Nitrogen continues to confound convention

Ah nitrogen…  It is the most abundant molecule in the air that we breathe (close to 80%), yet plants always seem to be starving for it.  Annually, nitrogen fertilizers are a $75 billion dollar industry. The problem is that the nitrogen gas that we breathe (N2) is very nonreactive, because the two nitrogen atoms are held together by a massively powerful triple bond.  So N2 must be broken down to some other more usable form (such as ammonia) – a process we call nitrogen fixation.  Most nitrogen fixers are microorganisms that live in soils or symbiotically within plants.  Unfortunately, N-fixation is energetically very costly, so even organisms that can fix nitrogen will generally happily use nitrogen compounds from the soil or leaf litter (the layer of fallen leaves above the soil) if they are available, rather than expending enormous energy to fix it for themselves. The general formula for nitrogen fixation (ignoring protons, electrons and energy transfers) is…

DynarskiEquation

A few years ago Scott Morford, Benjamin Houlton and Randy Dahlgren (the first two are co-authors of the present study) stunned the ecological world by identifying a previously unsuspected source of nitrogen – weathering of bedrock such as the mica schist pictured below. This bedrock was formed from seabeds which were rich in organic matter and had a high concentration of nitrogen compounds When the rock breaks down, both carbon and nitrogen compounds leach into the soil. Katherine Dynarski became interested in nitrogen fixation as an undergrad at Villanova University, so it was natural for her to move to the University of California at Davis to begin her graduate work with Morford and Houlton on how nitrogen cycles through ecosystems.

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Nitrogen-rich mica schist bedrock. Credit: Katherine Dynarski.

Dynarski got involved in this specific project essentially by accident. She was helping a fellow graduate student collect rocks at adjacent forests on contrasting bedrock (one high-N mica schist, and one low-N basalt), and figured that while she was out there, she might as well measure some N-fixation rates. In leaf litter and the soil below, most N-fixation is done by free-living soil bacteria. Dynarski expected higher N-fixation rates in the litter collected above the N-poor bedrock, reasoning that the microorganisms would need to fix nitrogen from the air, because there was little present in the litter.  In contrast, she expected to find lower N-fixation rates in litter collected above the N-rich bedrock, reasoning that the micro-organisms could save considerable energy by using existing nitrogen that had leached into the soil and leaf litter layer. She was shocked when she ran the samples and found exactly the opposite of her expectation, which led her to develop a more substantial project looking at the relationship between bedrock and N fixing microbes.

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Katherine Dynarski conducting gas incubations to measure N-fixation rates in the field. Credit: Scott Mitchell.

Working in northern California and Western Oregon, Dynarski and her colleagues identified sites whose bedrock was low in nitrogen (below 500 parts per million N) or high in nitrogen (above 500 ppm N). The researchers used soil and leaf litter samples from 14 paired sites – high N bedrock with nearby low N bedrock. They analyzed soil and leaf litter samples from each plot for concentration of nitrogen, carbon (C), phosphorus (P) and molybdenum (Mo) – the latter two elements have been shown in other systems to limit the rate of N-fixation.  The researchers also collected samples of underlying bedrock and analyzed N and Mo content of these rocks.

Recall that the conventional paradigm is that microorganisms should have lower N-fixation rates in N-rich environments.  There was negligible N-fixation occurring in the soil, but considerable N-fixation in the leaf litter above.  Thus the conventional prediction was that N-fixation rates would be higher in leaf litter above low-N bedrock. As I mentioned previously, Dynarski found the exact opposite to be true in one site; would this unconventional finding be confirmed by the 14 sites explored in this study?

The answer is yes!  Considerably more N-fixation was detected in leaf litter above high N bedrock than in leaf litter above low N bedrock.

DynarskiFig3

Mean leaf litter N-fixation rates and low-N and High_N bedrock sites.  Error bars are one standard deviation. P = 0.014.

You will notice the large error bars above the graph.  As it turns out, N-fixation rates vary dramatically – even on a very small spatial scale, which is why the researchers took multiple samples from each site. Some sample sites (hotspots) have unusually high rates of N-fixation.  These hotspots are also strongly correlated with high carbon concentration, with greater C in the leaf litter associated with much higher rates of N-fixation.

