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APPLICATIONS OF INTENSIVE DNA BARCODING PROJECTS FOR ADDRESSING GAPS IN FUNGAL BIODIVERSITY KNOWLEDGE PowerPoint Presentation
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APPLICATIONS OF INTENSIVE DNA BARCODING PROJECTS FOR ADDRESSING GAPS IN FUNGAL BIODIVERSITY KNOWLEDGE
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  1. APPLICATIONS OF INTENSIVE DNA BARCODING PROJECTS FOR ADDRESSING GAPS IN FUNGAL BIODIVERSITY KNOWLEDGE Matteo M. Garbelotto1, Vincent Robert2, Conrad Schoch3, Todd Osmundson1 1 University of California, Berkeley, CA, USA 2 Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands 3 National Center for Biotechnology Information, Bethesda, MD, USA

  2. MatteoU.C. Berkeley Forest Pathology & Mycology Lab

  3. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS Fungi are diverse and poorly-known Most are cryptic over the majority of their life cycle Many are economically important: - Pathogens - Mutualists - Foods, medicines, industrial products

  4. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Unknowns: • Diversity of life cycle stages (e.g., endophyte to pathogen transitions) • Host ranges • Geographic ranges / biogeography • Community ecology

  5. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Unknowns: • Diversity of life cycle stages (e.g., endophyteto pathogen transitions) • Host ranges • Geographic ranges / biogeography • Community ecology All of these characters are relevant to understanding ecology and evolution of fungi, and tracking the spread of fungal species.

  6. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Unknowns: • Diversity of life cycle stages (e.g., endophyte to pathogen transitions) • Host ranges • Geographic ranges / biogeography • Community ecology DNA Barcoding is a useful and important tool for identifying fungi (rapid • able to detect cryptic fungi) and thereby understanding these factors…

  7. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Unknowns: • Diversity of life cycle stages (e.g., endophyte to pathogen transitions) • Host ranges • Geographic ranges / biogeography • Community ecology … However, utility of this tool is dependent upon existence of comprehensive sequence databases and methods for confidently assigning taxonomic identities via sequence comparisons.

  8. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS Large-scale barcoding projects can aid in closing the sequencing gap. Types of foci: - Institutional - Geographic - Ecological

  9. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • 2 recent projects: • Barcoding the Venice Museum of Natural History Fungal Collection (Institutional) • The MooreaBiocode Project (Geographic ) • Issues/Questions: • What types of target are most effective? • What types of sampling are most effective? • What are the implications for barcoding strategy?

  10. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Barcoding the Venice Fungal Collection • Institutional strategy – barcode large number of samples from a single herbarium • Strengths: • Links to Italy’s largest amateur mycological society (AssociazioneMicologicaBresadola) • Collaboration with taxonomic experts • Taxonomic diversity of collections • Collections relatively recent • Habitat diversity (Alps  plains  Apennines  Mediterranean)

  11. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS Sampling Localities

  12. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Sampling : • Focus on breadth • Attempt to obtain Barcode for every macrofungalspecies in the collection (~ 6000) • Replication for well-represented species • Good coverage for Agaricales; additional coverage within Basidiomycota & Ascomycota

  13. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS Results ~31K collections ~5K collections sampled 2763 specimens PCR positive [Age; taxon] • 1400 specimens Sequence positive [Sequencing failure; contamination; paralogy]: • 1100 double-stranded • ~300 single-stranded

  14. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Collection age affects sequencing success: Pearson Chi-square test of independence (N=2648, DF=2): p < 0.0001

  15. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Taxon (controlled for collection age) affects sequencing success: 1980s-90s 2000s Pearson Chi-square tests of independence): p < 0.0001

  16. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Classification potential of ITS barcodes: • Can ITS barcodes predict membership in genera and families based on overall similarity? • Distance / UPGMA approach based on ITS1 + ITS2 distance matrices • Functionalities available in BioloMICS software (www.bio-aware.com)

  17. UPGMA tree showing sequence assignment to genera and families; genus Cortinarius (family Cortinariaceae) shown

  18. Cluster-based (NMDS) assignments to genera

  19. Cluster-based (NMDS) assignments to families

  20. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Classification potential of ITS barcodes: • May reveal errors in classification; e.g., segregate genera in Coprinoid fungi

