Speciation & Reproductive Isolation in Columbines
by J. Phil Gibson
University of Oklahoma
Columbine Case Questions:
• How do flowers, pollinators, and pollination systems
work?
• How can we identify and test different for
reproductive isolation in plants?
• What mechanisms can act to unify members of a
species and maintain separatations between
members of different species in plants?
Aquileqia caerulea – Colorado columbine
Clicker Question #1
The diagram to the right is a phylogeny
for 5 species in the same genus. This
phylogeny shows. . .
A. the degree to which organisms
look like one another.
B. how different organisms are
related through common
ancestors
C. the sequence in which species
evolved from left to right.
D. the amount of change each
species has experienced.
E. which species are most closely
related to the species at point M.
1 2 3 4 5
Clicker Question #2
What do the lines in this phylogeny
represent?
A. The passage of time
B. Lineages of organisms
C. Each line represents a single
species
D. Which species evolved first,
second, third, etc.
E. All of the above
1 2 3 4 5
Clicker Question #3
What do the nodes (indicated by G, P,
R, and M) represent in the
phylogeny?
A. Where two species
hybridized.
B. Lineages of organisms.
C. Common ancestors of
different species.
D. The sequence in which
species 1-5 evolved.
E. None of the above.
1 2 3 4 5
G
P
R
M
Clicker Question #4
What does the point labeled R indicate?
A. Species 5 in the past.
B. Species 2 in the past
C. Where species 2 and species 5
hybridized.
D. The common ancestor of species
2, 3, 4 & 5..
E. Where species 5 became species
2.
1 2 3 4 5
G
P
R
M
Clicker Question #5
Which statement(s) below accurately
describe what is happening at the nodes
indicated by MRPG? (choose all that apply)
A. Speciation is occurring
B. Mutation and natural selection are
making the best new species
C. One species is turning into another
species.
D. Gene pools are becoming separated
and isolated.
E. Two species are coming together to
make a new species.
1 2 3 4 5
G
P
R
M
Pre-case Thought Questions.
• How do flowers work? How do they play a role in
maintaining separate species? Make a labeled
diagram to summarize your thoughts.
• What are the important aspects of the
interactions and relationship between flowering
plants and their pollinators?
• What is the relationship among sister species, a
common ancestor to the sister species, and
reproductive isolation? Make a labeled diagram
to summarize your thoughts.
• What is a phylogeny and what does it tell us
about species?
Phylogenies are hypotheses about
relationships among taxa.
Branches in a phylogeny are called clades.
Members of a clade share a number of
features, such as similar flower structure,
due to common ancestry.
1 2 3 4 5
Nodes indicate common ancestors and
points of lineage divergence (speciation).
Lines represent separate lineages. Traits
can change in a lineage resulting in
unique traits shared by members of that
lineage.
Reproductive isolation maintains separate lineages.
Flowering plants are a clade that
share a unique feature, flowers.
sepals
petals
stamen
filament
{
pistil
ovary
style
anther
stigma
What Do Pollinators Do?
In flowering plants, pollination is the transfer of pollen
from an anther to a receptive stigma by an abiotic or
biotic vector. Wind is a common means of abiotic
pollination. Biotic pollination is typically a mutualistic
relationship between a plant and an animal in which
the plant attracts the animal to a flower to pick up
pollen and then carry it to another flower for sexual
reproduction.
Clicker Question #6
What do you think is the most
important feature of a flower for
attracting pollinators?
A. Flower color
B. Flower shape
C. Flower orientation
D. A reward, such as nectar
E. A reward, such as pollen
POLLINATOR ATTRACTANTS
PRIMARY ATTRACTANTS
• Food (nectar, pollen)
• Shelter (solar tracking flowers)
• Other needed materials (e.g. waxes,
pheromones, repellants)
SECONDARY ATTRACTANTS
• Odor (scents & fragrances)
• Visual cues (color, shape, nectar
guides)
Biotic Pollination Syndromes
• Cantherophily (beetles): white or drab, sweet scent, open bowl
shape
• Myophily (flies): variable
• Sapromyophily (carion and dung flies): purple-brown, stink of
decaying protein, deep traps
• Melittophily (bees): variable color/no pure red, sweet scent
• Sphignopily (hawkmoths): white or pale green, strong sweet
scent, deep narrow tubes with nectar
• Psychophily (butterflies): red, yellow, blue, moderately strong
sweet, deep narrow tubes with nectar
• Ornithophily (birds): bright red, no scent, wide deep tubes with
nectar
• Chiropterophily (bats): dull white or green, strong fermented
scent, brush or bowl shaped
Flowers and Pollinators:
A structural-functional-behavioral
interaction
In flowering plants, floral structure has been
suggested as an important mechanism for achieving
reproductive isolation and promoting speciation by
affecting either pollinator behavior (behavioral
isolation) or pollen transfer (mechanical isolation)
(Grant 1949).
