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Primates

, Volume 60, Issue 1, pp 7–13 | Cite as

Bonobos’ saliva remaining on the pith of terrestrial herbaceous vegetation can serve as non-invasive wild genetic resources

  • Shintaro Ishizuka
  • Yoshi Kawamoto
  • Kazuya Toda
  • Takeshi Furuichi
News and Perspectives

Abstract

Evaluating the genetic diversity of natural populations of endangered species is important for conservation. Although the genetic analysis of wildlife usually requires collecting DNA non-invasively, the variety of non-invasive DNA sampling methods is limited for each species. We present a method to obtain DNA of an endangered species, the bonobo (Pan paniscus), in which the pith of the terrestrial herbaceous vegetation (THV) that they consumed was newly utilized. We investigated the (1) frequency of encountering remnant saliva on three types of THV pith; (2) concentrations of DNA in the saliva samples by the real-time quantitative PCR; and (3) rates of positive PCR, accurate genotyping, and allelic drop out by analyzing two autosomal microsatellite loci (D7s817 and D9s910). The number of remnant saliva samples was recorded by following the bonobo groups on a daily basis. The frequency of encountering DNA samples was higher in saliva samples than in fecal samples. More than half of the saliva samples remaining on two types of THV pith provided sufficient concentrations of bonobo DNA (> 200 pg/μl). Rates of positive PCR and accurate genotyping were high, and allelic drop out rate was low when the amount of template DNA was above 200 pg per reaction. Our results suggest that the remnants of bonobo saliva on the pith of THV are a potential resource for obtaining DNA, and better than other kinds of samples from the perspective of the abundant sampling opportunities.

Keywords

Bonobo Pan paniscus Conservation Non-invasive DNA sampling Saliva Terrestrial herbivorous vegetation 

Notes

Acknowledgements

We thank the Research Centre for Ecology and Forestry and the Ministry of Scientific Research of the Democratic Republic of the Congo for permitting our research. We also thank Dr. T. Sakamaki and local assistants at Wamba for help in our fieldwork, and Dr. T. Yumoto for providing valuable comments on terrestrial herbivorous vegetation, and Dr. H. Imai, Dr. G. Hanya, Dr. N. Suzuki-Hashido, Dr. T. Hayakawa, Ms. M. Hakukawa, and Ms. K. Takano for help in our genetic analysis. We also thank the editor and two reviewers for their reviewing processes and helpful comments for our manuscript. This study was financially supported by Japan Society for the Promotion of Science Grant-in-aid for JSPS fellows (17J09827 to S.I.), and the Leading Graduate Program in Primatology and Wildlife Science of Kyoto University.

Author contributions

SI designed this study, collected DNA samples of animals, took photos of THV, conducted DNA experiments, analyzed the genetic data, wrote and revised the manuscript. KT helped DNA sampling of animals. YK and TF supervised SI to design this study. All authors revised the manuscript and gave final approval for publication.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was approved by the Research Centre for Ecology and Forestry and the Ministry of Scientific Research of the Democratic Republic of the Congo, and followed all laws in the Democratic Republic of the Congo. This study was conformed to the Guidelines for Field Research established by the Ethics Committee of the Primate Research Institute, Kyoto University.

