Discussion

Genetic health assessment of populations
The protection of species genetic diversity has always been the core of species protection (Frankham, 2005). The evaluation of the genetic diversity within the protected species can allow conservators to predict the probability of population extinction or survival when under stress and to provide an theoretical basis for the effective conservation of population. In this study, the genetic diversity of Mabian (Ho = 0.6324, He = 0.5773), Meigu (Ho = 0.598, He = 0.502) and Heizhugou (Ho = 0.466, He = 0.555) populations were lower than the diversity of Wolong wild population (Ho = 0.644, He = 0.684) and Shaanxi captive population (Ho = 0.610, He = 0.593) (Huang, 2015), but higher than the diversity of the wild Qinling Mountains population (Ho = 0.451, He = 0.439) (Ji, 2014). The D-loop region of mitochondrial DNA (mtDNA) is characterized by high base replacement rate (Yu et al., 2004), which is suitable for analyzing the genetic characteristics of a population. Haplotype diversity (h) and nucleotide diversity (π) are two important indicators to measure the level of population genetic variation. We found that mean h and π values from the three reserves were significantly lower than Qionglai, Qinling and Minshan wild populations, and also lower than Wolong, Chengdu and Shaanxi captive populations, only higher than that of Daxiangling and Xiaoxiangling wild populations (Table 5). Genetic diversity analysis based on microsatellite markers and mitochondrial control region sequences showed that the genetic diversity level of giant pandas in three Liangshan mountains populations was at a medium-low level, and the presences of rare alleles and inbreeding may further reduce their genetic diversity levels. These results show it is necessary to introduce new genetic resource into the three populations or enhance gene exchange between the three populations and/or other populations.
Serious genetic imbalance may lead to the loss of genetic diversity and population decline (Kvist et al., 2015). The Hardy-Weinberg equilibrium is often used as an assessment of genetic balance within a population (Guo et al., 1992). The Hardy-Weinberg equilibrium test results showed that four of the seven microsatellite loci in the Mabian population deviated from the Hardy-Weinberg equilibrium (P < 0.01), while three deviated in the Meigu population and two deviated from the Hardy-Weinberg equilibrium in the Heizhuguo population. Almost all loci that deviated from the Hardy-Weinberg equilibrium showed significant heterozygote deficiencies and significant inbreeding. Inbreeding may be the main cause of deviations from the Hardy-Weinberg equilibrium. Our results showed that the three giant panda populations, especially Mabian, are genetically unbalanced and there is the risk of further loss of genetic diversity.
Fecal samples were most frequently collected in roughly two geographical clusters. Feces that were frequently found in Mabian reserve were far away from these collection sites of Feces in Meigu and Heizhugou reserves. This geographical clustering was reflected in genetic structural units and differentiation of the three giant panda populations. The giant pandas in three reserves were clearly divided into two genetic structural units. The Meigu and Heizhugou populations formed a genetic structural unit, while the Mabian population was a relatively independent genetic structural unit (Figure 5), indicating limited gene exchange between Mabian and two other populations. Further support for observed clustering was the high genetic differentiation of Mabian population (Fst: 0.13320, 0.15880) with the other two populations. The genetic clustering also confirms that the geographical clusters were likely indications of higher panda activity and not an affect of sampling method.
The genetic and geographical clustering of the three populations suggests that there is a barrier preventing genetic exchange between the two areas. Feng (2015) found that suitable habitats were fragmented in central and northern Mabian Nature Reserve. Unsuitable habitats might be caused by deforestation, road construction and livestock invasion (Feng, 2015; Zhao et al., 2017; Zhang et al., 2018). Fragmented suitable habitats and unsuitable habitats could influence the habitat selection and migration of giant panda. These unsuitable habitats are mainly concentrated in the western margin and northern sections of the Mabian Nature Reserve (Feng,2015) and this resulted in giant panda have moved southward. This change might has occurred between the 3rd (1999-2002) and 4th (2011-2014) national panda surveys because the distribution of giant pandas in Mabian moved southward at 4th national panda surveys compared to 3rd surveys (State Forestry Administration, 2006; Sichuan Forestry Department, 2015). This increased geographically distance and potentially barrier effect between Mabian population and other two populations formed the genetic isolation of Mabian population from other two populations.
Conclusively, the level of genetic diversity of three giant panda populations was medium to low, while the genetic diversity of Mabian giant pandas was the lowest. The existence of genetic isolation, a high number of rare alleles, inbreeding and significant deviations from the Hardy-Weinberg equilibrium indicated that these three populations were genetically unstable, and inbreeding may further result in the loss of genetic resources (Wang, 2019).
