Bone 40 (2007) 1152–1158www.elsevier.com/locate/bone

Case Report

Do egg-laying crocodilian (Alligator mississippiensis)archosaurs form medullary bone?

M.H. Schweitzer a,b,⁎, R.M. Elsey c, C.G. Dacke d, J.R. Horner e, E.-T. Lamm e

a Department of Marine, Earth, Atmospheric Sciences, North Carolina State University, Raleigh, NC 27695, USAb North Carolina Museum of Natural Sciences, Raleigh, NC 27695, USA

c Louisiana Department of Wildlife and Fisheries, Rockefeller Wildlife Refuge, Grand Chenier, LA 70643, USAd School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, PO1 2DT, UK

e Museum of the Rockies, Montana State University, Bozeman, MT 59717, USA

Received 20 July 2006; revised 26 September 2006; accepted 3 October 2006Available online 12 January 2007

Abstract

It is beyond question that Mesozoic dinosaurs, like Aves and Crocodylia, are archosaurs. However, within the archosaurian clade, the originand distribution of some major features are less clear, particularly with respect to reproductive physiology. Medullary bone, a highly mineralized,bony reproductive tissue present in the endosteal cavities of all extant egg-laying birds thus far examined, has recently been reported inTyrannosaurus rex. Its presence or absence in extant crocodilians, therefore, may shed light on the timing of its evolutionary appearance. Ifmedullary bone is present in all three taxa, it arose before the three lineages diverged. However, if medullary bone arose after this divergence, itmay be present in both extinct dinosaurs and birds, or in birds only. If present in extinct dinosaurs and birds, but not crocodilians, it would indicatethat it arose in the common ancestor of this clade, thus adding support to the closer phylogenetic relationship of dinosaurs and birds relative tocrocodilians. Thus, the question of whether the crocodilian Alligator mississippiensis forms medullary bone during the production of eggs hasimportant evolutionary significance. Our examination of long bones from several alligators (two alligators with eggs in the oviducts, one that hadproduced eggs in the past but was not currently in reproductive phase, an immature female and an adult male) shows no differences on theendosteal surfaces of the long bones, and no evidence of medullary bone, supporting the hypothesis that medullary bone first evolved in thedinosaur–bird line, after the divergence of crocodilians from this lineage.© 2006 Elsevier Inc. All rights reserved.

Keywords: Medullary bone; Archosaur; Bird; Dinosaur; Crocodile; Reproduction

Introduction

The phylogenetic relationship between theropod dinosaursand birds is robustly supported by more than 300 derivedcharacteristics shared between these two clades and not found inother taxa, including their closest extant sister taxon, Crocodylia(e.g. [1–5]). Because of this evolutionary proximity, it is oftenargued that physiological strategies may also be shared betweenthe two groups, setting them apart from Crocodylia [1–5].

⁎ Corresponding author. Department of Marine, Earth and AtmosphericSciences, Room 2148 Jordan Hall, Campus Box 8208, North Carolina StateUniversity, Raleigh, NC 27695, USA. Fax: +1 919 515 7802.

E-mail address: schweitzer@ncsu.edu (M.H. Schweitzer).

8756-3282/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.bone.2006.10.029

Whether this extends to reproductive strategies has beendebated [5,6], but new evidence presented here supports thiscontention.

Birds, dinosaurs, crocodiles and many reptiles are distin-guished from other vertebrate classes by their ability to lay eggswith hard, calcified shells. Egg-laying birds have developed aunique physiological strategy to provide, over a short period oftime, the large quantities of calcium required for eggshellmineralization. They do not resorb calcium directly fromcortical or endochondral bone, as do other vertebrates. Toprotect their thin, flight-adapted bone cortices from debilitatingosteoporosis, the endosteal membrane lining the cavities of theirlong bones gives rise to large-scale deposits of a highly labile,vascular, easily mobilized bone tissue within the endosteal

Fig. 1. Eggs within the reproductive tracts of two Alligator mississippiensisspecimens used in this study.

