How Genetics Has Aided and Informed Our Understanding of Pangolins

Introduction

The word “Pangolin” is derived from the phrase “Pengaulling” which is a Malayan phrase meaning to curl up. Pangolins are mammals that hail from the order of Pholidota. They are nocturnal mammals which have timid behavioural characteristics, being non-aggressive and usually shy solitary creatures (Thapa, 2014). There are currently only eight living species of pangolins in the world as the others have become extinct due to the wildlife trade. Pangolins are highly sought after for their scales and meat as the meat is used for consumption and scales used in traditional medicine in eastern countries such as India and China (Zhang et al., 2015).

Of the eight living species there are four, which are in the genus Manis, which reside in Asia, these are;

• Thick-tailed Pangolin (Manis crassicaudata);
• Formosan Pangolin (Manis pentadactyla);
• Sunda Pangolin (Manis javanica);
• and Philippine Pangolin (Manis culionensis).

The other four remaining species can be found in Africa;

• Giant Pangolin (Smutsia gigantea);
• Cape Pangolin (Smutsia temminckii);
• Long-tailed Pangolin (Phatagunis tetradactyla);
• and the Tree Pangolin (PhataginusPhataginusPhatagunis tricuspis).

Due to advances in genetics we are able to learn more about pangolins, such as their phylogeny, genetic diversity, dispersal and conservation (Thapa, 2014).  Fossil records of pangolins are small due to their preferred environment being tropical forests which do not do well to preserve remains. Pangolins are the only mammals known to have protective scales made from keratin covering their entire bodily surface for protection. (Gaudin, Emry and Wible, 2009). In this report I will be reviewing scientific papers which have studies various aspects of pangolins such as pangolin phylogeny, genetic diversity in pangolins, and the conservation of pangolins using genetics. Reviewing these studies will show what is already known about pangolins, what needs to be improved about our understanding and what else needs to be done to have an in depth understanding of these mammals. 

Pangolin phylogeny

Pangolins are from the order Pholidota where they are the only members within that order. Of the eight species of pangolins which are still alive they are able to be classified into three genera; Manis if for the four Asian species of pangolin, Formosan Pangolin (Manis pentadactyla); Sunda Pangolin (Manis javanica); Philippine Pangolin (Manis culionensis); and the Thick-tailed Pangolin (Manis crassicaudata). The African tree pangolins are classified with Phatagunis, Tree Pangolin (Phataginus Phataginus Phatagunis tricuspis) and the Long-tailed Pangolin (Phatagunis tetradactyla). The two African ground pangolins are classified with Smutsia, Giant Pangolin (Smutsia gigantea); and the Cape Pangolin (Smutsia temminckii).

This study had sequenced the entire mitochondrial genome of all three of the genera of Pholidota. They had also compared the mitochondrial DNA that was provided by GenBank including sequences from five markers and the genomes of Manis pentadactyla and Phatagunis tetradactyla. DNA was extracted from tissue culture cells in this study from Manis javanica and samples of musclefrom Phatagunis tricuspis and also from Smutsia temminckii where a QIAGEN DNeasy Tissue Kit was used. Polymerase chain reactions were used with previously published about primers and four primers which were new and designed for this experiment to amplify the mitochondrial genome fragments. using sanger sequencing on both strands the PCR products were sequenced and then edited with sequencher 5.1. (Hassanin, Hugot and van Vuuren, 2015).

After being sequenced and edited, the mitochondrial genomes were analyzed and then the data provided was constructed into a phylogenic tree with four genera which had four orders of Laurasiatheria to help root the tree. On the tree there are ascension numbers of DNA sequences provided by GenBank. In the study it is  shown that MUSCLE (Edgar, 2004) was used to align complete mitochondrial genomes and then adjusted further by eye.

Fig.1. this figure is the phylogenetic tree that was produced by the study where mitochondrial DNA from Manis pentadactyla, Manis javanica, Smutsia temminckii, Phataginus tetradactyla, and Phataginus tricuspis were all compared after being sequenced. The values present on the branches of the tree represent the bootstrap percentages (Hassanin, Hugot and van Vuuren, 2015).

