Genetic diversity analysis of CWR in Portugal

Joana Magos Brehm, Brian V. Ford-

Lloyd, Nigel Maxted, Maria Amélia

Martins-Loução

Joint PGR Secure/ECPGR workshop:

Conservation strategies for European CWR and LR diversity

7–9 September 2011, Palanga, Lithuania

• Aims of this study

• National approach

• Genetic diversity study

• Selection of populations for conservation

• Relevant points and conclusions

OVERVIEW

• To evaluate the genetic diversity and population

genetic differentiation of priority species

throughout its distribution area in Portugal as a

means of obtaining genetic baseline information

for future conservation.

• To use genetic, demographic and threat data in

order to prioritise populations for conservation.

AIMS OF THIS STUDY

Inventory

NATIONAL APPROACH: PORTUGAL

Prioritisation at species level- 8 criteria, 4 methods-

2262 CWR 20 priorities

Priority species

In situ conservation

National distribution

Legislation

Global distribution

Threatened category

Ex situ conservation

Economic value

Native status

Dianthus cintranus Boiss. & Reut. subsp. barbatus R.

Fern. & Franco

Allium victorialis L.

20 priorities

NATIONAL APPROACH: PORTUGAL

Ecogeographic survey

Dianthus cintranus Boiss. & Reut. subsp.

cintranus

Dianthus laricifolius Boiss. & Reut. subsp. marizii (Samp.)

Franco

Field workPriority species

NATIONAL APPROACH: PORTUGAL

Prioritisation at population level

Amplified Fragment

Length Polymorphism

(AFLP)

Priority populations Demographic data

Threat data

Genetic data

Dianthus cintranus Boiss. & Reut. subsp. barbatus R. Fern. &

Franco

Priority species

1. Dianthus cintranus Boiss. & Reut. subsp. barbatus R. Fern. & Franco

Genetic diversity study

• Related crop: carnations (ornamentals)• Habitat: outcrops, mainly limestones• Global distribution: Portugal• National distribution: 5 provinces• In situ conservation: not active but part of its distribution occurs

within conservation areas• Ex situ conservation: none• Legislation: none

Dianthus cintranus subsp. barbatus

• IUCN category (2001): Endangered (EN)

• Threats: low precipitation, fires, invasive species, construction, trampling, grazing, trash deposition

#

#

### ####

##

################################

#

######

• breeding system?

• Dianthus spp. mainly insect pollinated ‐

(beetles, bees, butterflies, moths…)

• some inbreeding in Dianthus spp.

• some Dianthus spp. are facultative

outbreeders

• seed dispersal mechanisms? but seed

release by the wind

• genetic diversity?

Dianthus cintranus subsp. barbatus

2. Methodology

Genetic diversity study

• 5 populations, 20 plants/population

• 2 selective MesI and EcoRI primer

pairs

• loci 56-440 bp

Amplified Fragment Length Polymorphism (AFLP)

Descriptive stats:

• allele frequencies - Bayesian

approach suggested by Zhivotovsky

(1999)

• % polymorphic loci/population

• genetic diversity - Lynch & Milligan

(1994) for dominant markers

• # private alleles

Genetic data analysis

Population structure and differentiation:• Wright’s FST (1951) - Lynch & Milligan

(1994)

• dendrograms - agglomerative hierarchical clustering using UPGMA

• PCoA

• AMOVA - based on Euclidean pairwise genetic distances

• Bayesian clustering method - for cross-breeders and where isolation by distance not detected

Isolation by distance:

• Mantel test [pairwise FST

transformed to FST/(1 F‐ ST) versus

log transformed geographic ‐

distance] (GENALEX v. 6.0)

Dianthus cintranus subsp. barbatus

Dcb1

Dcb2

Dcb3

Dcb4Dcb5

Natura 2000 sites

Dcb3

Dcb1

Dcb4

Dcb2

Dcb5Dcb5↓ Hj=0.149↓ PL=46%↓ PA=2%

Dcb3↑Hj=0.170↑PL=53%PA=3%

Descriptive stats

•total genetic diversity:

Ht=0.163 (moderate)

