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      Genetic Evidence Reveals Distinct Lineage of Chinese Pangolin in Nepal: Insights From Scat and Blood Samples for Conservation and Wildlife Forensics

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          ABSTRACT

          Pangolins face critical threats from illegal trade and habitat loss, making their conservation a global priority. Despite their ecological and conservation significance, these elusive creatures remain poorly understood, particularly regarding their phylogeography and genetic diversity. This study successfully isolated DNA from two types of pangolin scat samples (whole scat and swabs) and blood that had been stored frozen for up to 3 years. A mitochondrial cytochrome b (424 bp) sequence was reliably generated from both types of scat samples, irrespective of whether the scat appeared in ‘good’ or ‘poor’ condition at the time of collection, and from the blood samples. Sanger sequencing identified four novel cytochrome b haplotypes, with distinct variations observed across sampling regions in Nepal (central and east). A comparison with reference sequences from China, Taiwan and Thailand revealed that the Chinese pangolins in Nepal represent a genetically distinct variant, differing by 15–19 base pairs from these other populations. These findings underscore significant genetic differentiation of Chinese pangolins in Nepal, with implications for the species' conservation and management. The methods described in this study are robust and adaptable, offering a valuable framework for broader genetic studies of pangolin populations across Nepal. Such approaches could facilitate the genetic mapping of pangolin variations, aiding in the identification of significant populations, the evaluation of conservation interventions and forensic applications to combat illegal poaching. This work emphasises the critical role of genetics in understanding and protecting pangolins, offering insights that are vital for their long‐term conservation.

          Abstract

          In this study, we utilised DNA isolated from stored pangolin scats and blood to amplify the cytochrome b region. Comparisons with reference sequences from pangolins originating in China, Taiwan and Thailand revealed a distinct variant of the Chinese pangolin in Nepal. These findings are significant, providing a foundation for future population genetic studies of pangolins in Nepal and informing conservation efforts to combat illegal poaching.

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          Most cited references51

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          popart: full-feature software for haplotype network construction

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            Evolution of the cytochrome b gene of mammals.

            With the polymerase chain reaction (PCR) and versatile primers that amplify the whole cytochrome b gene (approximately 1140 bp), we obtained 17 complete gene sequences representing three orders of hoofed mammals (ungulates) and dolphins (cetaceans). The fossil record of some ungulate lineages allowed estimation of the evolutionary rates for various components of the cytochrome b DNA and amino acid sequences. The relative rates of substitution at first, second, and third positions within codons are in the ratio 10 to 1 to at least 33. For deep divergences (greater than 5 million years) it appears that both replacements and silent transversions in this mitochondrial gene can be used for phylogenetic inference. Phylogenetic findings include the association of (1) cetaceans, artiodactyls, and perissodactyls to the exclusion of elephants and humans, (2) pronghorn and fallow deer to the exclusion of bovids (i.e., cow, sheep, and goat), (3) sheep and goat to the exclusion of other pecorans (i.e., cow, giraffe, deer, and pronghorn), and (4) advanced ruminants to the exclusion of the chevrotain and other artiodactyls. Comparisons of these cytochrome b sequences support current structure-function models for this membrane-spanning protein. That part of the outer surface which includes the Qo redox center is more constrained than the remainder of the molecule, namely, the transmembrane segments and the surface that protrudes into the mitochondrial matrix. Many of the amino acid replacements within the transmembrane segments are exchanges between hydrophobic residues (especially leucine, isoleucine, and valine). Replacement changes at first and second positions of codons approximate a negative binomial distribution, similar to other protein-coding sequences. At four-fold degenerate positions of codons, the nucleotide substitutions approximate a Poisson distribution, implying that the underlying mutational spectrum is random with respect to position.
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              Mammalian mitogenomic relationships and the root of the eutherian tree.

              The strict orthology of mitochondrial (mt) coding sequences has promoted their use in phylogenetic analyses at different levels. Here we present the results of a mitogenomic study (i.e., analysis based on the set of protein-coding genes from complete mt genomes) of 60 mammalian species. This number includes 11 new mt genomes. The sampling comprises all but one of the traditional eutherian orders. The previously unrepresented order Dermoptera (flying lemurs) fell within Primates as the sister group of Anthropoidea, making Primates paraphyletic. This relationship was strongly supported. Lipotyphla ("insectivores") split into three distinct lineages: Erinaceomorpha, Tenrecomorpha, and Soricomorpha. Erinaceomorpha was the basal eutherian lineage. Sirenia (dugong) and Macroscelidea (elephant shrew) fell within the African clade. Pholidota (pangolin) joined the Cetferungulata as the sister group of Carnivora. The analyses identified monophyletic Pinnipedia with Otariidae (sea lions, fur seals) and Odobenidae (walruses) as sister groups to the exclusion of Phocidae (true seals).
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                Author and article information

                Contributors
                fiona.hogan@federation.edu.au
                Journal
                Ecol Evol
                Ecol Evol
                10.1002/(ISSN)2045-7758
                ECE3
                Ecology and Evolution
                John Wiley and Sons Inc. (Hoboken )
                2045-7758
                16 February 2025
                February 2025
                : 15
                : 2 ( doiID: 10.1002/ece3.v15.2 )
                : e70982
                Affiliations
                [ 1 ] Future Regions Research Centre Federation University Australia Victoria Australia
                [ 2 ] Biodiversity Research Institute (University of Oviedo, Principado of Asturias, Spanish National Research Council), Mieres Campus University of Oviedo Mieres Asturias Spain
                [ 3 ] National Trust for Nature Conservation Lalitpur Nepal
                [ 4 ] IUCN SSC Pangolin Specialist Group Zoological Society of London London UK
                [ 5 ] Department of Biology University of Washington Seattle Washington USA
                Author notes
                [*] [* ] Correspondence:

                Fiona Hogan ( fiona.hogan@ 123456federation.edu.au )

                Author information
                https://orcid.org/0000-0001-6934-3720
                Article
                ECE370982 ECE-2024-09-01825.R1
                10.1002/ece3.70982
                11830433
                39958816
                9bdb95c1-8b03-46df-bd56-fa7f6a1b2a3b
                © 2025 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 19 December 2024
                : 03 September 2024
                : 08 January 2025
                Page count
                Figures: 8, Tables: 3, Pages: 13, Words: 7300
                Categories
                Conservation Genetics
                Research Article
                Research Article
                Custom metadata
                2.0
                February 2025
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.5.3 mode:remove_FC converted:16.02.2025

                Evolutionary Biology
                mitochondrial dna,non‐invasive genetic sampling,pangolin,scat
                Evolutionary Biology
                mitochondrial dna, non‐invasive genetic sampling, pangolin, scat

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