Fig. 2. Mid-shaft cross sections of femora of (A) 254 cm (Specimen A) femalewith calcified eggshells; (B) 206 cm (Specimen B) female with partiallycalcified shells ; (C) 191 cm sexually mature female not in active reproduction(Specimen C); (D) 137 cm sexually immature female (Specimen D).

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cavities of the long bones. This endosteal bone, known asmedullary bone [7–9], is a non-structural woven bone withlarge surface area-to-volume ratio that facilitates rapid mobili-zation of calcium mineral to the bloodstream [10].

Medullary bone had only been reported and figured in extantneognaths until its recent identification in egg-laying paleog-nath ratites [11]. With the exception of one unsubstantiatedanecdotal report suggesting its presence in female crocodiles[12], this tissue is limited to birds among extant taxa in theliterature. As a reproductive tissue, medullary bone is foundprimarily within the long bones of egg-laying birds, and hasbeen observed in all extant [13] and some fossil [14] bird taxastudied.

When the discovery of a brooding oviraptor (theropod) wasannounced [15], Martill et al. [16] proposed that avian-typemedullary bone might be found on the endosteal surfaces of longbones from this specimen; but the bones were not examined, andcalcium mobilization strategies in dinosaurs remained specula-tive. However, in 2005, Schweitzer et al. [11] described thepresence of avian-type medullary bone lining the endostealsurfaces of a 68-million-year-old Tyrannosaurus rex femur,estimated to have been approximately 18 years of age at the timeof death [17]. Morphological characteristics of this novel tissuewere compared with medullary bone from extant ratites (ostrichand emu). As the most basal of extant bird lineages, ratites retainmore characters in common with theropod dinosaurs, includingT. rex [1–5, and references therein] than do the more derivedextant neognaths.

The presence of medullary bone in dinosaurs, thoughhighly significant, does not address the question of dinosaur–bird reproductive and physiological similarities unless thistissue can be shown conclusively not to exist in extant

crocodilians, the closest extant taxon to both dinosaurs andliving birds [1–5]. Its absence in crocodilians was givenpreliminary support when Elsey and Wink [24] injectedimmature alligators with estradiol, but results were inconclusive(see Discussion).

Biomechanical and reproductive (shelling physiology) con-straints may make it difficult to recognize medullary bone inCrocodylia (including alligators, crocodiles, and caiman), ifpresent. Whereas extant birds shell one egg at a time, lay one ormore clutches of eggs per year, and rest at least 24 h betweeneach successive ovulation [19,20], crocodilian archosaurs shelltheir eggs en masse [21,22]. Thus, if it existed, medullary bonewould be present for a much shorter time in crocodiles than inbirds, even though their calcium metabolism is very intensiveduring this period [23–25]. Therefore, to optimize the chancesof finding this reproductive tissue in non-avian archosaurs, weexamined the bones of female alligators known to have eggspresent in the oviducts (Fig. 1), and compared them with non-reproducing specimens.

Materials and methods

Femora of two female American alligators (A. mississippiensis) captured onRockefeller Wildlife Refuge in Grand Chenier, LA during the summer layingseason, both with a full clutch of eggs in the oviducts, were used in the presentstudy. In one female, 254 cm in length (Specimen A), eggshells were fullycalcified, and in the other, 206 cm in length (Specimen B), the eggs presentedwith partially calcified shells (Figs. 1A, B). A third sexually mature female191 cm in length (Specimen C), with no eggs in the reproductive tract butevidence of having produced them in a previous season, and a fourth, a 137 cmsexually immature female (Specimen D), were also examined. For comparison, aground section from the femur of a mature male, prepared as part of anotherstudy, was included.

Bones were photographed, then fixed in 10% buffered formalin. Each studybone was cut at mid-diaphysis in transverse section, and longitudinal cuts weremade of the remaining ends, splitting the proximal end into cranial and caudalhalves, and the distal end into medial and lateral halves. Open cut surfaces werephotographed, and remaining soft tissues were dissected from the bone, leavingsurface ligament attachments.

