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DOI: 10.1017/pab.2016.47

Can secondary osteons be used as ontogenetic indicators in

sauropods? Extending the histological ontogenetic stages
into senescence

Jessica Mitchell, P. Martin Sander, and Koen Stein

Abstract.Sauropod bone histology has provided a great deal of insight into the life history of these enor-
mous animals. However, because of high growth rates, annual growth rings are not common in sauropod
long bones, so directly measuring growth rates and determining sexual maturity require alternative mea-
sures. Histological ontogenetic stages (HOS) have been established to describe the changes in bone histology
through development for basal Macronaria and Diplodocoidea, and subsequently for Titanosauria. Despite
this, the current HOS model is not able to discriminate bone tissues in late ontogeny, when sauropods had
reached asymptotic size and continued to live into senescence but their long bones became extensively
remodeled by secondary osteons and all primary bone was destroyed. Here we establish remodeling stages
(RS) to characterize the Haversian bone development through ontogeny in eight sauropod taxa
(Apatosaurinae, Giraffatitan brancai, Camarasaurus spp., Dicraeosaurus spp., Ampelosaurus atacis, Phuwiango-
saurus sirindhornae, Magyarosaurus dacus, and Alamosaurus sanjuanensis) and nd signicant correlation of RS
with corresponding femur length (CFL) for the studied taxa, with the exception of Dicraeosaurus and
Magyarosaurus. Remodeling stages are based on the maximum number of observable generations of cross-
cutting osteons from the innermost, mid-, and outermost part of the cortex. The correlation with CFL
indicates that secondary osteons present an ontogenetic signal that could extend the histological ontogenetic
stages. Remodeling stages also provide additional insight into the changes in histology through ontogeny for
Sauropoda. This method has the potential to be used in other taxa, such as thyreophorans and many
ornithischians, that develop Haversian tissue through development.

Jessica Mitchell and P. Martin Sander. Steinmann-Institut fr

Mineralogie, Geologie und Palontologie, Universitt Bonn, Nussallee 8, D-53115, Bonn, Germany.

Koen Stein. Earth System SciencesAMGC, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium,
and Royal Belgian Institute of Natural Sciences, Directorate Earth and History of Life, rue
Vautier, 29, 1000 Brussels, Belgium. E-mail:

Accepted: 10 October 2016

Introduction Bone histology, however, changes through

ontogeny, and juveniles can usually be differ-
Since the seminal studies of Armand de entiated from adult forms.
Ricqls (Ricqls 1968a,b, 1983), bone histology Skeletochronology, the study of growth lines
has proven to be an invaluable tool in under- in bone, is one of the few disciplines that allows
standing the biology of sauropod dinosaurs, individual age estimation in vertebrates (Cas-
providing insights into growth rates, species tanet 1994). Many authors have estimated the
determination, and age of these unique age of dinosaurs by calculating missing lines of
dinosaurs (Sander 2000; Erickson 2005; Sander arrested growth (LAGs) or estimating bone
et al. 2006, 2011; Griebeler et al. 2013; Stein and apposition rates (Curry 1999; Sander and
Prondvai 2014). Fossil bone histology plays a Tckmantel 2003; Erickson et al. 2004; Horner
major role in determining whether different and Padian 2004; Bybee et al. 2005; Klein and
body sizes reect ontogenetic or taxonomic Sander 2007; Wings et al. 2007; Griebeler et al.
differences. Morphological data are, in many 2013). In contrast to those of most dinosaurs
cases, insufcient to determine whether small- (including more basal sauropodomorphs), the
sized fossil animals are juveniles or different, highly vascularized long bones of sauropods
smaller adult forms (Sander et al. 2006; usually do not exhibit annual cycles, although
Klein and Sander 2008; Griebeler et al. 2013). some sauropods do have LAGs late in

2017 The Paleontological Society. All rights reserved. 0094-8373/17

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ontogeny. In some adult sauropods, rest lines Klein and Sander (2008) concluded that sexual
can be observed in a thin peripheral layer of maturity was generally attained around
avascular bone in the outermost cortex, known HOS 8, coinciding with a decrease in vascular-
as the external fundamental system (EFS) ization and an assumed decrease in growth
(sensu Ham 1953). The EFS indicates that the rate. They also concluded that a close relation-
animal attained full size. However, the annual ship of body size and HOS indicates a lack of
character of the rest lines within the EFS is not developmental plasticity, similar to modern
conrmed because of their compactness (San- mammals and birds.
der et al. 2011; Griebeler et al. 2013). The The histological ontogenetic stages were
general lack of annual cycles makes age originally established for growth series of basal
estimation in sauropods from their long bones Macronaria and Diplodocoidea (e.g., Apato-
difcult (Sander et al. 2011). The lack of growth saurus, Camarasaurus). Since then, the histolo-
marks in long bones does, however, suggest gical ontogenetic stages have also been applied
that sauropods grew relatively quickly without to titanosaurs (Klein et al. 2009; Woodward
any major slowdown in growth for noticeably and Lehman 2009; Stein et al. 2010; Company
long periods (Sander et al. 2011; Klein et al. 2011; Klein et al. 2012; Ghilardi et al. 2016),
2012; Waskow and Sander 2014). The incom- an indeterminate mamenchiasaurid, and
plete expression of growth marks in long bones Plateosaurus engelhardti, a basal sauropodo-
is corroborated by their presence in ribs of the morph (Griebeler et al. 2013). In general, the
same individual, where they provide a much titanosaurs all show an atypical type of
better age estimate than the long bones primary bone, termed modied laminar
(Waskow and Sander 2014). The disadvantage bone (Klein et al. 2012) and different amounts
of using ribs for aging is that body size of remodeling, with the exception of Alamo-
cannot be estimated from ribs. This means that saurus (Woodward and Lehman 2009; Klein
a rib to be used for aging needs to be associated et al. 2012) and an indeterminate titanosaur from
with at least a single propodial (humerus or Brazil (Ghilardi et al. 2016), which had similar
femur). In addition, the growth record pre- histology to Macronaria and Diplodocoidea
served in a given rib cannot be used to (Fig. 1). Magyarosaurus, as described by Stein
construct a growth curve for the individual et al. (2010), showed extreme cortical remodel-
the rib is derived from, because local apposi- ing within a growth series, which did not
tional growth is not only related to overall
body size increase but also to local morpho-
genesis (Waskow and Sander 2014).

Histologic Ontogenetic Stages (HOS)

Despite the common absence of LAGs in
sauropod long bones, histological changes can
be used to determine the relative maturity of an
individual. These histological changes were
documented by Klein and Sander (2008), who
developed histological ontogenetic stages.
There are 13 histological ontogenetic stages,
based on bone tissue type (e.g., brolamellar
bone), organization of vascular canals, degree
of vascularization, presence of primary
osteons, presence of growth marks, the appear-
ance of the EFS, and to a limited extent, the
degree of Haversian bone development. A
FIGURE 1. Phylogenetic relationships of Sauropodomorpha
fourteenth HOS was established by Stein et al. after Wilson (2002), Sander (2013), and Tschopp et al.
(2010) for the dwarf titanosaur Magyarosaurus. (2015). Taxa used in this study are in boldface type.

