Patrick Meirmans, Hans den Nijs & Peter van Tienderen meirmans@science.uva.nl

1. Institute for Biodiversity and Ecosystem Dynamics Universiteit van Amsterdam Changes in genotype frequencies of asexual dandelions in a declining population over a 14 year period. Patrick Meirmans, Hans den Nijs & Peter van Tienderen meirmans@science.uva.nl

2. Dandelions(Taraxacum) • Evolution of sex, problematic • Diploid sexuals and triploid asexuals co-occur in mixed populations • Ratio sexuals/asexuals dependent on ecology: • More disturbance => more asexuals • Gene flow => New asexual lineages • What maintains sexuality? • What maintains clonal diversity?

3. Why sex? Why are there still sexual dandelions? • >20 theories explaining the maintenance of sex • Ecological differentiation-Asexuals better colonisers-Sexuals better competitors

4. Diversity in asexuals What maintains the huge diversity in clonal genotypes? • Frozen niche variation: every clone has own ecological niche • Hybridisation with sexuals, through pollen from asexuals

5. Approach • Look at changes in a dandelion population after a 14 year period • Have a close look at ecology and population genetics

6. Sampled population • Population in Odenwald, Germany, intensively studied in 1986 and 2000 • Distribution of two reproductive types over the field • Vegetation analysis • In both years ~360 plants, sexuals and asexuals, were sampled for allozyme analysis • In 2000 also 5 microsatellite loci analysed

7. Ecology Sexuals vs. Asexuals Ecology

8. Changes after 14 years • If there is a big change in the ratio of sexuals to asexuals, there is probably some ecological difference between them • If asexuals are better colonisers and sexuals better competitors: increase in sexuals • If competitively equal: increase in asexuals through hybridisations (slow process) • Look at changes in ploidy level, and check for association with ecology

9. -80% Changes in dandelion density Maximum number of plants found in single 2.5 x 2.5 m. subplot: 1986 273 2000 56 Strong decrease in population density of dandelions Has this decrease been equal for sexuals and asexuals?

10. -80% 2000* 11.6% 8.4% Changes in dandelion density Sexuals Asexuals 1986 33.5% 66.5% 2000 55.9% 44.1%

11. -65% -87% -7.3% -13.7% annual Changes in dandelion density Sexuals Asexuals 1986 33.5% 66.5% 14 years 2000* 11.6% 8.4%

12. (Partial) Mantel tests for association between ploidy level (sexuals are diploid, asexuals triploid), vegetation and distance A * B corrected for Mantel's r sig ploidy * vegetation -0.090 *** ploidy * distance 0.055 *** vegetation * distance -0.150 *** ploidy * vegetation distance -0.083 *** ploidy * distance vegetation 0.042 ** Habitat differentiation ploidy * vegetation distance -0.083 *** • Sexuals and asexuals differ in ecology • This difference is not caused by spatial autocorrelation in both the ploidy data and the vegetation data Mantel test: 999 permutations, Vegetation: 360 relevees, Steinhaus' coefficient of similarity (S17)

13. CCA-score abundance Anthoxanthum odoratum -0.16 143.14 Cardamine pratensis -0.15 5.95 Cerastium fontanum -0.12 65.07 Rumex acetosa 0.12 53.15 Dactylis glomerata 0.19 33.27 Alopecurus pratensis 0.77 6.53 Rumex obtusifolius 0.85 7.05 Sexuals Asexuals Shown are only species with abundance>5, which showed high affinity with either sexual diploid (negative CCA-scores) or asexual triploid (positive CCA-scores) dandelions Habitat differentiation Results of a Canonical Correspondance Analysis, relating ploidy of dandelions to vegetation data (2000 data, p=0.02)

14. Take home message • In the long run asexuals loose (within this population)

15. Genetics Asexuals: Genetic drift / Selection

16. Changes after 14 years • Are there any changes in allele frequencies after 14 years of population decrease? • Has selection caused a sorting of asexual genotypes during the population decrease?

