1. Carl Barker, Fay Voller, Paul Ashton
Incidence and structure of asexual reproduction in a temperate canopy tree species across its UK range
Carl Barker, Fay Voller, Paul Ashton

2. Clonality Many organisms partially or entirely asexual
Particularly common in angiosperms (~80% spp.)[1]; often maintain mixed reproductive systems
Despite this, study of sexual reproduction more common
Possibly because of assumptions that underlie standard population genetic analyses? E.g. admixture, non-overlapping generations etc.
Authors, left to right; public domain images x2; Alistair J. Cullum; Thom Dallimore; Stan Shebs, used under CC BY-SA 3.0 license
1: Klimes et al. 1997

3. Effects of clonality in angiosperms
Population persistence
Large amount clonality may lead to increased mutational load[1]
Reduction in long-term genetic diversity (maybe[2])
Population structure
Arrangement of genetic variation
Outcrossing success[3]
Last points key here – should be considered
Many invasive plant species clonal (e.g. Japanese knotweed)
1: Silvertown, J., The Evolutionary Maintenance of Sexual Reproduction: Evidence from the Ecological Distribution of Asexual Reproduction in Clonal Plants. Int. J. Plant Sci. 169, 157–168.
2: Balloux, F., Lehmann, L., de Meeus, T., The population genetics of clonal and partially clonal diploids. Genetics 164, 1635–1644.
3: Charpentier, A., Consequences of clonal growth for plant mating. Evol. Ecol. 15, 521–530.

4. Tilia cordata (Small leaved lime)
Current EGG favourite!
Temperate canopy tree, ancient woodland indicator
Mixed reproductive mode, entomophilous
Low self-fertility
Suggestion of persistence at range edge due to clonality
Although pop. gen. studies no examination of clonality directly

5. Study
Aims
Assess extent and character of asexual reproduction and effect on spatial genetic structure across UK range
Study area
7 sampling sites spread across latitudinal gradient
All semi-natural ancient woodland, coppiced

6. Sampling 30 m x 30 m random quadrat Mapped, sampled every lime tree
Actually just wanted minimum sampling density for population differentiation study so that I didn’t get clonal replicates, hypocrite!

7. Lab work 174 distinct genotypes, 111 repeat First step: genotyping
11 SSR markers[1]
CTAB PCR, or ‘direct’ PCR
Fragment analysis on AB3500
174 distinct genotypes, 111 repeat
Add trace here for visual interest
1: Phuekvilai, P., Wolff, K., Characterization of Microsatellite Loci in Tilia platyphyllos (Malvaceae) and Cross-Amplification in Related Species. Appl. Plant Sci. 1,

8. Identifying clones Identical genotype = clone?
[3]
Identical genotype = clone?
Calculate likelihood of distinct zygote origin
All identical are below threshold [Psex(FIS)<0.01][1, 2]
What about inverse (distinct = not clone)?
Scoring error/somatic mutation creates distinct genotype in clone
Remove distinct locus; recalculate Psex(FIS)
Slide is boring. Add reference and picture (formula and Pigott 1991 distribution map?)
1: Young, A.., Hill, J.., Murray, B.., Peakall, R., Breeding system, genetic diversity and clonal structure in the sub-alpine forb Rutidosis leiolepis F. Muell. (Asteraceae). Biol. Conserv. 106, 71–78.
2: Arnaud-Haond, S., Duarte, C.M., Alberto, F., Serrão, E.A., Standardizing methods to address clonality in population studies. Mol. Ecol. 16, 5115–39.
3: Pigott, C.D., Tilia cordata Miller. J. Ecol. 79, 1147–1207.