1154 M.H. Schweitzer et al. / Bone 40 (2007) 1152–1158

Bone segments to be sectioned were incubated in a 10% solution of Zout®for 3 days to solubilize associated fat. Bones were then dehydrated in anethanol series (70% and 85% for 2 days each, then 100% EtOH for 1 week).Samples were air-dried, re-photographed, and then cleared in xylene for 7 h.Samples were air-dried for 2 additional days prior to embedding. To embed,bones were infiltrated with Epo-Thin® epoxy resin (Buehler Ltd.) with catalystomitted, and placed under vacuum for 2 days total to allow for maximuminfiltration. Specimens were then embedded in Epo-Thin® resin with catalystunder vacuum for 15 min to allow for infiltration of the catalyst into the resin-saturated bone. Blocks polymerized overnight under refrigeration, with novacuum. Wafers, approx. 2.0 mm thick, were cut using custom diamond blades(Norton) on a water-cooled tile saw (Felker 41-AR), or with ethanol aslubricant on an Isomet® 1000 Precision Saw (Buehler Ltd.). Penetrant/Stabilizer glue (PALEO-BOND™), Epo-Thin® resin, and/or (Triad®) – amethyl methacrylate based bonding agent – were all used to first coat andstabilize the ‘mounting-side’ of the cut wafers. The ‘mounting-side’ of thewafers were then ground to a 600-grit finish using an Ecomet® 4 Grinder-Polisher (Buehler Ltd.) with silicon carbide abrasive papers (Buehler Ltd.).Prepared cut sections were next mounted onto frosted plastic slides with 100–750 cps. cyanoacrylate glue (PALEO-BOND™). Mounted cut sections werethen ground and polished with silicon carbide papers of decreasing coarseness(60 to 600 grit) on an Ecomet® 4 Grinder-Polisher in water, ethanol and/orIsocut fluid (Buehler Ltd.). Finally, thin sections were polished using siliconcarbide papers (800 and 1200 grit).

Results

Gross and microscopic analyses of cross sections of thefemora of these alligator specimens are shown in Figs. 2 and 3,respectively. Fig. 2 shows gross transverse sections of thefemora of the females before embedding. Endosteal cavitiesremain free of bony infilling, but some fatty tissues fill orpartially fill some cavities (Fig. 2A). The endosteal surfaces ofthe bone on gross examination are similar in all specimens, andno evidence of new, endosteally derived bone growth is seen inany of these specimens, although large erosion rooms areapparent. Microscopic observation likewise does not demon-strate the presence of medullary bone extending into the longbone cavities.

Fig. 3 shows ground sections of femora of gravid alligatorfemales before oviposition (A–D), an adult female not in activereproduction (E, F), an immature female (G, H) and a malealligator (I, J). At higher magnifications (Figs. 3B, D, F, H, J),the endosteal surfaces of all specimens are smooth, althougherosion rooms are obvious (ER, arrows in lower magnificationpanels). In all specimens studied, lines of arrested growth(LAGs) were apparent, consistent with known annual patternsof bone deposition [17,26–29, and references therein]. Theextensive erosion rooms indicate metabolic turnover, regardlessof gender and reproductive phase. However, erosion rooms aremore abundant in ovulating females, similar to previousobservations [18,24,25].

The histological appearance of these bones, or alligatorbones is similar, regardless of reproductive phase or gender.This differs from extant ratites (Figs. 4A, B; Schweitzer et al.,2005), in which crystalline deposits of medullary bone form onendosteal bone surfaces during ovulation. Similar deposits havebeen noted on the endosteal surface of a T. rex (Figs. 4C, D;Schweitzer et al., 2005). As in extant birds, the dinosaur tissuesare highly vascular, and formed from rapidly deposited wovenbone derived from the endosteal lining tissues. While cortical

bone (Fig. 4D) shows dense Haversian bone with remodelling,typical of mature dinosaur bone, the medullary bone layershows dense vascularization, and the matrix fibers are lessorganized than those in the cortical bone.

