Amniote vertebral microanatomy - what are the major trends?

Amniote vertebral microanatomy – what are themajor trends?

ALEXANDRA HOUSSAYE1,2*, PAUL TAFFOREAU3 and ANTHONY HERREL2,4

1Steinmann Institut für Geologie, Paläontologie und Mineralogie, Universität Bonn, Nussallee 8,53115 Bonn, Germany2UMR 7179 du CNRS, Département Ecologie et Gestion de la Biodiversité, Muséum Nationald’Histoire Naturelle, 57 rue Cuvier CP-55, 75000 Paris, France3European Synchrotron Radiation Facility, BP220, 6 rue Jules Horowitz, 38043 Grenoble Cedex,France4Evolutionary Morphology of Vertebrates, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent,Belgium

Received 1 January 2014; revised 24 January 2014; accepted for publication 6 March 2014

This contribution qualitatively and quantitatively analyses vertebral microanatomical features based on virtualsections of numerous amniote dorsal vertebrae obtained from conventional and synchrotron X-raymicrotomographic investigations. It demonstrates the great diversity of amniote vertebral microanatomy andhighlights that it reflects structural, phylogenetic and ecological signals. Various microanatomical parametersappear to be strongly correlated with overall body size, which seems to be the principal structural constraint. Aphylogenetic signal was detected but appears rather low. This study also reveals the peculiarity of squamatesamong amniotes, and notably of squamate fossorial taxa that show clearly distinct trends from those of the otherfossorial amniotes, probably as they essentially use movements of the vertebral column rather than the legs to dig.Analyses based on habitat reveal several trends and two main tendencies concerning the tightness of the spongiosa(squamates excluded): a low number of relatively thick trabeculae in arboreal, flying and fossorial taxa, versus ahigh number of relatively thin trabeculae in aquatic forms. It also suggests that comparisons based on functionalrequirements, rather than habitat, would be more relevant. © 2014 The Linnean Society of London, BiologicalJournal of the Linnean Society, 2014, 112, 735–746.

ADDITIONAL KEYWORDS: habitat – phylogenetic signal – size effect – squamate peculiarity – vertebrae.

INTRODUCTION

Bone is a living structure and as such it recordsinformation about the biology and ecology of anorganism. According to the constructional morphologymodel of Seilacher (Seilacher, 1970; Gould, 2002;Cubo, 2004), biological features are considered as theoutcome of phylogenetic, adaptative, and architec-tural constraints, referred to as historical, functionaland structural constraints by Gould (2002). This isalso the case for bone microanatomical features, i.e.bone internal organization.

It appears of particular interest to analyse thesevarious constraints in several different bones underdifferent functional constraints. These signals havepreviously been investigated to some degree inamniote long bones (e.g. Germain & Laurin, 2005;Kriloff et al., 2008; Quemeneur, de Buffrénil &Laurin, 2013) but also, more recently, in vertebrae ofmammals to analyse differences linked with the sec-ondary adaptation to an aquatic life (Dumont et al.,2013), and in squamates (Houssaye et al., 2010;2013). The present contribution proposes to investi-gate the diversity in vertebral microanatomy amongamniotes in general and notably to test if significantdifferences are observable depending on ecology,taking into consideration the presence of a potential*Corresponding author. E-mail: [email protected]

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Biological Journal of the Linnean Society, 2014, 112, 735–746. With 3 figures

© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 735–746 735

mailto:[email protected]

phylogenetic signal in the data. We predict that thevertebrae of fossorial taxa will show a higher com-pactness than those of terrestrial and arboreal taxa,whereas flying taxa will be much lighter. We alsopredict different microanatomical specializations insemi-aquatic and aquatic taxa, depending on theirfunctional requirements.

MATERIAL AND METHODS

The material consists of mid-dorsal vertebrae of 72amniote species (see Table 1). These vertebrae,located above the lungs, were chosen to analyse thebiological and ecological adaptations of the thorax,from a bone microanatomical perspective. The taxawere sampled to encompass the diversity of amniotesfrom both phylogenetic (see Fig. 1) and ecologicalperspectives. Both longitudinal (in the mid-sagittalplane) and transverse (in the neutral transverseplane; see de Buffrénil et al., 2008) thin sections wereanalysed for the present study.

All new sections were obtained from microtomo-graphic investigations, allowing a non-destructiveimaging of the three-dimensional outer and innerstructure of the samples. Both conventional and syn-chrotron X-ray microtomography were used: (1) high-resolution computed tomography (Gephoenix|X-rayv|tome|xs 180 and 240; reconstructions performedusing datox/res software) at the Steinmann-Institut,University of Bonn (Germany) and (2) third-generationsynchrotron microtomography (Tafforeau et al., 2006)at the European Synchrotron Radiation Facility(ESRF, Grenoble, France) at the ID17 and ID19 beamlines (reconstruction performed using filtered back-projection algorithm with the ESRF PyHST software).It has previously been observed that these distincttechniques do not imply artefacts and do not biasinterpretation of the results for comparative analyses(A.H., pers. observ.; M. Dumont; pers. comm.). Imagesegmentation and visualization were performedusing VGStudioMax 2.0 and 2.1 (Volume Graphics).Some additional sections, either histological thinsections or virtual thin sections, come from previousstudies (Houssaye et al., 2010, 2013; Hayashi et al.,2013).

