Y-chromosomal gene flow of Macaca fascicularis (Cercopithecidae) between the insular and mainland peninsula of Penang state, Malaysia

DNA sequencing of the Y-chromosome testis-specific protein locus (TSPY) and sex-determining region (SRY) was conducted on 27 Macaca fascicularis individuals from eight insular and two peninsula localities of Penang State, Malaysia. Five haplotypes were discovered from the Penang samples, of which four were unique for that population. These haplotypes constituted of related individuals as observed in the Y-chromosomal gene flow within and between the insular and mainland regions. The occurrence of a dominant haplotype shared amongst M. fascicularis from southern Thailand, the Malay Peninsula, and Sumatra could be the result of a recent dispersal event from the common Pleistocene refugia, which had most likely been located in the Malay Peninsula. A combination of nucleotides at 12 sites distinguished the M. fascicularis from the northern region of the Isthmus of Kra from those of the southern region including our samples. The molecular phylogenetic tree confirmed that, unlike conspecific populations from regions north of the Isthmus of Kra, M. fascicularis from the southern region of the isthmus were free of introgression of Y-chromosome from M. mulatta. We dated the last common ancestors shared by the fascicularis group at approximately 1.5 million years ago (mya). Also, we estimated the bifurcation between the insular and the continental lineages of M. fascicularis as approximately 0.7 mya, which had been estimated as 0.4 mya in a previous study. Finally, similarly to the observations on various taxa by previous authors we recognise the role of the Isthmus of Kra area as a genetic barrier to the dispersal of and to gene flow in M. fascicularis

Expediting the sampling, decalcification, and forensic DNA analysis of large elephant ivory seizures to aid investigations and prosecutions

The illegal ivory trade continues to drive elephant poaching. Large ivory seizures in Africa and Asia are still commonplace. Wildlife forensics is recognised as a key enforcement tool to combat this trade. However, the time and resources required to effectively test large ivory seizures is often prohibitive. This limits or delays testing, which may impede investigations and/or prosecutions. Typically, DNA analysis of an ivory seizure involves pairing and sorting the tusks, sampling the tusks, powdering the sample, decalcification, then DNA extraction. Here, we optimize the most time-consuming components of this process: sampling and decalcification. Firstly, using simulations, we demonstrate that tusks do not need to be paired to ensure an adequate number of unique elephants are sampled in a large seizure. Secondly, we determined that directly powdering the ivory using a Dremel drill with a high-speed cutter bit, instead of cutting the ivory with a circular saw and subsequently powdering the sample in liquid nitrogen with a freezer mill, produces comparable results. Finally, we optimized a rapid 2 -h decalcification protocol that produces comparable results to a standard 3-day protocol. We tested/ optimised the protocols on 33 raw and worked ivory samples, and demonstrated their utility on a case study, successfully identifying 94% of samples taken from 123 tusks. Using these new rapid protocols, the entire sampling and DNA extraction process takes less than one day and requires less-expensive equipment. We expect that the implementation of these rapid protocols will promote more consistent and timely testing of ivory seizures suitable for enforcement action

DNA forensic case study: species identification from suspected crocodile penis

Department of Wildlife and National Parks (PERHILITAN) began developing the capacity on wildlife DNA forensic since 2009 to assist in law enforcement activities. Most of the forensic cases require DNA species identification of animal parts where key morphological characters are missing. Among the cases frequently confiscated are from traditional Chinese medicine (TCM), which often claim to use animal parts such as reproductive organs. Dried crocodile penises, in particular, are believed to have medicinal benefits and are highly demanded in TCM industries since millennials ago. In this case study, we analysed four enforcement cases comprising of 44 exhibits which resemble crocodile penis using the partial cytochrome b gene of the mitochondrial DNA. Sequence similarity searches were conducted using both the BLAST search engines of GenBank and also PERHILITAN’s MyWILDNA database to identify the species. Out of 44 exhibits, 22 items produced DNA sequences in which three were found to be derived from Crocodylus porosus while the remaining was identified as Bos taurus, Bos javanicus, and Bos indicus. This case study showed that most of TCM which claimed to be derived from crocodile penis turned out to be counterfeit products

Are Pangolins Scapegoats of the COVID-19 Outbreak-CoV Transmission and Pathology Evidence?

The COVID‐19 outbreak has infected over 6 million people across the world. The origin of COVID‐19 coronavirus (CoV) remains unknown, although pangolins have been suggested as potential hosts. We investigated two pangolins seized in Guangdong Province, China. Molecular screening revealed CoV in one pangolin (“Dahu”), while another (“Meidong”) was infected by Ehrlichia ruminantium. Dahu exhibited difficulty breathing, infections of lung, intestines, and nostrils, as revealed by computed tomography imaging and necropsy. Previous phylogenetic analyses showed bat coronavirus RaTG13 is closer to COVID‐19 CoV compared to pangolin coronavirus. Over 20 caregivers have had close physical contact with CoV‐positive Dahu, but none became infected with CoV. Our data suggest that pangolins are unlikely the natural reservoir or secondary hosts of COVID‐19 CoV. Pangolins seems to be victims infected by CoV carried by a not yet unidentified natural reservoir host species, perhaps due to their weakened immune system

Mitochondrial DNA diversity of the long-tailed macaque (Macaca fascicularis) from the northern region of Peninsular Malaysia

