Studies On The Conservation Genetics Of Black Muntjac (Muntiacus Crinifrons)

Black muntjac (Muntiacus crinifrons) is a rare species endemic to China and nowadays exists only in the mountains of eastern China. Due to habitat destruction and hunting pressure etc., the distribution range and population size of the animal have been continued declining in the past hundred years. Therefore, The black muntjac is currently listed in Category I of the State Key Protected Wildlife List in China and is also listed in Appendix I of the Convention on International Trade in Endangered Species (CITES). In this study, for the first time, we used mtNDA d-loop region and nucleotide DNA microsatellite as genetic markers to conduct a general survey on the genetic diversity, population subdivision, gene flow and phylogeography of the wild population and also on the genetic background of the single captive population. The aims were to provide essential genetic information for reasonsable conservation and management stratagies to be devised for the species.1. Length of the complete sequence of black muntjac varied from 919 to 925 bp due to a heterogeneous motif T_1-3C_8-11 exsited in the right region. Throuth comparison among the homologous regions of the black muntjac and other two congeneric species Chinese muntjac and India muntjac, we supposed that T_1-3C_8-11 might be characterized by species of genus Muntiacus.2. Only 3 variable sites were identified in 424 bp fragment of the d-loop region sequence from 18 individuals of the captive population. Both haplotype diversity (h) and nucleotide diversity (π) were very low with 0.569 for h and 0.0015 for π. Three haplotypes were defined based on the three variable sites, suggesting that only three female black muntjac of the founders geneticly contributed to the captive population. The three haplotypes showed distinct allelic frequency bias with haplotype h15 being the dominant allele and h3 being the rare one, implying that the three genetic contribution founders had different genetic contribution to the pupulation.3. A total of 18 haplotypes were defined based on 22 variable sites found in thetarget sequence for the wild population (n=47). Sequence divergence between haplotypes varied from 0.002 to 0.029 with average value 0.013. For the whole population, haplotype diversity and nucleotide diversity were 0.913 and 0.00867 respectively. Compared with other deer species, the black muntjac had still sustained moderate to high haplotype diversity though the nucleotide diversity was rather low. Of the three subpopulations, Suichang had the highest values of both diversity indicies, and Huangshan and Tianmushan showed the similar level in both genetic diversities. High sequence homology and low nucleotide diversity might implied that the species historically had low female effective population size. We suggested two possible reasons for the inference. One is that the black muntjac had narrower space niches than that of other deer species and had permanently limited by unique habitat factors, which resulted in the animal having little chance to disperse. Another factor is that the species might have histrorically suffered from several founder effects due to periodical glaciations in the Pleistocene.4. The AMOVA analysis within and among populations revealed that 16% of the total variation was present among populations (0st = 0.16, P< 0.001). The subdivision estimator 0st indicated that the Suichang population was significantly differentiated from Huangshan and Tianmushan populations (0st 0.16772, P < 0.001;0St = 0.20022, P< 0.001, respectively), suggesting that Suichang population separated from the other two populations along matriline. While the samples from Huangshan and Tianmushan populations showed a slight differentiation from each other (0st = 0.05628, P = 0.125), indicating that female-mediated gene transfer might be frequent between these two groups in the past. However, many private haplotypes were identified in Huangshan (3 ones) and Tianmushan (4 ones), possibly impling barriers to gene flow between the two populations also occurrence in the history.5. Both phylogenetic trees and haplotype network indicated that the mtDNA haplotypes of the black muntjac were split into two well divergent clades (Clade I and Clade II). MtDNA haplotype of the wild population have not evolved intoreciprocally monophylogenetic groups though there were two distinct haplotype clades coexisting in Suichang population.6. The nested clade analysis showed significant association between geographic region and haplotype distribution at three different hierarchical levels, and contiguous range expansion and past fragmentation followed by range expansion were inferred to account for the observed phylogeographic patterns. The examination of mismatch distribution and the tests of neutrality provided further evidence for historical population expansion in this species. Therefore, these analyses suggested that the coexistence of distinct haplotypes in Suichang population was induced by historical post-fragmentation population expansion. Regarding the significant population differentiation, the current restricted range of this species implied that it should be attributed to localized isolation by distance and habitat fragmentation and loss in the recent past.7. 