eg fracture displacement callus) andinflammation of bone (eg enlargements formed during re-active bone growth necrotic cavities) suggests the congenitalorigin of the fusion

The fused two anterior trunk vertebrae ZIN PH 4243(Fig 9) are characterized by the enlargements on the lateralsurfaces of the centra and the hypapophyses along the suturebetween vertebrae and by deformation of the hypapophyses

wwwfoss-recnet211592018 Foss Rec 21 159ndash169 2018

164 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 7 The fused atlas and the first trunk vertebra ZINPH 1832242 of Eoscapherpeton asiaticum (a) Dorsal view(b) ventral view (c) anterior view (d) dorsal view (digital restora-tion) (e) ventral view (digital restoration) (f) anterior view (digitalrestoration) with the location of longitudinal the microCT digitalsection (g) left lateral view (digital restoration) with the locationsof the microCT digital sections (h) digital longitudinal section (ver-tical plane) (i) digital longitudinal section (horizontal plane) in dor-sal view and (k) digital transverse section in posterior view Bro-ken lines show the border between atlas and trunk vertebra Arrowsshow bone enlargements Abbreviations AT ndash atlas TV ndash trunkvertebra

CT data show that the more posterior vertebra is slightly dis-placed in relation to the more anterior vertebrae (Fig 9l) sothe trauma took place before the forming of pathological en-largements The internal structure of enlarged areas is highlyporous and contains large necrotic cavities

The fused two anterior trunk vertebrae ZIN PH 6243(Fig 10) are characterized by shortening of the centra asym-metry of the centra (due to the unequal development of theanterior and posterior portions) and asymmetric arrangementof the transverse processes (widely separated on the left sideand closely spaced on the right side of the fused centra) Thebases of neural arches are fused and a large spinal nerve fora-men is present

Similarly to the specimen ZIN PH 6243 the fused twoposterior trunk vertebrae ZIN PH 7243 (Fig 11) exhibit thevertebral shortening with asymmetry of the centra and asym-metric arrangement of the transverse processes The absenceof any traces of trauma and inflammation of bone suggeststhe congenital origin of the fusion The asymmetric arrange-ment of the transverse processes and vertebral shortening

Figure 8 The fused atlas and the first trunk vertebra ZINPH 1831242 of Eoscapherpeton asiaticum (a) anterior view(b) posterior view (c) dorsal view (d) ventral view (e) left lat-eral view (f) anterior view (digital restoration) with the locationof longitudinal the microCT digital section (g) ventral view (digi-tal restoration) with the location of longitudinal the microCT digi-tal section (h) right lateral view (digital restoration) (i) left lateralview (digital restoration) (k) digital longitudinal section (horizon-tal plane) in dorsal view and (l) digital longitudinal section (verticalplane) Broken lines show the border between atlas and trunk verte-bra Abbreviations AT ndash atlas TV ndash trunk vertebra

could be the result of abnormal segmentation during embryo-genesis

The specimen ZIN PH 5243 consists of three fused ante-rior trunk vertebrae (Fig 12) The vertebrae exhibit the verte-bral shortening and do not display any signs of trauma andorinflammation of bone We suggest the congenital origin ofthe fusion in ZIN PH 5243

The neural arches of two trunk vertebrae ZIN PH 8243(Fig 13) are fused without any trace of suture The more an-terior neural arch is lower than the more posterior one Thereare no pathological enlargements (no signs of periosteal re-active growth) and the fusion of these neural arches is mostlikely of congenital origin

36 Femoral pathologies

The distal fragment of the medium-sized femur ZINPH 9243 (Fig 14andashh) displays the presence of large smoothfocal bone enlargement (ie periosteal elevation) in themetaphyseal region The internal structure of this bone en-largement is porous due to the presence of large cavities ofirregular shape (ie erosion bays) (Fig 14fndashh) The tissue

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 165

Figure 9 The fused two anterior trunk vertebrae ZIN PH 4243 ofEoscapherpeton asiaticum (a) Dorsal view (b) ventral view (c) an-terior view (d) right lateral view (e) left lateral view (f) anteriorview (digital restoration) (g) dorsal view (digital restoration) withthe location of longitudinal the microCT digital section (h) rightlateral view (digital restoration) with the location of longitudinalthe microCT digital section (i) left lateral view (digital restoration)(k) digital longitudinal section (vertical plane) and (l) digital lon-gitudinal section (horizontal plane) in dorsal view Blue lines showthe original median line of the centra prior to the trauma Arrowsshow bone enlargements Abbreviations NC ndash necrotic cavity

of the enlargement is entirely composed of a parallel-fiberedprimary bone of periosteal origin The parallel-fibered pri-mary bone in the outer part of the enlargement is more or-ganized than in the inner porous part and is similar to thatof the normal cortical bone of this femur (Fig 14h) No evi-dence of fracture was revealed by CT scanning and histolog-ical analysis Similar bone enlargement in modern reptileswas identified as hematoma (Rothschild et al 2010 2012)We also interpret the femoral pathology on ZIN PH 9243 ashematoma that could have been caused by trauma (traumaticorigin)

All other femoral specimens in the sample (two distal frag-ments ZIN PH 10-11243 of medium-sized femora and onedistal fragment ZIN PH 12243 of large femora Table 1Figs 14indasho 15) display the signs of healed fractures thatare traceable in CT images Externally all the specimensdemonstrate displacements of the distal end of the bone andhave large smooth focal bone enlargements (ie calluses) inthe metaphyseal region that were formed during the healingof traumatic fractures The internal structure of the callusesis porous and contains numerous longitudinal and obliquely

Figure 10 The fused two anterior trunk vertebrae ZIN PH 6243of Eoscapherpeton asiaticum (a) Dorsal view (b) ventral view(c) anterior view (d) left lateral view (e) right lateral view (f) ven-tral view (digital restoration) with the location of longitudinalthe microCT digital sections (g) anterior view (digital restora-tion) with the location of longitudinal the microCT digital section(h) left lateral view (digital restoration) (i) right lateral view (digitalrestoration) (kndashl) digital longitudinal sections (vertical plane) and(m) digital longitudinal section (horizontal plane) in ventral viewBroken lines show the border between trunk vertebrae Abbrevia-tions NA ndash neural arch TP1 ndash transverse process of more anteriorvertebra TP2 ndash transverse process of more posterior vertebra

oriented vascular canals that are typical for these structures(Fig 15nndashp)

4 Discussion

41 General discussion

Eoscapherpeton asiaticum demonstrates a wide spectrum ofosseous anomaliespathologies resulting from trauma pos-sible infection due to trauma and congenital disorders Trau-matic origins of pathologies are suggested for the dentaryZIN PH 2243 (the presence of the fracture and the callus)and for femoral fragments ZIN PH 9-12243 (hematoma andhealed fractures with the calluses on the distal portions of thebones) (Table 1) The possible traumatic-infectious patholo-gies are associated with bone enlargements formed duringreactive bone growth andor the presence of necrotic cavitiesare suggested for the prearticular ZIN PH 1243 the anteriortrunk vertebra ZIN PH 13243 the fused atlas and the firsttrunk vertebrae ZIN PH 1832242 and the fused two ante-

wwwfoss-recnet211592018 Foss Rec 21 159ndash169 2018

166 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 11 The fused two posterior trunk vertebrae ZIN PH 7243 ofEoscapherpeton asiaticum (a) Ventral view (b) dorsal view (c) an-terior view (d) right lateral view (e) left lateral view (f) ventralview (digital restoration) with the location of the microCT digitalsections (g) anterior view (digital restoration) with the location oflongitudinal the microCT digital section (h) right lateral view (dig-ital restoration) (i) left lateral view (digital restoration) (k) digitallongitudinal section (vertical plane) (l) digital transverse section inanterior view and (m) digital longitudinal section (horizontal plane)in dorsal view Broken lines show the border between trunk verte-brae Abbreviations NA ndash neural arch TP1 ndash transverse process ofmore anterior vertebra TP2 ndash transverse process of more posteriorvertebra

rior trunk vertebrae ZIN PH 4243 (Table 1) The congenitalpathologies of E asiaticum are represented by the hemiver-tebra (specimen ZIN PH 182924 see discussion below) andby fusion of two (fused atlas and anterior trunk vertebra ZINPH 1831242 fused anterior trunk vertebrae ZIN PH 6243fused posterior trunk vertebrae ZIN PH 7243 and for fusedneural arches 8243) or three (fused anterior trunk vertebraeZIN PH 5243) vertebrae (Table 1) The total number of thesevertebral congenital pathologies in E asiaticum is very low(n= 5) in comparison with thousands of collected vertebraeof this salamander (only collection of atlases ZIN PH 242contains 1909 specimens while there are only two speci-mens of congenital pathologies with atlas ndash ZIN PH 182924and ZIN PH 1831242 This frequency of vertebral congen-ital pathologies in E asiaticum (for congenital pathologieswith atlas lt 01 ) is in accordance with the previous estima-tions for PaleozoicndashMesozoic temnospondyls and Holocenefrogs (Rothschild and Laub 2013 Witzmann et al 2013)

Figure 12 The fused three anterior trunk vertebrae ZIN PH 5243of Eoscapherpeton asiaticum (a) Ventral view (b) dorsal view(c) left lateral view (d) right lateral view (e) ventral view (digi-tal restoration) with the location of longitudinal the microCT digitalsection (f) left lateral view (digital restoration) with the locationof longitudinal the microCT digital section (g) right lateral view(digital restoration) (h) digital longitudinal section (vertical plane)and (i) digital longitudinal section (horizontal plane) in dorsal viewBroken lines show the border between trunk vertebrae

42 Hemivertebra in Eoscapherpeton asiaticum

A hemivertebra is a congenital malformation in which oneor more lateral halves of vertebrae are intercalated betweenneighboring vertebrae Formation of hemivertebrae is a resultof abnormal segmentation during embryogenesis (ie fail-ure of somitogenesis before chondrification and ossificationof the vertebral anlagen eg Witzmann et al 2008 2013Burnham et al 2013) In amniots single vertebrae are de-rived from the sclerotome of two adjacent somites and thisprocess is widely known as resegmentation (Piekarski andOlsson 2014 and references therein) Resegmentation havebeen proposed to be present in all modern lissamphibians butembryological and molecular evidence for this process wasprovided only for one salamander species ndash ambystomatidAmbystoma mexicanum (Mexican axolotl) (Piekarski andOlsson 2014) The development of the described hemiver-tebra in more basal cryptobranchid salamander Eoscapher-peton asiaticum can also be explained by a failure of reseg-mentation If this is correct then it is significant for the fol-

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 167

Figure 13 The fused neural arches of two trunk vertebrae ZINPH 8243 of Eoscapherpeton asiaticum (a) Dorsal view (b) rightlateral view (c) left lateral view (d) left lateral view (digital restora-tion) (e) right lateral view (digital restoration) (f) dorsal view (digi-tal restoration) with the location of longitudinal the microCT digitalsection and (g) digital longitudinal section (vertical plane)

lowing reasons (1) it is the oldest occurrence of resegmen-tation in salamanders and (2) it is evidence that resegmen-tation is a common process for salamanders (including rela-tively basal cryptobranchids and advanced ambystomatids)

A hemivertebra in E asiaticum was found in a relativelylarge (according to the large size of the atlas that was co-ossified with hemivertebra) and presumably adult individual(Fig 6) The presence of a hemivertebra in a large individualsuggests that this pathology did not cause its death

The hemivertebrae have been reported earlier for somePaleozoic and Mesozoic temnospondyls (Witzmann et al2013) The finding of hemivertebra in the Late Cretaceouscryptobranchid E asiaticum is the oldest (and the first de-scribed) occurrence of this pathology among modern groupsof amphibians (ie lissamphibians salamanders frogs andcaecilians)

