Using mtDNA to distinguish species

By Roya ShariatiANU College of Archaeology

and Anthropology Supervisor: Prof. Colin Groves

Carl Linnaeus 250 years ago

Carl Linnaeus is best known for creating the system of classifying living organisms that became the international standard.

“You say tomato, I say Lycopersicon esculentum. You say potato, I saySolanum tuberosum. But Carl Linnaeus was the real plant buff.”

Often called the father of classification, Swedish naturalist Linnaeus established the familiar dual Latin names by which all creatures are now known.

DNA Barcoding A DNA barcode is

a short gene sequence

taken from standardized

portions of the genome, used to identify

species

An organism’s chromosome complement

is its karyotype

Category Genes

NADH dehydrogenase(complex I)

MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6

Coenzyme Q - cytochrome c reductase/Cytochrome b(complex III)

MT-CYB

cytochrome c oxidase(complex IV)

MT-CO1, MT-CO2, MT-CO3

ATP synthase MT-ATP6, MT-ATP8

Amino Acid 3-Letter 1-Letter MT DNA

Alanine Ala A MT-TA

Arginine Arg R MT-TR

Asparagine Asn N MT-TN

Aspartic acid Asp D MT-TD

Cysteine Cys C MT-TC

Glutamic acid Glu E MT-TE

Glutamine Gln Q MT-TQ

Glycine Gly G MT-TG

Histidine His H MT-TH

Isoleucine Ile I MT-TI

Leucine Leu LMT-TL1, MT-TL2

Lysine Lys K MT-TK

Methionine Met M MT-TM

Phenylalanine Phe F MT-TF

Proline Pro P MT-TP

Serine Ser SMT-TS1, MT-TS2

Threonine Thr T MT-TT

Tryptophan Trp W MT-TW

Tyrosine Tyr Y MT-TY

Valine Val V MT-TV

Transport chain

rRNAMitochondrial rRNA is encoded by MT-RNR1 (12S) and MT-RNR2 (16S).

tRNAThe following genes encode tRNA:

The Mitochondrial Genome

Cyt b

D-Loop

ND5

H-strand

ND4

ND4LND3

COIII

COICOIL-strand

ND6

COI

ND2

ND1

COII

Small ribosomal RNA Large

ribosomal RNA

ATPase subunit 8ATPase subunit

6

C O M PA R I S O N O F M I T O C H O N D R I A L G E N E O R D E R S A M O N G T Y P I C A L

V E R T E B R AT E S ,   E . P E L E C A N O I D E S , A N D   S . L AV E N B E R G I . F I V E B L O C K S O F

G E N E R E G I O N S ( A , T R N A G L U – T R N A G L N ; B , AT P 8 - N D 3 ; C , T R N A L E U ( C U N ) – N D 6 ;

D , T R N A I L E – T R N A L Y S ; A N D E , T R N A A R G – T R N A S E R ( A G Y ) G E N E R E G I O N S ) R E TA I N

T H E T Y P I C A L V E R T E B R AT E G E N E O R D E R W I T H T H E E X C E P T I O N O F

S E V E R A L T R N A G E N E S ( A R R O W H E A D S ) . T H E PA R T I A L M T D N A S E Q U E N C E S

F O R F O U R G E N E J U N C T I O N S ( V E R T I C A L B A R : N D 1 - AT P 8 , N D 3 - N D 5 , N D 6 -

N D 2 , A N D N D 4 - C Y T   B   R E G I O N S ) , T H E G E N E O R D E R O F W H I C H G R E AT LY

D I F F E R F R O M T H AT O F O T H E R T Y P I C A L V E R T E B R AT E S , W E R E

D E T E R M I N E D F O R F O U R A D D I T I O N A L   E . P E L E C A N O I D E S   I N D I V I D U A L S

( D ATA AVA I L A B L E F O R M D D B J / E M B L / G E N B A N K W I T H A C C E S S I O N

N U M B E R S A B 0 4 6 4 7 5 – A B 0 4 6 4 9 0 )

A remarkably short DNA sequence can contain more than enough information to resolve 10 or even 100 million species. For example, a 600-nucleotide segment of a protein-coding gene contains 200 nucleotides that are in the third position within a codon. At these sites, substitutions are (usually) selectively neutral and mutations accumulate through random drift. Even if a group of organisms was completely biased to either adenosine or thymine (or alternatively, to either guanosine or cytosine) at third nucleotide positions there would still be 2200 , or 1060 , possible sequences based on third-position nucleotides alone. DNA sequence analysis of a uniform target gene to enable species identification has been termed DNA bar-coding, by analogy with the Uniform Product Code barcodes on manufactured goods.

