- Email: [email protected]
Tropical-forest mammals as detected by environmental DNA at natural saltlicks in Borneo
Biological Conservation 210 (2017) 281–285
Contents lists available at ScienceDirect
Biological Conservation journal homepage: www.elsevier.com/locate/biocon
Short communication
Tropical-forest mammals as detected by environmental DNA at natural saltlicks in Borneo
MARK
Taichiro Ishigea, Masaki Miyab, Masayuki Ushioc,d, Tetsuya Sadob, Masaharu Ushiodae, Kaori Maebashie, Risako Yonechie, Peter Laganf, Hisashi Matsubayashie,⁎ a
NODAI Genome Research Center, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya, Tokyo, 156-8502, Japan Department of Ecology and Environmental Sciences, Natural History Museum and Institute, Chiba, 955-2, aoba-cho, Chuo, Chiba, 260-8682, Japan c Center for Ecological Research, Kyoto University, 2-509-3, Hirano, Otsu, Shiga 520-2113, Japan d PRESTO, Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama, 332-0012, Japan e Faculty of Agriculture, Tokyo University of Agriculture, 1737, Funako, Atsugi, Kanagawa, 243-0034, Japan f Sabah Forestry Department, Locked Bag 68, 90009, Sandakan, Sabah, Malaysia b
A R T I C L E I N F O
A B S T R A C T
Keywords: Borneo Endangered species Environmental DNA Natural saltlick NGS
Although tropical forests are among the most species-rich ecosystems on earth, 42% of mammal species in tropical forests are endangered because of overhunting and/or unsustainable exploitation. Camera-trap surveys have shown that natural saltlicks can be used to determine mammalian fauna, especially medium to large endangered species in tropical forests; establishment of camera traps, however, is time and effort intensive. Furthermore, the photographic range and detectable size of species are often restricted. Environmental DNA (eDNA) metabarcoding is a powerful approach that might provide a better way to study terrestrial animals in tropical forests. In this study, we examined whether eDNA from natural saltlicks comprehensively represented species composition in a Bornean tropical forest. We collected 100–150-mL water samples from natural saltlicks in Sabah, Malaysian Borneo. We constructed amplicon libraries for MiSeq sequencing using eDNA extracted from the water samples. Six endangered species were detected using this method, including Bornean orangutan (Pongo pygmaeus), Bornean banteng (Bos javanicus lowi), Asian elephant (Elephas maximus), Sunda pangolin (Manis javanica), sambar deer (Rusa unicolor) and bearded pig (Sus barbatus). However, most small and minor species were not detected, with low sequence identity (80–96%). Therefore, we propose that more species of tropical forest mammals should have their sequences deposited in DNA databases. This study is the first to report the endangered mammals of a tropical forest detected using eDNA from natural saltlicks.
1. Introduction Tropical forests are among the most species-rich ecosystems on earth (Wilkie et al., 2011). However, 42% of mammal species in tropical forests are endangered because of overhunting and/or unsustainable exploitation (Wilkie et al., 2011). The first step in wildlife and habitat conservation is a detailed inventory. To survey mammalian fauna in tropical forests, camera traps (infrared sensor cameras) have been used to observe mammals on the forest floor that would otherwise be difficult to observe among dense understorey vegetation. The usefulness of camera traps for surveying mammals in tropical forests is demonstrated at natural saltlicks (Ampeng et al., 2016; Blake et al., 2013; Loken et al., 2013; Matsubayashi et al., 2007a, 2011; Matsuda et al., 2015). These studies showed that natural saltlicks are a stable species-rich site that are used by endangered species, such as Bornean
⁎
Corresponding author. E-mail address: [email protected] (H. Matsubayashi).
http://dx.doi.org/10.1016/j.biocon.2017.04.023 Received 8 February 2017; Received in revised form 19 April 2017; Accepted 21 April 2017 0006-3207/ © 2017 Elsevier Ltd. All rights reserved.
orangutans (Pongo pygmaeus), and are important physiological and social sites for animals. However, camera traps take time and cannot detect all species at natural saltlicks because of the restricted photographic range and size of species (i.e., mammals with small body size cannot be detected by automated camera traps). Environmental DNA (eDNA) is genetic material that is derived from the habitat of an organism. Thomsen and Willerslev (2015) suggested that eDNA approaches does offer some great advantages (standardisation, non-invasiveness, sensitivity, cost-effectiveness and independence of weather conditions) over traditional methods in biodiversity monitoring. Although earlier studies used quantitative PCR and speciesspecific primers to amplify a particular region of eDNA, researchers have begun to apply massively parallel sequencing technology and universal primer sets to eDNA studies (the eDNA metabarcoding approach). Previous eDNA metabarcoding approaches using fish-tar-
