Basi botaniche: nomi, caratteri e analisi filogenetica in ...€¦ · New characters: DNA markers...

Basi botaniche: nomi, caratteri e analisi filogenetica in relazione alle

piante

DNA-Barcoding

Ana Crespo

Universidad Complutense de Madrid M a s t e r Fi t o

t e r a p i a 2 0 1 3

Why to identify species?

M a s t e r Fi t o

t e r a p i a 2 0 1 3

THEY ARE THERE!!

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Some reasons for species identification

•  Basic research: evolution, ecology, systematic etc.

•  Species conservation: protected / endangered

•  To ensure an accurate identification for industrial uses

•  Crop pests, biological control

•  Disease control: knowing vectors and pathogens

•  Ability of seed banks and culture collections

•  Meet invasive species (eg in enclosed waters)

•  Know the indicators of environmental quality

•  Sustainable management of agricultural crops

•  Consumer protection and fraud control M a s t e r Fi t o

t e r a p i a 2 0 1 3

The identification of species based on phenotypic chatacters

“Ci sono stati Zoologi o Botanici migliori. Ma non c'è stato nessuno che ha scritto più libri, in modo più corretto, più metodico, dalla migliore esperienza. Non c'è nessuno che abbia cambiato una scienza più profondamente e che più chiaramente abbia iniziato una nuova era. Nessuno scienziato è stato più conosciuto di lui in tutto il mondo ... ". (c. 1770)

M a s t e r Fi t o

t e r a p i a 2 0 1 3

To study and to identify species are keeping in international open Herbaria

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Papaver somniferum L. a plant used, at least from 2500 b. c.

One species one name

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Papaver somniferum L.

Papaver generic epithet used for all species within de genus

somniferum arbitrary epithet used as characteristic name for the species

L. first letter of the name of the first author recognising the species (here C. Linneus)

NO CAPITAL LETTER !!!!

CAPITAL LETTER !!!! M a s t e r Fi t o

t e r a p i a 2 0 1 3

Recognising Papaver somniferum L.

M a s t e r Fi t o

t e r a p i a 2 0 1 3

BASED ON CHARACTERS ABOUT 100 SPECIES OF PAPAVER HAVE BEEN RECOGNISED.

P. rhoeas P. dubium P. hybridum P. argemone

Opium poppy, Papaver somniferum,

Characters are tools for identification

M a s t e r Fi t o

t e r a p i a 2 0 1 3

A part of pharmacological characters, some characters are:

Habitus (including histological and micromorphological vegetative traits)

Leaves form and distribution on stems, presence /absence of stipules

Stems (including indument qualities, latex characteristic)

Flowers: floral formula ! K 2 C 2+2 A ∞ G (3) - (18)

All mentioned characters are called �classic characters�

Flowers:

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Character A

Character B

Character C

Character D

Character F

Plant 1 + + _

+ +

Plant 2 + _ + _

+

Plant 3 + + + +

+

Analysing Characters

( + - state of the character)

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Maximum likelyhood (cladistic representation in trees)

Plant 1 Plant 2 Plant 3

Plant 1 5 2 4

Plant 2 2 5 3

Plant 3 4 3 5

1 3 2

1 3 2

The systematic arrangement by dendrograms

Cladistic method stablish that in phylogeny (evolution) one new character makes a new node M a s t e r F

i t ot e r a p i a 2 0 1 3

Closer branches share lower number of differences and plants would look more similar

More similar plants should be more closely evolutionary related

More related species are in the same genus (PAPAVER)

More related genera are in the same family (PAPAVERACEAE)

More related families are in the same order (RANUNCULALES)

IN THAT WAY IT WAS BUILD THE SYSTEMATIC

M a s t e r Fi t o

t e r a p i a 2 0 1 3

THE IDENTIFICATION OF SPECIES IS NOT SO OBVIOUS TASK

Small or microbial organisms are difficult (small number of characters)

In many circunstances specimens for identification are not complete samples: e. g. toxicology, forensic work

In most biological groups there are also �cryptic species�

M a s t e r Fi t o

t e r a p i a 2 0 1 3

New characters: DNA markers

DNA as a source of identificative characters for all organisms

DNA sequences provide numerous characters that are transmissible and they can relate all organims

Closer organisms share higher number of DNA characters

Nucleotidic bases A, G, T, C provide excellent source of characters to identify organisms

M a s t e r Fi t o

t e r a p i a 2 0 1 3

To study and to identify species are keeping in international open Herbaria

M a s t e r Fi t o

t e r a p i a 2 0 1 3

To produce DNA analyses a molecular laboratory has been associated to modern Herbaria M a s t e r F

i t ot e r a p i a 2 0 1 3

ESTRAZIONE DEL DNA

M a s t e r Fi t o

t e r a p i a 2 0 1 3

AMPLIFICAZIONE DEL DNA

C

Denaturazione iniziale 94-96°C 3-5 min Denaturazione del DNA (94-96°C 1 min) Allineamento degli iniziatori (48-60°C 1-2 min Estensione della polimerasi 72°C 5-10min

M a s t e r Fi t o

t e r a p i a 2 0 1 3

DNA nucleare

DNA mitocondriale

M a s t e r Fi t o

t e r a p i a 2 0 1 3

SEQUENZIAMENTO

M a s t e r Fi t o

t e r a p i a 2 0 1 3

ALLINEAMENTO DELLE SEQUENZE

Programmi di allineamento multipli

M a s t e r Fi t o

t e r a p i a 2 0 1 3

ALLINEAMENTO DELLE SEQUENZE

Sequenza dello spaziatore ITs1 in differenti specie di funghi lichenizzati

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Characters representation

( + - are states)

