Novel real-time PCR species identification assays for British and Irish bats and their application to a non-invasive survey of bat roosts in Ireland


Detection and monitoring of extant bat populations are crucial for conservation success. Non-invasive genetic analysis of bat droppings collected at roosts could be very useful in this respect as a rapid, costefficient monitoring tool. We developed species-specific real-time PCR assays for 18 British and Irish bat species to enable non-invasive, large-scale distribution monitoring, which were then applied to a field survey in Ireland. One hundred and sixty-four DNA samples were collected from 95 bat roosts, of which 73% of samples were identified to species, and the resident bat species were identified at 89% of roosts. However, identification success varied between roost types, ranging from 22% for underground sites to 92% for bat boxes. This panel of DNA tests will be especially useful in cases where roosts contain multiple species, where the number of bats present is small, or bats are otherwise difficult to directly observe. The methodology could be applied to the surveillance of proposed development sites, post development mitigation measures, distribution surveys, bat box schemes and the evaluation of agri-environmental bat box schemes.

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  1. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402.

  2. Artyushin, I.V., Bannikova, A.A., Lebedev, V.S., Kruskop, S.V., 2009. Mitochondrial DNA relationships among North Palaearctic Eptesicus (Vespertilionidae, Chiroptera) and past hybridization between Common Serotine and Northern Bat. Zootaxa 2262, 40–52.

  3. Barlow, K.E., Briggs, P.A., Haysom, K.A., Hutson, A.M., Lechiara, N.L., Racey, P.A., Walsh, A.L., Langton, S.D., 2015. Citizen science reveals trends in bat populations: The National Bat Monitoring Programme in Great Britain. Bio. Conserv. 182, 14–26.

  4. Barratt, E.M., Deaville, R., Burland, T.M., Bruford, M.W., Jones, G., Racey, P.A., Wayne, R.K., 1997. DNA answers the call of pipistrelle bat species. Nature 379, 138–139.

  5. Bat Conservation Ireland. (2015). Bats and bat boxes: Guidance notes for Agri-environmental schemes.

  6. Beja-Pereira, A., Oliveira, R., Alves, P.C., Schwartz, M.K., Luikart, G., 2009. Advancing ecological understandings through technological transformations in noninvasive genetics. Mol. Ecol. Res. 9, 1279–1301.

  7. Benda, P., Hulva, P., Gaisler, J., 2004. Systematic status of African populations of Pipistrellus pipistrellus complex (Chiroptera: Vespertilionidae), with a description of a new species from Cyrenaica, Libya. Acta Chiropterol. 6, 193–217.

  8. Berthier, P., Excoffier, L., Ruedi, M., 2006. Recurrent replacement of mtDNA and cryptic hybridization between two sibling bat species Myotis myotis and Myotis blythii. Proc. R. Soc. B 273, 3101–3109.

  9. Boston, E.S.M., Hanrahan, N., Puechmaille, S.J., Ruedi, M., Buckley, D.J., Lundy, M.G., Scott, D.D., Prodöhl, P.A., Montgomery, W.I., Teeling, E.C., 2011. A rapid PCR-based assay for identification of cryptic Myotis spp. (M. mystacinus, M. brandtii and M. alcathoe). Conserv. Genet. Res. 3, 557–563.

  10. Boston, E.S.M., Puechmaille, S.J., Scott, D.D., Buckley, D.J., Lundy, M.G., Montgomery, I.W., Prodöhl, P.A., Teeling, E.C., 2012. Empirical assessment of non-invasive population genetics in bats: comparison of DNA quality from faecal and tissue samples. Acta Chiropterol. 14, 45–52.

  11. Clark, K., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Sayers, E.W., 2016. GenBank. Nucleic Acids Research. 44, D67–D72.

  12. Collins, J. (ed.) (2016) Bat Surveys for Professional Ecologists–Good Practice Guidelines (3rd Edition). The Bat Conservation Trust, London.

  13. Croose, E., Birks, J.D., O’Reilly, C., Turner, P., Martin, J., MacLeod, E.T., 2016. Sample diversity adds value to non-invasive genetic assessment of a pine marten (Martes martes) population in Galloway Forest, southwest Scotland. Mamm. Res. 61, 131–139.

  14. Dietz, C., Von Helversen, O., Nill, H., 2009. Bats of Britain, Europe and Northwest Africa. A. and C. Black Publishers Ltd., London, U.K.

