Forest canopy fixes nitrogen shortage

The two billion hectares of forest canopy remaining on our planet are ideal habitat for nitrogen fixing microorganisms that can convert N2 to ammonia.


View of the forest canopy at the research site. Credit: D. Stanton.

The forest canopy tends to be nutrient-poor because there is no access to nutrients that accumulate in the soils on the forest floor, and rainfall can leach away any nutrients that do accumulate in the canopy from atmospheric deposition. So if you are a microbe, and you want to enjoy the view from the canopy, it is to your advantage to be able to fix atmospheric nitrogen so you can build essential molecules such as proteins and ATP.

As I mentioned in a previous post (Nitrogen continues to confound convention) both phosphorus (P) and molybdenum (Mo) are essential nutrients for biological nitrogen fixation.  Daniel Stanton and his colleagues hypothesized that nitrogen fixation in the canopy might be limited by the availability of P and Mo, so they designed a series of experiments to explore the role of these nutrients at the San Lorenzo Canopy Crane in San Lorenzo National Park in the Republic of Panama.  The crane provides about 1 ha of canopy access to non-acrophobic ecologists.


The crane at the research site: Credit: D. Stanton.

In one experiment, Stanton and his colleagues filled thin nylon stockings with vermiculite to form 40 cm long cylinders of 4 cm diameter.  Each cylinder was then soaked in either pure water (control), a molybdenum (Mo) compound, a phosphorus (P) compound, or a combination of Mo and P,  thus establishing four treatments. They attached each of these stockings to five different trees and allowed them to reside in the canopy for six months, to be colonized by microorganisms.


Nylon stockings treated with nutrients (or untreated controls) and affixed to branches in the canopy. Credit: D. Stanton.

The researchers measured the rate of nitrogen fixation by cutting a 50 cm2 rectangle from the area of densest growth on each stocking, and incubating it (along with the colonizing microorganisms) in a closed bottle that they had inoculated with heavy nitrogen (15N).  They then measured how much 15N the colonizers took up during a 12 hr incubation period.


Samples incubating for 12 hr to measure the rate of nitrogen fixation. Credit: D. Stanton.

The most common colonizers were nitrogen fixing filamentous cyanobacteria. These cyanobacteria fixed nitrogen at a somewhat (but not statistically significant) higher rate with Mo addition and at a much higher rate with P addition, and even more so with Mo + P addition.


Nitrogen fixation rates for each experimental treatment. C = control.  Note that the y-axis is logarithmic, so these differences in fixation rates are substantial.  Non-overlapping lowercase letters above the bars indicate significant differences between the means.

Nitrogen fixation is complex and costly.  Part of the complexity arises because nitrogenase, the enzyme that catalyzes the reaction, cannot tolerate oxygen.  To deal with this problem, cyanobacteria have evolved heterocysts, which are specialized anaerobic cells where nitrogen fixation occurs.  How does nutrient addition influence heterocyst abundance and function?

There are actually two aspects to this story.  One finding is that Mo addition had no effect on heterocyst abundance, while P addition had a pronounced effect.


Heterocyst frequency for each experimental treatment.

A second aspect is that Mo addition had a pronounced effect on the efficiency of nitrogen fixation.  For one analysis the researchers compared the nitrogen fixation rate per heterocyst for the phosphorus addition treatments either without or with Mo addition (in other words, they compared the P added treatment to the Mo + P treatment). Nitrogen fixation rates were much higher in the Mo + P treatments.  So while Mo does not increase heterocyst abundance, it does dramatically increase heterocyst fixation efficiency.


Quantity of N fixed per heterocyst per day in relation to absence (left bar) or presence (right bar) of Mo.  P was added for both treatments.  Dark horizontal lines are the median values, quartile range is represented by top and bottom of each box, and the whiskers represent the range of values for each treatment.

Phosphorus acts by markedly increasing the overall cyanobacterial growth.  It increases the amount of cyanobacteria that colonizes the canopy and also increases heterocyst density per filament. In contrast molybdenum’s effect is more nuanced as it increases the efficiency of the nitrogen fixation reaction without having any (obvious) effect on cyanobacterial structure.

How do these findings influence our understanding of tropical forests in the western hemisphere?  It turns out that episodes of nutrient addition actually happen in nature, courtesy of vast plumes of nutrient-rich rock-derived dust that periodically blow over the Atlantic Ocean from the Sahara desert in western Africa. Preliminary estimates by Stanton and his colleagues indicate that nutrient enrichment from these dust plumes is sufficient to  profoundly increase the rates of nitrogen fixation in tropical forests.  This may require us to reconsider our understanding of how nitrogen cycles within and between ecosystems.

note: the paper that describes this research is from the journal Ecology. The reference is Stanton, D. E., S. A. Batterman, J. C. Von Fischer, and L. O. Hedin. 2019. Rapid nitrogen fixation by canopy microbiome in tropical forest determined by both phosphorus and molybdenum. Ecology 100(9):e02795. 10.1002/ecy.2795. Thanks to the Ecological Society of America for allowing me to use figures from the paper. Copyright © 2019 by the Ecological Society of America. All rights reserved.