43 Femoral pathologies in Eoscapherpeton possibleinsight into behavior

The traumatic origin of pathologies on the distal parts of thefemora (Figs 14ndash15) could be evidence of frequent occur-rence of hind limb injuries in Eoscapherpeton asiaticum Inmodern cryptobranchids such as Cryptobranchus limb in-juries are the most common physical abnormalities in wildpopulations (Miller and Miller 2005) The proposed maincause of limb injuries in Cryptobranchus is intraspecific ag-

Figure 14 The distal parts of the femora ZIN PH 9243 (andashh) andZIN PH 10243 (indasho) of Eoscapherpeton asiaticum Specimen ZINPH 9243 (a) ventral view (b) dorsal view (c) ventral view (dig-ital restoration) (d) dorsal view (digital restoration) (e) anterioror posterior view (digital restoration) with the locations of trans-verse histological sections and longitudinal the microCT digital sec-tion (f) digital longitudinal section and (gndashh) histological trans-verse section The arrow shows the border between cortical boneand pathological tissue Specimen ZIN PH 10243 (i) ventral view(k) dorsal view (l) ventral view (digital restoration) with the loca-tions of the microCT digital sections (m) anterior or posterior view(digital restoration) (n) digital longitudinal section and (o) digitaltransverse section Blue lines show the original bone position priorto the break Abbreviations BE ndash bone enlargement C ndash callus CBndash cortical bone

gression during the reproductive season (eg defending ofnesting sites) because fresh injuries were found only dur-ing the reproductive season (namely in August and Septem-ber) (Hiler et al 2005 Miller and Miller 2005) Takinginto account the phylogenetic position of Eoscapherpetonas a member of the cryptobranchid clade and their aquaticlifestyle (see Skutschas 2009 2013) the reason for the fre-quent occurrence of hind limb injuries in E asiaticum couldbe the same (intraspecific aggression during reproductiveseason) and this behavioral element could have appearedearly in the evolution of cryptobranchids ie at least in theLate Cretaceous

wwwfoss-recnet211592018 Foss Rec 21 159ndash169 2018

168 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 15 The distal parts of the femora ZIN PH 11243 (andashf) andZIN PH 12243 (gndashp) of Eoscapherpeton asiaticum Specimen ZINPH 11243 (a) ventral view (b) dorsal view (c) dorsal view (dig-ital restoration) with the locations of the microCT digital sections(d) anterior or posterior view (digital restoration) (e) digital lon-gitudinal section and (f) digital transverse section Specimen ZINPH 12243 (g) ventral view (h) dorsal view (i) ventral view (dig-ital restoration) (k) dorsal view (digital restoration) (l) anterior orposterior view (digital restoration) with the locations of transversehistological sections and longitudinal the microCT digital section(m) digital longitudinal section and (nndashp) transverse histologicalsections Blue lines show the original bone position prior to thebreak Abbreviations C ndash callus

5 Conclusions

The study revealed diverse osseous anomaliespathologies inEoscapherpeton asiaticum which were interpreted as trau-matic possible traumatic-infectious and congenital in originpertaining to bones of the lower jaw vertebrae and femoraThe hemivertebra and fused vertebrae (without any traces oftrauma and inflammation) document the earliest occurrenceof vertebral congenital pathologies in crown-group salaman-ders The frequency of vertebral congenital pathologies in Easiaticum is low and in accordance with the previous estima-tions for temnospondyls and frogs The numerous traumaticpathologies of femora (hematoma and healed fractures) sug-gest the survival of accidents and possibly the presence of in-

traspecific aggression during the reproductive season in thissalamander (like in modern cryptobranchids)

Data availability Micro-CT data are archived at the Department ofVertebrate Zoology of the Saint Petersburg State University (SaintPetersburg Russia) and can be accessed through the present authorsfor the purpose of scientific study

Author contributions PS designed the project PS and VK per-formed the research VK and IK prepared 3-D reconstructions VKand EB prepared figures and PS wrote the manuscript All authorscommented on the manuscript

Competing interests The authors declare that they have no conflictof interest

Acknowledgements We are grateful to the staff of the SaintPetersburg State University Research Centre for X-ray DiffractionStudies (Saint Petersburg Russia) for their help with using theLeica 2500P microscope and for CT scanning of specimens Theauthors thank Judith Pardo-Peacuterez and Nadia Froumlbisch for providinghelpful comments that improved the quality of the manuscript Thisstudy was fulfilled under support of the Russian Scientific Fundproject 14-14-00015

Edited by Constanze BickelmannReviewed by Nadia Froumlbisch and Judith Pardo Perez

References

Burnham D A Rothschild B M Babiarz J P and Martin LD Hemivertebrae as pathology and as a window to behavior inthe fossil record Palarchrsquos Journal of Vertebrate Palaeontology10 1ndash6 2013

Francillon-Vieillot H de Buffreacutenil V Castanet J Geraudie JMeunier F Sire J Y Zylenberberg L and de Ricqles A DMicrostructure and mineralization of vertebrate skeletal tissuesin Skeletal biomineralization Patterns Processes and Evolu-tionary Trends Vol 1 edited by Carter J G Van NostrandReinhold New York 471ndash530 1990

Garciacutea R A Cerda I A Heller M Rothschild B M and Zur-riaguz V The first evidence of osteomyelitis in a sauropod di-nosaur Lethaia 50 227ndash236 httpsdoiorg101111let121892016

Hanna R R Multiple injury and infection in a sub adult thero-pod dinosaur Allosaurus fragilis with comparisons to allosaurpathology in Cleveland-Lloyd Dinosaur Quarry Collection JVertebr Paleontol 22 76ndash90 httpsdoiorg1016710272-4634(2002)022[0076MIAIIA]20CO2 2002

Hiler W R Wheeler B A and Trauth S E Abnormalities inthe Ozark Hellbender (Cryptobranchus alleganiensis bishopi) inArkansas A Comparison between Two Rivers with a Historical

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 163

Figure 5 The anterior trunk vertebra ZIN PH 13243 of Eoscapher-peton asiaticum (a) Anterior view (b) posterior view (c) dor-sal view (d) left lateral view (e) anterior view (digital restora-tion) (f) posterior view (digital restoration) (g) dorsal view (digitalrestoration) (h) left lateral view (digital restoration) with the loca-tions of the microCT digital sections (i) digital transverse section inanterior view and (k) digital longitudinal section (horizontal plane)in dorsal view Abbreviations TP ndash transverse process R ndash rib

of the atlantal centrum is oriented posterolaterally Thehemivertebral centrum is narrow wedge-like and located atthe posterior left portion of the specimen The hemivertebralcentrum bears a large transverse process The bases of neu-ral arches of the atlas and hemivertebra are not fused Exter-nally the boundary between atlantal and the hemivertebralcentra is clearly traceable as a groove on the lateral surfaceof the hypapophysis Internally the boundary is not clearlyevident but some differences in the densities of atlantal andthe hemivertebral centra are visible in some virtual slices(Fig 6i) The inner structure of the enlarged transverse pro-cess demonstrates the presence of a large cavity (ie chan-nel)

35 Fusion of vertebrae

The sample of fused vertebrae (all vertebrae are relativelylarge in size) includes two specimens of fused atlas andthe first trunk vertebrae (ZIN PH 1831-1832242) twospecimens of the fused two anterior trunk vertebrae (ZINPH 4243 6243) one specimen of the fused three anteriortrunk vertebrae (ZIN PH 5243) one specimen of the fusedtwo posterior trunk vertebrae (ZIN PH 7243) and one spec-

Figure 6 The co-ossified atlas and hemivertebra ZIN PH 1829242of Eoscapherpeton asiaticum (a) Anterior view (b) posterior view(c) ventral view (d) dorsal view (e) dorsal view (digital restora-tion) (f) ventral view (digital restoration) (g) left lateral view (dig-ital restoration) with the locations of the microCT digital sectionsand (hndashi) digital longitudinal sections (horizontal plane) in ventralview Broken lines and an arrow show the border between atlas andhemivertebra Abbreviations HV ndash hemivertebra PC ndash posterioratlantal cotyle

imen of the fused neural arches of two trunk vertebrae (ZINPH 8243) (total number of specimens ndash 7 Table 1)

The specimen ZIN PH 1832242 (Fig 7) exhibits en-largements on the lateral surfaces of the centra and thehypapophyses along the suture between the atlas and thefirst trunk vertebrae that were formed during reactive bonegrowth The internal structure of these enlarged areasis highly porous The enlargements could be caused byosteomyelitis after trauma (traumatic-infectious nature ofpathology)

The specimen ZIN PH 1831242 (Fig 8) displays the fu-sion of the atlas and the first trunk vertebrae without notablepathological enlargements on the lateral surfaces of the cen-tra and the hypapophyses (only low ridge is present along thesuture between atlas and the first trunk vertebrae) This sug-gests the absence of the periosteal reactive bone growth Bothvertebrae are shortened in comparison with normal vertebraeor that in the specimen (ZIN PH 1832242) The absence ofany traces of trauma (eg fracture displacement callus) andinflammation of bone (eg enlargements formed during re-active bone growth necrotic cavities) suggests the congenitalorigin of the fusion

The fused two anterior trunk vertebrae ZIN PH 4243(Fig 9) are characterized by the enlargements on the lateralsurfaces of the centra and the hypapophyses along the suturebetween vertebrae and by deformation of the hypapophyses

wwwfoss-recnet211592018 Foss Rec 21 159ndash169 2018

164 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 7 The fused atlas and the first trunk vertebra ZINPH 1832242 of Eoscapherpeton asiaticum (a) Dorsal view(b) ventral view (c) anterior view (d) dorsal view (digital restora-tion) (e) ventral view (digital restoration) (f) anterior view (digitalrestoration) with the location of longitudinal the microCT digitalsection (g) left lateral view (digital restoration) with the locationsof the microCT digital sections (h) digital longitudinal section (ver-tical plane) (i) digital longitudinal section (horizontal plane) in dor-sal view and (k) digital transverse section in posterior view Bro-ken lines show the border between atlas and trunk vertebra Arrowsshow bone enlargements Abbreviations AT ndash atlas TV ndash trunkvertebra

CT data show that the more posterior vertebra is slightly dis-placed in relation to the more anterior vertebrae (Fig 9l) sothe trauma took place before the forming of pathological en-largements The internal structure of enlarged areas is highlyporous and contains large necrotic cavities

The fused two anterior trunk vertebrae ZIN PH 6243(Fig 10) are characterized by shortening of the centra asym-metry of the centra (due to the unequal development of theanterior and posterior portions) and asymmetric arrangementof the transverse processes (widely separated on the left sideand closely spaced on the right side of the fused centra) Thebases of neural arches are fused and a large spinal nerve fora-men is present

Similarly to the specimen ZIN PH 6243 the fused twoposterior trunk vertebrae ZIN PH 7243 (Fig 11) exhibit thevertebral shortening with asymmetry of the centra and asym-metric arrangement of the transverse processes The absenceof any traces of trauma and inflammation of bone suggeststhe congenital origin of the fusion The asymmetric arrange-ment of the transverse processes and vertebral shortening

Figure 8 The fused atlas and the first trunk vertebra ZINPH 1831242 of Eoscapherpeton asiaticum (a) anterior view(b) posterior view (c) dorsal view (d) ventral view (e) left lat-eral view (f) anterior view (digital restoration) with the locationof longitudinal the microCT digital section (g) ventral view (digi-tal restoration) with the location of longitudinal the microCT digi-tal section (h) right lateral view (digital restoration) (i) left lateralview (digital restoration) (k) digital longitudinal section (horizon-tal plane) in dorsal view and (l) digital longitudinal section (verticalplane) Broken lines show the border between atlas and trunk verte-bra Abbreviations AT ndash atlas TV ndash trunk vertebra

could be the result of abnormal segmentation during embryo-genesis

The specimen ZIN PH 5243 consists of three fused ante-rior trunk vertebrae (Fig 12) The vertebrae exhibit the verte-bral shortening and do not display any signs of trauma andorinflammation of bone We suggest the congenital origin ofthe fusion in ZIN PH 5243