References

  1. Arandjelovic M, Guschanski K, Schubert G, Harris TR, Thalmann O, Siedel H, Vigilant L (2009) Two-step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples. Mol Ecol Resour 9:28–36CrossRefGoogle Scholar
  2. Badrian N, Malenky RK (1984) Feeding ecology of Pan paniscus in the Lomako Forest, Zaire. In: Susman RL (ed) The pygmy chimpanzee, evolutionary biology and behavior. Plenum Press, New York, pp 275–299CrossRefGoogle Scholar
  3. Basabose AK, Inoue E, Kamungu S, Murhabale B, Akomo-Okoue EF, Yamagiwa J (2015) Estimation of chimpanzee community size and genetic diversity in Kahuzi-Biega National Park, Democratic Republic of Congo. Am J Primatol 77:1015–1025CrossRefGoogle Scholar
  4. Beja-Pereira A, Oliveira R, Schwartz MK, Luikart G (2009) Advancing ecological understanding through technological transformation in noninvasive genetics. Mol Ecol Res 9:1279–1301CrossRefGoogle Scholar
  5. Beringer V, Deschner T, Möstl E, Selzer D, Hohmann G (2012) Stress affects salivary alpha-amylase activity in bonobos. Physiol Behav 105:476–482CrossRefGoogle Scholar
  6. Blejwas KM, Williams CL, Shin GT, McCullough DR, Jaeger MM (2006) Salivary DNA evidence convicts breeding male coyotes of killing sheep. Wildl Manag 70:1087–1093CrossRefGoogle Scholar
  7. Bowen-Jones E, Pendry S (1999) The threat to primates and other mammals from the bushmeat trade in Africa, and how this threat could be diminished. Oryx 33:233–246Google Scholar
  8. Cheah PY, Bernstein H (1990) Modification of DNA by bile acids: a possible factor in the etiology of colon cancer. Cancer Lett 49:207–210CrossRefGoogle Scholar
  9. Demeke T, Adams RP (1992) The effects of plant polysaccharides and buffer additives on PCR. Biotechniques 12:332–334Google Scholar
  10. Deuter R, Pietsch S, Hertel S, Muller O (1995) A method for preparation of fecal DNA suitable for PCR. Nucleic Acids Res 23:3800–3801CrossRefGoogle Scholar
  11. Dirzo R, Young HS, Galetti M, Ceballos G, Isaac NJ, Collen B (2014) Defaunation in the Anthropocene. Science 401:401–406CrossRefGoogle Scholar
  12. Dunay E, Apakupakul K, Leard S, Palmer JL, Deem SL (2018) Pathogen transmission from humans to great apes is a growing threat to primate conservation. EcoHealth 2050:1–15Google Scholar
  13. Eriksson J, Siedel H, Lukas D, Kayser M, Erler A, Hashimoto C, Hohmann G, Boesch C, Vigilant L (2006) Y-chromosome analysis confirms highly sex-biased dispersal and suggests a low male effective population size in bonobos (Pan paniscus). Mol Ecol 15:939–949CrossRefGoogle Scholar
  14. Evans TS, Barry PA, Gilardi KV, Goldstein T, Deere JD, Fike J et al (2015) Optimization of a novel non-invasive oral sampling technique for zoonotic pathogen surveillance in nonhuman primates. PLoS Negl Trop Dis 9:1–17Google Scholar
  15. Frankham R (2005) Genetics and extinction. Biol Conserv 126:131–140CrossRefGoogle Scholar
  16. Frankham R (2010) Challenges and opportunities of genetic approaches to biological conservation. Biol Conserv 143:1919–1927CrossRefGoogle Scholar
  17. Frankham R, Ballou JD, Briscoe DA (2002) Introduction to conservation genetics. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  18. Fruth B, Hickey JR, André C, Furuichi T, Hart J, Hart T, Kuehl H, Maisels F, Nackoney J, Reinartz G, Sop T, Thompson J, Williamson EA (2016) Pan paniscus, IUCN red list of threatened species, version 3.1. IUCN Google Scholar
  19. Gerloff U, Hartung B, Fruth B, Hohmann G, Tautz D (1999) Intracommunity relationships, dispersal pattern and paternity success in a wild living community of bonobos (Pan paniscus) determined from DNA analysis of faecal samples. Proc R Soc Lond B 266:1189–1195CrossRefGoogle Scholar
  20. Hart JA, Grossmann F, Vosper A, Ilanga J (2008) Human hunting and its impact on bonobos in the Salonga National Park, Democratic Republic of Congo. In: Furuichi T, Thompson JM (eds) The bonobos: behavior, ecology and conservation. Springer, New York, pp 245–271CrossRefGoogle Scholar
  21. Hashimoto C, Takenaka O, Furuichi T (1996) Matrilineal kin relationship and social behavior of wild bonobos (Pan paniscus): sequencing the D-loop region of mitochondrial DNA. Primates 37:305–318CrossRefGoogle Scholar
