Optimisation of a pollen DNA metabarcoding method for diet analysis of flying-foxes (Pteropus spp.)
Karen L. Bell
A B E , Kathryn L. Batchelor A , Matt Bradford C , Adam McKeown D , Stewart L. Macdonald C and David Westcott C
+ Author Affiliations
- Author Affiliations
A CSIRO Health and Biosecurity, Floreat, WA 6014, Australia.
B University of Western Australia, School of Biological Sciences, Crawley, WA 6008, Australia.
C CSIRO Land and Water, Atherton, Qld 4883, Australia.
D CSIRO Land and Water, Waite Campus, Adelaide, SA 5064, Australia.
E Corresponding author. Email: karen.bell@uwa.edu.au
Australian Journal of Zoology - https://doi.org/10.1071/ZO20085
Submitted: 19 October 2020 Accepted: 4 October 2021 Published online: 28 October 2021
Journal Compilation © CSIRO 2021 Open Access CC BY-NC-ND
Abstract
Determining the diet of flying-foxes can increase understanding of how they function as pollinators and seed dispersers, as well as managing any negative impacts of large roosts. Traditional methods for diet analysis are time consuming, and not feasible to conduct for hundreds of animals. In this study, we optimised a method for diet analysis, based on DNA metabarcoding of environmental DNA (eDNA) from pollen and other plant parts in the faeces. We found that existing eDNA metabarcoding protocols are suitable, with the most useful results being obtained using a commercial food DNA extraction kit, and sequencing 350–450 base pairs of a DNA barcode from the internally transcribed spacer region (ITS2), with ~550 base pairs of the chloroplast rubisco large subunit (rbcL) as a secondary DNA barcode. A list of forage plants was generated for the little red flying-fox (Pteropus scapulatus), the black flying-fox (Pteropus alecto) and the spectacled flying-fox (Pteropus conspicillatus) from our collection sites across Queensland. The diets were determined to comprise predominantly Myrtaceae species, particularly those in the genera Eucalyptus, Melaleuca and Corymbia. With more plant genomes becoming publicly available in the future, there are likely to be further applications of eDNA methods in understanding the role of flying-foxes as pollinators and seed dispersers.
Keywords: pollen, DNA metabarcoding, DNA barcoding, environmental DNA, Pteropus, nectar, Myrtaceae, plant-animal interactions, diet, foraging.
References
Aziz, S. A., Clements, G. R., Peng, L. Y., Campos-Arceiz, A., McConkey, K. R., Forget, P.-M., and Gan, H. M. (2017). Elucidating the diet of the island flying fox (Pteropus hypomelanus) in Peninsular Malaysia through Illumina Next-Generation Sequencing. PeerJ 5, e3176.| Elucidating the diet of the island flying fox (Pteropus hypomelanus) in Peninsular Malaysia through Illumina Next-Generation Sequencing.Crossref | GoogleScholarGoogle Scholar | 28413729PubMed |
Aziz, S. A., McConkey, K. R., Tanalgo, K., Sritongchuay, T., Low, M.-R., Yong, J. Y., Mildenstein, T. L., Nuevo-Diego, C. E., Lim, V.-C., and Racey, P. A. (2021). The critical importance of Old World fruit bats for healthy ecosystems and economies. Frontiers in Ecology and Evolution 9, 641411..
Bayly, M. J., and Ladiges, P. Y. (2007). Divergent paralogues of ribosomal DNA in eucalypts (Myrtaceae). Molecular Phylogenetics and Evolution 44, 346–356.
| Divergent paralogues of ribosomal DNA in eucalypts (Myrtaceae).Crossref | GoogleScholarGoogle Scholar | 17188000PubMed |
Bell, K. L., Fowler, J., Burgess, K. S., Dobbs, E. K., Gruenewald, D., Lawley, B., Morozumi, C., and Brosi, B. J. (2017a). Applying pollen DNA metabarcoding to the study of plant–pollinator interactions. Applications in Plant Sciences 5, 1600124.
