APPLICATIONS OF INTENSIVE DNA BARCODING PROJECTS FOR ADDRESSING GAPS IN FUNGAL BIODIVERSITY KNOWLEDGE

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