Part 3 of the 2010 ACEBB seminar series, Dr Paul Rymer presents "Pollinator-mediated floral evolution and speciation in southern African Iridaceae." Abstract: Explaining the rapid diversification of flowering plants remains one of the greatest challenges facing evolutionary biologists. The pollinator-shift hypothesis developed by Grant (1949) and Stebbins (1970) is the most widely accepted explanation. However, pollinator mediated selection is yet to be shown to result in speciation. The focus of my investigation has been biodiversity hotspots in southern Africa, primarily because they harbour exceptional plant species diversity and endemism, and therefore the promise of detecting speciation in action. In an attempt to unravel the processes driving the evolution of floral traits and speciation, I have taken a multi-faceted approach. I will present my findings from three very different studies: 1. Macroevolution in Sparaxis (Iridaceae), 2. Population genetics in Gladiolus carinatus species complex (Iridaceae), 3. Mating patterns in Gladiolus longicollis (Iridaceae). These studies highlight the role of pollination in recent and continuous speciation events.

1. The Environment Institute
The Australian Centre for Evolutionary Biology and Biodiversity
ACEBB Seminar Series
Pollinator-mediated floral evolution and speciation in
southern African Iridaceae
Dr Paul Rymer

2. Pollinator-mediated floral
evolution and speciation in
southern African Iridaceae
Paul D. Rymer 1,2, Vincent Savolainen 1,2
John C. Manning 3, Peter Goldblatt 4, Steven D. Johnson 5
1 Imperial College London, Silwood Park, UK
2 Royal Botanic Gardens Kew, Jodrell Laboratory, UK
3 South African National Biodiversity Institute, Kirstenbosch, S. Africa
4 Missouri Botanical Garden, St. Louis, U.S.A.
5 University of Kwa-Zulu-Natal, Botany and Zoology, S. Africa
EUROPEAN COMMISSION
Marie Curie Actions - IIF

3. Talk outline
• Diversification of flowering plants
– biodiversity hotspots in southern Africa
• Macroevolution
– Sparaxis (Iridaceae)
• Population genetics
– Gladiolus carinatus species complex (Iridaceae)
• Mating patterns
– Gladiolus longicollis (Iridaceae)
• Current and future research
– Another ‘Great Southern Land’

4. Diversification of flowering plants
• The “abominable mystery” Charles Darwin
Associated with a shift from wind
to animal mediated pollination

5. Evolution of floral traits
Whittall & Hodges (2007) Nature, 447: 706-10

6. Biodiversity hotspots
• Southern Africa
– Cape Floristic Region
– Succulent Karoo
Vitals CFR SK
Hotspot Original Extent (km 2) 78.555 102,691
Hotspot Vegetation Remaining (km 2) 15.711 29,780
Endemic Plant Species 6.210 2,439
Area Protected (km 2) 10.859 2,567
Area Protected (km 2) in Categories I-IV* 10.154 1,890
http://www.biodiversityhotspots.org/

7. Macroevolution
• Evolution of traits across a phylogeny
– Avoid bias and false trends (Felsenstein 1985)
Without controlling for phylogeny, one or a
few species-rich clades with a trait could
strongly bias our interpretations and create an
apparent trend where none actually exists

8. Sparaxis (Iridaceae)

9. Phylogenetic analysis
• Taxonomic relationships
– Monophyly of Sparaxis
– Species delimitation
• Evolution of traits
– Floral traits and pollination syndromes
Sampling (54 taxa) Sequencing
• Outgroups (15) – Plastid loci (3106 bp)
• 100% species (16) +  trnV, trnQ, rpl32
subspecies (6) – Nuclear loci (569 bp)
• Multiple accessions  RPB2
(1-4/taxa)

10. Sparaxis molecular phylogeny
Sparaxis is a
monophyletic
genus, sister to
Duthiastrum
RAxML tree based on plastid and nuclear loci

11. Sparaxis molecular phylogeny
• 2 major clades
• 5 subclades
• Most species
relationships
remain
unresolved
RAxML tree based on plastid and nuclear loci

12. Pollination syndromes

13. Ancestral trait reconstruction
Ancestral state = Bee pollination
Independent transitions – bee (to generalist) to beetle
– bee to long proboscid fly

14. Evolutionary trait shifts
Tube elongation Tube elongation
Long proboscis fly Long proboscis fly
Bee
Symmetry Colour
Bee + beetle Beetle

15. Macroevolution
Summary
• Developed a partially resolved molecular
phylogeny for the genus Sparaxis
– Confirm monophyly and sister-species
• Reconstructed ancestral characters
– Two major transitions from bee to fly or beetle

