Paleohistological estimation of bone growth rate in extinct archosaurs

Paleohistological estimation of bone growth rate inextinct archosaurs

Jorge Cubo, Nathalie Le Roy, Cayetana Martinez-Maza, and Laetitia Montes

Abstract.—The clade Archosauria contains two very different sister groups in terms of diversity(number of species) and disparity (phenotypic variation): Crurotarsi (taxa more closely related tocrocodiles than to birds) and Ornithodira (pterosaurs and dinosaurs including birds). The extantspecies of Crurotarsi may constitute a biased sample of past biodiversity regarding growth patternsand metabolic rates. Bone histological characters can be conserved over hundreds of millions of yearsin the fossil record and potentially contain information about individual age at death, age at sexualmaturity, bone growth rates, and basal metabolic rates of extinct vertebrates. Using a sample of extantamniotes, we have constructed a paleobiological model to estimate bone growth rate from bonehistological traits. Cross-validation tests show that this model is reliable. We then used it to estimatebone growth rates in a sample of extinct archosaurs including Crurotarsi and Ornithodira. Aftertesting for phylogenetic signal, optimization of femoral growth rates through squared changeparsimony onto a time-calibrated tree of amniotes shows two divergent evolutionary trends: whereasbone growth rates increase from the last common ancestor of Ornithodira to extant birds, theydecrease from the last common ancestor of Crurotarsi to extant crocodiles. However, we conclude, onthe basis of recent evidence for unidirectional airflow in the lungs of alligators, that crocodiles mayhave retained the capacity of growing at high rates.

Jorge Cubo.* UPMC, Universite Paris 06, UMR 7193, ISTeP; and CNRS, UMR 7193, ISTeP, 4 place Jussieu,BC 19, F-75005, Paris, France. E-mail: [email protected]. *Corresponding author

Nathalie Le Roy. CNRS, UMR 5561, Biogeosciences, Universite de Bourgogne 6, boulevard Gabriel, Dijon21000, France

Cayetana Martinez-Maza. UPMC, Universite Paris 06, UMR 7193, ISTeP; and CNRS, UMR 7193, ISTeP,4 place Jussieu, BC 19, F-75005, Paris, France. Present address: Department of Paleobiology, Museo deCiencias Naturales–CSIC. Jose Gutierrez Abascal 2, 28006, Madrid, Spain

Laetitia Montes. UPMC, Universite Paris 06, UMR 7193, ISTeP; and CNRS, UMR 7193, ISTeP, 4 placeJussieu, BC 19, F-75005, Paris, France

Accepted: 7 October 2011Supplementary materials deposited at Dryad: doi:10.5061/dryad.j2m25n82

Introduction

Bone histological characters can be con-served over hundreds of millions of yearsin the fossil record. They have been usedextensively to estimate growth trajectories inbody mass (Erickson 2005; Bybee et al. 2006),age at sexual maturity (Lee and Werning2008), bone growth rates (Curry 1999; Horneret al. 1999, 2000, 2001; Sander 2000; Padianet al. 2001, 2004; Ricqles et al. 2003, 2008;Cooper et al. 2008), and metabolic rates(Ricqles 1978; Padian and Horner 2002), andcould potentially be used to estimate biome-chanical constraints (Lee 2004; Margerie et al.2005; Ponton et al. 2007). Paleobiologicalestimations of bone growth rate are basedon Amprino’s rule, according to whichvariation in periosteal bone tissues is primar-ily the expression of different periosteal bonegrowth rates (Amprino 1947). Starck and

Chinsamy (2002) noted that a given bonetissue type is associated with a wide range ofbone growth rates, depending on bone ele-ment, mechanical function, environmentalconditions, individual age and phylogenetichistory. Amprino’s rule has received wideempirical support in birds (Castanet et al.1996, 2000; Margerie et al. 2002, 2004) and hasbeen used to infer bone growth rates ofextinct diapsids, assuming the actualistichypothesis that similar bone tissue types areconstructed under similar conditions in ex-tant and extinct vertebrates.

Ricqles et al. (2003) proposed the hypoth-esis that high bone growth rates may haveevolved in archosauromorphs, before theappearance of Ornithodira (pterosaurs, dino-saurs, and their relatives). Further examina-tion of the bone histology of non-archosaurianarchosauromorphs has supported this hy-pothesis to some extent (Ricqles et al. 2008).

Paleobiology, 38(2), 2012, pp. 335–349

’ 2012 The Paleontological Society. All rights reserved. 0094-8373/12/3802–0010/$1.00

In this paper we test the hypothesis (derivedfrom the evolutionary trends inferred byRicqles et al. 2003, 2008) of a possible loss ofthe capacity of producing bone at highgrowth rates in the evolutionary history ofCrurotarsi (taxa more closely related tocrocodiles than to birds), leading to therelatively slow, cyclical growth observed inextant crocodiles. For this we use a newquantitative phylogenetic approach that im-proves the state of the art of paleobiologicalestimation of bone growth rates in threeimportant respects:

1. Previous paleobiological studies have usedexperimental data from species belongingto only a single clade, Neornithes (Castanetet al. 1996, 2000), to infer bone growth ratesof extinct members of a much larger clade(the diapsids). This is problematic because,according to the cladistic principle ofparsimony, at least the first two extantsister groups of an extinct taxon should beused to estimate character states of thisfossil taxon, a concept formalized byWitmer (1995) as the extant phylogeneticbracket. This would suggest that to esti-mate the bone growth rate of, for instance,a sauropod dinosaur, we would need toknow the relevant character states of theextant phylogenetic bracket of this taxon(birds and crocodiles). Here we present apaleobiological model of estimation ofbone growth rates constructed using ex-perimental data obtained from sixteenspecies of extant amniotes (inclusiveclade). This model is used to estimate bonegrowth rate of extinct archosaurs (internalclade).

