1. 1 Department of Phytology, Technical University in Zvolen, Zvolen, Slovakia 2 National Forestry Centre, Forestry Research Institute, Zvolen, Slovakia 3 State Forests of the Slovak Republic, General Directorate, Banská Bystrica, Slovakia Factors affecting effective population size estimation in a seed orchard: a case study of Pinus sylvestris Dušan Gömöry 1, Roman Longauer 2, Ladislav Paule 1, Rudolf Bruchánik 3

2. Ideal seed orchard: Randomly mating Mendelian population Contribution of genotypes to male and female gamete pools proportionate to their frequencies No change of allelic frequencies over generations

3. Real clonal seed orchards of European conifers: Marker studies: Generally no or small deviations of genotypic frequencies from HWE expectations Low levels of self-fertilization but (1) Sometimes significant differences in allelic frequencies between seed orchard and the crop but (2) Marker studies cannot easily be done everywhere

4. Ideal clonal seed orchard: Equal contribution of parental clones to the gene pool of the seed crop Reality: Deviations from equal contribution because of: unequal female gamete contributions unequal male gamete contributions phenological isolation pollen dispersal in space (distribution of clones – distance-dependent dispersal, wind direction, turbulences) genetic incompatibilities

5. Consequences: loss of genetic diversity in seed orchard crops increased coancestry (potentially higher inbreeding in the following generation) Monitoring the genetic diversity in seed orchard crops: Effective population size (e.g. effective number of clones) Status number (Lindgren et al. 1996: Silvae Genet. 45: 52–59). N S = 0.5/Θ = number of unrelated non-inbred genotypes having the same average coancestry as the considered population

6. Case study: three Scots pine seed orchards CharacteristicSeed orchard KolkáreňHájSýkorová Geographical coordinates Altitude Year of establishment Census number of clones Coefficient of variation of clone representation Number of ramets Number of wrongly planted ramets Number of alien genotypes Number of alien ramets Spacing Average cone yield per ramet over the period 1995 to 1999 Average cone yield per ramet in 1999 48°46‘N 19°16‘E 450 m 1982/1987 43 40.8% 589 117 160 5 m × 7 m 0.47 kg 0.89 kg 48°15'N 19°19'E 350 m 1979 30 43.0% 574 199 63 81 8 m × 8 m 2.38 kg 2.93 kg 48°29'N 18°42'E 475 m 1979 35 59.4% 372 56 45 7 m × 8 m 2.80 kg 3.17 kg

7. Assessed characteristics (for each ramet): Female gamete contribution – cone weighting Male gamete contribution – counting and size assessment of male strobili Flowering phenology: 3 dates in Háj and Sýkorová, 5 dates in Kolkáreň, 5 developmental stages for male strobili, 6 stages for female strobili Spatial dispersal of pollen: negative exponential dispersal function (Adams & Birkes 1991)

8. Alien genotypes present in all three seed orchards (rootstocks with aborted scions, unregistered clones, …) as proved by allozyme genotyping Three situations considered: (1) Alien material removed (2) Alien ramets not removed but not harvested (3) Cones harvested from all ramets

9. Effective number, status number and coancestry estimates Háj old Aliens removed Aliens not harvested All harvested ΘNΘNΘN Census Eff. clones N S : F FM FMP FMPD 0.0198 0.0236 0.0241 0.0248 30 25.5 25.2 21.2 20.8 20.1 0.0171 0.0207 0.0213 93 34.1 29.2 24.1 23.5 0.0150 0.0186 0.0190 93 34.1 33.3 26.9 26.3 F – female contributions assessed, M – male contributions assessed, P – phenology assessed, D – pollen flow considered

10. Effective number, status number and coancestry estimatesHáj

11. Effective number, status number and coancestry estimates Sýkorová old Aliens removed Aliens not harvested All harvested ΘNΘNΘN Census Eff. clones N S : F FM FMP FMPD 0.0205 0.0237 0.0234 35 26.1 24.4 21.1 21.4 0.0181 0.0199 0.0198 0.0201 80 33.5 27.6 25.1 25.3 24.9 0.0159 0.0177 0.0176 0.0178 80 33.5 31.5 28.3 28.4 28.0 F – female contributions assessed, M – male contributions assessed, P – phenology assessed, D – pollen flow considered

12. Effective number, status number and coancestry estimatesSýkorová

13. Effective number, status number and coancestry estimates Kolkáreň young Aliens removed Aliens not harvested All harvested ΘNΘNΘN Census Eff. clones N S : F FM FMP FMPD 0.0159 0.0214 0.0241 0.0263 43 36.9 31.4 23.4 20.7 19.0 0.0122 0.0162 0.0177 0.0197 203 68.0 40.9 30.9 28.2 25.4 0.0095 0.0123 0.0148 0.0165 203 68.0 52.4 40.7 33.8 30.4 F – female contributions assessed, M – male contributions assessed, P – phenology assessed, D – pollen flow considered

14. Effective number, status number and coancestry estimatesKolkáreň

15. Conclusions In old, fully fructificating seed orchards of Scots pine in Central Europe, effective number of clones is a sufficient estimator of the effective population size Phenology and spatially-dependent pollen dispersal do not considerably affect N S estimates and need not be assessed for practical purposes Young seed orchard are unbalanced and phenologically not synchronized, consequently, effective number of clones is a poor estimator of effective size and status number is affected by all factors (male + female fertility, phenology, spatial design)

16. Thank you for your attention