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Enabling pinniped conservation by means of non-invasive genetic population analysis

Abstract

Conservation and management of protected species, particularly of elusive species such as pinnipeds, is hampered by knowledge gaps. In the case of studies using genetic data these are often attributed to a lack of representative samples. Therefore, there is a pressing need for the development of minimally invasive sampling protocols suitable for genetic analyses of pinnipeds. The present study evaluated the applicability of various protocols for the collection and processing of samples from harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus), encompassing seven source sample types (blood, skin, hair (plucked/moulted), urine, buccal swabs, scat) and three different extraction methods. Protocols were designed for minimally invasive sampling, but also to evaluate differences in their performance based on cost and time of execution in comparison to traditional sampling approaches. The performance of each protocol was measured following successful DNA isolation, molecular sex determination and sequencing of a mitochondrial DNA fragment (control region). Protocols using plucked hair, urine and buccal swab samples proved effective for collection from individuals in captivity, whereas scat was most applicable for non-invasive sampling in the wild. Furthermore, following a pilot study on scat samples, DNA was found to be viable for genetic analysis after exposure to ambient conditions for up to four weeks. This study provides a useful assessment of the suitability of various minimal and non-invasively collected samples for DNA isolation, amplification and mitochondrial sequencing, enabling the effective design of future sampling strategies and a significant increase of samples available for genetic analysis of pinnipeds.

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Data availability

Data are available via the Electronic Supplementary Material.

Code availability

All R code can be provided upon request by contacting the corresponding author.

References

  • Abadía-Cardoso A, Freimer NB, Deiner K, Garza JC (2017) Molecular population genetics of the northern elephant seal Mirounga angustirostris. J Hered 108:618–627

    PubMed  PubMed Central  Google Scholar 

  • Alonso A, Albarrán C, Martín P, Garcia P, Garcia O, de la Rúa C, Alzualde A, De Simón LF, Sancho M, Piqueras JF (2003) Multiplex–PCR of short amplicons for mtDNA sequencing from ancient DNA. Int Congress series 1239:585–588

    CAS  Google Scholar 

  • Bajwa AA, Cuff JP, Imran M, Islam S, Mansha R, Ashraf K, Khan A, Rashid MI, Zahoor MY, Khan WA (2019) Assessment of nematodes in Punjab Urial (Ovis vignei punjabiensis) population in Kalabagh Game Reserve: development of a DNA barcode approach. Eur J Wildlife Res 65:63

    Google Scholar 

  • Beal A, Kiszka J, Wells R, Eirin-Lopez JM (2019) The Bottlenose dolphin Epigenetic Aging Tool (BEAT): a molecular age estimation tool for small cetaceans. Front Marine Sci 6:561

    Google Scholar 

  • Bickham, J. W., Dupont, J., O'hara, T. Godard-Codding, C. (2013) Genetics and preliminary hormone analyses in Western Gray whale biopsy samples collected off Sakhalin Island in 2011. Paper SC/65a/BRG23 presented to the IWC Scientific Committee,(Cambridge, MA: International Whaling Commission).

  • Bourgeois, S., Kaden, J., Senn, H., Bunnefeld, N., Jeffery, K. J., Akomo-Okoue, E. F., Ogden, R. Mcewing, R. (2019) Improving cost-efficiency of faecal genotyping: New tools for elephant species. PloS one, 14.

  • Bowles E, Trites AW (2013) Faecal DNA amplification in Pacific walruses (Odobenus rosmarus divergens). Polar Biol 36:755–759

    Google Scholar 

  • Caudron AK, Negro SS, Muller CG, Boren LJ, Gemmell NJ (2007) Hair sampling and genotyping from hair follicles: a minimally-invasive alternative for genetics studies in small, mobile pinnipeds and other mammals. Marine Mammal Sci 23:184–192

    Google Scholar 

  • Cole TL, Waters JM, Shepherd LD, Rawlence NJ, Joseph L, Wood JR (2018) Ancient DNA reveals that the ‘extinct’Hunter Island penguin (Tasidyptes hunteri) is not a distinct taxon. Zool J Linnean Soc 182:459–464

    Google Scholar 

  • Collins CJ, Chilvers BL, Osborne A, Taylor M, Robertson BC (2017) Unique and isolated: population structure has implications for management of the endangered New Zealand sea lion. Conserv Genet 1-13.