  15. Dufresnes, C., Remollino, N., Stoffel, C., Manz, R., Weber, J.-M., Fumagalli, L., 2019. Two decades of non-invasive genetic monitoring of the grey wolves recolonizing the Alps support very little dog introgression. Sci. Rep. UK 9, 148.

  16. Elliott, J., 2018, August 30–31. Design of agri-environmental schemes evidence from the monitoring and evaluation of GLAS in Ireland. 166th Seminar of the European Association of Agricultural Economists.

  17. Farrell, L.E., Roman, J., Sunquist, M.E., 2000. Dietary separation of sympatric carnivores identified by molecular analysis of scats. Mol. Ecol. 9, 1583–1590.

  18. Gaisler, J., Chytil, J., 2002. Mark-recapture results and changes in bat abundance at the cave of Na Turoldu, Czech Republic. Folia Zool. 51, 1–10.

  19. García-Mudarra, J.L., Ibáñez, C., Juste, J., 2009. The Straits of Gibraltar: barrier or bridge to Ibero-Moroccan bat diversity? Biol. J. Linn. Soc. 96, 434–450.

  20. Glover, A.M., Altringham, J.D., 2008. Cave selection and use by swarming bat species. Biol. Conserv. 141, 1493–1504.

  21. Goldberg, C.S., Turner, C.R., Deiner, K., Klymus, K.E., Thomsen, P.F., Murphy, M.A., Cornman, R.S. 2016. Critical considerations for the application of environmental DNA methods to detect aquatic species. Met. in Ecol. and Evol. 7, 1299–1307

  22. Hamilton, P., Webster, T.U., Basiewicz, M., Kennedy, E., De-Bastos, E., Mathews, F., 2015. A rapid PCR-based test for identification of fifteen British bat species. Conser. Genet. Res. 7, 651–657.

  23. Harper, L.R., Griffiths, N.P., Lawson Handley, L., Sayer, C.D., Read, D.S., Harper, K.J., Blackman, R.C., Li, J., Hanfling, B., 2019, Development and application of environmental DNA surveillance for the threatened crucian carp (Carassius carassius). Freshw. Rev. 64, 93–107

  24. Hulva, P., Benda, P., Hanak, V., Evin, A., Horacek, I., 2007, New mitochondrial lineages within the Pipistrellus pipistrellus complex from Mediterranean Europe. Folia Zool. 56, 378–388

  25. Ibáñez, C., García-Mudarra, J.L., Ruedi, M., Stadelmann, B., Juste, J., 2006. The Iberian contribution to cryptic diversity in European bats. Acta Chiropterol. 8, 277–297.

  26. Juste, J., Benda, P., García-Mudarra, J.L., Ibáñez, C., 2013. Phylogeny and systematics of Old World serotine bats (genus Eptesicus, Vespertilionidae, Chiroptera): an integrative approach. Zool. Scr. 42, 441–457.

  27. Juste, J., Ruedi, M., Puechmaille, S.J., Salicini, I., Ibáñez, C., 2018. Two new cryptic bat species within the Myotis nattereri species complex (Vespertilionidae, Chiroptera) from the western palaearctic. Acta Chiropterol. 20, 285–300.

  28. Kanuch, P., Hajkova, P., Rehak, Z., Bryja, J., 2007. A rapid PCR-based test for species identification of two cryptic bats, Pipistrellus pipistrellus and P. pygmaeus, and its application on museum and dropping samples. Acta Chiropterol. 9, 277–282.

  29. Kokurewicz, T., Ogórek, R., Pusz, W., Matkowski, K., 2016. Bats increase the number of cultivable airborne fungi in the “Nietoperek” bat reserve in western Poland. Microb. Ecol. 72, 36–48.

  30. Lisón, F., Palazón, J.A., Calvo, J.F., 2013. Effectiveness of the Natura 2000 network for the conservation of cave-dwelling bats in the Mediterranean region. Anim. Conserv. 16 (528), 537.

  31. Lundy, M.G., Aughney, T., Montgomery, W.I., Roche, N., 2011. Landscape conservation for Irish bats and species specific roosting characteristics. Bat Conservation Ireland.

  32. Marnell, F., Presetnik, P., 2010. Protection of overground roosts for bats (particularly roosts in buildings of cultural heritage importance). EUROBATS Publication Series No. 4 (English version). UNEP / EUROBATS Secretariat, Bonn, Germany, 57 pp.