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All equipment was sterilized, filtered pipet tips were used, and preand post-PCR methods were conducted separately to avoid crosscontamination. We also used negative controls to monitor contamination during the procedures. The first PCR was conducted using a 12-μL reaction volume containing 6-μL 2 × KAPA HiFi HotStart Ready Mix (KAPA Biosystems, Wilmington, WA, USA), 0.7-μL of each primer (5 μM) (Table A1), 2.6-μL sterilized H2O, and 2-μL template DNA. When the first PCR was multiplexed (i.e., when it was treated using MiMammal-Mix), the final concentration of each primer (MiMammalU/E/B) (Table A1) was 0.3 μM (0.9 μM total concentration of primers). The PCR program used was as follows: 95 °C initial denaturation for 3 min, followed by 35 cycles of denaturation at 98 °C for 20 s, annealing at 65 °C for 15 s, extension at 72 °C for 15 s; and a final extension at 72 °C for 5 min. We performed each first-PCR in four replicates, and the replicates were pooled to mitigate PCR dropouts. PCR products were then purified by using AMPure XP beads solution (× 0.8) (Beckman Coulter, Brea, CA, USA) and 10-fold diluted first-PCR products were used as templates for the index PCR. The index PCR was conducted using a 24-μL reaction volume containing 12-μL × KAPA HiFi HotStart Ready Mix, 1.4-μL of each primer (5 μM) (Table A1), 7.2-μL sterilized H2O, and 2-μL first-PCR product. The PCR program used was as follows: 95 °C initial denaturation for 3 min, followed by 12 cycles of denaturation at 98 °C for 20 s, annealing and extension at 72 °C for 15 s; and a final extension at 72 °C for 5 min. The index PCR products were then purified using AMPure XP beads solution (×0.8). Twenty-μL of 2nd PCR products were added to the combined library for samples with higher concentrations of 2nd PCR products, while 10-μL of 2nd PCR products were added to the combined library for samples with lower concentrations of 2nd PCR products to normalize sequence reads per sample. The pooled library was size-selected from around 380 bp using 2% E-Gel Size Select agarose gel (Invitrogen, Carlsbad, CA, USA). The size-selected libraries were sequenced as paired-end, 150 bp reads using a MiSeq (Illumina, San Diego, CA, USA) following the manufacturer instructions. All sequence data associated with this project have been submitted to the DNA Data Bank of Japan Sequence Reads Archive (DRA) (accession number DRA005516). The overall quality of the MiSeq reads were evaluated, and the reads were assembled using the software FLASH with a minimum overlap of 10 bp (Magoč and Salzberg, 2011). The assembled reads were further filtered and cleaned, and the pre-processed reads were clustered and taxonomically assigned. The pre-processed reads from the above custom pipeline were dereplicated using UCLUST (Edgar, 2010). Those
geted universal primers (MiFish primers) enabled the detection of > 230 fish species from a single sample of seawater (Miya et al., 2015). Subsequently, Ushio et al. (2016) demonstrated the utility of eDNA in forest pond water for the detection of common terrestrial mammals in cool temperate forests. We hypothesized that species composition might be better reflected in frequently visited sites, such as natural saltlicks, as the frequency of visitation rates recorded by camera traps varies greatly among species, but only a few species dominate the habitat (Matsubayashi et al., 2007a). Furthermore, we expect that eDNA can greatly improve wildlife surveys in tropical forest ecosystems, which are species-rich, but where direct observation of wildlife is difficult. However, one concern is that eDNA might be rapidly degraded by enzyme activities (secreted by microorganisms) in the high temperatures and humidity of tropical forests. In this study, we examined whether eDNA comprehensively detected the composition of mammalian species at natural saltlicks in Bornean tropical forest.
2. Material and methods We collected 14 water samples on 22–25 August 2016 from 4 natural saltlicks (S1: 05°19′N 117°28′E; S2: 05°21′N 117°31′E; S3: 05°19′N 117°34′E; S4: 05°16′N 117°31′E) in Deramakot Forest Reserve, Sabah, Malaysian Borneo to examine mammalian eDNA using the metabarcoding approach (Fig. 1). As the water volume of natural saltlicks was small and scattered, samples were collected from several points per site. Approximately 100–150-mL water samples were collected from natural saltlicks using 50-mL sterile syringes (Thermo Co., Tokyo, Japan) and were filtered using 0.22-μm Sterivex filters cartridge (Millipore, MA, USA). Approximately 3-mL of RNAlater (Thermo Fisher Scientific, MA, USA) was injected into the cartridge, which was stored at 4 °C for 12 d, then kept at −20 °C until eDNA extraction. Environmental DNA was extracted from the cartridge using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). Before lysis, 20-μL proteinase-K solution, 220-μL PBS (phosphate buffered saline), and 200-μL AL buffer were mixed and added to the Sterivex filter. The Sterivex filter was placed on a rotary shaker in a pre-heated incubator at 56 °C and shaken at a speed of 20 rpm for 20 min. After incubation, the Sterivex filter was centrifuged at 5000g for 1 min to collect DNA. The collect DNA was purified using a DNeasy blood and tissue purification kit (Qiagen, GmbH, Hilden, Germany) following the manufacture protocols (Miya et al., 2016).
Fig. 1. Bornean orangutan was recorded at the natural saltlicks (S1) in Deramakot.
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283
0 0 0 0 0 160 14 174 0 0 0 0 0 0 8,883 8,883 0 0 0 0 0 57 86,291 86,348 0 0 0 0 0 64 10,354 10,418 0 0 0 0 0 0 62 62 0 0 588 0 0 0 67 655 0 170 273 0 0 0 6,093 6,536 0 0 770 0 27 582 95,069 96,448 6,655 0 0 0 28 7,310 95,009 109,002 12,639 0 0 0 88 1,442 150,929 165,098 8,376 0 0 0 57 8,465 150,907 167,805 4,114 0 0 0 60 375 180,238 184,787 Critically endangered Critically endangered Endangered Endangered Vulnerable Vulnerable Bornean orangutan (Pongo pygmaeus) Sunda pangolin (Manis javanica) Asian elephant (Elephas maximus) Bornean banteng (Bos javanicus lowi) Sambar deer (Rusa unicolor) Bearded pig (Sus barbatus) Other sequences Total sequences
17,668 0 0 0 36 1,838 121,726 141,268
Point2 Point1 Point2 Point1 Point2 Point1 Point1 Point2
Red list assessments Species (Scientific name)
Table 1 Sequence counts detected species from water samples in natural saltlicks, Deramakot.