The neural arches of two trunk vertebrae ZIN PH 8243(Fig 13) are fused without any trace of suture The more an-terior neural arch is lower than the more posterior one Thereare no pathological enlargements (no signs of periosteal re-active growth) and the fusion of these neural arches is mostlikely of congenital origin

36 Femoral pathologies

The distal fragment of the medium-sized femur ZINPH 9243 (Fig 14andashh) displays the presence of large smoothfocal bone enlargement (ie periosteal elevation) in themetaphyseal region The internal structure of this bone en-largement is porous due to the presence of large cavities ofirregular shape (ie erosion bays) (Fig 14fndashh) The tissue

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 165

Figure 9 The fused two anterior trunk vertebrae ZIN PH 4243 ofEoscapherpeton asiaticum (a) Dorsal view (b) ventral view (c) an-terior view (d) right lateral view (e) left lateral view (f) anteriorview (digital restoration) (g) dorsal view (digital restoration) withthe location of longitudinal the microCT digital section (h) rightlateral view (digital restoration) with the location of longitudinalthe microCT digital section (i) left lateral view (digital restoration)(k) digital longitudinal section (vertical plane) and (l) digital lon-gitudinal section (horizontal plane) in dorsal view Blue lines showthe original median line of the centra prior to the trauma Arrowsshow bone enlargements Abbreviations NC ndash necrotic cavity

of the enlargement is entirely composed of a parallel-fiberedprimary bone of periosteal origin The parallel-fibered pri-mary bone in the outer part of the enlargement is more or-ganized than in the inner porous part and is similar to thatof the normal cortical bone of this femur (Fig 14h) No evi-dence of fracture was revealed by CT scanning and histolog-ical analysis Similar bone enlargement in modern reptileswas identified as hematoma (Rothschild et al 2010 2012)We also interpret the femoral pathology on ZIN PH 9243 ashematoma that could have been caused by trauma (traumaticorigin)

All other femoral specimens in the sample (two distal frag-ments ZIN PH 10-11243 of medium-sized femora and onedistal fragment ZIN PH 12243 of large femora Table 1Figs 14indasho 15) display the signs of healed fractures thatare traceable in CT images Externally all the specimensdemonstrate displacements of the distal end of the bone andhave large smooth focal bone enlargements (ie calluses) inthe metaphyseal region that were formed during the healingof traumatic fractures The internal structure of the callusesis porous and contains numerous longitudinal and obliquely

Figure 10 The fused two anterior trunk vertebrae ZIN PH 6243of Eoscapherpeton asiaticum (a) Dorsal view (b) ventral view(c) anterior view (d) left lateral view (e) right lateral view (f) ven-tral view (digital restoration) with the location of longitudinalthe microCT digital sections (g) anterior view (digital restora-tion) with the location of longitudinal the microCT digital section(h) left lateral view (digital restoration) (i) right lateral view (digitalrestoration) (kndashl) digital longitudinal sections (vertical plane) and(m) digital longitudinal section (horizontal plane) in ventral viewBroken lines show the border between trunk vertebrae Abbrevia-tions NA ndash neural arch TP1 ndash transverse process of more anteriorvertebra TP2 ndash transverse process of more posterior vertebra

oriented vascular canals that are typical for these structures(Fig 15nndashp)

4 Discussion

41 General discussion

Eoscapherpeton asiaticum demonstrates a wide spectrum ofosseous anomaliespathologies resulting from trauma pos-sible infection due to trauma and congenital disorders Trau-matic origins of pathologies are suggested for the dentaryZIN PH 2243 (the presence of the fracture and the callus)and for femoral fragments ZIN PH 9-12243 (hematoma andhealed fractures with the calluses on the distal portions of thebones) (Table 1) The possible traumatic-infectious patholo-gies are associated with bone enlargements formed duringreactive bone growth andor the presence of necrotic cavitiesare suggested for the prearticular ZIN PH 1243 the anteriortrunk vertebra ZIN PH 13243 the fused atlas and the firsttrunk vertebrae ZIN PH 1832242 and the fused two ante-

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166 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 11 The fused two posterior trunk vertebrae ZIN PH 7243 ofEoscapherpeton asiaticum (a) Ventral view (b) dorsal view (c) an-terior view (d) right lateral view (e) left lateral view (f) ventralview (digital restoration) with the location of the microCT digitalsections (g) anterior view (digital restoration) with the location oflongitudinal the microCT digital section (h) right lateral view (dig-ital restoration) (i) left lateral view (digital restoration) (k) digitallongitudinal section (vertical plane) (l) digital transverse section inanterior view and (m) digital longitudinal section (horizontal plane)in dorsal view Broken lines show the border between trunk verte-brae Abbreviations NA ndash neural arch TP1 ndash transverse process ofmore anterior vertebra TP2 ndash transverse process of more posteriorvertebra

rior trunk vertebrae ZIN PH 4243 (Table 1) The congenitalpathologies of E asiaticum are represented by the hemiver-tebra (specimen ZIN PH 182924 see discussion below) andby fusion of two (fused atlas and anterior trunk vertebra ZINPH 1831242 fused anterior trunk vertebrae ZIN PH 6243fused posterior trunk vertebrae ZIN PH 7243 and for fusedneural arches 8243) or three (fused anterior trunk vertebraeZIN PH 5243) vertebrae (Table 1) The total number of thesevertebral congenital pathologies in E asiaticum is very low(n= 5) in comparison with thousands of collected vertebraeof this salamander (only collection of atlases ZIN PH 242contains 1909 specimens while there are only two speci-mens of congenital pathologies with atlas ndash ZIN PH 182924and ZIN PH 1831242 This frequency of vertebral congen-ital pathologies in E asiaticum (for congenital pathologieswith atlas lt 01 ) is in accordance with the previous estima-tions for PaleozoicndashMesozoic temnospondyls and Holocenefrogs (Rothschild and Laub 2013 Witzmann et al 2013)

Figure 12 The fused three anterior trunk vertebrae ZIN PH 5243of Eoscapherpeton asiaticum (a) Ventral view (b) dorsal view(c) left lateral view (d) right lateral view (e) ventral view (digi-tal restoration) with the location of longitudinal the microCT digitalsection (f) left lateral view (digital restoration) with the locationof longitudinal the microCT digital section (g) right lateral view(digital restoration) (h) digital longitudinal section (vertical plane)and (i) digital longitudinal section (horizontal plane) in dorsal viewBroken lines show the border between trunk vertebrae

42 Hemivertebra in Eoscapherpeton asiaticum

A hemivertebra is a congenital malformation in which oneor more lateral halves of vertebrae are intercalated betweenneighboring vertebrae Formation of hemivertebrae is a resultof abnormal segmentation during embryogenesis (ie fail-ure of somitogenesis before chondrification and ossificationof the vertebral anlagen eg Witzmann et al 2008 2013Burnham et al 2013) In amniots single vertebrae are de-rived from the sclerotome of two adjacent somites and thisprocess is widely known as resegmentation (Piekarski andOlsson 2014 and references therein) Resegmentation havebeen proposed to be present in all modern lissamphibians butembryological and molecular evidence for this process wasprovided only for one salamander species ndash ambystomatidAmbystoma mexicanum (Mexican axolotl) (Piekarski andOlsson 2014) The development of the described hemiver-tebra in more basal cryptobranchid salamander Eoscapher-peton asiaticum can also be explained by a failure of reseg-mentation If this is correct then it is significant for the fol-

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P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 167

Figure 13 The fused neural arches of two trunk vertebrae ZINPH 8243 of Eoscapherpeton asiaticum (a) Dorsal view (b) rightlateral view (c) left lateral view (d) left lateral view (digital restora-tion) (e) right lateral view (digital restoration) (f) dorsal view (digi-tal restoration) with the location of longitudinal the microCT digitalsection and (g) digital longitudinal section (vertical plane)

lowing reasons (1) it is the oldest occurrence of resegmen-tation in salamanders and (2) it is evidence that resegmen-tation is a common process for salamanders (including rela-tively basal cryptobranchids and advanced ambystomatids)

A hemivertebra in E asiaticum was found in a relativelylarge (according to the large size of the atlas that was co-ossified with hemivertebra) and presumably adult individual(Fig 6) The presence of a hemivertebra in a large individualsuggests that this pathology did not cause its death

The hemivertebrae have been reported earlier for somePaleozoic and Mesozoic temnospondyls (Witzmann et al2013) The finding of hemivertebra in the Late Cretaceouscryptobranchid E asiaticum is the oldest (and the first de-scribed) occurrence of this pathology among modern groupsof amphibians (ie lissamphibians salamanders frogs andcaecilians)

43 Femoral pathologies in Eoscapherpeton possibleinsight into behavior

The traumatic origin of pathologies on the distal parts of thefemora (Figs 14ndash15) could be evidence of frequent occur-rence of hind limb injuries in Eoscapherpeton asiaticum Inmodern cryptobranchids such as Cryptobranchus limb in-juries are the most common physical abnormalities in wildpopulations (Miller and Miller 2005) The proposed maincause of limb injuries in Cryptobranchus is intraspecific ag-

Figure 14 The distal parts of the femora ZIN PH 9243 (andashh) andZIN PH 10243 (indasho) of Eoscapherpeton asiaticum Specimen ZINPH 9243 (a) ventral view (b) dorsal view (c) ventral view (dig-ital restoration) (d) dorsal view (digital restoration) (e) anterioror posterior view (digital restoration) with the locations of trans-verse histological sections and longitudinal the microCT digital sec-tion (f) digital longitudinal section and (gndashh) histological trans-verse section The arrow shows the border between cortical boneand pathological tissue Specimen ZIN PH 10243 (i) ventral view(k) dorsal view (l) ventral view (digital restoration) with the loca-tions of the microCT digital sections (m) anterior or posterior view(digital restoration) (n) digital longitudinal section and (o) digitaltransverse section Blue lines show the original bone position priorto the break Abbreviations BE ndash bone enlargement C ndash callus CBndash cortical bone

gression during the reproductive season (eg defending ofnesting sites) because fresh injuries were found only dur-ing the reproductive season (namely in August and Septem-ber) (Hiler et al 2005 Miller and Miller 2005) Takinginto account the phylogenetic position of Eoscapherpetonas a member of the cryptobranchid clade and their aquaticlifestyle (see Skutschas 2009 2013) the reason for the fre-quent occurrence of hind limb injuries in E asiaticum couldbe the same (intraspecific aggression during reproductiveseason) and this behavioral element could have appearedearly in the evolution of cryptobranchids ie at least in theLate Cretaceous

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168 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 15 The distal parts of the femora ZIN PH 11243 (andashf) andZIN PH 12243 (gndashp) of Eoscapherpeton asiaticum Specimen ZINPH 11243 (a) ventral view (b) dorsal view (c) dorsal view (dig-ital restoration) with the locations of the microCT digital sections(d) anterior or posterior view (digital restoration) (e) digital lon-gitudinal section and (f) digital transverse section Specimen ZINPH 12243 (g) ventral view (h) dorsal view (i) ventral view (dig-ital restoration) (k) dorsal view (digital restoration) (l) anterior orposterior view (digital restoration) with the locations of transversehistological sections and longitudinal the microCT digital section(m) digital longitudinal section and (nndashp) transverse histologicalsections Blue lines show the original bone position prior to thebreak Abbreviations C ndash callus

5 Conclusions

The study revealed diverse osseous anomaliespathologies inEoscapherpeton asiaticum which were interpreted as trau-matic possible traumatic-infectious and congenital in originpertaining to bones of the lower jaw vertebrae and femoraThe hemivertebra and fused vertebrae (without any traces oftrauma and inflammation) document the earliest occurrenceof vertebral congenital pathologies in crown-group salaman-ders The frequency of vertebral congenital pathologies in Easiaticum is low and in accordance with the previous estima-tions for temnospondyls and frogs The numerous traumaticpathologies of femora (hematoma and healed fractures) sug-gest the survival of accidents and possibly the presence of in-

traspecific aggression during the reproductive season in thissalamander (like in modern cryptobranchids)