Urban frogs shed no blood

Life is a series of tradeoffs. As one example, we humans have the opportunity (if we are fortunate enough to be given choices) to opt for an urban or rural existence. The urban life is quicker-paced, offers more cultural opportunities, and can be annoyingly noisy and polluted. The rural existence is more laid-back, has fewer cultural opportunities, and may provide a peaceful and relatively unpolluted environment. These different environments can profoundly affect how we feel, with some people being stressed-out by cities and others by farms. On a personal level, I was born in New York City and now live in a very small town in Virginia – I was one of the fortunate ones who was given a choice.


Panama City skyline. Credit: Mariordo (Mario Roberto Duran Ortiz)

Heat, light and noise pollution are common in and near cities, and can influence the distribution and behavior of individuals of many different species. But these factors don’t only operate individually; they can work interactively. In other words, someone might not be annoyed by flashing light nor by loud noise, but might find the combination of the two very disturbing. In addition, these factors might not only operate on individuals; they can also affect relationships, or interactions. For example, loud noises generated by natural gas wells have been shown to influence the abundance of seed predators and seed dispersers, ultimately reducing the number of newly-established pine trees.

Armed with this understanding of interactions and relationships, Taegan McMahon and her colleagues wondered how the combination of heat, light and noise pollution might affect urban túngara frogs (Engystomops pustulosus) in comparison to their more rural counterparts. McMahon had observed that urban frogs were not being swarmed by small Corethrella midges that bite them and suck their blood in more rural and forested areas. These midges carry parasites, and if her observation was correct, urban frogs might have lower exposure to some diseases than do their rural counterparts.


Corethrella midge biting a túngara frog. Credit: Taegan McMahon.

The researchers surveyed 49 túngara frog calling sites in urban (Panama City) and rural (near the small town of Gamboa) areas. At each site they counted the number of frogs, number of midges on or above the frogs, the number of frog egg masses (in foam nests), and measured the air temperature, and the light and sound intensity. As expected, urban calling sites were lighter, noisier and warmer. There were slightly more (statistically insignificant) frogs at the urban sites and considerably more egg masses at rural sites. But the dramatic finding was that there were no midges to be seen on or near any urban frogs. So it might have been hot, bright and noisy, but at least those urban frogs were unbitten!

Factor Urban Rural
Light intensity 0.16 ± 0.02 lx 0.11 ± 0.02 lx
Noise intensity 69.0 ± 0.80 dB 59.2 ± 1.00 dB
Temperature 27.6° ± 0.09°C 25.9° ± 0.04°C
Túngara frog abundance 6.09 ± 2.63 frogs/site 4.05 ± 1.11 frogs/site
Foam nest abundance 0.24 ± 0.23 nests/site 2.06 ± 0.74 nests/site
Frog-biting midge abundance 0.00 ± 0.00 midges/site 67.75 ± 43.27 midges/site

Values are means ± standard error.

Analysis of the field survey data showed that temperature did not influence midge abundance but that light and noise were both important. Interestingly, light and noise interacted with each other in an interesting way. At low sound levels (below 65 db) light was important, in that midge abundance decreased at higher light intensity (Figure A). But at high sound levels, it could be pitch black and you would still have no midges (Figure B).


Log(number of midges) in relation to light levels, in field surveys in which noise levels were (A) below 65 db or (B) above 65 db.

Did light and noise somehow influence a midge’s ability to locate a frog? The researchers set up an experiment to see whether midges were attracted to frog calls at low, medium and high light intensities, and low, medium and high sound intensities. The sounds were recordings of Panama City traffic noise. At the same time, the researchers also broadcast the mating calls of túngara frogs at their normal calling intensity (which is remarkably loud for a small animal). They then counted the number of midges attracted to these traps, which were positioned in a rural setting.


Two calling túngara frogs competing for a female’s attention. Credit: Taegan McMahon.

At low light intensities, many midges were attracted to the recorded frog calls, but city noise (low or high) greatly reduced this attraction. At medium light intensity, fewer midges were attracted to frog calls, and again city noise reduced this attraction. Finally, at high light, even fewer midges were attracted to frog calls, regardless of noise.


Number of midges attracted to recordings of túngara frog calls in relation to light and sound intensity.

McMahon and her colleagues conclude that city noise and light pollution work together to disrupt the frog-biting midges host-parasite interaction. However, the overall impact of urbanization on túngara frogs is unclear at this point. Frogs can lose up to 10% of their blood volume to midges in a night of active calling. Frog-biting midges can transmit blood parasites such as Trypanosoma tungarae to túngara frogs, so urban frogs may be liberated from this scourge. A midge-free existence may allow urban male túngara frogs to call louder and for longer periods of time, which would make them more attractive to females. However, loud and long calling has also been shown to attract the túngara frogs’s mortal enemy, the voracious frog-eating bat. The researchers call for more research on how urbanization can affect species interactions, and for greater consideration of how different forms of pollution can interact to influence ecosystem dynamics.

note: the paper that describes this research is from the journal Ecology. The reference is McMahon, T. A., Rohr, J. R., & Bernal, X. E. (2017). Light and noise pollution interact to disrupt interspecific interactions. Ecology, 98(5), 1290-1299. Thanks to the Ecological Society of America for allowing me to use figures from the paper. Copyright © 2017 by the Ecological Society of America. All rights reserved.