Point3
Point4 Point1
MiSeq sequencing of the 14 samples from natural saltlicks generated 1,047,938 reads (Table 1), and the negative control generated 458 reads. There were no processed reads in the negative control. Six endangered species, Bornean orangutan (Pongo pygmaeus), Sunda pangolin (Manis javanica), Asian elephant (Elephas maximus), Bornean banteng (Bos javanicus lowi), sambar deer (Rusa unicolor) and bearded pig (Sus barbatus) were detected (Table 1). Sambar deer and bearded pig were the dominant species documented by camera traps at natural saltlicks in Deramakot (Matsubayashi et al., 2007a, 2007b, 2011) and eDNA only identified bearded pig at all the natural saltlicks. Bornean orangutans, Bornean banteng, and Asian elephants were also detected at all natural saltlicks with camera traps (Matsubayashi et al., 2007a, 2011). About Bornean orangutans, the highest number was recorded at S1 with camera traps and also eDNA. As these three species are highly endangered (Table 1), efficiently detecting their key habitat areas is very important for their conservation. Sunda pangolin was detected at S2 both with camera traps and eDNA. However, its frequency in the camera trap was very low. The eDNA was highly efficient in that it could detect the pangolin with only a single sample. These results showed that eDNA metabarcoding was an efficient approach to detect tropical forest mammals from natural saltlick water. However, there were two problems: (1) Misidentification of mammalian species due to highly similar sequences, (2) lack of mtDNA sequences (in NCBI) of rare mammalian species. First, sambar deer (Rusa unicolor), Javan deer (Rusa timorensis), white-lipped deer (Cervus albirostris), sika deer (Cervus nippon) and red deer (Cervus elaphus) were detected by this study and the similarity between target region of these species are > 97% (Fig. A1, Table A2). Since these species were not distinguishable by eDNA, reads assigned to these species were judged to be derived from sambar deer, only do habitat in Borneo (Timmins et al., 2015). Thus, at present, a combination of eDNA and reference data based on traditional survey methods is needed for accurate interpretation. Second, 12S ribosomal RNA (rRNA) sequences of rare and most rainforest species in Borneo are not deposited in the NCBI database. For example, the Sunda clouded
S1 (25-August 2016)
3. Results and discussion
S1 (22-August 2016)
S2 (23-August 2016)
Point3
S3 (23-August 2016)
Point3
S4 (24-August 2016)
Point3
sequences represented by > 10 identical reads were subjected to the downstream analyses, and the remaining under-represented sequences (those with < 10 identical reads) were subjected to pairwise alignment using UCLUST. If the latter sequences (observed from < 10 reads) showed at least 99% identity with one of the former reads (i.e., no > 1 or 2 nucleotide differences), they were operationally considered as identical (owing to sequencing or PCR errors and/or actual nucleotide variations in the populations). The processed reads were subjected to local BLASTN searches against a custom-made database (Camacho et al., 2009). The custommade database was generated by downloading all mitogenome sequences from Sarcopterygii deposited in NCBI Organelle Genome Resources (http://www.ncbi.nlm.nih.gov/genomes/Organelle Resource.cgi?taxid=8287). As of 15 March 2016, the database covered 1881 species across a wide range of families and genera (Ushio et al., 2016). The top BLAST hit with a sequence identity of at least 80% and E-value threshold of 10− 5 was applied to assign species to each representative sequence. The species assignments of each representative sequence were subjected to local BLASTN searches against the NCBI (nt) nucleotide database (ftp://ftp.ncbi.nih.gov/blast/db/25-102016). The top BLAST hit with a sequence identity of at least 97% and E-value threshold of 10− 5 was used to assign a species to each representative sequence, and a sequence identity of 80–96% was treated as an ‘other sequence’ that did not match a known reference. The reads that matched non-terrestrial or extinct animals were considered contamination and were discarded, and reads matching animals not found in Borneo according to the IUCN red list were treated as ‘other sequences’ in this study. We follow the nomenclature and the conservation status of IUCN Red List of Threatened Species (2016).
0 0 51,451 742 0 217 18,044 70,454
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18,044 14 8,883 67
62
10,354 6,093 95,069
86,291
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0
0 0 0 0 0 0 0 0 0
2,228 0 0
0 18,044 0 0 0 0 0 0 14 0 0 0 0 0 0 8,883 0 0 0 0 0 0 29 0 0 0 38 0
0 62 0 0 0 0 0
0 10,354 0 0 0 0 0 0 1,791 0 0 0 4,302 0 0 91,697 33 0 3,311 28 0
Although these species inhabit in Borneo, the values of species identity were low. This means the original sampling region might be different.
In this study, we demonstrated that eDNA can be used to detect mammals visiting natural saltlicks in tropical forests. Compared with traditional survey methods, such as direct visual census and automated camera methods, the eDNA method is inexpensive and can be conducted over a shorter timescale. Traditional survey methods relied on observing each species, and individual researcher experience, while the eDNA method relies on nucleotide sequences, which are universally conserved, resulting in using same analysis pipeline obtain same result. However, the eDNA method has some drawbacks, such as classification of related species and lacks of genetic information in species assignment databases. Finally, this study is the first to apply the eDNA metabarcoding approach to natural saltlicks in tropical forests; we propose that this approach is an effective tool for snapshot measurements of mammal biodiversity at natural saltlicks in tropical forests. Acknowledgements This research conducted a collaborative research with Sabah Forestry Department and supported by the Japan Society for the Promotion of Science (JSPS) Core-to-Core Program, A. Advanced Research Networks (Wildlife Research Centre of Kyoto University), the Ministry of Education, Culture, Sports, Science and Technology (MEXT; S1311017) and the Japan Science and Technology Agency (JST) CREST Program (JPMJCR13A2). We are grateful to the individuals who have provided the opportunities for this study. From Sabah Forestry Department: Datuk Sam Mannan, Director of Sabah Forestry Department, Jaimi Mujit, Azny Ahmad, Edward Thomas, Johny Kissing, all staff of the Deramakot District Forestry Office. We also thank Hiroki Yamanaka at Department of Environmental Solution Technology/The Research Center for Satoyama Research in Ryukoku University for providing an opportunity to use Illumina MiSeq platform. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.biocon.2017.04.023. References
a
95,009 150,929 180,238 121,726
150,907
0 0 0 0 0 583 0 20 4,306 95–96 89 89–95 Vulnerable Lower Risk Least Concern
0 0 0
0 0 109
12 94,966 31 0 0 0 0 762 149,304 54 226 0 0 0 14 175,848 50 0 0 0 0 99–100 99 99 95 90–91 90–91 98 Vulnerable Vulnerable Least concern Least concern Vulnerable Least concern Least concern
Javan deer (Rusa timorensis) White-lipped deer (Cervus albirostris) Sika deer (Cervus nippon) Red deer (Cervus elaphus) Clouded leopard (Neofelis nebulosa) Masked palm civeta (Paguma larvata) Hill long-tongued fruit bat (Macroglossus sobrinus) Ear-spot squirrela (Callosciurus adamsi) Red-cheeked squirrel (Dremomys rufigenis) Red and white giant flying squirrel (Petaurista alborufus) Total sequences
0 121,649 58 0 0 0 19
0 150,718 60 20 0 0 0
Point3 Point2 Point1
Point3
Point4
S1 (25-August 2016) Point1 S1 (22-August 2016) Red list assessments
vs Detected speciese identity (%)
leopard (Neofelis diardi) occurs in Borneo (Wilting et al., 2007, 2011); it is a vulnerable species but has no 12S rRNA sequence deposited in NCBI. However, the clouded leopard (Neofelis nebulosa) in China is a close relative of the Sunda clouded leopard, and while it does not occur in Borneo, it has a complete mtDNA sequence deposited in NCBI. Analyses of molecular (Buckley-Beason et al., 2006; Wilting et al., 2007) and morphological data (Christiansen, 2008; Kitchener et al., 2006) demonstrated that Bornean and Sumatran clouded leopards are clearly distinct from those on the continental mainland (Wilting et al., 2011). Given that the eDNA reads assigned to the clouded leopard from China had low similarity (90–91%) (Table 2), these reads may have been derived from the Sunda clouded leopard; however, given that they could not be confirmed using the NCBI database, we judged these reads as ‘other sequences’. On the other hand, the hill long-tongued fruit bat (Macroglossus sobrinus) was detected with high sequence identity (98%) by using eDNA (Table 2) and does not occur in Borneo. Thomsen and Willerslev (2015) suggested that identification of eDNA sequences depends crucially on reliable reference DNA-sequence database, then future DNA databases focus on complete mitochondrial for much wider applications than traditional DNA barcoding. Therefore, we propose that more sequences from more species of tropical forest mammals should be identified and deposite in the NCBI database. 4. Conclusion
Species (Scientific name)
Table 2 Detected other sequences from the water samples in natural saltlicks, Deramakot.