The study seems to be quite certain that the phylogenetic tree is accurate as the tree is based upon the alignment of 14,926 base pairs which has maximal base pairs that shows interspecific relationships within the Manidae family. The two groups of pangolins (Asian and African) are monophyletic which was always suspected as they are too similar to be distant on the phylogenetic tree. The length of the branches of the Manis terminal branches are surprisingly shorter than expected in fig.1.

This shows that the Manis javanica and Manis pentadactyla have mitochondrial genomes that are 99% identical to the other as suggested by the study Qin et al., 2012 but Hassanin, Hugot and van Vuuren, 2015 suggests that one of their samples was misidentified and so they had tried to resolve the issue by comparing the mitochondrial genomes to the homologous fragments which are on GenBank for Pholidota. The NJ clustering method was used to analyze five mtDNA fragments. The analysis of the fragments suggests that the NC_016008 genome was incorrectly identified as Manis pentadactyla and should have actually been reported as Manis javanica in Qin et al., 2012. The sample of Manis javanica had been collected in Guangxi which is a province in China where it was thought only to have Manis pentadactyla but there are currently two theories to Manis javanica being found there.

The first suggested by the study is that it was illegally imported into the country for the meat or scales of a pangolin which is rumored to have special healing properties in east Asian countries.

The second theory that is proposed by the study is that the two species live in symbiosis in the province of Guangxi, which would suggest that the current geographical range for Manis javanica is incorrect and that the occupancy of Manis javanica needs to be reassessed. This problem was found within another paper Arnason et al., 2002 when looking at the Phatagunis tetradactyla mitochondrial genome it was found to be 99% identical to the mitochondrial genome of Phataginus tricuspis and its suggested that the most likely possibility is the incorrect identification of the sample.

There is also the result of a high nucleotide divergence of the specimen selected for the study and others that had been collected from Gahanna, Nigeria, and Cameroon (Hassanin, Hugot and van Vuuren, 2015). 

Genetic diversity in pangolins 

Due to pangolins being an endangered species there is a trade ban on the export or import of any part of the animal. Due to this trade ban items suspected of being of pangolin origin must be tested using genetic tools to assess if they are in fact pangolin. In one genetic study some scales ceased from an illegal trade have been genetically tested and have identified from which region the scales originated from by comparing the genetic diversity of the scales to known populations of Indian pangolins.

This study had used 15 different batches of seized samples of scales that were collected between 2011 and 2014. They had taken the scales to extract the DNA by initially cleaning them with ethanol and 10X Phosphate buffered saline (PBS) twice, then 0.5mm of the surface of the samples were removed with a surgical grade blade as to remove any contaminants which may have been on the surface then washed with the 10X PBS again. Small parts of the pangolin scales were then cut and transferred to a 2µl Eppendorf tube so that the concentration of DNA could be quantified.

Absorbance was measured at the wavelength of 260 nm using 1 µl samples. The samples were then put through PCR amplification to increase the number of partial fragments of the Cyt b gene and 16s rRNA gene. There was a total reaction volume of 15 µl which consisted of a PCR Master Mix, each primer pair, Q solution (which is supplied with the PCR kit), DNA elutant and RNase-free water.

When they felt that there was enough of the partial fragments needed they were then purified using ExoSAP and sequenced using a genetic analyser. The sequences that they had gotten from the 15 samples were compared with known pangolin species samples which are publicly accessible from GenBank (http://blast.ncbi.nlm.nih.gov/).

To identify where the scales have come from they used both rarefaction and sample coverage techniques (Zhang et al., 2015). They had used these methods to estimate the number of pangolin individuals and the COI haplotypes as the COI haplotypes can be used as barcode regions that are specific to species of pangolins so can be used to identify where the scales have come from.

Using these genetic tools, they have been able to separate the African from the Asian species of pangolin looking at the COI haplotype (Hebert et al., 2003). This study shows that there is a large amount of genetic diversity of species of pangolins as from the COI haplotypes they are able to distinguish where the scales have come from, other factors had hinted as to where they had come from as they could rule out African pangolins as the scales had inter-scale bristles which are only found on Asian pangolins.