The Phylogenetic species concept

tokogenetic versus phylogenetic relationships

The phylogenetic species concept

Speciation and phylogenetic relationships

Applied tool for identifying regulated species:Disease vectors, agricultural pests, invasiveEnvironmental indicators, protected species Using minimal samples, damaged specimens, gut

contents, droppings

Research tool for improving species-level taxonomy:Associating all life history stages, gendersTesting species boundaries, finding new variants

“Triage” tool for flagging potential new species:Undescribed and cryptic species

Uses of DNA Barcodes

Using DNA Barcodes

Establish reference library of barcodes from identified voucher

specimens

If necessary, revise species limits

Then:

Identify unknowns by searching against reference sequences

Look for matches (mismatches) against ‘library on a chip’

Before long: Analyze relative abundance in multi-species

samples

Reactions to Barcoding: 2004 From ecologists and other users:

“This is what we need! How soon can we get started?”

From traditional taxonomists:

“Species should be based on lots of characters,

not just barcodes”

From forward-looking taxonomists:

“Using molecular data as species diagnostics isn’t new, but standardization

and broad implementation are great!”

From barcoding practitioners:

“I had my doubts at the beginning, but it really works as a tool for

identification (96% accurate in a recent mollusc paper) and it is at least as good as

traditional approaches to discovering new species.”

What DNA Barcoding is NOT

Barcoding is not DNA taxonomy; no single gene (or character)

is adequate

Barcoding is not Tree of Life; barcode clusters are not

phylogenetic trees

Barcoding is not just COI; standardizing on one region has

benefits and limits

Molecules in taxonomy is not new; but large-scale and

standardization are new

Barcoding can help to create a 21st century research

environment for taxonomy

What DNA Barcoding is NOT

Barcoding is not DNA taxonomy; no single gene (or character)

is adequate

Barcoding is not Tree of Life; barcode clusters are not

phylogenetic trees

Barcoding is not just COI; standardizing on one region has

benefits and limits

Molecules in taxonomy is not new; but large-scale and

standardization are new

BUT…Barcoding can help to create a 21st century research

environment for taxonomy

Barcode Sequence

Voucher Specime

n

Species Name

Specimen

Metadata

Literature(link to content

or citation)

BARCODE Data Standard

Indices - Catalog of Life - GBIF/ECAT

Nomenclators - Zoo Record - IPNI

NameBank

Publication links - New species

GeoreferenceHabitatCharacter setsImagesBehaviorOther genes Trace

filesOther Database

sPhylogeneticPop’n GeneticsEcological

Primers

Barcoding projects have four components:1.The Specimen Collection: Desired

Specimen must be collected for which we

want to generate DNA Barcodes.

2.The Laboratory Analysis: Barcoding

protocols as described in the Materials and

Method Section can be followed to obtain

DNA Barcode sequences from these

collected specimens.

3. The Database: One of the most

important components of the Barcode

Initiative is the construction of a public

library of species identifiers which could be

used to assign unknown specimens to known

species

Consortium for the Barcode of Life (CBOL)

First barcoding publications in 2002

Cold Spring Harbor planning workshops in 2003

Sloan Foundation grant, launch in May 2004

Secretariat opens at Smithsonian, September 2004

First international conference February 2005

Now an international affiliation of:

130+ Members Org’s, 40 countries, 6 continents

Natural history museums, biodiversity organizations

Users: e.g., government agencies

Private sector biotech companies, database providers

IDS – Identification System

DNA from identified voucher

Create BARCODE reference

record

ID unknownsRefine taxonomy of

group

DNA from identified adult

voucher

Create BARCODE reference records

Associate immatures with

names

ID unknowns

Refine taxonomy of

group

DNA from unidentified

immature specimen

Repository of provisional vouchers

Add names to vouchered immatures

DIAGNOSIS OF NEW SPECIES . TAXONOMY IS RAPIDLY ABSORBING GENETICS INTO ITS PANOPLY

OF APPROACHES. BARCODING SHOULD BE A USEFUL ADDITION TO THE EXISTING TOOLS FOR SPECIES

IDENTIFICATION, BUT IT IS NOT INTENDED TO REPLACE THEM. IN MANY GROUPS, ALPHA

TAXONOMY REQUIRES DATA FROM MORPHOLOGY, BEHAVIOR, ECOLOGY, NATURAL HISTORY, AND

GEOGRAPHIC VARIATION. THESE DATA CAN ONLY BE ENHANCED BY COMPLEMENTARY INFORMATION

REGARDING DNA SEQUENCES

RESULT

http://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=An%20external%20file%20that%20holds%20a%20picture%2C%20illustration%2C%20etc.%0AObject%20name%20is%20zpq0040609290003.jpg%20%5BObject%20name%20is%20zpq0040609290003.jpg%5D&p=PMC3&id=1348015_zpq0040609290003.jpg

 References  

1. Fang SG, Wan QH, Fijihara N. 2002. Formalin removal from archival tissue by critical point drying. BioTechniques 33:604-611.

2. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 1994 3:294-299.

3. Hebert PDN, Cywinska A, Ball SL, deWaard JR. 2003a. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London, Series B 270:313-322.

4. Hebert PDN, Ratnasingham S, deWaard JR. 2003b. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London, Series B