0 84,030 33 0 0 0 0
Point3 Point2 Point1 Point3 Point1 Point2 Point1
Point2
S3 (23-August 2016) S2 (23-August 2016)
S4 (24-August 2016)
T. Ishige et al.
Ampeng, A., Shukor, M.N., Sahibin, A.R., Idris, W.M.R., Ahmad, S., Mohammad, H.,
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Matsubayashi, H., Ahmad, A.H., Wakamatsu, N., Nakazono, E., Takyu, M., Majalap, N., Lagan, P., Sukor, J.R.A., 2011. Natural-licks use by orangutans and conservation of their habitats in Bornean tropical production forest. Raffles Bull. Zool. 59, 109–115. Matsuda, I., Ancrenaz, M., Akiyama, Y., Tuuga, A., Majalap, N., Bernaed, H., 2015. Natural licks are required for large terrestrial mammals in degraded riparian forest, Sabah, Borneo, Malaysia. Ecol. Res. 30, 191–195. Miya, M., Sato, Y., Fukunaga, T., Sado, T., Poulsen, J.Y., Sato, K., Minamoto, T., Yamamoto, S., Yamanaka, H., Araki, H., Kondoh, M., Iwasaki, W., 2015. MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species. R. Soc. Open Science 2, 150088. Miya, M., Minamoto, T., Yamanaka, H., Oka, S., Sato, K., Yamamoto, S., Sado, T., Doi, H., 2016. Use of a filter cartridge for filtration of water samples and extraction of environmental DNA. J. Vis. Exp. 117, e54741. Thomsen, P.F., Willerslev, E., 2015. Environmental DNA–an emerging tool in conservation for monitoring past and present biodiversity. Biol. Conserv. 183, 4–18. Timmins, R., Kawanishi, K., Giman, B., Lynam, A., Chan, B., Steinmetz, R., Sagar Baral, H., Samba Kumar, N., 2015. Rusa unicolor. (Errata Version Published in 2015) The IUCN Red List of Threatened Species 2015: e.T41790A85628124. (Downloaded on 28 January 2017). Ushio, M., Fukuda, H., Inoue, T., Makoto, K., Kishida, O., Sato, K., Murata, K., Nikaido, M., Sado, T., Sato, Y., Takeshita, M., Iwasaki, W., Yamanaka, H., Kondoh, M., Miya, M., 2016. Environmental DNA enables detection of terrestrial mammals from forest pond water. bioRxiv. pp. 068551. Wilkie, D.S., Bennett, E.L., Peres, C.A., Cummimgham, A.A., 2011. The empty forest revisited. Ann. N. Y. Acad. Sci. 1223, 120–128. Wilting, A., Buckley-Beason, V.A., Feldhaar, H., Gadau, J., O'brien, S.J., Linsenmair, K.E., 2007. Clouded leopard phylogeny revisited: support for species recognition and population division between Borneo and Sumatra. Front. Zool. 4, 15. Wilting, A., Christiansen, P., Kitchener, A.C., Kemp, Y.J., Ambu, L., Fickel, J., 2011. Geographical variation in and evolutionary history of the Sunda clouded leopard (Neofelis diardi) (Mammalia: Carnivora: Felidae) with the description of a new subspecies from Borneo. Mol. Phylogenet. Evol. 58, 317–328.
Madeline, G.P., Ali, N., Bujamg, M., Hashim, I., Bujamg, A., Md-Zain, B.M., 2016. Patterns of mineral lick use by northwest Bornean orangutans (Pongo pygmaeus) in the Lanjak Entimau wildlife sanctuary, Sarawak, Malaysia. Eur. J. Wildl. Res. 62, 147–150. Blake, J.K., Mosquera, D., Salvador, J., 2013. Use of mineral licks by mammals and birds in hunted and non-hunted areas of Yasuní National Park, Ecuador. Anim. Conserv. 16, 420–437. Buckley-Beason, V.A., Johnson, W.E., Nash, W.G., Stanyon, R., Menninger, J.C., Driscoll, C.A., Stone, G., Martelli, P.P., Wen, C., Ling, L., Duraisingam, R.K., Lam, P.V., O'Brien, S.J., 2006. Molecular evidence for species-level distinctions in clouded leopards. Curr. Biol. 16, 2371–2376. Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., Madden, T.L., 2009. BLAST +: architecture and applications. BMC Bioinf. 10, 421. Christiansen, P., 2008. Species distinction and evolutionary differences in the clouded leopard (Neofelis nebulosa) and Diard's clouded leopard (Neofelis diardi). J. Mammal. 89, 1435–1446. Edgar, R.C., 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461. IUCN, 2016. The IUCN Red List of Threatened Species. http://www.iucnredlist.org/ technical-documents/red-list-documents (visited October 25, 2016). Kitchener, A.C., Beaumont, M.A., Richardson, D., 2006. Geographical variation in the clouded leopard, Neofelis nebulosa, reveals two species. Curr. Biol. 16, 2377–2383. Loken, B., Spehar, S., Rayadin, Y., 2013. Terrestriality in the Bornean orangutan (Pongo pygmaeus morio) and implications for their ecology and conservation. Am. J. Primatol. 75, 1129–1138. Magoč, T., Salzberg, S.L., 2011. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27, 2957–2963. Matsubayashi, H., Lagan, P., Majalap, N., Tangah, J., Sukor, J.R.A., Kitayama, K., 2007a. Importance of natural licks for the mammals in Bornean inland tropical rain forests. Ecol. Res. 22, 742–748. Matsubayashi, H., Lagan, P., Sukor, J.R.A., Kitayama, K., 2007b. Seasonal and daily use of natural licks by sambar deer (Cervus unicolor) in a Bornean tropical rain forest. Tropics 17, 81–86.
