Food for Sex Or How to Attract a Pollinator: Co-Evolution and Speciation in the Monkey-Flower

Food for sex or how to attract a pollinator: co-evolution and speciation in the monkey-flower (Mimulus)

Introduction

Many plants depend on pollination for their reproductive success. Co-evolution of a plant with its pollinator is often seen in flowering plants. Flowers provide pollinators with a sweet, high energy meal (nectar). Pollinators in turn collect the flower pollen on their body while feeding and transport it to the next flower they feed on, assisting in dispersal of the male gametes and in fertilization. Pollinators with a strict preference for one type of flower are therefore responsible for creating a pre-zygotic barrier and contribute to the reproductive isolation of sympatric flowers. Reproductive isolation then can lead to speciation events and the formation of closely related sister taxa.

When Charles Darwin collected an orchid in Madagascar with a very long spur (tubular corolla), Agraecum sesquipedale, he predicted the existence of a moth with a very long proboscis (moth’s tongue), which would have been needed to pollinate the flower.

"I fear that the reader will be wearied, but I must say a few words on the Angræcum sesquipedale, of which the large six-rayed flowers, like stars formed of snow-white wax, have excited the admiration of travellers in Madagascar. A whip-like green nectary of astonishing length hangs down beneath the labellum. In several flowers sent me by Mr. Bateman I found the nectaries eleven and a half inches long, with only the lower inch and a half filled with very sweet nectar. What can be the use, it may be asked, of a nectary of such disproportional length? We shall, I think, see that the fertilisation of the plant depends on this length and on nectar being contained only within the lower and attenuated extremity. It is, however, surprising that any insect should be able to reach the nectar: our English sphinxes have probosces as long as their bodies: but in Madagascar there must be moths with probosces capable of extension to a length of between ten and eleven inches!"

Darwin, 1862

Five years later, Wallace predicted the type of moth that should be found in Madagascar.

"I have carefully measured the proboscis of a specimen of Macrosila cluentius from South America in the collection of the British Museum, and find it to be nine inches and a quarter long! One from tropical Africa (Macrosila morganii) is seven inches and a half. A species having a proboscis two or three inches longer could reach the nectar in the largest flowers of Angræcum sesquipedale, whose nectaries vary in length from ten to fourteen inches. That such a moth exists in Madagascar may be safely predicted; and naturalists who visit that island should search for it with as much confidence as astronomers searched for the planet Neptune,--and they will be equally successful!"

Wallace, 1867

This Malagasy moth, Xanthopan (Macrosila) morgani, was discovered thirty six years later.

Xanthopan morgani praedicta and the star orchid Angræcum sesquipedale

http://perso.orange.fr/cryptozoo/dossiers/moth.htm

Pre-Laboratory activity

The flowering plants of the genus Bauhinia exhibit a great diversity in flower form, size, and color. Several Bauhinia species can be found in Venezuela, inhabiting very different forest ecosystems. Some Bauhinia species are tree-form while others are liana-form. Studies of the pollination of Bauhinia have shown that some species are bat pollinated, while others are pollinated by moths, insects or bird.

Seven species of closely related Bauhinia from Venezuela have been studied and have shown great differences in flower morphology, physiology and nectar production. Several possible pollinators are present in their community.

After studying the plant morphology and physiology, you are asked to make a prediction for the type of pollinator most likely associated with each plants.

The following potential pollinators are found in the forests associated with Bauhinia flowers:

Moths (Sphingidae and Noctuidae): nocturnal. Pollen and nectar are their source of protein.

Bats (Phyllostomidae): nocturnal, cannot assimilate sucrose, some eat insects and do not require proteins in nectar.

Diurnal insects: butterflies (Pieridae), wasps and bees. Pollen and nectar are their main source of protein. Prefer sucrose rich nectar.

Hummingbirds (Trochilidae): Nectar is their source of protein. Prefer sucrose rich nectar.

What do pollinators gain when they visit a flower?

What do the flowers gain when they attract a pollinator?

Fill out this table with the possible pollinators. Justify your answer.