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conform to the normal histology of other sequence is initiated in which preosteoclasts

sauropods. The smallest bone in the series was are recruited to the site and fuse into multi-
still placed at HOS 12. Many of the large nucleated osteoclasts (Jaworski et al. 1981;
individuals had several generations of second- Partt 1994). Upon arrival, the osteoclasts
ary osteons, up to three or four. This is why begin tunneling through bone, resorbing the
the additional HOS, HOS 14, was added to mineral and collagen in what is referred to as
differentiate between highly remodeled bone the cutting cone (Partt 1994). Following the
with more than one generation in the outer osteoclasts is a reversal zone, with cells that
cortex and moderately remodeled bone, which deposit a cementing substance, visible as the
only had one generation in the outer cortex. cementing line in cross section, which
This rst attempt to differentiate between surrounds the osteon. The osteoblasts then
generations of secondary osteons inspired the centripetally deposit bone lamellae, termed the
establishment of the remodeling stages in the closing cone (Partt 1994). The vascular space
current work. in the center, the Haversian canal, is then lled
In most cases in sauropods, after the EFS has with blood vessels, connective tissue, and
been deposited, bone remodeling continues to nerves (Partt 1994). Some osteoblasts will
extend into the entire cortex, destroying any become buried within the matrix they are
previously deposited primary bone and all its secreting and become osteocytes. They are
histological features. Therefore, once old age is usually visible as small, black, attened to
reached, secondary osteons are the only plump shapes in thin section, connected to
remaining histological feature. Because of the each other by a network of canaliculi that
lack of primary bone, old individuals have not allows for communication and the exchange of
been studied in detail, as they are assumed to nutrients. The remainder of the osteoblasts will
be less informative about ontogeny. To under- either die or become lining cells (Francillon-
stand growth rates, age, and sexual maturity, Vieillot et al. 1990; Partt 1994). In cross
most researchers have focused on juveniles, section, secondary osteons have a round to
with rapid growth, and their transition to oval appearance, and osteocyte lacunae and
adults, with markedly slowed-down growth, individual concentric lamellae are usually
as indicated by decreased vascularization and visible. Secondary osteons are aligned along
the EFS. the dominant loading direction, suggesting
that they are mechanically regulated (Ascenzi
and Bonucci 1967, 1968; van Oers et al. 2008).
Haversian Bone Formation Additionally, secondary osteon formation is
Haversian or remodeled bone is deposited primarily associated with fatigue damage and
in units called secondary osteons (Gross 1934; microcracks (Burr et al. 1985; Mori and Burr
Francillon-Vieillot et al. 1990) in areas where 1993; Bentolila et al. 1998; Martin 2000).
tissue has previously been resorbed. It is very Primary osteons, by contrast, form by deposi-
common in large dinosaurs, medium to large tion of bone on a preexisting surface; resorp-
mammals, and many birds (Chinsamy-Turan tion is absent in the process. As this study only
2005). In the formation of secondary osteons, addresses secondary osteons, we will mostly
two cellular components, osteoclasts and refer to them as osteons.
osteoblasts, interact dynamically in discrete In long bones, as the animal grows and
packets known as a basic multicellular unit the cortex thickens, the innermost cortex is
(Frost 1969). Osteoclasts are responsible for the resorbed, resulting in medullary cavity expan-
resorption of bone, whereas osteoblasts are sion. This optimizes the weight and strength of
responsible for the deposition of new bone to the bone. In sauropods, as with other dino-
replace the resorbed bone (Partt 1994; van saurs, while the medullary cavity is expanding,
Oers et al. 2008). Remodeling in compact bone osteons begin to form in the innermost cortex
begins when osteocytes send signals for the and, through ontogeny, extend toward the
need for remodeling by emitting proteins and outer cortex. This process continues until the
other factors (Bonewald 2011). An activation animal dies, leading to crosscutting osteons

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(Haversian tissue formation) (e.g., Horner et al. Singh and Gunberg 1970; Thompson 1979;
2000; Stein et al. 2010; Klein et al. 2012; Samson and Branigan 1987; Walker et al.
Werning 2012; Mitchell and Sander 2014). 1994; Thomas et al. 2000; Streeter 2010). Similar
Remodeling rates vary between animals, but in to Kerley, most studies relied on the density of
sauropods, cortical remodeling usually does not Haversian bone and primary bone. Further
start until later in ontogeny; secondary osteons research was done on whether histology varied
appear usually around HOS 68. Presumably, in different ethnic groups and by gender (e.g.,
this is when the individuals are becoming Richman et al. 1979; Burr et al. 1990), while
sexually mature (Klein and Sander 2008). How- other studies focused on different aspects of
ever, Curry Rogers et al. (2016) noted evidence remodeling, such as size and shape of second-
of secondary osteons in a titanosaur perinate ary osteons (Britz et al. 2009; Castrogiovanni
(Rapetosaurus krausei), and the entire ontogenetic et al. 2011). A few studies on modern nonhu-
series of the titanosaur Magyarosaurus is remo- man mammals and comparative works on
deled with secondary osteons (Stein et al. 2010). different taxa have been done to assess aging
This is not the case for all titanosaurs, Alamo- in different species (e.g., Wu et al. 1970; Havill
saurus being a notable exception (Woodward 2003; Mulhern and Ubelaker 2003; Hillier and
and Lehman 2009). In fully grown specimens (as Bell 2007). As evidenced by work with humans,
indicated by the presence of an EFS), sauropods which like sauropods, lack distinctive growth
show nearly complete remodeling of primary marks and have strong remodeling, bone
bone (Ricqls 1980; Klein and Sander 2008, histology can be used to determine age. The
Mitchell and Sander 2014). extent to which Haversian bone can be used as
an ontogenetic indicator for sauropods is tested
here using several sauropod growth series.
Age Determination and Forensic Science Institutional abbreviations: BYU, Museum of
The strongest line of evidence for correlating Earth Sciences, Brigham Young University,
age with the development of Haversian tissue Provo, Utah, U.S.A.; CM, Carnegie Museum
comes from the eld of forensic science. Bone of Natural History, Pittsburgh, Pennsylvania,
histology has proven to be a useful tool in U.S.A.; FGGUB, Faculty of Geology and
helping uncover the age of individual human Geophysics of the University of Bucharest,
remains. Although histological studies on Romania; LCE, Muse de Cruzy, Cruzy, France;
humans had been done for well over a century, MAFI, Geological Survey of Hungary,
Kerley (1965) was the rst to determine the age Budapest, Hungary; MDE, Muse des Dino-
of human bone using histology. His method, saures, Esperaza, France; MFN, Museum fr
known as the Kerley method, involves count- Naturkunde, Berlin, Germany; OMNH, Sam
ing the number of secondary osteons, second- Noble Oklahoma Museum of Natural History,
ary osteon fragments (parts of older osteons in University of Oklahoma, Norman, Oklahoma,
the interstices of younger osteons, also known U.S.A.; PC.DMR, Department of Mineral
as interstitial lamellae), and non-Haversian Resources, Khon Kaen Province, Kalasin,
canals, and estimating the amount of primary Thailand; SMA, Sauriermuseum Aathal, Canton
lamellar bone in four regions of the outer cortex Zrich, Switzerland; TMM, Vertebrate Paleon-
of the midshaft of femora, tibiae, and bulae. tology Collection, Texas Memorial Museum,
A regression analysis of 126 individuals, from University of Texas, Austin, Texas, U.S.A.
birth to 95 years of age, allowed the age at
death to be estimated within 10 years 95% of
the time. Secondary osteons and osteon Materials and Methods
fragments were shown to be much more
correlative to age than primary lamellar bone Studied Sauropod Taxa
and non-Haversian canals (Kerley 1965; Kerley A total of 79 specimens were analyzed from
and Ubelaker 1978). Several other researchers the following eight neosauropod taxa:
have subsequently adopted and modied Apatosaurinae, Giraffatitan brancai, Dicraeosaurus
this method (e.g., Ahlqvist and Damsten 1969; spp., Camarasaurus spp., Phuwiangosaurus

