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Genetic variation plays a significant role in maintaining the evolutionary potential of a species. Comparing the patterns of adaptive and neutral diversity in extant populations is useful for understanding the local adaptations of a species. In this study, we determined the fine-scale genetic structure of 6 extant populations of the giant panda (Ailuropoda melanoleuca) using mtDNA and DNA fingerprints, and then overlaid adaptive variations in 6 functional Aime-MHC class II genes (DRA, DRB3, DQA1, DQA2, DQB1, and DQB2) on this framework. We found that: (1) analysis of the mtDNA and DNA fingerprint-based networks of the 6 populations identified the independent evolutionary histories of the 2 panda subspecies; (2) the basal (ancestral) branches of the fingerprint-based Sichuan-derived network all originated from the smallest Xiaoxiangling (XXL) population, suggesting the status of a glacial refuge in XXL; (3) the MHC variations among the tested populations showed that the XXL population exhibited extraordinary high levels of MHC diversity in allelic richness, which is consistent with the diversity characteristics of a glacial refuge; (4) the phylogenetic tree showed that the basal clades of giant panda DQB sequences were all occupied by XXL-specific sequences, providing evidence for the ancestor-resembling traits of XXL. Finally, we found that the giant panda had many more DQ alleles than DR alleles (33∶13), contrary to other mammals, and that the XXL refuge showed special characteristics in the DQB loci, with 7 DQB members of 9 XXL-unique alleles. Thus, this study identified XXL as a glacial refuge, specifically harboring the most number of primitive DQB alleles. [ABSTRACT FROM AUTHOR]

The Père David's deer is a highly inbred, but recovered, species, making it interesting to consider their adaptive molecular evolution from an immunological perspective. Prior to this study, genomic sequencing was the only method for isolating all functional MHC genes within a certain species. Here, we report a novel protocol for isolating MHC class II loci from a species, and its use to investigate the adaptive evolution of this endangered deer at the level of multi-locus haplotypes. This protocol was designated "HURRAH" based on its various steps and used to estimate the total number of MHC class II loci. We confirmed the validity of this novel protocol in the giant panda and then used it to examine the Père David's deer. Our results revealed that the Père David's deer possesses nine MHC class II loci and therefore has more functional MHC class II loci than the eight genome-sequenced mammals for which full MHC data are currently available. This could potentially account at least in part for the strong survival ability of this species in the face of severe bottlenecking. The results from the HURRAH protocol also revealed that: (1) All of the identified MHC class II loci were monomorphic at their antigen-binding regions, although DRA was dimorphic at its cytoplasmic tail; and (2) these genes constituted two asymmetric functional MHC class II multi-locus haplotypes: DRA1*01 ∼ DRB1 ∼ DRB3 ∼ DQA1 ∼ DQB2 (H1) and DRA1*02 ∼ DRB2 ∼ DRB4 ∼ DQA2 ∼ DQB1 (H2). The latter finding indicates that the current members of the deer species have lost the powerful ancestral MHC class II haplotypes of nine or more loci, and have instead fixed two relatively weak haplotypes containing five genes. As a result, the Père David's deer are currently at risk for increased susceptibility to infectious pathogens.