Microsatellite Loci Analysis Reveals Post-bottleneck Recovery of Genetic Diversity in the Tibetan Antelope

The Tibetan antelope (chiru, Pantholops hodgsoni) is one of the most endangered mammals native to the Qinghai-Tibetan Plateau. The population size has rapidly declined over the last century due to illegal hunting and habitat damage. In the past 10 years, the population has reportedly been expanding due to conservation efforts. Several lines of evidence suggest that the Tibetan antelope has undergone a demographic bottleneck. However, the consequences of the bottleneck on genetic diversity and the post-bottleneck genetic recovery remain unknown. In this study, we investigate the genetic variation of 15 microsatellite loci from two Tibetan antelope populations sampled in 2003 (Pop2003) and 2013 (Pop2013). A higher level of genetic diversity (NA, 13.286; He, 0.840; PIC, 0.813; I, 2.114) was detected in Pop2013, compared to Pop2003 (NA, 12.929; He, 0.818; PIC, 0.789; I, 2.033). We observe that despite passing through the bottleneck, the Tibetan antelope retains high levels of genetic diversity. Furthermore, our results show significant or near significant increases in genetic diversity (He, PIC and I) in Pop2013 compared with Pop2003, which suggests that protection efforts did not arrive too late for the Tibetan antelope.

genetic consequences of intervention strategies remain unknown. Herein, we address this gap in knowledge by using 15 microsatellite loci to investigate temporal changes in the genetic diversity of Tibetan antelope during an 11-year period. The aim of this study was to examine the trends in this change and to what extent the populations have been restored at the genomic level. Our results provide guidance for future conservation and management strategies.

Materials and Methods
Materials. Skin tissue samples were obtained from Qinghai Forest Bureau in 2003, which confiscated Tibetan antelopes that were poached in the Hoh Xil National Nature Reserves (hereafter referred to as Pop2003, n = 47). Placental tissues were collected from the Zhuonaihu Lake area in Hoh Xil National Nature Reserve in 2013 (hereafter referred to as Pop2013, n = 47). All samples were washed three times with deionized water, sucked with 95% ethanol and stored at − 80 °C. All necessary approvals for collection and experimentation were acquired for the described field study from the Forestry Department of Qinghai Province, China. All procedures were in accordance with the guidelines of the regulations of experiments on animals and were approved by the China Zoological Society.
Microsatellite loci and primers. A total of 15 polymorphic microsatellite loci were screened. Seven loci (locus P1, locus P9, locus P17, locus P24, locus P113, locus P154 and locus P160) were obtained using the FIASCO method 8 , and another 8 loci (locus P6, locus P63, locus P67, locus P73, locus P75, locus P78, locus P90 and locus P96) were searched and developed from the Tibetan antelope genome using Perl script and MISA software (http://pgrc.ipk-gatersleben.de/misa/misa.html) for extracting the microsatellite DNA sequences from genome DNA of chiru (http://www.ncbi.nlm.nih.gov/Traces/wgs/?val = AGTT01#contigs). The forward microsatellite primers were labeled by FAM and synthesized by Sangon Biotech (Shanghai). The primers specific for 15 microsatellite loci are shown in Table 1. DNA extraction. Genomic DNA was extracted using the standard SDS-Phenol method 9 with minor modifications. The concentration and purity of genomic DNA were measured by Nano 2000C, and template DNA was diluted to 50 ng/μ L. All loci were amplified in each of the 47 samples of Pop2003 and Pop2013. The PCR amplification was conducted in a 20-μ L reaction mixture, which contained 0.1 mmol/L dNTPs, 0.2 μ mol/L each Statistical analysis of genetic diversity parameters. Original data were analyzed and manually corrected to validate the accurate peak shape and allele size using GeneMarker V1.75. Micro-Checker was used to validate the availability of genotype data 10 . The transformations for genotype data formats were conducted by Convert V1.3.1 11 for subsequent analysis. Linkage disequilibrium (LD) test was analyzed by Genepop V4.4 12,13 . Number of Alleles (NA) and Shannon index (I) were calculated by Microsatellite Analyzer V4.05 14 . We calculated observed heterozygosity (Ho), expected heterozygosity (He) and analyzed the Hardy-Weinberg equilibrium (HWE) by implementing Arlequin V3.5 15 . Polymorphism information content (PIC) was estimated by modified PowerStats Worksheet 16 . BOTTLENECK V1.2.02 17,18 was used to validate whether the population had undergone the bottleneck effect. Under a two-phase model (TPM), we constrained the model by defining 90% of mutations as conforming to a stepwise mutation model and 10% as multi-step. Furthermore, the change in Ne was estimated under the infinite allele model according to the formula Θ (theta) = 4Ne × Mu, where Ne is the effective population size and Mu is the microsatellite mutation ratio.

