In this study, we investigated the variability of microsatellite loci to understand how different factors of genetic diversification such as isolation by distance, isolation by geographical barriers could affect the genetic diversity and population structure of Siberian roe deer in Northern Asia. Our study is based on samples from extensive geographic areas of Northern Asia, from Ural Mountains to the Korean Peninsula and Jeju Island, covering most of the species’ range to clarify the genetic relationships among populations from different geographical locations. Autosomal nuclear markers of microsatellites were employed to investigate the levels of genetic variation and genetic structuring of Siberian roe deer populations.
Genetic diversity of Siberian roe deer
Relative comparison of genetic diversity estimates among other roe deer species/populations would be informative to understanding of the present genetic status of Siberian roe deer. Although different sets of microsatellite loci were employed, apart from populations in Jeju Island, South Korea (SKJ), most of Siberian roe deer populations revealed moderate levels of genetic diversity (H
E = 0.522 to 0.628), compared to those previously reported for European roe deer. Microsatellite diversity of European roe deer ranged from 0.17 to 0.79 in several locations from Italy, Britain and northern Germany (H
E = 0.17 to 0.58 [11], H
E = 0.59 to 0.62 [18], and H
E = 0.74 to 0.79 [25], respectively). However, because the different sets of microsatellites were employed in diversity estimates and this may cause an inherent ascertainment bias that can vary among primer pairs, especially in different species, it should be interpreted with caution.
During the 20th century, many of the local Siberian roe deer populations were significantly abated as a result of human interference [26-30]. However, present data on the genetic diversity of Siberian roe deer suggests that the historical population reduction was transient, and its effects on the genetic diversity of the populations were insignificant. Result of bottleneck test also supported the lack of evidence for bottleneck event, except in the Jeju Island population (See below), indicating general stability of Siberian roe deer populations in continental Asia.
Different measures of microsatellite variability are consistently high in populations from East and Central Asia compared to West Siberia (Table 1). One reasonable assumption is that areas to the south and east of Siberia have function as refugia for roe deer during glacial periods. Several vertebrate species were also reported to have high levels of mitochondrial DNA variations in eastern Russia compared with those of surrounding areas [31]. Combination of cold open steppes with forested areas in south and east of Siberia may have resulted in highly diverse faunas [32], which could provide preservation and diversification of genetic lineages. However, phylogeographic and archaeological inference with additional samples from different geographical regions, using various marker systems, such as mtDNA and nuclear genes, should be implemented to precisely determine the role of this region as refugia.
Roe deer from Jeju Island, South Korea (SKJ) showed the lowest level of genetic diversity among Siberian roe deer that were sampled in this study. This presumably is due to the geographic isolation and historical population fluctuations on Jeju Island. Roe deer inhabited in Jeju Island during the last glacial maximum (LGM) when there was a bridge between the island and the Korean peninsula. It is probable that a relatively small group of animals was founded in the island after the last glacial periods, which led to reduced genetic diversity due to processes such as founder effect and genetic drift. Human interference, such as excessive hunting and poaching, could be another possible cause of the genetic deprivation in Jeju population. The roe deer population in Jeju gradually declined to near extinction in the early 1970s because of continuous hunting and poaching [33]. Since the 1980s, Jeju Special Self-Governing Province and Jeju citizens has been active in conservation for roe deer such as providing food during winter, removing traps, and clamping down on poaching [34, 35]. Consequently, the roe deer population in Jeju increased to 5,000 individuals in 1992 and climbed to 12,881 individuals in 2009 [33]. The effect of recent fluctuations of roe deer population in Jeju Island on its genetic diversity is supported by the Bottleneck tests (Table 4). Therefore, continuous monitoring of genetic diversity would be essential for effective management and conservation of Siberian roe deer in Jeju Island.