66 microsatellite primer pairs originated from bovine and corvine animals were tested in black muntjac, of which, 70% could give PCR product but only 30% showed polymorphic. 11 most polymorphic and easily scorable loci were choosed in this study. The lowest value of PIC (polymorphic information content) of the 11 loci is 0.645 with average value of 0.794, which is much higher than 0.5. Furthermore, value of cumulative discrimination power (CDP) across 11 loci is larger than 1.0 and cumulative paternity exclusion (CPE) value across the 11 loci was up to 0.9993 under the condition that information of both parents is unknow. The high values of PIC, CDP and CPE indicate that the loci screened in this study are highly diagnostic.8. For the wild population (n=42), a total of 99 distinct alleles were observed at eleven loci over the complete data set, of which 23 were private alleles. The number of alleles per locus ranged from 7 to 11 with an average of 9 and the average heterozygosity across 11 loci was up to 0.78. So the microsatellite variation observed in black muntjac was rather higher compared with that of other ungulate species analyzed, which contrasted a rather low mtDNA diversity revealed by mtDNA d-loop sequence variation depicted above. Which showedthat mtDNA analyses of genetic variation have not always provided a reliable indicator of nuclear genome diversity due to different inherited patterns. Low mitochondrial variation coincident with high nuclear variation of the black muntjac might be explained in terms of small femal effective population size of the species historically. We suggested that the black muntjac is a very young species compared with other ungulates.9. A total of 59 alleles were recorded and no private allele was found in the captive population (n=14). For the captive population, the mean number of alleles per locus was 5.4 and the average heterozygosity across 11 loci was 0.68. Both values were significantly lower than that of wild population. Furthermore, the population also show distinct bias in allele frequency of nucleotide gene and average inbreeding efficient of the population was up to 0.13 and. All these parameters suggested that the population began to show characterizes of genetic deterioration for small population.10. Homogeneity test showed significant difference in allele frequency distributions between Suichang subpopulation and the other two subpopulations. Both AMOVA and individual assignment test supported that Suichang had significantly differentiated from Huangshan and Tianmushan. As was consistent with the result of analysis using mtDNA control region sequence variations. Thus, despite difference in the genetic pattern and degree of differentiation, both types of markers roughly supported that the extant black muntjac population was genetically structured into two parts, i.e., Huangshan/Tianmushan and Suichang.11. Both types analyses revealed the relationship of genetic differentiation and geographical distance was not significant (p<0.05), implying that genetic differentiation might result from many factors.12. Taking the degree of difference in estimates of Fst between mtDNA control region and nuclear microsatellite markers into account, genetic differentiation based on mtDNA (overall 0sj=OA6) is approximately three times greater than that based on microsatellite data (overall Fsf=0.053), suggesting a male-biased gene flow in black muntjac.13. Both mtDNA d-loop and nucleotide DNA microsatellite supported that the extant black muntjac population were genetically structured into two parts, i.e., Huangshan/Tianmushan and Suichang. However, mtDNA haplotype of the wild population have not evolved into reciprocally monophylogenetic groups. Therefore, we suggest that the current black muntjac should be taken as one evolutionary significant unit (ESU) but Huangshan/Tianmushan and Suichang should be protected as different management unit (MU).14. Our results indicated that the captive population has already showed genetic deterioration. So we suggested that two measures should be considered to maintain a healthy captive population into the future. One is a new scientifically managed captive breeding program that should be specially stipulated for the captive population to prevent further improper management. Another suggestion is to introduce some new founders (10-15) to the existing captive population to alleviate the potential inbreeding depression.15. Since the extant black muntjac were genetically structured into two parts and the current captive population was founded with individuals from Huangshan, it genetically represented Huangshan/Tianmushan population but not Suichang. Therefore, to maximum levels of genetic diversity of the extant black muntjac in the wild we strongly recommend that a new captive population for conservation purpose with individuals of SC population as founders source be established.