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
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• 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