| Applying pollen DNA metabarcoding to the study of plant–pollinator interactions.Crossref | GoogleScholarGoogle Scholar |
Bell, K. L., Loeffler, V. M., and Brosi, B. J. (2017b). An rbcL reference library to aid in the identification of plant species mixtures by DNA metabarcoding. Applications in Plant Sciences 5, 1600110.
| An rbcL reference library to aid in the identification of plant species mixtures by DNA metabarcoding.Crossref | GoogleScholarGoogle Scholar |
Bell, K. L., Burgess, K. S., Botsch, J. C., Dobbs, E. K., Read, T. D., and Brosi, B. J. (2019). Quantitative and qualitative assessment of pollen DNA metabarcoding using constructed species mixtures. Molecular Ecology 28, 431–455.
| Quantitative and qualitative assessment of pollen DNA metabarcoding using constructed species mixtures.Crossref | GoogleScholarGoogle Scholar | 30118180PubMed |
Bengtsson-Palme, J., Ryberg, M., Hartmann, M., Branco, S., Wang, Z., Godhe, A., De Wit, P., Sánchez-García, M., Ebersberger, I., de Sousa, F., Amend, A. S., Jumpponen, A., Unterseher, M., Kristiansson, E., Abarenkov, K., Bertrand, Y. J. K., Sanli, K., Eriksson, K. M., Vik, U., Veldre, V., and Nilsson, R. H. (2013). Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data. Methods in Ecology and Evolution 4, 914–919.
| Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data.Crossref | GoogleScholarGoogle Scholar |
Bista, I., Carvalho, G. R., Tang, M., Walsh, K., Zhou, X., Hajibabaei, M., Shokralla, S., Seymour, M., Bradley, D., Liu, S., Christmas, M., and Creer, S. (2018). Performance of amplicon and shotgun sequencing for accurate biomass estimation in invertebrate community samples. Molecular Ecology Resources 18, 1020–1034.
| Performance of amplicon and shotgun sequencing for accurate biomass estimation in invertebrate community samples.Crossref | GoogleScholarGoogle Scholar |
Bradford, M., Venz, M., Hogan, L., Smith, G., Bell, K. L., McKeown, A., Vanderduys, E., Macdonald, S. L., Ford, A., Eyre, T., and Westcott, D. (2020). The diet of the little red flying-fox (Pteropus scapulatus) in Queensland. In ‘The little red flying-fox: ecology and management of Australia’s most enigmatic flying-fox’. (Eds D. Westcott, et al.) pp. 245–277. A report to the Queensland Department of Environment and Science, CSIRO.
Breitwieser, F. P., Pertea, M., Zimin, A. V., and Salzberg, S. L. (2019). Human contamination in bacterial genomes has created thousands of spurious proteins. Genome Research 29, 954–960.
| Human contamination in bacterial genomes has created thousands of spurious proteins.Crossref | GoogleScholarGoogle Scholar | 31064768PubMed |
Brooker, M. I. H., and Kleinig, D. A. (2004). ‘Field Guide to Eucalypts. Volume 3. Northern Australia.’ (Bloomings Books: Melbourne, Australia.)
Callahan, B. J., McMurdie, P. J., Rosen, M. J., Han, A. W., Johnson, A. J. A., and Holmes, S. P. (2016). DADA2: high-resolution sample inference from Illumina amplicon data. Nature Methods 13, 581–583.
| DADA2: high-resolution sample inference from Illumina amplicon data.Crossref | GoogleScholarGoogle Scholar | 27214047PubMed |
CBOL Plant Working Group (2009). A DNA barcode for land plants. Proceedings of the National Academy of Sciences of the United States of America 106, 12794–12797.
| A DNA barcode for land plants.Crossref | GoogleScholarGoogle Scholar | 19666622PubMed |
Centre for Australian National Biodiversity Research (2020). ‘EUCLID Eucalyptus of Australia.’ 4th edn. (Centre for Australian Biodiversity Research (CANBR): Canberra.)
