Genetic Diversity of Large Japanese Field Mouse Apodemus speciosus Populations and Identification of their Food Plant Resources using DNA Barcoding in an Industrial Green Space

Estimates of the genetic diversity of Large Japanese field mouse Apodemus speciosus populations and identification of their plant food resources were conducted in an industrial green space, where were constructed on reclaimed land and belonged to the Aichi Refinery of Idemitsu Kosan Co., Ltd., in Aichi Prefecture, Japan. A total of six mitochondrial D-loop haplotypes were identified in 50 mice. Habitat condition with the highest number of captured individuals had abundant broad-leaved trees and understory vegetation. A minimum spanning network, which did not form a ring-shaped network, revealed that the hereditary population structure was weak. The low genetic diversity observed in the study area was thus attributed to isolation from other populations once the population in the study area by sea and road, which is more than 30 m wide. In order to identify which plant food resources were utilized by mice captured inside the industrial green space, partial chloroplast rbcL sequences were amplified by PCR from DNA extracted from 43 feces samples. Calculations of sample completeness curve revealed that 25 of the taxa identified in this study comprised approximately 90% of the food plant resources in the study area. Of the 21 plant families identified from the obtained rbcL sequences, members of the Rosaceae (28.0%), Fagaceae (17.2%), Lauraceae (14.2%) and Oleaceae (7.7%) were dominant. To ensure the continued survival of A. speciosus population in this industrial green space would be to preferentially conserve plant species that are used as food resources by this species. DOI : 10.14302/issn.2637-6075.jpae-18-2062 Corresponding author: Motoyasu Minami, Graduate School of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi, 487-8501, Japan, Fax number : +81-568-51-6297, E-mail : minami@isc.chubu.ac.jp. Running title: Apodemus speciosus in industrial green space


Introduction
In industrial areas, green spaces are primarily used as buffers for industrial pollution and for accident prevention.Since industrial green spaces also function as habitats for local wildlife, these areas are considered to contribute positively towards regional biodiversity conservation.Given this background, we previously surveyed the wildlife species that inhabit the industrial green space of the Aichi Refinery of Idemitsu Kosan Co., Ltd. in Aichi Prefecture, Japan 1 .This industrial green space comprises a very large forest zone connected to smaller industrial green spaces, which are the favored habitats of native medium-sized mammals, such as the Raccoon dog (Nyctereutes procyonoides), Japanese hare (Lepus brachyurus), and the Japanese red fox (Vulpes vulpes japonica) reported once local extinction in the southern part of the Aichi Prefecture, which are representative mammal species of Japan 1 .Considering that the native mammals of Japan are threatened by extensive habitat destruction, mainly due to urbanization, this industrial green space have contributed positively towards maintaining viable populations of mammals from this region.Since Idemitsu Kosan Co., Ltd. has never experienced any loss due to these mammals inhabiting its property, the company has promoted the utilization of this industrial green space for native medium-sized mammal conservation.
Of the small mammals in Japan, Large Japanese field mouse Apodemus speciosus is dominant in this industrial green space 1 .Consequently, in order to ensure the successful conservation of the carnivores in this industrial green space, it is important to understand the ecological status of the murids that are preyed upon by these predators.Maintaining genetic variation, the most basic level of biological diversity, is at the center of conserving species, populations, and ecosystems.This is because maintaining high levels of genetic diversity is crucial for ensuring the fitness and survival of a species and its ability to adapt to environmental change 2 .In addition, to increase the potential of this industrial green space for supporting populations of A. speciosus, it is important to understand their food resources, especially plants.However, the genetic diversity and diets of this murid species in the industrial green space have not yet been investigated in any detail.
We inferred the genetic diversity of A. speciosus populations by examining sequence variation in the mitochondrial D-loop region; the most variable mitochondrial DNA region 3 .In addition, we identified the plant food resources utilized by A. speciosus populations in this industrial green space by DNA barcoding of the chloroplast rbcL gene.To predict the ratio of the number of identified plant species to the number of plant species used for food in this industrial green space, a sample completeness curve was calculated for this species.
Consequently, our genetic approach proposed manage the population of A. speciosus in this industrial green space.

Trapping
The industrial green space of the study area were constructed on reclaimed land and belonged to the Aichi Refinery of Idemitsu Kosan Co., Ltd.(34°58'30"N, 136°50'28"E) (Fig. 1).Since this industrial area opened in the 1970s, this industrial green space has been separated from the surrounding area by Ise Bay to the west and the Chita industrial road, which is more than 30 m wide, to the east.The industrial green space is belt-shaped, with a total length of ca. 2 km and a width of ca. 100 m.Approximately 450,000 trees were planted when the refinery was built; the vegetation at the industrial green space currently consists of a mixture of typical evergreen broad-leaved forest and deciduous broad-leaved trees.In addition, bamboo groves and artificial grasslands have been established on part of the area.The footpaths (width ca. 2 to 5 m) for business activities and nature observation are maintained.
Trapping was conducted at approximately To detect DNA polymorphisms in the D-loop, the obtained DNA sequences were aligned using the MEGA 6.06 software package 4 .Calculation of haplotype diversity and construction of the minimum spanning network (MSN) was performed using ARLEQUIN ver.