  22. Higham JP, Vitale AB, Mas-Rivera A, Ayala JE, Maestripieri D (2010) Measuring salivary analytes from free-ranging monkeys. Physiol Behav 101:601–607CrossRefGoogle Scholar
  23. Huijbregts B, De Wachter P, Obiang LSN, Akou ME (2003) Ebola and the decline of gorilla Gorilla gorilla and chimpanzee Pan troglodytes populations in Minkebe Forest, north-eastern Gabon. Oryx 37:437–443CrossRefGoogle Scholar
  24. Idaghdour Y, Broderick D, Korrida A (2003) Faeces as a source of DNA for molecular studies in a threatened population of great bustards. Conserv Genet 4:789–792CrossRefGoogle Scholar
  25. Idani G, Mwanza N, Ihobe H, Hashimoto C, Tashiro Y, Furuichi T (2008) Changes in the status of bonobos, their habitat, and the situation of humans at Wamba in the Luo Scientific Reserve, Democratic Republic of Congo. In: Furuichi T, Thompson JM (eds) The bonobos: behavior, ecology and conservation. Springer, New York, pp 291–302CrossRefGoogle Scholar
  26. Inoue E, Inoue-Murayama M, Takenaka O, Nishida T (2007) Wild chimpanzee infant urine and saliva sampled noninvasively usable for DNA analyses. Primates 48:156–159CrossRefGoogle Scholar
  27. Inoue E, Akomo-Okoue EF, Ando C, Iwata Y, Judai M, Fujita S, Hongo S, Nze-Nkogue C, Inoue-Murayama M, Yamagiwa J (2013) Male genetic structure and paternity in western lowland gorillas (Gorilla gorilla gorilla). Am J Phys Anthropol 151:583–588CrossRefGoogle Scholar
  28. Ishizuka S, Kawamoto Y, Sakamaki T, Tokuyama N, Toda K, Okamura H, Furuichi T (2018) Paternity and kin structure among neighbouring groups in wild bonobos at Wamba. R Soc Open Sci 5:171006CrossRefGoogle Scholar
  29. Kano T (1992) The last ape: pygmy chimpanzee behavior and ecology. Stanford University Press, StanfordGoogle Scholar
  30. Kano T, Mulavwa M (1984) Feeding ecology of the pygmy chimpanzees (Pan paniscus) of Wamba. In: Susman RL (ed) The pygmy chimpanzee, evolutionary biology and behavior. Plenum Press, New York, pp 233–274CrossRefGoogle Scholar
  31. Kawamoto Y, Takemoto H, Higuchi S, Sakamaki T, Hart JA, Hart TB, Tokuyama N, Reinartz GE, Guislain P, Dupain J, Cobden AK, Mulavwa MN, Yangozene K, Darroze S, Devos C, Furuichi T (2013) Genetic structure of wild bonobo populations: diversity of mitochondrial DNA and geographical distribution. PLoS One 8:e59660CrossRefGoogle Scholar
  32. Keller LF, Waller DM (2002) Inbreeding effects in wild populations. Trends Ecol Evol 17:230–241CrossRefGoogle Scholar
  33. Kuroda S (1979) Grouping of the pygmy chimpanzees. Primates 20:161–183CrossRefGoogle Scholar
  34. Leroy EM, Rouquet P, Formenty P, Souquiere S, Kilbourne A, Froment JM, Bermejo M, Smit S, Karesh W, Swanepoel R, Zaki SR, Rollin PE (2004) Multiple Ebola virus transmission events and rapid decline of central African wildlife. Science 303:387–390CrossRefGoogle Scholar
  35. Malenky RK, Stiles EW (1991) Distribution of terrestrial herbaceous vegetation and its consumption by Pan paniscus in the Lomako forest, Zaire. Am J Primatol 23:153–169CrossRefGoogle Scholar
  36. Matsubara M, Basabose AK, Omari I, Kaleme K, Kizungu B, Sikubwabo K, Kahindo M, Yamagiwa J, Takenaka O (2005) Species and sex identification of western lowland gorillas (Gorilla gorilla gorilla), eastern lowland gorillas (Gorilla beringei graueri) and humans. Primates 46:199–202CrossRefGoogle Scholar
  37. Morin PA, Chambers KE, Boesch C, Vigilant L (2001) Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees (Pan troglodytes verus). Mol Ecol 10:1835–1844CrossRefGoogle Scholar
  38. Mulavwa M, Furuichi T, Yangozene K, Yamba-Yamba M, Motema- Salo B, Idani G, Ihobe H, Hashimoto C, Tashiro Y, Mwanza N (2008) Seasonal changes in fruit production and party size of bonobos at Wamba. In: Furuichi T, Thompson J (eds) The bonobos: behavior, ecology, and conservation. Springer, New York, pp 121–134CrossRefGoogle Scholar
  39. Murphy MA, Waits LP, Kendall KC (2003) The influence of diet on faecal DNA amplification and sex identification in brown bears (Ursus arctos). Mol Ecol 12:2261–2265CrossRefGoogle Scholar
