bioRxiv preprint doi: https://doi.org/10.1101/068551; this version posted August 9, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Environmental DNA enables detection of terrestrial mammals from forest pond water Masayuki Ushio,1, 2 Hisato Fukuda,1 Toshiki Inoue,1 Kobayashi Makoto,3 Osamu Kishida,4 Keiichi Sato,5 Koichi Murata,6, 7 Masato Nikaido,8 Tetsuya Sado,9 Yukuto Sato,10 Masamichi Takeshita,11 Wataru Iwasaki,11 Hiroki Yamanaka,1, 12 Michio Kondoh,1 and Masaki Miya9 1Department of Environmental Solution Technology, Faculty of Science and Technology, Ryukoku University, 1-5 Yokotani, Seta Oe-cho, Otsu, Shiga 520-2194, Japan 2Joint Research Center for Science and Technology, Ryukoku University, Otsu, Shiga 520-2194, Japan 3Teshio Experimental Forest, Field Science Center for Northern Biosphere, Hokkaido University, 098-2943, Horonobe, Japan 4Tomakomai Experimental Forest, Field Science Center for Northern Biosphere, Hokkaido University, 053-0035, Japa 5Okinawa Churashima Research Center, Okinawa 905-0206, Japan 6College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan 7Yokohama Zoological Gardens ZOORASIA, 1175-1 Kamishiranecho Asahi-ku, Yokohama, Kanagawa 241-0001, Japan 8School of Life Science and Technology, Tokyo Institute of Technology, Tokyo 152-8550, Japan 9Department of Zoology, Natural History Museum and Institute, Chiba 260-8682, Japan 10Tohoku Medical Megabank Organization, Tohoku University, Miyagi 980-8573, Japan 11Department of Biological Sciences, The University of Tokyo, Tokyo 113-0032, Japan 12The Research Center for Satoyama Studies, Ryukoku University, 1-5 Yokotani, Seta Oe-cho, Otsu, Shiga 520-2194, Japan Terrestrial animals must have frequent contact with water to maintain their lives, implying that environmental DNA (eDNA) originating from terrestrial animals should be detectable from places containing water in terrestrial ecosystems. Aiming to detect the presence of terrestrial mammals using forest water samples, we applied a set of universal PCR primers (MiMammal, a modified version of fish universal primers) for metabarcoding mammalian eDNA. After verifying the primers’ usefulness in silico and using water samples from zoo cages of animals with known species composi- tions, we collected five 500-ml water samples from ponds in two cool-temperate forests in Hokkaido, northern Japan. Using eDNA extracted from the water samples, we constructed amplicon libraries using MiMammal primers for Illumina MiSeq sequencing. MiMammal metabarcoding yielded a to- tal of 75,214 reads, which we then subjected to data pre-processing and taxonomic assignment. We thereby detected species of mammals common to the sampling areas, including deer (Cervus nippon), mouse (Mus musculus), vole (Myodes rufocanus), raccoon (Procyon lotor), rat (Rattus norvegicus) and shrew (Sorex unguiculatus). Previous applications of the eDNA metabarcoding approach have mostly been limited to aquatic/semiaquatic systems, but the results presented here show that the approach is also promising even in forest mammal biodiversity surveys. Keywords: metabarcoding; forest; environmental DNA; mitogenome; mammal; community ecology I. INTRODUCTION non-fish vertebrates such as giant salamanders [6]. Those studies have shown that the use of eDNA can be a promis- ing approach as an efficient non-invasive monitoring tool Environmental DNA (eDNA) is a genetic material that for biodiversity in aquatic ecology. is found in an environment and derived from organisms living in that habitat, and researchers have recently been Although earlier studies used quantitative PCR and using eDNA to detect the presence of macro-organisms, species-specific primers to amplify a particular region particularly those living in an aquatic environment. In of eDNA [2–4, 6, 7], researchers have begun to ap- the case of macro-organisms, eDNA originates from var- ply massively parallel sequencing technology (e.g., Illu- ious sources such as metabolic waste, damaged tissue, mina MiSeq) and universal primer sets to eDNA stud- or sloughed skin cells [1], and the eDNA contains infor- ies (eDNA metabarcoding approach) [8, 9]. This ap- mation about the species identity of organisms that pro- proach greatly improved the efficiency and sensitivity of duced it. Ficetola et al. [2] first demonstrated the useful- eDNA detection. A previous study demonstrated that an ness of eDNA for detecting the presence of an aquatic ver- eDNA metabarcoding approach using fish-targeting uni- tebrate (American bullfrog invasive in France) in natural versal primers (MiFish primers) enabled the detection of wetlands. Subsequently, eDNA in aquatic ecosystems has more than 230 fish species from seawater in a single study been repeatedly used as a monitoring tool for the distri- [8]. Accordingly, the eDNA metabarcoding approach has butions of fish species in ponds, rivers and seawater [3–5] become a cost- and labor-effective approach for capturing as well as the distributions of other