Data availability Micro-CT data are archived at the Department ofVertebrate Zoology of the Saint Petersburg State University (SaintPetersburg Russia) and can be accessed through the present authorsfor the purpose of scientific study

Author contributions PS designed the project PS and VK per-formed the research VK and IK prepared 3-D reconstructions VKand EB prepared figures and PS wrote the manuscript All authorscommented on the manuscript

Competing interests The authors declare that they have no conflictof interest

Acknowledgements We are grateful to the staff of the SaintPetersburg State University Research Centre for X-ray DiffractionStudies (Saint Petersburg Russia) for their help with using theLeica 2500P microscope and for CT scanning of specimens Theauthors thank Judith Pardo-Peacuterez and Nadia Froumlbisch for providinghelpful comments that improved the quality of the manuscript Thisstudy was fulfilled under support of the Russian Scientific Fundproject 14-14-00015

Edited by Constanze BickelmannReviewed by Nadia Froumlbisch and Judith Pardo Perez

References

Burnham D A Rothschild B M Babiarz J P and Martin LD Hemivertebrae as pathology and as a window to behavior inthe fossil record Palarchrsquos Journal of Vertebrate Palaeontology10 1ndash6 2013

Francillon-Vieillot H de Buffreacutenil V Castanet J Geraudie JMeunier F Sire J Y Zylenberberg L and de Ricqles A DMicrostructure and mineralization of vertebrate skeletal tissuesin Skeletal biomineralization Patterns Processes and Evolu-tionary Trends Vol 1 edited by Carter J G Van NostrandReinhold New York 471ndash530 1990

Garciacutea R A Cerda I A Heller M Rothschild B M and Zur-riaguz V The first evidence of osteomyelitis in a sauropod di-nosaur Lethaia 50 227ndash236 httpsdoiorg101111let121892016

Hanna R R Multiple injury and infection in a sub adult thero-pod dinosaur Allosaurus fragilis with comparisons to allosaurpathology in Cleveland-Lloyd Dinosaur Quarry Collection JVertebr Paleontol 22 76ndash90 httpsdoiorg1016710272-4634(2002)022[0076MIAIIA]20CO2 2002

Hiler W R Wheeler B A and Trauth S E Abnormalities inthe Ozark Hellbender (Cryptobranchus alleganiensis bishopi) inArkansas A Comparison between Two Rivers with a Historical

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

164 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 7 The fused atlas and the first trunk vertebra ZINPH 1832242 of Eoscapherpeton asiaticum (a) Dorsal view(b) ventral view (c) anterior view (d) dorsal view (digital restora-tion) (e) ventral view (digital restoration) (f) anterior view (digitalrestoration) with the location of longitudinal the microCT digitalsection (g) left lateral view (digital restoration) with the locationsof the microCT digital sections (h) digital longitudinal section (ver-tical plane) (i) digital longitudinal section (horizontal plane) in dor-sal view and (k) digital transverse section in posterior view Bro-ken lines show the border between atlas and trunk vertebra Arrowsshow bone enlargements Abbreviations AT ndash atlas TV ndash trunkvertebra

CT data show that the more posterior vertebra is slightly dis-placed in relation to the more anterior vertebrae (Fig 9l) sothe trauma took place before the forming of pathological en-largements The internal structure of enlarged areas is highlyporous and contains large necrotic cavities

The fused two anterior trunk vertebrae ZIN PH 6243(Fig 10) are characterized by shortening of the centra asym-metry of the centra (due to the unequal development of theanterior and posterior portions) and asymmetric arrangementof the transverse processes (widely separated on the left sideand closely spaced on the right side of the fused centra) Thebases of neural arches are fused and a large spinal nerve fora-men is present

Similarly to the specimen ZIN PH 6243 the fused twoposterior trunk vertebrae ZIN PH 7243 (Fig 11) exhibit thevertebral shortening with asymmetry of the centra and asym-metric arrangement of the transverse processes The absenceof any traces of trauma and inflammation of bone suggeststhe congenital origin of the fusion The asymmetric arrange-ment of the transverse processes and vertebral shortening

Figure 8 The fused atlas and the first trunk vertebra ZINPH 1831242 of Eoscapherpeton asiaticum (a) anterior view(b) posterior view (c) dorsal view (d) ventral view (e) left lat-eral view (f) anterior view (digital restoration) with the locationof longitudinal the microCT digital section (g) ventral view (digi-tal restoration) with the location of longitudinal the microCT digi-tal section (h) right lateral view (digital restoration) (i) left lateralview (digital restoration) (k) digital longitudinal section (horizon-tal plane) in dorsal view and (l) digital longitudinal section (verticalplane) Broken lines show the border between atlas and trunk verte-bra Abbreviations AT ndash atlas TV ndash trunk vertebra

could be the result of abnormal segmentation during embryo-genesis

The specimen ZIN PH 5243 consists of three fused ante-rior trunk vertebrae (Fig 12) The vertebrae exhibit the verte-bral shortening and do not display any signs of trauma andorinflammation of bone We suggest the congenital origin ofthe fusion in ZIN PH 5243

The neural arches of two trunk vertebrae ZIN PH 8243(Fig 13) are fused without any trace of suture The more an-terior neural arch is lower than the more posterior one Thereare no pathological enlargements (no signs of periosteal re-active growth) and the fusion of these neural arches is mostlikely of congenital origin

36 Femoral pathologies

The distal fragment of the medium-sized femur ZINPH 9243 (Fig 14andashh) displays the presence of large smoothfocal bone enlargement (ie periosteal elevation) in themetaphyseal region The internal structure of this bone en-largement is porous due to the presence of large cavities ofirregular shape (ie erosion bays) (Fig 14fndashh) The tissue

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 165

Figure 9 The fused two anterior trunk vertebrae ZIN PH 4243 ofEoscapherpeton asiaticum (a) Dorsal view (b) ventral view (c) an-terior view (d) right lateral view (e) left lateral view (f) anteriorview (digital restoration) (g) dorsal view (digital restoration) withthe location of longitudinal the microCT digital section (h) rightlateral view (digital restoration) with the location of longitudinalthe microCT digital section (i) left lateral view (digital restoration)(k) digital longitudinal section (vertical plane) and (l) digital lon-gitudinal section (horizontal plane) in dorsal view Blue lines showthe original median line of the centra prior to the trauma Arrowsshow bone enlargements Abbreviations NC ndash necrotic cavity

of the enlargement is entirely composed of a parallel-fiberedprimary bone of periosteal origin The parallel-fibered pri-mary bone in the outer part of the enlargement is more or-ganized than in the inner porous part and is similar to thatof the normal cortical bone of this femur (Fig 14h) No evi-dence of fracture was revealed by CT scanning and histolog-ical analysis Similar bone enlargement in modern reptileswas identified as hematoma (Rothschild et al 2010 2012)We also interpret the femoral pathology on ZIN PH 9243 ashematoma that could have been caused by trauma (traumaticorigin)

All other femoral specimens in the sample (two distal frag-ments ZIN PH 10-11243 of medium-sized femora and onedistal fragment ZIN PH 12243 of large femora Table 1Figs 14indasho 15) display the signs of healed fractures thatare traceable in CT images Externally all the specimensdemonstrate displacements of the distal end of the bone andhave large smooth focal bone enlargements (ie calluses) inthe metaphyseal region that were formed during the healingof traumatic fractures The internal structure of the callusesis porous and contains numerous longitudinal and obliquely

Figure 10 The fused two anterior trunk vertebrae ZIN PH 6243of Eoscapherpeton asiaticum (a) Dorsal view (b) ventral view(c) anterior view (d) left lateral view (e) right lateral view (f) ven-tral view (digital restoration) with the location of longitudinalthe microCT digital sections (g) anterior view (digital restora-tion) with the location of longitudinal the microCT digital section(h) left lateral view (digital restoration) (i) right lateral view (digitalrestoration) (kndashl) digital longitudinal sections (vertical plane) and(m) digital longitudinal section (horizontal plane) in ventral viewBroken lines show the border between trunk vertebrae Abbrevia-tions NA ndash neural arch TP1 ndash transverse process of more anteriorvertebra TP2 ndash transverse process of more posterior vertebra

oriented vascular canals that are typical for these structures(Fig 15nndashp)

4 Discussion

41 General discussion

Eoscapherpeton asiaticum demonstrates a wide spectrum ofosseous anomaliespathologies resulting from trauma pos-sible infection due to trauma and congenital disorders Trau-matic origins of pathologies are suggested for the dentaryZIN PH 2243 (the presence of the fracture and the callus)and for femoral fragments ZIN PH 9-12243 (hematoma andhealed fractures with the calluses on the distal portions of thebones) (Table 1) The possible traumatic-infectious patholo-gies are associated with bone enlargements formed duringreactive bone growth andor the presence of necrotic cavitiesare suggested for the prearticular ZIN PH 1243 the anteriortrunk vertebra ZIN PH 13243 the fused atlas and the firsttrunk vertebrae ZIN PH 1832242 and the fused two ante-

wwwfoss-recnet211592018 Foss Rec 21 159ndash169 2018

166 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 11 The fused two posterior trunk vertebrae ZIN PH 7243 ofEoscapherpeton asiaticum (a) Ventral view (b) dorsal view (c) an-terior view (d) right lateral view (e) left lateral view (f) ventralview (digital restoration) with the location of the microCT digitalsections (g) anterior view (digital restoration) with the location oflongitudinal the microCT digital section (h) right lateral view (dig-ital restoration) (i) left lateral view (digital restoration) (k) digitallongitudinal section (vertical plane) (l) digital transverse section inanterior view and (m) digital longitudinal section (horizontal plane)in dorsal view Broken lines show the border between trunk verte-brae Abbreviations NA ndash neural arch TP1 ndash transverse process ofmore anterior vertebra TP2 ndash transverse process of more posteriorvertebra

rior trunk vertebrae ZIN PH 4243 (Table 1) The congenitalpathologies of E asiaticum are represented by the hemiver-tebra (specimen ZIN PH 182924 see discussion below) andby fusion of two (fused atlas and anterior trunk vertebra ZINPH 1831242 fused anterior trunk vertebrae ZIN PH 6243fused posterior trunk vertebrae ZIN PH 7243 and for fusedneural arches 8243) or three (fused anterior trunk vertebraeZIN PH 5243) vertebrae (Table 1) The total number of thesevertebral congenital pathologies in E asiaticum is very low(n= 5) in comparison with thousands of collected vertebraeof this salamander (only collection of atlases ZIN PH 242contains 1909 specimens while there are only two speci-mens of congenital pathologies with atlas ndash ZIN PH 182924and ZIN PH 1831242 This frequency of vertebral congen-ital pathologies in E asiaticum (for congenital pathologieswith atlas lt 01 ) is in accordance with the previous estima-tions for PaleozoicndashMesozoic temnospondyls and Holocenefrogs (Rothschild and Laub 2013 Witzmann et al 2013)

Figure 12 The fused three anterior trunk vertebrae ZIN PH 5243of Eoscapherpeton asiaticum (a) Ventral view (b) dorsal view(c) left lateral view (d) right lateral view (e) ventral view (digi-tal restoration) with the location of longitudinal the microCT digitalsection (f) left lateral view (digital restoration) with the locationof longitudinal the microCT digital section (g) right lateral view(digital restoration) (h) digital longitudinal section (vertical plane)and (i) digital longitudinal section (horizontal plane) in dorsal viewBroken lines show the border between trunk vertebrae