QUANTITATIVE ANALYSES

Measurements were taken directly on the sectionsusing, except for ‘centrum length’ (CL), ImageJ(Abramoff, Magelhaes & Ram, 2004) and an in-housedeveloped software routine (‘LineTrab’, available fromthe corresponding author upon request). The meas-urements taken were: (a) the length of the centrumbetween the condylar and cotylar rims (CL), which is

used as an indicator of specimen size; (b) the globalcompactness of the centrum in longitudinal section(Cls), calculated as the total area of the centrumminus the area occupied by cavities multiplied by 100and divided by the total area of the centrum; (c) theglobal compactness in transverse section (Cts), calcu-lated as the total sectional area minus the area occu-pied by cavities and the neural canal multiplied by100 and divided by the total area minus the areaoccupied by the neural canal; (d) the centrum com-pactness in transverse section (CtsC), calculated asthe total sectional area minus the area occupied bycavities multiplied by 100 and divided by the totalarea, in an area defined as the transverse sectionalarea below a horizontal line located at the base of theneural canal; (e) the total number of cavities in lon-gitudinal section (TNCL); (f) the total number ofcavities in transverse section (TNCT); (g) the areaoccupied by the neural canal (SNC), calculated asthe area occupied by the neural canal multiplied by100 and divided by the total sectional area; (h) thenumber of trabeculae in the centrum longitudinalsection (NTCL), calculated as an average value basedon three dorso-ventral cuts made along the centrumat about the neutral transverse plane (NTP) and attwo planes equidistant from the NTP, the anterior onebeing at about one-quarter of the centrum length; (i)the absolute mean cortical thickness in transversesection (AMCT), calculated as the average of tenmeasurements of the thickness of the peripheral layerof periosteal bone taken rather regularly along thecentrum; (j) the relative mean cortical thickness intransverse section (RMCT), calculated as AMCT mul-tiplied by 100 and divided by the centrum height; (k)the mean thickness of the bone layer surrounding theneural canal (MTNCP) in transverse section, calcu-lated as the average of ten measurements of thethickness of this layer taken rather regularly over360°.

It was decided not to define the lines where thevalues for the indices i and k were calculated alongperfect regular intervals (e.g. every 36° for MTNCP),as this would be biased, notably because of thecomplex geometry of the vertebrae and the unwantedinclusion of trabeculae connected to the layers ofinterest.

(l) The absolute mean trabecular thickness(AMTT), calculated as the thickness of bone dividedby the number of trabeculae, taken on the same threecuts as NTCL; (m) the relative mean trabecular thick-ness (RMTT), calculated as the thickness of bonedivided by the length of the cut, multiplied by 100 anddivided by the number of trabeculae, taken on thesame three cuts as NTCL.

AMTT and RMTT correspond to the mean valuesobtained from the three cuts.

736 A. HOUSSAYE ET AL.

© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 735–746

Tab

le1.

Lis

tof

the

mat

eria

lan

alys

edw

ith

corr

espo

ndi

ng

indi

ces

Fam

ily

Taxo

nA

b.C

oll.

no.

Res

olu

tion

(μm

)E

colo

gyC

L(m

m)

Cls

Cts

Cts

CT

NC

LT

NC

TS

NC

NT

CL

AM

CT

RM

CT

MT

NC

PA

MT

TR

MT

T

Squ

amat

aS

cin

cida

eS

cin

cus

scin

cus

Ss

MN

HN

SQ

-Ver

t5

XF

os3.

050

.862

.353

.111

734

.74.

754

8.6

17.1

253.

381

.98.

0Te

iida

eTu

pin

ambi

ste

guix

inT

tM

NH

NA

C19

2010

816

.7Te

r6.

851

.659

.439

.944

4015

.82.

718

32.9

76.4

1249

.223

0.0

12.8

Am

phis

baen

idae

Am

phis

baen

aal

baA

aM

NH

NA

C19

8602

04X

Fos

4.9

73.1

81.5

79.0

4327

26.0

4.7

1787

.922

.394

1.1

219.

614

.5Ig

uan

idae

Am

blyr

hyn

chu

scr

ista

tus

Ac

ZF

MK

1083

4X

SA

13.2

55.8

44.6

57.6

128

–7.

26.

715

43.8

28.6

502.

618

2.6

4.8

An

guid

aeA

ngu

isfr

agil

isA

fM

NH

NA

C19

9601

99X

Fos

3.8

47.5

65.2

57.7

1726

27.4

4.0

317.

49.

628

2.9

93.0

8.4

Var

anid

aeVa

ran

us

ben

gale

nsi

sV

bM

NH

NS

Q-V

ert

XTe

r11

.259

.858

.147

.584

4610

.94.

011

12.2

37.1

313.

627

0.0

9.5

Trop

idop

hii

dae

Trac

hyb

oabo

ule

nge

riT

bA

HS

0001

9.4

Fos

3.9

82.9

83.1

69.1

101

1114

.93.

345

7.3

24.1

424.

648

7.9

21.7

Pyt

hon

idae

Bot

hro

chil

us

boa

Bb

ZF

MK

5203

25.7

Ter

5.2

73.0

86.9

73.0

6820

22.4

4.0

836.

327

.076

0.3

478.

016

.2A

croc

hor

dida

eA

croc

hor

du

sja

van

icu

sA

jM

NH

NS

Q-V

ert

14X

Aq

8.4

66.8

77.5

69.3

3512

19.9

4.0

1538

.013

.921

73.3

539.