Plant name, plant form, location
Plant description / Possible pollinators
Bauhinia aculeata, tree form, dry premontane forest
Large, white flower. Open asynchronously at night and at dusk and dawn. High nectar production both night and day. Nectar rich in sucrose. Some proteins in nectar.
Bauhinia multinervia, tree form, dry premontane forest
Large, white flower. Open at night. High nectar production at night. Nectar rich in hexose. Few proteins in nectar.
Bauhinia pauletia, tree form, dry premontane forest
Large, white flower. Open at night. High nectar production at night. Nectar rich in hexose. Few proteins in nectar.
Bauhinia ungulata, tree form, tropical dry forest
Large, white flower, some red flower. Open at night. High nectar production at night. Nectar rich in hexose. Few proteins in nectar.
Bauhinia glabra, liana form, dry premontane forest
White, smaller flower with purple lines. Open in daylight.
Very small nectar production but sticky (concentrated), rich in sucrose and glucose, contains proteins
Bauhinia guianensis, liana form, humid tropical forest
White or clear yellow smaller flower. Open in daylight.
Small production of concentrated nectar rich in both sucrose and hexose.
Bauhinia rutilans, liana form found high in the canopy of very humid tropical forest
Pink or yellow-greenish smaller flower. Open in daylight. Small production of concentrated nectar

How would the pollinator’s choice of flower influence the morphological and physiological feature of the flower they visit?

What are the evolutionary implication of the relationship between pollinator and pollinated flower?

Laboratory activities:

The purpose of the laboratory activities is to model the effect of pollinators on the reproductive success of mimulus flowers. We will explore the effect (s) of gene mutation affecting coloration and/or nectar production. We will also explore the effect of pollinator preferences on flower reproductive success.

We can also model the condition(s) that would lead to the reproductive isolation (and speciation) of Mimulus flowers.

The monkey-flowers (Mimulus) are a group of closely related sister taxa, varying widely in shape, structure and coloration. Field studies have been conducted on two sympatric species of Mimulus (Mimulus cardinalis and Mimulus lewisii) to determine the adaptive value of alternative alleles at a single locus. One of the allele under study (YUP allele) is an allele controlling the presence or absence of yellow carotenoid pigments in the petal of the mimulus flower.

Mimulus lewisii flowers possess the dominant YUP allele, which prevents the formation of carotenoids, so the flower petals only express pink anthocyanin pigments, leading to a pink flower coloration. Mimulus lewisii also produces low amount of nectar and have wide flattened petals. The amount of nectar produced is mostly controlled by a single gene. The allele responsible for high nectar production is dominant.

Mimulus cardinalis flowers possess the recessive yup allele, which allows carotenoid deposition into the petals, leading to a red coloration of the flower. Mimulus cardinalis also produces high amount of nectar and have narrow petals.

Exercise 1: Define all the glossary terms and make a concept map relating terms together.

Glossary: allele, recessive allele, dominant allele, pure-breed, parent generation, F1 generation, F2 generation, pre-zygotic barrier, reproductive isolation, speciation, sympatric, pollination, pollinator.

Exercise 2:

Determine the genotype and the phenotype of two sympatric species of Mimulus flowers, using the abbreviation used in the simulation. Both Mimulus species are pure breeding.

The following abbreviations are used in the simulation:

Abbreviation used for the genotype:

Gene controlling pink coloration (dominant): P

Gene controlling red coloration (recessive): p

Gene controlling high nectar production: N

Gene controlling low nectar production: n

Abbreviation used for the phenotype:

Pink flower: P

Red flower: r

High nectar production: H

Low nectar production: l

Mimulus cardinalis genotype: ______

Mimulus cardinalis phenotype: ______

Mimulus lewisii genotype: ______

Mimulus lewisii phenotype: ______

Exercise 3:

Mimulus cardinalis and Mimulus lewisii can be found in sympatric locations. What would be the resulting flower phenotypes and genotypes if we crossed these two pure-breed species together?

Genotype (s) of Mimulus cardinalis gametes: ______

Genotype (s) of Mimulus lewisii gametes: ______

Draw the Punnett square of the cross: Mimulus cardinalis X Mimulus lewisii

List all the phenotype (s) and genotype (s) of the F1 generation. Give the ratio for each genotype and each phenotype.

Genotype (s): ______

Phenotype (s) ______

What would be the resulting flower phenotypes and genotypes of the F2 generation if we crossed the F1 generation together?

Genotype (s) of Mimulus F1 gametes: ______

Draw the Punnett square of the cross: Mimulus F1 X Mimulus F1

List all the phenotype (s) and genotype (s) of the F2 generation and give the ratio for each genotype and phenotype.

Genotype (s): ______

______

Phenotype (s) ______

Compare the phenotypes of the F2 generation with the phenotypes of the F1 and the parental generation. Explain why some of the F2 phenotypes are different from the parent phenotypes. Are these new species? Why or why not?