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sirindhornae, Ampelosaurus atacis, Magyarosaurus Klein et al. (2009). Alamosaurus sanjuanensis is a

dacus, and Alamosaurus sanjuanensis. The phylo- derived large-bodied titanosaur from the Maas-
genetic relationships of the sampled taxa are trichtian of the southern Rocky Mountain region
shown in Figure 1. Note that many of the of the United States, and its long bone histology
samples have been used in previous studies, as was described by Woodward and Lehman
pointed out on a taxon-by-taxon basis below. (2009) and Klein et al. (2012). Our sample was
Two sauropods, Dicraeosaurus spp. and originally acquired for the latter study.
Giraffatitan brancai, formerly Brachiosaurus We used thin sections of ontogenetic series of
brancai (see Taylor 2009), come from the Late femora and humeri, most of which have been
Jurassic Tendaguru Formation, Tanzania. used in previous studies, as noted above. Most
Dicraeosaurus is a medium-sized diplodocoid samples were obtained by coring in the mid-
sauropod (Wilson 2002) known from two shaft region following the protocol of Sander
species, D. hansemanni and D. sattleri, both of (2000), using the method described in detail by
which are represented in our sample and were Stein and Sander (2009). Samples were taken
histologically described by Sander (2000). from the midshaft of the long bones, because
Giraffatitan is one of the larger sauropods, this region contains the most complete growth
belonging to Brachiosauridae, and is the sister record, as long bones grow appositionally from
taxon to the North American Brachiosaurus the central region of the femur and humerus
altithorax (Taylor 2009). For a general descrip- (Francillon-Vieillot et al. 1990; Sander 1999,
tion of Giraffatitan and Dicraeosaurus histology, 2000; Stein and Sander 2009). Some complete
see Sander (2000). cross sections were obtained as well, and all
Camarasaurus is a medium-sized sauropod samples were processed into thin sections
from the Late Jurassic Morrison Formation in using standard thin-sectioning procedures
the United States. It is the most common (see Enlow and Brown 1956; Chinsamy and
sauropod in the formation and is considered a Raath 1992; Wilson et al. 1994). The standard
basal macronarian. Specimens of Apatosaurinae coring location for femora is the anterior side of
can also found in the Morrison Formation and the midshaft, whereas the standard coring
are considered diplodocid sauropods. Although location in humeri is the posterior side of the
described as the genus Apatosaurus in Klein and midshaft (Sander 2000; Klein and Sander 2008;
Sander (2008), the recent publication of Tschopp Sander et al. 2011). Most samples were taken
et al. (2015) has put into question the true afnity from these standard locations, but a few were
of some of the Morrison diplodocids. Hence, we not (see Table 1 for details). All samples were
assign the specimens used here to Apatosaur- included in the analysis to compare histovaria-
inae. Among the sampled taxa, it is most closely bility of Haversian bone development between
related to Dicraeosaurus (Fig. 1; Wilson 2002). standard and nonstandard regions.
A general description of histology for Apato- The Dicraeosaurus specimens include ve
saurinae and Camarasaurus can be found in Klein long bones from D. sattleri and one from
and Sander (2008). D. hansemanni. The latter was included because
The last four sauropods are currently regarded its histology and size are similar to those of
as titanosaurs. Magyarosaurus dacus is a dwarf D. sattleri. Two morphotypes of Camarasaurus
titanosaur from the early Maastrichtian of were distinguished using histological ontoge-
Romania with a maximum length of 6 m. Its netic stages (Klein and Sander 2008). They
long bone histology was described by Stein et al. were combined for statistical analyses in this
(2010). Ampelosaurus atacis is a derived medium- study. The bone histology of all taxa has been
sized late Campanian to early Maastrichtian described in previous studies (see Table 1), and
titanosaur from southern France, and histologi- the thin sections are stored in the thin-section
cal descriptions are found in Klein et al. (2012). collection in the Steinmann Institute of the
Phuwiangosaurus sirindhornae is a basal titanosaur University of Bonn, Germany.
or basal somphospondylian and comes from the Femur length is commonly used as a proxy
Early Cretaceous Sao Khua Formation in Thai- for body size in sauropods (e.g., Bonnan 2004).
land. Its long bone histology was described by In femora, the length was measured directly on

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TABLE 1. Specimens with their respective HOS (histologic ontogenetic stages), RS (remodeling stages), and measure-
ments for CFL (corresponding femur length), Avg. ODm. (average osteon diameter), and OPD (osteon population den-
sity). Note some values are NA due to poor specimen preservation. For detailed histology see Klein and Sander (2008):
Apatosaurinae, Camarasaurus; Sander (2000): Dicraeosaurus, Giraffatitan; Klein et al. (2009): Phuwiangosaurus; Woodward
and Lehman (2009): Alamosaurus; Stein et al.(2010): Magyarosaurus; Klein et al. (2012): Ampelosaurus. Asterisks indicate
femur sampled on posterior side (*) and humerus sampled on anterior side (**).

Bone Bone length CFL Avg. ODm OPD

Specimen Taxon element (mm) (mm) HOS RS (m) (n/mm2)
OMNH 01279 Apatosaurinae Femur 340 5 0 0 0.000
OMNH 01278 Humerus 258 396 5 0 0 0.000
CM 33976 Femur 725 6 2.5 189 0.197
BYU 601 17103 Femur 830 7 2 378 0.414
BYU 725-17014 Femur 970 7 3 222 0.572
CM 30766 Femur* 1302 10 6 308 2.701
BYU 681-11940 Femur 1330 9 6 332 3.293
BYU 681-4749 Humerus 880 1350 8 4.5 351 2.248
BYU 681-17328 Femur* 1580 12 2 272 2.484
BYU 681-17328 Femur 1580 12 8 262 5.540
OMNH 4020 Femur 1800 13 15 323 7.946
MFN XX19 Giraffatitan brancai Humerus 690 663 6 4 263 0.611
MFN IX 1 Femur 880 9 3 221 0.493
MFN T8 Humerus 1280 1231 9.5 3 203 2.484
MFN dd 452 Femur 1350 10 5 198 4.259
MFN T7 Humerus 1530 1471 9.5 5 223 1.755
MFN Nr 305 Femur 1560 11 6 185 5.067
MFN II 22e Humerus 1760 1692 11 8 210 6.506
MFN ST 291 Femur 1830 10.5 7 270 4.140
MFN XV Femur 2190 12 9 218 7.748
OMNH 2115 Camarasaurus spp. Humerus 227 292 3.5 0 0 0.000
CM 21772 Femur 550 6 0 0 0.000
OMNH 1794 Femur 600 6 0 0 0.000
CM 33963 Humerus 508 654 8 3 318 1.636
BYU 681-4742 Humerus 615 794 7 3 224 1.518
SMA K11-29-1 Femur 830 12 4.5 292 NA
SMA 0002 Femur 935 11 1.5 238 1.242
SMA 0002 Humerus 705 935 11 7 276 6.211
BYU 725-12173 Femur 1330 8 3 280 1.498
CM 38320 Humerus 1040 1342 10.5 4 274 2.169
CM 36664 Femur 1452 12 4 272 3.017
CM 36664 Humerus 1172 1452 12 5 237 6.565
OMNH 02113 Humerus 1204 1554 10 3 301 0.375
CM 11393 Femur 1566 12 5 270 2.228
MFN ab10 Dicraeosaurus spp. Humerus 580 934 6 1 206 0.355
MFN O2 Femur 980 8 2 219 0.375
MFN O3 Humerus** 610 982 7 1 214 0.552
MFN ab2 Humerus 620 998 8 3 216 2.741
MFN M1 Femur 1120 9 3 268 1.065
MFN dd3032 Femur 1140 9 3 234 0.848
MAFI Ob 3092 Magyarosaurus dacus Humerus 222 288.6 12 8 226 NA
FGGUB r1220 Femur 346 14 9 135 NA
FGGUB r1195 Humerus 266 346 13 9 215 NA
FGGUB r1246 Humerus 320 416 14 9 168 NA
FGGUB r1511 Femur 466 13 8 276 NA
MAFI Ob 3089 Humerus 365 474.5 14 10 160 NA
FGGUB v13492 Humerus 372 483.6 13 9 166 NA
FGGUB r1046 Femur 525 14 11 182 NA
FGGUB r1992 Femur 540 14 11 174 NA
MAFI Ob 3128 Humerus 432 561.6 14 9 222 NA
MDE C3 270 Ampelosaurus atacis Humerus 180 203 8 2 0 0.118
MDE C3 977 Humerus 190 214 9 2.5 227 0.887
MDE C3 238 Humerus 340 384 13 11 240 4.830
LCE Cruzy 4 Femur* 550 12 6 270 3.391
LCE Cruzy 2 Femur* 630 13 15 201 NA
MDE C3 582 Femur* 635 13 11 193 5.954
MDE C3 527 Femur 680 13 11 217 7.512
MDE C3 1182 Femur 695 13 9 165 5.639