DynarskiFig4

Litter N-fixation rates in relation to % soil carbon at N-fixation hotspots. Hotspots are defined as having fixation rates greater than 1 kg N per hectare per year.

Dynarski and her colleagues also discovered that, in general, leaf litter above high-N bedrock tended to have more C and P than did leaf litter above low-N bedrock.  Given this finding (along with the hotspot finding) we are now ready to explore the question of why microbes are expending more energy to fix nitrogen in regions where more nitrogen is naturally available.

The researchers considered two hypotheses.  First, it takes N to make N.  N-fixation is catalyzed by N-rich enzymes. It may be that leaf litter above low-N bedrock is too N-poor to provide microbes with enough nitrogen make these enzymes. So the additional nitrogen from high-N bedrock is just enough to allow microbes to produce the N-fixation enzymes.

The second hypothesis is that the litter above low-N bedrock is also low in C, P and Mo, all of which are required for N-fixation. Thus the positive effect of these nutrients overwhelms the negative effect of additional nitrogen on the rate of nitrogen fixation.  According to this hypothesis, the conventional paradigm of high nitrogen availability reducing the rate of N-fixation is correct, but other factors may be equally or more important in natural ecosystems.

Fortunately, this conundrum is easily resolved.  Dynarski and her colleagues took some leaf litter samples and added a small amount of nitrogen to them.  These N-additions significantly reduced N-fixation rates at both low and high bedrock N sites.  Thus environmental N does reduce biological N-fixation, but other factors, such as the availability of other essential nutrients, can overwhelm the inhibitory effect of environmental nitrogen in natural ecosystems

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A Douglas fir forest in the Oregon Coast Range, where some of this research was conducted.  Credit: Katherine Dynarski.

The researchers conclude that nitrogen input from bedrock weathering leads to increased C storage and P retention, ultimately enhancing rates of N-fixation. About 75% of Earth’s surface is underlain by rocks with substantial N reservoirs, so we need to continue exploring the effects of weathering bedrock on ecosystem processes and functioning.

note: the paper that describes this research is from the journal Ecology. The reference is Dynarski, K. A., S. L. Morford, S. A. Mitchell, and B. Z. Houlton. 2019. Bedrock nitrogen weathering stimulates biological nitrogen fixation. Ecology 100(8):e02741. 10.1002/ ecy.2741. 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.

Light levels limit lake phytoplankton response to fertilization

One might naively think that because we humans are land-dwelling creatures, our impact on aquatic ecosystems might be relatively minor. Unfortunately, this assumption is incorrect, as human activities are changing aquatic environments in profound ways that influence how aquatic species survive and interact. Global warming is increasing lake and river temperatures, uncontrolled development is causing some streams to run dry and others to flood, and agricultural practices are adding nutrients to many lakes and streams. Because these human impacts occur simultaneously, it is difficult to evaluate how each factor contributes to the observed changes in species relations.

In northern Sweden, lakes vary naturally in the amount of dissolved organic carbon (DOC) they contain. DOC comes from runoff of decaying plant matter, so lakes surrounded by substantial vegetation, or that experience a great deal of water input (runoff) from the surrounding area, would have higher DOC than other lakes. DOC is potentially very important to lakes, because DOC tends to discolor a lake, which reduces light penetration and slows down photosynthesis. On the positive side, carbon may bond to other molecules such as phosphorus and nitrogen, which are important nutrients that may be in short supply in these relatively infertile lakes.   Anne Deininger and her colleagues focused their studies on two factors: DOC and nitrogen. Most lakes have too much nitrogen, a result of excessive use of nitrogen fertilizers that run off into lakes, so these relatively low-nitrogen lakes provided the researchers with a unique opportunity to see how these two factors, DOC and nitrogen, interacted in a natural ecosystem.

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Low DOC control lake. Credit: M. Klaus

The researchers selected six lakes that varied naturally in DOC levels: two low (~7 mg DOC/liter), two medium (~11 mg DOC/liter), and two high (~20 mg DOC/liter). In 2011 they measured everything possible about each lake: abundance of all of the life forms, DOC, temperature, light levels, nutrients and photosynthetic rates. In 2012 and 2013, they supplemented one of each pair of lakes with nitrogen compounds every one to two weeks. The added nitrogen was equivalent to the higher nitrogen inputs that are experienced by lakes in southern Sweden. And, as you might expect, the researchers continued measuring all factors of interest in both the experimental (fertilized) and control (unfertilized) lakes throughout the year – at least until the lakes froze over.