Clicker Question #7
Which form of reproductive isolation do you think would be
most effective for flowering plants?
A. Ecological isolation due to plants growing in different
habitats
B. Behavioral isolation due to floral traits influencing pollinator
behavior
C. Hybrids being sterile and unable to produce functional
gametes or not being able to survive in populations
D. Biochemical isolation due to failed interaction between
pollen and stigma.
E. Mechanical isolation due to pollen not being successfully
gathered or delivered.
Aquilegia spp. (Columbines) - Spurs & Species
All columbine flowers have nectar spurs and other
structural similarities.
stigmas
anthers
nectar
spurs
Clicker Question #8
In columbines, color would be an example of a
_________ attractant and nectar would be
an example of a _________ attractant.
A. primary/secondary
B. floral/pollinator
C. secondary/primary
D. bird/insect
E. reward/visual
Floral nectar spurs are
considered innovations
that have promoted
speciation and
reproductive isolation
in columbines through
shifts in pollinators
related to changes in
nectar spur length. How
could this work?
bee pollinated
hummingbird pollinated
hawkmoth pollinated
hummingbird & hawkmoth pollinated
Aquilegia formosa
Aquilegia pubescens
Aquilegia fomosa and A. pubescens are species that evolved from a
recent common ancestor.
What differences between A. formosa and A. pubescens flowers may
be important for interacting with pollinators?
Why might their sharing a recent common ancestor be important?
Clicker Question #9
Because they share a common ancestor. . .
A. both columbine species share relatively few traits.
B. both columbine species share some traits, but
also have other unique features that define and
reinforce each species’ as a separate lineage.
C. both species are completely different from one
another and share no traits.
D. both columbine species will attract the same
pollinators and grow in the same places.
E. both columbine species are reproductively
isolated from one another.
Spurs & species
Given the proposed importance of nectar
spurs, how would you test their influence on
columbine speciation?
Develop an hypothesis and design an experiment to
to explore potential causes and function of
behavioral isolation and mechanical isolation in A.
pubescens and A. formosa flowers.
In an initial study, researchers presented both columbine
species in a hexagonal array with 9 of each spp. Arrays were
placed near A. pubescens and A. formosa populations and
pollinator visits recorded.
Why did they do this?
A. formosa
A. pubescens
What would you expect?
Mean visits per flower per hour by different pollinators to A.
formosa & A. pubescens
visits/flower/hour (+/- s.e.)
1.2
1
0.8
A. formosa
A. pubescens
0.6
0.4
0.2
0
hummingbird
hawkmoth
fly
pollinator species
bee
Experimental Results
Mean visits per flower per hour by different pollinators to A.
formosa & A. pubescens
visits/flower/hour (+/- s.e.)
0.3
0.25
0.2
A. formosa
A. pubescens
0.15
0.1
0.05
0
hummingbird
hawkmoth
fly
pollinator species
bee
Experimental Results: Pollinators identified
Aquilegia formosa
Selasphorus rufus
Aquilegia pubescens
Hyles lineata
Experimental Data: Pollinator preferences for
A. formosana and A. pubescens
Visits to
hummingbirds
hawkmoths
bees
A. formosa
81
0
85
A. pubescens
9
115
19
Χ2
57.6
115
41.88
p
<0.0001
<0.0001
<0.0001
What can the researchers conclude so far?
What data should
they collect? Why?
What will the data
tell them?
Aquilegia pubescens
Next, the researchers focused on A. pubescens. In this study, they
planted two arrays of modified and control (unmodified) flowers.
Array 1: pedicels for ½ of the A. pubescens flowers staked to make
flowers pendant (point downwards)
Array 2: spurs for ½ of the A. pubescens flowers shortened
(squeezed nectar from bottom of spurs, tied & clipped spur)
A. pubescens Flower Manipulations
Measure pollen removal as an
indicator of effective pollinator
visitation.
Unmanipulated
Pendant
Shortened
Clicker Question #10
Which of these hypotheses are the researchers potentially
testing?