Chan, A. A. Q., Aziz, S. A., Clare, E. L., and Coleman, J. L. (2021). Diet, ecological role and potential ecosystem services of the fruit bat, Cynopterus brachyotis, in a tropical city. Urban Ecosystems 24, 251–263.
| Diet, ecological role and potential ecosystem services of the fruit bat, Cynopterus brachyotis, in a tropical city.Crossref | GoogleScholarGoogle Scholar |
Chen, S., Yao, H., Han, J., Liu, C., Song, J., Shi, L., Zhu, Y., Ma, X., Gao, T., Pang, X., Luo, K., Li, Y., Li, X., Jia, X., Lin, Y., and Leon, C. (2010). Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One 5, e8613.
| Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species.Crossref | GoogleScholarGoogle Scholar | 20062805PubMed |
Costion, C. M., Lowe, A. J., Rossetto, M., Kooyman, R. M., Breed, M. F., Ford, A., and Crayn, D. M. (2016). Building a plant DNA barcode reference library for a diverse tropical flora: an example from Queensland, Australia. Diversity 8, 5.
| Building a plant DNA barcode reference library for a diverse tropical flora: an example from Queensland, Australia.Crossref | GoogleScholarGoogle Scholar |
Cristescu, M. E. (2014). From barcoding single individuals to metabarcoding biological communities: towards an integrative approach to the study of global biodiversity. Trends in Ecology & Evolution 29, 566–571.
| From barcoding single individuals to metabarcoding biological communities: towards an integrative approach to the study of global biodiversity.Crossref | GoogleScholarGoogle Scholar |
Davis, N. M., Proctor, D. M., Holmes, S. P., Relman, D. A., and Callahan, B. J. (2018). Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. Microbiome 6, 226.
| Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data.Crossref | GoogleScholarGoogle Scholar | 30558668PubMed |
Dormontt, E. E., van Dijk, K.-j., Bell, K. L., Biffin, E., Breed, M. F., Byrne, M., Caddy-Retalic, S., Encinas-Viso, F., Nevill, P. G., Shapcott, A., Young, J. M., Waycott, M., and Lowe, A. J. (2018). Advancing DNA barcoding and metabarcoding applications for plants requires systematic analysis of herbarium collections – an Australian perspective. Frontiers in Ecology and Evolution 6, 134.
| Advancing DNA barcoding and metabarcoding applications for plants requires systematic analysis of herbarium collections – an Australian perspective.Crossref | GoogleScholarGoogle Scholar |
Fahner, N. A., Shokralla, S., Baird, D. J., and Hajibabaei, M. (2016). Large-scale monitoring of plants through environmental DNA metabarcoding of soil: recovery, resolution, and annotation of four DNA markers. PLoS ONE 11, e0157505.
| Large-scale monitoring of plants through environmental DNA metabarcoding of soil: recovery, resolution, and annotation of four DNA markers.Crossref | GoogleScholarGoogle Scholar | 27310720PubMed |
Fleming, T. H., Geiselman, C., and Kress, W. J. (2009). The evolution of bat pollination: a phylogenetic perspective. Annals of Botany 104, 1017–1043.
| The evolution of bat pollination: a phylogenetic perspective.Crossref | GoogleScholarGoogle Scholar | 19789175PubMed |
Gómez-Rodríguez, C., Crampton-Platt, A., Timmermans, M. J. T. N., Baselga, A., and Vogler, A. P. (2015). Validating the power of mitochondrial metagenomics for community ecology and phylogenetics of complex assemblages. Methods in Ecology and Evolution 6, 883–894.