215

.9E

lapi

dae

Oph

ioph

agu

sh

ann

ahO

hM

NH

NA

C20

0242

32.3

Ter

12.5

75.2

72.1

59.9

8526

14.9

4.7

519.

114

.060

1.0

598.

211

.9Te

stu

din

esE

myd

idae

Em

ysor

bicu

lari

sE

oM

NH

NR

EP

32X

Ter

2.8

58.0

81.6

67.6

81–

24.1

4.7

395.

29.

928

5.6

94.1

14.0

Cro

cody

lia

Cro

cody

lida

eC

roco

dyl

us

nil

otic

us

Cn

MN

HN

AC

1964

-403

73.1

SA

9.5

67.8

–55

.641

6–

–20

.725

6.5

6.6

–14

2.2

3.2

All

igat

orid

aeA

llig

ator

mis

siss

ippi

ensi

sA

mS

TIP

BR

599b

60.1

SA

17.5

52.9

83.4

74.7

134

1129

.59.

378

8.0

17.9

449.

623

7.0

4.7

Ave

sA

nat

idae

An

ser

anse

rA

aS

TIP

BR

629

30.9

Fly

18.4

31.2

38.2

30.2

4217

29.2

5.7

235.

16.

031

0.8

190.

55.

0P

has

ian

idae

Gal

lus

gall

us

Gg

ST

IPB

R63

032

.4Te

r14

.142

.954

.147

.927

2327

5.3

424.

29.

236

7.1

306.

46.

9P

has

ian

us

colc

hic

us

Pc

Hou

ssay

eP

C46

.9Te

r13

.335

.545

.332

.625

2338

.73.

323

7.0

11.8

138.

315

7.6

6.4

Mel

eagr

isga

llop

avo

Mg

ST

IPB

R46

257

.5Te

r16

.928

.724

.533

.184

373.

37.

049

1.7

5.1

213.

125

9.2

2.9

Sph

enis

cida

eA

pten

odyt

espa

tago

nic

us

Ap

Un

nu

mbe

red

UP

MC

45.4

SA

21.2

25.5

77.8

67.4

116

7745

.916

.350

0.4

8.8

419.

813

7.1

2.5

Ch

arad

riif

orm

esIn

det

.C

iS

TIP

BR

461

31.4

Fly

31.4

33.7

68.5

39.6

2516

722.

710

8.1

6.4

80.2

634.

49.

1S

trig

idae

Ind

et.

Si

Taff

orea

uP

CF

ly4.

823

.832

.323

.451

3743

.43.

087

.23.

110

9.1

58.5

4.2

Col

um

bida

eC

olu

mba

palu

mbu

sC

pH

ouss

aye

PC

46.9

Fly

8.7

30.7

45.8

46.9

1313

32.2

3.3

179.

58.

211

2.6

130.

15.

8P

sitt

acid

aeA

rach

loro

pter

aA

cS

TIP

BR

536

29.4

Fly

7.3

22.3

29.7

28.8

3059

28.4

4.3

27.5

2.1

43.0

80.7

2.6

Mam

mal

iaO

rnit

hor

hyn

chid

aeO

rnit

hor

hyn

chu

san

atin

us

Oa

MH

NL

5000

0986

5.1

SA

4.5

4439

.828

.033

1429

.58

87.7

2.7

165.

119

7.9

6.2

Did

elph

idae

Did

elph

isvi

rgin

ian

aD

vM

6585

34.5

Arb

6.8

39.3

49.1

42.4

2110

13.5

5.3

307.

99.

394

5.2

218.

46.

1M

acro

podi

dae

Mac

ropu

sru

fus

Mr

ZF

MK

8324

36.4

Ter

16.4

40.2

–40

.110

9–

–14

.341

4.9

4.9

–17

0.1

2.0

Tric

hec

hid

aeTr

ich

ech

us

man

atu

sT

mZ

FM

K73

.223

121.

5A

q32

.366

.363

.148

.562

558

416

.557

1493

.45.

514

53.5

284.

61.

0C

hry

soch

lori

dae

Ch

loro

talp

ale

uco

rhin

aC

mM

HN

L50

0000

565.

1F

os2.

635

.533

.630

.410

411

14.8

9.3

58.0

4.1

46.8

40.7

2.9

Bra

dypo

dida

eB

rad

ypu

sB

Un

nu

mbe

red

ZF

MK

30.7

Arb

10.4

47.9

–51

.656

––

13.3

263.

34.

8–

158.

52.

6D

asyp

odid

aeD

asyp

us

nov

emci

nct

us

Dn

ST

IPB

M25

2346

.9Te

r7.

459

.468

.058

.756

8934

.412

.741

3.7

11.2

266.

713

7.1

3.8

Cas

tori

dae

Cas

tor

fibe

rC

fZ

FM

K20

06.0

0728

.5S

A7.

923

.952

.433

.537

226

25.1

20.7

263.

73.

665

3.3

77.7

1.1

Sci

uri

dae

Sci

uru

svu

lgar

isS

vTa

ffor

eau

PC

5.1

Arb

4.5

33.9

44.5

27.9

4763

49.1

9.3

84.2

4.4

109.

776

.43.

6B

ath

yerg

idae

Het

eroc

eph

alu

sgl

aber

Hg

ST

IPB

M10

8820

.7F

os2.

641

.827

.824

.814

615

.34.

780

.57.

334

.249

.94.

6M

yoca

stor

idae

Myo

cast

orco

ypu

sM

cM

HN

L50

0007

0830

.3S

A6.