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Contents lists available at ScienceDirect
Biological Conservation journal homepage: www.elsevier.com/locate/biocon
Short communication
Tropical-forest mammals as detected by environmental DNA at natural saltlicks in Borneo
MARK
Taichiro Ishigea, Masaki Miyab, Masayuki Ushioc,d, Tetsuya Sadob, Masaharu Ushiodae, Kaori Maebashie, Risako Yonechie, Peter Laganf, Hisashi Matsubayashie,⁎ a
NODAI Genome Research Center, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya, Tokyo, 156-8502, Japan Department of Ecology and Environmental Sciences, Natural History Museum and Institute, Chiba, 955-2, aoba-cho, Chuo, Chiba, 260-8682, Japan c Center for Ecological Research, Kyoto University, 2-509-3, Hirano, Otsu, Shiga 520-2113, Japan d PRESTO, Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama, 332-0012, Japan e Faculty of Agriculture, Tokyo University of Agriculture, 1737, Funako, Atsugi, Kanagawa, 243-0034, Japan f Sabah Forestry Department, Locked Bag 68, 90009, Sandakan, Sabah, Malaysia b
A R T I C L E I N F O
A B S T R A C T
Keywords: Borneo Endangered species Environmental DNA Natural saltlick NGS
Although tropical forests are among the most species-rich ecosystems on earth, 42% of mammal species in tropical forests are endangered because of overhunting and/or unsustainable exploitation. Camera-trap surveys have shown that natural saltlicks can be used to determine mammalian fauna, especially medium to large endangered species in tropical forests; establishment of camera traps, however, is time and effort intensive. Furthermore, the photographic range and detectable size of species are often restricted. Environmental DNA (eDNA) metabarcoding is a powerful approach that might provide a better way to study terrestrial animals in tropical forests. In this study, we examined whether eDNA from natural saltlicks comprehensively represented species composition in a Bornean tropical forest. We collected 100–150-mL water samples from natural saltlicks in Sabah, Malaysian Borneo. We constructed amplicon libraries for MiSeq sequencing using eDNA extracted from the water samples. Six endangered species were detected using this method, including Bornean orangutan (Pongo pygmaeus), Bornean banteng (Bos javanicus lowi), Asian elephant (Elephas maximus), Sunda pangolin (Manis javanica), sambar deer (Rusa unicolor) and bearded pig (Sus barbatus). However, most small and minor species were not detected, with low sequence identity (80–96%). Therefore, we propose that more species of tropical forest mammals should have their sequences deposited in DNA databases. This study is the first to report the endangered mammals of a tropical forest detected using eDNA from natural saltlicks.
1. Introduction Tropical forests are among the most species-rich ecosystems on earth (Wilkie et al., 2011). However, 42% of mammal species in tropical forests are endangered because of overhunting and/or unsustainable exploitation (Wilkie et al., 2011). The first step in wildlife and habitat conservation is a detailed inventory. To survey mammalian fauna in tropical forests, camera traps (infrared sensor cameras) have been used to observe mammals on the forest floor that would otherwise be difficult to observe among dense understorey vegetation. The usefulness of camera traps for surveying mammals in tropical forests is demonstrated at natural saltlicks (Ampeng et al., 2016; Blake et al., 2013; Loken et al., 2013; Matsubayashi et al., 2007a, 2011; Matsuda et al., 2015). These studies showed that natural saltlicks are a stable species-rich site that are used by endangered species, such as Bornean
⁎
Corresponding author. E-mail address: [email protected] (H. Matsubayashi).
http://dx.doi.org/10.1016/j.biocon.2017.04.023 Received 8 February 2017; Received in revised form 19 April 2017; Accepted 21 April 2017 0006-3207/ © 2017 Elsevier Ltd. All rights reserved.
orangutans (Pongo pygmaeus), and are important physiological and social sites for animals. However, camera traps take time and cannot detect all species at natural saltlicks because of the restricted photographic range and size of species (i.e., mammals with small body size cannot be detected by automated camera traps). Environmental DNA (eDNA) is genetic material that is derived from the habitat of an organism. Thomsen and Willerslev (2015) suggested that eDNA approaches does offer some great advantages (standardisation, non-invasiveness, sensitivity, cost-effectiveness and independence of weather conditions) over traditional methods in biodiversity monitoring. Although earlier studies used quantitative PCR and speciesspecific primers to amplify a particular region of eDNA, researchers have begun to apply massively parallel sequencing technology and universal primer sets to eDNA studies (the eDNA metabarcoding approach). Previous eDNA metabarcoding approaches using fish-tar-