  21. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Is there a ‘barcoding gap’? • Compared number of nucleotide differences within and between identified taxa • Assessed 2 types of potential error: • - False negative (same ITS for different species) • - False positive (>1 ITS for one species)

  22. Is there a ‘percent similarity’ threshold for assignment to genera’? Minimum Similarity

  23. Within- and between-species nucleotide divergence (bp) Frequency of pairwise comparisons Nucleotide differences (bp)

  24. Within-species nucleotide divergence (bp) Frequency of pairwise comparisons Nucleotide differences (bp) ~1-2%

  25. “Barcoding gap” ratio (within/between species counts) by level of nucleotide dissimilarity Ratio within / between species Nucleotide differences (bp)

  26. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • When differentiation fails (same ITS sequence for species identified as different): • 60 pairs with 0 bp difference but not belonging to the same species: • 59 congeners (synonyms, species complexes or ‘minor’ misidentifications) • 1 epigeous - sequestrate confamilial pair (Leucoagaricusmedioflavoides + Endoptychumagaricoides)

  27. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • When differentiation fails (same ITS sequence for species identified as different): • 77 pairs with 1 bp difference but not belonging to the same species: • 73 congeners • 2 ‘major’ misidentifications or mixed samples (Boletus vs. Inocybe; Sarcosphaera vs. Psathyrella) • 1 ‘moderate’ misidentification (Pholiotina vs. Galerina)

  28. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Examination of ITS1 and ITS2 as “mini-barcodes” • Improved sequencing success rate: • ITS1 amplified using primers ITS1F + ITS2 for 30 randomly-selected samples previously negative for full-length PCR amplifications (using ITS1F + ITS4) from 3 large genera: • GENUSPCR-POSITIVESEQUENCE • - Cortinarius 30/30 (100%) 24/30 (80%) • - Russula30/30 (100%) 27/30 (90%) • - Mycena 27/30 (90%) 4/30 (13%)

  29. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Examination of ITS1 and ITS2 as “mini-barcodes” • Classification potential remains strong • ITS1 exhibits higher correlation to full-length ITS

  30. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Conclusions, Venice Herbarium Project: • Similarity-based taxonomic assignment using ITS sequences works reasonably well at genus and family levels (but not flawless) • At species level, incorrect assignments (assessed for morphospecies) in both “false negative” and “false positive” directions. • A 1-2% divergence cutoff eliminates most “false positives” (>1 ITS sequence per morphospecies); however, “false negatives” (>1 taxon per ITS sequence occur even at 0-1 nucleotide difference.

  31. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Conclusions, Venice Herbarium Project (continued): • “Mini-barcodes” improve success rates and retain ability to classify sequences; ITS1 outperformed ITS2 for the taxa examined

  32. INTRODUCTION VENICE HERBARIUM MOOREA BIOCODE CONCLUSIONS • Conclusions , Venice Herbarium Project (continued): • Value of herbaria in facilitating a large collection approach to barcoding • Value to herbaria (increase ‘relevance,’ streamline use of collections)

  33. INTRODUCTION VENICEHERBARIUM MOOREA BIOCODE CONCLUSIONS • THE MOOREA BIOCODE PROJECT • Geographic/ecological strategy: barcoding an entire biome

  34. A relatively simplified tropical ecosystem, due to age, size, isolation and location in pacific biodiversity gradient Species richness

  35. Goal: to develop a fully-characterized tropical island model ecosystem through intensive biotic surveys and generation of DNA barcode libraries

  36. INTRODUCTION VENICEHERBARIUM MOOREA BIOCODE CONCLUSIONS • THE MOOREA BIOCODE PROJECT • South Pacific islands an undersampled region for fungi • Moore Foundation-funded, multi-taxon ATBI. • Approach: threefold – • Voucher-based collection of macromycetes • Environmental DNA sampling • Culture-based sampling

  37. INTRODUCTION VENICEHERBARIUM MOOREA BIOCODE CONCLUSIONS • Sampling Approach: • Field collection • Voucher information, photos, and DNA sequence linked together and made public • Collaboration with BioMatters, Inc.– GeneiousMooreaBiocodeworkbench / data pipeline

  38. Macrofungal richness

  39. 62 percent of sequences exhibit less than 97% best match in Genbank; 23% exhibit best BLAST match to environmental sequence (cultured or uncultured) 62% of sequences exhibit < 97% identity to sequences currently in GenBank. This project will therefore make an important contribution to GenBank taxon coverage. 97%