Discussion

Medullary bone was first described by Foote [30], andrediscovered by Kyes and Potter [31] in pigeons. The tissue,which forms shortly before laying begins, is triggered by thesurge in blood estrogen levels that precedes ovulation [32]. Thehighly mineralized medullary bone is rapidly resorbed at theend of the reproductive period, and shows cyclical fluctuationsas successive clutches of eggs are deposited. Approximately40% of the calcium within the hen's eggshell is derived from theskeleton [33], and the vast majority comes directly frommedullary bone [34]. Because this reproductive bone tissueforms in response to rising gonadal steroid levels, it can beinduced to form in adult male birds by injection of estrogens orin immature birds by injection of estrogens combined withandrogenic steroids [7,13,32,35].

Medullary bone has not been described in non-archosaurianreptiles [7,13]. Edgren [36] investigated seasonal changes inbone density in the female musk turtle (Sternothaerus odoratus),but did not find any avian-type medullary bone within thistaxon. In a study of the slider turtle (Pseudemys scripta elegans),Suzuki [37] examined bones of 17 females with shelled eggsinside the oviduct. In these reproducing turtles, the cortical boneof the femora appeared thinner than in non-laying turtles of anequivalent size, and was described as having an “osteoporoticappearance” but new, endosteally derived bone was notobserved. Attempts have been made to induce medullary boneformation in various Chelonia by injections with gonadalsteroids [13,37,38]. Magliola [38] found that doses of 17β-estradiol sufficient to cause profound hypercalcaemia in malebox turtles (Terrapene carolina triunguis) had no discernibleeffect on skeletal growth or uptake of radio-labelled calcium inseveral skeletal elements including the plastron (shell), whencompared with control, untreated animals, and it is generallyaccepted that non-archosaurian reptiles do not produce thisreproductive tissue.

Like the anuran amphibians, some, but not all reptiles(notably small geckos and iguanid lizards), have developedthe use of greatly extended endolymphatic calcium depositsas a reproductive strategy. These were first described inPhilippine house lizards (Cosmybotus platurus) [39] andreviewed by Simkiss [13] and Dacke [7]. There are no reportsthat larger lizards or extant, non-avian archosaurs havedeveloped endolymphatic calcium deposits to such an extent.

Crocodilians are the closest living relatives of avian anddinosaurian archosaurs [1–5], and because they lay calcifiedeggs, it is reasonable to hypothesize that they may also haveevolved medullary bone. Like birds, adult female alligatorsresorb structural bone during eggshell formation [18,24,25].Therefore, Elsey and Wink [24] hypothesized that estrogentreatment might initiate medullary bone formation in alligatorsas it does in male and/or immature female birds [32]. In light of

Fig. 3. Light micrographs of ground sections at low and high magnifications showing endosteal surface, endosteal laminar bone (ELB), and erosion rooms (ER,arrows). (A) Low and (B) high magnifications of Specimen A, large female alligator with calcified eggs; (C) Low and (D) high magnifications of Specimen B, withpartially calcified eggs; (E) Low and (F) high magnifications of Specimen C, no eggs in body cavity; (G) Low and (H) high magnifications of Specimen D, immaturefemale, no eggs; (I) Low and (J) high magnifications of adult male alligator. No endosteal bone is observed in any case. Scale bars as indicated.

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Fig. 4. Low magnification fresh fracture of extant female emu (A, B) in reproductive phase, and Tyrannosaurus rex (C, D), with similar bone tissues on the endostealsurface of the tibia. In fresh fracture (C) and in ground section (D), the newly deposited bone tissues can be seen on the endosteal surfaces of the dinosaurian corticalbone. CB, cortical bone; ELB, endosteal laminar bone; MB, medullary bone, T, trabecular bone. Scale bars as indicated.

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this, attempts were made to invoke the production of medullarybone with injection of estradiol into juvenile (3.5 years old)alligators of both genders. Although these animals showedsignificant rises in plasma calcium levels within one week oftreatment, there was no change in femoral bone structure up to amonth after injection, when compared with controls [24].Prosser and Suzuki [40] reported similar results when theyinjected estradiol valerate into hatchling and juvenile Caimansclerops. However, because in all cases the experimentalanimals were sexually immature, this treatment may havebeen insufficient to stimulate medullary bone production [8],because both estrogens and testosterone are needed to induce itsformation in immature birds [7,13,35]. The question ofmedullary bone production in non-avian archosaurs remainedopen, and required a re-examination of this issue in animalsreproducing under natural conditions.