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All equipment was sterilized, filtered pipet tips were used, and preand post-PCR methods were conducted separately to avoid crosscontamination. We also used negative controls to monitor contamination during the procedures. The first PCR was conducted using a 12-μL reaction volume containing 6-μL 2 × KAPA HiFi HotStart Ready Mix (KAPA Biosystems, Wilmington, WA, USA), 0.7-μL of each primer (5 μM) (Table A1), 2.6-μL sterilized H2O, and 2-μL template DNA. When the first PCR was multiplexed (i.e., when it was treated using MiMammal-Mix), the final concentration of each primer (MiMammalU/E/B) (Table A1) was 0.3 μM (0.9 μM total concentration of primers). The PCR program used was as follows: 95 °C initial denaturation for 3 min, followed by 35 cycles of denaturation at 98 °C for 20 s, annealing at 65 °C for 15 s, extension at 72 °C for 15 s; and a final extension at 72 °C for 5 min. We performed each first-PCR in four replicates, and the replicates were pooled to mitigate PCR dropouts. PCR products were then purified by using AMPure XP beads solution (× 0.8) (Beckman Coulter, Brea, CA, USA) and 10-fold diluted first-PCR products were used as templates for the index PCR. The index PCR was conducted using a 24-μL reaction volume containing 12-μL × KAPA HiFi HotStart Ready Mix, 1.4-μL of each primer (5 μM) (Table A1), 7.2-μL sterilized H2O, and 2-μL first-PCR product. The PCR program used was as follows: 95 °C initial denaturation for 3 min, followed by 12 cycles of denaturation at 98 °C for 20 s, annealing and extension at 72 °C for 15 s; and a final extension at 72 °C for 5 min. The index PCR products were then purified using AMPure XP beads solution (×0.8). Twenty-μL of 2nd PCR products were added to the combined library for samples with higher concentrations of 2nd PCR products, while 10-μL of 2nd PCR products were added to the combined library for samples with lower concentrations of 2nd PCR products to normalize sequence reads per sample. The pooled library was size-selected from around 380 bp using 2% E-Gel Size Select agarose gel (Invitrogen, Carlsbad, CA, USA). The size-selected libraries were sequenced as paired-end, 150 bp reads using a MiSeq (Illumina, San Diego, CA, USA) following the manufacturer instructions. All sequence data associated with this project have been submitted to the DNA Data Bank of Japan Sequence Reads Archive (DRA) (accession number DRA005516). The overall quality of the MiSeq reads were evaluated, and the reads were assembled using the software FLASH with a minimum overlap of 10 bp (Magoč and Salzberg, 2011). The assembled reads were further filtered and cleaned, and the pre-processed reads were clustered and taxonomically assigned. The pre-processed reads from the above custom pipeline were dereplicated using UCLUST (Edgar, 2010). Those
geted universal primers (MiFish primers) enabled the detection of > 230 fish species from a single sample of seawater (Miya et al., 2015). Subsequently, Ushio et al. (2016) demonstrated the utility of eDNA in forest pond water for the detection of common terrestrial mammals in cool temperate forests. We hypothesized that species composition might be better reflected in frequently visited sites, such as natural saltlicks, as the frequency of visitation rates recorded by camera traps varies greatly among species, but only a few species dominate the habitat (Matsubayashi et al., 2007a). Furthermore, we expect that eDNA can greatly improve wildlife surveys in tropical forest ecosystems, which are species-rich, but where direct observation of wildlife is difficult. However, one concern is that eDNA might be rapidly degraded by enzyme activities (secreted by microorganisms) in the high temperatures and humidity of tropical forests. In this study, we examined whether eDNA comprehensively detected the composition of mammalian species at natural saltlicks in Bornean tropical forest.
2. Material and methods We collected 14 water samples on 22–25 August 2016 from 4 natural saltlicks (S1: 05°19′N 117°28′E; S2: 05°21′N 117°31′E; S3: 05°19′N 117°34′E; S4: 05°16′N 117°31′E) in Deramakot Forest Reserve, Sabah, Malaysian Borneo to examine mammalian eDNA using the metabarcoding approach (Fig. 1). As the water volume of natural saltlicks was small and scattered, samples were collected from several points per site. Approximately 100–150-mL water samples were collected from natural saltlicks using 50-mL sterile syringes (Thermo Co., Tokyo, Japan) and were filtered using 0.22-μm Sterivex filters cartridge (Millipore, MA, USA). Approximately 3-mL of RNAlater (Thermo Fisher Scientific, MA, USA) was injected into the cartridge, which was stored at 4 °C for 12 d, then kept at −20 °C until eDNA extraction. Environmental DNA was extracted from the cartridge using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). Before lysis, 20-μL proteinase-K solution, 220-μL PBS (phosphate buffered saline), and 200-μL AL buffer were mixed and added to the Sterivex filter. The Sterivex filter was placed on a rotary shaker in a pre-heated incubator at 56 °C and shaken at a speed of 20 rpm for 20 min. After incubation, the Sterivex filter was centrifuged at 5000g for 1 min to collect DNA. The collect DNA was purified using a DNeasy blood and tissue purification kit (Qiagen, GmbH, Hilden, Germany) following the manufacture protocols (Miya et al., 2016).
Fig. 1. Bornean orangutan was recorded at the natural saltlicks (S1) in Deramakot.
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0 0 0 0 0 160 14 174 0 0 0 0 0 0 8,883 8,883 0 0 0 0 0 57 86,291 86,348 0 0 0 0 0 64 10,354 10,418 0 0 0 0 0 0 62 62 0 0 588 0 0 0 67 655 0 170 273 0 0 0 6,093 6,536 0 0 770 0 27 582 95,069 96,448 6,655 0 0 0 28 7,310 95,009 109,002 12,639 0 0 0 88 1,442 150,929 165,098 8,376 0 0 0 57 8,465 150,907 167,805 4,114 0 0 0 60 375 180,238 184,787 Critically endangered Critically endangered Endangered Endangered Vulnerable Vulnerable Bornean orangutan (Pongo pygmaeus) Sunda pangolin (Manis javanica) Asian elephant (Elephas maximus) Bornean banteng (Bos javanicus lowi) Sambar deer (Rusa unicolor) Bearded pig (Sus barbatus) Other sequences Total sequences
17,668 0 0 0 36 1,838 121,726 141,268
Point2 Point1 Point2 Point1 Point2 Point1 Point1 Point2
Red list assessments Species (Scientific name)
Table 1 Sequence counts detected species from water samples in natural saltlicks, Deramakot.
