DOI: 10.5061/dryad.6wwpzgn3p
title: The sacred deer conflict of management after a 1000-year history: Hunting in the name of conservation or loss of their genetic identity

description: 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.
description: 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 km<sup>2</sup>), 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).
description: # The sacred deer conflict of management after a 1000-year history:
 Hunting in the name of conservation or loss of their genetic identity
 Author Information Toshihito Takagi Fukushima University, Faculty of
 Symbiotic Systems Science, Fukushima City, Fukushima, Japan.
 [takagitosihito@gmail.com](mailto:takagitosihito@gmail.com) Harumi Torii
 Center for Natural Environment Education, Nara University of Education,
 Nara City, Nara, Japan. Shingo Kaneko Fukushima University, Faculty of
 Symbiotic Systems Science, Fukushima City, Fukushima, Japan.
 [kaneko.shingo@gmail.com](mailto:kaneko.shingo@gmail.com) Hidetoshi B.
 Tamate Yamagata University, Faculty of Science, Yamagata City, Yamagata,
 Japan. Date of data collection: 2018-7-26 to 2023-3-31 ## File List:
 Supplemental_Info_NaraDeer.xls Sample ID: sample name Sampling Area: area
 name in the deer management plan in Nara city Sub-population:
 sub-population name Sampling Date: date of sampling GPS: sampling point
 data Distance from Nara Park: distance of sampling points from the centre
 of Nara Park (km) Haplotype: mitochondrial DNA haplotype excluding tandem
 repeats. Genbank accessions LC730667-C730801 Repetype: tandem repeat-only
 mitochondrial DNA haplotypes. Genbank accessions LC730667-C730801 Q1: Q1
 indicate the attribution rate to cluster1 from genetic clustering analysis
 using STRUCTURE Q2: Q2 indicate the attribution rate to cluster1 from
 genetic clustering analysis using STRUCTURE BM4107_1: allele 1 SSR marker
 BM4107 BM4107_2: allele 2 SSR marker BM4107 BOVIRBP_1: allele 1 SSR marker
 BOVIRBP BOVIRBP_2: allele 2 SSR marker BOVIRBP BM203_1: allele 1 SSR
 marker BM203 BM203_2: allele 2 SSR marker BM203 BM6438_1: allele 1 SSR
 marker BM6438 BM6438_2: allele 2 SSR marker BM6438 IDVGA29_1: allele 1 SSR
 marker IDVGA29 IDVGA29_2: allele 2 SSR marker IDVGA29 TGLA127_1: allele 1
 SSR marker TGLA127 TGLA127_2: allele 2 SSR marker TGLA127 BL42_1: allele 1
 SSR marker BL42 BL42_2: allele 2 SSR marker BL42 BMC1009_1: allele 1 SSR
 marker BMC1009 BMC1009_2: allele 2 SSR marker BMC1009 RME095_1: allele 1
 SSR marker RME095 RME095_2: allele 2 SSR marker RME095 CSSM019_1: allele 1
 SSR marker CSSM019 CSSM019_2: allele 2 SSR marker CSSM019 BM888_1: allele
 1 SSR marker BM888 BM888_2: allele 2 SSR marker BM888 ETH225_1: allele 1
 SSR marker ETH225 ETH225_2: allele 2 SSR marker ETH225 ABS12_1: allele 1
 SSR marker ABS12 ABS12_2: allele 2 SSR marker ABS12 OarFCB193_1: allele 1
 SSR marker OarFCB193 OarFCB193_2: allele 2 SSR marker OarFCB193