Approach For Conservation Genetics Of Brown-eared Pheasant (crossoptilon Mantchuricum)

Brown-Eared pheasant (Crossoptilon mantchuricum)is a rare endemic species to China and listed in the 2000 IUCN Red List of Threatened Species as'Vulnerable'. It is also listed in Appendix I of the Convention on International Trade in Endangered Species (CITES). Due to habitat destruction and hunting pressure, it only distributes in the Lvliang mountain of Shanxi province, and Xiaowutai mountain of Hebei province, China. In this study, we used mtDNA control 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. Furthermore, we also performed the analysis on the genetic background of the single captive population. The aims were to provide essential genetic information for reasonable conservation and management strategies to be devised for the species.1. A total of 14 haplotypes were defined based on 18 variable sites found in fragment of the d-loop region sequence for the wild population (n=43). For the whole population, haplotype diversity (h) and nucleotide diversity (π) were 0.833 and 0.00378, respectively. Compared with other pheasant species, the genetic diversity of Brown-Eared pheasant was rather low. Of the three subpopulations, Xiaowutaishan had the lowest values of both genetic diversity, Luyashan and Pangquangou showed the similar level but slightly higher than Xiaowutaishan. Low genetic diversity might implied that the species historically had low female effective population size. Only 7 variable sites were identified in 713 bp fragment of the target sequence from 20 individuals of the captive population. Both haplotype diversity (h) and nucleotide diversity (π) were very low with 0.611 for h and 0.00369 forπ. Three haplotypes were defined based on the seven variable sites, suggesting that only three female Brown-Eared pheasants of the founders genetically contributed to the captive population. The three haplotypes showed distinct allelic frequency bias with haplotype h13 being the dominant allele and h4 being the rare one, implying that the three genetic contribution founders had different genetic contribution to the population.2. The subdivision estimator Fst in the AMOVA analysis indicated that the Xiaowutaishan subpopulation was significantly differentiated from Luyashan and Pangquangou subpopulations (Fst=0.13008, p<0.001, Fst=0.40979,p<0.001,respectively), suggesting that Xiaowutaishan subpopulation separated from the other two subpopulations along matrilineage. While the samples from Luyashan and Pangquangou subpopulations had not differentiation from each other (Fst=-0.01003 , p=0.46847), indicating that female-mediated gene transfer might be frequent between these two subpopulations in the past. Both phylogenetic trees and haplotype network indicated that the mtDNA haplotypes of the Brown-Eared pheasant were split into two well divergent clades (CladeⅠand CladeⅡ). mtDNA haplotypes of the wild population have not evolved into reciprocally monophylogenetic groups.3. 28 microsatellite primer pairs were tested in Brown-Eared pheasant, of which, 70% could give PCR product. 6 most polymorphic loci were chosen in this study. The lowest value of PIC (polymorphic information content) of the 6 loci is 0.635 with average value of 0.736, which is much higher than 0.5. For the wild population (n=43), a total of 44 distinct alleles were observed at six loci over the complete data set, of which 5 were private alleles. The average of alleles per locus was 7.33, and the average expected heterozygosity across 6 loci was 0.773. So the microsatellite variation observed indicated that in genetic diversity of Brown-Eared pheasant in nuclear DNA was rather higher level, which contrasted a rather lower mtDNA diversity revealed by mtDNA d-loop sequence variation depicted above, which showed that 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 Brown-Eared pheasant might be explained in terms of small female effective population size of the species historically, which suggested that the Brown-Eared pheasant is a very young species. A total of 28 alleles were recorded and no private allele was found in the captive population (n=20). For the captive population, the mean number of alleles per locus was 4.57 and the expected heterozygosity across 6 loci was 0.39. Both values were lower than that of wild population. Furthermore, the population also showed distinct bias in allele frequency of nucleotide gene and inbreeding efficient of the population was 17.3%. All these parameters showed that the population began to show characterizes of genetic deterioration for small population.4. AMOVA supported that Xiaowutaishan subpopulation significantly differentiated from Pangquangou subpopulation, but not significantly from Luyashan subpopulation. Combined with the result of analysis from mtDNA control region sequence variations, we considered that the extant Brown-Eared pheasant population was genetically structured into two parts, i.e., Hebei Xiwaowutaishan population/Shanxi Lvliang population. Therefore, we suggested that the current Brown-Eared pheasant should be taken as two different management unit (MU). For captive population, due to the small founder number with unclearing pedigree in breeding process and lack of scientific management, it had shown characterizes of genetic deterioration for small population. In order to enable the genetic diversity of the captive population to be maintained long time, we proposed the well pedigree of this population should be established as soon as possible.