Point3
Point4 Point1
MiSeq sequencing of the 14 samples from natural saltlicks generated 1,047,938 reads (Table 1), and the negative control generated 458 reads. There were no processed reads in the negative control. Six endangered species, Bornean orangutan (Pongo pygmaeus), Sunda pangolin (Manis javanica), Asian elephant (Elephas maximus), Bornean banteng (Bos javanicus lowi), sambar deer (Rusa unicolor) and bearded pig (Sus barbatus) were detected (Table 1). Sambar deer and bearded pig were the dominant species documented by camera traps at natural saltlicks in Deramakot (Matsubayashi et al., 2007a, 2007b, 2011) and eDNA only identified bearded pig at all the natural saltlicks. Bornean orangutans, Bornean banteng, and Asian elephants were also detected at all natural saltlicks with camera traps (Matsubayashi et al., 2007a, 2011). About Bornean orangutans, the highest number was recorded at S1 with camera traps and also eDNA. As these three species are highly endangered (Table 1), efficiently detecting their key habitat areas is very important for their conservation. Sunda pangolin was detected at S2 both with camera traps and eDNA. However, its frequency in the camera trap was very low. The eDNA was highly efficient in that it could detect the pangolin with only a single sample. These results showed that eDNA metabarcoding was an efficient approach to detect tropical forest mammals from natural saltlick water. However, there were two problems: (1) Misidentification of mammalian species due to highly similar sequences, (2) lack of mtDNA sequences (in NCBI) of rare mammalian species. First, sambar deer (Rusa unicolor), Javan deer (Rusa timorensis), white-lipped deer (Cervus albirostris), sika deer (Cervus nippon) and red deer (Cervus elaphus) were detected by this study and the similarity between target region of these species are > 97% (Fig. A1, Table A2). Since these species were not distinguishable by eDNA, reads assigned to these species were judged to be derived from sambar deer, only do habitat in Borneo (Timmins et al., 2015). Thus, at present, a combination of eDNA and reference data based on traditional survey methods is needed for accurate interpretation. Second, 12S ribosomal RNA (rRNA) sequences of rare and most rainforest species in Borneo are not deposited in the NCBI database. For example, the Sunda clouded
S1 (25-August 2016)
3. Results and discussion
S1 (22-August 2016)
S2 (23-August 2016)
Point3
S3 (23-August 2016)
Point3
S4 (24-August 2016)
Point3
sequences represented by > 10 identical reads were subjected to the downstream analyses, and the remaining under-represented sequences (those with < 10 identical reads) were subjected to pairwise alignment using UCLUST. If the latter sequences (observed from < 10 reads) showed at least 99% identity with one of the former reads (i.e., no > 1 or 2 nucleotide differences), they were operationally considered as identical (owing to sequencing or PCR errors and/or actual nucleotide variations in the populations). The processed reads were subjected to local BLASTN searches against a custom-made database (Camacho et al., 2009). The custommade database was generated by downloading all mitogenome sequences from Sarcopterygii deposited in NCBI Organelle Genome Resources (http://www.ncbi.nlm.nih.gov/genomes/Organelle Resource.cgi?taxid=8287). As of 15 March 2016, the database covered 1881 species across a wide range of families and genera (Ushio et al., 2016). The top BLAST hit with a sequence identity of at least 80% and E-value threshold of 10− 5 was applied to assign species to each representative sequence. The species assignments of each representative sequence were subjected to local BLASTN searches against the NCBI (nt) nucleotide database (ftp://ftp.ncbi.nih.gov/blast/db/25-102016). The top BLAST hit with a sequence identity of at least 97% and E-value threshold of 10− 5 was used to assign a species to each representative sequence, and a sequence identity of 80–96% was treated as an ‘other sequence’ that did not match a known reference. The reads that matched non-terrestrial or extinct animals were considered contamination and were discarded, and reads matching animals not found in Borneo according to the IUCN red list were treated as ‘other sequences’ in this study. We follow the nomenclature and the conservation status of IUCN Red List of Threatened Species (2016).
0 0 51,451 742 0 217 18,044 70,454
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18,044 14 8,883 67
62
10,354 6,093 95,069
86,291
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0
0 0 0 0 0 0 0 0 0
2,228 0 0
0 18,044 0 0 0 0 0 0 14 0 0 0 0 0 0 8,883 0 0 0 0 0 0 29 0 0 0 38 0
0 62 0 0 0 0 0
0 10,354 0 0 0 0 0 0 1,791 0 0 0 4,302 0 0 91,697 33 0 3,311 28 0
Although these species inhabit in Borneo, the values of species identity were low. This means the original sampling region might be different.
In this study, we demonstrated that eDNA can be used to detect mammals visiting natural saltlicks in tropical forests. Compared with traditional survey methods, such as direct visual census and automated camera methods, the eDNA method is inexpensive and can be conducted over a shorter timescale. Traditional survey methods relied on observing each species, and individual researcher experience, while the eDNA method relies on nucleotide sequences, which are universally conserved, resulting in using same analysis pipeline obtain same result. However, the eDNA method has some drawbacks, such as classification of related species and lacks of genetic information in species assignment databases. Finally, this study is the first to apply the eDNA metabarcoding approach to natural saltlicks in tropical forests; we propose that this approach is an effective tool for snapshot measurements of mammal biodiversity at natural saltlicks in tropical forests. Acknowledgements This research conducted a collaborative research with Sabah Forestry Department and supported by the Japan Society for the Promotion of Science (JSPS) Core-to-Core Program, A. Advanced Research Networks (Wildlife Research Centre of Kyoto University), the Ministry of Education, Culture, Sports, Science and Technology (MEXT; S1311017) and the Japan Science and Technology Agency (JST) CREST Program (JPMJCR13A2). We are grateful to the individuals who have provided the opportunities for this study. From Sabah Forestry Department: Datuk Sam Mannan, Director of Sabah Forestry Department, Jaimi Mujit, Azny Ahmad, Edward Thomas, Johny Kissing, all staff of the Deramakot District Forestry Office. We also thank Hiroki Yamanaka at Department of Environmental Solution Technology/The Research Center for Satoyama Research in Ryukoku University for providing an opportunity to use Illumina MiSeq platform. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.biocon.2017.04.023. References
a
95,009 150,929 180,238 121,726
150,907
0 0 0 0 0 583 0 20 4,306 95–96 89 89–95 Vulnerable Lower Risk Least Concern
0 0 0
0 0 109
12 94,966 31 0 0 0 0 762 149,304 54 226 0 0 0 14 175,848 50 0 0 0 0 99–100 99 99 95 90–91 90–91 98 Vulnerable Vulnerable Least concern Least concern Vulnerable Least concern Least concern
Javan deer (Rusa timorensis) White-lipped deer (Cervus albirostris) Sika deer (Cervus nippon) Red deer (Cervus elaphus) Clouded leopard (Neofelis nebulosa) Masked palm civeta (Paguma larvata) Hill long-tongued fruit bat (Macroglossus sobrinus) Ear-spot squirrela (Callosciurus adamsi) Red-cheeked squirrel (Dremomys rufigenis) Red and white giant flying squirrel (Petaurista alborufus) Total sequences
0 121,649 58 0 0 0 19
0 150,718 60 20 0 0 0
Point3 Point2 Point1
Point3
Point4
S1 (25-August 2016) Point1 S1 (22-August 2016) Red list assessments
vs Detected speciese identity (%)
leopard (Neofelis diardi) occurs in Borneo (Wilting et al., 2007, 2011); it is a vulnerable species but has no 12S rRNA sequence deposited in NCBI. However, the clouded leopard (Neofelis nebulosa) in China is a close relative of the Sunda clouded leopard, and while it does not occur in Borneo, it has a complete mtDNA sequence deposited in NCBI. Analyses of molecular (Buckley-Beason et al., 2006; Wilting et al., 2007) and morphological data (Christiansen, 2008; Kitchener et al., 2006) demonstrated that Bornean and Sumatran clouded leopards are clearly distinct from those on the continental mainland (Wilting et al., 2011). Given that the eDNA reads assigned to the clouded leopard from China had low similarity (90–91%) (Table 2), these reads may have been derived from the Sunda clouded leopard; however, given that they could not be confirmed using the NCBI database, we judged these reads as ‘other sequences’. On the other hand, the hill long-tongued fruit bat (Macroglossus sobrinus) was detected with high sequence identity (98%) by using eDNA (Table 2) and does not occur in Borneo. Thomsen and Willerslev (2015) suggested that identification of eDNA sequences depends crucially on reliable reference DNA-sequence database, then future DNA databases focus on complete mitochondrial for much wider applications than traditional DNA barcoding. Therefore, we propose that more sequences from more species of tropical forest mammals should be identified and deposite in the NCBI database. 4. Conclusion
Species (Scientific name)
Table 2 Detected other sequences from the water samples in natural saltlicks, Deramakot.