42 Hemivertebra in Eoscapherpeton asiaticum

A hemivertebra is a congenital malformation in which oneor more lateral halves of vertebrae are intercalated betweenneighboring vertebrae Formation of hemivertebrae is a resultof abnormal segmentation during embryogenesis (ie fail-ure of somitogenesis before chondrification and ossificationof the vertebral anlagen eg Witzmann et al 2008 2013Burnham et al 2013) In amniots single vertebrae are de-rived from the sclerotome of two adjacent somites and thisprocess is widely known as resegmentation (Piekarski andOlsson 2014 and references therein) Resegmentation havebeen proposed to be present in all modern lissamphibians butembryological and molecular evidence for this process wasprovided only for one salamander species ndash ambystomatidAmbystoma mexicanum (Mexican axolotl) (Piekarski andOlsson 2014) The development of the described hemiver-tebra in more basal cryptobranchid salamander Eoscapher-peton asiaticum can also be explained by a failure of reseg-mentation If this is correct then it is significant for the fol-

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 167

Figure 13 The fused neural arches of two trunk vertebrae ZINPH 8243 of Eoscapherpeton asiaticum (a) Dorsal view (b) rightlateral view (c) left lateral view (d) left lateral view (digital restora-tion) (e) right lateral view (digital restoration) (f) dorsal view (digi-tal restoration) with the location of longitudinal the microCT digitalsection and (g) digital longitudinal section (vertical plane)

lowing reasons (1) it is the oldest occurrence of resegmen-tation in salamanders and (2) it is evidence that resegmen-tation is a common process for salamanders (including rela-tively basal cryptobranchids and advanced ambystomatids)

A hemivertebra in E asiaticum was found in a relativelylarge (according to the large size of the atlas that was co-ossified with hemivertebra) and presumably adult individual(Fig 6) The presence of a hemivertebra in a large individualsuggests that this pathology did not cause its death

The hemivertebrae have been reported earlier for somePaleozoic and Mesozoic temnospondyls (Witzmann et al2013) The finding of hemivertebra in the Late Cretaceouscryptobranchid E asiaticum is the oldest (and the first de-scribed) occurrence of this pathology among modern groupsof amphibians (ie lissamphibians salamanders frogs andcaecilians)

43 Femoral pathologies in Eoscapherpeton possibleinsight into behavior

The traumatic origin of pathologies on the distal parts of thefemora (Figs 14ndash15) could be evidence of frequent occur-rence of hind limb injuries in Eoscapherpeton asiaticum Inmodern cryptobranchids such as Cryptobranchus limb in-juries are the most common physical abnormalities in wildpopulations (Miller and Miller 2005) The proposed maincause of limb injuries in Cryptobranchus is intraspecific ag-

Figure 14 The distal parts of the femora ZIN PH 9243 (andashh) andZIN PH 10243 (indasho) of Eoscapherpeton asiaticum Specimen ZINPH 9243 (a) ventral view (b) dorsal view (c) ventral view (dig-ital restoration) (d) dorsal view (digital restoration) (e) anterioror posterior view (digital restoration) with the locations of trans-verse histological sections and longitudinal the microCT digital sec-tion (f) digital longitudinal section and (gndashh) histological trans-verse section The arrow shows the border between cortical boneand pathological tissue Specimen ZIN PH 10243 (i) ventral view(k) dorsal view (l) ventral view (digital restoration) with the loca-tions of the microCT digital sections (m) anterior or posterior view(digital restoration) (n) digital longitudinal section and (o) digitaltransverse section Blue lines show the original bone position priorto the break Abbreviations BE ndash bone enlargement C ndash callus CBndash cortical bone

gression during the reproductive season (eg defending ofnesting sites) because fresh injuries were found only dur-ing the reproductive season (namely in August and Septem-ber) (Hiler et al 2005 Miller and Miller 2005) Takinginto account the phylogenetic position of Eoscapherpetonas a member of the cryptobranchid clade and their aquaticlifestyle (see Skutschas 2009 2013) the reason for the fre-quent occurrence of hind limb injuries in E asiaticum couldbe the same (intraspecific aggression during reproductiveseason) and this behavioral element could have appearedearly in the evolution of cryptobranchids ie at least in theLate Cretaceous

wwwfoss-recnet211592018 Foss Rec 21 159ndash169 2018

168 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 15 The distal parts of the femora ZIN PH 11243 (andashf) andZIN PH 12243 (gndashp) of Eoscapherpeton asiaticum Specimen ZINPH 11243 (a) ventral view (b) dorsal view (c) dorsal view (dig-ital restoration) with the locations of the microCT digital sections(d) anterior or posterior view (digital restoration) (e) digital lon-gitudinal section and (f) digital transverse section Specimen ZINPH 12243 (g) ventral view (h) dorsal view (i) ventral view (dig-ital restoration) (k) dorsal view (digital restoration) (l) anterior orposterior view (digital restoration) with the locations of transversehistological sections and longitudinal the microCT digital section(m) digital longitudinal section and (nndashp) transverse histologicalsections Blue lines show the original bone position prior to thebreak Abbreviations C ndash callus

5 Conclusions

The study revealed diverse osseous anomaliespathologies inEoscapherpeton asiaticum which were interpreted as trau-matic possible traumatic-infectious and congenital in originpertaining to bones of the lower jaw vertebrae and femoraThe hemivertebra and fused vertebrae (without any traces oftrauma and inflammation) document the earliest occurrenceof vertebral congenital pathologies in crown-group salaman-ders The frequency of vertebral congenital pathologies in Easiaticum is low and in accordance with the previous estima-tions for temnospondyls and frogs The numerous traumaticpathologies of femora (hematoma and healed fractures) sug-gest the survival of accidents and possibly the presence of in-

traspecific aggression during the reproductive season in thissalamander (like in modern cryptobranchids)

Data availability Micro-CT data are archived at the Department ofVertebrate Zoology of the Saint Petersburg State University (SaintPetersburg Russia) and can be accessed through the present authorsfor the purpose of scientific study

Author contributions PS designed the project PS and VK per-formed the research VK and IK prepared 3-D reconstructions VKand EB prepared figures and PS wrote the manuscript All authorscommented on the manuscript

Competing interests The authors declare that they have no conflictof interest

Acknowledgements We are grateful to the staff of the SaintPetersburg State University Research Centre for X-ray DiffractionStudies (Saint Petersburg Russia) for their help with using theLeica 2500P microscope and for CT scanning of specimens Theauthors thank Judith Pardo-Peacuterez and Nadia Froumlbisch for providinghelpful comments that improved the quality of the manuscript Thisstudy was fulfilled under support of the Russian Scientific Fundproject 14-14-00015

Edited by Constanze BickelmannReviewed by Nadia Froumlbisch and Judith Pardo Perez

References

Burnham D A Rothschild B M Babiarz J P and Martin LD Hemivertebrae as pathology and as a window to behavior inthe fossil record Palarchrsquos Journal of Vertebrate Palaeontology10 1ndash6 2013

Francillon-Vieillot H de Buffreacutenil V Castanet J Geraudie JMeunier F Sire J Y Zylenberberg L and de Ricqles A DMicrostructure and mineralization of vertebrate skeletal tissuesin Skeletal biomineralization Patterns Processes and Evolu-tionary Trends Vol 1 edited by Carter J G Van NostrandReinhold New York 471ndash530 1990

Garciacutea R A Cerda I A Heller M Rothschild B M and Zur-riaguz V The first evidence of osteomyelitis in a sauropod di-nosaur Lethaia 50 227ndash236 httpsdoiorg101111let121892016

Hanna R R Multiple injury and infection in a sub adult thero-pod dinosaur Allosaurus fragilis with comparisons to allosaurpathology in Cleveland-Lloyd Dinosaur Quarry Collection JVertebr Paleontol 22 76ndash90 httpsdoiorg1016710272-4634(2002)022[0076MIAIIA]20CO2 2002

Hiler W R Wheeler B A and Trauth S E Abnormalities inthe Ozark Hellbender (Cryptobranchus alleganiensis bishopi) inArkansas A Comparison between Two Rivers with a Historical

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 165

Figure 9 The fused two anterior trunk vertebrae ZIN PH 4243 ofEoscapherpeton asiaticum (a) Dorsal view (b) ventral view (c) an-terior view (d) right lateral view (e) left lateral view (f) anteriorview (digital restoration) (g) dorsal view (digital restoration) withthe location of longitudinal the microCT digital section (h) rightlateral view (digital restoration) with the location of longitudinalthe microCT digital section (i) left lateral view (digital restoration)(k) digital longitudinal section (vertical plane) and (l) digital lon-gitudinal section (horizontal plane) in dorsal view Blue lines showthe original median line of the centra prior to the trauma Arrowsshow bone enlargements Abbreviations NC ndash necrotic cavity

of the enlargement is entirely composed of a parallel-fiberedprimary bone of periosteal origin The parallel-fibered pri-mary bone in the outer part of the enlargement is more or-ganized than in the inner porous part and is similar to thatof the normal cortical bone of this femur (Fig 14h) No evi-dence of fracture was revealed by CT scanning and histolog-ical analysis Similar bone enlargement in modern reptileswas identified as hematoma (Rothschild et al 2010 2012)We also interpret the femoral pathology on ZIN PH 9243 ashematoma that could have been caused by trauma (traumaticorigin)

All other femoral specimens in the sample (two distal frag-ments ZIN PH 10-11243 of medium-sized femora and onedistal fragment ZIN PH 12243 of large femora Table 1Figs 14indasho 15) display the signs of healed fractures thatare traceable in CT images Externally all the specimensdemonstrate displacements of the distal end of the bone andhave large smooth focal bone enlargements (ie calluses) inthe metaphyseal region that were formed during the healingof traumatic fractures The internal structure of the callusesis porous and contains numerous longitudinal and obliquely

Figure 10 The fused two anterior trunk vertebrae ZIN PH 6243of Eoscapherpeton asiaticum (a) Dorsal view (b) ventral view(c) anterior view (d) left lateral view (e) right lateral view (f) ven-tral view (digital restoration) with the location of longitudinalthe microCT digital sections (g) anterior view (digital restora-tion) with the location of longitudinal the microCT digital section(h) left lateral view (digital restoration) (i) right lateral view (digitalrestoration) (kndashl) digital longitudinal sections (vertical plane) and(m) digital longitudinal section (horizontal plane) in ventral viewBroken lines show the border between trunk vertebrae Abbrevia-tions NA ndash neural arch TP1 ndash transverse process of more anteriorvertebra TP2 ndash transverse process of more posterior vertebra

oriented vascular canals that are typical for these structures(Fig 15nndashp)

4 Discussion

41 General discussion

Eoscapherpeton asiaticum demonstrates a wide spectrum ofosseous anomaliespathologies resulting from trauma pos-sible infection due to trauma and congenital disorders Trau-matic origins of pathologies are suggested for the dentaryZIN PH 2243 (the presence of the fracture and the callus)and for femoral fragments ZIN PH 9-12243 (hematoma andhealed fractures with the calluses on the distal portions of thebones) (Table 1) The possible traumatic-infectious patholo-gies are associated with bone enlargements formed duringreactive bone growth andor the presence of necrotic cavitiesare suggested for the prearticular ZIN PH 1243 the anteriortrunk vertebra ZIN PH 13243 the fused atlas and the firsttrunk vertebrae ZIN PH 1832242 and the fused two ante-

wwwfoss-recnet211592018 Foss Rec 21 159ndash169 2018

166 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 11 The fused two posterior trunk vertebrae ZIN PH 7243 ofEoscapherpeton asiaticum (a) Ventral view (b) dorsal view (c) an-terior view (d) right lateral view (e) left lateral view (f) ventralview (digital restoration) with the location of the microCT digitalsections (g) anterior view (digital restoration) with the location oflongitudinal the microCT digital section (h) right lateral view (dig-ital restoration) (i) left lateral view (digital restoration) (k) digitallongitudinal section (vertical plane) (l) digital transverse section inanterior view and (m) digital longitudinal section (horizontal plane)in dorsal view Broken lines show the border between trunk verte-brae Abbreviations NA ndash neural arch TP1 ndash transverse process ofmore anterior vertebra TP2 ndash transverse process of more posteriorvertebra

rior trunk vertebrae ZIN PH 4243 (Table 1) The congenitalpathologies of E asiaticum are represented by the hemiver-tebra (specimen ZIN PH 182924 see discussion below) andby fusion of two (fused atlas and anterior trunk vertebra ZINPH 1831242 fused anterior trunk vertebrae ZIN PH 6243fused posterior trunk vertebrae ZIN PH 7243 and for fusedneural arches 8243) or three (fused anterior trunk vertebraeZIN PH 5243) vertebrae (Table 1) The total number of thesevertebral congenital pathologies in E asiaticum is very low(n= 5) in comparison with thousands of collected vertebraeof this salamander (only collection of atlases ZIN PH 242contains 1909 specimens while there are only two speci-mens of congenital pathologies with atlas ndash ZIN PH 182924and ZIN PH 1831242 This frequency of vertebral congen-ital pathologies in E asiaticum (for congenital pathologieswith atlas lt 01 ) is in accordance with the previous estima-tions for PaleozoicndashMesozoic temnospondyls and Holocenefrogs (Rothschild and Laub 2013 Witzmann et al 2013)