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TABLE 1. Continued

Bone Bone length CFL Avg. ODm OPD

Specimen Taxon element (mm) (mm) HOS RS (m) (n/mm2)
MDE C3 1506 Humerus 620 700 11 8 225 NA
MDE C3 203 Femur 780 12 9 195 8.222
MDE C3 175 Humerus 700 790 13 11 205 NA
MDE C3 261 Femur 840 12 11 192 6.467
PC. DMR K16-20 Phuwiangosaurus Femur 390 5 0 0 0.000
PC.DMR PW5 A-1 Femur 410 5 0 0 0.000
PC.DMR K4-428 Humerus** 710 800 10.5 5 180 5.737
PC.DMR K4-162 Humerus 730 820 10 3 201 4.200
PC.DMR K1-28 Humerus 770 865 10 4 196 3.431
PC.DMR K4-366 Femur 930 7 1 0 0.059
PC.DMR K11-1 Femur* 1000 7 1 0 0.217
PC. DMR no Femur 1030 10.5 1 0 0.000
PC.DMR K4-69 Femur* 1050 12 7 NA NA
PC.DMR K4-69 Femur 1050 12 7.5 177 NA
PC.DMR K 21 Femur 1120 11 4 209 2.188
PC.DMR PW1-8 Humerus 1020 1150 11.5 4 186 8.261
PC.DMR KD2-1 Humerus 1100 1240 10.5 5 209 3.352
TMM 45600-1 Alamosaurus Humerus 460 495 3 0 0 0.000
TMM 43600-2 Humerus 915 984 7 2 0 0.118
TMM 43090-1 Humerus 1300 1398 9 6 307 2.622
TMM No #ab Humerus 1350 1452 12.5 8 212 6.960

the bones. In humeri, the measured length Quantifying Remodeling

had to be converted to a corresponding length Three different methods were used to quan-
based on the average humerus/femur ratio for tify the remodeling progress in our specimens.
each taxon (Klein and Sander 2008). Corre- These methods are based on forensic age
sponding femur length (CFL, measured in determination of humans as noted above, but
millimeters) is used as the proxy for body size have been modied to suit the available types
and to describe the length of femora and of sections. Many studies of age determination
humeri, because both long bones are used in human bone measure several bone histolo-
together in regression analyses. This is justi- gical features, including Haversian bone
ed, because they show the same HOS at the density (whole osteons and fragments),
same body size (Klein and Sander 2008), which primary lamellar bone, and non-Haversian
is not always the case in nonhomologous vascular canals. Because the goal of this
elements (Padian et al. 2016). Merging the research is to determine ontogenetic signals in
femur and humerus samples also increases the mature sauropods in which only Haversian
sample size of specimens. All examined speci- bone is present, only secondary osteons were
mens are listed in Table 1. taken into consideration for measurements.
Thin sections were examined with standard Osteon Size.Secondary osteon size has
light microscopic techniques (plane polarized, been correlated with age in humans (Currey
cross-polarized light, and lambda waveplate) 1964; Ortner 1975; Britz et al. 2009). Therefore,
with a Leica (Wetzlar, Germany) DMLP com- we took size measurements to test this
pound microscope (2.5 to 40 objective correlation in sauropods. The short-axis
lenses). The photographs were taken with a diameter was measured (see Fig. 2B), because
Leica DFC420 camera attachment, and mea- secondary osteons can appear to be more
surements were taken with the ImageAccess elliptical than circular in shape, and this may
easyLab7 software. Statistical analyses were be a result of the thin section being cut at a
performed with the statistical program R, slight angle to the transverse plane or
Version 3.2.3 (R Core Team 2015). secondary osteons forming at a slight angle to

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FIGURE 2. A, Core section of an Apatosaurinae femur showing how the cortex was divided to determine the RS. Within
each section, the maximum number of generations was recorded (Table 2). B, Micrograph showing the measurement of
osteon diameter (ODm). C, Micrograph showing the counting of osteon generations for Apatosaurinae OMNH 4020,
in which six generations are preserved, the most generations observed in any specimen.

a bones long axis. The short-axis diameter was Osteon Density.The osteon population
thus measured for the most conservative density (OPD) (Kerley 1965; Ahlqvist and
estimate of size. Measured osteons had to be Damsten 1969; Ericksen 1991) was estimated
completely or mostly visible (not partially by counting all visible osteons in a total area of
replaced by younger osteons), but aside from 50 mm2 (the total area of four microscope
this necessity, osteons from the entire cortex, images at 2.5 ). Because more osteons are
when possible, were used. For each specimen, usually situated in the inner cortex of most
the number of osteons measured varied due to specimens, different areas of the cortex were
the amount of osteons present and the used to better represent the distribution; these
preservation of the thin section. If there were areas were chosen randomly but represented
very few (<20 visible osteons), all were areas from the innermost to outermost cortex.
measured. If there were many, then dozens of Resorption spaces that may represent early
osteons were measured. Once all the stages of osteon formation with little to no
measurements of osteon diameter (ODm) deposited lamellae were excluded, because
were taken, the average diameter was some resorption spaces become part of the
calculated for the entire cortex of each expanding medullary region during growth,
specimen (Table 1). A regression analysis was failing to develop into secondary osteons. The
used to test whether average ODm was OPD for each specimen is reported in Table 1.
correlated with the CFL. The density of osteons was then compared with

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FIGURE 3. Micrographs showing examples of remodeling in certain studied sauropod taxa. A, BYU 681-4742;
Apatosaurinae humerus, HOS 8, RS 4 (plane light). Near the inner cortex, at the bottom of the image, several secondary
osteons are forming. Primary bone is visible in the upper half. Similar histology is seen in Camarasaurus, Giraffatitan,
and Alamosaurus. B, MfN dd3032; Dicraeosaurus femur, HOS 9, RS 3, innermost cortex (cross-polarized light with
lambda waveplate). Note the large resorption spaces with sporadic secondary osteons, which are different from the
pattern in other diplodocoideans and macronarians. C, Maygarosaurus dacus, FGGUB r1220 HOS 14, RS 9, mid- to inner
cortex. Example of a region of poor preservation typical for many Magyarosaurus specimens, which prevented complete
analysis in some cases. D, PC.DMR K4-69; Phuwiangosaurus, HOS 12, RS 7, innermost cortex (cross-polarized light).
Example of extensive remodeling, typical of Magyarosaurus and Ampelosaurus, but also in the late ontogenetic stages of
all taxa, particularly in the inner to midcortex (with the exception of Dicraeosaurus).

the CFL in a linear regression to test for a mid-, and innermost cortex, representing the
correlation. Due to poor preservation of most youngest to oldest primary bone (Fig. 2A). In
Magyarosaurus specimens (see Fig. 3C), an some specimens, the transition to the medullary
accurate OPD could not always be determined. area (usually occupied by cancellous bone) is
Therefore, a regression analysis was not smoother than in others (where very little
performed for Magyarosaurus. cancellous bone is present and the medullary
Number of Osteon Generations.As noted area is either nearly empty or simply not
earlier, the number of generations of osteons preserved). In cases in which the boundary
was used by Stein et al. (2010) to establish HOS between medullary region and cortical region is
14. This approach is adopted here to observe gradual, the cortical region was dened as the
whether it can provide an even more detailed region where the bone represents more than half
ontogenetic signal. For determination of the the area and pore space is less than 50%.
number of osteon generations, the cortex was The maximum number of generations was
divided into three equal segments to analyze determined by documenting the highest number
variation across the core sample, the outermost, of crosscutting (i.e., overlapping) relationships