Genetic structure and gene flow
Present studies of genetic structure and differentiation among Siberian roe deer populations clearly display the existence of genetically distinct three clusters which comprise of the southeastern group (SKM, RPR, RSO and MGN), northwestern group (RUL, RKU and RNO) and Jeju Island population in Korea (SKJ). Such pattern of genetic structure is well in accordance with distribution of the two subspecies, C. p. pygargus and C. p. tianschanicus, suggested by previous study [36]. Recently, mitochondrial DNA sequence and nuclear IRBP (Interphotoreceptor retinoid binding protein) data has been presented that Jeju Island population to another subspecies, C. p. ochracea [37]. The genetic makeups of the two populations (RYA and RAL) are indicative of admixture of the two groups (southeastern and northwestern groups); however, a small sample size limits ultimate defining of their genetic status.
A previous study [2] proposed three major factors that may limit the geographical distribution of Siberian roe deer. The first factor is geographical barriers consisting of major mountain ridges (Altai, Sayans and Stanovoye) and the Lake Baikal (Fig. 1), which also delineate geographical ranges of two subspecies (C. p. pygargus and C. p. tianschanicus). The second factor is the depth of snow and duration of the snowy period [2, 38, 39] and last factor is the predominant vegetation type of the region, such as taiga, tundra, and desert [2]. These three factors and their interaction presumably limited further spread of roe deer, but probably first factor is the most important for the formation of genetic groups or subspecies. The other possible reason of it is that the mountain ridges could serve as refugia during periods of climate change (e. g. during the glacial maximums). In the periods of climatic optimums different genetic lineages could spread from the mountains in different areas resulting in formation of genetically different groups, possibly subspecies. However, this assumption need to additional phylogenetic studies will be required.
Barrier analysis that detected change genetic composition was also support limited gene flow in the major mountain ridges (Fig. 5). Southeastern group (SKM, RPR, RSO and MGN) and Northwestern group (RUL, RKU and RNO) supported relatively high frequency and fallowed by genetically admixed two populations (RYA and RAL) in the border areas. Besides, results of the Isolation by distance (IBD) (Fig. 6b) displayed that about 38 % of the genetic variation is explained by geographical distances between locations over the entire continent of Asia, which fits the hierarchical island model, suggesting modern genetic structure resulted from natural processes [2, 10, 40, 41]. Additionally, different pattern of distribution in the IBD scatter plot between and within groups (southeastern and northwestern groups) ascertains the effect of mountains ridges on the restricted level of gene flow between groups. Thus, mountain ridges of the southern Siberia have limited gene flow between Southeastern (SKM, RPR, RSO and MGN) and Northwestern (RUL, RKU and RNO) groups, leading to current genetic structure.
It should be noted that the Altay population (RAL) is located in the border area of two subspecies and shows the admixed pattern of two genetic clusters. This population is genetically related to both groups (Southeastern and Northwestern) and likely has historical and ongoing gene flow with adjacent locations (Fig. 1). A previous study of mitochondrial DNA [42] proposed that roe deer in Altai Mountain might experience multiple population replacements, stressing the role of the Altai Mountain as a physical boundary separating C. p. pygargus and C. p. tianschaniscus. This speculation is based on the genetic heterogeneity of Siberian roe deer in the Altai Mountains, and relatively stable climatic conditions of the region compared to other Siberian regions during the Pleistocene [42]. However, to resolve the question of border area, additional population genetic studies with more samples from areas at a finer geographic scale will be required.
Roe deer population in Yakutia, Russia (RYA), were established as a result of natural radiation from the southern parts of geographical range and could originate from both C. p. pygargus and C. p. tianschaniscus [43]. This assumption complies with the genetic structure of the Yakutian population obtained in this study and is also confirmed by the previous studies using morphology and karyotype [44, 45].
