AFRICAN OR ASIATIC ORIGIN? GENOME ANALYSES SOLVED THE MYSTERY OF THE HYBRID ORIGIN OF THE RESCUED LION CUB

Abstract Species assignment of any seized material using DNA analysis has been a routine and widely accepted standard procedure in providing scientific advisory for the legal prosecution of wildlife cases. Scientific advancements and rigorous application of genetic tools have led to the development of a variety of molecular markers with their defined efficacy in wildlife forensics. However, in a few unusual cases where a hybrid needs to be identified or assignment need to be made at sub-species level, mitochondrial markers often fail or else provide biased results, which can affect the overall judgment in the court of law. Here, we report one such challenging case of lion cub rescued by the law enforcement from illegal trafficking. Phylogenetic assessment based on complete mitogenome assigned rescued lion cub with African lion (Panthera leo leo). However, the TSPY gene of the Y chromosome established that the lion cub shared its paternal lineage from Asiatic lion (Panthera leo persica). With the use of maternally and paternally inherited markers, we conclude a hybrid origin of the rescued lion cub which shared ancestry from both Asiatic as well as African lion. The present study exhibits the application of genome sequencing in thinking beyond routine identification and contributes to the operating procedures of wildlife forensics. Introduction Forensic identification of confiscated wildlife and their products are of utmost importance to the field of wildlife forensics. Law enforcement agencies and the conservation authorities rely on the taxonomic or genetic assessment of species identification and therefore, it becomes crucial for scientists or forensic experts to meticulously investigate the seized materials. Unlike laboratories conducting human identification, there are only a few accredited laboratories worldwide conducting wildlife forensic investigations (Amorim et al., 2020). One of the most overlooked issues in wildlife forensic investigations during the past few decades has been the application of combined markers including mitochondrial, autosomal and sex chromosomes. For example, the use of maternally inherited mitochondrial genes for species identification is a routine practice and included in several standard operating procedures (SOP) of wildlife forensics (Gaur and Reddy, 2017, Beiglböck et al., 2019). In large vertebrates, especially in mammals and birds, where hybridization is well reported in the wild as well as in captivity (Willis et al., 2004, Hill et al., 2019, Robinson et al., 2005), the sole use of mitochondrial genes for species identification may be inadequate or lead to erroneous species assignment (Amorim et al., 2020). However, a few studies advocated applying autosomal microsatellites (Miller et al., 2014, Eo et al., 2016, Curry et al., 2019, Smitz et al., 2018) and SNPs (Philipp et al., 2010) in forensic assignment and population assessment of feline species. These studies also established subspecies level identity but lack information on how these tools may be applied for identifying hybrids in forensic casework. Further, the drawback of applying only mitochondrial genes is that they reveal only the maternal origin of the confiscated specimen. However, the paternal ancestry is equally important as they may have different protection status under the local legislations. This proves to be an advantageous loophole in the legal system of wildlife protection as many countries do not assign any protection status to hybrids (Espinoza et al., 2012, Linacre and Tobe, 2013). Hence, it is necessary to consider the prevalence of hybrids when assigning the species from confiscated wildlife and their products. In this context, the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) has assigned the protection to hybrids at the level of highest protected parental species (Animal hybrids, https://cites.org/eng/res/10/10-17R14.php). Mitochondrial DNA (mtDNA) markers like cytochrome b (cytb) and cytochrome c oxidase I (COI) have been most widely used tools in forensic investigation and evolutionary studies (Cronin et al., 1991, Hsieh et al., 2001, Branicki et al., 2003, Tobe and Linacre, 2010, Linacre et al., 2011, Ahlers et al., 2017). Despite, universal utility of mtDNA in identifying species of forensic cases, the role of mtDNA was reported as being contentious due to its uniparental signature (Rubinoff and Holland 2005). In a recent study, Lorenzini et al. (2020) advocated the combined approach of using multiple marker system in assigning individuals to one or other parental gene pools. Thus, species assignment particularly in those species among which hybridization is well reported e.g., wild boar, golden jackal, wolf etc. (Lorenzini et al., 2020, Iacolina et al., 2018, Galov et al., 2015, Torres et al., 2017) should be undertaken with the use of both mtDNA and nuclear genes otherwise seizure may be assigned to maternal origin of the species. Recent improvements and the development of massive parallel sequencing has revolutionized genetic data generation and, with the large-scale availability of high-quality genomic data, forensic interpretation can be greatly enhanced and the several such issues may be answered.