DNA Barcoding in Plants: Biodiversity Identification and Discovery

Transcription

1 DNA Barcoding in Plants: Biodiversity Identification and Discovery University of Sao Paulo December 2009 W. John Kress Department of Botany National Museum of Natural History Smithsonian Institution

2 New Technologies for Taxonomy DNA Barcodes

3 UNITED STATES NATIONAL HERBARIUM 4.7 Million Specimens

4 NATIONAL MUSEUM OF NATURAL HISTORY 124 Million Specimens

5 DNA Barcodes A short universal gene sequence taken from a standardized portion of the genome used to identify species

6 Uses of DNA Barcodes 1. Research tool for taxonomists: To aid identification of species To expand species diagnoses to all life history stages, including fruits, seeds, dimorphic sexes, damaged specimens, gut contents, scats To test consistency of species definitions with a DNA measure of variability 2. Applied tool for users of taxonomy: To identify regulated species, including invasives To test purity and identity of biological products To assist ecologists in field studies of poorly known organisms 3. Discovery tool: To flag potentially new species, especially undescribed and cryptic species

7 The Barcoding Process - 2 parts 1. Populate the barcode library with known species Collect tissue from voucher specimen Extract DNA PCR/Amplify/cycle sequence gene(s) Sequence Database to answer compelling scientific 3. Put barcode sequences to work questions Ecological forensics 2. BLAST an unidentified Community ecology and specimen against the barcode phylogenetics library Sequence comparison New searching technologies Ultimately - handheld device?

8 Smithsonian s National Museum of Natural History Caribbean Sponges

9 Select plant material DNA Barcode Pipeline DNA Extraction PCR Robotic Sequencing Finished Barcode Data Editing L i b r a r y

10 The Primary Choice for Barcoding in Animals: the Mitochondrial Genome Cyt b D-Loop Small ribosomal RNA Large ribosomal RNA ND5 ND6 L-strand COI ND1 ND2 H-strand ND4 ND4L ND3 COIII COI COII ATPase subunit 8 ATPase subunit 6

11 What about Plants? Why were plants behind? Finding the right gene regions Mobilizing a consensus in the botanical community Finally. Consensus on gene regions Moving ahead

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13 Criteria for DNA Barcoding Contains sufficient variation to discriminate between species Conserved flanks for universal primers All land plants Short, bp Limited by current sequencing technology, cost consideration (= 1 read length), and ability to use degraded samples Sequence Quality

14 Three Genomes of Plant Cells for Barcode Candidates Chloroplast *High copy number *Conserved structure *Diversity of substitution rates across genes, introns, and intergenic spacers Nuclear *Contain the most variable loci *Problems with multigene families *Single-copy genes often technically difficult Mitochondrial *Locus of choice for animal barcoding is mitochondrial COI *Limitations with plants -Low divergence -Rapid genome rearrangements

15 Atropa vs. Nicotiana Chloroplast Genomes Complete Schmitz- Linneweber et al. 2002

16 Atropa vs. Nicotiana Chloroplast Genomes 1% divergence

17 Atropa vs. Nicotiana Chloroplast Genomes trnl-f trnv-atpe atpb-rbcl psbm-trnd ycf6-psbm trnc-ycf6 trnk-rps16 rpl36-rps8 2% difference 2% divergence trnh-psba

18 Top Plant Barcode Candidate: Intergenic Spacer trnh-psba CRITERIA FOR BARCODING Short, bp trnh-psba = 450 bp Conserved flanks for universal primers trnh-psba = % success Contains sufficient variation to discriminate between species trnh-psba = 1.17%

19 A SINGLE-LOCUS PLANT BARCODE Option #1: Best Candidate Plastid Non-Coding trnh-psba Many Other Regions Proposed: accd, matk, ndhj, rbcl, rpoc1, rpob2, trnl, YCF5, UPA, ITS, CO1

20 SAMPLING AND PCR SUCCESS: 39 Orders of Land Plants

21 A SINGLE-LOCUS PLANT BARCODE: Comparative Results

22 A TWO-LOCUS PLANT BARCODE Hierarchical and Complementary rbcl = the Anchor (Plastid Coding Gene) + trnh-psba = the Identifier (Plastid Noncoding Spacer)

23 INTERGENIC SPACERS Indels, Alignment, and Repeats: Problems or Assets? Spacers for Identification (and localscale phylogenetics) Indels as added characters for ID Partial sequences are useful New Informatics Tools for Searching the Reference Database New technologies for solving problems Indel variation in segment of trnh-psba spacer among 57 species Do we need a coding gene??

24 An Alternative Two-Locus Plant Barcode CBOL Plant Working Group U n i v e r s a l i t y Conclusion: rbcl + matk with trnh-psba & other spacers as alternative barcodes D i s c r i m i n a t i o n 156 Cryptogams 81 Gymnosperms 170 Angiosperms

25 A THREE-LOCUS PLANT BARCODE Hierarchical and Complementary matk rbcl = the Anchor (Plastid Coding Gene) + trnh-psba = the Identifier (Plastid Noncoding Spacer) + matk (Plastid Coding Gene)

26 Major Medicinal Plants of the World: An Applied Test of DNA Barcoding What is a medicinal plant? We used a consensus of four sources that list medicinal plants, primarily: World Economic Plants - A Standard Reference

27 Major Medicinal Plants of the World: An Applied Test of DNA Barcoding How we assembled our set: Selected ~1150 species Requested USDA germplasm USBG living collection Local gardens NMNH herbarium What we have: 768 species >168 Genera 113 Plant Families 4 accessions per species