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Anne Deininger (in orange) and Sonja Prideaux collect samples from a lake. Credit: M. Deininger.

Deininger and her colleagues were most interested in differences in the abundance of phytoplankton – small free-floating photosynthetic organisms, because these are the primary producers – the organisms that produce the chemical energy (via photosynthesis) that enters food webs. There are many different types or groups of these phytoplankton; some are flagellated, with hair-like processes that allow them to navigate in the water column. Some are exclusively autotrophs, producing their own energy from photosynthesis, some are primarily hetrotrophic, eating other organisms or the remains of dead organisms, while others are mixotrophs, using both strategies to produce energy. Cyanobacteria are photosynthetic bacteria, while picophytoplankton are phytoplankton of unusually small size.

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Flagellated phytoplankton (Cryptomonas). Illustration by Anne Deininger.

Many important findings are summarized in the graph below. “B” represents the year before fertilization (2011), while “A1” is 2012 (after fertilization – 1st year) and “A2” is 2013 (after fertilization – 2nd year). Remember only the N-lakes were fertilized; the control lakes were simply monitored all three years. One finding is that in 2011, the high DOC lakes had the lowest phytoplankton abundance.  A second is that the low and medium DOC lakes had both flagellated and non-flagellated phytoplankton, while the high DOC lakes were dominated by flagellated phytoplankton.

Moving to the years after fertilization (A1 and A2), you can see that nitrogen fertilization increased phytoplankton abundance, but more so for the low-DOC lake. However, fertilization had little impact on the types of phytoplankton found in each lake; rather it simply increased the abundance of already existing groups.

DeiningerFig1

Mean biomass of major phytoplankton groups in relation to DOC.  Recall that B refers to 2011 (the year before fertilization), while A1 and A2 refer to the two years after fertilization (2012, 2013).

The data can be organized so we can get a better view of what is happening quantitatively. Fertilization increases phytoplankton biomass, but much more for lakes with low DOC levels. In addition DOC appears to decrease phytoplankton abundance.

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Deininger and her colleagues conclude that in these northern lakes, phytoplankton production is nutrient-limited at low DOC levels, but becomes limited by light availability in more murky waters. So adding nitrogen increases phytoplankton abundance to a greater extent in low DOC lakes. High DOC lakes have more flagellated autotrophs, as these species can swim to the top of the water column where there is more light for photosynthesis. As needed, flagellated phytoplankton can move lower in the water column where nutrients are more abundant.

The researchers emphasize that the nitrogen experiments were only conducted for two years. They don’t know if, for example, the types of species would change if fertilization continued for more than two years. They also don’t know if after 2013, the communities reverted to their pre-fertilization state, or if biomasses remained higher when nitrogen fertilization stopped. These types of questions are important to pursue because we humans are making drastic changes to most of our aquatic systems in a very uncontrolled manner. We need to understand the effects of these changes to the aquatic environment, and also how we can reverse the effects should they prove to be highly detrimental.

note: the paper that describes this research is from the journal Ecology. The reference is Deininger, A., Faithfull, C. L., & Bergström, A. K. (2017). Phytoplankton response to whole lake inorganic N fertilization along a gradient in dissolved organic carbon. Ecology98(4), 982-994. 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.

Nitrogen nurses

Alfred Lord Tennyson puzzled over the conflict between love as a foundation of Christianity, and the apparent violence of the natural world.

Who trusted God was love indeed

And love Creation’s final law

Tho’ nature, red in tooth and claw

With ravine, shriek’d against his creed

The good poet would be relieved to learn that modern ecologists have uncovered a softer, gentler side of the natural world – facilitative interactions, in which one species (the facilitator) helps out a second species. In many, but not all, cases, the second species also helps out the first species. Ecologists describe these mutually-beneficial interactions as mutualisms. As an example, Mimosa luisana is a mutualist with Rhizobium bacteria, providing the bacteria with root nodules to live in and carbohydrates as an energy source, while receiving ammonia (NH3) that the bacteria fix (convert) from atmospheric N2. A second type of mutualism, a mycorrhizal association, is a very common facilitative interaction between plants and fungi, which grow alongside or within the plant roots. In many mycorrhizal associations, the plant provides carbohydrates to the fungi, which import and share nutrients and water.