2. Previous paleobiological studies of bonegrowth rate estimation have used aqualitative approach based on a typolog-ical classification of bone tissue in whicha given bone tissue type (e.g., laminarfibrolamellar bone tissue) is associatedwith a mean bone growth rate valueobserved in model species. Here wemeasured a set of bone histologicalquantitative features and used them asvariables explaining observed bonegrowth rates. In this way, we circumvent

problems associated with the use of atypological classification, because it ismore realistic to assume a histologicalcontinuum rather than well-defined bonetissue types.

3. It has been shown recently that both bonehistological variability and bone growthrate contain a significant phylogeneticsignal (Cubo et al. 2005, 2008). That is tosay, closely related species may have astatistically higher correlation of bonetissue features than would be expectedfrom a random sample of taxa. Theseresults allow the analysis of evolutionarypatterns using squared-change parsimony,for which the use of a time-calibratedphylogeny including branch length infor-mation is optimal (Laurin 2004). Thus,unlike previous studies of bone growthrate estimation, we include in our analysesnot only the phylogenetic positions of bothextant and extinct species, but also geolog-ical divergence times, which allow morereliable estimations of ancestral bonegrowth rates at nodes.

Material and Methods

Extant Species.—We sampled 52 specimensbelonging to 16 species of extant amniotes toconstruct three paleobiological models (hu-merus, femur, tibia) aimed at estimating bonegrowth rates from bone tissue features. Ofthese, 12 species (three mammals, threetestudines, four lizards, one crocodile, andone bird) were used in two previous studies(Montes et al. 2007; Cubo et al. 2008).Considering that the mode of developmentis an important factor explaining bone growthrate variation (altricial birds allocate mostresources to growth whereas precocial birdsallocate an important fraction of resources tolocomotion, thermoregulation, search for ali-ments), but that body mass also explains afraction of bone growth rate variation, weadded four species in the analyses to con-struct our predictive models: Struthio camelusand Dromaius novaehollandiae (previously usedin the study by Castanet et al. [2000] andincluded in this study to expand the range ofbody masses of the sample), and Turdus

336 JORGE CUBO ET AL.

merula and Streptopelia decaocto (new experi-mental work performed to include altricialbirds in the sample). All the experimentalprocedures were carried out in accordancewith the European Communities CouncilDirective (86/609/EEC).

Extinct Species.—These predictive modelswere used to estimate bone growth rates inthe following extinct archosaurs: PostosuchusUCMP 28353 (humerus), CalyptosuchusUCMP 25914 (femur), Rutiodon UCMP 25921(femur), and Typothorax UCMP 25905 (femur)among Crurotarsi; and Lesothosaurus QR 3076(femur), Maiasaura MOR 005 (tibia), Coelophy-sis AMNH 27435 (tibia), Allosaurus UUVP3694 (femur) and UUVP 154 (tibia), andTroodon MOR 748 (femur) among Ornitho-dira.

Phylogeny.—Figure 1 shows the phyloge-netic relationships and the divergence times(mostly based on fossil evidence) among thespecies of the entire sample. The topology forTestudines was compiled from Gaffney andMeylan (1988). In our sample, Trachemys(Emydidae) and Pelodiscus (Trionychidae)are sister groups, and this clade is the sistergroup of Macrochelodina (Pleurodira). For thesquamates, the topology was compiled fromEstes (1982), Estes et al. (1988), Rieppel (1988),and Caldwell (1999). The placement of Tes-tudines is still controversial (Rieppel andReisz 1999; Zardoya and Meyer 2001); wedecided to follow the generally acceptedposition that Testudines is the sister groupof Diapsida, as numerous paleontologicalstudies have argued (Laurin and Reisz 1995;Lee 2001). Considering that the oldest knownamniote (Hylonomus lyelli) comes from the lateBashkirian (Marjanovic and Laurin 2007), weused a divergence time between mammalsand sauropsids (last common ancestor ofAmniota) of 310 Ma. Divergence times be-tween lepidosaurs and crocodylians (lastcommon ancestor of Diapsida, 252 Ma) andbetween crocodiles and birds (last commonancestor of Archosauria, 243 Ma) were takenfrom Reisz and Muller (2004). These diver-gence times are reliable estimations becauseof the high quality of the fossil record beforeand after the first occurrence of these taxa(Reisz and Muller 2004). Of course, all less

inclusive nodes containing extinct taxa asterminal branches (Postosuchus, Calyptosuchus,Rutiodon, Typothorax, Lesothosaurus, Maiasaura,Coelophysis, Allosaurus, and Troodon) were alsocalibrated using the fossil record. Finally,divergence time between Lacertidae andVaranidae (189 Ma) and the age of the lastcommon ancestor of dinosaurs (230 Ma) wererespectively taken from Evans (2003: Fig. 8)and Langer et al. (2010), both obtained fromthe fossil record. Other divergence times weretaken from Pyron (2010), who used a molec-ular approach calibrated by the four well-constrained fossil dates obtained by Mullerand Reisz (2005). This was the case fordivergence times between Paleognathae andNeognathae (last common ancestor of mod-ern birds, 112 Ma), and between Anas andTurdus (last common ancestor of Neognathae,77 Ma). These molecular clock estimates arecongruent with vicariance biogeography andfossil evidence respectively.