Figure 12 The fused three anterior trunk vertebrae ZIN PH 5243of Eoscapherpeton asiaticum (a) Ventral view (b) dorsal view(c) left lateral view (d) right lateral view (e) ventral view (digi-tal restoration) with the location of longitudinal the microCT digitalsection (f) left lateral view (digital restoration) with the locationof longitudinal the microCT digital section (g) right lateral view(digital restoration) (h) digital longitudinal section (vertical plane)and (i) digital longitudinal section (horizontal plane) in dorsal viewBroken lines show the border between trunk vertebrae

42 Hemivertebra in Eoscapherpeton asiaticum

A hemivertebra is a congenital malformation in which oneor more lateral halves of vertebrae are intercalated betweenneighboring vertebrae Formation of hemivertebrae is a resultof abnormal segmentation during embryogenesis (ie fail-ure of somitogenesis before chondrification and ossificationof the vertebral anlagen eg Witzmann et al 2008 2013Burnham et al 2013) In amniots single vertebrae are de-rived from the sclerotome of two adjacent somites and thisprocess is widely known as resegmentation (Piekarski andOlsson 2014 and references therein) Resegmentation havebeen proposed to be present in all modern lissamphibians butembryological and molecular evidence for this process wasprovided only for one salamander species ndash ambystomatidAmbystoma mexicanum (Mexican axolotl) (Piekarski andOlsson 2014) The development of the described hemiver-tebra in more basal cryptobranchid salamander Eoscapher-peton asiaticum can also be explained by a failure of reseg-mentation If this is correct then it is significant for the fol-

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 167

Figure 13 The fused neural arches of two trunk vertebrae ZINPH 8243 of Eoscapherpeton asiaticum (a) Dorsal view (b) rightlateral view (c) left lateral view (d) left lateral view (digital restora-tion) (e) right lateral view (digital restoration) (f) dorsal view (digi-tal restoration) with the location of longitudinal the microCT digitalsection and (g) digital longitudinal section (vertical plane)

lowing reasons (1) it is the oldest occurrence of resegmen-tation in salamanders and (2) it is evidence that resegmen-tation is a common process for salamanders (including rela-tively basal cryptobranchids and advanced ambystomatids)

A hemivertebra in E asiaticum was found in a relativelylarge (according to the large size of the atlas that was co-ossified with hemivertebra) and presumably adult individual(Fig 6) The presence of a hemivertebra in a large individualsuggests that this pathology did not cause its death

The hemivertebrae have been reported earlier for somePaleozoic and Mesozoic temnospondyls (Witzmann et al2013) The finding of hemivertebra in the Late Cretaceouscryptobranchid E asiaticum is the oldest (and the first de-scribed) occurrence of this pathology among modern groupsof amphibians (ie lissamphibians salamanders frogs andcaecilians)

43 Femoral pathologies in Eoscapherpeton possibleinsight into behavior

The traumatic origin of pathologies on the distal parts of thefemora (Figs 14ndash15) could be evidence of frequent occur-rence of hind limb injuries in Eoscapherpeton asiaticum Inmodern cryptobranchids such as Cryptobranchus limb in-juries are the most common physical abnormalities in wildpopulations (Miller and Miller 2005) The proposed maincause of limb injuries in Cryptobranchus is intraspecific ag-

Figure 14 The distal parts of the femora ZIN PH 9243 (andashh) andZIN PH 10243 (indasho) of Eoscapherpeton asiaticum Specimen ZINPH 9243 (a) ventral view (b) dorsal view (c) ventral view (dig-ital restoration) (d) dorsal view (digital restoration) (e) anterioror posterior view (digital restoration) with the locations of trans-verse histological sections and longitudinal the microCT digital sec-tion (f) digital longitudinal section and (gndashh) histological trans-verse section The arrow shows the border between cortical boneand pathological tissue Specimen ZIN PH 10243 (i) ventral view(k) dorsal view (l) ventral view (digital restoration) with the loca-tions of the microCT digital sections (m) anterior or posterior view(digital restoration) (n) digital longitudinal section and (o) digitaltransverse section Blue lines show the original bone position priorto the break Abbreviations BE ndash bone enlargement C ndash callus CBndash cortical bone

gression during the reproductive season (eg defending ofnesting sites) because fresh injuries were found only dur-ing the reproductive season (namely in August and Septem-ber) (Hiler et al 2005 Miller and Miller 2005) Takinginto account the phylogenetic position of Eoscapherpetonas a member of the cryptobranchid clade and their aquaticlifestyle (see Skutschas 2009 2013) the reason for the fre-quent occurrence of hind limb injuries in E asiaticum couldbe the same (intraspecific aggression during reproductiveseason) and this behavioral element could have appearedearly in the evolution of cryptobranchids ie at least in theLate Cretaceous

wwwfoss-recnet211592018 Foss Rec 21 159ndash169 2018

168 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 15 The distal parts of the femora ZIN PH 11243 (andashf) andZIN PH 12243 (gndashp) of Eoscapherpeton asiaticum Specimen ZINPH 11243 (a) ventral view (b) dorsal view (c) dorsal view (dig-ital restoration) with the locations of the microCT digital sections(d) anterior or posterior view (digital restoration) (e) digital lon-gitudinal section and (f) digital transverse section Specimen ZINPH 12243 (g) ventral view (h) dorsal view (i) ventral view (dig-ital restoration) (k) dorsal view (digital restoration) (l) anterior orposterior view (digital restoration) with the locations of transversehistological sections and longitudinal the microCT digital section(m) digital longitudinal section and (nndashp) transverse histologicalsections Blue lines show the original bone position prior to thebreak Abbreviations C ndash callus

5 Conclusions

The study revealed diverse osseous anomaliespathologies inEoscapherpeton asiaticum which were interpreted as trau-matic possible traumatic-infectious and congenital in originpertaining to bones of the lower jaw vertebrae and femoraThe hemivertebra and fused vertebrae (without any traces oftrauma and inflammation) document the earliest occurrenceof vertebral congenital pathologies in crown-group salaman-ders The frequency of vertebral congenital pathologies in Easiaticum is low and in accordance with the previous estima-tions for temnospondyls and frogs The numerous traumaticpathologies of femora (hematoma and healed fractures) sug-gest the survival of accidents and possibly the presence of in-

traspecific aggression during the reproductive season in thissalamander (like in modern cryptobranchids)

Data availability Micro-CT data are archived at the Department ofVertebrate Zoology of the Saint Petersburg State University (SaintPetersburg Russia) and can be accessed through the present authorsfor the purpose of scientific study

Author contributions PS designed the project PS and VK per-formed the research VK and IK prepared 3-D reconstructions VKand EB prepared figures and PS wrote the manuscript All authorscommented on the manuscript

Competing interests The authors declare that they have no conflictof interest

Acknowledgements We are grateful to the staff of the SaintPetersburg State University Research Centre for X-ray DiffractionStudies (Saint Petersburg Russia) for their help with using theLeica 2500P microscope and for CT scanning of specimens Theauthors thank Judith Pardo-Peacuterez and Nadia Froumlbisch for providinghelpful comments that improved the quality of the manuscript Thisstudy was fulfilled under support of the Russian Scientific Fundproject 14-14-00015

Edited by Constanze BickelmannReviewed by Nadia Froumlbisch and Judith Pardo Perez

References

Burnham D A Rothschild B M Babiarz J P and Martin LD Hemivertebrae as pathology and as a window to behavior inthe fossil record Palarchrsquos Journal of Vertebrate Palaeontology10 1ndash6 2013

Francillon-Vieillot H de Buffreacutenil V Castanet J Geraudie JMeunier F Sire J Y Zylenberberg L and de Ricqles A DMicrostructure and mineralization of vertebrate skeletal tissuesin Skeletal biomineralization Patterns Processes and Evolu-tionary Trends Vol 1 edited by Carter J G Van NostrandReinhold New York 471ndash530 1990

Garciacutea R A Cerda I A Heller M Rothschild B M and Zur-riaguz V The first evidence of osteomyelitis in a sauropod di-nosaur Lethaia 50 227ndash236 httpsdoiorg101111let121892016

Hanna R R Multiple injury and infection in a sub adult thero-pod dinosaur Allosaurus fragilis with comparisons to allosaurpathology in Cleveland-Lloyd Dinosaur Quarry Collection JVertebr Paleontol 22 76ndash90 httpsdoiorg1016710272-4634(2002)022[0076MIAIIA]20CO2 2002

Hiler W R Wheeler B A and Trauth S E Abnormalities inthe Ozark Hellbender (Cryptobranchus alleganiensis bishopi) inArkansas A Comparison between Two Rivers with a Historical

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

166 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 11 The fused two posterior trunk vertebrae ZIN PH 7243 ofEoscapherpeton asiaticum (a) Ventral view (b) dorsal view (c) an-terior view (d) right lateral view (e) left lateral view (f) ventralview (digital restoration) with the location of the microCT digitalsections (g) anterior view (digital restoration) with the location oflongitudinal the microCT digital section (h) right lateral view (dig-ital restoration) (i) left lateral view (digital restoration) (k) digitallongitudinal section (vertical plane) (l) digital transverse section inanterior view and (m) digital longitudinal section (horizontal plane)in dorsal view Broken lines show the border between trunk verte-brae Abbreviations NA ndash neural arch TP1 ndash transverse process ofmore anterior vertebra TP2 ndash transverse process of more posteriorvertebra

rior trunk vertebrae ZIN PH 4243 (Table 1) The congenitalpathologies of E asiaticum are represented by the hemiver-tebra (specimen ZIN PH 182924 see discussion below) andby fusion of two (fused atlas and anterior trunk vertebra ZINPH 1831242 fused anterior trunk vertebrae ZIN PH 6243fused posterior trunk vertebrae ZIN PH 7243 and for fusedneural arches 8243) or three (fused anterior trunk vertebraeZIN PH 5243) vertebrae (Table 1) The total number of thesevertebral congenital pathologies in E asiaticum is very low(n= 5) in comparison with thousands of collected vertebraeof this salamander (only collection of atlases ZIN PH 242contains 1909 specimens while there are only two speci-mens of congenital pathologies with atlas ndash ZIN PH 182924and ZIN PH 1831242 This frequency of vertebral congen-ital pathologies in E asiaticum (for congenital pathologieswith atlas lt 01 ) is in accordance with the previous estima-tions for PaleozoicndashMesozoic temnospondyls and Holocenefrogs (Rothschild and Laub 2013 Witzmann et al 2013)

Figure 12 The fused three anterior trunk vertebrae ZIN PH 5243of Eoscapherpeton asiaticum (a) Ventral view (b) dorsal view(c) left lateral view (d) right lateral view (e) ventral view (digi-tal restoration) with the location of longitudinal the microCT digitalsection (f) left lateral view (digital restoration) with the locationof longitudinal the microCT digital section (g) right lateral view(digital restoration) (h) digital longitudinal section (vertical plane)and (i) digital longitudinal section (horizontal plane) in dorsal viewBroken lines show the border between trunk vertebrae