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between osteons in each segment (Fig. 2A,C). stages were subsequently regressed against
The highest number was recorded for the three CFLs of the different sauropod taxa to test
segments of the samples. The more generations whether an increase in CFL could be correlated
observed, the more time must have passed, thus with an increase in RS. Because the remodeling
the older the specimen (Kerley 1965). stages are ordinal data, a simple linear
Remodeling Stages.The number of regression was not appropriate, but instead an
generations in a given cortex segment ordered logistic regression (proportional-odds
presented a relatively consistent pattern, with model) was performed in R, using the package
a higher number in the innermost cortex VGAM (Yee 2016). The ordered logistic
and a lower number in the outermost regression calculates the log odds of being in a
cortex (Table 2). Remodeling stages were higher RS (response) for an increase in CFL
developed independently from the histological (predictor). It assesses the probability of the
ontogenetic stages to describe the maximum z-test statistic to be greater than observed in the
number of generations of osteons in cross null hypothesis (i.e., z-test statistic for a given
section. The remodeling stage (RS) represents a predictor not signicantly different from 0),
particular combination of values for the where the z-test statistic is the ratio of the
maximum numbers of observed generations of coefcient to the standard error of the predictor
osteons for the innermost, mid-, and outermost (CFL). Alamosaurus could not be analyzed with
cortex. Remodeling stages begin with a value 0, an ordered logistic regression, because the
representing no Haversian bone development sample size (four) was too small. Instead, using
to 15, the highest amount of observed Haversian the stats package in R (R Core Team 2015), a
bone development (six generations in the Kendalls b test was performed, because it can
midcortex). If more generations of osteons are be used for ordinal associations. It measures the
observed in future specimens, the remodeling rank correlation coefcient between RS and CFL
stages can be extended as needed. For example, for Alamosaurus.
if the innermost cortex had a maximum of three
generations, the midcortex two generations, and
the outermost cortex one, then the RS would be Results
six (see Table 2; Fig. 2A). These remodeling
Size of Osteons (ODm) and OPD
TABLE 2. Remodeling stages (RS). Each stage describes Table 3 includes the results of the regression
the progress of remodeling as the maximum number of analysis for the different sauropod taxa for
observed generations of osteons in the innermost, mid-,
and outermost cortex, based on the ontogenetic series of
osteon size and OPD. For the ODm, only
sauropods. Note that in the higher RS, no more than four Dicraeosaurus showed a signicant correlation
or ve generations of secondary osteons were observed in with CFL (R2 = 0.69 p = 0.0399). OPD signi-
the innermost cortex.
cantly increased with CFL for Apatosaurinae
Maximum number of generations of secondary osteons (R2 = 0.79, p = 0.0014), Giraffatitan (R2 = 0.81,
Innermost cortex Midcortex Outermost cortex RS p = 0.0010), and Ampelosaurus (R2 = 0. 82,
0 0 0 0
p = 0.0008), whereas the other taxa showed no
1 0 0 1 signicant correlation. These measures were
1 1 0 2 thus deemed to be of lesser or no value for
2 1 0 3
2 1 1 4
determining ontogenetic stage.
2 2 1 5
3 2 1 6
3 2 2 7 Remodeling Stages
3 3 2 8
4 3 2 9 Using the ordered logistic regression (Table 4),
4 3 3 10 a signicant correlation was found between
4 4 3 11
45 4 4 12
CFL and RS for Apatosaurinae (p = 0.0039),
45 5 4 13 Giraffatitan (p = 0.0174), Camarasaurus (p =
45 5 5 14 0.0174), Phuwiangosaurus (p = 0.0111), and Ampe-
45 6 5 15
losaurus (p = 0.0300) but not for Magyarosaurus

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TABLE 3. Results of linear regression of avg. ODm compared with CFL and OPD compared with
CFL for each sauropod taxon. *p < 0.05.

Results from avg. ODm vs. CFL Results from OPD vs. CFL
Species R p-value R2 p-value
Apatosaurinae 0.06927 0.4938 0.78622 0.0014*
Giraffatitan brancai 0.0261 0.6780 0.8082 0.0010*
Camarasaurus spp. 0.26581 0.2531 0.00182 0.9201
Dicraeosaurus spp. 0.69229 0.0399* 0.01634 0.8093
Magyarosaurus dacus 0.00017 0.9714 NA NA
Ampelosaurus atacis 0.28869 0.1092 0.81666 0.0008*
Phuwiangosaurus sirindhornae 0.1768 0.1525 0.22124 0.1701
Alamosaurus sanjuanensis 0.72393 0.1492 0.59972 0.2256

Poor preservation of Magyarosaurus specimens prevented adequate estimate of OPD

TABLE 4. Results from the ordered logistic regression for each sauropod taxon. The coefcient estimate indicates the
expected change in log odds of being in a higher RS category for every unit increase in CFL. The odds ratio indicates
the ratio of the odds of an increase in CFL over the odds of no increase in CFL. A signicant p-value (Pr(> |z|)) indicates
the probability of the z-test statistic (ratio of coefcient to standard error) being statistically different from the null
hypothesis. *p < 0.05.

Ordered logistic regression (proportional-odds model)

Species Number of specimens Coefcient estimate Standard error Odds ratio Pr( > |z|)
Apatosaurinae 11 0.0073 0.0025 1.0073 0.0039*
Giraffatitan brancai 9 0.0074 0.0028 1.0075 0.0080*
Camarasaurus spp. 14 0.0036 0.0015 1.0036 0.0174*
Dicraeosaurus spp. 6 0.3346 0.304 1.3974 0.271
Magyarosaurus dacus 10 0.0161 0.009 1.0162 0.0749
Ampelosaurus atacis 12 0.0074 0.0034 1.0074 0.0300*
Phuwiangosaurus sirindhornae 13 0.0069 0.0027 1.0069 0.0111*
Alamosaurus sanjuanensis 4 0.8434 NA 2.3243 NA

Unable to calculate regression statistics for Alamosaurus because of the small sample size.

(p = 0.0749) or Dicraeosaurus (p = 0.2710). For for Phuwiangosaurus; other femoral sections,

Alamosaurus, the value of from Kendalls test both anteriorly and posteriorly sampled, had
equaled one (p = 0.04), which indicates very very low remodeling stages (see Table 1). The
strong agreement between CFL and RS. Ampelosaurus femur specimens MDE C3 582
(CFL = 635 mm; HOS 13; RS 11) and LCE Cruzy
2 (CFL = 630; HOS 13; RS 15) were sampled on
Differences in Sampling Locations the posterior side and had different remodeling
In the Apatosaurinae specimen BYU 601- stages even though the CFLs are similar. The
17328, two samples were taken, one posteriorly specimens MDE C3 527 (CFL = 680 mm) and
and the other anteriorly. Although the HOS MDE C3 1182 (CFL = 695) with anteriorly
for both sections was the same (HOS 12, as sampled femora had RS 11 and 9, respectively.
indicated by the presence of an EFS), remodel- The posteriorly sampled LCE Cru 4 (CFL = 530;
ing was very different. The anterior side HOS 12; RS 6) had a much lower RS, but no
showed signicantly more remodeling (RS 8) anteriorly sampled femurs of similar size were
than the posterior side (RS 2). The highly available.
remodeled Phuwiangosaurus specimen PC. Two Camarasaurus specimens (SMA 0002;
DMR k4-69, in which the bone was cored from HOS 11; and CM 36664; HOS 12) had both
the anterior straight through to the posterior humerus and femur sampled. The RS for the
side, had the same HOS of 12 and only a slight femur of SMA 0002 (RS 1.5) was much lower
difference in RS between posterior (RS 7) and than the humerus (RS 7). Whereas in CM
anterior (RS 7.5). This RS was particularly high 36664, the femur had an RS of 4 and the