Vicariance biogeography data are congru-ent with the age estimated for the lastcommon ancestor of modern birds (112 Ma).Morphological phylogeny of ratite birdssuggests that New Zealand species (moasand kiwis) are the sister group of all otherratites and that, within this last clade, Aepyor-nithidae (Madagascar) is the sister group of theclade Struthio-Rheidae-Casuariidae-Dromaiidae(Bourdon et al. 2009). Considering that ratitesare flightless birds, the vicariance event inwhich the Madagascar/India block driftedaway from Antarctica, which occurred in theEarly Cretaceous between 130 and 110 Ma(Scotese 2001), may date the divergence be-tween Aepyornithidae and the clade Struthio-Rheidae-Casuariidae-Dromaiidae. Thus, con-trary to most molecular clock estimates, thedivergence date estimated by Pyron (2010) forthe last common ancestor of modern birds(112 Ma) appears conservative.

On the other hand, divergence time betweenAnas and Turdus (77 Ma) is well sustained bythe finding of definitive evidence of fossilneognathes in the Cretaceous (Olson andParris 1987; Chatterjee 2002; Hope 2002; Clarkeat al. 2005). In particular, the finding of Vegavisiaai (Aves, Neognathae, Anseriformes) in theMiddle to Upper Maastrichtian (ca. 66–68 Ma)

EVOLUTION OF GROWTH RATES IN ARCHOSAURS 337

FIGURE 1. Phylogenetic relationships and divergence times among the species of the entire sample reconstructedfollowing the references cited in the main text. The bottom edge contains a time calibration (in Ma). Divergence dates(also in Ma) are noted at nodes.


of Cape Lamb, Vega Island (Clarke at al.2005), a species closely related to Anasplatyrhynchos (in our sample), suggests thatthe molecular clock estimate by Pyron(2010) of the divergence time between Anasand Turdus (77 Ma) is realistic and highlyconservative.

Ontogenetic Control.—A marked ontogenet-ic variation of both bone growth rates andbone tissue types has been documented. Forthis reason, ontogenetic control is indispens-able in any comparative analysis. Periostealgrowth in bone can be modeled usingsigmoid equations (e.g., Gompertz, von Ber-talanffy, logistic). These equations contain aphase of growth acceleration and a phase ofgrowth deceleration. Although mathematical-ly these phases are separated by an inflectionpoint, biologically there is a phase of sus-tained high growth rate between the end ofthe acceleration phase and the beginning ofthe deceleration one (see, for instance, Fig. 2of Bybee et al. 2006, in which circumference-to-age plots of many Allosaurus long bonescould be well modeled by straight lines ofconstant growth rate starting at about sixyears). In the samples analyzed in our study,bone tissue formed during the accelerationphase is no longer present because it has beenremoved during the process of perimedullaryresorption. On the other hand, bone tissueformed during the deceleration phase, whenpresent, is easily identifiable near the boneperiphery: bone vascularization progressively

decreases and even disappears, and bonematrix becomes more parallel-fibered andprogressively less woven-fibered. We alwaysmeasured bone growth rate in regions formedduring the phase of sustained high growthrate. We selected these regions on the basis ofthe following criterion: bone tissues formedbefore and after the selected region arehistologically similar and were formed at thesame rate as bone tissue of the selectedregion. Considering that the shape of thebone growth curve differs between males andfemales in a given species (Cubo and Manosa1999) and between ectothermic and endother-mic species (Lee and Werning 2008), andthat important biological events such as theacquisition of female reproductive maturityoccurs at different phases in different taxo-nomic groups (Lee and Werning 2008), it isdifficult, and biologically unjustified, to de-fine a standardized ‘‘growth stage.’’ Rather,as stated above, we compared growth ratesduring the phase of sustained high growthrate, after the end of the acceleration phase,and before the beginning of the decelerationphase. Whereas in extant species sampledbone formed at sustained high growth rate islocated at bone periphery (specimens wereactively growing when they were eutha-nized), in extinct taxa bone formed at sus-tained high growth rate is located in the deepcortex (i.e., fossil specimens were older thanthose belonging to extant species). We wereable to place some fossil specimens of oursample on published growth trajectories.Bybee et al. (2006), who observed five andretrocalculated eight lines of arrested growth(LAGs) in Allosaurus femur UUVP 3694, gavean estimated age of 13 years for this speci-men. These authors observed two and retro-calculated five LAGs in Allosaurus tibia UUVP154, and they estimated an age of 7 years forthis specimen. According to Figure 2 in Bybeeet al. (2006), these two specimens (UUVP 3694and UUVP 154) were actively growing bothwhen the bone tissue measured was formed(in the deep cortex) and at death. Ericksonet al. (2007) found no LAGs in Troodon femurMOR 748. However, they concluded that thisspecimen was 18 years old on the basis of theanalysis of a metatarsal IV and a tibia.

FIGURE 2. Method of determining vascular orientation(inspired by Margerie 2002). Under ImageJ we obtainedan angle formed by the major axis of the largest ellipseinserted into each vascular canal and a vector tangent tobone periphery. This continuous variation was trans-formed into three discrete types of orientation (radial,circular, and oblique). Longitudinal orientation cannot bedetermined using this method.