445

.066

.047

.499

181

31.7

17.7

298.

77.

542

4.5

129.

12.

4C

avii

dae

Cav

iapo

rcel

lus

Cp

ST

IPB

M61

7348

.6Te

r3.

661

.047

.833

.019

1030

.28

138.

96.

013

8.1

132.

86.

0C

ynoc

eph

alid

aeC

ynoc

eph

alu

svo

lan

sC

vM

NH

NA

C(n

ore

f.)

28.4

Arb

2.9

52.5

54.3

39.4

3031

27.2

9.3

129.

35.

417

5.8

83.9

3.9

Cer

copi

thec

idae

Mac

aca

sp.

Ms

Un

nu

mbe

red

UP

MC

35.2

Ter

8.4

46.7

50.8

3018

144

23.5

14.7

193.

83.

936

8.4

70.5

1.3

Eri

nac

eida

eE

rin

aceu

seu

ropa

eus

Ee

Taff

orea

uP

C10

.1Te

r4.

341

.633

.225

.592

7728

.89.

714

0.7

6.1

134.

110

1.3

3.7

Sor

icid

aeS

orex

aran

eus

Sa

MN

HN

1961

-593

5.1

Ter

0.9

68.0

42.5

24.7

115

47.0

6.7

32.9

4.7

34.2

34.2

4.5

AMNIOTE VERTEBRAL MICROANATOMY 737


Tab

le1.

Con

tin

ued

Fam

ily

Taxo

nA

b.C

oll.

no.

Res

olu

tion

(μm

)E

colo

gyC

L(m

m)

Cls

Cts

Cts

CT

NC

LT

NC

TS

NC

NT

CL

AM

CT

RM

CT

MT

NC

PA

MT

TR

MT

T

Talp

idae

Gal

emys

pyre

nai

cus

Gp

MH

NL

5000

0020

5.1

SA

1.2

33.5

34.6

20.9

8437

39.3

6.7

57.6

4.8

62.0

36.0

2.9

1.2

33.5

47.4

23.9

8426

41.9

–73

.56.

181

.3S

calo

pus

aqu

atic

us

Sa

MH

NL

5000

0050

5.1

Fos

2.1

27.9

28.9

18.7

3636

31.0

454

.04.

244

.463

.64.

52.

429

.834

.022

.09

1528

.56.

762

.43.

759

.840

.32.

6T

alpa

euro

paea

TeM

NH

N19

96-5

365.

1F

os1.

931

.230

.020

.842 69

6 431

.57.

35.

368

.15.

757

.032

.447

.144

.54.

23.

5F

elid

aeP

anth

era

leo

Pl

ZF

MK

2006

.031

65.0

Ter

32.2

43.3

45.9

43.4

569

908

26.2

57.7

526.

72.

445

8.6

127.

70.

6F

elis

sylv

estr

isF

sU

nn

um

bere

dZ

FM

K27

.3Te

r7.

738

.8–

43.6

46–

–10

381.

010

.6–

148.

23.

9C

anid

aeVu

lpes

vulp

esV

vU

nn

um

bere

dU

PM

C60

.1Te

r13

.845

.260

.252

.664

7938

.86

525.

712

.528

7.5

294.

46.

8C

anis

lupu

sC

lH

ouss

aye

PC

51.9

Ter

16.8

45.2

42.8

36.1

215

213

19.2

25.7

306.

03.

113

2.1

179.

61.

8U

rsid

aeTr

emar

ctos

orn

atu

sTo

ZF

MK

97.2

7560

.7Te

r34

.043

.861

.248

.430

398

416

.661

.311

15.8

4.3

938.

114

2.1

0.5

Urs

us

mar

itim

us

Um

ZF

MK

2005

.356

78.2

SA

37.0

48.7

52.5

48.1

786

2442

11.7

79.7

719.

02.

153

2.5

184.

90.

5O

tari

idae

Zal

oph

us

cali

forn

ian

us

Zc

ZF

MK

49.9

846

.8A

q22

.920

.835

.429

.646

237

14.8

43.7

147.

70.

643

5.1

95.8

0.4

Ota

ria

byro

nia

Ob

MN

HN

AC

(no

ref.

)50

.7A

q17

.045

.5–

44.5

232

––

4024

2.3

2.5

–90

.20.

9P

hoc

idae

Ph

oca

vitu

lin

aP

vS

TIP

BM

6064

.4A

q24

.837

.632

.426

.823

227

532

.038

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atic

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t.



Figure 1. Consensus phylogenetic tree (essentially from Motokawa, 2004; Livezey & Zusi, 2007; Lee, 2009; Kan et al.,2010; Meredith et al., 2011; Yu et al., 2011; Hedges, 2012; Wiens et al., 2012; Lee, 2013; Yang et al., 2013).



All data were log10-transformed prior to analyses(except SNC for which √SNC was used) to meetassumptions of normality and homoscedasticityrequired for parametric analyses. Only NTCL couldnot be transformed to meet these assumptions. Para-metric analyses are considered robust enough for thisparameter to remain of interest, although the resultsshould be interpreted with caution.