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humerus an RS of 5. In Phuwiangosaurus, the Dicraeosaurus cortical bone has highly vascu-

humeral sections had notably higher remodel- larized primary bone as well, but it also has a
ing stages than the femora (see Table 1) for distinct vascularization, which is not observed
most specimens. The other species did not in Giraffatitan or other Tendaguru sauropods
show very different trends between humerus (Sander 2000). Large erosion cavities extend
RS and femur RS, although individual differ- into the midcortex of some specimens (Fig. 3B).
ences were observed (see following section and Not only was its primary bone different, but
Table 1). Dicraeosaurus had different Haversian bone
development from the other sauropods
studied. Remodeling was always restricted to
Remodeling Patterns by Taxon the innermost to midpart of the cortex for most
The primary bone in our sample of Apato- specimens. The histological ontogenetic stages
saurinae is dominated by highly vascularized were determined (after Klein and Sander 2008)
laminar to plexiform brolamellar bone to be between 6 and 9, but in other sauropods
(woven-parallel complex of Prondvai et al. remodeling usually has already reached the
2014) with the onset of Haversian bone devel- outermost cortex by HOS 8 or 9. Remodeling
opment around HOS 7. Figure 3A gives an stages did not correlate well with ontogeny
example of the typical remodeled tissue of (Table 4). However, poor correlation is prob-
Apatosaurinae. A clear increase in remodeling ably due to a small sample size and the bone
stages is evident throughout Apatosaurinae lengths being quite similar.
ontogeny (Fig. 4). A signicant correlation Primary bone in Camarasaurus is laminar to
between CFL and RS was observed (Table 4). plexiform and highly vascularized. Most of the
The largest Apatosaurinae (OMNH 4020 FL cortices in Camarasaurus were not completely
1800 mm) was observed to have a maximum of remodeled. It was noted in Klein and Sander
six generations in the middle part of the cortex, (2008) that two morphotypes could be
with an RS of 15, representing the highest observed based on the relative size and HOS.
number of generations observed (Fig. 2C). The Both types are labeled in Figure 4, but for the
inner cortex of this specimen did not have more ordered logisitic regression, all specimens were
than four, possibly ve, observable genera- used together. No distinct morphotypes could
tions. OMNH 4020 is classied as a HOS 13 be observed in remodeling stages. A signicant
sensu Klein and Sander (2008) and HOS 14 correlation was observed between RS and CFL
sensu Stein et al. (2010). There is a large gap (Table 4).
between the largest specimen and second- The bone histology of the dwarf titanosaur
largest specimen in terms of Haversian bone Magyarosaurus is quite different from other
development, suggesting there may have been taxa, as even small individuals have nearly
a signicant age gap at time of death (Fig. 4). completely remodeled bone (Fig. 3D), as
Laminar to plexiform highly vascularized already noted by Stein et al. (2010). The
primary bone is dominant in Giraffatitan, with primary bone is modied laminar bone (sensu
the rst secondary osteons occurring around Klein et al. 2012). None of the samples show a
HOS 8. Giraffatitan has a signicant correlation clear EFS (Stein et al. 2010), though Klein et al.
of osteon generations with ontogeny (Table 4; (2012) noted a pair of closely spaced LAGs
Fig. 4). The highest number of observed in the outermost cortex of one specimen. In
generations in the largest femur MFN XV (FL general, there is no signicant increase in RS
2190 mm) was four generations, with an RS of with bone length (Table 4). Many of the
9, notably less than the largest Apatosaurinae. specimens were poorly preserved with many
In the two smallest humeral sections (MFN cracks and diagenetic features that obscured
XX19 and MFN T8), the amount of remodeling the bone tissue (Fig. 3C); thus, determining the
did not differ (RS of 3), but MFN T8 has a number of generations within each section of
humerus of almost twice the length and a cortex was difcult. In general, crosscutting
much higher HOS (9 compared with 6 of relationships of osteons could only be assessed
MFN XX19). in small areas. Because of the issues with

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FIGURE 4. HOS and RS compared with CFL for each sauropod taxon. Of the taxa studied, Apatosaurinae, Giraffatitan,
Camarasaurus, Ampelosaurus, Phuwiangosaurus, and Alamosaurus show a signicant correlation of RS to CFL (compare
with Table 4 for regression results). Note, however, that the remodeling stages do not match up with any particular
HOS. Camarasaurus is marked with both morphotypes, as observed in Klein and Sander (2008).

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preservation, it could be that the assigned observed with increasing bone length and
remodeling stages are incorrect and there is correlated well with the HOS (Table 4).
indeed a signicant correlation with size.
FGGUB R1046 (CFL 500 mm) was determined
to have an RS of 11, whereas some of the less Discussion
well-preserved specimens were estimated The goal of this research was to determine
between RS 8 and 10. These estimates may be whether secondary osteons provide an onto-
lower than the true number of observable genetic signal to extend histological ontoge-
generations because of limited observable area. netic stages into late adulthood or senescence
The poor correlation between RS and size of sauropods, when primary bone is nearly to
mirrors the poor correlation between HOS completely absent. Of the three methods used
and size (Fig. 4). here, the number of generations of secondary
The main bone tissue type in Phuwiango- osteons proved to be the most useful. A
saurus is modied laminar bone (Klein et al. secondary outcome of this research is a more
2012), but the remodeling was peculiar in this detailed look into the variability of Haversian
taxon. Particularly in the femora, Haversian bone development in these different taxa.
bone development is much lower compared When comparing the ODm and the CFL of
with the smaller humeri. The smaller humeri each species, no correlation was observed, with
have RS 3 through 5. With the exception of PC. the exception of Dicraeosaurus. Britz et al. (2009)
DMR k4-69 (CFL = 1050 mm, RS 7), the other observed in humans that ODm decreases with
femora of similar size had an RS of 1 and the age but also reported that weight was nega-
largest femur specimen (PC.DMR K 21; CFL = tively related to ODm and females had smaller
1120 mm) had an RS of 4. Even with notable osteons than males. Havill (2003) also noted the
differences in humeri and femora, a signicant disparity in osteon area between male and
correlation between RS and CFL was obtained female macaques (Macaca mulatta) but no
when both humeri and femora were analyzed difference in osteon size between immature
together (Table 4; Fig. 4). and mature individuals. Thus, it appears that
Primary bone of Ampelosaurus consists of other factors may have a more important
modied laminar bone (sensu Klein et al. 2012). inuence on osteon size, which may explain
Many Ampelosaurus specimens show an extre- the poor correlation in the sauropods.
mely high degree of remodeling (Fig. 3D), with Density of secondary osteons (OPD)
at least six generations observed in the poster- increases with ontogeny for Apatosaurinae,
ior section LCE Cruzy 02 (CFL 630 mm) in the Giraffatitan, and Ampelosaurus. However, mea-
midcortex, making it one of the highest suring variability of densities in late ontoge-
observed RS at 15. Like Magyarosaurus, for netic stages is difcult with few samples. The
some Ampelosaurus specimens, poor preserva- area chosen to sample osteon density may not
tion lead to an underestimation of the true have been large enough. Additionally,
remodeling stage. Nonetheless, a signicant measurements are subject to bias, because the
correlation with ontogeny was observed for amount of Haversian bone, particularly in the
RS in Ampelosaurus (Fig. 4; Table 4), including mid- to outermost cortex, is unevenly distrib-
specimens that were not sampled in the uted, meaning a sample taken from one area
standard location (femora LCE Cruzy 4, LCE may result in no osteons but an area only a
Cruzy 2 and MDE C3 582). couple of millimeters away may be almost
For Alamosaurus, only humeri samples were entirely osteonal. Ideally, the entire cortex of
available for examination. The primary bone is the cored section would be used to determine
mainly laminar, and the remodeling pro- osteon density, but this becomes very time-
gressed strikingly similar to that observed in consuming in practice, particularly for the large
Giraffatitan and Apatosaurinae (Fig. 4) as sauropod sections. With more specimens from
opposed to other titanosaurs, which were late ontogeny, it may be possible to observe
much more remodeled (e.g., Ampelosaurus and differences in osteon density. However, Walker
Magyarosaurus). An increase in RS was et al. (1994) studied the histomorphometry of

