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.