The amount of phylogenetic signal was investigatedfor the different parameters analysed. Statistical testswere performed using a consensus phylogeny derivedfrom several published phylogenies (Motokawa, 2004;Livezey & Zusi, 2007; Lee, 2009, 2013; Kan et al., 2010;Meredith et al., 2011; Yu et al., 2011; Hedges, 2012;Wiens et al., 2012; Yang et al., 2013; Fig. 1). We calcu-lated the K-statistic following Blomberg, Garland &Ives (2003), which compares the observed phylogeneticsignal in a trait with the signal under a Brownianmotion model of trait evolution. A K-value lower than1 implies less similarity between relatives thanexpected under Brownian motion. We also performedrandomization tests and random tree generationtests following Germain & Laurin (2005) to test thephylogenetic signal of each parameter. Analyses werefirst performed independently based on all data avail-able for each parameter, and then on the taxon dataavailable for all parameters. Species means were usedwhen several specimens were available for the samespecies.

We tested the influence of size (using CL as ourestimate of size) on the various microanatomicalparameters using linear regression analyses. As aphylogenetic signal was generally detected, we calcu-lated independent contrasts and forced regressionsthrough the origin. We also analysed the correlationbetween Cts and CtsC to test if the compactness ofthe centrum was a good estimate of the compactnessof the whole section. All these analyses were per-formed using the statistic software R (R DevelopmentCore Team, Vienna, Austria).

A principal component analysis (PCA), also usingthe statistical software R, was conducted on thedataset for which all variables are available toexplore the distribution of the different taxa inmorphospace.

To test the impact of habitat on the vertebralmicroanatomical features, the sampled taxa wereclassified into six habitat categories: fossorial, terres-trial and generalist, arboreal, flying, semi-aquatic,aquatic. ANOVAs, ANCOVAs (when a size effectwas detected) and phylogenetic ANOVAs (when aphylogenetic effect was detected) and ANCOVAs(when both size and phylogenetic effects weredetected) were performed. All analyses were per-formed using R (R Development Core Team), exceptphylogenetic ANCOVAs, which required the use of the

PDSIMUL and PDANOVA routines implemented inPDAP (Garland et al., 1993). In the PDSIMULprogram, we used Brownian motion as our modelfor evolutionary change and ran 1000 unboundedsimulations to create an empirical null distributionagainst which the F-value from the original datacould be compared.

RESULTSQUANTITATIVE DATA

The K statistics calculated are all much lower than 1(0.32 < K < 0.64) except for RMTT (K = 1.05 and 1.09).However, the randomization tests and the randomtree generation indicate a significant phylogeneticsignal for all parameters.

Linear regressions on the independent contrastdata show an impact of size on the parameters TNCL[adjusted R2 (aR2) = 0.38, P << 0.0001], TNCT (aR2 =0.43; P << 0.0001), NTCL (aR2 = 0.31; P << 0.0001),AMCT (aR2 = 0.46; P << 0.0001), MTNCP (aR2 = 0.41;P << 0.0001), RMTT (aR2 = 0.35; P << 0.0001; nega-tive relationship) and AMTT, but not Cls (aR2 = –0.01;P = 0.96), Cts (aR2 = 0.01; P = 0.21), CtsC (aR2 = 0.03;P = 0.08), SNC (aR2 = 0.04; P = 0.07) and RMCT(aR2 = –0.0084; P = 0.52). Moreover, Cts and CtsC arestrongly correlated (r = 0.86; P << 0.0001).

The PCA (see Fig. 2) shows that the two mainaxes explain 74.7% of the variance (41.4 and 33.3%,respectively). It notably highlights the peculiarity ofsquamates among amniotes. Indeed, despite differentecologies, all squamates group together, away fromthe other amniotes sampled (as highlighted by thegreen area in Fig. 2A). This result is in accordancewith previous studies on squamate vertebral micro-anatomy that suggested a peculiar microanatomicalorganization within this group (Houssaye et al., 2010,2013). This essentially is related to the variablesRMCT, RMTT, Cls and Cts, which quantify the rela-tive thickness of the bone layer surrounding theperiphery of the bone and the relative thickness ofthe trabeculae within the centrum, respectively, andthe compactness in longitudinal and transverse sec-tions. This is consistent with the description of a thickperipheral layer in extant squamates, a low numberof trabeculae that are thus relatively thick, and therelatively high inner compactness observed (cf.Houssaye et al., 2010, 2013). Because of this result,which would bias the analysis of the dataset in lightof ecology, another PCA was conducted excludingsquamates. On the second analysis, the two mainaxes explain 72.6% of the variance (49.6 and 23.0%respectively; Fig. 2B).

These two analyses also enable us to determinewhich variables co-varied. This is, for example,



clearly the case for Cls and Cts, which show that,despite the fact that the longitudinal section displaysbone of both endochondral and periosteal origin incontrast to the transverse neutral section, which onlydisplays bone of periosteal origin, the compactnessseems to vary rather homogeneously within the wholebone. MTNCP and AMCT also vary together. Theyexpress the absolute thickness of the bone layerssurrounding the neural canal and the centrumperiphery, respectively. NTCL, TNCL and TNCT logi-cally combine their action as they all express thetightness of the trabecular network in the spongiosa.Although the various ecologies clearly overlap onthe graphs resulting from the PCA, some tendenciesare nevertheless clear. Fossorial taxa clearly grouptogether. They are characterized by a relativelylow compactness, a low number of relatively thicktrabeculae, a rather large neural canal, and therather low absolute thickness of the bone layers sur-rounding the neural canal and the centrum periphery.Although they do not group together, all the flyingand arboreal taxa are distributed on only one part ofthe graph, on the same side as the fossorial taxa.They are characterized by similar microanatomicalfeatures (see above). Terrestrial and semi-aquatictaxa are distributed rather randomly.