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femora for humans aged 5195 and found no and 1 year (Partt 1994), and osteon formation
correlation between age and density of second- rate (number of osteons/mm2/yr) decreases
ary osteons and osteon fragments. Thus, as over time (Frost 1969; Stout and Paine 1994).
adults enter into senescence, the ontogenetic With three or four generations more in Apato-
signal that secondary osteon density provides saurinae OMNH 4020 than in BYU 681-17328
seems to decrease in resolution. (RS 15 and RS 8, respectively), it is likely that a
few years would be needed to form those
additional generations. Additionally, the
Remodeling Stages as an Ontogenetic Signal activation frequency, that is, the number of
The highest number of observed generations osteons formed per square millimeter per year
proved to be a simple and useful way of for macaques (Havill 2003) and humans (Frost
documenting an ontogenetic signal in Haver- 1969; Stout and Paine 1994) decreased from
sian bone development. The remodeling stages approximately 8 to 12 osteons/mm2/yr in
correlate well with CFL in six out of eight skeletally immature macaques and young
species studied. With each increase in RS, from humans (1019 years of age) to 1 to 2
0 to 1 to 2 and so forth, we can infer an increase osteons/mm2/yr in skeletally mature maca-
in age of these animals. Dicraeosaurus lacked ques and adult (2089 years of age) humans.
signicant correlation with CFL. The low It would be reasonable to assume that a
number of samples and all individuals of this decrease in activation frequency of osteons in
taxon being close to the same body size likely mature individuals is the norm for animals that
prevented a good correlation. With better- develop Haversian bone. When animals reach
preserved specimens of Magyarosaurus, it too adulthood, overall growth stops or dramati-
may show better correlation with CFL. Even cally slows, so the amount of energy put into
though most histological ontogenetic stages primary bone development decreases; it could
were high (HOS 11 or higher) for Ampelosaurus, be that secondary bone formation also
the RS showed a correlation with CFL. Even decreases, as observed in primates. If senescent
with the relatively low sample size of the sauropods had similar activation frequencies
sauropod taxon, an ontogenetic signal is to adult primates, it is reasonable to assume
present, suggesting that there may be a way that for an observed density of 8 osteons/mm2,
to differentiate older, highly remodeled indivi- 4 to 8 years would have been needed to create
duals of sauropods from younger ones. all the observed osteons.
Although the circumference has been shown
to be a better proxy for mass in quadrupedal
mammals and reptiles compared with length Limitations to Remodeling Stages
(Campione and Evans 2012), in sauropods, One of the major issues with the use of
femur length has an isometric relationship with the remodeling stages is providing a reliable
circumference (Bonnan 2004; Kilbourne and account for the highest amount of observable
Makovicky 2010). Thus, the femur length is osteons in different parts of a thin section.
used for our taxa; when applying this method Particularly, the innermost cortex, which is the
to other taxa, circumference may be the better oldest bone and thus has had more time to
choice. undergo remodeling, should by this reasoning
We cannot say with any certainty how much have more secondary osteons but also more
time has passed from one RS to another, generations of osteons (see also Mitchell and
nevertheless we can estimate that, for example, Sander 2014). However, because the innermost
the largest Apatosaurinae (1800 mm femur cortex usually comprises large resorption
length), which has an RS much higher than spaces, many of the older osteon generations
the second-largest Apatosaurinae individual, are destroyed, which could then lead to a lower
was probably many years older when it died. observable number (see Table 1). In highly
We know that for humans, osteon formation remodeled bone, the innermost cortex no
(from onset of resorption to the osteoid be- longer exceeds the number of observed gen-
coming mineralized) takes between 6 months erations in the mid- to outermost cortex after

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about three or four generations. An alternative not always correlate with a particular HOS, and
reason could be that the amount of secondary thus reects the fact that bone remodeling is
osteons in any given region of bone becomes independent from primary bone formation. This
more uniform over time. Once old age is is expected based on observations of different
reached, bone turnover may no longer proceed timings of secondary osteon appearance, such as
from innermost to outermost cortex, decreas- observed in the perinate from Curry Rogers
ing the resolution of RS. The maximum et al. (2016).
number of generations observed was six in
Apatosaurinae and Ampelosaurus. Although
the actual maximum number of possible Integration of Remodeling Stages with
observable generations has not been deter- Histological Ontogenetic Stages
mined, six may well be it. Thus, RS 15 may A better understanding of how remodeling
actually represent a plateau where ontogenetic changes through ontogeny is established with
time is no longer recorded accurately. RS, potentially providing a more extensive
Another issue, which we encountered in toolbox to describe histological differences in
many sections, was poor preservation, which ontogeny in addition to HOS. However, a
can also lead to an underestimate of RS. synthesis between HOS and RS is required,
Diagenetic stains and fractures obstructed the because secondary osteons are part of the
view of crosscutting relationships in many description in several histological ontogenetic
samples. An approximation of RS had to be stages. A possible solution would be to use
made for many of the titanosaurs, particularly HOS to describe primary bone and RS to
the long bones of Magyarosaurus, because only describe secondary bone. Once the onset of
small areas were clear enough to observe remodeling commences, both HOS and RS can
crosscutting relationships be used, and once primary bone is no longer
visible, RS can be used to describe the extent of
remodeling. This way, an additional parameter
Comparing Histological Ontogenetic Stages is gained to describe the histological develop-
and Remodeling Stages ment of sauropods. This can be helpful, for
The largest Giraffatitan and Apatosaurinae example, to compare a specimen with HOS 7
individuals (HOS 12 and 13, respectively) have and RS of 3 with another specimen that has a
very different remodeling stages (9 and 15, HOS 7 but an RS of 6. The combination of HOS
respectively), even though both have nearly and RS suggests that the primary vascular
completely remodeled cortices. This indicates canals and primary tissue are the same but the
the limit of histological ontogenetic stages and Haversian bone development is different in the
demonstrates the potential of remodeling two specimens, one being younger than
stages for distinguishing further stages late in the other.
ontogeny. However, remodeling stages tend to Converting the remodeling stages into addi-
increase at a different rate than HOS (See tional histological ontogenetic stages to
Fig. 4). In some cases, the HOS can increase by describe late ontogeny (e.g., make RS of 10
two or three stages between two specimens, into HOS 15 and so forth) would not make
but the RS stays more or less the same (e.g., in sense, because there was no one-to-one rela-
Giraffatitan in Fig. 4) or the remodeling tionship between RS and HOS. Alternatively,
stages are very different between similarly extending the histological ontogenetic stages
sized individuals or similar histological may be possible by incorporating the max-
ontogenetic stages (e.g., Phuwiangosaurus in imum observed number of generations in the
Fig.4). Nevertheless, the RS does show indivi- outermost cortex into their denition, modify-
dual differences in these highly remodeled speci- ing and extending the denition of HOS 14 of
mens, something that cannot be accomplished Stein et al. (2010). HOS 13 is dened as nearly
with the current HOS scheme. Importantly, the completely remodeled bone, and based on
remodeling stages are clearly independent from observations in the current study, this usually
the histological ontogenetic stages: the RS does means at least two generations of osteons in the

