1. 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

2. Aims of this study National approach Genetic diversity study Selection of populations for conservation Relevant points and conclusions

3. 2262 CWR (mainland) (~77% of Portuguese Flora) <1% in situ conservation ~12% ex situ conservation (mostly Leguminosae, Poaceae) ~14% threatened spp. ~6% protected by legislation

5. 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.

6. Prioritisation at species level Inventory Priority species Prioritisation at population level Priority populations for conservation Demographic data Threat data Genetic data

7. Inventory 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

8. Dianthus cintranus Boiss. & Reut. subsp. barbatus R. Fern. & Franco Allium victorialis L. 20 priorities Ecogeographic survey Dianthus cintranus Boiss. & Reut. subsp. cintranus Dianthus laricifolius Boiss. & Reut. subsp. marizii (Samp.) Franco Field work Priority species

9. 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

11. 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 IUCN category (2001): Endangered (EN) Threats: low precipitation, fires, invasive species, construction, trampling, grazing, trash deposition

12. 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?

14. 5 populations, 20 plants/population 2 selective MesI and EcoRI primer pairs loci 56-440 bp

15. Descriptive stats: allele frequencies - Bayesian approach suggested by Zhivotovsky (1999) % polymorphic loci/population genetic diversity - Lynch & Milligan (1994) for dominant markers # private alleles Population structure and differentiation: Wright’s F ST (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 F ST transformed to F ST /(1‐F ST ) versus log‐transformed geographic distance] (GENALEX v. 6.0)

16. Dcb1 Dcb2 Dcb3 Dcb4 Dcb5 Natura 2000 sites

17. Dcb3 Dcb1 Dcb4 Dcb2 Dcb5 ↓ 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

18. F st = 0.038 => populations are little genetically different AMOVA (Analysis of Molecular Variance): Among populations: 8% Within populations: 92% OUTBREEDER Population genetic structure and differentiation Dcb3 Dcb1 Dcb4 Dcb2 Dcb5

19. 978 671 1000 Dcb3 Dcb1 Dcb4 Dcb2 Dcb5 NO GEOGRAPHIC PATTERN Isolation by distance Dcb3 Dcb1 Dcb4 Dcb2 Dcb5 Mantel test => NO ISOLATION BY DISTANCE (no restriction to geneflow)

20. GENETICALLY VERY HOMOGENOUS! Dcb3 Dcb1 Dcb4 Dcb2 Dcb5

22. DEMOGRAPHIC DATA: population size THREAT DATA : number of threats GENETIC DATA: 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)

23. 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)

25. TAXONPOP. POP. SZ. St THREATS St Hj StD St# PL St# cl StSUM PRIORITY (%) D. cintranus subsp. barbatus Dcb10,501,000,950,500,990,003,9478,80 Dcb21,000,500,910,500,980,083,9779,40 Dcb31,000,001,00 5,00100,00 Dcb41,000,500,890,100,890,433,8176,20 Dcb51,000,750,880,100,870,464,0681,20

26. 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 OUTBREEDER Ex situ and in situ: Dcb3!

28. Genetic diversity = evolutionary potential of a species to evolve and adapt to a changing environment 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?

29. 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

30. 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