Another study (LUO et al., 2006) had used microsatellites to assess genetic diversity between populations of pangolins. They had isolated 32 polymorphic microsatellite markers in Manis javanica and the cross-species applicability in Manis pentuadactyla and Manis tricuspis. DNA was extracted from a sample of muscle tissue of Manis tricuspis, using this sample a microsatellite-enriched genomic library was produced.

After being sequenced with both forward and back primers, 48 samples out of 288 clones had sequences that were of good enough quality for primer pairs to be designed. 32 of the 48 primer pairs had produced microsatellite amplicons in 24 Manis javanica which was then tested using 12 Manis pentuadactyla and two Manis tricuspis. Polymerase chain reaction products were then amplified in a 10 µl system. This system went through a polymerase chain reaction, then the microsatellite patterns had been size-fractionated and scored using genescan 2.1 and genetyper 2.5. high genetic variability was detected in Manis javanica as all 32 loci were polymorphic with 19 loci containing 10 or more alleles with the range of alleles going from 2-21 at each locus.

The observed heterozygosity of Manis javanica had an average of 0.708 while the expected heterozygosity had an average of 0.805. 27 of the 32 loci had successfully been amplified with Manis pentuadactyla and 18 of the loci on Manis tricuspis, this is more than likely to be due to the divergence time between the three pangolin species (LUO et al., 2006). This study had shown that the Malayan pangolin (Manis javanica) seemingly has a higher genetic diversity than the Chinese pangolin (Manis pentuadactyla) and the African Tree pangolin (Manis tricuspis). This high genetic diversity was shown with all 32 loci being polymorphic and having at least 10 alleles on 19 loci.

Conservation of pangolins using genetic tools

Genetic tools are used to aid the conservation of pangolins by providing evidence to make appropriate decisions to manage and improve the conservation of pangolins. The type of things that genetics have been used for in the conservation of species is, to stop wildlife trading on illegal species, to aid in breeding for conservation including genetically rescuing populations and captive breeding (Nash et al., 2018). Pangolins are one of the highest illegally trafficked mammals in the wildlife trade on the globe. It has been raised that pangolins are a priority to be genetically researched for their own conservation action plan (Challender et al. 2020).

Multiple methods can be used to gather data to aid in the conservation of pangolins. Single nucleotide polymorphisms (SNPs) are used as one of these methods which are able to provide information in much more detail such as microsatellites. Next generation sequencing methods have been used in looking into the population and structure of some mammals, with methods like a double digest restriction site sequencing (Knowles et al., 2016).

The Sunda pangolins whole genome was sequenced and published about a few years ago which will aid in further genetic studies for the Sunda pangolin which would help in deciding action plans in the conservation of the species. For example there is a study which suggests that unlike previously known, there may be in fact more than one species (Manis javanica) of pangolin the Indonesia but this is debated as the mitochondrial control region is a single marker which may have introgression and selective sweeps due to bias (Wirdateti et al. 2017).

One conservation study had collected number of Sunda pangolin samples where 89 out of the total 97 samples were from Indonesian samples of dead pangolin muscle tissue with specialists on pangolins there to confirm all samples were from Sunda pangolins by the morphological features like the hind scale counts. DNA was then extracted from the tissue samples using DNeasy blood and tissue kits. The yields of DNA were measured using fluorometric quantitation, for the low yields of DNA they had reextracted it with phenol chloroform.

Single nucleotide polymorphisms (SNPs) were used to along with sequencing and restriction enzymes Mspl and EchoRI-HF as they had seemed to work well for other studies. They had also chosen to use fragments of DNA with a range of 250-650 base pairs. Molecular grade water was used was a control to assure that there had been no incident of contamination. Using the results a mitochondrial tree and a haplotype network (Fig.2.) was created. There are differences in the SNP results and the haplotype network that shows that there are signs of introgression in Borneo and Sumatra. It is discussed that in the tests for secondary gene flow there were signs of introgression as shown in the figure below as the test result for MZBR 0270 had shown introgression.