According to Figure 1G in Erickson et al.(2007), this specimen almost reached theasymptote at death (i.e., it was nearly adult).In this study, we quantified histologicalfeatures in the deep cortex (formed whenTroodon femur MOR 748 was still activelygrowing), to get data comparable to thoseused to construct the predictive model (ob-tained experimentally in extant taxa).

Finally, testudines, squamates, and croco-diles are characterized by annual cycles ofbone formation, each of which can be mod-eled using a sigmoid equation. In thesegroups, measurements were carried out dur-ing the phase of sustained high growth rate—after the end of the acceleration phase andbefore the beginning of the deceleration phaseof the first year. See Supplementary Table 1for information on the body mass ranges ofthe analyzed specimens.

Variables

Periosteal Bone Growth Rate.—This is thedependent variable to be estimated in extinctspecies, and it was quantified in extantspecies through in vivo fluorescent labelingusing the methodology described by Monteset al. (2007). Fluorescent dyes, xylenol-orange(XO) and fluoresceine (DCAF), specificallycolor the mineralizing zone of growing bonetissue, in deep orange and green respectively.Animals were euthanized after a given timeinterval (from two to several weeks depend-ing on the species), which was used tocompute bone growth rate. Femora, tibiae,and humeri were then removed from eachindividual. Bones were dehydrated in gradedethanol and degreased in acetone and trichlo-roethylene before being embedded in apolyester resin (Matrajt et al. 1967). Trans-verse sections 100 6 10 mm thick were madeacross the diaphysis using a diamond-tippedcircular saw. Each thin section was groundand polished before being mounted on aslide, and then observed under ultravioletlight (Zeiss Axiovert 35; Jena, Germany) anddigitally imaged (Olympus, Japan). The high-est density across the fluorescent label wasused as the endpoint of the fluorescent mark.Bone growth rates were measured in mm/dayusing image analysis in Image J (a computer

program by W. Rasband available at http://rsb.info.nih.gov/ij/) as the distance (in mm)between two consecutive fluorescent labels,or between the last label and the boneperiphery (the period with higher growthrate was selected), divided by the elapsedtime (days). For each bone section, thisdistance was measured as the differencebetween the radius of the external circle(delimited either by the external label or bybone periphery) and the radius of the internalone (delimited by the internal label).

Vascular Orientation.—Blood vessels in thebones were lost during sample preparation inextant species, and during the fossilizationprocess in the extinct taxa. Thus, this variablemeasures the orientation of the cavities(called vascular canals) that had containedthe blood vessels and associated connectivetissues. The orientation of each vascularcavity was determined using Image J. Weinserted the largest ellipse into each vascularcavity. To improve repeatability, the orienta-tion of each vascular cavity was measuredusing the radial index published by Boefand Larsson (2007). The orientation of thesecavities was computed as the angle betweenthe major axis of each ellipse and a vectortangent to bone periphery. Thus vascularcanals running parallel to bone peripheryhave angles approaching 0u, and those run-ning parallel to the radius of bone cross-section have angles approaching 90u (Boefand Larsson 2007). Vascular canal orientationis a continuously varying trait that wetransformed into discrete orientation classesusing the criteria shown in Figure 2 (inspiredby Margerie 2002). Three types of standardvascular canal orientation were defined in ourtransverse sections: circular canals (C), whichrun roughly parallel to the bone periphery (0u+ 22.5u; 180u–22.5u); radial canals (R), whichrun roughly orthogonal to the bone periphery(90u 6 22.5u); and oblique canals (O), i.e.,those canals excluded from the intervalscorresponding to radial canals and to circularones. These types of vascular orientation areillustrated in Margerie (2002), Margerie et al.(2004), and Boef and Larsson (2007). We usedthree variables to describe the major vascularorientations found in each bone section:


proportion of circular canals (C/C + R + O),proportion of radial canals (R/C + R + O), andproportion of oblique canals (O/C + R + O). Inavascular bones, the proportions of circular,radial and oblique canals were considered aszero.

Vascular Density.—Cubo et al. (2005) mea-sured vascular density as the ratio of totalvascular canal area to primary bone area.Here we measured the number of canalsdivided by mm2 because the osteons are notyet filled in our sample of extant taxa (theindividuals were still growing). Sectionsshowing a single vascular canal were consid-ered to be avascular because this singlevascular canal most probably corresponds toa ‘‘canal nourricier’’ (a blood vessel runningfrom the periosteum to the endosteum).

Cellular Variables.—Cellular shape, size, anddensity were carefully measured outside theosteons both in extant taxa (in which osteonsare not yet filled because they are still growing)and in extinct taxa (in which osteons arealready filled). Like vascular canals, osteocyteswere lost during the preparation of bonesamples in extant species and during theprocess of fossilization in extinct taxa. Thus,we measured the shape, size, and density ofcavities (osteocyte lacunae) that contained bonecells (the osteocytes). When possible in extantspecies (i.e., when the bone section containedenough osteocyte lacunae), we measured 120osteocyte lacunae/bone section.

Cellular shape was quantified as the ratiobetween the major and the minor axes ofthese cavities (0 , shape # 1). A value of 1means that the lacunae are perfectly circular.

Cellular size was computed using the majorand minor axes of osteocyte lacunae andassuming the geometry of an ellipse ( p 3 L/23 l/2).

Cellular density was quantified as the num-ber of lacunae divided by the surface in mm2.

All measurements were carried out using amicroscope focused on a single layer ofosteocyte lacunae. Thus, the measurementsrefer to a single layer of osteocyte lacunaewhatever the thickness of the ground section.Cellular density was computed including allosteocyte lacunae of the quoted single layer.Following Organ et al. (2007), we measured

only the largest osteocyte lacunae in this layerto compute cell size and shape, to ensure thatcell lacunae were measured near the mid axis.