Character A

Character G

Character T

Character C

Character lack

Plant 1 + + _

+ +

Plant 2 + _ + _

+

Plant 3 + + + +

+

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Maximum likelyhood (cladistic representation in trees)

Plant 1 Plant 2 Plant 3

Plant 1 5 2 4

Plant 2 2 5 3

Plant 3 4 3 5

1 3 2

1 3 2

M a s t e r Fi t o

t e r a p i a 2 0 1 3

METODI DI ANALISI FILOGENETICA

Dato un insieme di caratteri e stati di caratteri (matrice) si può ricostruire la filogenia osservando i cambiamenti che sono avvenuti nelle diverse posizioni della sequenza

Cladogramma Filogramma M a s t e r Fi t o

t e r a p i a 2 0 1 3

Metodo della distanza (Neighbor Joining, UPGMA) Massima Parsimonia (MP) Massima similitudine (ML) Analisi bayesiana (BAY)

M a s t e r Fi t o

t e r a p i a 2 0 1 3

MASSIMA PARSIMONIA (MP)

M a s t e r Fi t o

t e r a p i a 2 0 1 3

ANALISI COMBINATA DI GENI

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Closer branches share lower number of differences and plants would look more similar

More related species are in the same genus (PAPAVER)

More related genera are in the same family (PAPAVERACEAE)

More related families are in the same order (RANUNCULALES)

IN THAT WAY IT IS BUILT THE MODERN SYSTEMATIC

More similar plants –more similar DNA- should be more closely related

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Phylogenetics of Papaver and Related Genera Based on DNA Sequences from ITS Nuclear Ribosomal DNA and Plastid trnL Intron and trnL–F Intergenic Spacers. Carolan J. C .& al 2006 Annals of Botany 98: 141–155.

M a s t e r Fi t o

t e r a p i a 2 0 1 3

M a s t e r Fi t o

t e r a p i a 2 0 1 3

THE IDENTIFICATION OF SPECIES IS NOT SO OBVIOUS TASK

Small or microbial organisms are difficult (small number of characters)

In many circunstances specimens for identification are not complete samples: e. g. toxicology, forensic work

In most biological groups there are also �cryptic species�

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Burns, J & al . 2008. PNAS:105 (17): 6350–6355

NJ tree based on Kimura two-parameter distances for COX1 DNA barcodes

Le farfalle salterine

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Razza (Dipturus batis)

Iglésias, S.P. & al 2009. Marine and Freshwater Ecosystems

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Barcode for plants: Nuclear ribosomal ITS (internal transcribed spacer ) region and the plastid trnH-psbA intergenic spacer. Other like two chloroplast genes, rbcL and matK, taken together, are also possible. Barcode for fungi: The ITS (n rDNA) also has been selected for fungi but a complementary loci is still necessary.

What is DNA barcoding

DNA barcoding is a technique for characterizing species of organisms using a short DNA sequence from a standard and agreed-upon position in the genome.

DNA barcode sequences are very short relative to the entire genome and they can be obtained reasonably quickly and cheaply.

The cytochrome c oxidase subunit 1 mitochondrial region (COI -cytochrome c oxidase I gene) is emerging as the standard barcode region for higher animals. It is 648 nucleotide base pairs long in most groups, a very short sequence relative to 3 billion base pairs in the human genome, for example.

M a s t e r Fi t o

t e r a p i a 2 0 1 3

38

DNA barcoding is the brainchild of Paul Herbert (University of Ontario, Canada) expressed in 2003. The idea is to generate a unique identification tag for each species, including any organism, from the unicellular to the hominids, based on a short sequenceof DNA.

The article in The Economist used to highlight the importance of this project the study of 390 species of mosquitoes including transmitting the germ that causes malaria.

DNABarcoding can easily recognize the different species, some of which transmit malaria in different type or severity.

M a s t e r Fi t o

t e r a p i a 2 0 1 3

To reduce confusion and also possible fraud

M a s t e r Fi t o

t e r a p i a 2 0 1 3

How to perform a DNA-BARCODING

ESPECÍMENS

DNA ISOLATION SELECTED REGION USING SELECTED PRIMERS FOR PCR

SECUENCIATION ALIGNEMENT

COMPARING BANK FOR

DNA-BARCODING

SMALL PIECE

ND3 COIII

ND2 ND1

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Organizzazioni membri del CBOL: 2009

•  200+ organizzazioni membri, 50 paesi

•  35+ organizazioni membri 20+ paesi in via di sviluppo

SPAGNA: MADRID E CANARIE

M a s t e r Fi t o

t e r a p i a 2 0 1 3

•  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

M a s t e r Fi t o

t e r a p i a 2 0 1 3

THE IDENTIFICATION OF SPECIES MUST BE AN OBVIOUS TASK

LIKELY …

M a s t e r Fi t o

t e r a p i a 2 0 1 3

Next future date: 201?

IDENTIFICATION

•  Data from seconds to minutes

•  Few hundredths of a cost

•  Connecting to the database reference

•  A taxonomic GPS

•  For use by non-specialists

M a s t e r F

i t ot e r a p i a 2 0 1 3