The link between the microanatomical features andecology was subsequently analysed using ANOVAsand, because of the important impact of size on several

variables, ANCOVAs. These analyses were conductedwith squamates both included and excluded. Somegeneral trends could be observed based on these analy-ses (see Table 2). Cls appears slightly lower in flyingorganisms; without squamates, this is the case for bothfossorial and flying taxa. Cts appears lower in aquatictaxa, and this result is confirmed when squamates areremoved, but with the addition of much lower valuesfor fossorial taxa. Without squamates, CtsC alsoclearly shows lower values for fossorial taxa (but notfor aquatic ones). TNCT and TNCL appear muchhigher in aquatic taxa, and rather low in fossorial,arboreal and flying taxa (with and withoutsquamates). SNC shows higher values for flying taxaand lower values for aquatic taxa. NTCL shows par-ticularly low values for fossorial, arboreal and flyingtaxa, and high values for aquatic taxa. AMCT showslower values for flying taxa; when squamates areremoved lower values are also displayed by fossorialtaxa. RMCT shows lower values for aquatic forms.MTNCP shows high values for aquatic forms and lowvalues for flying taxa, as well as for fossorial taxa whensquamates are removed. AMTT shows lower values forflying and fossorial taxa. RMTT shows much lowervalues for aquatic taxa and rather high values forfossorial, arboreal and flying taxa.

Beyond these trends, only a few differences in ver-tebral microanatomical features depending on habitatwere revealed to be significant by the analyses of

Figure 2. Microanatomical clusters obtained by PCAs. Graphs showing the distribution of the variance in all taxaexamined for which all parameters were available according to the PCA1 and PCA2 axes. A, based on all taxa. B, withsquamates excluded from the analysis. Abbreviations for the taxa in the PCA graphs are as in Table 1.



(co)variance. An impact of ecology on microanatomicalfeatures, when the six habitats were taken into con-sideration (see Table 3), was revealed for (1) Cls,AMCT and RMCT; (2) TNCT and SNC (only withsquamates included); and (3) AMTT (only withsquamates excluded), and for Cls, RMCT and SNCwhen the phylogeny was taken into consideration.Based on the general trends observed, the analyseswere redone with only three habitat categories: (a)terrestrial and semi-aquatic taxa, (b) arboreal,fossorial and flying taxa, and (c) aquatic forms. Theseanalyses revealed significant differences for (1) Ctsand AMTT (when the phylogeny was not taken intoconsideration), (2) AMCT and (3) RMCT (only whenthe phylogeny was taken into consideration). The Fvalues obtained for the phylogenetic ANCOVAs andANOVAs are variably higher or lower than thoseobtained in the traditional analyses. This shows theoccurrence of some phylogenetic signal in the data,

but also suggests that it is rather low. Only AMCTshows a significant ecological signal in all analyses,even when the phylogeny is taken into consideration.

QUALITATIVE DATA

A wide range of microanatomical organization isobserved within our sample. Differences can thus behighlighted. Because of the peculiarity of squamatevertebrae, the following part only describes the non-squamate amniotes. Squamate features have beendescribed by Houssaye et al. (2010, 2013).

Variation is observed among terrestrial and gener-alist taxa, from the general pattern described byHayashi et al. (2013), with some taxa showing athicker cortex and fewer trabeculae (e.g. Felis, Sorex,Philantomba), Emys displaying a particularly thickcortex, and birds showing a rather light structure(notably Phasianus and Meleagris). Some trends areobserved for the other habitats (Fig. 3). Fossorial taxashow a rather ‘hollow’ structure with a peculiarly lownumber of trabeculae (Fig. 3A, D). This is also thecase for arboreal and flying taxa (Fig. 3B, C, E, F),except Bradypus and Pteropus, which show a tight-ness of the spongiosa similar to that of terrestrialtaxa. Flying taxa also show a larger neural canal(Fig. 3F). Semi-aquatic taxa show diverse trends: atight spongiosa with a rather thick cortex (e.g. inUrsus, Hippopotamus; Fig. 3J), a lighter structure(with fewer and/or thinner trabeculae and corticallayer; e.g. in Castor, Galemys; Fig. 3G) and a particu-larly thick cortex (e.g. in Crocodylus, Aptenodytes;Fig. 3H, K). Aquatic taxa clearly show a trendtowards an increase in the tightness of the spongiosawith no increase in cortical thickness (Fig. 3I, L),although to a lesser extent in Enhydra, which is theleast efficient diver in our sample. In contrast to thisgeneral trend, Trichechus displays a strong increasein the thickness of the bone layers surrounding theneural canal and the vertebral periphery (seeHayashi et al., 2013, fig. 12A inside); this is consistentwith its mode of life as, contrary to the others,Trichechus is a poorly efficient shallow diver charac-terized by bone mass increase (see Houssaye, 2009).

DISCUSSIONSPECIFICITY OF SQUAMATES

As suggested by previous studies (Houssayeet al., 2010; 2013), squamates display peculiar micro-anatomical features among amniotes. Differencesbetween the analyses conducted with squamatesincluded or excluded notably highlight the peculiarityof squamate fossorial taxa, which clearly show trendsdistinct from those of the other fossorial amniotes.Fossorial squamates indeed show a much higher

Table 2. Peculiar microanatomical features linked tosome habitats

Habitat Features

Fossorial Cls lowCts lowCtsC lowTNCT lowTNCL lowNTCL lowAMCT lowMTNCP lowAMTT lowRMTT high

Arboreal TNCT lowTNCL lowNTCL lowRMTT high

Flying Cls lowTNCT lowTNCL lowSNC highNTCL lowAMCT lowMTNCP lowAMTT lowRMTT high

Aquatic Cts lowTNCT highTNCL highSNC lowNTCL highRMCT lowMTNCP highRMTT low



Tab

le3.