  • Creel S, Spong G, Sands JL, Rotella J, Zeigle J, JOE L, Murphy KM, Smith D (2003) Population size estimation in Yellowstone wolves with error‐prone noninvasive microsatellite genotypes. Mol Ecol 1.

  • Fain SR, Lemay JP (1995) Gender identification of humans and mammalian wildlife species from PCR amplified sex linked genes. Proc Am Acad Forensic Sci 1:34

    Google Scholar 

  • Fietz K, Galatius A, Teilmann J, Dietz R, Frie AK, Klimova A, Palsbøll PJ, Jensen LF, Graves JA, Hoffman JI (2016) Shift of grey seal subspecies boundaries in response to climate, culling and conservation. Mol Ecol 25:4097–4112

    PubMed  Google Scholar 

  • Foote AD, Vilstrup JT, de Stephanis R, Verborgh P, Abel Nielsen SC, Deaville R, Kleivane L, Martin V, Miller PJO, Øien N (2011) Genetic differentiation among North Atlantic killer whale populations. Mol Ecol 20:629–641

    PubMed  Google Scholar 

  • Gentry-Shields J, Wang A, Cory RM, Stewart JR (2013) Determination of specific types and relative levels of QPCR inhibitors in environmental water samples using excitation–emission matrix spectroscopy and PARAFAC. Water Res 47:3467–3476

    CAS  PubMed  Google Scholar 

  • Gosch M, Hernandez-Milian G, Rogan E, Jessopp M, Cronin M (2014) Grey seal diet analysis in Ireland highlights the importance of using multiple diagnostic features. Aquat Biol 20:155–167

    Google Scholar 

  • Hausknecht R, Gula R, Pirga B, Kuehn R (2007) Urine—a source for noninvasive genetic monitoring in wildlife. Mol Ecol Notes 7:208–212

    CAS  Google Scholar 

  • Holubová N, Sak B, Hlásková L, Květoňová D, Hanzal V, Rajský D, Rost M, Mcevoy J, Kváč M (2018) Host specificity and age-dependent resistance to Cryptosporidium avium infection in chickens, ducks and pheasants. Exp Parasitol 191:62–65

    PubMed  Google Scholar 

  • Ices (2018) Report of the Workshop on MSFD biodiversity of species D1 aggregation (WKDIVAGG), 1–4 May 2018, ICES HQ, Copenhagen, Denmark. ICES CM 2018/ACOM:47. 53 pp. 2018.

  • Karamanlidis AA, Gaughran S, Aguilar A, Dendrinos P, Huber D, Pires R, Schultz J, Skrbinšek T, Amato G (2016) Shaping species conservation strategies using mtDNA analysis: the case of the elusive Mediterranean monk seal (Monachus monachus). Biol Conserv 193:71–79

    Google Scholar 

  • Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX, Wilson AC (1989) Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proceed Nat Acad Sci 86:6196–6200

    CAS  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016) Mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    CAS  Google Scholar 

  • Lampa S, Gruber B, Henle K, Hoehn M (2008) An optimisation approach to increase DNA amplification success of otter faeces. Conserv Genet 9:201

    CAS  Google Scholar 

  • Masland EDP, Sweezy MA, Ono KA (2010) Molecular methods for differentiating grey seal (Halichoerus grypus) and harbour seal (Phoca vitulina) scat. Mol Ecol Resour 10(1):214–217

