76 A. Khan et al.


TABLE 1 List of sequences (of domestic dog Canis familiaris, leopard Panthera pardus, lion Pathera leo and six subspecies of tiger Panthera tigris) from the National Center for Biotechnology Information database used in this analysis.


Accession numbers


NC_051810.1: 68724255–68726124


AB048589.1 MF136765.1 OQ117410.1 MN124244.1 AB211408.1


AB211409.1 AB211410.1 AB211411.1


AF053031.1 AF053033.1 AF053034.1 FJ895266.1


Species Canis familiaris Canis familiaris


Comment Numt


Numt


Panthera pardus Numt Panthera pardus Panthera leo


Numt P. tigris altaica


P. tigris altaica P. tigris altaica P. tigris altaica


P. tigris altaica P. tigris altaica P. tigris altaica P. tigris altaica


MH893763.2:14173–15312 P. tigris altaica MN624080.1:14530–15669 P. tigris altaica AF053053.1 AF053019.1 AF053024.1 AF053025.1


JF357967.1:14172–15311 JF357968.1:14172–15311


P. tigris tigris P. tigris tigris P. tigris tigris P. tigris tigris P. tigris tigris P. tigris tigris


KF892541.1:14173–15312 P. tigris tigris AF053050.1 AF053051.1


JF357971.1:14077–15216 JF357972.1:13017–14156 FJ403465.1


FJ403466.1 FJ403467.2


MH290773.1


OQ601561.1 OQ601562.1


P. tigris corbetti P. tigris corbetti P. tigris corbetti P. tigris corbetti P. tigris sondaica


P. tigris sondaica P. tigris sondaica P. tigris sondaica


P. tigris sondaica P. tigris sondaica


HM589214.1:15069–16208 P. tigris amoyensis HM589215.1:15113–16252 P. tigris amoyensis NC_014770.1:15113–16252 P. tigris amoyensis AF053054.1 AF053045.1 AF053046.1 AF053047.1 AF053048.1


JF357970.1:14173–15312 OQ629466.1 OQ629467.1 OQ629468.1 OQ629469.1 OQ629470.1 OQ629471.1


Numt


Large amount of missing data


Large amount of missing data


DNA extract re-sequencing


The Numt sequences were amplified because of stochastic binding of the primers to the Numt region instead of the mitochondrial cytB region (Sui et al., 2024) in Wirdateti et al. (2024); we attempted to rectify this through repeated PCR and sequencing (Fig. 1). In Round 1 we amplified the DNA extract using 3, 4, 5 and 7 μl templates in a 30 μl PCR, using the conditions described in Wirdateti et al. (2024). The amplicons from these reactions were not visible in agarose gel electrophoresis. Thus, we further amplified the 3 and 5 μl products from all four PCRs (henceforth referred to as ‘amplicon templates’) in the previous step using the same PCR conditions in a 30 μl volume (leading to a total of eight reactions in this Round 2). Five of the eight reactions in Round 2 yielded bands on the agarose gel, four of which were from the 5 μl amplicon template and in the PCR in which the 7 μl original template was used. The 3 μl amplicon template in the second PCR also yielded bands. We amplified all templates on different days to avoid cross-contamination. The reaction with the 5 μl original template in Round 1


Large amount of missing data


Large amount of missing data


and the 5 μl amplicon template in Round 2 of PCR yielded the c. 900 bp-long DNA fragment from the test hair strand sequence that is presented here. We aligned these new sequences to the sequences listed in Table 1 using MAFFT


We built neighbour joining trees using MAFFT.


We chose the default option for multiple sequence align- ment. For building the tree we chose the conserved sites option, which retained 252 sites for Dataset 1, 937 sites for Dataset 2 and 240 sites for Dataset 3. We used raw dif- ferences for the substitution model and performed boot- strap resampling 1,000 times.


P. tigris sumatrae Numt P. tigris sumatrae P. tigris sumatrae P. tigris sumatrae P. tigris sumatrae P. tigris sumatrae P. tigris sumatrae P. tigris sumatrae P. tigris sumatrae P. tigris sumatrae P. tigris sumatrae P. tigris sumatrae


FIG. 1 DNA amplification strategy used in this study.We performed two rounds of PCR. In Round 1 we used DNA extracts from samples as templates, and in Round 2 we used the amplicons from Round 1 as templates. The numbers on the arrows indicate the volumes of the templates used for each step. (Readers of the printed journal are referred to the online article for a colour version of this figure.)


Oryx, 2025, 59(1), 75–80 © The Author(s), 2025. Published by Cambridge University Press on behalf of Fauna & Flora International doi:10.1017/S0030605324001297


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