Some major episodes in the history of life.

Trends in Ecology & EvolutionFeb. 2003. 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 al4. A plea for DNA taxonomy Tautz et al5. The encyclopedia of life Edward Wilson

DNA barcodinga 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

ND3COIII

L-strand

ND6

ND2

ND1

COII

Small ribosomal RNA

ATPase subunit 8

ATPase subunit 6

Cytochrome b

COICOI

The Mitochondrial Genome

• Copy number. There are 100-10,000 more copies ofmitochondrial than nuclear DNA per cell, making recovery,especially from small or partially degraded samples, easierand 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 extraction CO1 gene DNA sequencing Trace file

Database of Barcode Records

Collecting

ND3

COIII

ND2

ND1

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!Advocates Opposition

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 andDatabasing

Central Nodes

Developing Nodes

Regional Nodes

Curation andIdentification

Sequencing MirroredDatabases

Data Analysisand 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 VertebratesWG1.2 Land PlantsWG1.3 FungiWG1.4 Animal Parasites, Pathogens & VectorsWG1.5 Agricultural & Forestry Pests & ParasitoidsWG1.6 PollinatorsWG1.7 Freshwater Bio-SurveillanceWG1.8 Marine Bio-SurveillanceWG1.9 Terrestrial Bio-SurveillanceWG1.10 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 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: 2009

• 200+ Member organizations, 50 countries

• 35+ Member organizations from 20+ developing countries

NBII, 25 February 2009

GenBank, EMBL, and DDBJGlobal, Open Access to Barcode Data

http://www.insdc.org/

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 referenceHabitat

Character setsImages

BehaviorOther genes

Trace files

Other Databases

PhylogeneticPop’n

GeneticsEcological

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 throughputLarge 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

samadrin@uniandes.edu.co

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 PartnersPartner Organizations Scientists Target groups

Royal Botanic Gardens, Kew, UK Robyn Cowan Mark Chase

Conostylis, Pinus, Equisetum, Dactylorhiza maculata/incarnata

complex

Natural History Museum (London), UK Mark Carine Tortella, Ptychomniaceae, Asplenium,

Natural History Museum, Denmark Gitte Petersen Hordeum, Scalesia, Crocus

New York Botanical Garden, USA Kenneth Cameron Elaphoglossum, Cupressus, Labordia

Royal Botanic Garden Edinburgh, UK Peter Hollingsworth Podocarpus, Araucaria, Asterella,

Anastrophyllum

South African National Biodiversity Institute, (Cape Town), South Africa Ferozah Conrad Encephalartos, Mimetes

Universidad de los Andes, Colombia Santiago Madriñán Lauraceae

Instituto de Biologia UNAM, Mexico Gerardo A. Salazar Agave

Universidade Estadual de Feira de Santana, Brasil

Cássio van den Berg Laelia, Cattleya

University of Cape Town, South Africa Ter ry Hedderson Anastrophyllum- Barbilophozia,

Bryum

Imperial College, UK (& RBG Kew) Timothy Barraclough (Data analysis)

Plant Barcode Proposals

04/22/23 45

Molecules and their useful rangesin phylogenetic relationships

Species Genera Family Order Class Divisions

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

groupsPlastid DNA• Monomorphic

• High copy number

• Highly diagnostic

Regions and PrimersGene Primer Direction Sequence 5'- 3'

