Individual and sexual identification for the wild black muntjac (Muntiacus crinifrons) based on fecal DNA

The black muntjac (Muntiacus crinifrons), endemic to China, was categorized as a Grade I National Key Protected Animal by the Ministry of Agriculture of China and classified as Vulnerable (VU) by IUCN. Recent years, studies had been conducted on this species mainly focusing on habitat selection, food habit, gene flow etc., only with a few reports on the population dynamics. Individual identification of wild animals is one of the most important subjects in the population dynamic research. Of various molecular markers, microsatellite DNA fingerprinting has been used most frequently and successfully on many kinds of animals. Here, we constructed identification system for the black muntjac using 8 microsatellite loci. 31 black muntjacs were identified from 141 fecal samples, whereas 43 samples could be used for PCR after repeated trials. Further, the sequencing for Cytb gene was also conducted for convincing us the identity of fecal samples. The results, highly consistent between sequencing consequence and sequence data from Genebank, implied that those experienced local people are of the convincing knowledge about wild animals, especially at the respects of identification to black muntjac’ feces pellets. Moreover, we detected the specificity of identification system to black muntjac. BM1225 was the only one locus that unsuccessful PCR for the muscle samples of Muntiacus reevesi was observed, which suggested that our identification system could be used for excluding the non-researched objects in some cases. Analyses using softwares CERVUS 3.0 and POPGENE 1.21 showed that the present identification system had strong discrimination power: 0.938 per loci (DP) or 0.999 in total (CDP). The mean observed number of alleles (Na), mean effective number of alleles (Ne), and mean excepted heterogosity (He) were 8.875, 6.375, and 0.829 respectively. Considering that the change of sex ratio in population could exert significant impact on population growth, density to some extent, we also analyzed the sex ratio of those individuals that had been identified based on fecal samples from filed using SRY (Sex-determining region Y) gene amplification, which identified 19 males and 12 females.