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Electronic Supplementary Material for:
Experimental Perspective on Fallback Foods and Dietary
Adaptations in Early Hominins
Jeremiah E. Scott, Kevin R. McAbee, Meghan M. Eastman, Matthew J. Ravosa
1. Expanded Materials and Methods
(a) Animal care
All procedures used in this study were approved by the University of Notre Dame’s
Institutional Animal Care and Use Committee (IACUC). Our sample consisted of n = 30 male
New Zealand white rabbits (Oryctolagus cuniculus) obtained at weaning (five weeks old) from
Harlan Laboratories (www.harlan.com) and housed at the University of Notre Dame’s animal
care facility, Friemann Life Science Center. Both institutions are USDA-licensed and AAALACaccredited and subject to periodic inspections. The purchase and usage of controlled substances
for all experimental subjects was monitored by the DEA. Day-to-day care of these experimental
subjects, including monitoring of their health, was handled by trained veterinary staff. The
animals were raised for 48 weeks, making them 53 weeks old (i.e., fully adult) at the conclusion
of the experimental period. In white rabbits, weaning occurs at about four to five weeks of age,
with skeletal maturity attained at approximately 26 weeks old. At the end of the experimental
protocol, all subjects were sacrificed using the following procedure: subjects were anesthetized
using a cocktail of ketamine (25 mg/kg), xylazine (5 mg/kg), and acepromazine (2.5 mg/kg)
administered via intramuscular injection to the quadriceps femoris and then given a pentobarbital
overdose (100 mg/kg) via cardiac puncture, with bilateral thoracotomy used as a secondary
means of assuring death. Sacrifices were performed by veterinary staff following established
standards and IACUC guidelines.
(b) Micro-CT imaging
The rabbits were scanned in Bioscan/Mediso X-CT using the following settings: 70 kVp
and 100 μA, with a 71-μm reconstructed isometric voxel size. Prior to scanning, animals were
anesthetized using the ketamine-xylazine-acepromazine cocktail described above.
(c) Morphometric data collection
Reconstructed scans were opened in the program PMOD version 3.3 (PMOD
Technologies Ltd.) and oriented so that the sagittal plane was parallel to the computer monitor
and the occlusal plane was horizontal. Following orientation, maximum cranial length and bone
cross-sectional areas were collected using the measurement and segmenting tools available in
PMOD. Variables are listed and described in supplementary table S5. Longitudinal data are
complete for 27 of the 30 rabbits.
(d) Bootstrap procedure
We performed among-cohort comparisons at selected time points using a bootstrap test
on size-adjusted bone cross-sectional areas—i.e., logged (base e) shape ratios computed by
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dividing the square root of each subject’s cross-sectional area by its cranial length at a given time
point. The bootstrap procedure was implemented as follows, using the palate shape ratio for the
control and annual cohorts at the first time point (i.e., Week 0) as an example:
Step 1. At Week 0, bootstrap (i.e., resample with replacement) from the palate shape
ratios for the control rabbits 1,000 times, with each bootstrap sample being
identical in size to the original sample (i.e., n = 10).
Step 2. Compute the mean palate shape ratio for each of the 1,000 bootstrap samples.
Step 3. Perform steps 1 and 2 on the annual cohort.
Step 4. Randomly pair the 1,000 mean shape ratios for the control rabbits with those for
the annual rabbits.
Step 5. For each pairing, subtract the mean shape ratio for the control rabbits from the
mean shape ratio for the annual rabbits. This step produces a distribution of
pairwise differences for the palate shape ratio.
Step 6. Centre the distribution of pairwise differences on zero by subtracting the mean of
the 1,000 pairwise differences generated in the Step 5 from each pairwise
difference. This step is necessary because the distribution of pairwise differences
will be centred on the observed difference between the control and annual rabbits.
In order to derive a p-value for the observed difference between the two cohorts,
the distribution must be centred on—i.e., the mean of the distribution must
equal—zero.
Step 7. Using the zero-centred distribution, count the number of values that are as
extreme as or more extreme than the observed difference between the control and
annual rabbits. The resulting value is M. At Week 0, the sign of the differences
were disregarded, making the test two-tailed, as there is no a priori reason to
expect the annual rabbits to have relatively larger palate cross-sectional areas at
this time point. In subsequent weeks, however, M represents counts of only the
positive differences, making the test one-tailed, as we expected the annual rabbits
to have relatively larger palate cross-sectional areas than the control rabbits (see
above).
Step 8. Use the following formula to obtain the p-value for the comparison: p = (M +
1)/(N + 1), where M is as above, N is the total number of bootstrap differences
(i.e., 1,000), and one is added to M and N to include the observed difference.
Tests that returned marginally significant p-values were repeated using 10,000 iterations.