•genetic diversity within

populations: Hw=0.152

•inter‐population genetic

diversity: Hb=0.006

OUTBREEDER

Dianthus cintranus subsp. barbatus

Fst = 0.038 => populations are little genetically different

AMOVA (Analysis of Molecular Variance):

Among populations: 8%

Within populations: 92%

OUTBREEDER

Population genetic structure and differentiation

Dianthus cintranus subsp. barbatus

Dcb3

Dcb1

Dcb4

Dcb2

Dcb5

0.001

Dcb3

Dcb1

Dcb2

Dcb4

Dcb5

978

671

1000

Dcb3

Dcb1

Dcb4

Dcb2

Dcb5

NO GEOGRAPHIC PATTERN

Isolation by distance

Dianthus cintranus subsp. barbatus

Dcb3

Dcb1

Dcb4

Dcb2

Dcb5

Mantel test => NO

ISOLATION BY

DISTANCE (no

restriction to geneflow)

GENETICALLY VERY

HOMOGENOUS!

Dianthus cintranus subsp. barbatus

Dcb3

Dcb1

Dcb4

Dcb2

Dcb5

Selection of populationsfor conservation

1. Criteria used

DEMOGRAPHIC DATA: population size

THREAT DATA : number of threats

GENETIC DATA:

Criteria

• genetic diversity (measure of molecular diversity)

• # polymorphic alleles (measure of genepool richness)

• # common (population frequency >0.05) and localised

alleles (in <50% of populations) (modified from Marshall &

Brown 1975)

• inter‐population genetic distance (measure of how similar

populations are)

• Standardisation to 1, integrated in a sum per

population and transformed into % using the

highest score as the reference value of 100%

• Priorities: populations with higher %

(Delgado et al. 2008)

Integration of all data

Selection of populationsfor conservation

2. Results

Integration of all data

TAXON POP. POP. SZ. St THREATS St Hj St D St # PL St # cl St SUM

PRIORITY (%)

D. cintranus

subsp.

barbatus

Dcb1 0,50 1,00 0,95 0,50 0,99 0,00 3,94 78,80

Dcb2 1,00 0,50 0,91 0,50 0,98 0,08 3,97 79,40

Dcb3 1,00 0,00 1,00 1,00 1,00 1,00 5,00 100,00

Dcb4 1,00 0,50 0,89 0,10 0,89 0,43 3,81 76,20

Dcb5 1,00 0,75 0,88 0,10 0,87 0,46 4,06 81,20

Dcb3

Dcb1

Dcb4

Dcb2

Dcb5

Dcb3 (Condeixa-a-

Nova)

↑Hj=0.170

↑D=0.005

↑PL=53%

PA=3%

↑#cl=37

<50 plants

Unknown threats

Outside conservation

area

OUTBREEDE

R

Ex situ and in situ:

Dcb3!

Dianthus cintranus subsp. barbatus

Relevant points and conclusions

Genetic diversity = evolutionary potential of a species

to evolve and adapt to a changing environment

Neutral versus adaptive diversity

ADAPTIVE DIVERSITY

• evolution

• reflects the species potential

ability to adapt to changing

environments

NEUTRAL DIVERSITY

• migration, mutation, genetic

drift…

• no direct effect on species

fitness

• not affected by natural selection

Correlation between neutral and adaptive

diversity?

• AFLP successfully used to obtain genetic baseline information

on priority CWR:

D. cintranus subsp. barbatus is an outbreeder, with genetically

homogenous populations and moderate values of genetic

diversity, low but significant levels of genetic differentiation,

most genetic variation within populations

• Genetic + demographic + threat basic data => suggest target

populations for in situ and ex situ conservation of Portuguese

priority CWR:

D. cintranus subsp. barbatus: Dcb3

Conclusions

Genetic diversity analysis of CWR in Portugal

Joana Magos Brehm, Brian V. Ford-

Lloyd, Nigel Maxted, Maria Amélia

Martins-Loução

Joint PGR Secure/ECPGR workshop:

Conservation strategies for European CWR and LR diversity

7–9 September 2011, Palanga, Lithuania