A: Mimulus lewisii B: Mimulus F1 hybrid C: Mimulus cardinalis

D-L: Mimulus F2 hybrids

Simulation exercises:

Open the excel file labeled “Mimulus Pollination.xls”. You will first see your starting population genotype and phenotype table. The frequencies should all be set to 0.

Genotypes / PPNN / PpNN / ppNN / PPNn / PpNn / ppNn / PPnn / Ppnn / ppnn
Phenotype / PH / PH / rH / PH / PH / rH / Pl / Pl / rl
Frequency / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0

You will see your pollinator (bee or bird) pollination preference tables. The frequencies should also all be set to 0.

Bee Preference / Pink / Red
Hi nectar / 0 / 0
Lo nectar / 0 / 0

You will also know which pollinator is actively pollinating (Visit table). Each bee visit is as effective as a bird visit for pollination efficiency. In most of our simulation we will have an equal number of bee and bird visits, so the setting should be 1 for each, unless specified.

Bee visits / 1
Bird visits / 1

The goal of the simulations is to see what will happen to your population of flowers after 20 generations when you modify either the population genotype frequencies, the bee or bird pollination preferences, or the presence or absence of one or all the pollinators.

Simulation 1: Changes in frequencies of the different flower population (s) in the presence of two pollinators with fixed preferences.

a)  Set up the pollinator preferences to the following settings:

Bee Preference / Pink / Red
Hi nectar / 0.01 / 0
Lo nectar / 0.98 / 0.01
Bird Preference / Pink / Red
Hi nectar / 0.01 / 0.98
Lo nectar / 0 / 0.01

Make sure that all the frequencies add up to 1. If they do not, you will see a red number on the right of the table.

Based on the preference setting above, you can determine the pollinator preference.

With these preference settings, which flower (s) will be more likely pollinated by bees (give the genotype(s) and phenotype(s))?

With these preference settings, which flower (s) will be more likely pollinated by birds (give the genotype(s) and phenotype(s))?

Set up the population table so that the only type of flower present is : Mimulus lewisii

Genotypes / PPNN / PpNN / ppNN / PPNn / PpNn / ppNn / PPnn / Ppnn / ppnn
Phenotype / PH / PH / rH / PH / PH / rH / Pl / Pl / rl
Frequency / 0 / 0 / 0 / 0 / 0 / 0 / 1 / 0 / 0

Make sure that all the frequencies add up to 1. If they do not, you will see a red number on the right of the table.

After 20 generations, which type of flower (s) are present in the environment?

Predict what would happen if all the birds died? Explain your prediction.

Predict what would happen if all the bees died? Explain your prediction.

Do a simulation to test your prediction.

Did your prediction agree with the simulation results? Why or why not?

If your hypothesis did not agree with the simulation results, what would you need to change in the simulation settings to have your hypothesis agree with your prediction.

b) A single mutation resulting in a change in the coloration of the flower occurs.

Modify the population frequency to reflect the apparition of this mutation in the population.

Genotypes / PPNN / PpNN / ppNN / PPNn / PpNn / ppNn / PPnn / Ppnn / ppnn
Phenotype / PH / PH / rH / PH / PH / rH / Pl / Pl / rl
Frequency / 0 / 0 / 0 / 0 / 0 / 0 / 0.98 / 0.01 / 0.01

At the beginning of the simulation, which type of flower (s) are present in the environment (give the ratio)?

After 20 generations, which type of flower (s) are present in the environment (give the ratio)?

Predict what would happen if all the birds died? Explain your prediction.

Predict what would happen if all the bees died? Explain your prediction.

Do a simulation to test your prediction.

Did your prediction agree with the simulation results? Why or why not?

If your hypothesis did not agree with the simulation results, what would you need to change in the simulation settings to have your hypothesis agree.

c)  Another mutation resulting in a change in the increase in the production of nectar occurs.

Predict what would happen if the mutation affecting nectar production occurred in the pink flower?

In order to run a simulation of this mutation in the pink flower, you need to use the setting in a) (pink flower only) and set up the frequency at 0.01 for all genotypes leading to a pink flower-high nectar production phenotype. Remember that all the frequencies must add to 1. See below for the table settings:

Genotypes / PPNN / PpNN / ppNN / PPNn / PpNn / ppNn / PPnn / Ppnn / ppnn
Phenotype / PH / PH / rH / PH / PH / rH / Pl / Pl / rl
Frequency / 0.01 / 0.01 / 0 / 0.01 / 0.01 / 0 / 0.96 / 0 / 0

Predict what would happen if you were to change the frequency of the high nectar gene?