| Validating the power of mitochondrial metagenomics for community ecology and phylogenetics of complex assemblages.Crossref | GoogleScholarGoogle Scholar |
Halpin, K., Young, P. L., Field, H., and Mackenzie, J. S. (1999). Newly discovered viruses of flying foxes. Veterinary Microbiology 68, 83–87.
| Newly discovered viruses of flying foxes.Crossref | GoogleScholarGoogle Scholar | 10501164PubMed |
Hebert, P. D. N., Cywinska, A., Ball, S. L., and DeWaard, J. R. (2003). Biological identifications through DNA barcodes. Proceedings. Biological Sciences 270, 313–321.
| Biological identifications through DNA barcodes.Crossref | GoogleScholarGoogle Scholar |
Ji, Y., Huotari, T., Roslin, T., Schmidt, N. M., Wang, J., Yu, D. W., and Ovaskainen, O. (2020). SPIKEPIPE: a metagenomic pipeline for the accurate quantification of eukaryotic species occurrences and intraspecific abundance change using DNA barcodes or mitogenomes. Molecular Ecology Resources 20, 256–267.
| SPIKEPIPE: a metagenomic pipeline for the accurate quantification of eukaryotic species occurrences and intraspecific abundance change using DNA barcodes or mitogenomes.Crossref | GoogleScholarGoogle Scholar | 31293086PubMed |
Kolter, A., Gemeinholzer, B., and Boatwright, J. S. (2021). Plant DNA barcoding necessitates marker-specific efforts to establish more comprehensive reference databases. Genome 64, 265–298.
| Plant DNA barcoding necessitates marker-specific efforts to establish more comprehensive reference databases.Crossref | GoogleScholarGoogle Scholar | 32649839PubMed |
Kress, W. J., Erickson, D. L., Jones, F. A., Swenson, N. G., Perez, R., Sanjur, O., and Bermingham, E. (2009). Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama. Proceedings of the National Academy of Sciences of the United States of America 106, 18621–18626.
| Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama.Crossref | GoogleScholarGoogle Scholar | 19841276PubMed |
Lang, D., Tang, M., Hu, J., and Zhou, X. (2019). Genome‐skimming provides accurate quantification for pollen mixtures. Molecular Ecology Resources 19, 1433–1446.
| Genome‐skimming provides accurate quantification for pollen mixtures.Crossref | GoogleScholarGoogle Scholar | 31325909PubMed |
Lewin, H. A., Robinson, G. E., Kress, W. J., Baker, W. J., Coddington, J., Crandall, K. A., Durbin, R., Edwards, S. V., Forest, F., Gilbert, M. T. P., Goldstein, M. M., Grigoriev, I. V., Hackett, K. J., Haussler, D., Jarvis, E. D., Johnson, W. E., Patrinos, A., Richards, S., Castilla-Rubio, J. C., van Sluys, M.-A., Soltis, P. S., Xu, X., Yang, H., and Zhang, G. (2018). Earth BioGenome Project: sequencing life for the future of life. Proceedings of the National Academy of Sciences of the United States of America 115, 4325–4333.
| Earth BioGenome Project: sequencing life for the future of life.Crossref | GoogleScholarGoogle Scholar | 29686065PubMed |
Li, D.-Z., Gao, L.-M., Li, H.-T., Wang, H., Ge, X.-J., Liu, J.-Q., Chen, Z.-D., Zhou, S.-L., Chen, S.-L., Yang, J.-B., Fu, C.-X., Zeng, C.-X., Yan, H.-F., Zhu, Y.-J., Sun, Y.-S., Chen, S.-Y., Zhao, L., Wang, K., Yang, T., and Duan, G.-W. (2011). Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants. Proceedings of the National Academy of Sciences of the United States of America 108, 19641–19646.
| Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants.Crossref | GoogleScholarGoogle Scholar | 22100737PubMed |
Lim, V.-C., Clare, E. L., Littlefair, J. E., Ramli, R., Bhassu, S., and Wilson, J.-J. (2018a). Impact of urbanisation and agriculture on the diet of fruit bats. Urban Ecosystems 21, 61–70.