Bone Size.—This variable was measuredonly in extant taxa, as bone cross-sectionalarea (bone surface encircled by the perioste-um in mm2).

Phylogenetic Comparative Methods

Constructing Paleobiological Models of BoneGrowth Rate Estimation.—The paleobiologicalgrowth rate inference models were constructedusing multiple linear regression tested for signi-ficance using permutations (Legendre et al.1994). We used the computer program Per-mute! distributed by P. Casgrain and obtained astandardized coefficient for each variable (a9),each of which was un-standardized into a rawcoefficient (a) by using the expression

a1~a’1 � sY=sX1ð Þ,where sY is the standard deviation of thedependent variable (bone growth rate) and sX1

is the standard deviation of the independentvariable under analysis. The intercept wascomputed using the following equation:

b~Ymean{a1 � X1 mean{a2 � X2 mean

{ . . . :: an � Xn mean:

Once constructed, we applied the models toestimate bone growth rate in each extinctspecies of our sample.

Cross-Validation Tests.—In regression anal-ysis, any equation will fit better on the dataset used to create it than on a new data set.This effect is known as shrinkage. In ourstudy, predictive equations are optimized forthe sample of extant species used to createthem, and will not perform as well in othersamples (e.g., for estimating bone growth rateof extinct taxa). Thus we performed cross-validation tests to determine whether we canbe confident in the reliability of our models.For each bone, the sample of extant speciesfor which bone growth rates are known wassplit into two subsamples. A predictiveregression (of bone growth rate) was comput-ed from the first subsample and used toestimate predicted scores for the species inthe second subsample. Then, predicted scores(predicted growth rates) were correlated with


observed scores (experimentally measuredgrowth rates).

Computing Confidence Intervals for Estima-tions at Tree Nodes.—Confidence interval forthe root node was computed using the PDAPmodule for Mesquite (Midford et al. 2003). Con-fidence intervals for nodes other than the rootnode were computed following Laurin (2004),also using the PDAP module for Mesquite.

Computing Confidence Intervals for Bone GrowthRate Estimations in Fossils.—Each extinct taxonof our sample was transformed into a node bysplitting it into two sister taxa of standardizedbranch lengths of 2 Myr. Values of the twonewly created terminal taxa were empiricallymodified in order to obtain, at the newlycreated node (occupying the phylogeneticposition of our extinct taxon) the bone growthrate estimated for this extinct taxon. Then, a95% confidence interval was computed for eachextinct taxon following the method of Laurin(2004) for nodes other than the root node, alsousing the PDAP module for Mesquite.

Optimization of Bone Growth Rates onto thePhylogenetic Tree.—We used both the values ofbone growth rate measured in extant species,and those estimated for extinct species toperform an optimization through squaredchange parsimony using the computer pro-gram Mesquite (Maddison and Maddison2009). This method allows the analysis ofpatterns of character evolution. However, asrecommended by Cubo et al. (2002) and Laurinet al. (2004), before performing these optimi-zations on the phylogenetic trees, we tested theexistence of a significant phylogenetic signal inthe variation of the characters under study.

Testing for Phylogenetic Signal.—The phy-logeny of our sample of amniotes (Fig. 1) wasexpressed in the form of principal coordinates(Diniz-Filho et al. 1998) to be used asexplanatory variables in tests of phylogeneticsignal. Considering that we obtained as manyprincipal coordinates (PC) as species wereincluded in the analyses, a selection proce-dure was necessary. We retained and used asexplanatory variables the phylogenetic PCssignificantly related to the dependent variable(Desdevises et al. 2003).

Phylogenetically Independent Contrasts.—Thismethod was used to test whether bone

growth rate (expressed in mm/day or inmm2/day) is significantly related to bone size(measured as bone cross-sectional area, i.e.,the surface encircled by the periosteum).Analyses were performed using the PDAPmodule for Mesquite (Midford et al. 2003).The phylogenetically independent contrastsmethod allows testing of these relationshipsby circumventing the problem of the nonindependence of observations due to thephylogeny (Felsenstein 1985).

Results

Data are available in the online supplemen-tary materials. Supplementary Table 1 showsraw data (histological traits, bone growth rate)measured in our sample of extant amnioteswith the aim of constructing three paleobiolog-ical bone growth rate inference models: one forthe femur, one for the humerus, and one for thetibia. Mean species values were used instatistical analyses. Supplementary Table 2shows data obtained from the fossil specimens.The dependent variable (to be estimated inextinct archosaurs) is bone growth rate. Asindependent variables we used all histologicalvariables (the multiple regression method takesinto account the redundancy of information,called colinearity, among independent vari-ables). We obtained the following predictivemodel for femora (r2 5 0.850; p 5 0.0091) testedcomputing 9999 permutations (the b coeffi-cients and the intercept are not standardized):

Femoral growth rate 5 213.5753–0.2574 3

Vascular density + 20,474.2882 3 Cellulardensity 2 101.5837 3 Cellular shape +1.9660 3 Cellular size + 60.0438 3 Propor-tion of circular canals 2 34.7885 3 Propor-tion of oblique canals 2 30.2255 3 Propor-tion of radial canals