Fig 2. This figure is a haplotype network formed from the results of looking at 97 samples of Sunda javanic. Suggesting the Sunda javanica from Singapore and Borneo have been brought over illegally through the wildlife trade and breeding leading to introgression (Nash et al., 2018).

This study could have revealed some possibility of illegal trade and the directions as shown in figure 3 (to the left) as it shows the number of samples of Sunda javanica and the trade routes that are suggested by where they were collected and where they were identified to originate from (Nash et al., 2018). It is also suggested that the lineages of the Sunda pangolin may be ecologically and genomically different, due to looking at the genetic clusters (also shown in figure 3). It is suggested that the study should be used to aid in the taxonomic classification of pangolins and the data gathered shows divergences in the genome which may be supported by a phenotypic inquiry (Nash et al., 2018).

References

Zhang, H., Miller, M., Yang, F., Chan, H., Gaubert, P., Ades, G. and Fischer, G. (2015). Molecular tracing of confiscated pangolin scales for conservation and illegal trade monitoring in Southeast Asia. Global Ecology and Conservation, 4, pp.414-422.

Gaudin, T., Emry, R. and Wible, J. (2009). The Phylogeny of Living and Extinct Pangolins (Mammalia, Pholidota) and Associated Taxa: A Morphology Based Analysis. Journal of Mammalian Evolution, 16(4), pp.235-305.

Thapa, P. (2014). An Overview of Chinese Pangolin (Manis pentadactyla): Its General Biology, Status, Distribution and Conservation Threats in Nepal. The Initiation, 5, pp.164-170.

Hassanin, A., Hugot, J. and van Vuuren, B. (2015). Comparison of mitochondrial genome sequences of pangolins (Mammalia, Pholidota). Comptes Rendus Biologies, 338(4), pp.260-265.

Hebert, P., Cywinska, A., Ball, S. and deWaard, J. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B: Biological Sciences, 270(1512), pp.313-321.

Edgar, R. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32(5), pp.1792-1797.

Qin, X., Dou, S., Guan, Q., Qin, P. and She, Y. (2012). Complete mitochondrial genome of theManis pentadactyla(Pholidota, Manidae): Comparison ofM. pentadactylaandM. tetradactyla. Mitochondrial DNA, 23(1), pp.37-38.

Arnason, U., Adegoke, J., Bodin, K., Born, E., Esa, Y., Gullberg, A., Nilsson, M., Short, R., Xu, X. and Janke, A. (2002). Mammalian mitogenomic relationships and the root of the eutherian tree. Proceedings of the National Academy of Sciences, 99(12), pp.8151-8156.

LUO, S., CAI, Q., DAVID, V., ZHANG, L., MARTELLI, P., LIM, N., FERRAND, N., CHIN, S., GAUBERT, P., RAMOS, M., O’BRIEN, S., ANTUNES, A. and JOHNSON, W. (2006). Isolation and characterization of microsatellite markers in pangolins (Mammalia, Pholidota,Manisspp.). Molecular Ecology Notes, 7(2), pp.269-272.

Nash, H., Wirdateti, Low, G., Choo, S., Chong, J., Semiadi, G., Hari, R., Sulaiman, M., Turvey, S., Evans, T. and Rheindt, F. (2018). Conservation genomics reveals possible illegal trade routes and admixture across pangolin lineages in Southeast Asia. Conservation Genetics, 19(5), pp.1083-1095.

Challender, D., Willcox, D.H.A., Panjang, E., Lim, N., Nash, H., Heinrich, S. & Chong, J. 2019. Manis javanica . The IUCN Red List of Threatened Species 2019: e.T12763A123584856. Downloaded on 05 January 2020.

Knowles, L., Massatti, R., He, Q., Olson, L. and Lanier, H. (2016). Quantifying the similarity between genes and geography across Alaska's alpine small mammals. Journal of Biogeography, 43(7), pp.1464-1476.

Wirdateti, Semiadi G (2017) Genetic variation of confiscated pangolins of Sumatra, Java, and Kalimantan based on control region mitochondrial DNA. J Vet 18(2):181–191