| Impact of urbanisation and agriculture on the diet of fruit bats.Crossref | GoogleScholarGoogle Scholar |
Lim, V.-C., Ramli, R., Bhassu, S., and Wilson, J.-J. (2018b). Pollination implications of the diverse diet of tropical nectar-feeding bats roosting in an urban cave. PeerJ 6, e4572.
| Pollination implications of the diverse diet of tropical nectar-feeding bats roosting in an urban cave.Crossref | GoogleScholarGoogle Scholar | 29607265PubMed |
Lima, R. A. F. d., Oliveira, A. A. d., Colletta, G. D., Flores, T. B., Coelho, R. L. G., Dias, P., Frey, G. P., Iribar, A., Rodrigues, R. R., Souza, V. C., and Chave, J. (2018). Can plant DNA barcoding be implemented in species-rich tropical regions? A perspective from São Paulo State, Brazil. Genetics and Molecular Biology 41, 661–670.
| Can plant DNA barcoding be implemented in species-rich tropical regions? A perspective from São Paulo State, Brazil.Crossref | GoogleScholarGoogle Scholar |
Markus, N., and Hall, L. (2004). Foraging behaviour of the black flying-fox (Pteropus alecto) in the urban landscape of Brisbane, Queensland. Wildlife Research 31, 345–355.
| Foraging behaviour of the black flying-fox (Pteropus alecto) in the urban landscape of Brisbane, Queensland.Crossref | GoogleScholarGoogle Scholar |
Marshall, A. G. (1983). Bats, flowers and fruit: evolutionary relationships in the Old World. Biological Journal of the Linnean Society 20, 115–135.
| Bats, flowers and fruit: evolutionary relationships in the Old World.Crossref | GoogleScholarGoogle Scholar |
Marshall, A. G. (1985). Old World phytophagous bats (Megachiroptera) and their food plants: a survey. Zoological Journal of the Linnean Society 83, 351–369.
| Old World phytophagous bats (Megachiroptera) and their food plants: a survey.Crossref | GoogleScholarGoogle Scholar |
Moorhouse-Gann, R. J., Dunn, J. C., de Vere, N., Goder, M., Cole, N., Hipperson, H., and Symondson, W. O. C. (2018). New universal ITS2 primers for high-resolution herbivory analyses using DNA metabarcoding in both tropical and temperate zones. Scientific Reports 8, 8542.
| New universal ITS2 primers for high-resolution herbivory analyses using DNA metabarcoding in both tropical and temperate zones.Crossref | GoogleScholarGoogle Scholar | 29867115PubMed |
Oksanen, J., Blanchet, F.G., Friendly, M., Kindt, R., Legendre, P., McGlinn, D., Minchin, P.R., O’Hara, R.B., Simpson, G.L., Solymos, P., Stevens, M.H.H., Szoecs, E., and Wagner, H. (2019). Community Ecology Package Version 2.6. Available at: https://cran.r-project.org/web/packages/vegan/vegan.pdf
Palmer, C., Price, O., and Bach, C. (2000). Foraging ecology of the black flying fox (Pteropus alecto) in the seasonal tropics of the Northern Territory, Australia. Wildlife Research 27, 169.
| Foraging ecology of the black flying fox (Pteropus alecto) in the seasonal tropics of the Northern Territory, Australia.Crossref | GoogleScholarGoogle Scholar |
Palmieri, L., Bozza, E., and Giongo, L. (2009). Soft fruit traceability in food matrices using real-time PCR. Nutrients 1, 316–328.
| Soft fruit traceability in food matrices using real-time PCR.Crossref | GoogleScholarGoogle Scholar | 22253987PubMed |
Parmentier, I., Duminil, J., Kuzmina, M., Philippe, M., Thomas, D. W., Kenfack, D., Chuyong, G. B., Cruaud, C., and Hardy, O. J. (2013). How effective are DNA barcodes in the identification of African rainforest trees? PLoS One 8, e54921.
| How effective are DNA barcodes in the identification of African rainforest trees?Crossref | GoogleScholarGoogle Scholar | 23565134PubMed |
Parry-Jones, K. A., and Augee, M. L. (2001). Factors affecting the occupation of a colony site in Sydney, New South Wales by the grey-headed flying-fox Pteropus poliocephalus (Pteropodidae). Austral Ecology 26, 47–55.