0 84,030 33 0 0 0 0
Point3 Point2 Point1 Point3 Point1 Point2 Point1
Point2
S3 (23-August 2016) S2 (23-August 2016)
S4 (24-August 2016)
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Matsubayashi, H., Ahmad, A.H., Wakamatsu, N., Nakazono, E., Takyu, M., Majalap, N., Lagan, P., Sukor, J.R.A., 2011. Natural-licks use by orangutans and conservation of their habitats in Bornean tropical production forest. Raffles Bull. Zool. 59, 109–115. Matsuda, I., Ancrenaz, M., Akiyama, Y., Tuuga, A., Majalap, N., Bernaed, H., 2015. Natural licks are required for large terrestrial mammals in degraded riparian forest, Sabah, Borneo, Malaysia. Ecol. Res. 30, 191–195. Miya, M., Sato, Y., Fukunaga, T., Sado, T., Poulsen, J.Y., Sato, K., Minamoto, T., Yamamoto, S., Yamanaka, H., Araki, H., Kondoh, M., Iwasaki, W., 2015. MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species. R. Soc. Open Science 2, 150088. Miya, M., Minamoto, T., Yamanaka, H., Oka, S., Sato, K., Yamamoto, S., Sado, T., Doi, H., 2016. Use of a filter cartridge for filtration of water samples and extraction of environmental DNA. J. Vis. Exp. 117, e54741. Thomsen, P.F., Willerslev, E., 2015. Environmental DNA–an emerging tool in conservation for monitoring past and present biodiversity. Biol. Conserv. 183, 4–18. Timmins, R., Kawanishi, K., Giman, B., Lynam, A., Chan, B., Steinmetz, R., Sagar Baral, H., Samba Kumar, N., 2015. Rusa unicolor. (Errata Version Published in 2015) The IUCN Red List of Threatened Species 2015: e.T41790A85628124. (Downloaded on 28 January 2017). Ushio, M., Fukuda, H., Inoue, T., Makoto, K., Kishida, O., Sato, K., Murata, K., Nikaido, M., Sado, T., Sato, Y., Takeshita, M., Iwasaki, W., Yamanaka, H., Kondoh, M., Miya, M., 2016. Environmental DNA enables detection of terrestrial mammals from forest pond water. bioRxiv. pp. 068551. Wilkie, D.S., Bennett, E.L., Peres, C.A., Cummimgham, A.A., 2011. The empty forest revisited. Ann. N. Y. Acad. Sci. 1223, 120–128. Wilting, A., Buckley-Beason, V.A., Feldhaar, H., Gadau, J., O'brien, S.J., Linsenmair, K.E., 2007. Clouded leopard phylogeny revisited: support for species recognition and population division between Borneo and Sumatra. Front. Zool. 4, 15. Wilting, A., Christiansen, P., Kitchener, A.C., Kemp, Y.J., Ambu, L., Fickel, J., 2011. Geographical variation in and evolutionary history of the Sunda clouded leopard (Neofelis diardi) (Mammalia: Carnivora: Felidae) with the description of a new subspecies from Borneo. Mol. Phylogenet. Evol. 58, 317–328.
Madeline, G.P., Ali, N., Bujamg, M., Hashim, I., Bujamg, A., Md-Zain, B.M., 2016. Patterns of mineral lick use by northwest Bornean orangutans (Pongo pygmaeus) in the Lanjak Entimau wildlife sanctuary, Sarawak, Malaysia. Eur. J. Wildl. Res. 62, 147–150. Blake, J.K., Mosquera, D., Salvador, J., 2013. Use of mineral licks by mammals and birds in hunted and non-hunted areas of Yasuní National Park, Ecuador. Anim. Conserv. 16, 420–437. Buckley-Beason, V.A., Johnson, W.E., Nash, W.G., Stanyon, R., Menninger, J.C., Driscoll, C.A., Stone, G., Martelli, P.P., Wen, C., Ling, L., Duraisingam, R.K., Lam, P.V., O'Brien, S.J., 2006. Molecular evidence for species-level distinctions in clouded leopards. Curr. Biol. 16, 2371–2376. Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., Madden, T.L., 2009. BLAST +: architecture and applications. BMC Bioinf. 10, 421. Christiansen, P., 2008. Species distinction and evolutionary differences in the clouded leopard (Neofelis nebulosa) and Diard's clouded leopard (Neofelis diardi). J. Mammal. 89, 1435–1446. Edgar, R.C., 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461. IUCN, 2016. The IUCN Red List of Threatened Species. http://www.iucnredlist.org/ technical-documents/red-list-documents (visited October 25, 2016). Kitchener, A.C., Beaumont, M.A., Richardson, D., 2006. Geographical variation in the clouded leopard, Neofelis nebulosa, reveals two species. Curr. Biol. 16, 2377–2383. Loken, B., Spehar, S., Rayadin, Y., 2013. Terrestriality in the Bornean orangutan (Pongo pygmaeus morio) and implications for their ecology and conservation. Am. J. Primatol. 75, 1129–1138. Magoč, T., Salzberg, S.L., 2011. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27, 2957–2963. Matsubayashi, H., Lagan, P., Majalap, N., Tangah, J., Sukor, J.R.A., Kitayama, K., 2007a. Importance of natural licks for the mammals in Bornean inland tropical rain forests. Ecol. Res. 22, 742–748. Matsubayashi, H., Lagan, P., Sukor, J.R.A., Kitayama, K., 2007b. Seasonal and daily use of natural licks by sambar deer (Cervus unicolor) in a Bornean tropical rain forest. Tropics 17, 81–86.
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