Fan

dP

valu

esob

tain

edfo

rth

eva

riou

san

alys

esof

(co)

vari

ance

Mea

sure

men

tC

lsC

tsC

tsC

TN

CT

TN

CL

SN

C

T.A

nA

nP.

An

An

P.A

nA

nP.

An

An

cP.

An

cA

nc

P.A

nc

An

P.A

n6H

F5,

66=

4.83

F5,

52=

1.59

F5,

66=

1.65

F5,

50=

4.00

F5,

50=

3.97

F5,

66=

3.49

F5,

66=

4.61

F5,

51=

4.19

Sq

P=

0.03

P=

0.21

P=

0.20

P=

0.05

P=

0.05

P=

0.07

P=

0.11

P=

0.05

IP

=0.

18P

=0.

88P

=0.

73P

=0.

156H

F5,

56=

5.81

F5,

42=

2.24

F5,

56=

2.49

F5,

41=

3.36

F5,

41=

4.44

F5,

56=

2.59

F5,

56=

4.84

F5,

41=

3.17

Sq

P=

0.02

P=

0.14

P=

0.12

P=

0.07

P=

0.14

P=

0.11

P=

0.29

P=

0.08

EP

=0.

44P

=0.

30P

=0.

20P

=0.

273H

F2,

53=

3.76

F2,

45=

4.61

F2,

53=

3.58

F2,

38=

1.80

F2,

38=

6.80

F2,

53=

0.59

F2,

53=

0.59

F2,

38=

6.99

Sq

P=

0.06

P=

0.04

P=

0.06

P=

0.19

P=

0.48

P=

0.45

P=

0.45

P=

0.79

EP

=0.

30P

=0.

31P

=0.

22P

=0.

27

Mea

sure

men

tN

TC

LA

MC

TR

MC

TM

TN

CP

AM

TT

RM

TT

T.A

nA

nc

P.A

nc

An

cP.

An

cA

nP.

An

An

cP.

An

cA

nc

P.A

nc

An

cP.

An

c6H

F5,

66=

0.14

F5,

66=

4.56

F5,

66=

13.6

4F

5,66

=4.

65F

5,66

=6.

43F

5,53

=1.

88F

5,53

=4.

26F

5,66

=3.

26F

5,66

=4.

59F

5,66

=0.

01F

5,66

=4.

64S

qP

=0.

71P

=1.

00P

<0.

01P

<0.

01P

=0.

01P

=0.

18P

=0.

35P

=0.

08P

=0.

15P

=0.

94P

=1.

00I

P=

0.08

6HF

5,56

=0.

05F

5,56

=4.

35F

5,56

=28

.02

F5,

56=

4.60

F5,

56=

7.24

F5,

43=

3.73

F5,

43=

4.98

F5,

56=

5.38

F5,

56=

4.56

F5,

56=

0.01

F5,

56=

4.43

Sq

P=

0.82

P=

1.00

P<

0.01

P<

0.01

P=

0.01

P=

0.06

P=

0.10

P=

0.02

P=

0.02

P=

0.92

P=

1.00

EP

=0.

173H

F2,

53=

1.26

F2,

53=

6.48

F2,

53=

16.5

9F

2,53

=7.

41F

2,53

=10

.37

F2,

40=

1.92

F2,

40=

6.67

F2,

53=

4.87

F2,

53=

7.19

F2,

53=

0.76

F2,

53=

6.73

Sq

P=

0.27

P=

0.61

P<

0.01

P<

0.01

P<

0.01

P=

0.17

P=

0.42

P=

0.03

P=

0.14

P=

0.39

P=

0.73

EP

=0.

03

T.A

n,

type

ofan

alys

is;

An

,A

NO

VA

;A

nc,

AN

CO

VA

;P.

An

,ph

ylog

enet

icA

NO

VA

;P.

An

c,ph

ylog

enet

icA

NC

OV

A.

H,

hab

itat

;S

QI,

squ

amat

esin

clu

ded;

SQ

E,

squ

amat

esex

clu

ded.



compactness (Cls, Cts and CtsC values), as well as ahigher absolute thickness of the layers surroundingthe neural canal and the vertebral periphery. Thesedifferences are probably due to the fact that fossorialsquamates essentially dig with movements of thevertebral column (e.g. Roscito & Rodrigues, 2013)whereas the other amniotes essentially use their legs.As a result, the vertebrae of fossorial squamates needto be relatively compact and resistant to accommo-date the transmission of high forces from the animalto the surrounding environment (O’Reilly, Ritter &Carrier, 1997).

SIZE EFFECT

This study shows clearly that various micro-anatomical features are correlated with overall body

size. The observation of a positive correlation withsize for TNCT, TNCL and NTCL and of a negative onefor RMTT is not surprising, as it was also suggestedin previous studies (e.g. Houssaye et al., 2010;Dumont et al., 2013) that the tightness of thespongiosa increases with specimen size, withtrabeculae becoming relatively thinner but morenumerous. This study reveals that the absolute thick-ness of the layers surrounding the neural canal andthe bone periphery also increases with size, as doesabsolute mean trabecular thickness. This last resultinterestingly shows that, despite the general trend ofa relative reduction of trabecular thickness, the abso-lute trabecular thickness increases with size. Hereagain, these observations suggest strong structuralconstraints, depending on overall size, on vertebralmicroanatomy.

