ABSTRACT
The Sunda pangolin (Manis javanica) is the most widely distributed Asian pangolin species, occurring across much of Southeast Asia and in southern China. It is classified as Critically Endangered and is one of the most trafficked mammals in the world, which not only negatively impacts wild Sunda pangolin populations but also poses a potential disease risk to other species, including humans and livestock. Here, we aimed to investigate the species' phylogeography across its distribution to improve our understanding of the species' evolutionary history, elucidate any taxonomic uncertainties and enhance the species' conservation genetic management and potential wildlife forensics applications. We sequenced mtDNA genomes from 23 wild Sunda pangolins of known provenance originating from Malaysia to fill sampling gaps in previous studies, particularly in Borneo. To conduct phylogenetic and population genetic analyses of Sunda pangolins across their range, we integrated these newly generated mitochondrial genomes with previously generated mtDNA and nuclear DNA data sets (RAD-seq SNP data). We identified an evolutionarily distinct mtDNA lineage in north Borneo, estimated to be ~1.6 million years divergent from lineages in west/south Borneo and the mainland, comparable to the divergence time from the Palawan pangolin. There appeared to be mitonuclear discordance, with no apparent genetic structure across Borneo based on analysis of nuclear SNPs. These findings are consistent with the 'out of Borneo hypothesis', whereby Sunda pangolins diversified in Borneo before subsequently migrating throughout Sundaland, and/or a secondary contact scenario between mainland and Borneo. We have elucidated possible taxonomic issues in the Sunda/Palawan pangolin complex and highlight the critical need for additional georeferenced samples to accurately apportion its range-wide genetic variation into appropriate taxonomic and conservation units. Additionally, these data have improved forensic identification testing involving these species and permit the implementation of geographic provenance testing in some scenarios.
ABSTRACT
The use of carbon dioxide (CO2) exposure as a means of animal euthanasia has received considerable attention in mammals and birds but remains virtually untested in reptiles. We measured the behavioral responses of four squamate reptile species (Homalopsis buccata, Malayopython reticulatus, Python bivitattus, and Varanus salvator) to exposure to 99.5% CO2 for durations of 15, 30, or 90 minutes. We also examined alterations in plasma corticosterone levels of M. reticulatus and V. salvator before and after 15 minutes of CO2 exposure relative to control individuals. The four reptile taxa showed consistent behavioral responses to CO2 exposure characterized by gaping and minor movements. The time taken to lose responsiveness to stimuli and cessation of movements varied between 240-4260 seconds (4-71 minutes), with considerable intra- and inter-specific variation. Duration of CO2 exposure influenced the likelihood of recovery, which also varied among species (e.g., from 0-100% recovery after 30-min exposure). Plasma corticosterone concentrations increased after CO2 exposure in both V. salvator (18%) and M. reticulatus (14%), but only significantly in the former species. Based on our results, CO2 appears to be a mild stressor for reptiles, but the relatively minor responses to CO2 suggest it may not cause considerable distress or pain. However, our results are preliminary, and further testing is required to understand optimal CO2 delivery mechanisms and interspecific responses to CO2 exposure before endorsing this method for reptile euthanasia.