Cross-validation tests show that the femo-ral equation performs well. Our sample ofextant species was split into two subsamples.A predictive regression (of bone growth rate)was computed from the first subsample(including Cavia porcellus, Mus musculus,Trachemys scripta, Chelodina siebenrocki, Lacertavivipara, Varanus exanthematicus, Crocodylusniloticus, Dromaius novaehollandiae, Struthiocamelus, Anas platyrhynchos, and Turdus mer-


ula). This equation (r2 5 0.974; p 5 0.02551,tested computing 9999 permutations) wasused to estimate predicted scores for thespecies of the second subsample (includingMicrocebus murinus, Pelodiscus sinensis, Podar-cis muralis, Varanus niloticus, and Streptopeliadecaocto). Finally, predicted scores (predictedgrowth rates for the five species of the secondsubsample) were correlated with observedscores (experimentally measured growthrates in the five species of the secondsubsample). We obtained a significant rela-tionship (r2 5 0.690; p 5 0.0110, testedcomputing 9999 permutations), suggestingthat the femoral equation performs ratherwell. Thus the femoral equation obtainedfrom the global sample of extant species wasused for estimating bone growth rate in oursample of extinct archosaurs from theirhistological features. We obtained the follow-ing results (estimations and 95% confidenceintervals): Typothorax: 0 mm/day (0–8.13 mm/day), Calyptosuchus: 34.91 mm/day (26.89–42.93), Rutiodon: 7.32 mm/day (0–15.22), Le-sothosaurus: 89.42 mm/day (81.41–97.43), Allo-saurus: 76.75 mm/day (68.79–84.71), and Troo-don: 131.56 mm/day (123.55–139.57).

In contrast, cross-validation tests show thatneither the equation obtained for the tibiae,nor that obtained for the humeri, performswell enough to be used in paleobiologicalinference. In the case of the tibiae, thepredictive equation obtained from the firstsubsample was significant (r2 5 0.966; p 5

0.0365 tested computing 9999 permutations).However, the correlation between predictedgrowth rates (for the five species of thesecond subsample) and observed scores (ex-perimentally measured growth rates, also insecond subsample), was not significant. In thecase of the humeri, the predictive equationobtained from the first subsample was notsignificant.

The variation of femoral growth rate con-tains a highly significant phylogenetic signal(p , 0.001) both in the sample including fossils(n 5 22) and in that including only extantspecies (n 5 16). Phylogeny explained 84.4%and 97.1% of bone growth rate variationrespectively. Concerning the other bonesanalyzed, although phylogenetic signal in

bone growth rate variation was highly signif-icant in tibiae (r2 5 0.967; p , 0.001), it was notsignificant in humeri (r2 5 0.977; p 5 0.055).For tibiae and humeri these tests wereperformed on the sample of extant species (n5 16 in both cases).

The optimization onto the phylogenetic treeof values of femoral growth rate (1) measuredin extant amniotes and (2) estimated forextinct archosaurs using our predictive equa-tion, allows the analysis of evolutionarypatterns. Two conditions are required toperform optimizations onto a phylogenetictree. First, the trait under analysis mustcontain a significant phylogenetic signal(otherwise the optimization may be meaning-less). As noted above, variation in the rate offemoral and tibial bone growth contained asignificant phylogenetic signal. Second, whendealing with values estimated for extinctspecies, cross-validation tests must show thatthe predictive equations perform well. Takinginto account the poor performances obtainedin cross-validation tests by equations forhumerus and tibia, and the high performanceobtained in these tests by the equation forfemur, only estimations of femoral growthrates were optimized onto the phylogenetictree (Fig. 3). The estimated condition for bonegrowth rate of the last common ancestor ofarchosaurs is 31 mm/day. The optimizationshows two divergent evolutionary trends:whereas there is a reduction toward the lastcommon ancestor of Crurotarsi (19.71 mm/day) and extant crocodiles (Crocodylus niloti-cus, 9.54 mm/day), there is an increase towardthe last common ancestor of Dinosauria(51.54 mm/day) and the last common ancestorof modern birds (95.90 mm/day).

Analyses of phylogenetically independentcontrasts show that femoral appositionalgrowth rate, expressed in mm/day, is signifi-cantly related to bone size (mm2): r2 5 0.625; p ,

0.001; n 5 16 species, 15 contrasts, df 5 14.Likewise, when expressed in mm2/day, it is alsosignificantly related to bone size (mm2): r2 5

0.996; p , 0.001; n 5 16 species, 15 contrasts, df5 14 (Fig. 4). In spite of these positive results,bone size was not included in the predictivemodel because we lack this information for anumber of fossil specimens in our sample.


FIGURE 3. Optimization through squared change parsimony of femoral growth rates, empirically measured in oursample of extant amniotes and inferred in our sample of extinct diapsids. Estimated growth rate values and 95%confidence intervals are given for nodes and for extinct taxa. Mean values, standard deviations (sd), and sample sizes(n) are given for the sample of extant species used to construct the predictive model.


Discussion

This paper is aimed at testing the hypothe-sis, derived from the evolutionary trendsfound by Ricqles et al. (2003, 2008), that extantCrurotarsi (crocodiles) may have lost thecapacity for high bone growth rate. To testfor this, we estimated bone growth rates in asample of extinct archosaurs using a newquantitative phylogenetic approach that cir-cumvents some of the problems encounteredin previous studies. First, unlike previousstudies of bone growth rate estimation, ourpaleobiological inferences remain within theextant phylogenetic bracket (Witmer 1995) ofextant amniotes sampled to construct thepredictive model. Second, we quantified avariety of detailed bone histological traits,circumventing problems associated with theuse of the classical qualitative typologicalapproach. Third, our model includes not onlythe phylogenetic position but also divergencetimes among extant species and the extincttaxa. Thus, in principle, our predictions shouldbe more reliable than those made previously.

