The sacred deer conflict of management after a 1000-year history: Hunting in the name of conservation or loss of their genetic identity

The conservation of wildlife involves different stakeholders with different values and to resolve conflicts and develop solutions, management plans need to be based on scientific knowledge. In Nara city in Japan, wild sika deer (Cervus nippon) are considered sacred and have been protected for more than 1,000 years, giving them a unique genetic identity. However, when those sacred deer exited the sanctuary and began causing agricultural damage, there was debate as to whether they could be lethally treated as pests. Here, we used mitochondrial DNA (mtDNA) and nuclear simple sequence repeat (SSR) markers to detect the origin of deer in the management areas and verify the validity of the current zoning. As a result, two genetic clusters of deer were detected in Nara city. In the protected area, we detected only one specific mtDNA haplotype (S4). On the other hand, seven haplotypes, including S4, were detected in the management area. SSR analysis also suggested that the deer in the management area may be an admixed population of multiple origins. In the future, interbreeding populations may expand into sanctuaries, and unique genetic populations may disappear. Therefore, various stakeholders must promote discussion, including the need to conserve sacred deer in Nara.

Methods
Study area
Nara city (34°41ʹ N, 135°50ʹ E) is in the northern part of Nara Prefecture on central Honshu Island, Japanese archipelago (Fig. 2). The total area of the city is approximately 276.9 km2, and the total human population is about 353,000 (Nara city 2021). Nara Park (6.6 km2), located in the center of Nara city, contains historical buildings, such as Todaiji Temple and Kasuga Taisha Shrine, which are World Heritage Sites of the Ancient Capital of Nara, and the virgin forest of Mount Kasuga (UNESCO World Heritage Conservation). The park attracted approximately 17 million tourists in FY2019 (Nara city 2021). As mentioned above, Nara city is zoned according to the Japanese sika deer management plan. This plan has defined three areas: the protected area (districts A and B), the management area (district D), and the buffer area (district C) (Fig. 2, Table S1, Nara Prefecture 2019b; Nara Prefecture 2022a). 

Sample collection and genetic analysis
Muscle or fecal samples were collected from 167 deer between 2017 and 2019 at 9 sites in districts A, B, and D in Nara city (Fig. 2, Table S1). In District A, fecal samples were collected simultaneously at 2 sites (Todai-ji, NP1 and Tobihino, NP2) in Nara Park on September 6, 2018. In District B, fecal samples were collected at Nara University of Education (NUE) on December 29, 2018. One sample of one individual fecal pellet group was taken and genotyping further confirmed that there were no duplicates of individuals. After collection, fecal samples were immediately chilled on dry ice and stored at -30°C after delivery to the laboratory. In District D, muscle samples were collected from 6 sites (Ohyagyuu, OY; Tahara, TH; Youri, TU; Seika, SI; Semakawa, SM; and Yagyuu, YG) between 2017 and 2019. The samples from District D were collected from individuals captured in accordance with the sika deer management plan for Nara City (Nara Prefecture 2019a, 2022a). Deer was treated following the guidelines of the management plan and laws on hunting in Japan. The culling points of sika deer in district D may contain slight deviations due to the hunters' reporting system. See supplementary electronic material for the year of sampling and GPS data of the sampling point for each sample. In this study, we consider the nine sampling sites as sub-populations for our analysis. Muscle samples were stored in 70–90% ethanol at room temperature. DNA was extracted from feces using DNeasy Stool Mini Kit (Qiagen, Hilden, Germany), and from muscles using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany).

For sequence determination, a DNA fragment spanning the mtDNA control region (mtDNA-CR) was amplified by polymerase chain reaction (PCR) using the primer pair L15926 (Nagata et al. 1998) and H12S-112 (Sato et al. 2013). PCR was performed using Takara EX Taq HS (Takara Bio Inc., Kusatsu, Japan) and PCR protocols for mtDNA-CR described in previous studies (Takagi et al. 2020, 2023). After removing excess primers and dNTPs using ExoSAP-IT Express (Thermo Fisher Scientific Inc., MA, USA), PCR products were subjected to dye terminator cycle sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc., Waltham, USA). Seven sequencing primers (L15926, LD5, LD13, LD15, HD8, HD6, and H597) designed by the previous study (Nagata et al. 1998 and Nabata et al. 2004) were used to determine the mtDNA-CR haplotypes. DNA sequencing was performed using SeqStudio Genetic Analyzer (Thermo Fisher Scientific Inc., MA, USA).

To determine the multi-locus genotypes of nuclear SSR loci, we used 14 markers described in a previous study (Takagi et al. 2020): ABS12 (Pfister-Genskow et al. 1995); BL42, BMC1009, BM203, BM888, BM4107, BM6438, and RM095 (Bishop et al. 1994); BOVIRBP (Abernethy 1994); CSSM019 (Moore et al. 1994); ETH225 (Steffen et al. 1993); IDVGA29 (Mezzelani 1995); OarFCB193 (Buchanan and Crawford 1993); and TGLA127 (Georges and Massey 1992). PCR was performed using Takara EX Taq HS (Takara Bio Inc., Kusatsu, Japan) and PCR protocols for SSR described in previous studies (Takagi et al. 2020, 2023). The fragment size of PCR products and their genotypes were determined using SeqStudio Genetic Analyzer (Thermo Fisher Scientific Inc., Waltham, USA).