Use of DNA metabarcoding to identify plants from environmental samples: comparisons with traditional approaches Christine E. Edwards 1, Denise L. Lindsay 2, Thomas Minckley 3, and Richard F. Lance 2 1 Center for Conservation and Sustainable Development, Missouri Botanical Garden, St. Louis, MO, 63166 2- US Army Engineer Research and Development Center, Environmental Laboratory, Vicksburg, MS, 39180 3 - University of Wyoming, Department of Geography and Program in Ecology, 1000 E. University Avenue, Laramie, WY, 82071
What is DNA metabarcoding? High-throughput, DNA-based identification of species from an environmental sample Biodiversity assessment (from an environmental sample) Diet analysis (from a fecal sample) Approach: Collect sample and extract DNA Use PCR with taxon-specific, DNA-barcoding primers to amplify DNA regions of target taxa Carry out next-generation DNA sequencing of samples Compare the resulting sequences to those in DNA sequence databases (NCBI) to identify the species of origin for each sequence.
Can we use DNA metabarcoding to understand the diversity of plants utilized by a pollinator? Nectar-feeding bats in the southwest US: The Mexican longtongued Bat (Choeronyctris mexicana) The lesser long-nosed bat (Leptonycteris curasoae)
Nectar-feeding bats in Southwest AZ Bat populations migrate in response to seasonal variation in food: Migration Northward along coast in Spring following blooming of Columnar cacti Populations roost in Southwestern US in summer-fall the migrate Southward inland in Fall following Agave sp. What plant species do they eat/pollinate while in AZ?
Questions: Can we use DNA metabarcoding to identify the plants utilized by two species of nectar-feeding bats? Can DNA metabarcoding detect likely food plant species from fecal or environmental samples? How do estimates of plant diversity using DNA metabarcoding compare to visual identification of pollen in samples? If different types of samples are used for metabarcoding, do they produce different estimates of diversity?
Sample Collection SPLAT: 5 mixed-species samples from different locations/dates Wren Bridge Fort Huachuca, AZ Pyeatt Cave Fort Huachuca, AZ
Sample collection-pollen swabs: 37 population samples from different locations and dates
Methods to quantify plant diversity: Visual identification of pollen DNA Metabarcoding: CTAB DNA extraction PCR of DNA regions used for plant DNA barcoding rbcl, psba-trnh, trnl-f Next gen sequencing: Each group of samples (from different sites, dates, and collection methods) was tagged to distinguish them in the resulting pool of sequences Samples pooled DNA sequenced using ¼-plate run on a 454 DNA sequencer Bioinformatics: Sorted sequences by tag Assembly to form contigs Annotation using BLAST searches of NCBI s nr database
99.7% Agave 0.3% Yucca Results-Visual identification Trace amounts of pollen from Anacardiaceae, Malvaceae, Asteraceae
Results-DNA metabarcoding 63,849 sequences with amplicon length >170bp Sequences were recovered from 4 of 5 SPLAT samples and 21 of 37 pollen-swab samples Pollen swab samples: 8,621 sequences (13.5% of total) 97.3% of pollen-swab sequences were plant targets SPLAT samples: 54,936 sequences (86% of total) 19.1% of SPLAT sequences were plant targets
Results-DNA metabarcoding Pollen-swab sequences SPLAT sequences agave Allium Amaranthaceae Anacardiaceae Apiaceae Asteraceae Brassicaceae Cactaceae conifers Fabaceae Hyacinthaceae Lamiids Lemna and wolffia Musa other agavaceae Poaceae Rosaceae Yucca Misc.
Questions: Can DNA metabarcoding detect likely food plant species from fecal or environmental samples? Probable food plants for bats were identified from both types of samples How do estimates of plant diversity using DNA metabarcoding compare to visual identification of pollen in samples? DNA metabarcoding detected more species, some of which may be food plants, some may be by-catch If different types of samples are used for metabarcoding, do they produce different estimates of diversity? Pollen-swab samples and visual identification of pollen recovered similar results Likely species from which bats are consuming pollen and nectar Species composition in SPLAT samples differed May be biased by variation in number of chloroplasts present in plant tissue consumed (i.e. fruit may have more plastids than pollen and may bias results) May more fully represent the total diet of the bats, and diet may be more diverse than previously thought
Conclusions DNA metabarcoding is potentially a powerful approach for understanding plant-animal interactions and quantifying plant diversity in an environmental sample Strengths: Can process more samples, faster, than visual pollen identification More sensitive than visual pollen identification Able to quantify diversity from unidentifiable samples (fecal samples) Weaknesses: Identification of species is dependent on the number and accuracy of NCBI databases May be prone to environmental or lab contamination Absolute accuracy is unknown Use of chloroplast DNA may bias results because chloroplasts are not present equally across species or different plant tissues Success of this approach will depend on: Continued development of DNA sequence databases Careful sample processing and negative controls Additional research to quantify accuracy
Future directions How accurate is DNA metabarcoding for diet analysis? Test accuracy (diversity and proportion) using zoo populations fed a known diet Test relative accuracy of chloroplast vs. nuclear markers Can we use DNA metabarcoding of fecal samples to accurately quantify multiple aspects of bat populations? Species identification Sex of individual Infection with White-Nose Syndrome Diet Parasite Diversity and Load Population Size
Potential applications of plant DNA metabarcoding for land management Understand diet resources of wildlife Temporal sampling can help understand long-term or seasonal changes in diet Protect plant communities or species that are utilized by endangered wildlife Use animals or insects to sample flowering plant diversity to conduct vegetation surveys Monitor for the presence of endangered or invasive plant species Use stratified samples from lake sediments to understand temporal changes in vegetation communities
Acknowledgments Funding: US Army 6.1 Basic Research Program Permitting and access: Sheridan Stone, Wildlife Biologist, Fort Huachuca Lab assistance: Michael Jung (ERDC) Xin Guan (ERDC) Sample collection: Eric Britzke (ERDC) Ronnie Sidner (Environmental Consultant) George Ferguson (University of Arizona) Pamela Bailey (ERDC)
Can we use DNA metabarcoding to identify the plants utilized by two species of nectarfeeding bats? - DNA metabarcoding successfully identified plant DNA sequences from both types of samples How do estimates of plant diversity using DNA metabarcoding compare to visual identification of pollen in samples? DNA metabarcoding detected more plant species than visual identification Some additional species are likely food resources and some may be by-catch from incidental transfer
If different types of samples are used for metabarcoding, do they produce different estimates of diversity? DNA sequencing of pollen-swab samples and visual identification of pollen recovered similar results Likely represent the species from which bats are consuming pollen and nectar Species composition in SPLAT samples differed May more accurately represent the total diet of the bats, and diet may be more diverse than previously thought
How do estimates of plant diversity using DNA metabarcoding compare to visual identification of pollen in samples? DNA metabarcoding detected more plant species than visual identification Some additional species are likely food resources and some may be by-catch from incidental transfer
Study Design 2 species of nectar-feeding bats The lesser long-nosed bat (Leptonycteris curasoae) The Mexican long-tongued Bat (Choeronyctris mexicana) 2 ways of collecting pollen Fecal samples Fur swabs 2 approaches for analyzing pollen diversity Visual identification of pollen DNA metabarcoding