EFN nectar production and herbivore defense tradeoffs in Passiflora biflora

“But those who wait for the LORD shall renew their strength, they shall mount up with wings like eagles, they shall run and not be weary, they shall walk and not faint.”

            ~ Isaiah 40:31

The final bit of our program focused on independent research.  We each formulated a research proposal, and then collected data for four weeks while in our homestays before returning to the station for the data analysis and report.  I studied extra floral nectar production and other herbivore defense mechanisms in a species of passion vine (Passiflora biflora).  This was my first real taste of research (a very different experience from a research assistant).  I decided I wanted to be a biologist in 6^th grade when I joined Science Olympiad, now here I am 10 years later conducting an independent research project in Costa Rica!  Here is a summary of my study and findings.

Passion vine (Passiflora biflora) growing in the
Selvatura Butterfly Garden in Monteverde, Costa Rica

Leaf toughness universally protects plants from herbivory, however newly formed leaves require time to toughen before this defense is effective.  Passion vines produce cyanide to deter generalist herbivores from damaging young leaves.  Additionally, many specialized defense mechanisms have evolved that target parasitic Heliconiine butterfly larvae.  In Passiflora biflora, this includes nectar production in extra floral nectaries (EFNs) that attracts ants that prey upon the Heliconiine caterpillars.  Increased nectar production may compensate defenses for leaves with low cyanide concentrations, or young leaves that lack toughness, to deter herbivory.  Alternatively, variation in nectar volume may by due to morphological differences in leaves that result in more surface area or leaf water content.  Defense mechanisms are costly; therefore the fitness of the plant may be compromised if resources are allocated excessively where defense is not needed.  My study examined evidence for investment tradeoffs in P. biflora, focusing on leaf toughness, cyanide concentration, and nectar production as the primary defense mechanisms.  Additionally I looked at factors potentially influencing nectar production in P. biflora, including leaf morphology, branch position, and active leaf production.

Nectar produced by EFNs on a leaf of P. biflora

I conducted this study in the Monteverde and Selvatura Butterfly Gardens in Monteverde, Costa Rica, where P. biflora can be found in large enclosures with Heliconiine butterflies.  Branches of P. biflora were chosen that varied in leaf morphology, branch position, and new leaf production.  Two different types of leaf morphology were observed in individual plants of P. biflora in this study, and were characterized as ‘wide morph’ and ‘thin morph.’ Branch position was determined as the spatial arrangement of the tip of the branch.  Branches climbing vertically were noted as ‘up’, branches hanging vertically as ‘down’, and branches growing horizontally as ‘flat.’  Finally, presence or absence of new growth was examined for each branch.

Thin (left) and wide (right leaf morphologies in P. biflora vines

Leaf age was measured by relative leaf position.  The first fully expanded leaf at the tip of each branch was youngest and was denoted as position 1, and the position of each consecutive leaf determined as the number of leaf nodes from position 1.  Leaf width was measured as the widest portion of the leaf.  Number of EFNs present was counted, and percent herbivory on each leaf was recorded.  Finally, nectar was collected using microcapillary tubes and the sucrose content was measured using a sucrose refractometer.

Nectar collected using microcapillary tubes

From there I went to the lab and measured leaf toughness using a leaf penetrometer.  Leaves were placed between two metal plates with a hole in the center allowing a rod to enter through the hole and rest on top of the leaf.  The rod supported a platform, which held a cup that was gradually filled with water until the rod punctured the leaf.  The total weight supported by the leaf was recorded and used as a measure of toughness.

Leaf penetrometer

Cyanide concentrations were determined using a sodium picrate test.  Test strips were exposed to cyanide in crushed leaf tissue, and then dipped in water to elute the cyanide.  The concentration was measured using a spectrophotometer and calculated using a standard curve developed by a previous student.

Sodium picrate test

Pairwise correlations indicated strong evidence for tradeoffs in defense.  Leaf age, percent herbivory, nectar volume, sucrose concentration, toughness, and cyanide concentrations were all correlated in leaf samples of P. biflora.  Percent herbivory, percent sucrose, and toughness were all positively correlated with leaf age.

Alternatively, nectar volume and cyanide concentrations were both negatively correlated with age.

Leaf morphology was found to impact nectar production in P. biflora.  Leaves exhibiting the wide morphology were found to produce significantly more nectar than leaves exhibiting the thin morphology for a given leaf age.

Branch placement also impacted nectar production.  Leaves on branches hanging down produced more nectar than leaves on branches in either the up or flat positions.

Finally, leaves on branches actively producing new leaves were not found to produce significantly more or less nectar than leaves on branches without new growth.

Evidence for trade-offs in herbivore defenses is clear in P. biflora, as correlations were found between leaf age, toughness, percent herbivory, cyanide concentrations, nectar volume, and percent sucrose.  Leaf toughness increases with leaf age, making youngest leaves most susceptible to herbivory because they have not had adequate time to toughen.  Older leaves exhibited higher herbivory because herbivory effects are cumulative and will be preserved through the life of the leaf.  As leaves age and become less susceptible to herbivory, investment in defense is reduced and those resources are allocated to newer leaves, which was displayed by higher cyanide concentrations and nectar volumes in young leaves.

Nectar production was influenced by leaf morphology and branch position.  Leaves of the wide morph produced more nectar than leaves with thin morphology.  Visually, wide leaves were observed to have greater leaf area than thin leaves of comparable ages.  Larger leaf area corresponds to more photosynthetic potential, more surface area for transpiration, and higher water storage capacity in leaves capable of producing nectar, all of which could account for increased nectar production in the wide morph.  Additionally, branches hanging down could have experienced increased EFN output because nectar flow is influenced by gravity, and water moving through branches oriented downward would allow more water to collect in EFNs on those leaves.

While tradeoffs in defense are observable, limitations in these trends occur.  Nectar production was not correlated with cyanide concentration, indicating the highly variable nature of these defenses. Additionally, some variation in nectar production appears to be beyond the plant’s control, as physics also plays a role.  This highlights the importance of multiple defenses in young leaves most susceptible to herbivory, possibly making cyanide a more reliable deterrent given the variable nature of EFN output.  As leaves toughen to avoid herbivory, cyanide concentrations were found to significantly decrease, while nectar production was not correlated with leaf toughness, and many young leaves failed to produce nectar at all.  This could reflect investment decisions by the plant: unprotected leaves are “cheaters” and butterflies may not be able to recognize this.  Alternatively nectar production may be induced by herbivore activity, thereby saving the plant resources until they are needed.

Defense mechanisms in plants are constantly being rewritten under pressures by herbivores.  Eventually leaves toughen, but until they do cyanide and EFN nectar are primary defenses.  Nonetheless these defenses are not all encompassing, and some leaves remain unprotected from herbivory.  This is especially true for leaves with thin leaf morphology, or on branches that are climbing, as these leaves secrete less nectar.  These leaves could be more likely to suffer from herbivore attack, or they may benefit from other leaves displaying protection while avoiding resource investment themselves.  Tradeoffs in defense are prevalent, but not perfect, allowing the cycle of plant defense and herbivore response to continue to coevolve in conjunction with one another.