  • Miles KA, Holtz MN, Lounsberry ZT, Sacks BN (2015) A paired comparison of scat-collecting versus scat-swabbing methods for noninvasive recovery of mesocarnivore DNA from an arid environment. Wildlife Soc Bull 39:797–803

    Google Scholar 

  • Mitelberg A, Vandergast AG (2016) Non-invasive genetic sampling of southern mule deer (Odocoileus hemionus fuliginatus) reveals limited movement across California State route 67 in San Diego county. Western Wildlife 3:8–18

    Google Scholar 

  • Mizuno M, Sasaki T, Kobayashi M, Haneda T, Masubuchi T (2018) Mitochondrial DNA reveals secondary contact in Japanese harbour seals, the southernmost population in the western Pacific. PloS One 13:e0191329

    PubMed  PubMed Central  Google Scholar 

  • Murphy MA, Waits LP, Kendall KC, Wasser SK, Higbee JA, Bogden R (2002) An evaluation of long-term preservation methods for brown bear (Ursus arctos) faecal DNA samples. Conserv Genet 3:435–440

    CAS  Google Scholar 

  • Olsen MT, Islas V, Graves JA, Onoufriou A, Vincent C, Brasseur S, Frie AK, Hall AJ (2017) Genetic population structure of harbour seals in the United Kingdom and neighbouring waters. Aquat Conserv: Marine Freshwater Ecosys 27:839–845

    Google Scholar 

  • Panasci M, Ballard WB, Breck S, Rodriguez D, Densmore LD III, Wester DB, Baker RJ (2011) Evaluation of fecal DNA preservation techniques and effects of sample age and diet on genotyping success. J Wildlife Manag 75:1616–1624

    Google Scholar 

  • Peralta DM, Ibañez EA, Lucero S, Cappozzo HL, Túnez JI (2020) A new minimally invasive and inexpensive sampling method for genetic studies in pinnipeds. Mammal Res 65:11–18

    Google Scholar 

  • Peters KJ, Ophelkeller K, Bott NJ, Goldsworthy SD (2015) PCR-based techniques to determine diet of the Australian sea lion (Neophoca cinerea): a comparison with morphological analysis. Marine Ecol 36:1428–1439

    CAS  Google Scholar 

  • Pilliod DS, Goldberg CS, Arkle RS, Waits LP (2013) Estimating occupancy and abundance of stream amphibians using environmental DNA from filtered water samples. Can J Fisher Aquat Sci 70:1123–1130

    CAS  Google Scholar 

  • Pinfield R, Dillane E, Runge AKW, Evans A, Mirimin L, Niemann J, Reed TE, Reid DG, Rogan E, Samarra FIP (2019) False-negative detections from environmental DNA collected in the presence of large numbers of killer whales (Orcinus orca). Environ DNA 1:316–328

    Google Scholar 

  • Polanowski AM, Robbins J, Chandler D, Jarman SN (2014) Epigenetic estimation of age in humpback whales. Mol Ecol Res 14:976–987

    CAS  Google Scholar 

  • Reed JZ, Tollit DJ, Thompson PM, Amos W (1997) Molecular scatology: the use of molecular genetic analysis to assign species, sex and individual identity to seal faeces. Mol Ecol 6:225–234

    CAS  PubMed  Google Scholar 

  • Robinson SJ, Waits LP, Martin ID (2009) Estimating abundance of American black bears using DNA-based capture–mark–recapture models. Ursus 20:1–12

    Google Scholar 

  • RStudio Team (2019) RStudio: integrated development for R. RStudio. PBC, Boston, MA. http://www.rstudio.com/

  • Stenglein JL, de Barba M, Ausband DE, Waits LP (2010) Impacts of sampling location within a faeces on DNA quality in two carnivore species. Mol Ecol Res 10:109–114

    CAS  Google Scholar 

  • Swanson BJ, Kelly BP, Maddox CK, Moran JR (2006) Shed skin as a source of DNA for genotyping seals. Mol Ecol Res 6:1006–1009

