Some major episodes in the history of life.

Trends in Ecology & Evolution Feb Vol. 18, Iss. 2 3 of these 5 are the most downloaded papers in TREE 1. Taxonomy: renaissance or Tower of Babel? Jim Mallet et al 4. A plea for DNA taxonomy Tautz et al 5. The encyclopedia of life Edward Wilson

DNA barcoding a new diagnostic tool for rapid species recognition identification, and discovery James Hanken, Museum of Comparative Zoology, Harvard University, USA New Scientist, 26 June, 2004

BARCODING LIFE Mark Stoeckle, The Rockefeller University; Paul E. Waggoner, Connecticut Agricultural Experiment Station; Jesse H. Ausubel, Alfred P. Sloan Foundation Barcoding is a standardized approach to identifying plants and animals by minimal sequences of DNA, called DNA barcodes. DNA Barcode: A short DNA sequence, from a uniform locality on the genome, used for identifying species.

By harnessing advances in electronics and genetics, barcoding will help many people quickly and cheaply recognize known species and retrieve information about them speed discovery of the millions of species yet to be named provide vital new tools for appreciating and managing the Earth’s immense and changing biodiversity. Standardization accelerate construction of a comprehensive, consistent reference library of DNA sequences speedy development of economical technologies for species identification. The goal is that anyone, anywhere, anytime be able to identify quickly and accurately the species of a specimen whatever its condition. Results so far suggest that a mitochondrial gene will enable identification of most animal species. For plants, mitochondrial genes do not differ sufficiently to distinguish among closely related species. Promising approaches to standardize plant identification use one or possibly more barcode regions are under development.

An Internal ID System for All Animals Typical Animal Cell Mitochondrion DNA mtDNA D-Loop ND5 H-strand ND4 ND4L ND3 COIII L-strand ND6 ND2 ND1 COII Small ribosomal RNA ATPase subunit 8 ATPase subunit 6 Cytochrome b CO I The Mitochondrial Genome

Copy number. There are ,000 more copies of mitochondrial than nuclear DNA per cell, making recovery, especially from small or partially degraded samples, easier and cheaper. Relatively few differences within species in most cases. Small intraspecific and large interspecific differences signal distinct genetic boundaries between most species, enabling precise identification with a barcode. Introns, which are non-coding regions interspersed between coding regions of a gene, are absent from mitochondrial DNA of most animal species, making amplification straightforward. Nuclear genes are often interrupted by introns, making amplification difficult or unpredictable. Why barcode animals with mitochondrial DNA? Mitochondria, energy-producing organelles in plant and animal cells, have their own genome. Twenty years of research have established the utility of mitochondrial DNA sequences in differentiating among closely-related animal species. Four properties make mitochondrial genomes especially suitable for identifying species * Greater differences among species

How Barcoding is Done From specimen to sequence to species Voucher Specimen DNA extractionCO1 geneDNA sequencing Trace file Database of Barcode Records Collecting ND3ND3 COIIICOIII ND2ND2 ND1ND1

What are the main limits to barcoding encountered so far? Groups with little sequence diversity Resolution of recently diverged species Hybrids Nuclear pseudogenes

What do barcode differences among and within animal species studied so far suggest? Barcodes identify most animal species unambiguously. Approximately 2-5% of recognized species have shared or overlapping barcodes with closely-related species. Many of the species with overlapping barcodes hybridize regularly. In all groups studied so far, distinct barcode clusters with biologic co- variation suggest cryptic species.

Barcoding North American birds highlights probable cryptic species

Barcodes affirm the unity of the species Homo sapiens. Comparisons show we differ from one another by only 1 or 2 nucleotides out of 648, while we differ from chimpanzees at 60 locations and gorillas at 70 locations.

Can barcodes aid understanding history of animal and plant species?

What isn’t DNA barcoding? It is not intended to, in any way, supplant or invalidate existing taxonomic practice. It is not DNA-taxonomy; it does not equate species identity, formally or informally, with a particular DNA sequence. It is not intended to duplicate or compete with efforts to resolve deep phylogeny, e.g., Assembling the Tree of Life (ATOL).

“the role of any molecular diagnostic is to aid research, not to serve as an end in itself. Barcoding … is independent of questions as to whether individual taxa are species, what species are (or should be), and where they fit in a unified tree of life…. Barcoding is not an end in itself, but will boost the rate of discovery. The unique contribution of DNA barcoding to … taxonomy and systematics is a compressed timeline for the exploration and analysis of biodiversity.”

Sequence data Barcoding must adhere to standards for specimen and data management Voucher specimens and electronic databases Digital images

Strengths Offers alternative taxonomic identification tool for situations in which morphology is inconclusive. Focus on one or a small number of genes provides greater efficiency of effort. Cost of DNA sequencing is dropping rapidly due to technical advances. Potential capacity for high throughput and processing large numbers of samples. Once reference database is established, can be applied by non-specialist.