  33. Mayer, F., von Helversen, O., 2001. Cryptic diversityin European bats. Proc. R. Soc. Lond. B 268, 1825–1832.

  34. McAney, K., 1999. Mines as roosting sites for bats: their potential and protection. Biol. Environ. 99B, 63–65.

  35. McAney, K., Hanniffy, R., 2015. The Vincent Wildlife Trust’s Irish Bat Box Schemes. The Vincent Wildlife Trust, Donaghpatrick, Headford, Ireland, 56 pp.

  36. Mitchell-Jones, A.J., 2004. Bat Mitigation Guidelines. English Nature, Peterborough, UK.

  37. Meyer, C.F.J., 2015. Methodological challenges in bat monitoring population- and assembly- level changes for anthropogenic impact assessment. Mamm. Biol. 80, 159–169.

  38. Monterroso, P., Godinho, R., Oliveira, T., Ferreras, P., Kelly, M.J., Morin, D.J., Waits, L.P., Alves, P.C., Mills, L.S., 2019. Feeding ecological knowledge: the underutilised power of faecal DNA approaches for carnivore diet analysis. Mam. Rev. 49, 97–112.

  39. Moran, S., 2009. Non-invasive genetic identification of small mammals and investigating multiple paternity in the European hedgehog. Unpublished PhD thesis, Waterford Institute of Technology, Ireland.

  40. Moran, S., Turner, P.D., O’Reilly, C., 2008. Non-invasive genetic identification of small mammal species using real-time polymerase chain reaction. Mol. Ecol. Res. 8, 1267–1269.

  41. Mullins, J., Statham, M.J., Roche, T., Turner, P.D., O’Reilly, C., 2010. Remotely plucked hair genotyping: a reliable and non-invasive method for censusing pine marten (Martes martes, L. 1758) populations. Eur. J. Wildlife Res. 56, 443–453.

  42. Murphy, M.A., Kendall, K.C., Robinson, A., Waits, L.P., 2007. The impact of time and field conditions on brown bear (Ursus arctos) faecal DNA amplification. Conserv. Genet. 8, 1219–1224.

  43. National Parks and Wildlife Service, 2019. The status of EU protected habitats and species in Ireland. Volume 3: Species. Unpublished NPWS report.

  44. O’Mahony, D.T., Powell, C., Power, J., Hanniffy, R., Marnell, F., Turner, P., O’Reilly, C., 2017. Non-invasively determined multi-site variation in pine marten Martes martes density, a recovering carnivore in Europe. Eur. J. Wildl. Res. 63, 48.

  45. O’Mahony, D.T., Turner, P., O’Reilly, C., 2015. Pine marten (Martes martes) abundance in an insular mountainous region using non-invasive techniques. Eur. J. Wildl. Res. 61, 103–110.

  46. O’Meara, D.B., McDevitt, A.D., O’Neill, D., Harrington, A.P., Turner, P., Carr, W., Desmond, M., Lawton, C., Marnell, F., Rubalcava, S., Sheehy, E., Sleeman, D.P., Tosh, D.G., Waters, C., O’Reilly, C., 2018. Retracing the history and planning the future of the red squirrel (Sciurus vulgaris) in Ireland using non-invasive genetics. Mamm. Res. 63, 173–184.

  47. O’Meara, D.B., Sheehy, E., Turner, P., O’Mahony, D., Harrington, A., Denman, H., Lawton, C., MacPherson, J., O’Reilly, C., 2014. Non-invasive multispecies monitoring: real-time PCR detection of small mammal and squirrel prey DNA in pine marten (Martes martes) scats. Acta Theriol. 59, 111–117.

  48. O’Meara, D.B., Turner, P.D., Coffey, L., O’Reilly, C., 2012. Taqman assays for species identification of the red squirrel (Sciurus vulgaris) and the grey squirrel (Sciurus carolinensis). Conserv. Genet. Res. 4, 603–604.

  49. O’Neill, D., Turner, P.D., O’Meara, D.B., Chadwick, E.A., Coffey, L., O’Reilly, C., 2013. Development of novel real-time Taqman PCR assays for the species and sex identification of otter (Lutra lutra) and their application to noninvasive genetic monitoring. Mol. Ecol. Res. 13, 877–883.