If floral structural traits in A. pubescens are important for
reproductive isolation, then we should expect. . .
A. the floral modifications will have no effect on
pollinator visitation and pollen removal
B. hawkmoths will be better able to remove pollen from
modified flowers.
C. hummingbirds will be attracted to the modified
flowers less than unmodified flowers.
D. the unmodified flowers will have higher visitation and
pollen removal by hawkmoths than modified flowers.
Clicker Question #11
In the floral manipulation experiment, you should expect . . .
A. unmanipulated flowers will have more pollen removed by
hawkmoths than the others.
B. unmanipulated flowers will have less pollen removed by
hawkmoths than the others.
C. hawkmoths will visit pendant flowers more than the
others.
D. hawkmoths will remove pollen from shortened spur
flowers more than the others.
E. None of these are expected outcomes for this experiment.
Clicker Question #12
Experimental Data: Visits by Hyles lineata to A.
pubescens with differing floral orientation.
Visits to
Number of
observed visits
upright
51
pendent
5
Χ2
45.5
p
<0.0001
These results are consistent with the
researchers’ prediction.
A. True B. False
Clicker Question #13
Experimental Data: Visits by Hyles lineata to A.
pubescens with long or short nectar spurs.
Visits to
Number of
observed visits
long
17
short
19
Χ2
0.11
p
Not significant
These results are consistent with the
researchers’ prediction.
A. True B. False
Clicker Question #14
Mean number of pollen grains remaining in A. pubescens anthers
after hawkmoth visitation.
mean pollen grains remaining in anther (+/- se)
4500
4000
These results are
consistent with
the researchers’
prediction.
A. True
B. False
3500
3000
2500
2000
1500
1000
500
0
control (long)
shortened
treatment
Clicker Question #15
From these studies we can conclude that
A. Orientation and color promote mechanical isolation and
floral structure promotes behavioral isolation.
B. The species have few floral features that would promote
reproductive isolation.
C. Spur length is a primary attractant and color is a secondary
attractant.
D. Orientation and color promote behavioral isolation and
floral structure promotes mechanical isolation.
E. Floral structure causes reproductive isolation after
pollination occurs.
What about hybrids and habitats?
A. pubescens typically grows at higher
elevations and in more xeric habitats.
A. formosa tends to grow at lower
elevations and in more mesic (moist)
habitats.
Although they have these differences, hybrid populations of viable,
reproductively functioning plants with floral traits and molecular
markers characteristic of both species have been identified at
intermediate elevations and habitats!
How could that happen?
What does it mean biologically? Evolutionarily?
Researchers measured floral (A & C) and genetic (B & D)
traits in hybrid populations along elevation (A & B) and
habitat transects (C & D). What do the data show?
A. pubescens
spur color
blade color
spur length
blade length
A. formosa
A. pubescens
orientation
A. formosa
genetic markers
A. pubescens
genetic markers
A. formosa
Clicker Question #16
The occurrence of hybrids indicates. . .
A. that A. formosa and A. pubescens are really just
one species.
B. reproductive isolation does not matter for plant
species.
C. pollinator behavior is not important for
maintaining species.
D. reproductive isolating barriers are not always
absolute between species.
E. that habitat is not important for maintaining
species.
Clicker Question #17
Based upon the data in the floral manipulation
studies, spurs act to maintain separate Aquilegia
species by. . .
A. causing flowers to grow in different habitats
B. offering different rewards to different pollinators
C. influencing pollen removed from and depositing
on flowers.
D. attracting different pollinators.
Clicker Question #18
Based upon the data in the hybrid studies, habitat
selection acts to maintain A. formosa and A.
pubescens as separate species by. . .
A. causing plants of the separate species to grow
better in distinct habitats.
B. offering different rewards to different pollinators
based upon the habitat.
C. causing hybrid individuals to survive as well as
non-hybrid individuals in any habaitat.
D. causing the pollinators living in different habitats
to have access to different rewards.
Species Concepts
•
•
•
•
Morphological species concept
– Based on physical characteristics.
– Simple, clear, but does not address mechanisms that separate or maintain species.
Biological species concept
– Species is a group of individuals whose members have the potential to interbreed
with one another in nature to produce viable, fertile offspring but cannot
successfully interbreed with members of other species
– Not good for plants, asexual reproducers, hybrids, or extinct species.
