No evidence of extant Javan tiger 73
(NC_056676.1:5,568,675-5,569,645) and second best (NC_056676.1:5,585,283-5,586,253) matched regions of OQ601561.1 and OQ601562.1 are both located on an auto- somal scaffold corresponding to tiger chromosome F2. The nucleotide sequence identities of these matches are all .97.4% across the 971 bp trimmed sequences, whereas the similarity to the tiger mtDNA (NC_010642.1)is#92.5%. In contrast, mtDNA segments from a previously published Javan tiger sequence (Maza0008; Sun et al., 2023) and the Sumatran tigers Panthera tigris sumatrae acquired by the authors (OQ629467.1 and OQ629468.1) matched the tiger mtDNA, with . 98.75% sequence similarity (Fig. 1, Table 2). These results suggest that the two supposed Javan tiger sequences generated by the authors are not com- pletely derived from Cymt but more likely from Numt or a mixture of the two. Thirdly, the high variant rate in the putative Javan tiger
sequences prompted concerns related to data accuracy and quality control. There are 24 mismatches in the 971 bp se- quence between the hair and the museum Javan tiger speci- men, corresponding to a genetic distance of 2.473 × 10−2. The sequences are twice as divergent from one another as from the mean pairwise difference between other tiger mtDNA haplotypes, or 10 times more divergent than amongst Sunda tigers. In population genomic analyses in- cluding all tiger subspecies, only 196 variants were found across the mtDNA (15.5 kb in length with the control region removed), which is c. 12.6 variants per 1,000 bp (Liu et al., 2018). The genetic difference amongst the Javan, Bali Panthera tigris balica and Sumatran tigers from Sundaland is even lower, with 44 variants across the 15.5 kb mtDNA sequence, corresponding to c. 2.84 variants per 1,000 bp (Sun et al., 2023). For the tiger nuclear DNA, the single-nucleotide variant rate in different subspecies varies, ranging between 0.026% and 0.072%, which equates to 0.26–0.72 variants per 1,000 bp (Liu et al., 2018). Regardless of their mitochondrial or nuclear DNA origins, the presence of such a large number of variant sites between the putative Javan tiger sequences generated by the authors is unusual for two homologous sequences that are both from tigers, and this is indicative of data unreliability. There are various potential reasons for these errors, but
they cannot be identified based on the information provided by Wirdateti et al. (2024). Processing DNA from single hair or museum specimens requires stringent precautions, in- cluding contamination prevention, elimination of potential inhibitors, multiple replications to exclude non-specific sto- chastic amplifications from trace amounts of the DNA template, and data quality measurements, amongst others. For instance, residual hair keratin could inhibit PCR and Sanger sequencing reactions, hence reducing the efficiency and quality of DNA sequence analysis (Schrader et al., 2012). However, we are not able to determine from the article whether the DNA extraction and downstream
experiments were handled with the precautions that are required for working with degraded genetic material, nor how such precautions might have been implemented. As few details with regard to quality control are provided, it is inappropriate to use these sequences to draw conclusions regarding the existence of the Javan tiger. The report of the rediscovery of the Javan tiger by
Wirdateti et al. (2024) garnered widespread attention from the general public as well as amongst scientists and conser- vationists. We would all be thrilled to learn that the Javan tiger is not extinct and we agree with the authors that ‘[w]hether the Javan tiger actually still occurs in the wild needs to be confirmed with further genetic and field studies’ (Wirdateti et al., 2024,p. 472). However, the authors’ initial conclusions based on DNA analysis of one putative tiger hair sample are more likely to be erroneous than to reflect the survival of the Javan tiger, because of the flawed experi- mental design employed and the lack of scientific strin- gency. Clear and reliable visual, physical or genetic evidence will be required to conclude that the Javan tiger still survives in Java nearly half a century since the last confirmed sighting.
Author contributions Study design: S-JL; data analysis: Z-YS, S-JL; writing: all authors.
Acknowledgements We thank the general public, media, scientists and the conservation community for their support of our efforts to defend scientific rigor.
Conflicts of interest None.
Ethical standards This research abided by the Oryx guidelines on ethical standards.
Data availability All the data are published, available inGenBank and on Github at
github.com/xinsun1/Xin_etal_2023_NEE_TigerPopGen.
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CREECY, J., COIL,B.&HICKEY,K.(2024) Enzymatic removal of Numts from Panthera tigris DNA samples. Forensic Science International: Animals and Environments, 5, 100088.
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Oryx, 2025, 59(1), 69–74 © The Author(s), 2024. Published by Cambridge University Press on behalf of Fauna & Flora International doi:10.1017/S0030605324001248
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