Figure 3. Schematic drawings illustrating the various microanatomical patterns observed in: A, Heterocephalus glaberSTIPB M1088B; B, Sciurus vulgaris Tafforeau PC; C, Anser anser STIPB R629; D, Scalopus aquaticus MHNL 50000050;E, Cynocephalus volans Unnumbered MNHN AC; F, Ara chloroptera STIPB R536; G, Castor fiber ZFMK 2006 007; H,Crocodylus niloticus MNHN AC 1964 403; I, Delphinapterus leucas MNHN 1971 156; J, Ursus maritimus ZFMK 2005 356;K, Aptenodytes patagonicus Unnumbered UPMC; L, Zalophus californianus ZFMK 49 98. A–C, G–I, longitudinal sections;D–F, J–L, transverse sections. Scale bars = 1 mm.



ECOLOGICAL SIGNAL

Despite the occurrence of only a few significant dif-ferences in vertebral microanatomy with ecology(except for AMCT, which showed a significant signalin all the analyses performed), based on the habitatswe defined, some trends could clearly be highlighted.Two main tendencies are observed concerning thetightness of the spongiosa: a low number of relativelythick trabeculae (low TNCT, TNCL and NTCL values;high RMTT values) in arboreal, flying and fossorialtaxa, versus a high number of relatively thintrabeculae in aquatic forms. Contrary to what isobserved in squamates, and in contrast to our originalassumption, fossorial amniotes generally show arather low inner compactness, a trend much strongerthan for flying taxa (at least in our sample). Bothfossorial and flying taxa are characterized by lowerabsolute thickness of the layers surrounding theneural canal and bone periphery (MTNCP andAMCT), and absolute thickness of the bone trabeculae(AMTT). It also appears that flying taxa display alarger neural canal, whereas the reverse is observedin aquatic taxa, which also show a lower relativecortical thickness but a relatively thick layer of bonesurrounding the neural canal (Table 2).

Unsurprisingly, the semi-aquatic taxa do not grouptogether. Under the name ‘semi-aquatic’ taxa aregrouped animals with very distinct ecologies and withvery different functional adaptations. Previous analy-ses on microanatomical features of some amniotelong bones also did not succeed in distinguishingsemi-aquatic taxa from terrestrial or aquatic taxa(Germain & Laurin, 2005; Kriloff et al., 2008;Canoville & Laurin, 2010). As done here they inte-grated all semi-aquatic taxa into a single ecologicalcategory rather than distinguishing them based ontheir distinct functional requirements.

PHYLOGENETIC SIGNAL

This study has revealed a phylogenetic signal in themicroanatomical parameters analysed but also sug-gests that it is rather weak. This signal probablyreflects the fact that only a few lineages adapted tosome peculiar ecologies. For example, there are notmany lineages of aquatic or flying amniotes, so thatmany taxa displaying these ecologies were forminggroups on the phylogeny.

CONCLUSION

This study demonstrates the great diversity ofamniote vertebral microanatomical features andshows that these features reflect structural,phylogenetic and ecological signals. The structuralconstraint on amniote vertebral microanatomical fea-

tures is strong and appears mainly to be caused by anadjustment to overall body size. The phylogeneticsignal is rather weak. The peculiarity of squamatesamong amniotes was clearly highlighted in this study.Some trends depending on habitat could clearly beobserved, notably rather similar tendencies forfossorial, arboreal and flying taxa, generally opposedto those observed in aquatic forms. However, differ-ences were often not significant in our quantitativeanalyses. As previously suggested, the same habitatcan be shared by taxa with different functionalrequirements, so that it would probably be muchmore relevant to distinguish categories based on func-tional requirements rather than habitat.

ACKNOWLEDGEMENTS

We are particularly grateful to C. Bens, C. Lefèvreand S. Bailon (MNHN, Paris, France), D. Berthet(MHNL, Lyon, France), W. Böhme and R. Hutterer(ZFMK, Bonn, Germany), E. Gardin, C. Guintard(Ecole Nationale Vétérinaire, de l’Agroalimentaireet de l’Alimentation, Nantes France), O. Golle, andthe LPG (Nantes) for the loan of and help withmaterial. We thank the ESRF (Grenoble, France) andSteinmann Institut (University of Bonn, Germany)for providing beamtime and support. We are verygrateful to N. Steichen for development of the soft-ware ‘LineTrab’, and to A-C. Fabre (UniversityCollege London) for her help with statistics. Manythanks to D. Germain (MNHN, Paris, France) and M.Dumont (Max-Planck-Institut für EisenforschungGmbH, Düsseldorf, Germany) for fruitful commentsthat improved the manuscript and to J. Allen and S.Moore for editorial work. Al.H. acknowledges finan-cial support from the A. v. Humboldt Foundationand the ANR-13-PDOC-0011. Author contributions:Research conception and design: Al.H. Data acquisi-tion: Al.H. Data analysis and interpretation: Al.H.Help with data acquisition and analysis: An.H. & P.T.Drafting of the manuscript: Al.H. Critical revision ofthe manuscript: An.H. & P.T.

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Amniote vertebral microanatomy - what are the major trends?