28 Major Medicinal Plants of the World: An Applied Test of DNA Barcoding Two-locus approach: create backbone of tree with rbcl as the Anchor; then separate individuals species in smaller groups with trnh-psba as the Identifier Lamiales: Mentha Results: >94% success with rbcl/ trnh-psba rbcl Anchor trnh-psba Identifier

29 50-ha Forest Dynamics Plot on Barro Colorado Island, Panama Vital statistics of BCI Island in Panama Canal Premier Ecological Plot Research 296 tree Institute species Forest Science 1035 specimens (~3 accession/species) 180 Genera bal Earth Observatories 49 Families O) ~50% of genera have one species = easy test of barcoding forest research: monitoring mate change Why DNA Barcoding on BCI? Species identification *forensic/ecological Phylogenetic applications *species/community phylogenies *functional trait mapping

30 50-ha Forest Dynamics Plots Field Information Management System Collection Data Tab Geographic Data Tab Tissue Data Tab

31 50-ha Forest Dynamics Plot on Barro Colorado Island, Panama Institute nce ervatories h: monitoring Barcode Success trnh-psba* matk rbcla pcr seq pcr seq pcr seq 98% 95% 85% 69% 94% 94% ID Freq ID Freq ID Freq 95% 99% 75% *Note: ~8% of sequences are partial

32 50-ha Forest Dynamics Plot on Barro Colorado Island, Panama Species Identification = BLAST (Basic Local Alignment Search Tool) Designed to search for similarity among sequences Can quantify rates of resolution Use 281 barcode sequences as both library and query RESULTS rbcla + trnh-psba + matk: 98% of all samples could be assigned to correct Species All ambiguity was in 4 genera: Psychotria, Ficus, Inga, Piper 100% of sequences were assigned to correct Genus Partial sequences were assigned correctly

33 Barcodes and Forensic Ecology Barcode

34 Barcodes and Community Ecology The Components of Biodiversity Swenson 2009

35 Building a Community Phylogeny with Phylomatic Phylogenetically clustered = High Plateau, Low Plateau and Young Habitats Phylogenetically Overdispersed = Swamp and Slope Habitats Phylogenetically Random = Stream and Mixed Habitats

36 Building a Community Phylogeny with Barcodes: A Supermatrix of rbcl, matk, and trnh-psba rbcla *aligns unambiguously matk *aligned with backtranslation (AA) trnh-psba *aligned within ORDERS (Muscle), then orders placed within rbcla alignment with missing data coded for other Orders (MacClade) Trees *constructed with Parsimony (PAUP) and ML (Garli: GTR+I+Ѓ)

37 50-ha Forest Dynamics Plot on BCI, Panama (281 species): Community Phylogeny using a Supermatrix Approach with rbcl/trnhpsba/matk

38 A Comparison of Ordinal and Family Relationships on BCI Asterids 50-ha Forest Dynamics Plot on BCI, Panama (282 (281 species): Community Phylogeny of 23 Orders using using a a Supermatrix Approach with rbcl/trnh-psba psba/matk

39 Barcodes vs. Phylomatic vs. 50-ha Forest Dynamics Plot on BCI, Panama (282 (281 species): Community Phylogeny of 23 Orders using using a a Supermatrix Approach with rbcl/trnh-psba psba/matk Overall Rubiaceae Tree: < 50% resolution vs >97% resolution

40 Barcodes vs. Phylomatic 50-ha Forest Dynamics Plot on BCI, Panama (282 (281 species): Community Phylogeny using a Supermatrix Approach with rbcl/trnh-psba psba/matk Phylomatic Phylogeny: Phylogenetically clustered = High Plateau, Low Plateau and Young Habitats Phylogenetically Overdispersed = Swamp and Slope Habitats Phylogenetically Random = Stream and Mixed Habitats Barcode Phylogeny: Phylogenetically clustered = Low Plateau and Slope Habitats Phylogenetically Overdispersed = High Plateau, Mixed and Young Habitats = Phylogenetically Random = Stream and Swamp Habitats Net Relatedness Index (NRI)

41 50-ha Forest Dynamics Plot on BCI, Panama (281 species): Community Phylogeny using a Supermatrix Approach with rbcl/trnhpsba/matk Functional Trait Analysis

42 Phylogenies and Community Ecology Community Assembly, Productivity, Stability, Functional Trait Evolution Swenson 2009

43 Center for Tropical Forest Science Smithsonian Institution Global Earth Observatories (SIGEO) 22 Established Sites (Black) 12 Candidate Sites (Blue) Barcoding Initiated (Red) Smithsonian Tropical Research Institute Center for Tropical Forest Science ** * * * * Smithsonian Institution Global * Earth Observatories (SIGEO) * * * * * A global program of long-term forest research: monitoring * the impact of climate change Purpose: *Forest Dynamics *Climate Change Expanding *Conservation the network!

44 Smithsonian Center for Tropical Institution Forest Global Science Earth Smithsonian Observatories Institution (SIGEO) Global Earth Observatories (SIGEO)

45 DNA Barcoding in Plants: Biodiversity Identification and Discovery Dave Erickson Ken Wurdack Liz Zimmer Dan Janzen Lee Weigt Ling Zhang Nate Swenson Andy Jones Oris Sanjur Jamie Whitaker Ida Lopez Stuart Davies Joe Wright Biff Bermingham Scott Miller W. John Kress Department of Botany National Museum of Natural History Smithsonian Institution University of Sao Paulo December 2009