16. Population genetics
• Fills the gap between phylogenetics and
experimental ecology
• Coalescent analysis
– Estimate divergence times, historical gene flow &
ancestral population sizes
• Genome scans
– Detect genetic signatures of selection
– Identify candidate loci

17. Gladiolus carinatus species complex
GcB GcY
G. carinatus (blue and yellow morph)
Gg Gq
G. griseus G. quadrangulus

19. GcB GcY Gg Gq
GcB 1 0.33* 0.86 0.86
GcY 1 0.00 0.67
Gg 1 0.29
Gq 1

21. Floral differentiation
Species differentiation – Gq isolated
– Gg and Gc partial overlap

22. Coalescent analysis
“Isolation-with-Migration” model (Hey & Nielsen 2004)

23. Genome scans
Genetic signatures of selection can be elucidated
from large genomic datasets, where genes with...
increased population differentiation... may be
candidates for selected loci (Nielsen 2005)

24. Genome scans - species

25. Genome scans - morphs

26. Speciation candidate loci
Genetic divergence (Fst) Association with floral traits
Fragment Global Gq / Gq / Gg / Gg / Gg / GcB /
timing number size shape colour
size-label analysis Gg Gc Gc GcB GcY GcY
106-B species * 0.875 0.823 † -0.050 0.011 0.040 NS NS ** ** **
134-G species * 0.580 0.884 † 0.168 0.148 0.241 0.016 ** NS ** ** **
†
species,
91-G , 0.394 0.867 ‡ 0.308 † 0.314 ‡ 0.200 -0.002 NS NS ** ** **
morphs
‡
324-B morphs * -0.014 0.561 0.390 † 0.362 † 0.508 † 0.051 NS NS ** ** NS
186-G morphs * -0.030 0.273 0.318 † 0.292 ‡ 0.318 ‡ -0.017 NS NS <0.001 <0.001 NS
169-B morphs ‡ 0.487 0.044 0.352 † 0.332 ‡ 0.247 -0.017 <0.001 <0.001 NS NS NS
89-B morphs ‡ 0.149 0.009 § 0.256 ‡ 0.347 † 0.200 0.017 NS NS NS NS NS
225.5-G morphs ‡ 0.329 0.034 0.232 ‡ 0.328 † 0.115 0.079 ** ** NS NS NS
187-G morphs † 0.077 0.172 0.416 * 0.332 ‡ 0.374 -0.021 NS NS <0.001 NS NS
154-B morphs † -0.037 § 0.316 0.348 † 0.314 ‡ 0.332 -0.024 NS NS ** <0.001 NS
291-B morphs ‡ 0.281 -0.004 § 0.219 -0.011 0.508 † 0.393 * NS NS NS NS **

27. Neutral and selected loci
Population structure, but no differentiation of Gc-Gg

28. Neutral and selected loci
All species form distinct clusters

29. Population genetics
Summary
• Evidence for recent and continuous speciation
in the face of gene flow
• Facilitated by a few loci associated with shifts
in floral traits

30. Mating patterns
• Gene flow needs to be halted for speciation to
proceed to completion
• Assortative mating
– Sympatric / secondary contact
• Floral traits may be ‘magic traits’

31. Gladiolus longicollis (Iridaceae)
Corolla tube 8 – 12 cm
Hawkmoth syndrome
• white
• tubular flowers
• open at night
Corolla tube 3 – 6 cm
• scented

32. Hawkmoth pollinators

33. Pollinator behaviour
Long morph
Nectar Pollen Visits Seed
10.1 ± high high high
1.9 mm
0.6 ± high density high
0.4 mm
Short morph
Nectar Pollen Visits Seed
2.7 ± none density none
0.5 mm
2.7 ± high high high
0.5 mm

34. Predictions
• Intermediate morphs will have reduced
reproductive success
• Most pollination events will be within morphs
• Between morph pollination events will be
infrequent and vary in reproductive success
– Density dependent

35. Distribution pattern

36. Study population

37. Population
census
• 2 flowering seasons
• 4 day/night census
• Map & tag plants
• Measure traits
– Corolla tube length
– Dorsal tepal length
– Height of flower
– Leaf length
– Colour (subsample)
– Shape (subsample)
– Scent (subsample)
• Reproductive success
– Capsule maturation
– Seed production

38. Genetic analysis
• Developed SSR markers
– 5 loci (Combined non-exclusion probability 0.00119)
Locus N # alleles Ho He
GL17 859 16 0.733 0.781
GL35 836 57 0.847 0.956
GL41 855 19 0.735 0.904
GL63 871 40 0.885 0.948
GL65 846 41 0.856 0.955
• Extracted DNA
– 128 (2007) and 321 (2008) flowering plants
– 30 (2007) and 32 (2008) progeny arrays
• 600 germinated seed (95-100%)