Ecological species concept
– Each species occupies an ecological niche, which is the unique set of habitat
resources that a species requires, as well as its influence on the environment and
other species.
– Can be hard to determine if species have broad habitat tolerances.
Evolutionary lineage (Phylogenetic) concept
– Species should be defined based on the separate evolution of lineages.
– May be hard to draw delimitation.
Clicker Question #19
Which species concept do you think is least applicable
for A. formosa and A. pubescens?
A. morphological
B. biological
C. ecological
D. phylogenetic
E. None of these really works.
Clicker Question #20
The two columbine species. . .
A. are really just one species because their flowers are
not different enough to be separate species.
B. represent different lineages that became isolated
from one another and diverged from a common
ancestor.
C. are tentatively two species until the pollinators can
differentiate them better.
D. represent different lineages but the pollinators’
inability to distinguish them will prevent further
speciation.
Epilogue
There are floral differences that influence pollinator
behavior.
However, there is some pollinator infidelity and hybrid
offspring between the two species are produced
and survive in habitats intermediate to the typical
conditions for each species.
Although floral features maintain some reproductive
isolation between species, habitat selection against
hybrids is also a strong force maintaining
reproductive isolation for both species.
End of Case Thought Questions
• Prepare a diagram that uses these columbines to show the
relationship between pollinators and flower structure and
function.
• Prepare a diagram that uses these columbines to
demonstrate the evolutionary and ecological factors shaping
the relationship between two sister species.
• Prepare a diagram that describes the history of these species
and their common ancestor, how they may have become
different, and how interactions with pollinators and habitat
have shaped speciation.
• If we assume these species evolved from a common ancestor
that was hummingbird pollinated, can you use a
phylogenetic tree to diagram how traits diverged in the
different ?
Image Credits
Slide 1: Clump of columbines Copyright © 2006 J. Phil Gibson
Slide 2: Aquilegia flower Copyright © 2006 J. Phil Gibson
Slide 10: Kalmia latifolia flower Copyright © 2006 J. Phil Gibson
Slide 11: Butterflies on a milkweed Copyright © 2012 J. Phil Gibson
Slide 13: Cactus flower with bee Copyright © 2012 J. Phil Gibson
Slide 17: Detail from Clump of columbines Copyright © 2006 J. Phil Gibson
Slide 20: A. formosa: http://species.wikimedia.org/wiki/File:Aquilegia_formosa_14962.JPG This file is licensed under the Creative
Commons Attribution-Share Alike 3.0 Unported license.
A. pubescens: Copyright © 2009 Barry Breckling http://calphotos.berkeley.edu/cgi/img_query?enlarge=0000+0000+1209+2492.
This image has a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 (CC BY-NC-SA 3.0) license.
Slide 26: A. formosa: http://species.wikimedia.org/wiki/File:Aquilegia_formosa_14962.JPG This file is licensed under the Creative
Commons Attribution-Share Alike 3.0 Unported license.
A. pubescens: Copyright © 2009 Barry Breckling http://calphotos.berkeley.edu/cgi/img_query?enlarge=0000+0000+1209+2492
This image has a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 (CC BY-NC-SA 3.0) license.
Rufus hummingbird: Selasphorus rufus http://en.wikipedia.org/wiki/File:Selasphorus_rufus.jpg
Hawkmoth: Hyles lineata http://en.wikipedia.org/wiki/File:White-lined_sphinx.JPG This file is licensed under the Creative
Commons Attribution-Share Alike 3.0 Unported license.
Slide 28: A. pubescens: Copyright © 2009 Barry Breckling
http://calphotos.berkeley.edu/cgi/img_query?enlarge=0000+0000+1209+2492 This image has a Creative Commons AttributionNonCommercial-ShareAlike 3.0 (CC BY-NC-SA 3.0) license.
Slide 36:
A. formosa: http://species.wikimedia.org/wiki/File:Aquilegia_formosa_14962.JPG This file is licensed under the Creative
Commons Attribution-Share Alike 3.0 Unported license.
A. pubescens: Copyright © 2009 Barry Breckling http://calphotos.berkeley.edu/cgi/img_query?enlarge=0000+0000+1209+2492
This image has a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 (CC BY-NC-SA 3.0) license.
Slide 37: Figure from from Hodges, SA and ML Arnold. 1994. Floral and Ecological isolation between Aquilegia formosa and
Aquilegia pubescens. Poc Nat Acad. Sci USA 91: 2493-2496. Permission needed.