Reanalysis of sequences of alleged Javan tiger highlights the difficulties in studying big cats and the need for high-throughput sequencing ANUBH AB KHAN * 1 , 2 , 3 ,YULIA NTO YULIANTO 4
S AB HR I N A GIT A ANIN T A 2 andWIRDATETI WIRDATETI 5
Abstract Big cats are of conservation concern throughout their range, and genetic tools are often employed to study them for various purposes. However, there are several diffi- culties in using genetic tools for big cat conservation that could be resolved by modern methods of DNA sequencing. Recent reports of the sighting of a putative Javan tiger Panthera tigris sondaica in West Java, Indonesia, highlight some of the difficulties of studying the genetics of big cats. We reanalysed the data of the original reports and found that the conclusions were drawn based on incorrect copies of the genes. Specifically, the nuclear copy of the mitochon- drial gene was analysed with the mitochondrial sequence, leading to discordance in the
results.However, re-sequencing of the remaining DNA confirms that the sighting could have been that of a tiger, but the subspecies cannot be confirmed. This work highlights the urgency of developing high- throughput sequencing infrastructure in the tropics and the need for reliable databases for the study of big cats.
Keywords Capacity building, DNA analysis, extinct, Javan tiger, Lazarus species, next-generation sequencing, Sanger sequencing, shed hair
Introduction
studied across their range, much remains unknown about them. Accurate species identification of big cats is important in research on matters such as poaching and trafficking, livestock depredation, ex situ breeding and dispersal events. Genetic tools are often used to identify species of large felids, but classical tools may be inadequate for this task. A recent study by Wirdateti et al. (2024) that reported the
B
*Corresponding author,
anubhabkhan@gmail.com 1Centre for Ecological Sciences, Indian Institute of Science, Bangalore, India 2Section for Computational and RNA Biology, Department of Biology,
University of Copenhagen, Copenhagen, Denmark 3Department of Biology, Pwani University, Kilifi, Kenya 4Research Center for Applied Zoology, National Research and Innovation
Agency (BRIN), Cibinong, Indonesia 5Research Center for Biosystematics and Evolution, National Research and Innovation Agency (BRIN), Cibinong, Indonesia
Received 17 April 2024. Revision requested 14 June 2024. Accepted 23 August 2024. First published online 23 January 2025.
ig cats are rare and elusive. Although they attract a sig- nificant amount of conservation funding and have been
presence of a Javan tiger Panthera tigris sondaica in West Java is an example of the inadequacy of these classical genetic tools. The Javan tiger was categorized as Extinct in 2008 (Jackson & Nowell, 2008), and has not been detected since the 1990s. However, in 2019 a local resident reported seeing atiger near avillage in West Java, and one of the authors of Wirdateti et al. (2024) collected a hair sample fromthe sighting location. To determine whether this sighting could be the extinct
Javan tiger, museum samples of Javan and Sumatran tigers Panthera tigris sumatrae were also collected and DNA was extracted from the hair and the museum samples (Wirdateti et al., 2024). The authors sequenced the cytochrome B (cytB) region of the samples and performed comparative phylogenetic analysis with previously published cytB se- quences of tigers and leopards, concluding that the hair be- longed to a Javan tiger. However, concerns were raised regarding the study (Emont, 2024; Sui et al., 2024). Here we reanalyse the sequences and repeat some of the experiments, to highlight the difficulties of studying big cat genetics and some potential solutions to these issues.
Phylogenetic tree reconstruction
We reanalysed the Javan tiger sequences in Wirdateti et al. (2024) by making a phylogenetic tree that included ad- ditional cytB and nuclear copies of mitochondrial pseudo- gene (Numt) sequences downloaded from the National Center for Biotechnology Information database (Table 1). We aligned the sequences using MAFFT (Katoh et al., 2002). We conducted three batches of analysis based on the length of sequences analysed and the type of sequences used. For Dataset 1 we removed sequences MH290773, AB211408–AB211411 and FJ403465 from the analysis because of the excess of missing data. We trimmed all regions with any missing data using Jalview (Waterhouse et al., 2009). This retained 36 sequences with 265 bp of data for analysis. For Dataset 2 we removed sequences AB211408–AB211411, FJ895266,FJ403466,FJ403467,MH290773 and FJ403465 from the analysis because of the excess of missing data. We trimmed all regions with any missing data using Jalview, retaining 33 sequences with 971 bp of data for analysis. For Dataset 3 we aligned the cytB Numt sequences using MAFFT. We trimmed sites with missing data using Jalview. This retained 453 sites with eight sequences.
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (
http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. 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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