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outermost cortex. Thus, HOS 13 could be more also noted in Lirainasaurus (Company 2011)
specically dened as a maximum of two and some other titanosaurs (DEmic and
generations of secondary osteons in the outer- Wilson 2012; Garcia et al. 2015).
most cortex; HOS 14, three generations of This trend, is not observed in all dwarf
secondary osteons in the outermost cortex; sauropods. The island dwarf sauropod Euro-
HOS 15, four generations, and so forth. The pasaurus shows very little Haversian bone
applicability of this approach needs testing by development, so little, in fact, that it was not
preferential sampling of large and old saur- included in this study. One reason for this
opod individuals. observed higher remodeling in the smaller
sauropods might be because remodeling
begins earlier in ontogeny. The Magyarosaurus
Extreme Remodeling in Magyarosaurus and specimens represent a fairly complete growth
Ampelosaurus series, and the smallest specimen already has
Figure 5 shows a graph of all species extensive Haversian bone development (Stein
together with RS on the y-axis and CFL on the et al. 2010). In addition, both Ampelosaurus and
x-axis. Most species plot in the same region, Magyarosaurus display modied laminar bone,
showing an overall increasing trend of RS with in which the typical woven bone aspect of
bone length. There is a distinct gap between the brolamellar bone is replaced with parallel-
majority of sampled species and Magyarosaurus bered bone, which may suggest slow growth
and Ampelosaurus, which both plot further left (Klein et al. 2012). This slower growth may
on the graph (due to a smaller relative bone have enabled more time for Haversian bone
length). Most notably, they plot, on average, to develop than in sauropods with typical
higher than the other sauropods, indicating brolamellar bone (Klein et al. 2012).
overall higher RS. This indicates a shift to an Mitchell and Sander (2014) noted that the
increase in Haversian bone development difference in remodeling between the two
with smaller body size, interpreted as island dwarfs Europasaurus and Magyarosaurus
dwarng in Magyarosaurus (Stein et al. 2010) resulted from a much more rapid resorption
but not in Ampelosaurus. Haversian remodeling front (expanding medullary cavity during
appears to be stronger in titanosaurs in general growth) in Europasaurus. Magyarosaurus, on
compared with more basal sauropods and was the other hand, has a slower resorption front

FIGURE 5. Graph of RS versus CFL with all taxa together in one plot. Note the correlation of the two parameters in
all taxa but that Ampelosaurus and Magyarosaurus are set off from the other sauropods, indicated by the overall
higher RS.

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(indicated by the much thicker cortex), which testing on a large sample of midshaft cross
allowed Haversian bone to develop and sections.
progress through the cortex. Variability occurs not only within a single
The remodeling stages divide the cortex into bone but across elements. In the Phuwiangosaurus
three equal parts: inner, mid- and outer cortex. and Camarasaurus (SMA 0002 and CM 36664)
Of course, over time the cortex thickens in specimens, the smaller humeri showed more
sauropods. New bone is deposited apposition- Haversian tissue development than the larger
ally and the oldest bone is removed or femora, and this may be a reection of relative
remodeled into cancellous bone. This means differences in modeling (apposition and resorp-
that through ontogeny, what was once the tion during growth) and remodeling (removal
outer cortex of a juvenile will be the midcortex and deposition of small packets of bone). If the
of a subadult and the inner cortex of an rate of remodeling is more or less constant in
adult. This method works well for sauropods, each limb but growth (both appositional deposi-
because the thick cortex allows for a very tion and resorption of the medullary region) is
prominent gradient in secondary osteon faster in the larger bone (to maintain proper
deposition that increases through ontogeny. proportions), then the observed effect could be
In taxa that have thinner cortices (or develop less Haversian tissue in larger bones (Mitchell
thin cortices through ontogeny), a division into and Sander 2014). Padian et al. (2016) suggest
three sections may not be feasible. But the RS that in large dinosaurs, smaller long bones
can be altered for a different taxonomic group. remodel more than large long bones because of
For example, the cortex can be divided into two differential growth. They hypothesize that the
instead of three sections, or no division is ow of metabolites must be the same in large
made, and the highest RS for the entire length long bones as is in smaller long bones of an
of the cortex in a specic location (e.g., anterior individual. The larger bones use these metabo-
region of femur) is used. lites for appositional growth, whereas smaller
bones, not needing to grow as much, use them
High Variability in Remodeling of Sauropods for remodeling (Padian et al. 2016). Alternatively,
Sampling location is important, because the increase in remodeling in smaller limb bones
variation in remodeling within a single bone may be related to mechanical loading. In modern
is well documented (e.g., Amprino and Marotti sheep, an increase in Haversian tissue is
1964; Frost 1969; Martin et al. 1980; Stout 1989; observed in the smaller, distal limbs compared
Pfeiffer et al. 1995; Padian et al. 2016). As seen with proximal limbs of juvenile and adult speci-
with the different sauropod taxa, the RS could mens, with adults exhibiting higher remodeling
vary by region of bone by a large factor. The rates than juveniles (Lieberman et al. 2003). The
variability in Haversian bone distribution is increased Haversian tissue is interpreted to be
attributed partially to differences in mechan- the result of lower safety factors and higher
ical loading and drifting (Francillon-Vieillot strains in distal limbs (Lieberman et al. 2003;
et al. 1990; Currey 2011). This means the extent Robling et al. 2006). Regardless of the underlying
and density is not entirely due to aging but mechanism, the differences in Haversian tissue
also to biomechanics. When exploring the development between limb bones as observed in
relationship of ontogeny with Haversian bone fossil or extant vertebrates need to be considered
development, maintaining the same sampling when comparing specimens.
location will reduce bias, but noting the
remodeling stages in different regions around Application to Other Taxa
the cortex can quantify the variation in Haver- Further investigation of this method to early
sian bone, which may be benecial for biome- sauropodomorphs is warranted. However,
chanical analyses. Padian et al. (2016) suggest Haversian tissue development is limited in
that smaller bones generally display more many taxa, such as Massospondylus (Chinsamy
Haversian bone, and the RS could be used as 1993) or Plateosaurus (Klein and Sander 2007)
a method to compare different elements. and most basal sauropodomorphs (Cerda et al.
However, this approach requires further 2016). But many other taxa display extensive

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Haversian tissue development, and the RS and other ontogenetic series of sauropodo-
could be easily applied to them. For example, morphs, would be benecial for testing the
thyreophorans (Hayashi et al. 2009; Stein robustness of the statistical outcome. Since
et al. 2013) typically have extremely dense variability of Haversian bone distribution is
Haversian tissue in long bones, which makes observable across many taxa, not just sauro-
using primary bone tissue to assess ontogenetic pods, the applicability of RS in other dinosaurs
age not possible. Using the RS, different highly but also other extinct large terrestrial vertebrates
remodeled specimens can be compared to is worthwhile to explore.
assess relative age. Even in taxa that have
LAGs (e.g., theropods: Chinsamy 1990; Varric-
chio 1993), understanding and quantifying
Haversian tissue development provide addi- The authors would like to thank O. Dlfer,
tional insight into ontogenetic changes. technician, for thin-section preparation, and
N. Klein for providing data on histological
ontogenetic stages and bone lengths for the
Conclusions sauropod specimens. We also thank A. Canoville
The study of sauropod life history from the for helpful discussions and A. H. van Heteren
propodials (humerus and femur) is challen- and E. Prondvai for advice on statistical ana-
ging. With a lack of direct evidence (i.e., well- lyses. We thank two reviewers for providing
developed annual growth lines), age cannot be insightful comments on an earlier version of this
reliably determined. Histologic ontogenetic article. We are forever grateful to all the curators
stages can accommodate this problem by who gave us permission to sample specimens
accounting for changes in histology over time. under their care: D. Berman (CM), R. Cifelli
However, sauropods that lived to old ages often and K. Davies (OMNH), Z. Csiki-Sava (FGGUB),
have completely remodeled bone in their W.-D Heinrich and H.-P. Schultz (MFN),
humeri and femora, with secondary osteons L. Krds (MAFI), J. Le Loeuff (MDE),
being the only feature. The number of cross- T. Rowe and the late W. Langston (TMM), K.
cutting relationships of these osteons can be Stadtman (BYU), H. J. Siber (SMA), and
quantied to describe late ontogenetic stages V. Suteethorn (PC.DMR). Funding was provided
within some sauropod taxa. We described the by several grants from the German Research
changes in number of secondary osteon genera- Foundation (DFG) to P.M.S. K.S. is currently
tions as remodeling stages and provide a funded by a postdoctoral fellowship of the
comparative ontogenetic scale of growth series Foundation for Scientic Research Flandres
of sauropod humeri and femora. Starting from (FWO).
the onset of remodeling to the occurrence of up This is contribution number 171 of the DFG
to six generations of crosscutting relations of Research Unit 533 Biology of the Sauropod
secondary osteons, the remodeling stages can be Dinosaurs.
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