42 Hemivertebra in Eoscapherpeton asiaticum

A hemivertebra is a congenital malformation in which oneor more lateral halves of vertebrae are intercalated betweenneighboring vertebrae Formation of hemivertebrae is a resultof abnormal segmentation during embryogenesis (ie fail-ure of somitogenesis before chondrification and ossificationof the vertebral anlagen eg Witzmann et al 2008 2013Burnham et al 2013) In amniots single vertebrae are de-rived from the sclerotome of two adjacent somites and thisprocess is widely known as resegmentation (Piekarski andOlsson 2014 and references therein) Resegmentation havebeen proposed to be present in all modern lissamphibians butembryological and molecular evidence for this process wasprovided only for one salamander species ndash ambystomatidAmbystoma mexicanum (Mexican axolotl) (Piekarski andOlsson 2014) The development of the described hemiver-tebra in more basal cryptobranchid salamander Eoscapher-peton asiaticum can also be explained by a failure of reseg-mentation If this is correct then it is significant for the fol-

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 167

Figure 13 The fused neural arches of two trunk vertebrae ZINPH 8243 of Eoscapherpeton asiaticum (a) Dorsal view (b) rightlateral view (c) left lateral view (d) left lateral view (digital restora-tion) (e) right lateral view (digital restoration) (f) dorsal view (digi-tal restoration) with the location of longitudinal the microCT digitalsection and (g) digital longitudinal section (vertical plane)

lowing reasons (1) it is the oldest occurrence of resegmen-tation in salamanders and (2) it is evidence that resegmen-tation is a common process for salamanders (including rela-tively basal cryptobranchids and advanced ambystomatids)

A hemivertebra in E asiaticum was found in a relativelylarge (according to the large size of the atlas that was co-ossified with hemivertebra) and presumably adult individual(Fig 6) The presence of a hemivertebra in a large individualsuggests that this pathology did not cause its death

The hemivertebrae have been reported earlier for somePaleozoic and Mesozoic temnospondyls (Witzmann et al2013) The finding of hemivertebra in the Late Cretaceouscryptobranchid E asiaticum is the oldest (and the first de-scribed) occurrence of this pathology among modern groupsof amphibians (ie lissamphibians salamanders frogs andcaecilians)

43 Femoral pathologies in Eoscapherpeton possibleinsight into behavior

The traumatic origin of pathologies on the distal parts of thefemora (Figs 14ndash15) could be evidence of frequent occur-rence of hind limb injuries in Eoscapherpeton asiaticum Inmodern cryptobranchids such as Cryptobranchus limb in-juries are the most common physical abnormalities in wildpopulations (Miller and Miller 2005) The proposed maincause of limb injuries in Cryptobranchus is intraspecific ag-

Figure 14 The distal parts of the femora ZIN PH 9243 (andashh) andZIN PH 10243 (indasho) of Eoscapherpeton asiaticum Specimen ZINPH 9243 (a) ventral view (b) dorsal view (c) ventral view (dig-ital restoration) (d) dorsal view (digital restoration) (e) anterioror posterior view (digital restoration) with the locations of trans-verse histological sections and longitudinal the microCT digital sec-tion (f) digital longitudinal section and (gndashh) histological trans-verse section The arrow shows the border between cortical boneand pathological tissue Specimen ZIN PH 10243 (i) ventral view(k) dorsal view (l) ventral view (digital restoration) with the loca-tions of the microCT digital sections (m) anterior or posterior view(digital restoration) (n) digital longitudinal section and (o) digitaltransverse section Blue lines show the original bone position priorto the break Abbreviations BE ndash bone enlargement C ndash callus CBndash cortical bone

gression during the reproductive season (eg defending ofnesting sites) because fresh injuries were found only dur-ing the reproductive season (namely in August and Septem-ber) (Hiler et al 2005 Miller and Miller 2005) Takinginto account the phylogenetic position of Eoscapherpetonas a member of the cryptobranchid clade and their aquaticlifestyle (see Skutschas 2009 2013) the reason for the fre-quent occurrence of hind limb injuries in E asiaticum couldbe the same (intraspecific aggression during reproductiveseason) and this behavioral element could have appearedearly in the evolution of cryptobranchids ie at least in theLate Cretaceous

wwwfoss-recnet211592018 Foss Rec 21 159ndash169 2018

168 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 15 The distal parts of the femora ZIN PH 11243 (andashf) andZIN PH 12243 (gndashp) of Eoscapherpeton asiaticum Specimen ZINPH 11243 (a) ventral view (b) dorsal view (c) dorsal view (dig-ital restoration) with the locations of the microCT digital sections(d) anterior or posterior view (digital restoration) (e) digital lon-gitudinal section and (f) digital transverse section Specimen ZINPH 12243 (g) ventral view (h) dorsal view (i) ventral view (dig-ital restoration) (k) dorsal view (digital restoration) (l) anterior orposterior view (digital restoration) with the locations of transversehistological sections and longitudinal the microCT digital section(m) digital longitudinal section and (nndashp) transverse histologicalsections Blue lines show the original bone position prior to thebreak Abbreviations C ndash callus

5 Conclusions

The study revealed diverse osseous anomaliespathologies inEoscapherpeton asiaticum which were interpreted as trau-matic possible traumatic-infectious and congenital in originpertaining to bones of the lower jaw vertebrae and femoraThe hemivertebra and fused vertebrae (without any traces oftrauma and inflammation) document the earliest occurrenceof vertebral congenital pathologies in crown-group salaman-ders The frequency of vertebral congenital pathologies in Easiaticum is low and in accordance with the previous estima-tions for temnospondyls and frogs The numerous traumaticpathologies of femora (hematoma and healed fractures) sug-gest the survival of accidents and possibly the presence of in-

traspecific aggression during the reproductive season in thissalamander (like in modern cryptobranchids)

Data availability Micro-CT data are archived at the Department ofVertebrate Zoology of the Saint Petersburg State University (SaintPetersburg Russia) and can be accessed through the present authorsfor the purpose of scientific study

Author contributions PS designed the project PS and VK per-formed the research VK and IK prepared 3-D reconstructions VKand EB prepared figures and PS wrote the manuscript All authorscommented on the manuscript

Competing interests The authors declare that they have no conflictof interest

Acknowledgements We are grateful to the staff of the SaintPetersburg State University Research Centre for X-ray DiffractionStudies (Saint Petersburg Russia) for their help with using theLeica 2500P microscope and for CT scanning of specimens Theauthors thank Judith Pardo-Peacuterez and Nadia Froumlbisch for providinghelpful comments that improved the quality of the manuscript Thisstudy was fulfilled under support of the Russian Scientific Fundproject 14-14-00015

Edited by Constanze BickelmannReviewed by Nadia Froumlbisch and Judith Pardo Perez

References

Burnham D A Rothschild B M Babiarz J P and Martin LD Hemivertebrae as pathology and as a window to behavior inthe fossil record Palarchrsquos Journal of Vertebrate Palaeontology10 1ndash6 2013

Francillon-Vieillot H de Buffreacutenil V Castanet J Geraudie JMeunier F Sire J Y Zylenberberg L and de Ricqles A DMicrostructure and mineralization of vertebrate skeletal tissuesin Skeletal biomineralization Patterns Processes and Evolu-tionary Trends Vol 1 edited by Carter J G Van NostrandReinhold New York 471ndash530 1990

Garciacutea R A Cerda I A Heller M Rothschild B M and Zur-riaguz V The first evidence of osteomyelitis in a sauropod di-nosaur Lethaia 50 227ndash236 httpsdoiorg101111let121892016

Hanna R R Multiple injury and infection in a sub adult thero-pod dinosaur Allosaurus fragilis with comparisons to allosaurpathology in Cleveland-Lloyd Dinosaur Quarry Collection JVertebr Paleontol 22 76ndash90 httpsdoiorg1016710272-4634(2002)022[0076MIAIIA]20CO2 2002

Hiler W R Wheeler B A and Trauth S E Abnormalities inthe Ozark Hellbender (Cryptobranchus alleganiensis bishopi) inArkansas A Comparison between Two Rivers with a Historical

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 167

Figure 13 The fused neural arches of two trunk vertebrae ZINPH 8243 of Eoscapherpeton asiaticum (a) Dorsal view (b) rightlateral view (c) left lateral view (d) left lateral view (digital restora-tion) (e) right lateral view (digital restoration) (f) dorsal view (digi-tal restoration) with the location of longitudinal the microCT digitalsection and (g) digital longitudinal section (vertical plane)

lowing reasons (1) it is the oldest occurrence of resegmen-tation in salamanders and (2) it is evidence that resegmen-tation is a common process for salamanders (including rela-tively basal cryptobranchids and advanced ambystomatids)

A hemivertebra in E asiaticum was found in a relativelylarge (according to the large size of the atlas that was co-ossified with hemivertebra) and presumably adult individual(Fig 6) The presence of a hemivertebra in a large individualsuggests that this pathology did not cause its death

The hemivertebrae have been reported earlier for somePaleozoic and Mesozoic temnospondyls (Witzmann et al2013) The finding of hemivertebra in the Late Cretaceouscryptobranchid E asiaticum is the oldest (and the first de-scribed) occurrence of this pathology among modern groupsof amphibians (ie lissamphibians salamanders frogs andcaecilians)

43 Femoral pathologies in Eoscapherpeton possibleinsight into behavior

The traumatic origin of pathologies on the distal parts of thefemora (Figs 14ndash15) could be evidence of frequent occur-rence of hind limb injuries in Eoscapherpeton asiaticum Inmodern cryptobranchids such as Cryptobranchus limb in-juries are the most common physical abnormalities in wildpopulations (Miller and Miller 2005) The proposed maincause of limb injuries in Cryptobranchus is intraspecific ag-

Figure 14 The distal parts of the femora ZIN PH 9243 (andashh) andZIN PH 10243 (indasho) of Eoscapherpeton asiaticum Specimen ZINPH 9243 (a) ventral view (b) dorsal view (c) ventral view (dig-ital restoration) (d) dorsal view (digital restoration) (e) anterioror posterior view (digital restoration) with the locations of trans-verse histological sections and longitudinal the microCT digital sec-tion (f) digital longitudinal section and (gndashh) histological trans-verse section The arrow shows the border between cortical boneand pathological tissue Specimen ZIN PH 10243 (i) ventral view(k) dorsal view (l) ventral view (digital restoration) with the loca-tions of the microCT digital sections (m) anterior or posterior view(digital restoration) (n) digital longitudinal section and (o) digitaltransverse section Blue lines show the original bone position priorto the break Abbreviations BE ndash bone enlargement C ndash callus CBndash cortical bone

gression during the reproductive season (eg defending ofnesting sites) because fresh injuries were found only dur-ing the reproductive season (namely in August and Septem-ber) (Hiler et al 2005 Miller and Miller 2005) Takinginto account the phylogenetic position of Eoscapherpetonas a member of the cryptobranchid clade and their aquaticlifestyle (see Skutschas 2009 2013) the reason for the fre-quent occurrence of hind limb injuries in E asiaticum couldbe the same (intraspecific aggression during reproductiveseason) and this behavioral element could have appearedearly in the evolution of cryptobranchids ie at least in theLate Cretaceous

wwwfoss-recnet211592018 Foss Rec 21 159ndash169 2018

168 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 15 The distal parts of the femora ZIN PH 11243 (andashf) andZIN PH 12243 (gndashp) of Eoscapherpeton asiaticum Specimen ZINPH 11243 (a) ventral view (b) dorsal view (c) dorsal view (dig-ital restoration) with the locations of the microCT digital sections(d) anterior or posterior view (digital restoration) (e) digital lon-gitudinal section and (f) digital transverse section Specimen ZINPH 12243 (g) ventral view (h) dorsal view (i) ventral view (dig-ital restoration) (k) dorsal view (digital restoration) (l) anterior orposterior view (digital restoration) with the locations of transversehistological sections and longitudinal the microCT digital section(m) digital longitudinal section and (nndashp) transverse histologicalsections Blue lines show the original bone position prior to thebreak Abbreviations C ndash callus

