Javan tiger DNA sequencing 77
FIG. 2 Neighbour joining tree of the test sample (hair strand of putative Javan tiger Panthera tigris sondaica), the Javan tiger museum sample (OQ601562_1_son) and other tiger sequences using (a) 36 sequences and 252 sites and (b) 33 sequences and 937 sites. We included a sequence of the nuclear copy of the cytochrome B pseudogene (Numt) in the analysis. (Readers of the printed journal are referred to the online article for a colour version of this figure.)
and trimmed them using Jalview. We conducted two batches of analysis on the basis of this trimming: one that retained 38 sequences and 264 bases and another that retained 35 sequences and 907 bases. We reanalysed the sequences generated by Wirdateti
et al. (2024) alongside a nuclear copy of the cytB pseudogene (Numt) sequence of the Bengal tiger Panthera tigris tigris (AF053053.1) and the cytB sequences of several other tiger subspecies. The clustering of the putative Javan tiger sequence (test sample in Fig. 2) and the museum sample of the Javan tiger (OQ601562 in Fig. 2) with the Numt sequence revealed that the sequences generated for the samples were Numts and not the cytB regions that they were being compared to (Fig. 2). We further added Numt sequences of other Panthera spe-
cies and the domestic dog Canis familiaris (as an outgroup) to test the potential of Numts for species identification (Fig. 3). Comparisons of the available annotated cytBNumt sequences of Panthera species in the National Center for Biotechnology
Information database demonstrate that Numt sequences can be highly divergent within the Panthera genus (Fig. 3). We also observed that the lion Panthera leo cytB Numt sequence ismore diverged fromother cats and froma canid cytBNumt. We re-sequenced DNA amplicons from the putative
Javan tiger hair strand and the museum specimen of Javan tiger, and we retained sequences closely related to the cytB sequences of the other tigers (Fig. 4). The 3 μl template in Round 1 and the 5 μl amplicon template in Round 2 yielded the Numt sequence (Fig. 1). However, the mitochondrial cytB DNA does not have the power to distinguish between Sumatran and Javan tigers (Fig. 4).
Discussion
Mitochondrial genomes are often used to delimit species and subspecies. However, they have several limitations, es- pecially for big cats. In big cats nuclear copies of mitochon- drial pseudogenes are a common problem in genetic
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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