  40. INTRODUCTION VENICEHERBARIUM MOOREA BIOCODE CONCLUSIONS • 62 percent of sequences exhibit less than 97% best match in Genbank: • Incomplete database? • Potential radiations? • Insular rapid evolution? • 23% exhibit best BLAST match to environmental sequence (cultured or uncultured) • Voucher-supported sequences will enhance ecological and biodiversity discovery; underscores importance of collections-based research

  41. INTRODUCTION VENICEHERBARIUM MOOREA BIOCODE CONCLUSIONS • For sequences with 98% or greater match: • Suggests recent arrival or slow evolution • If to named material: sequencing facilitates ID of Moorea taxa • If to environmental sequences: Voucher now exists corresponding to the environmental sequence

  42. INTRODUCTION VENICEHERBARIUM MOOREA BIOCODE CONCLUSIONS • MooreaBiocode Project: • Contributions include: • Augmenting geographical and taxonomic coverage in GenBank • Providing vouchers that match environmental sequences • Providing material for biogeographic studies

  43. INTRODUCTION VENICEHERBARIUM MOOREABIOCODE CONCLUSIONS • CONCLUSIONS: • Large-scale barcoding approaches based on institutional collections and ecosystems contribute to the study of fungal biodiversity, ecology, biogeography, and epidemiology. • Institutional targeting: • Adds barcodes for well vouchered and identified material • Adds value to herbarium collections • Allows assessment of barcode “behavior” (laboratory approaches, barcoding gaps, need for multilocus barcodes, etc.) for different taxonomic groups

  44. INTRODUCTION VENICEHERBARIUM MOOREABIOCODE CONCLUSIONS • CONCLUSIONS, continued: • Ecosystem targeting: • Increases representation of poorly-sampled biomes • Aids biogeographic studies • Barcoding speeds time to identification, facilitating biodiversity surveys • Both Approaches: • Exhibit advantages over focusing on specific taxonomic groups of expertise or interest • Offer opportunities for collaboration with taxonomic specialists and amateur enthusiasts

  45. INTRODUCTION VENICEHERBARIUM MOOREABIOCODE CONCLUSIONS • Implications of large-scale studies for DNA barcoding of fungi: • 1. Utility of ITS as a barcode locus: • Similarity-based searches are often sufficient for assignment to genera and families • Use as a species-specific marker problematic due to false negatives and positives • A firm “barcoding gap” is lacking, though some level of success is met using 1-2 bp differences; A 98% or 97% similarity threshold appears to be too low; most species-level similarity is on the order of 99% or higher

  46. INTRODUCTION VENICEHERBARIUM MOOREABIOCODE CONCLUSIONS • Implications of large-scale studies for DNA barcoding of fungi: • 2. “Environmental species”: • Formal (or semi-formal) recognition of entities known only from environmental DNA sequences; subject of a formal discussion session at 2011 MSA annual meeting • Appealing as a means to improve comparison across studies and progress from descriptive or comparative community studies (OTU-based) to a more functional understanding of communities and ecosystems

  47. INTRODUCTION VENICEHERBARIUM MOOREABIOCODE CONCLUSIONS • Implications of large-scale studies for DNA barcoding of fungi: • 2. “Environmental species” (continued): • Our results suggest that a simple identity- or similarity-based approach may be useful for group placement (genera or families), but species-level circumscription is unlikely.

  48. INTRODUCTION VENICEHERBARIUM MOOREABIOCODE CONCLUSIONS • Implications of large-scale studies for DNA barcoding of fungi: • 3. Multilocusbarcodes: • Already well-recognized that ITS is not a species-level barcode for some groups of Ascomycota • Our results strongly suggest that a similar issue exists for Agaricales (Basidiomycota) as well.

  49. INTRODUCTION VENICEHERBARIUM MOOREABIOCODE CONCLUSIONS Acknowledgements: Venice: Giovanni Robich Luca Mizzan Amy Smith Lydia Baker Moorea: Sarah Bergemann Neil Davies Chris Meyer Rikke Rasmussen Natalie Lowell Lydia Smith Lydia Baker Wesley Shipley Gordon & Betty Moore Foundation