    CAS  Google Scholar 

  • Tende T, Hansson B, Ottosson U, Bensch S (2014) Evaluating preservation medium for the storage of DNA in African lion Panthera leo faecal samples. Curr Zool 60:351–358

    CAS  Google Scholar 

  • Ushio M, Fukuda H, Inoue T, Makoto K, Kishida O, Sato K, Murata K, Nikaido M, Sado T, Sato Y (2017) Environmental DNA enables detection of terrestrial mammals from forest pond water. Mol Ecol Res 17:e63–e75

    CAS  Google Scholar 

  • Valiere N, Taberlet P (2000) Urine collected in the field as a source of DNA for species and individual identification. Mol Ecol 9:2150–2152

    CAS  PubMed  Google Scholar 

  • Valqui J, Hartl GB, Zachos FE (2010) Non-invasive genetic analysis reveals high levels of mtDNA variability in the endangered South-American marine otter (Lontra felina). Conserv Genet 11:2067–2072

    Google Scholar 

  • Valtonen M, Heino M, Aspi J, Buuri H, Kokkonen T, Kunnasranta M, Palo JU, Nyman T (2015) Genetic monitoring of a critically-endangered seal population based on field-collected placentas. Ann Zool Fennici 52:51–65

    Google Scholar 

  • Walker FM, Horsup A, Taylor AC (2009) Leader of the pack: faecal pellet deposition order impacts PCR amplification in wombats. Mol Ecol Res 9:720–724

    CAS  Google Scholar 

  • Wedrowicz F, Karsa M, Mosse J, Hogan FE (2013) Reliable genotyping of the koala (Phascolarctos cinereus) using DNA isolated from a single faecal pellet. Mol Ecol Res 13:634–641

    CAS  Google Scholar 

  • Wedrowicz F, Mosse J, Wright W, Hogan FE (2019) Isolating DNA sourced non-invasively from koala scats: a comparison of four commercial DNA stool kits. Conserv Genet Res 11:219–229

    Google Scholar 

  • Westlake RL, O’corry-Crowe GM (2002) Macrogeographic structure and patterns of genetic diversity in harbor seals (Phoca vitulina) from Alaska to Japan. J Mammal 83:1111–1126

    Google Scholar 

  • Zappes IA, Fabiani A, Sbordoni V, Rakaj A, Palozzi R, Allegrucci G (2017) New data on Weddell seal (Leptonychotes weddellii) colonies: A genetic analysis of a top predator from the Ross Sea, Antarctica. PloS One 12:e0182922

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors wish to thank Seal Rescue Ireland, DAERA, Seehundstation Norddeich and Seehundstation Friedrichskoog e.V. for providing samples, Proinsias Hernon for assistance during fieldwork and Javier Burgoa Cardas for assistance with DNA extractions. Prototypes for PB-200 were made available by DNA Genotek (Ottawa, Canada). Our thanks also go to the MFRC/GMIT staff and NPWS staff/rangers for their support.

Funding

Financial support was received from Galway-Mayo Institute of Technology (Research & Innovation Strategic Endowment (RISE) scholarship), the Irish National Parks and Wildlife Service (Grant REF SPU G07-2017) and the Irish Department of Housing, Local Government and Heritage.

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Correspondence to Kristina Steinmetz.

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Licences for sampling in the wild were obtained from the Irish National Parks and Wildlife Service (C151/16, C32/17, C33/17, C83/17, C121/17, C59/18, C86/18, C179/18).

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Steinmetz, K., Murphy, S., Ó Cadhla, O. et al. Enabling pinniped conservation by means of non-invasive genetic population analysis. Conservation Genet Resour 13, 131–142 (2021). https://doi.org/10.1007/s12686-020-01182-4

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  • DOI: https://doi.org/10.1007/s12686-020-01182-4

Keywords

  • Non-invasive
  • Population genetics
  • Grey seal
  • Harbour seal