Weaknesses Assumes intraspecific variation is negligible, or at least lower than interspecific values. No single gene will work for all taxa (e.g., COI is not appropriate for vascular plants, or even for some animals). Single-gene approach is less precise than using multiple genes; may introduce unacceptable error. Some of the most attractive aspects rely on future technology, e.g., handheld sequencer

Simple & Ambitious! AdvocatesOpposition ID all species Discover new species Speed up ID’s Revitalize biological collections Won’t work Destroy traditional systematics Service industry Pseudo taxonomy

A global science project ► 5 years ► 5M specimens ► 500K species Making Every Species Count

Official launch of iBOL – CN Tower, Toronto, September 25, 2010

iBOL launches with 1M records, 100K species

iBOL structure: participating nations Central Nodes ($25M)Regional Nodes ($5M)National Nodes ($1M)

Collection and Databasing Central Nodes Developing Nodes Regional Nodes Curation and Identification Sequencing Mirrored Databases Data Analysis and Access ICI is an alliance of researchers and biodiversity organisations in 21 nations. All nations active in specimen assembly, curation and data analysis. Sequencing and informatics support by regional and central nodes.

Theme 1: DNA Barcode Library WG1.1 Vertebrates WG1.2 Land Plants WG1.3 Fungi WG1.4 Animal Parasites, Pathogens & Vectors WG1.5 Agricultural & Forestry Pests & Parasitoids WG1.6 Pollinators WG1.7 Freshwater Bio-Surveillance WG1.8 Marine Bio-Surveillance WG1.9 Terrestrial Bio-Surveillance WG1.10 Polar Life

iBOL WG 1.5 Bringing genomics to the fight against plant pests and invasive species Assembling a DNA barcode reference library of pests and their parasitoids 2015 target: 25,000 of the most important pest species

BENEFITS OF DNA BARCODING DNA barcoding can speed up identification of new species. DNA barcodes can be linked to readily observable morphological characters. DNA barcoding can provide an avenue to encourage new participants into taxonomy. Applied taxonomic research areas will benefit from barcoding. Food adulteration

Promote barcoding as a global standard Build participation Working Groups BARCODE standard International Conferences Increase production of public BARCODE records Networks, Projects, Organizations Barcode of Life Community

CBOL Member Organizations: Member organizations, 50 countries 35+ Member organizations from 20+ developing countries

NBII, 25 February 2009 GenBank, EMBL, and DDBJ Global, Open Access to Barcode Data

Barcode Sequence Voucher Specimen Species Name Specimen Metadata Literature (link to content or citation) BARCODE Records in INSDC Indices - Catalogue of Life - GBIF/ECAT Nomenclators - Zoo Record - IPNI - NameBank Publication links - New species Geo reference Habitat Character sets Images Behavior Other genes Trace files Other Databases Phylogenetic Pop’n Genetics Ecological Primers Databases - Provisional sp.

NBII, 25 February 2009 Linkout from GenBank to BOLD

NBII, 25 February 2009 Linkout from GenBank to Taxonomy

NBII, 25 February 2009 Link from GenBank to Museums

Current Norm: High throughput Large labs, hundreds of samples per day ABI 3100 capillary automated sequencer Large capacity PCR and sequencing reactions

Emerging Norm: Table-top Labs Faster, more portable: Hundreds of samples per hour Integrated DNA microchips Table-top microfluidic systems

Producing Barcode Data: 201? Barcode data anywhere, instantly Data in seconds to minutes Pennies per sample Link to reference database A taxonomic GPS Usable by non- specialists

Adoption by Regulators Food and Drug Administration –Reference barcodes for commercial fish NOAA/NMFS –$100K for Gulf of Maine pilot project –FISH-BOL workshop with agencies, Taipei, Sept 2007 Federal Aviation Administration – $500K for birds Environmental Protection Agency –$250K pilot test, water quality bioassessment FAO International Plant Protection Commission –Proposal for Diagnostic Protocols for fruit flies CITES, National Agencies, Conservation NGOs –International Steering Committee, identifying pilot projects

Establishing a DNA Barcode for Land plants Santiago Madriñán Restrepo Universidad de los Andes, Bogotá, Colombia

COI or cox1 in Plants Low sequence divergence Other mitochondrial genes –Exhibit incorporation of foreign genes –Frequent transfer of some genes to the nuclear genome

Barcode representation of DNA fingerprints of Indian CASHEW varieties (Archak et al 2003

Project Partners

Plant Barcode Proposals

8/29/ Molecules and their useful rangesin phylogenetic relationships Species Genera FamilyOrderClassDivisions Spacers [ITS] mt DNA Nu rDNA Taylor, et al., 1991 ; more sufficient statistically significant results ; sufficient statistically significant results

Other Regions Internal transcribed spacer regions of nuclear ribosomal DNA (ITS) –often highly variable in angiosperms at the generic and species level –divergent copies are often present within single individuals Non-coding plastid regions –Highly length variable rbcL ( –Not variable enough at species level for many plant groups Plastid DNA Monomorphic High copy number Highly diagnostic

