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Fig. S1. Geographic distribution of fossil spotted hyenas in Far East Asia, showing Pleistocene hyena fossil sites in China.

Fig. S2. Schematic view of the 713 bp configs of the cyt b gene for the Pleistocene samples using nine overlapping PCR fragments.

Fig. S3. Phylogenetic tree for fossil and extant spotted hyenas.

Fig. S4. Different tests showing temporal signal in the datasets.

Fig. S5. Comparison of rates (a) and dates (b) estimates with 2 calibration dates for the Chinese samples.

Fig. S6. Photos of three Da'an Cave specimens.

Fig. S7. Posterior maximum likelihood estimators output from bayessc.

Fig. S8. Graph of time dependent mutation rate, showing (a) linear and (b) exponential decay relationships between the two calibration points.

Table S1. PCR primers for Crocuta crocuta ultima mitochondrial cytochrome b gene.

Table S2. Details on sequences used in this study.

Table S3. Variations in the newly obtained ancient sequences compared to living spotted hyenas.

Table S4. Probability of observing the paleontological fossil dates under the null hypothesis that the posterior distribution of the basal nodes are either, the externally (Miocene fossil dated) calibration model or the internally (AMS radiocarbon tip dated) calibration model.

Table S5. Akaike Information Criterion (AIC) values for the twelve different model/mutation rate combinations.

Table S6. Maximum Likelihood Estimators (MLE) with lower (2.5%) and upper (97.5%) confidence bounds for divergence times from the Bayesian Serial Simcoal (bayessc) analysis (for divergence models only – H1 and H2).

Table S7. Mutation rates used in the bayessc analysis, and the method used to calculate the time dependent mutation rates Additional references

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