Parsons, J. G., Cairns, A., Johnson, C. N., Robson, S. K. A., Shilton, L. A., and Westcott, D. A. (2006). Dietary variation in spectacled flying foxes (Pteropus conspicillatus) of the Australian Wet Tropics. Australian Journal of Zoology 54, 417–428.
| Dietary variation in spectacled flying foxes (Pteropus conspicillatus) of the Australian Wet Tropics.Crossref | GoogleScholarGoogle Scholar |
R Core Team (2016). ‘R: A Language and Environment for Statistical Computing.’ (R Foundation for Statistical Computing: Vienna, Austria.)
Schuster, T. M., Setaro, S. D., Tibbits, J. F. G., Batty, E. L., Fowler, R. M., McLay, T. G. B., Wilcox, S., Ades, P. K., and Bayly, M. J. (2018). Chloroplast variation is incongruent with classification of the Australian bloodwood eucalypts (genus Corymbia, family Myrtaceae). PLoS One 13, e0195034.
| Chloroplast variation is incongruent with classification of the Australian bloodwood eucalypts (genus Corymbia, family Myrtaceae).Crossref | GoogleScholarGoogle Scholar | 29668710PubMed |
Shapcott, A., Forster, P. I., Guymer, G. P., McDonald, W. J. F., Faith, D. P., Erickson, D., and Kress, W. J. (2015). Mapping biodiversity and setting conservation priorities for SE Queensland’s rainforests using DNA barcoding. PLoS One 10, e0122164.
| Mapping biodiversity and setting conservation priorities for SE Queensland’s rainforests using DNA barcoding.Crossref | GoogleScholarGoogle Scholar | 25803607PubMed |
Sickel, W., Ankenbrand, M. J., Grimmer, G., Holzschuh, A., Härtel, S., Lanzen, J., Steffan-Dewenter, I., and Keller, A. (2015). Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach. BMC Ecology 15, 20.
| Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach.Crossref | GoogleScholarGoogle Scholar | 26194794PubMed |
Tait, J., Perotto-Baldivieso, H. L., McKeown, A., and Westcott, D. A. (2014). Are flying-foxes coming to town? Urbanisation of the spectacled flying-fox (Pteropus conspicillatus) in Australia. PLoS One 9, e109810.
| Are flying-foxes coming to town? Urbanisation of the spectacled flying-fox (Pteropus conspicillatus) in Australia.Crossref | GoogleScholarGoogle Scholar | 25295724PubMed |
Tidemann, C. R., and Nelson, J. E. (2004). Long-distance movements of the grey-headed flying fox (Pteropus poliocephalus). Journal of Zoology 263, 141–146.
| Long-distance movements of the grey-headed flying fox (Pteropus poliocephalus).Crossref | GoogleScholarGoogle Scholar |
Westcott, D. A., Dennis, A. J., McKeown, A., Bradford, M., and Margules, C. R. (2001). ‘The Spectacled Flying Fox, Pteropus conspicillatus, in the Context of the World Heritage Values of the Wet Tropics World Heritage Area.’ (CSIRO Sustainable Ecosystems and the Rainforest Cooperative Research Centre: Atherton, Australia.)
White, T. J., Bruns, T., Lee, S., and Taylor, J. W. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In ‘PCR Protocols: a Guide to Methods and Applications’. (Eds M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White.) pp. 315–322. (Academic Press: New York.)
