Phylogenetic Relationship And Species Identification Of Manidae

Pangolins are endangered due to human poaching and illegal trade for their food and medicinal value.The study shows that the taxonomic relationship of pangolins is still unknown: whether there is a new species of Asian pangolin.In this study,we reconstructed the phylogenetic relationships of pangolin using mitochondrial genomes and nine nuclear genes.In addition,given the existing problems on the identification of pangolin species by molecular biological methods,we screened the optimal molecular markers from 9 nuclear genes and evaluated the effect of Cytochrome B(Cytb)gene sequence length on species identification.The main results are as follows.1.The reconstructed phylogenetic tree supports the current taxonomic system of pangolins: 2subfamilies and 3 genera.However,two individuals(MJ＿DCW＿89 and MJ＿DCW＿116)were monophyletic and formed sister groups with M.javanica and M.culionensis.The minimum genetic distance between them was 0.059,which was higher than the standard minimum genetic distance between species,0.05.The time of differentiation was 4.5 million years ago(95%HPD=2.5-15.7),which was earlier than that of M.javanica and M.culionensis.These results indicated that the clade represented by the two individuals should be a new species of pangolin.2.Eight different models were used to calculate the intraspecies and interspecific genetic distance of Pangolins.Only the minimum interspecific genetic distance(inter-d)(0.023)of Ts based on K2 P was much larger than the maximum intraspecies genetic distance(intra-d)(0.006).furthermore moreover,among the 9 nuclear genes in this model,only the minimum interspecific genetic distance(0.022 and 0.023)of FES and GNB1 was much higher than the maximum intraspecies genetic distance(0.014 and 0.006).These results indicated that FES and GNB1 were the most suitable molecular markers for identification.The similarity between98.6% of samples(n=481)and corresponding pangolin species was higher than 98%.This suggested that the value should be used as the criterion to identify pangolin species.The minimum inter-specific genetic distance(inter-d)was greater than the maximum Intra-specific genetic distance(intra-d)among five pangolins except M.javanica(211bp)and P.tricuspis(421bp and 211bp).The variation of sequence length and the imbalance of sample size could cause an unstable ratio.Phylogenetic trees showed that most pangolin species were monophyletic except M.javanica and M.culionensis.Shorter sequence led to change in their topological structure and confidence of some nodes.This study built a relatively comprehensive and complete framework for the phylogenetic relationship of pangolins and provided a foundation for pangolin’s conservation research.By identifying optimal molecular markers for identification and evaluating the effects of sequence length changes on identification results,we offer a new approach to tracking the global illegal trade of pangolin.