A recent study published in Oryx proposed that the extinct Javan tiger Panthera tigris sondaica may still survive on the Island of Java, Indonesia, based on mitochondrial DNA analysis of a single hair sample collected from a location where a tiger was reportedly encountered. However, upon reanalysing the genetic data presented in that study, we conclude that there is little support for this claim. The sequences of the putative tiger hair and Javan tiger museum specimens generated are not from tiger cytoplasmic mitochondrial DNA but more likely the nuclear pseudogene copies of mitochondrial DNA. In addition, the number of mismatches between the two Javan tiger sequences is unusually high for homologous sequences that are both from tigers, suggesting potential issues with data reliability. The paper provides insufficient details on quality control measures, making it impossible to rule out the possibility that errors were introduced during the analysis. Consequently, it is inappropriate to use the sequences presented in that study to infer the existence of the Javan tiger.

Mitochondrial ribosomal DNA is commonly used in DNA-based dietary analyses. In such studies, these sequences are generally assumed to be the only version present in DNA of the organism of interest. However, nuclear pseudogenes that display variable similarity to the mitochondrial versions are common in many taxa. The presence of nuclear pseudogenes that co-amplify with their mitochondrial paralogues can lead to several possible confounding interpretations when applied to estimating animal diet. Here, we investigate the occurrence of nuclear pseudogenes in fecal samples taken from bottlenose dolphins (Tursiops truncatus) that were assayed for prey DNA with a universal primer technique. We found pseudogenes in 13 of 15 samples and 1–5 pseudogene haplotypes per sample representing 5–100% of all amplicons produced. The proportion of amplicons that were pseudogenes and the diversity of prey DNA recovered per sample were highly variable and appear to be related to PCR cycling characteristics. This is a well-sampled system where we can reliably identify the putative pseudogenes and separate them from their mitochondrial paralogues using a number of recommended means. In many other cases, it would be virtually impossible to determine whether a putative prey sequence is actually a pseudogene derived from either the predator or prey DNA. The implications of this for DNA-based dietary studies, in general, are discussed.