matK 2.1 f CCTATCCATCTGGAAATCTTAG 2.1a f ATCCATCTGGAAATCTTAGTTC 5 r GTTCTAGCACAAGAAAGTCG 3.2 r CTTCCTCTGTAAAGAATTC rpoC 1 1 f GTGGATACACTTCTTGATAATGG 2 f GGCAAAGAGGGAAGATTTCG 3 r TGAGAAAACATAAGTAAACGGGC 4 r CCATAAGCATATCTTGAGTTGG rpoB 1 f AAGTGCATTGTTGGAACTGG 2 f ATGCAACGTCAAGCAGTTCC 3 r CCGTATGTGAAAAGAAGTATA 4 r GATCCCAGCATCACAATTCC accD 1 f AGTATGGGATCCGTAGTAGG 2 f GGRGCACGTATGCAAGAAGG 3 r TTTAAAGGATTACGTGGTAC 4 r TCTTTTACCCGCAAATGCAAT YCF5 1 f GGATTATTAGTCACTCGTTGG 2 f ACTTTAGAGCATATATTAACTC 3 r ACTTACGTGCATCATTAACCA 4 r CCCAATACCATCATACTTAC ndhJ 1 f CATAGATCTTTGGGCTTYGA 2 f TTGGGCTTCGATTACCAAGG 3 r ATAATCCTTACGTAAGGGCC 4 r TCAATGAGCATCTTGTATTTC

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 speciesSect. 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.labiata C. aclandiae S. perrinii S. sp. nov.

Cássio van den BergUniversidade 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

accD matK ndhJ rpoB rpoC1Actinodaphne glabra K-8202Actinodaphne pruinosa K-8203Aiouea dubia AN-417Beilschmiedia pendula BCI-065681Beilschmiedia pendula BCI-170421Beilschmiedia pendula BCI-257596Beilschmiedia tawa K-5519Caryodaphnopsis cogolloy JAUM-s.n.Cinnamomum camphora K-6469Cinnamomum dictyoneuron K-8201Cinnamomum obtusifolium K-8303Cinnamomum triplinerve BCI-206711Cinnamomum triplinerve BCI-240764Cinnamomum triplinerve SM-Ama-005Cinnamomum zeylanicum K-8306Cryptocarya triplinervis K-5522Dodecadenia grandiflora K-5520Endlicheria sp. 1 SM-Ama-006Endlicheria sp. 2 SM-Ama-003Lauraceae sp. 1 A-1535Lauraceae sp. 2 NN-BCI?Laurus azorica K-21989Laurus nobilis SM-s.n.Lindera benzoin K-16947Litsea cubeba K-15475Nectandra cissiflora BCI-216314Nectandra cissiflora BCI-233922Nectandra cissiflora BCI-244145Nectandra cissiflora BCI-269005Nectandra cuspidata FC-1579Nectandra cuspidata Gamboa-s.n.Nectandra 'fuzzy' BCI-036152Nectandra 'fuzzy' BCI-105750Nectandra lineata BCI-065446Nectandra lineata BCI-220065Nectandra purpurea BCI-151022Nectandra purpurea BCI-277412Nectandra purpurea BCI-415163Nectandra sp. 1 AN-410Nectandra sp. 1 AN-411Neolitsea aciculata K-17739Ocotea callophyla SM-s.n.Ocotea cernua BCI-206437Ocotea cernua BCI-215988Ocotea cernua BCI-412951Ocotea floribunda HD-1166Ocotea guianensis A-818Ocotea oblonga BCI-309078Ocotea oblonga BCI-403510Ocotea puberula BCI-146684Ocotea puberula BCI-272219Ocotea puberula BCI-716317Ocotea sp. 1 HD-1167Ocotea whitei BCI-008086Ocotea whitei BCI-306277Persea americana SM-s.n.Persea caerulea Sánchez-4911Persea rimosa K-8204Rhodostemonodaphne frontinoensis Brant-1387Rhodostemonodaphne kunthiana HD-1175Rhodostemonodaphne kunthiana Madriñán-717Rhodostemonodaphne penduliflora SM-s.n.Sassafras albidum K-16948

accD matK ndhJ rpoB rpoC1

Genera 17 17 16 17 17

Species 42 40 36 42 43

Specimens 47 58 42 48 49

Sp. w/ >1 specimen

5 11 6 6 6

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 LamiacaeaFabrizio 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, 20126 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

www.kew.org/barcoding

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