  50. O’Reilly, C., Statham, M.J., Mullins, J., Turner, P.D., O’Mahony, D., 2008. Efficient species identification of pine marten (Martes martes) and red fox (Vulpes vulpes) scats using a 5′ nuclease real-time PCR assay. Conserv. Genet. 9, 735–738.

  51. Piggott, M.P., Taylor, A.C., 2003. Extensive evaluation of faecal preservation and DNA extraction methods in Australian native and introduced species. Aust. J. Zool. 51, 341–355.

  52. Poulton, S., 2006. An analysis of the usage of batboxes in England, Wales and Ireland for the Vincent Wildlife Trust. Unpublished Report for the Vincent Wildlife Trust.

  53. Puechmaille, S.J., Mathy, G., Petit, E.J., 2007. Good DNA from bat droppings. Acta Chiropterol. 9, 269–276.

  54. Puechmaille, S.J., Teeling, E.C., 2014. Non-invasive genetics can help find rare species: a case study with Rhinolophus mehelyi and R. euryale (Rhinolophidae: Chiroptera) in Western Europe. Mammalia 78, 251–255.

  55. Roche, N., 1998. A survey for bat roosts in Church of Ireland churches. Report for the Heritage Council, Kilkenny, Ireland.

  56. Roche, N., Aughney, T., Lundy, M., Marnell, F., 2014. Irish Bats in the 21st Century. Bat Conservation Ireland.

  57. Ruedi, M., Mayer, F., 2001. Molecular systematics of the genus Myotis (Vespertilionidae) suggests deterministic ecomorphological convergences. Mol. Phylogenet. Evol. 21, 436–448.

  58. Russo, D., Bosso, L., Ancilotto, L., 2018. Novel perspectives on bat insectivory highlight the value of this ecosystem service in farmland: Research frontiers and management implications. Agr. Ecosyst. Environ. 266, 31–38.

  59. Russo, D., Jones, G., 2015. Bats as bioindicators: an introduction. Mamm. Biol. 80, 157–158.

  60. Sargent, G., 1995. Bats in churches project. The Bat Conservation Trust, London, U.K.

  61. Sheehy, E., Lawton, C., 2014. Population crash in an invasive species following the recovery of a native predator: the case of the American grey squirrel and the European pine marten in Ireland. Biodivers. Conserv. 23, 753–774.

  62. Sheehy, E., Sutherland C, O’Reilly C, Lambin X. 2018. The enemy of my enemy is my friend: native pine marten recovery reverses the decline of the red squirrel by suppressing grey squirrel populations. Proc. R. Soc. B. 285, 20172603.

  63. Stebbings, R.E., Yalden, D.W., Herman, J.S., 2007. Which Bat is it? The Mammal Society, London, UK.

  64. Stone, E.L., Jones, G., Harris, S., 2013. Mitigating the effect of development on bats in England with derogation licensing. Conserv. Biol. 27, 1324–1334.

  65. Tournayre, O., Leuchtmann, M., Filippi-Codaccioni, O., Trillat, M., Piry, S., Pontier, D., Charbonnel, N., Galan, M., 2019. In silico and empirical evaluation of twelve COI & 16S metabarcoding primer sets for insectivorous diet analyses. BioRxiv doi:

  66. Van der Meij, T., Van Strien, A.J., Haysom, K.A., Dekker, J., Russ, J., Biala, K., Bihari, Z., Jansen, E., Langton, S., Kurali, A., Limpens, H., Meschede, A., Petersons, G., Presetnik, P., Prüger, J., Reiter, G., Rodrigues, L., Schorcht, W., Uhrin, M., Vintulis, V., 2015. Return of the bats? A prototype indicator of trends in European bat populations in underground hibernacula. Mamm. Biol. 80, 170–177.

  67. White, S., O’Neill, D., O’Meara, D.B., Shores, C., Harrington, A.P., O’Reilly, C., Weyman, G., Sleeman, D.P., 2013. A non-invasive genetic survey of otters (Lutra lutra) in an urban environment: a pilot study with citizen scientists. IUCN Otter Specialist Group Bulletin. 30/02.

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Harrington, A.P., O’Meara, D.B., Aughney, T. et al. Novel real-time PCR species identification assays for British and Irish bats and their application to a non-invasive survey of bat roosts in Ireland. Mamm Biol 99, 109–118 (2019) doi:10.1016/j.mambio.2019.10.005

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  • Non-invasive genetics
  • Conservation
  • Bats
  • Roost
  • Detection
  • Real-time PCR