Roe deer from Jeju Island, South Korea (SKJ) are genetically divergent from all other Siberian roe deer, including those on the Korean mainland. The Jeju Island population was isolated from the mainland population since LGM, and as a result, there has been no gene flow between these two locations. Thus, the present genetic feature of the Jeju Island population was derived as a consequence of long-term geographical isolation and adaptation to island environment. Cases where Jeju island populations showing unique genetic and/or morphological features was also described for other mammal species such as wild boar (Sus scrofa), striped field mouse (Apodemus agrarius chejuensis) and Siberian weasel (Mustela sibirica) [46]. Future studies of this isolated population would contribute to understanding the effect of peripheral isolation on microevolution in Cervidae.
Our results do not coincide with the recent phylogeographic findings [19] that demonstrated no apparent geographical structuring for Siberian roe deer sampled from vast geographic areas of Eurasia. Variability of mtDNA control region suggested that the Siberian roe deer in Asia has undergone genetic admixture and appears to show no apparent geographic barriers to gene flow [19]. This difference could be due to the sensitivity of molecular markers and disparate interpretation owing to insufficient sample size and different modes of inheritance. The microsatellites are highly polymorphic and autosomal nuclear markers with biparental inheritance, and are more appropriate to delineate genetic structure of recently diverged populations.
Management and conservation Implications
Overall, this study suggests that at least three distinct management units may exist for the Siberian roe deer populations in Asia [47]: Northwest genetic group (RUL, RKU and RNO, partially corresponding to C. p. pygargus subspecies), southeast genetic group (SKM, RPR, RSO and MGN, corresponding to C. p. tianschanicus) and Jeju Island genetic group. Future planning of management and/or conservation policies, including ex situ population breeding, translocation and reintroduction programs, need to consider the distinctiveness of the three genetic groups in the Siberian roe deer species. Strict application of management unit concept for the two admixed populations (RYA and RAL) might be relaxed, or postponed until more detailed studies focusing on these populations are performed.
The roe deer population in Jeju Island, Korea (SKJ) needs special attention due to its low level of genetic diversity compared to those of continental populations. The Jeju Island population seems to be thriving at the present time, despite the low level of heterozygosity. The current size of the Jeju roe deer population is estimated to be around 12,881 [33] and considered to be over-populated in the island. However, considering the deprived level of genetic diversity, it is probable that the Jeju population might be vulnerable to epidemic diseases or any change of environment in the future. Therefore, it is recommended that both the genetic and physical health statuses of the population are closely monitored. Artificial translocation of roe deer individuals from the mainland Korea to Jeju Island to increase genetic diversity of Jeju population is not recommended because these two populations are genetically highly differentiated and should be regarded as separate management units.
Herbivorous animals such as roe deer play an important role in the ecosystem, providing a prey for large carnivores. Therefore, proper genetic management of Siberian roe deer populations and continuous monitoring of its genetic status is critical for maintaining healthy ecosystem. It is important to stress that systematic cooperation between countries where Siberian roe deer inhabit (Russia, Kazakhstan, Mongolia, China, North Korea and South Korea) is imperative for effective maintenance of genetic diversity and gene flow of Siberian roe deer. In particular, cooperative management of border area is important not only for the roe deer itself but also for a number of endangered large carnivore species.
For example, Siberian roe deer is one of the main prey animal of Amur leopard (Panthera pardus orientalis) in the border area among Russia, China and North Korea [48, 49]. Thus maintaining healthy roe deer population in this transboundary region is crucial for the survival of Amur leopard, which is one of the most severely endangered subspecies of large Felidae species in the world [49–53]. The status of the Siberian roe deer population in North Korea remains unknown and the gene flow has been discontinued along the Demilitarized Zone (DMZ) of North and South Korean border for more than five decades. This situation would have negative impacts on the long-term persistence of the Siberian roe deer in Korean peninsula and the restoration efforts of Amur leopard and tiger populations in this region. Siberian roe deer also serve as an important prey species for other carnivores like Amur tigers, gray wolves, lynxes, dholes, bears, as well as foxes, martens, eagles and wild boars [51, 54]. Thus, proper management of roe deer populations in northern Asian continent will also benefit many other species, and eventually, the biodiversity of the entire region.