17. Allele frequencies 1986 - 2000 6pgdh1 6pgdh2 mdh1 sexuals asexuals 1986 2000 a b c a b c d a b c

18. Differentiation 1986 - 2000 input #loci Fst sig asexuals 3 0.003 sexuals 3 -0.002 *p<=0.05, ** p<= 0.01, *** p<=0.001 • No difference in allele frequencies, both in sexuals and asexuals • Population sizes are so large that drift is negligible after 14 years • But how relevant are allele frequencies in asexuals? • Shouldn't you look at genotypes (clones)?

19. Triploid genotype frequencies Frequency (%) Genotype Only the 15 most frequent multilocus genotypes are shown

20. Differentiation 1986 - 2000 input #loci Fst sig asexuals 3 0.003 sexuals 3 -0.002 asexual genotypes 1 0.016 *** sexual genotypes 1 -0.001 *p<=0.05, ** p<= 0.01, *** p<=0.001 • Only 1.6% change in genotype frequencies in 14 year period • Amounts to 0.14% change per year, compared to annual decrease of 14% in population size of asexuals

21. Take home message • In the long run asexuals loose (within this population) • Genotypes freqs change a bit faster than allele freqs

22. Genetics Asexuals: Frozen niche

23. Genotypes and ecology • Is there a "frozen niche" for the genotypes: does every genotype have a separate niche? • Use genotypes based on microsatellites for this because of higher resolution

24. Cutoff Distances between clones due to "recombination" Distances within clones due to mutation Defining microsatellite genotypes Number of pairs Genetic distance between pairs of individuals Based on 5 microsatellite loci, distance is the total number of mutationsteps (under stepwise mutation model) that's needed to transform one genotype into the other

25. between clones within clones Microsatellite genotypes and vegetation Number of pairs Ecological (vegetation) distance between pairs of individuals 119 vegetation relevees, Steinhaus' coefficient of similarity (S17), transformed into distance (1-S), genotypes based on 5 hypervariable microsatellite loci

26. Between genotypes: Number of seedheads (2002): r2=0.47*** Date of seedhead opening (2002): r2=0.44*** First flowering date (2003): r2=0.75*** *p<=0.05, ** p<= 0.01, *** p<=0.001 Gene flow sexuals-asexuals • No association between genotype and vegetation • But when followed in greenhouse, there are differences: Sexuals vs. asexuals: First flowering date(2003) n.s.

27. Take home message • In the long run asexuals loose (within this population) • Genotypes freqs change a bit faster than allele freqs • Genotypes don't differ in ecological characters, but do in flowering characteristics (and fitness?)

28. Genetics Sexuals & Asexuals: Gene Flow

29. Gene flow • We know that gene-flow is going on between sexuals and asexuals • Can we make an estimate of the rate? • Is this rate symmetric? Is the flow from sexuals to asexuals as big as vice versa?

30. Gene flow sexuals-asexuals Theta (=4 * population size * mutation rate): • sexuals (diploid): 1.97 • asexuals (triploid): 3.22 Migration (=4 * population size * migration rate): • sexuals -> asexuals: 18.78 • asexuals -> sexuals: 14.41 p<0.001 Migrate (v. 1.7.3): analysis of gene-flow using coalescent theory, 60 individuals subsampled from both types, preliminary results from several runs (averaged), sometimes with slightly different search parameters.

31. Take home message • In the long run asexuals loose (within this population) • Genotypes freqs change a bit faster than allele freqs • Genotypes don't differ in ecological characters, but do in flowering characteristics (and fitness?) • High level of gene-flow between sexuals and asexuals

32. So...

33. Main results: ecology • Population decline twice as fast in asexuals as in sexuals • Diploids better competitors -> Red Queen? • Ecological differentiation: Asexuals in more disturbed parts of the population

34. Main results: genetics • Hard to estimate relative importance of different factors that may promote clonal diversity Recombination Frozen niche

35. This talk wouldn’t have been the same without: • Annemieke Kiers • Harold Lemereis • Jan Kirschner • Liesbeth Vlot • Ludek Tikovsky • Marc Stift • Peter Kuperus • Piet Oosterveld • Pieternella Luttikhuizen • Rob Bregman • Ron van der Hulst • Steph Menken • Stephanie Hamm • Everybody at the Hugo de Vries-lab