  40. Nater A, Nietlisbach P, Arora N, van Schaik CP, van Noordwijk MA, Willems EP, Singleton I, Wich SA, Goossens B, Warren KS, Verschoor EJ, Perwitasari-Farajallah D, Pamungkas J, Krutzen M (2011) Sex-biased dispersal and volcanic activities shaped phylogeographic patterns of extant orangutans (genus: Pongo). Mol Biol Evol 28:2275–2288CrossRefGoogle Scholar
  41. Nietlisbach P, Arora N, Nater A, Goossens B, van Schaik CP, Krutzen M (2012) Heavily male-biased long-distance dispersal of orangutans (genus: Pongo), as revealed by Y-chromosomal and mitochondrial genetic markers. Mol Ecol 21:3173–3186CrossRefGoogle Scholar
  42. Okamura H (2018) Inter-individual difference in forest utilization height in wild bonobos. Master’s thesis, Kyoto UniversityGoogle Scholar
  43. Pimm SL, Russell GJ, Gittleman JL, Brooks TM (1995) The future of biodiversity. Science 269:347–350CrossRefGoogle Scholar
  44. Pompanon F, Bonin A, Bellemain E, Taberlet P (2005) Genotyping errors: causes, consequences and solutions. Nat Rev Genet 6:847–859CrossRefGoogle Scholar
  45. Simons ND, Lorenz JG, Sheeran LK, Li JH, Xia DP, Wagner RS (2012) Noninvasive saliva collection for DNA analyses from free-ranging Tibetan macaques (Macaca thibetana). Am J Primatol 74:1064–1070CrossRefGoogle Scholar
  46. Smiley T, Spelman L, Lukasik-Braum M, Mukherjee J, Kaufman G, Akiyoshi DE, Cranfield M (2010) Noninvasive saliva collection techniques for free-ranging mountain gorillas and captive eastern gorillas. J Zoo Wildl Med 41:201–209CrossRefGoogle Scholar
  47. Sommer S (2005) The importance of immune gene variability (MHC) in evolutionary ecology and conservation. Front Zool 2:16CrossRefGoogle Scholar
  48. Sugiyama Y, Kawamoto S, Takenaka O, Kumazaki K, Miwa N (1993) Paternity discrimination and inter-group relationships of chimpanzees at Bossou. Primates 34:545–552CrossRefGoogle Scholar
  49. Taberlet P (1996) Reliable genotyping of samples with very low DNA quantities using PCR. Nucleic Acids Res 24:3189–3194CrossRefGoogle Scholar
  50. Taberlet P, Luikart G (1999) Non-invasive genetic sampling and individual identification. Biol J Linn Soc 68:41–55CrossRefGoogle Scholar
  51. Takemoto H (2017) Acquisition of terrestrial life by human ancestors influenced by forest microclimate. Sci Rep 7:1–8CrossRefGoogle Scholar
  52. Tashiro Y, Idani G, Kimura D, Bongori L (2007) Habitat changes and decreases in the bonobo population in Wamba, Democratic Republic of the Congo. Afr Study Monogr 28:99–106Google Scholar
  53. Terada S, Nackoney JR, Sakamaki T, Mulavwa MN, Yumoto T, Furuichi T (2015) Habitat use of bonobos (Pan paniscus) at Wamba: selection of vegetation types for ranging, feeding and night-sleeping. Am J Primatol 77:701–713CrossRefGoogle Scholar
  54. Waits LP, Paetkau D (2005) Noninvasive genetic sampling tools for wildlife biologists: a review of applications and recommendations for accurate data collection. J Wildl Manage 69:1419–1433CrossRefGoogle Scholar
  55. Wei T, Lu G, Clover G (2008) Novel approaches to mitigate primer interaction and eliminate inhibitors in multiplex PCR, demonstrated using an assay for detection of three strawberry viruses. J Virol Methods 151:132–139CrossRefGoogle Scholar
  56. White OL, Densmore LD III (1992) Mitochondrial DNA isolation. In: Hoelzel AR (ed) Molecular genetic analysis of populations, a practical approach, 1st edn. IRL Press, Oxford, pp 29–55Google Scholar
  57. Williams CL, Blejwas K, Johnston JJ, Jaeger MM (2003) A coyote in sheep ́s clothing: predator identification from saliva. Wildl Soc Bull 31:926–932Google Scholar
  58. Yamakoshi G (2004) Food seasonality and socioecology in Pan: are West African chimpanzees another bonobo? Afr Study Monogr 25:45–60Google Scholar

Copyright information

© Japan Monkey Centre and Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Primate Research InstituteKyoto UniversityInuyamaJapan
  2. 2.Japan Society for the Promotion of ScienceTokyoJapan
  3. 3.Nippon Veterinary and Life Science UniversityMusashino, TokyoJapan

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