aquatic/semiaquatic aquatic biodiversity. bioRxiv preprint doi: https://doi.org/10.1101/068551; this version posted August 9, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 2 The use of eDNA is, however, not necessarily limited to in silico and using extracted DNA and eDNA from zoo aquatic/semiaquatic vertebrates, because terrestrial ani- cages (see Figs. S1-S2 and Tables S1-S3). mals also have frequent opportunities to contact aquatic systems. For example, Rodgers and Mock [7] tested the potential of drinking water for mammals as a medium B. Water sampling and DNA extraction that contains eDNA originating from terrestrial mam- mals. They hypothesized that when a terrestrial mam- We collected five water samples from four ponds in mal (coyote; Canis latrans) drinks from a water source, two cool-temperate forests in Hokkaido, northern Japan DNA from its saliva and mouth tissues is shed and can (Teshio Experimental Forest, 44◦54’55” N, 142◦1’21” be used as a tool for species identification. In their study, E; Tomakomai Experimental Forest, 42◦39’32” N, they successfully amplified a specific region of eDNA us- 141◦36’19” E) to examine the use of the primers for ing coyote-specific primers. That study demonstrated metabarcoding mammalian eDNA from natural forest that eDNA is a potentially useful tool even for detect- ponds with unknown mammal compositions in an open ing and monitoring the presence of mammals living in a ecosystem (Fig.1). All sampling and filtering equipment terrestrial ecosystem if one can collect appropriate media was washed with a bleach solution before use. Approxi- that contain mammalian DNA. Mammals living in a for- mately 500-ml water samples were collected from forest est ecosystem often contact/utilize water in a pond [10], ponds using polyethylene bottles (Fig.1). and thus, a forest pond may provide an opportunity for The water samples were transported to the laboratory. researchers to efficiently collect mammals’ eDNA. While they were transported, the samples were kept at In the present study, we hypothesized that, when mam- 4◦C ((for up to 48 hours before filtration) to prevent mals utilize/contact water, they shed their tissues into eDNA degradation. The water samples were filtered and the water as a source of eDNA, and that this eDNA can stored at -20◦C until eDNA extraction. DNA was ex- be used to detect the presence of mammals living in a for- tracted from the filters using a DNeasy Blood and Tissue est ecosystem. To improve the sensitivity and efficiency Kit (Qiagen, Hilden, Germany). Detailed information on of the eDNA approach, we modified the previously devel- the DNA extraction method is available in [8] and Sup- oped fish-targeting universal primers (MiFish primers) [8] plementary information. by accommodating them to mammal-specific variation, and conducted mammalian eDNA metabarcoding. The versatility of the primers was tested in silico, and their C. Paired-end library preparation, MiSeq accuracy was further tested by analyzing water samples sequencing and sequence data processing from zoo cages containing animals of known species com- position. Finally, we examined the effectiveness of the Because a preliminary experiment suggested that new primer set using water samples from field sites. MiMammal-U primers could not effectively amplify ele- phant or bear sequences, two complementary primer sets for elephants and bears were included in the PCR II. MATERIALS AND METHODS (MiMammal-E and -B). The mixed MiMammal-U/E/B primers (hereafter, MiMammal-mix), combined with A. New primer sets and test of versatility MiSeq sequencing primers and six random bases (N), was used for the multiplex PCR (see Table S4 for detailed in- Mitochondrial DNA (mtDNA) was chosen as the genetic formation). marker because the copy number per cell of mtDNA is The first PCR amplified the target region using the greater than that of nuclear DNA, and the detection rate MiMammal-mix primer set. The product of the first PCR is therefore expected to be higher when using the for- was then purified and used as a template for the second mer. Our primers were designed by modifying previously PCR. A second PCR was performed to append MiSeq developed MiFish primers [8], which corresponded to re- adaptors and dual index sequences to the first PCR prod- gions in the mitochondrial 12S rRNA gene, and we named ucts (Table S4). The indexed second PCR products were our primers MiMammal-U (forward primer = GGG TTG pooled and purified, and the DNA library was sequenced GTA AAT TTC GTG CCA GC; reverse primer = CAT on the MiSeq platform using a MiSeq v2 Reagent Kit for AGT GGG GTA TCT AAT CCC AGT TTG). In ad- 2 150 bp PE (Illumina, San Diego, CA, USA). dition, we also designed MiMammal-E (forward primer The preprocessing of the obtained sequence, OTU clus- = GGA CTG GTC AAT TTC GTG CCA GC; reverse tering and taxa assignments were performed using the primer = CAT AGT GAG GTA TCT AAT CTC AGT pipeline described in Miya et al.