39. Mating patterns
MLTR (Ritland 1991) 2007 2008
Multilocus outcrossing = tm (SD) 0.979 (0.074) 0.992 (0.100)
Singlelocus outcrossing = ts (SD) 0.851 (0.025) 0.876 (0.031)
Biparental inbreeding = tm-ts (SD) 0.128 (0.068) 0.116 (0.086)
Correlation of outcrossing (SD) 0.129 (0.502) 0.923 (0.539)
1/(number of sires) = rp (SD) 0.290 (0.067) 0.383 (0.065)
High levels of outcrossing with some biparental inbreeding
Large variation in outcrossing rates in 2007

40. Dispersal curves
Frequent short dispersal with fat tale (max 3.94km)

41. Correlation of tube length
• 2007 (low density) assortative mating
• 2008 (high density) significant mating among morphs

42. Mating patterns
Summary
• Assortative mating among morphs
• Between morph pollination events are NOT
infrequent... especially at high densities

43. Current and future research
• Population genetic
– Hybrid determination
– Differential selection
• Reinforcement of traits
– Common garden experiment
• Coevolution of hawkmoth pollination
– Geographic mosaic

44. Current and future research
• Comparative analysis of hotspot evolution
– Western Cape
– Southwest Australia
• Convergent evolution of floral traits
• Role of standing genetic variation
• Adaptation to climate change

45. Acknowledgements
Kew NHM
• Mark Chase • Ian Kitching
• Felix Forest
• Jan Schnitzler Gladiolus longicollis
• Kit Strange • Steve Johnson
• Sandy-Lynn Steenhuisen
Silwood • Ruth Cozien
• Vincent Savolainen • Bruce Anderson
• Martyn Powell • Ronny Alexandersson
• Joaquin Hortal
• Celine Devaux Sparaxis
• Cuong Tang • John Manning
• Peter Goldblatt
• Clare Dean

46. General questions + comments

47. Pollination Ecoregion
Bee S. Karoo
Generalist Lowland
HBSPF Highland
LPF

48. Reinforcement
of traits
- coarse grained
• Compare virgin and
2nd contact areas
– Reproductive ecology
– Population genetics
– Common garden

49. Quantitative genetics
• Common garden of material collected from
virgin areas and secondary contact areas
– Detect shifts + heritability of floral traits

50. Scent analysis
(a) Linalool (b) Methyl benzoate (c) Benzaldehyde
100 50 70
t = 16.06 t = 2.35 t = 0.74
P < 0.001 60 P = 0.47
80 40 P = 0.02
50
60 40
30
40 30
20 20
20
10 10 1.5
0 0
Short-tubed morph
0
Long-tubed morph
Percentage of total ion count
Short Long Short Long Short Long 1.0
(d) Ocimene (e) Phenylacetaldehyde (f) Benzyl acetate
80 35 40
t = 6.43 t = 3.71 t = 5.12 0.5
P < 0.001 30 P = 0.002 P < 0.001
30
Dimension 2
60 25
20 20 0.0
40
15
20 10 10
5 -0.5
0 0
0
Short Long Short Long -1.0
Short Long
(g) Eugenol (h) Benzyl alcohol (i) Phenylethyl acetate -1.5
1.6 1.0 0.35
t = 1.53 t = 4.96 t = 4.89 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
1.4 0.30
P = 0.14 0.8 P < 0.001 P < 0.001
1.2 0.25 Dimension 1
1.0 0.6 0.20
0.8
0.4 0.15
0.6
0.10
0.4 0.2
0.2 0.05
0.0 0.00
0.0
Short Long Short Long Short Long

51. Bimodal pattern
floral tube and moth tongue length
Anderson et al submitted to Evolution

52. Selection against intermediates
Cross pollination by hand
Pollen limitation?
Natural pollination
Anderson et al submitted to Evolution

53. Is there any incompatibility
among morphs?
Pollen parent
Int Short Long Int Short Long Int Short Long
160
140 No significant reduction in seed set among crosses,
Intermediates can have high seed set
120 except long mothers crossed with short fathers
Mean seed set
AB
AB
100 BCD B
BC
AB AB
80
4 AD 26
60 AC 17
7
2 20
40
16
20 5
2
0
Int Int Int Short Short Short Long Long Long
Anderson et al submitted to Evolution Ovule parent

54. The Environment Institute
The Australian Centre for Evolutionary Biology and Biodiversity
Next seminar
Environmental Genomics
Dr Chris Hardy