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DOI: 10.1017/pab.2016.47
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.
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: kstein@vub.ac.be
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).
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SECONDARY OSTEONS IN SAUROPODS 3
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4 JESSICA MITCHELL ET AL.
(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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SECONDARY OSTEONS IN SAUROPODS 5
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6 JESSICA MITCHELL ET AL.
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 (**).
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SECONDARY OSTEONS IN SAUROPODS 7
TABLE 1. Continued
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8 JESSICA MITCHELL ET AL.
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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SECONDARY OSTEONS IN SAUROPODS 9
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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10 JESSICA MITCHELL ET AL.
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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SECONDARY OSTEONS IN SAUROPODS 11
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
2
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.
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12 JESSICA MITCHELL ET AL.
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SECONDARY OSTEONS IN SAUROPODS 13
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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14 JESSICA MITCHELL ET AL.
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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SECONDARY OSTEONS IN SAUROPODS 15
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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16 JESSICA MITCHELL ET AL.
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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SECONDARY OSTEONS IN SAUROPODS 17
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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18 JESSICA MITCHELL ET AL.
(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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SECONDARY OSTEONS IN SAUROPODS 19
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-
Acknowledgments
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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