First we will deal with the problem of thebody mass effect. Although Varanus niloticus,Crocodylus niloticus, and Struthio camelus (allincluded in our sample) are among the largestextant members of the Lepidosauria, Croco-dylia, and Aves, respectively, other extantamniotes used in our experimental studies aresmall relative to the extinct ones for which weperformed estimations of bone growth rates.We wanted to test whether this ‘‘body sizegap’’ could introduce distortions in ourestimations. Case (1978) suggested that largertetrapods of a given clade might grow athigher rates than smaller ones. Kirkwood et al.(1989) proposed, on the basis of theoreticalgrounds and empirical evidence, that this ruledoes not necessarily work for growth in lineardimensions such as bone growth rate (mm/day), which may be independent of bodymass. These authors analyzed the growth inlength (through endochondral ossification) ofthe tarsometatarsus in a sample of 88 speciesof birds ranging in body mass from 0.009 kg(Lonchura cuculata) to 120 kg (Struthio camelus),and found that bone growth rate (mm/day)

FIGURE 4. Regression of phylogenetically independent contrasts of femoral appositional growth rate (mm2/day) andcontrasts of bone size (bone cross-sectional area, i.e., bone encircled by the periosteum in mm2). r2 5 0.996; p , 0.001; n5 16 species, 15 contrasts, df 5 14.


was not significantly correlated with adultbody mass (Kirkwood et al. 1989). Cubo et al.(2000) also showed that tarsometatarsusgrowth rate in length (mm/day) was indepen-dent of adult body mass in a sample of sevenspecies of Ardeidae. In this paper we provideevidence, found in periosteal appositionalbone growth, against Kirkwood et al.’s (1989)conclusions, and evidence for Case’s (1978)position. Femoral appositional growth rates(expressed in mm/day or expressed in mm2/day) are significantly related to bone size(bone cross-sectional area encircled by theperiosteum, expressed in mm2; Fig. 4). Thesize effect on bone appositional growth rate isevident when evolutionary patterns in mod-ern birds are analyzed: with the exception ofTurdus, big birds grow faster than smallerones (Fig. 3). In fact, a mixture of a size effectand a developmental mode effect (altricialversus precocial) may explain growth ratevariation in birds. For instance, althoughTurdus merula is much smaller than Dromaiusnovaehollandiae, it grows at a higher rate thanDromaius, probably because it has an altricialdevelopmental mode (in which most resourc-es are allocated to growth), whereas Dromaiushas a precocial one in which a fraction ofresources are allocated to locomotion, searchfor food, etc.

Histological data were used to constructpredictive models of bone growth rate.Cross-validation tests suggest that the femo-ral equation performs rather well. In con-trast, as noted above, these tests show thatneither the equation obtained for the tibiaenor that obtained for the humeri performswell enough to be used in paleobiologicalinference. Thus only paleohistological esti-mations of bone growth rate obtained byusing the equation for femora will beanalyzed. As in previous studies (Padianet al. 2001, 2004), our optimizations onto thephylogenetic tree (Fig. 3) suggest divergentevolutionary trends in bone growth rates inCrurotarsi (taxa more closely related tocrocodiles than to birds) and Ornithodira(taxa more closely related to birds); whereasextant taxa of the former clade (crocodiles)show a bone tissue formed at low growthrates and characterized by a parallel-fibered

bone matrix containing mostly longitudinalvascular canals, Ornithodira show a typicalfibrolamellar complex formed at highergrowth rates. Although we stress the differ-ences between Crurotarsi and Ornithodira,this by no means assumes that Ornithodiraare homogeneous from an ontogenetic pointof view. For instance, Allosaurus, Troodon,and modern birds share high growth rates(of more than 60 mm/day). However, whileboth non-avian theropods grew over manyyears (Allosaurus for 13–19 years [Bybee et al.2006]; Troodon for at least 8 years [Ericksonet al. 2007]), and acquired sexual maturitybefore the end of the growth period, modernbirds generally complete growth in a fewweeks and become sexually mature well afterthey reach adult size.

To determine the polarity and the intensityof evolutionary trends of bone growth rates inarchosaurs, we need to determine the prim-itive condition for this clade. This is requiredto determine if the last common ancestor ofarchosaurs had a growth pattern more similarto that of terminal Crurotarsi or Ornithodira.The optimization of bone growth rates ontothe phylogenetic tree shows that the ancestralcondition for archosaurs is 31 mm/day(Fig. 3). The upper limit of the 95% confi-dence interval (CI) of this estimation(56.9 mm/day) is similar to the lower limit ofthe 95% CI of the estimation for the undoubt-edly endothermic last common ancestor ofextant birds (57.46 mm/day). In other words,the 95% CIs for the mean growth ratesestimated for the last common ancestors ofarchosaurs and for modern birds excludeeach other, but the upper limit of the firstone and the lower limit of the second one arevery close (Fig. 3). Thus, in this optimization,the estimated primitive condition for archo-saurs does not correspond to either a typicalectothermic or a typical endothermic thermo-metabolism, and the question remains unan-swered. Ricqles et al. (2008) analyzed the bonehistology of Erythrosuchus, a close outgroup ofArchosauria, and found radial bone tissue inmetatarsal bones of this species which, ac-cording to the results of Margerie et al. (2004),is formed at high growth rates (up to morethan 150 mm/day). The finding of high bone