5 Conclusions

The study revealed diverse osseous anomaliespathologies inEoscapherpeton asiaticum which were interpreted as trau-matic possible traumatic-infectious and congenital in originpertaining to bones of the lower jaw vertebrae and femoraThe hemivertebra and fused vertebrae (without any traces oftrauma and inflammation) document the earliest occurrenceof vertebral congenital pathologies in crown-group salaman-ders The frequency of vertebral congenital pathologies in Easiaticum is low and in accordance with the previous estima-tions for temnospondyls and frogs The numerous traumaticpathologies of femora (hematoma and healed fractures) sug-gest the survival of accidents and possibly the presence of in-

traspecific aggression during the reproductive season in thissalamander (like in modern cryptobranchids)

Data availability Micro-CT data are archived at the Department ofVertebrate Zoology of the Saint Petersburg State University (SaintPetersburg Russia) and can be accessed through the present authorsfor the purpose of scientific study

Author contributions PS designed the project PS and VK per-formed the research VK and IK prepared 3-D reconstructions VKand EB prepared figures and PS wrote the manuscript All authorscommented on the manuscript

Competing interests The authors declare that they have no conflictof interest

Acknowledgements We are grateful to the staff of the SaintPetersburg State University Research Centre for X-ray DiffractionStudies (Saint Petersburg Russia) for their help with using theLeica 2500P microscope and for CT scanning of specimens Theauthors thank Judith Pardo-Peacuterez and Nadia Froumlbisch for providinghelpful comments that improved the quality of the manuscript Thisstudy was fulfilled under support of the Russian Scientific Fundproject 14-14-00015

Edited by Constanze BickelmannReviewed by Nadia Froumlbisch and Judith Pardo Perez

References

Burnham D A Rothschild B M Babiarz J P and Martin LD Hemivertebrae as pathology and as a window to behavior inthe fossil record Palarchrsquos Journal of Vertebrate Palaeontology10 1ndash6 2013

Francillon-Vieillot H de Buffreacutenil V Castanet J Geraudie JMeunier F Sire J Y Zylenberberg L and de Ricqles A DMicrostructure and mineralization of vertebrate skeletal tissuesin Skeletal biomineralization Patterns Processes and Evolu-tionary Trends Vol 1 edited by Carter J G Van NostrandReinhold New York 471ndash530 1990

Garciacutea R A Cerda I A Heller M Rothschild B M and Zur-riaguz V The first evidence of osteomyelitis in a sauropod di-nosaur Lethaia 50 227ndash236 httpsdoiorg101111let121892016

Hanna R R Multiple injury and infection in a sub adult thero-pod dinosaur Allosaurus fragilis with comparisons to allosaurpathology in Cleveland-Lloyd Dinosaur Quarry Collection JVertebr Paleontol 22 76ndash90 httpsdoiorg1016710272-4634(2002)022[0076MIAIIA]20CO2 2002

Hiler W R Wheeler B A and Trauth S E Abnormalities inthe Ozark Hellbender (Cryptobranchus alleganiensis bishopi) inArkansas A Comparison between Two Rivers with a Historical

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

168 P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum

Figure 15 The distal parts of the femora ZIN PH 11243 (andashf) andZIN PH 12243 (gndashp) of Eoscapherpeton asiaticum Specimen ZINPH 11243 (a) ventral view (b) dorsal view (c) dorsal view (dig-ital restoration) with the locations of the microCT digital sections(d) anterior or posterior view (digital restoration) (e) digital lon-gitudinal section and (f) digital transverse section Specimen ZINPH 12243 (g) ventral view (h) dorsal view (i) ventral view (dig-ital restoration) (k) dorsal view (digital restoration) (l) anterior orposterior view (digital restoration) with the locations of transversehistological sections and longitudinal the microCT digital section(m) digital longitudinal section and (nndashp) transverse histologicalsections Blue lines show the original bone position prior to thebreak Abbreviations C ndash callus

5 Conclusions

The study revealed diverse osseous anomaliespathologies inEoscapherpeton asiaticum which were interpreted as trau-matic possible traumatic-infectious and congenital in originpertaining to bones of the lower jaw vertebrae and femoraThe hemivertebra and fused vertebrae (without any traces oftrauma and inflammation) document the earliest occurrenceof vertebral congenital pathologies in crown-group salaman-ders The frequency of vertebral congenital pathologies in Easiaticum is low and in accordance with the previous estima-tions for temnospondyls and frogs The numerous traumaticpathologies of femora (hematoma and healed fractures) sug-gest the survival of accidents and possibly the presence of in-

traspecific aggression during the reproductive season in thissalamander (like in modern cryptobranchids)

Data availability Micro-CT data are archived at the Department ofVertebrate Zoology of the Saint Petersburg State University (SaintPetersburg Russia) and can be accessed through the present authorsfor the purpose of scientific study

Author contributions PS designed the project PS and VK per-formed the research VK and IK prepared 3-D reconstructions VKand EB prepared figures and PS wrote the manuscript All authorscommented on the manuscript

Competing interests The authors declare that they have no conflictof interest

Acknowledgements We are grateful to the staff of the SaintPetersburg State University Research Centre for X-ray DiffractionStudies (Saint Petersburg Russia) for their help with using theLeica 2500P microscope and for CT scanning of specimens Theauthors thank Judith Pardo-Peacuterez and Nadia Froumlbisch for providinghelpful comments that improved the quality of the manuscript Thisstudy was fulfilled under support of the Russian Scientific Fundproject 14-14-00015

Edited by Constanze BickelmannReviewed by Nadia Froumlbisch and Judith Pardo Perez

References

Burnham D A Rothschild B M Babiarz J P and Martin LD Hemivertebrae as pathology and as a window to behavior inthe fossil record Palarchrsquos Journal of Vertebrate Palaeontology10 1ndash6 2013

Francillon-Vieillot H de Buffreacutenil V Castanet J Geraudie JMeunier F Sire J Y Zylenberberg L and de Ricqles A DMicrostructure and mineralization of vertebrate skeletal tissuesin Skeletal biomineralization Patterns Processes and Evolu-tionary Trends Vol 1 edited by Carter J G Van NostrandReinhold New York 471ndash530 1990

Garciacutea R A Cerda I A Heller M Rothschild B M and Zur-riaguz V The first evidence of osteomyelitis in a sauropod di-nosaur Lethaia 50 227ndash236 httpsdoiorg101111let121892016

Hanna R R Multiple injury and infection in a sub adult thero-pod dinosaur Allosaurus fragilis with comparisons to allosaurpathology in Cleveland-Lloyd Dinosaur Quarry Collection JVertebr Paleontol 22 76ndash90 httpsdoiorg1016710272-4634(2002)022[0076MIAIIA]20CO2 2002

Hiler W R Wheeler B A and Trauth S E Abnormalities inthe Ozark Hellbender (Cryptobranchus alleganiensis bishopi) inArkansas A Comparison between Two Rivers with a Historical

Foss Rec 21 159ndash169 2018 wwwfoss-recnet211592018

P Skutschas et al Osseous anomalies of the cryptobranchid Eoscapherpeton asiaticum 169

Perspective Journal of the Arkansas Academy of Science 5988ndash94 2005

Marais L C Ferreira N Aldous C and Le Roux T L BThe classification of chronic osteomyelitis South African Or-thopoedic Journal 13 22ndash28 2014

Martinelli A G Teixeira V P A Marinho T S FonsecaP H M Cavellani C L Araujo A J G Ribeiro LC B and Ferraz M L F Fused mid-caudal vertebraein the titanosaur Uberabatitan ribeiroi from the Late Creta-ceous of Brazil and other bone lesions Lethaia 48 456ndash462httpsdoiorg101111let12117 2015

Miller B T and Miller J R Prevalence of physical abnormali-ties in Eastern Hellbender (Cryptobranchus alleganiensis alle-ganiensis) populations of Middle Tennessee Southeast Nat 4513ndash520 2005

Nesov L A Cretaceous salamanders and frogs of KizylkumDesert (in Russian) Trudy Zoologicheskogo Instituta AN SSSR101 57ndash88 1981

Nesov L A Cretaceous nonmarine vertebrates of Northern Eura-sia Posthumous edited by Golovneva L B and Averianov AO Izdatelrsquostvo Sankt-Peterburgskogo Universiteta Saint Peters-burg 218 pp 1997 (in Russian)

Pardo-Peacuterez J M Kear B P Goacutemez M Moroni M andMaxwell E E Ichthyosaurian palaeopathology evidence of in-jury and disease in fossil ldquofish lizardsrdquo J Zool 304 21ndash332017

Peterson J E and Vittore C P Cranial pathologies in aspecimen of Pachycephalosaurus PLoS One 7 e36227httpsdoiorg101371journalpone0036227 2012

Piekarski N and Olsson L Resegmentation in the Mexican Ax-olotl Ambystoma Mexicanum J Morphol 275 141ndash152 2014

Ramiacuterez-Velasco A A Morales E Rivera R H andTanke D Spinal and rib osteopathy in Huehuecanauhtlustiquichensis (Ornithopoda Hadrosauroidea) from theLate Cretaceous in Mexico Hist Biol 29 208ndash222httpsdoiorg1010800891296320161147033 2016

Redman C M and Leighton L R Multivariate faunal analysisof the Turonian Bissekty Formation variation in the degree ofmarine influence in temporally and spatially averaged fossil as-semblages Palaios 24 18ndash26 2009

Rothschild B M Macroscopic recognition of non-traumatic os-seous pathology in the post-cranial skeletons of crocodiliansand lizards J Herpetol 44 13ndash20 httpsdoiorg10167008-2431 2010

Rothschild B M and Laub R S Epidemiology of anuran pathol-ogy in the holocene component of the Hiscock site rare or notsurvived J Herpetol 47 169ndash173 2013

Rothschild B M and Martin L D Skeletal impact of diseaseBull New Mexico Mus Natl Hist 33 1ndash230 2006

Rothschild B M and Tanke D Paleopathology of vertebratesinsights to lifestyle and health in the geological record GeosciCan 19 72ndash82 1991

Rothschild B M Schultze H-P and Pellegrini R Herpetologi-cal Osteopathology Annotated Bibliography of Amphibians andReptiles Heidelberg Springer 450 pp 2012

Skutschas P P Re-evaluation of Mynbulakia Nesov 1981 (Lis-samphibia Caudata) and description of a new salamander genusfrom the Late Cretaceous of Uzbekistan J Vertebr Paleontol29 1ndash6 2009

Skutschas P P Mesozoic salamanders and albanerpetontids ofMiddle Asia Kazakhstan and Siberia in Mesozoic and Ceno-zoic lissamphibian and squamate assemblages of Laurasia editedby Gardner J D and Nydam R L Palaeobiodiversity andPalaeoenvironments 93 444ndash457 2013

Waldron T Palaeopathology Cambridge Manuals in ArchaeologyCambridge University Press Cambridge 279 pp 2009

Wang Y Dong L and Evans S E Polydactyly and otherlimb abnormalities in the Jurassic salamander Chunerpeton fromChina Palaeobio Palaeoenv 96 49ndash59 2016

Witzmann F Asbach P Remes K Hampe O Hilger A andPaulke A Vertebral pathology in an ornithopod dinosaur ahemivertebra in Dysalotosaurus lettowvorbecki from the Juras-sic of Tanzania Anat Rec 291 1149ndash1155 2008

Witzmann F Rothschild B M Hampe O Sobral G Gubin YM and Asbach P Congenital malformations of the vertebralcolumn in ancient amphibians Anat Histol Embryol 43 90ndash102 2013

wwwfoss-recnet211592018 Foss Rec 21 159ndash169 2018