If medullary bone exists in non-avian archosaurs, itmight be expected to be more short-lived than in birds,because of differences in ovulation, shelling and ovipositiondiscussed above. Therefore, the best chance of determiningconclusively whether crocodilians have the physiologicalability to produce medullary bone requires direct observationof animals in the process of naturally producing and shellingeggs. If crocodilians produce medullary bone, its presence indinosaurs may not shed light on the evolution of reproductivephysiology within the archosaurian clade. However, if crocodi-lians do not produce medullary bone, this evolutionary strategymust have evolved after the divergence of lineages leading toextant crocodilians and birds (Early Triassic or even latestPermian [1]).

Reproductive physiology of non-avian archosaurs

Latitude affects the onset of sexual maturity in Americanalligators (A. mississippiensis), and those with more southerlyrange attain reproductive maturity earlier (∼10 years insouthwest Louisiana, USA) than those with a more northerlyrange (∼18 years, North Carolina [41]). Sexually immaturealligators show seasonal cycles of reproductive hormones [42];likewise, reproductive activity in sexually mature animals isseasonal [43]. Reproductively active females show elevatedconcentrations of circulating estradiol in both fall (September–November) and spring (March–May). When female alligatorswere examined in spring, ovaries with larger follicles wereobserved [41].

Female alligators lay one clutch of about 40 eggs in June[22,44]. These eggs are ovulated simultaneously, as in othernon-archosaurian reptiles, and eggshells are calcified inassembly-line fashion along the oviduct, followed by simulta-neous oviposition [6,22]. In contrast, all birds lay extendedclutches of eggs, often several times during the reproductiveseason [19,20]. Packard and Packard [45] studied themobilization of calcium, phosphorus and magnesium byembryonic alligators, and found that both yolk and shell areimportant sources of calcium for the developing embryo.

These observations imply that reproducing female alligatorsmust mobilize high concentrations of calcium over a relativelyshort period of time to meet the requirements for eggshellcalcification. Edgren [36] and Simkiss [13] speculated thatmedullary bone formation might be found in “higher reptiles”(sic) such as crocodilians. However, the source of this needed

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calcium is unidentified at this time. The possibilities that it maybe mobilized from 1) endolymphatic deposits, as in anurans andsome lizards; 2) short-lived medullary bone or analogousdeposits, as in birds; or 3) some other source such as theosteoderm layer of the integument or simply mobilization ofstructural bone, has not been conclusively determined and isstill debated.

However, using animals in active reproduction, we did notfind evidence for medullary bone in precisely the stages offemale crocodilians deemed most likely to present it, if it wereproduced. Regardless of their reproductive phase, the endostealsurfaces of bones of female alligators do not differ from non-reproducing, immature, or male members of this clade. Thesedata support the prior study of Wink and Elsey [25].

It must be noted that the data presented does not allow usto address the presence of medullary bone in other livingspecies of crocodilian archosaurs, or any extinct ones. It ispossible that this tissue was once present in other lineages butis now lost in living representatives. Only direct examinationof other fossil material can demonstrate the existence ofmedullary bone in other lineages, but to date, this is notsupported.

Therefore, the presence of this novel reproductive tissue indinosaurs [11] adds support to the theropod–bird relationship,and suggests that at some point after the divergence ofcrocodilians from this lineage, both groups attained physiolog-ical status that required the rapid mobilization of calcium forshelling over that of crocodilians.

Acknowledgments

We thank C. Simpson and H. Woodward (Museum of theRockies, Montana State University), for assistance in prepara-tion of the ground sections illustrated in this paper, and J.L.Wittmeyer (North Carolina State University), for photographicassistance. We also acknowledge W. Parke Moore III of theLouisiana Department of Wildlife and Fisheries for adminis-trative support, and Phillip “Scooter” Trosclair and DwayneLeJeune for assistance with collection and sampling ofalligators. This research was funded in part by NSF (EAR-0435626) and by the Discovery Channel and funds from NorthCarolina State University.

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