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Mimosa luisana. Credit: Leticia Soriano Flores, algunos derechos reservados (CC BY-NC)

Alicia Montesinos-Navarro and her colleagues, and researchers before them, noticed that in arid and semi-arid environments, plant-plant facilitation was most common between two plant species that were structurally and functionally very distinct, and that tended to be very distantly related to each other. In particular, M. luisana tends to associate with many different species of plants, including many cacti that look nothing like it, and are very distantly related. M. luisana is called a nurse plant, because other species tend to grow under its branches, which shade the soil and reduce water loss from evaporation. Recent work by Montesinos-Navarro and her colleagues showed another benefit of nursing – some plants receive nitrogen from these nurse plants via the network of mycorrhizal fungi.

Traditionally, ecologists have argued that associations between distantly-related plants occur because the plants have very different ecological niches, using different resources in different ways, so they are not competing with each other. Montesinos-Navarro and her colleagues argue that a second process might be important in this and other systems. Close relatives of M. luisana might tend to have high nitrogen levels and thus not benefit from nitrogen transfer from the nurse plant, while more distantly-related plants might tend to have lower nitrogen levels and thus benefit from any nitrogen arriving from M. luisana. They explored this hypothesis in the semi-arid Valley of Zapotitlan in the state of Puebla, Mexico.

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Study site dominated by the columnar cactus Neobuxbaimia tetezo, Credit: Alicia Montesinos-Navarro.

Measuring nitrogen transfer from the nurse plant to the recipient is not the world’s easiest task. Fortunately there is a rare form or isotope of nitrogen, 15N, which can be distinguished from the more common 14N. The researchers soaked M. luisana leaves in urea that was made up of primarily 15N, and the leaves took up the urea. Consequently, any exported nitrogen would contain a disproportionately high concentration of 15N, resulting in high 15N levels in the recipient plant. They then measured 15N levels in 14 different species of plants that used M. luisana as their nurse. The researchers were able to test two hypotheses. First, they could see whether close relatives to M. luisana tended to have higher N-levels than more distantly related species. Second they could see whether distant relatives tended to receive more nitrogen from nurse plants than did close relatives.

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Mimosa luisana branch taking up 15N-labeled urea. Credit: Alicia Montesinos-Navarro.

The graph below summarizes the results. The y-axis measures how much the 15N level in the facilitated species increased by the end of the experiment (15 days). The x-axis measures the evolutionary relationship between M. luisana and the facilitated species – more precisely how long ago the two species shared a common ancestor. Lastly, the size of the dot measures the initial difference in leaf N-levels between M. luisana and the facilitated plant.

Ecology Fig 2

Influence of evolutionary relationship between M. luisana and the facilitated species (x- axis) and nitrogen gradient – the initial difference in nitrogen levels between the two species (size of dots) on the amount of nitrogen imported by the facilitated species.

Several trends are evident. First, close relatives of M. luisana tended to have similar leaf nitrogen values to M. luisana (medium sized dots), while distant relatives tended to have much less nitrogen than M. luisana (largest dots). Second, the most distant relatives tended to have the greatest increase in their 15N levels, which indicates that they received the greatest nitrogen export from their nurses.

One question is how the nitrogen is transported. Montesinos-Navarro and her colleagues describe how they treated soil with a fungicide, presumably killing the mycorrhizae, which resulted in a substantial reduction in nitrogen transport. This suggests that the mycorrhizal network is important for nitrogen transport. But more pressing is what do the nurse plants get out of the relationship. The researchers suggest that the recipient plants may provide M. luisana with either water or phosphorus, both of which may be in short supply in arid environments.

This study indicates that we need to look beyond traditional niche theory, and may need to  dig deeper to understand the structure of plant communities, and how facilitative interactions can explain the coexistence of very distantly related plants.

note: the paper that describes this research is from the journal Ecology. The reference is MontesinosNavarro, A., Verdú, M., Querejeta, J. I., & ValienteBanuet, A. (2017). Nurse plants transfer more nitrogen to distantly related species. Ecology, 98(5), 1300-1310. 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.