Results
Hardy-Weinberg equilibrium. For  Discussions Genetic diversity. We examined 15 microsatellite loci in this study to assess the genetic variation in the Tibetan antelope. Our data show rich genetic diversity in Pop2003 and Pop2013 with high values in MNA, He and PIC. Although the results are not directly comparable because different microsatellite loci were used, comparisons of the Tibetan antelope to other Bovidae species suggest that genetic diversity in the Tibetan antelope ranks highest (Table 5), with values of 12.929/13.286 and 0.818/0.840 for MNA and He in Pop2003 and Pop2013, respectively. Compared to the domestic yak, which is herded in the QTP and the adjacent Asian highlands with a population of more than 14 million, the Tibetan antelope exhibits higher genetic variation (Table 5). These results are in agreement with a previous genetic analysis 20 and our previous study 21 , in which significant heterogeneity in the frequencies of mtDNA control region haplotypes was observed.
Bottleneck signature detection. Bottleneck detection is critical for interpreting the historical demography of populations and is informative for establishing conservation strategies for endangered animals.
Simulations inferred from mtDNA D-loop fragment show that the Tibetan antelope experienced a severe historical demographic decline since approximately five thousand years before present (B.P.) 21 . Although the Wilcoxon test detected no significant recent population bottleneck signature in the Tibetan antelope under the TPM and SMM via BOTTLENECK in the present study, a recent well-documented decline in the population size of the Tibetan antelope has occurred over the last century, with population size decreasing from a maximum of approximately 500,000-1,000,000 to a minimum of 50,000 2,3 . Heavy illegal poaching associated with a profitable fur trade could account for this severe demographic reduction. The bottleneck was not detected using the Wilcoxon test for heterozygous excess probably because the number of loci analyzed was small 17,18 or due to an insufficient sample size 17 .
Slow post-bottleneck recovery of genetic diversity. Demographic bottlenecks can result in a loss of genetic variation [22][23][24][25][26] due to the bottleneck effect and subsequent genetic drift, as has already been observed in African elephants 27 , black-footed ferrets 28 and Arctic foxes 29 . Rather than rapid genetic loss, the results presented   Table 5. Summary of genetic diversity parameters of microsatellite data of several Bovidae species.
here suggest a slow post-bottleneck recovery of genetic diversity (in terms of both allele numbers and heterozygosity) in the Pop2013 population in comparison to the Pop2003 population, with values from 12.800 to 13.133 and from 0.821 to 0.841 for MNA and He, respectively. Studies have indicated that factors such as dispersive capabilities [30][31][32][33][34] and effective population size 35-37 may affect the change in genetic variation. High dispersal potential due to migration of females in most populations each year to summer calving grounds 38,39 is assumed to promote frequent gene flow. Substantial gene flow was detected in our earlier investigation by examining mtDNA fragments 21 . Therefore, recovery of genetic variation via gene flow is expected, especially within the large populations. Moreover, starting in the 1990s, the Chinese government established seven Nature Reserves and constructed corridors for facilitating the migration of Tibetan antelope, both of which have likely facilitated gene flow among populations and reduced genetic loss in post-bottleneck populations.
We conclude that ample genetic diversity may still exist in the Tibetan antelope. Furthermore, the Tibetan antelope has shown a slight increase in genetic variation during the past 11-year period. In this sense, the results of the present study suggest that protection efforts did not arrive too late for the Tibetan antelope and provide molecular evidence for the effectiveness of conservation strategies.