Genetic management recommendations
Liangshan Mountains is one of the main distribution areas of living giant pandas and belongs to the southernmost distribution of giant pandas. Mabian, Meigu and Heizhugou reserves are located in the heartland of Liangshan Mountains, and are also the core distribution areas of giant pandas in the Liangshan Mountains. The effective protection of the three giant panda populations is crucial for the conservation of all Giant pandas in Liangshan Mountains. The results of our have shown that the three giant panda populations are at risk of decline or extinction given stochastic events, especially the Mabian population. It is therefore urgent to improve each population’s genetic status by increasing genetic resources. We recommend two strategies for improving the genetic status of three populations. Firstly, improve genetic diversity of three populations by the introduction of genetically distinct individuals. The China Conservation and Research Center for the Giant Panda and the Chengdu Research Base of the Giant Panda have the largest captive breeding populations of giant pandas in China. These captive populations are genetically stable and distantly related to populations from the Liangshan Mountains (Shan et al., 2014). Therefore, genetic introductions from the two captive breeding populations would increase genetic resources into these core populations of Liangshan Mountains giant panda. However, captive-bred introductions are difficult and require considerable resources and time (Yang et al., 2018), and therefore it should not be the only strategy for the improvement of genetic health.
Our second recommendation for improving the genetic status of the three populations is to increase connectivity and genetic exchange between the two geographically and genetically distinct panda groups. Although significant genetic differentiation between the two groups exists, no significant difference in behavior and morphology has been found. Similarly, there was no evidence that the Mabian population was subject to different geographical or climatic conditions and thus no unique or local adaptation. Therefore, there should be no genetic, behavioral or morphological impediment to breeding and risk of distant hybridization (Frankham, 2010). The fecal sample distribution and population genetics demonstrated there was limited genetic exchange between Mabian and two other populations. However, there is no topographical barrier between the two groups, and the limiting factor is likely from unsuitable habitat and habitat fragmentation due to disturbance and lack of bamboo vegetation (Feng, 2015; Zhao et al., 2017; Zhang et al., 2018). Consequently, we recommend that suitable habitat and continuity should be rehabilitated and restored. Recent roads should be reforested and prevented from new construction. Human activities, especially grazing and bamboo shoot collection, should be controlled and minimized. Existing natural forest (bamboo) should be protected from further damage and the non-bamboo areas should be rehabilitated. As a priority, restoration should focus on creating corridors through the ‘habitat barrier’ to increase panda movement as soon as possible and then expand the area and proportion of suitable habitat. Given that pandas begin moving and they breed, there should be an improvement in the genetic health and population stability of giant panda in Liangshan Mountains.
Although Wei et al. (2020) concluded that China’s Panda Protection System and nature reserves can achieve the goals of protecting their habitats and biodiversity, and most giant panda nature reserves have been established based on the distribution of giant pandas, however, the gaps, overlapping designations and disparities in management still exist (Xu et al., 2017; Xu et al., 2019). The reserves in the Liangshan Mountains were established early in China’s panda protection efforts and zoning was determined roughly according to predicted panda distributions and human activities. However, many factors have changed over time, and pandas have become more flexible in their habitat choices than previously thought (Hull et al., 2014). For example, space utilization by giant pandas gradually expanded outward between the third and fourth surveys. In addition, we found that a large amount of panda activity occurred outside the reserve (Figure 2), indicating gaps in the coverage of the reserve. Although the Giant Panda National Park offers an opportunity to promote more effective management and improve the management system by integrating and expanding the existing reserves, however, Liangshan Mountains is not included in the newly established Giant Panda National Park (National Forestry and Grassland Administration (National Park Administration), 2019). In this case, greater attention should be paid to the protection of the main extant population of wild giant panda. We strongly suggested that the scope of nature reserves in the Liangshan Mountains should be adjusted, by integrating surrounding suitable habitats into the reserve, better protect giant panda habitats, restore degraded habitat, increase gene exchange between populations, and ensure the population stability of giant pandas in Liangshan Mountains.
In conclusion, giant panda populations in Liangshan Mountains had medium-low genetic diversity, with a high number of rare alleles, significant heterozygote deficiencies and inbreeding. Three populations clustered into two geographically and genetically distinct groupings, with the Mabian population being separated from the other two by a large tract of unsuitable habitat. The giant panda population in Liangshan Mountains is genetically unstable and at risk of decline or extinction given stochastic events. It is therefore recommended that connectivity between populations be re-established by improving habitat quality and continuity, and genetic health be enhanced by the introduction of captive-bred distantly related individuals. These changes could be incorporated into the updated conservation plans for the Liangshan Mountains. Our study revealed that high attention should be paid to the protection of these giant panda populations outside the Giant panda national Park, to ensure them survival in their distribution areas, and can serve as a reference for the genetic management of Giant panda populations in other distribution areas and some key conservation species in China and world.