We examined the genetic diversity of 64 long-tailed macaques from the northern states of Peninsular Malaysia covering the states of Perlis and Kedah including the Langkawi Island using the complete control region (CR) segment of the mitochondrial DNA. Standard genetic diversity including nucleotide diversity, haplotype diversity and genetic divergence were calculated. Moderate nucleotide diversity (π = 0.021) was observed which is higher than a previous study on the Penang M. fascicularis population. Twenty-three haplotypes were detected with haplotype diversity, h of 0.936. Haplotype sharing was observed among Langkawi and Perlis macaques indicating historical connection between the island and the mainland. Phylogenetic trees constructed grouped the samples into 4 groups without any obvious populations structuring

Using Y-Chromosome to elucidate the evolution and dispersal pattern of the long-tailed macaques (Macaca fascicularis) in Southeast Asia

We employed a combined segment of the testis-specific protein (TSPY) and the sex determining region (SRY) of the Y-chromosome gene to elucidate the evolutionary pattern of the long-tailed macaques (Macaca fascicularis) in Southeast Asia. A maximum-likelihood (ML) tree and a phylogenetic network were constructed using 147 sequences of M. fascicularis from the Peninsular Malaysia and Sarawak including sequences from the other regions of the species range taken from the other previous studies. Measurements of standard genetic diversity indices were calculated. Our findings revealed that the M. fascicularis are separated into two major groups of the continental and the insular lineages. Furthermore, the continental lineage is separated into two faunal regions demarcated at the Isthmus of Kra. The Y-chromosome dataset revealed a dominant haplotype emerging at around 0.25 (±0.1) million years ago (mya) which was shared by 82 samples from the southern region of the Isthmus of Kra which ranges from Songkhla, Thailand, the Malay Peninsula and downwards to Sumatra, Indonesia. The insular lineage emerged at around 0.61 (±0.4) mya which occupied the island of Borneo and the Philippines. We also confirmed that the introgression of the M. mulatta Ychromosome into the Indochinese M. fascicularis (Vietnam and Cambodia) are absent in the M. fascicularis haplotypes from the southern region of the Isthmus of Kra. Keywords: Macaca fascicularis, Y-chromosome, phylogenetic tree and network, dispersal route, time estimates

No Evidence of Coronaviruses or Other Potentially Zoonotic Viruses in Sunda pangolins (Manis javanica) Entering the Wildlife Trade via Malaysia

The legal and illegal trade in wildlife for food, medicine and other products is a globally significant threat to biodiversity that is also responsible for the emergence of pathogens that threaten human and livestock health and our global economy. Trade in wildlife likely played a role in the origin of COVID-19, and viruses closely related to SARS-CoV-2 have been identified in bats and pangolins, both traded widely. To investigate the possible role of pangolins as a source of potential zoonoses, we collected throat and rectal swabs from 334 Sunda pangolins (Manis javanica) confiscatedin PeninsularMalaysia and Sabah between August 2009 andMarch 2019. Total nucleic acid was extractedfor viral molecular screening using conventional PCR protocols used to routinely identify known and novel viruses in extensive prior sampling (> 50,000 mammals). No sample yielded a positive PCR result for any of the targeted viral families—Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae and Paramyxoviridae. In the light of recent reports of coronaviruses including a SARS-CoV-2-related virus in Sunda pangolins in China, the lack of any coronavirus detection in our ‘upstream’ market chain samples suggests that these detections in ‘downstream’ animals more plausibly reflect exposure to infected humans, wildlife or other animals within the wildlife trade network. While confirmatory serologic studies are needed, it is likely that Sunda pangolins are incidental hosts of coronaviruses. Our findings further support the importance of ending the trade in wildlife globally

De novo sequencing, assembly and analysis of eight different transcriptomes from the Malayan pangolin

Pangolins are scale-covered mammals, containing eight endangered species. Maintaining pangolins in captivity is a significant challenge, in part because little is known about their genetics. Here we provide the first large-scale sequencing of the critically endangered Manis javanica transcriptomes from eight different organs using Illumina HiSeq technology, yielding ~75 Giga bases and 89,754 unigenes. We found some unigenes involved in the insect hormone biosynthesis pathway and also 747 lipids metabolism-related unigenes that may be insightful to understand the lipid metabolism system in pangolins. Comparative analysis between M. javanica and other mammals revealed many pangolin-specific genes significantly over-represented in stress-related processes, cell proliferation and external stimulus, probably reflecting the traits and adaptations of the analyzed pregnant female M. javanica. Our study provides an invaluable resource for future functional works that may be highly relevant for the conservation of pangolins

Pangolin genomes and the evolution of mammalian scales and immunity

Pangolins, unique mammals with scales over most of their body, no teeth, poor vision, and an acute olfactory system, comprise the only placental order (Pholidota) without a whole-genome map. To investigate pangolin biology and evolution, we developed genome assemblies of the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Strikingly, we found that interferon epsilon (IFNE), exclusively expressed in epithelial cells and important in skin and mucosal immunity, is pseudogenized in all African and Asian pangolin species that we examined, perhaps impacting resistance to infection. We propose that scale development was an innovation that provided protection against injuries or stress and reduced pangolin vulnerability to infection. Further evidence of specialized adaptations was evident from positively selected genes involving immunity-related pathways, inflammation, energy storage and metabolism, muscular and nervous systems, and scale/hair development. Olfactory receptor gene families are significantly expanded in pangolins, reflecting their well-developed olfaction system. This study provides insights into mammalian adaptation and functional diversification, new research tools and questions, and perhaps a new natural IFNE-deficient animal model for studying mammalian immunity.University of Malaya and Ministry of Education, Malaysia [UM.C/HIR/MOHE/08]; UMRG grant from the University of Malaya and Ministry of Education, Malaysia [RG541-13HTM]; Russian Ministry of Science [11.G34.31.0068]; NIH-NHGRI grant [5U54HG00307907]SCI(E)[email protected]