9. Clonal incidence All sites show mixed reproduction occurring14 49 41 34 78 28
285
All sites show mixed reproduction occurring
Wide range (0.195 – R)
Mean of ~50% clones
No clear geographic pattern

10. Clonal diversity Diverse2 3 2 5 2 2 2 4
Diverse
Not dominated by one clonal lineage; small groups (2 – 22 n; M = 2)
Very similar across all sites
I don’t think I need all of these Pareto things, too small; steepest and shallowest? That’d be Eaves Wood and Shrawley Wood. There’s a slight pattern of more southern = more even, but that doesn’t match the V scores.
Range and median for clonal group sizes I think. As boxplot?

11. Clonal arrangement
Clones often form exclusive groups, but these groups usually intermingled with unique genets
Max. distance between clones (Dmax) for ea. group varied but typically <10 m (0.60 – m, M = 4.58 m)

12. When does clonality stop affecting spatial genetic structure (SGS)?
Mean pairwise coancestry
coefficient (Fij)

13. Summary All sites have mixed reproductive modes
Ranges from % clonal; average of 47.7 %
High evenness across sites, small clonal groups (2 – 22 n; M = 2)
Not dominated by any particular clonal lineage – all trees have similar chance of reproducing asexually?
Clonal groups typically small (Dmax < 10 m), spread across area evenly
Clonality does not seem to affect SGS much beyond scale of groups

14. Conclusions
Observed higher levels of clonality than previously reported (~50% vs. 25%) for Tilia[1]; sampling method?
Levels of clonality intermediate relative to other tree spp.[2,3]; similar structure to other spp.[4]
Given weak SGS and small clonal group size, expect not limiting outcrossing success
More recruitment from seed than suspected?
1: Logan, S.A., Phuekvilai, P., Wolff, K., Ancient woodlands in the limelight: delineation and genetic structure of ancient woodland species Tilia cordata and Tilia platyphyllos (Tiliaceae) in the UK. Tree Genet. Genomes 11, 52.
2: Morris, A.B., Millsaps, N.K., Mason, K.Q., Howell, J.S., Speer, J.H., Mature beech trees (Fagus grandifolia; Fagaceae) are persistently clonal in coves and beech gaps in the Great Smoky Mountains. Am. J. Bot. 101, 381–8.
3: Alfonso-Corrado, C., Esteban-Jiménez, R., Clark-Tapia, R., Piñero, D., Campos, J.E., Mendoza, A., Clonal and genetic structure of two Mexican oaks: Quercus eduardii and Quercus potosina (Fagaceae). Evol. Ecol. 18, 585–599.

15. Further work Mixed sites (in progress)
Larger scale (in concert with above)
Relationship between management and clonality
Perhaps longer latitudinal cline (field trip to Italy anyone?)

16. Acknowledgements Fieldworkers
Mark Ashton, Robert Ashton, Jordan Bilsborrow, Thom Dallimore & others
Stakeholders for sampling permission (Natural England, landowners)

17. Thanks for listening
Any questions?

20. R vs. G/N
G / N as measure of genotypic richness is dependent on sample size
E.g. monoclonal stand with 20 ramets: G / N = 0.05; monoclonal stand with 100 ramets: G / N = 0.01
R = (G – 1) / (N – 1), so examples above are 0 & 0 resp., removing bias introduced by low n

21. Genetic diversity & differentiation
Pairwise GST
Roudsea Wood
Eaves Wood
Ivy Wood
Collyweston Great Wood
Shrawley Wood
Bovingdon Hall
Garnetts Wood -
0.33
0.37
0.27
0.22
0.31
0.18
0.29
0.16
0.28
0.34
0.19
0.49
0.50
0.36
0.32
Locus A
1-D
Hexp V
Tc6 11
0.74
0.73
Tc920 7
0.80
0.85
Tc937 8
0.58
0.62
Tc4 10
Tc943 4
0.49
Tc11 6
0.52
0.86
Tc963 27
0.93
0.82
Tc915 17
0.84
0.69
Tc5 12
0.70
Tc7 9
0.72
Tc951
0.78
mean
10.7
0.75

23. Genotypic richness (R)
Saplings
Understorey
Canopy
Legend