growth rates in a taxon closely related toArchosauria supports the hypothesis that theprimitive condition for archosaurs may be apattern of rapid growth (Ricqles et al. 2003,2008). Considering this hypothesis, and thefact that extant Crurotarsi grow at moderateto low growth rates (9.54 6 3.85 mm/day inour sample of crocodiles), we can wonderabout a possible loss of the primitive condi-tion (capacity for high growth rates) in extantCrurotarsi (crocodiles). The analysis of evo-lutionary patterns shows that although someTriassic Crurotarsi may have retained thecapacity for high growth rates (e.g., Calypto-suchus, estimated bone growth rate 34.91 mm/day; 95% CI 26.89–42.93), others (e.g., Ty-pothorax, Rutiodon) already showed slowergrowth rates, similar to the derived growthpattern characterized by the slow (relative toOrnithodira), cyclical growth observed inextant crocodiles (Fig. 3). The derived condi-tion shared by extant Crurotarsi (crocodiles)could be explained by the fact that they arepredators (occupying the top of the foodchain before the end of the ontogeny in manyhabitats), and it is known that carnivoresgrow more slowly than herbivores (Cooperet al. 2008). Even giant crocodiles such as theCretaceous Deinosuchus (8–10 m long, 2500to 5000 kg) grew at rates similar to thoseof smaller crocodylian taxa (Erickson andBrochu 1999). In this context, after analyz-ing bone thin sections of 80 alligators(both captive and wild), Woodward andHorner (2010) concluded that these animalswere unable to form truly fibrolamellar bonetissue.

Our conclusion of a high bone growth rateestimated for the last common ancestor ofarchosaurs, retained by Ornithodira and someTriassic Crurotarsi (e.g., Calyptosuchus) andpartly lost in other Triassic Crurotarsi (e.g.,Typothorax, Rutiodon) and extant ones (croco-diles), agrees with the conclusions of Ricqleset al. (2003, 2008). This is not surprisingconsidering that all three studies are basedon paleohistology (quantitative approach ap-plied in this study, qualitative approach instudies by Ricqles et al.). Interestingly, recentfindings about the anatomy of the respiratorysystem support this conclusion. Evidence for

the presence of air-sac systems anterior andposterior to the gas-exchange pulmonaryregion has been found in non-avian thero-pods (O’Connor and Claessens 2005) as wellas in pterosaurs (Claessens et al. 2009).Although these results were not evidence foreither unidirectional (like that of birds) orbidirectional intrapulmonary airflow, theysuggested a flow-through model for the lungsof both non-avian theropods and pterosaurs(O’Connor and Claessens 2005; Claessenset al. 2009). Furthermore, Farmer and Sanders(2010) recently published exciting resultssuggesting that extant alligators undoubtedlyshare with extant birds unidirectional airflowin the lungs, which is most likely inheritedfrom the last common ancestor of archosaurs.This anatomical disposition of the respiratorysystem is much more efficient than that inmammals (in which air moves into and out ofterminal gas-exchange sacs). It may haveallowed for the high bone growth ratesestimated in this study for the last commonancestor of archosaurs and for some TriassicCrurotarsi (Calyptosuchus). Also, althoughextant crocodiles are characterized by lowgrowth rates (0–5 mm/day for Alligator [Pa-dian et al. 2001, 2004]; 9.54 mm/day 6

3.85 mm/day for Crocodylus niloticus [thisstudy]), the efficient anatomical dispositionof their respiratory system may explain whycaptive alligators grow at such high rates (atleast twice those observed in wild individu-als) when kept at a constant temperature andfed ad libitum with a high-protein foodsupply (Padian et al. 2004).

We conclude that the evolutionary trendfrom the high bone growth rates estimated forthe last common ancestor of archosaurs andfor some Triassic Crurotarsi (e.g., Calyptosu-chus) toward a reduction of bone growth ratesin Crurotarsi is well supported. The predato-ry behavior shared by extant Crurotarsi(crocodiles and alligators) may be linked tothe low bone growth rates characterizingthese animals in the wild. On the other hand,the efficient anatomical disposition of theirrespiratory system may explain why captivealligators, when kept at favorable conditions,grow at rates at least twice those observed inwild individuals.


Acknowledgments

We thank K. Padian (University of California,Berkeley), J. R. Horner (Museum of the Rockies,Bozeman, Montana), and A. de Ricqles (Uni-versite Pierre et Marie Curie, Paris) for the loanof histological slides of fossil taxa and for usefulcomments on a preliminary draft of themanuscript. We also thank D. Marjanovic(Universite Pierre et Marie Curie, Paris) for hisnomenclatural and stylistic comments, M.Laurin (Museum National d’Histoire Naturelle,Paris) for fruitful discussions on methodologi-cal issues and for English edition, A. Lee (OhioUniversity, Athens) for preparing the femoralsection of Lesothosaurus and F. Knoll forallowing it to be sectioned. Thanks are alsogiven to the two editors who handled the draft,as well as to the reviewers, the comments ofwhich greatly improved the quality of thispaper. This study was partly funded by the PPFBiomineralisations of the Universite Pierre etMarie Curie – Paris VI. C.M.M. was funded bythe National Human Resources Mobility Pro-gramme under the I+D+i 2008–2011 NationalPlan of the Spanish Ministry of Science andInnovation (MICINN).

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Paleohistological estimation of bone growth rate in extinct archosaurs