Regions and Primers

Sister taxa: Cattleya and Sophronitis “Corsage orchids” Unifoliate species = 18 species, allopatric species “complex” Bifoliate species = 25 well-defined species, 6 species pairs Sophronitis: 63 spp. in 3 subgenera (as “sections”) Sect. Cattleyodes+Hadrolaelia – 17 well-defined species Sect. Parviflorae – 40 spp. messy complex, genetic data indicate ca. 15 spp. Sect. Sophronitis – 6 allopatric closely related species Cattleya: 43 spp. in 2 subgenera C.labiataC. aclandiaeS. perriniiS. sp. nov. C á ssio van den Berg Universidade Estadual de Feira de Santana, Brasil

% species discriminated ITS: 90.5% psbA-trnH: 60% matK: 33.3% ndhJ: 37.1% rpoB: 9.9% rpoC1:9.9% accD: 6.05 % Nuclear non-coding Plastid non-coding Plastid coding accD, rpoB, rpoC1: variation too low for use as a single barcode matK and ndhF: more variable but with great variation of rate among subgenera Non-coding regions (ITS and psbA-trnH spacer) performed better, but required great manual effort for indel alignment

Lauraceae Big family Largely unstudied VERY difficult to id. Economically important

Lauraceae accDmatKndhJrpoB rpoC 1 Genera Species Specimens Sp. w/ >1 specimen

matK 974 bp

ndhJ 428 bp

Cryptocarya triplinervis K-5522

Laurus nobilis SM-s.n.

A comparative study of different DNA barcoding markers for the identification of some members of Lamiacaea Fabrizio De Mattia, Ilaria Bruni, Andrea Galimberti, Francesca Cattaneo, Maurizio Casiraghi, Massimo Labra Università degli Studi di Milano Bicocca, ZooPlantLab, Dipartimento di Biotecnologie e Bioscienze, Piazza della Scienza 2, Milano, Italy Food Research International 44 (2011) 693–702 The objective is to evaluate the efficacy of a DNA barcoding approach as a tool for the recognition of commercial kitchen spices belonging to the Lamiaceae family that are usually sold as enhancers of food flavor. A total of 64 spices samples, encompassing six different genera (i.e. Mentha, Ocimum, Origanum, Salvia, Thymus and Rosmarinus) were processed with a classical DNA barcoding approach by amplifying and sequencing four candidate barcode regions (rpoB, rbcL, matK and trnH-psbA) with universal primers. Results suggest that the non-coding trnH-psbA intergenic spacer is the most suitable marker for molecular spices identification followed by matK, with interspecific genetic distance values ranging between about 0% to 7% and 0% to 5%, respectively. Both markers were almost invariably able to distinguish spices species from closest taxa with the exclusion of samples belonging to the genus Oregano. Moreover, in a context of food traceability the two markers are useful to identify commercial processed spice species (sold as dried plant material). We also evaluated the potential benefits of a multilocus barcode approach over a single marker and although the most suitable combination was the matK+trhH-psbA, the observed genetic distances values were very similar to the discriminatory performance of the trnH-psbA. Finally, this preliminary work provide clear evidences that the efficacy of a DNA barcoding approach to the recognition of commercial spices is biased by the occurrence of taxonomic criticisms as well as traces of hybridization events within the family amiaceae. For this reason, to better define a more practical and standardized DNA barcoding tool for spices traceability, the building of a dedicated aromatic plants database in which all species and cultivars are described (both morphologically and molecularly) is strongly required.

Fig. 1. Neighbor-joining reconstructions obtained with MEGA 4.0 for three out of the four molecular datasets produced in this study. Each tree encompasses all the samples analysed for the six taxonomical group considered: a) trnH-psbA, b) matK, c) rbcL. Bootstrap values lower than 70% not showed. Details on samples, species, cultivar, provenance and accession numbers for each marker can be retrieved from Table 1. Each taxonomic group has been shown on the tree with squared brackets.

Overall Results Standardized universal primers Different levels of variation in different groups at different taxonomic levels Variable ID success with a single region Score on basis of –Amplification success –Sequence variation

Non-COI regions for other taxa Land plants: –Chloroplast matK and rbcL approved Nov 09 –Non-coding plastid and nuclear regions being explored

What barcode providers want High PCR and sequencing success rates Bigger window into older, compromised samples Better software integration to eliminate bottlenecks Smaller labs/developing countries: –Lower equipment and maintenance costs –Simplification for techs with less training –Install anywhere without lab renovations –Willing to accept slower throughput

What barcode users want Answers to specific questions: –Is this thing on this list of species or not? –Is this thing a member of this genus/family? –Which of the species on this list is this thing? –What species is this thing? Production-scale capabilities: –Hundreds to thousands of installations –Lower but constant throughput –Rapid turnaround –The right price-point and limited life cycle costs

What barcode users would do with the reference libraries Inspection stations at every port and international airport for: –Agricultural pest control –Illegal trade in endangered species –Violations of trade quotas Regular Federal and State water quality surveys Federal, State and local food inspection Public health monitoring and diagnoses

IISR Barcoding of insect pests of Spices (IIHR, IISR initiative (TK Jacob) - COI Barcoding for checking adulterants in traded spices (Sasikumar B) – ITS, rbcL, Mat K etc