Plant Food Resource Identification by DNA Barcoding
To identify plant-derived food resources, total plant DNA was isolated from the dried feces (5.9 to 60. (5'-TATCTTGGCAGCATTCCGAGTAACTCC -3') and R3 (5'-GATTCGCAGATCCTCCAGACGTAGAGC -3') 6 .PCR amplification was performed using a DNA Thermal Cycler (GeneAmp PCR System 9700, Applied Biosystems) using an initial denaturation step of 98°C for 2 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 15 s, and extension at 68°C for 20 s.
Each PCR product was then cloned into a plasmid (pGEM-T Easy Vector System I, Promega, WI), which was then transformed into JM109 competent cells (Toyobo, Japan), as described by the manufacturer.
After incubation, we selected a maximum of 30 colonies per Petri dish and checked for insertion of the PCR To identify food plants, homology searches were performed by comparing the DNA sequences obtained from the feces samples against all of the published sequences deposited in the DDBJ using the BLASTN program.To ensure that the accuracy of the identification was sufficiently robust, plant species detected only once and plant species with a homology of less than 98% were excluded after the homology search.In the event that a given sequence was identified as assigned to two or more taxa with the same score, then that sequence was assigned to the highest taxonomic level that included both of those taxa 6 .As a result, some of the obtained sequences were assigned to the rank of genus and others to family 6 .In order to predict the ratio of the number of identified plant food resources to the total number of plant food resources in this industrial green space, a sample completeness curve was calculated using iNEXT package 7,8 .

Number of Captured Individuals and Haplotype Diversity
The number of captured individuals and intraspecific DNA D-loop polymorphisms observed at each trapping site are summarized in Table 1.A total of 50 mice were caught at nine trapping sites.The trapping Co., Ltd. in Aichi Prefecture, Japan.Each haplotype name in the square is shown in Table 1.
The number of vertical lines on branches represents the number of different bases (polymorphisms) between haplotypes.ring-shaped and had a radial form (Fig. 2).

Plant Food Resources Identification
The BLASTN search results obtained for the food plant sequences are summarized in Table 2.A total of 46 feces samples could be collected from 50 captured Sample completeness curve results revealed that 25 plant taxa detected in this study covered approximately 90% of the plant food resources in this industrial green space (Fig. 3).

Discussion
The areas around sites ID-5 to ID-9 (including site ID-6 with the highest number of captured individuals) had abundant broad-leaved trees and understory vegetation.These sites also had large amounts of organic litter in the form of fallen leaves and branches from cladoptosis that covered the ground, which would be used by the mice to find food and cover

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month intervals from May 2012 to November 2014 using Sherman-type live traps (6.5 × 5.5 × 16.0 cm, H. B. Sherman Traps, FL) baited with a mixture of barley and walnuts (15:1 w/w).Nine trapping sites (sites ID-1 to 9) were a set at intervals of 200 to 320 m, and five traps were set at 10-m intervals on each side of the footpaths at each trapping site to give a total of ten traps at each trapping site.Immediately after capture, body hair for D-loop analysis, and feces for identifying food plants, were collected from the mice and traps, respectively.The trapped individuals were then released at the site of www.openaccesspub.orgJPAE CC-license DOI : 10.14302/issn.2637-6075.jpae-18-2062Vol-1 Issue 2 Pg.no.-3 capture.All body hairs (including hair roots) and feces samples were frozen at -20°C until total DNA was extracted.This study was conducted in full compliance with the guidelines for the treatment of animals proposed by the Mammal Society of Japan and a license for capturing Large Japanese field mice was obtained from the Aichi Prefectural government.Detection of DNA Polymorphisms Total DNA was isolated from body hair using a DNeasy Blood & Tissue Kit (Qiagen, Germany) and purified using a Geneclean Spin Kit (MP-Biomedicals, CA).PCR reactions were performed in reaction mixtures of 50 μl containing 1 unit of MightyAmp DNA Polymerase Ver. 2 (Takara, Japan) and 0.32 µM of each primer according to the manufacturer's instructions.using a DNA Thermal Cycler (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA) using an initial denaturation step of 98°C for 2 min, followed by 30 cycles of denaturation for 10 s at 98°C, annealing for 15 s at 60°C, and extension for 30 s at 68°C.All PCR products were purified using a QIAquick PCR Purification Kit (Qiagen) and subjected to dye-terminator cycle sequencing using DTCS Quick Start Mix (Beckman Coulter, CA) and an automatic sequencer (CEQ 2000XL, Beckman Coulter).

Fig. 1 :
Fig. 1: Map and aerial photograph of the industrial green space examined in this study: Aichi Refinery of Idemitsu Kosan Co., Ltd.Solid line and dotted line indicates the industrial green space and the Chita industrial road, respectively.Map data were extracted from Google, Digital Globe.

Table 1 . 5 Fig. 2 :
Fig. 2: Minimum spanning network of haplotypes based on the D-loop region of mitochondrial DNA of Apodemus speciosus in industrial green space of the Aichi Refinery of Idemitsu Kosan

Fig. 3 :
Fig. 3: Sample completeness curve for rarefied samples (solid line segment) and extrapolation (dotted line segments) sampling curves with 95% confidence intervals (shaded areas) for the food resource data of Apodemus speciosus in industrial green space of the Aichi Refinery of Idemitsu Kosan Co., Ltd. in Aichi Prefecture, Japan.

Table 2 .
Homology search of obtained for the rbcL region of chloroplast DNA (257 -263 bp) in food plants utilized by in Apodemus speciosus in the industrial green space of Aichi Refinery of Idemitsu Kosan Co., Ltd., In addition, Hap 3 to 6, which were captured from sites ID-1 to 4, were not captured at sites ID-5 to ID-9.It was therefore considered that migration between sites www.openaccesspub.orgJPAE CC-license DOI : 10.14302/issn.2637-6075.jpae-18-2062Vol-1 Issue 2 Pg.no.-7