Warming Arctic forests diverge over nutrients

Humans continue their unique uncontrolled experiment to see how increased atmospheric carbon dioxide and the resulting warmer temperatures influence biomes worldwide.  One expected outcome of this global experiment is that trees in the extreme north will show improved growth resulting from a more benign physical environment.  As it turns out, some regions of the north do show this trend, while others don’t – this lack of consistent response is known as divergence.  For her graduate work, Sarah Ellison, working with Patrick Sullivan, Sean Cahoon and Rebecca Hewitt, wanted to document divergence in northern Alaska, and to figure out what might be causing it.

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The Wind River, near Arctic Village in the Arctic National Wildlife Refuge, is the easternmost site in the study. Credit: Patrick Sullivan.

The researchers established four study sites in four watersheds across the Brooks Range in northern Alaska.  They knew from previous studies that white spruce (Picea glauca) in the western Brooks Range have shown increased growth in response to climate warming, whereas those in the central and eastern Brooks Range have not responded. Some researchers hypothesized  that warmer temperatures caused moisture limitation in the eastern Brooks Range, but previous plant physiological studies done by this research team show no evidence of water stress, even in the extreme eastern portion of the range.

EllisonFig1

The four study sites within the Brooks Range from west to east: Agashashok, Kugururok, Dietrich and Wind River.

So what’s causing divergence?

At each study site, the researchers set up climate stations which collected continuous data on air and soil temperature, wind speed and direction, solar radiation, snow depth and precipitation. (Check out the following (you may need to copy and paste into your browser) for an entertaining look at the challenges of doing this research: https://youtu.be/ty6vwio9LvU).

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A weather station at the Wind River sight. Credit: Sarah Ellison.

They discovered that soil temperatures were consistently warmer in the western part of the range over the course of the season.

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Soil

Temp.

(deg. C)

 

 

Colder soils are often associated with low levels of available nutrients, because bacteria are less active at colder temperatures, and thus less capable of breaking down nutrients into a form that can be taken up by roots.  In 2014, Ellison and her colleagues measured soil nutrient levels at each site and found generally lower levels in the central (Dietrich) and eastern (Wind River) sites.

EllisonFig3

From top: ammonium, nitrate, phosphate and total free primary amine (TFPA – a proxy for amino acids) availability in the soils at the four sites during the 2014 growing season. Error bars are +/- 1 SE.

There were several other important physiological pieces to this puzzle.  Plants in the west grew more quickly than did plants in the east. Rapid growth was associated with greater photosynthetic rates in the western watersheds. The researchers measured needle nutrient concentration and found that it decreased from west to east. Each year, there was a strong correlation between needle nutrient concentration and branch extension (the measure of tree growth), but the correlation with phosphorus was generally stronger than the correlation with nitrogen.

EllisonFig5

Branch extension in relation to needle phosphorus concentration (left graph) and needle nitrogen concentration (right graph) for three years of the study.

Armed with these findings, Ellison and her colleagues decided to experimentally test whether nutrient availability was limiting growth, particularly in the eastern regions of the Brooks Range.  If so, this would support the hypothesis that cold temperatures and the resulting decrease in nutrient availability were primary factors causing divergence across this vast ecosystem. In June, 2015, the researchers fertilized five trees at each site with a mixture of nitrogen, phosphorus, and potassium fertilizer, and left five similar nearby trees as untreated controls. After one year, branch extension was greatly enhanced at the most eastern site, and only slightly (insignificantly) enhanced at the most western site.

EllisonFig7top

EllisonFig7bottom

Mean annual growth (branch extension) before and after fertilization experiment for fertilized trees (gray circles) and control trees (black circles). Bars are +/- 1 SE.  Fertilization occurred in 2015 (indicated by vertical dotted lines).

For many years, forest ecologists have believed that forests in young glacial soils are nitrogen limited.  This study, and a few other recent studies, thrust phosphorus into prominence as a factor that can limit forest productivity.  Over time, as the climate continues to warm, soils in the eastern Brooks Range will enjoy increased microbial activity, and may no longer suffer as much from nutrient limitation.

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The Agashashok mesic treeline sits on a gentle slope above the Agashashok river. Credit Sarah Ellison.

One surprising finding was that mycorrhizal growth on fine roots was more extensive in central and eastern watersheds.  Abundant mycorrhizae were associated with reduced branch extension, suggesting that these mycorrhizae may be parasitic, rather than mutualistic. The researchers are in the process of expanding their study to an even greater spatial extent of 20 sites distributed across the Brooks Range, which will allow them to further explore how general their findings are across this vast biome.

note: the paper that describes this research is from the journal Ecology. The reference is Ellison, S. B. Z.,  Sullivan, P. F.,  Cahoon, S. M. P., and  Hewitt, R. E..  2019.  Poor nutrition as a potential cause of divergent tree growth near the Arctic treeline in northern Alaska. Ecology100( 12):e02878. 10.1002/ecy.2878. 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.

Drought differentially diminishes ecosystem production

Sometimes, even the most carefully conceived experiment is thrown for a loop by Mother Nature.  Good scientists must embrace the unexpected.  Ellen Esch, David Lipson and Elsa Cleland set out to explore how plant communities responded to high, normal and low rainfall conditions.  The researchers set up rainfall manipulation plots that were covered with a clear plastic roof that would allow most light to pass through, but intercept all of the water.  They then reapplied the intercepted water, with each plot receiving either 50%, 100% or 150% of the fallen rain.  The plan was to simulate drought, normal and wet conditions. The natural world had other plans, however, as 2013-2016 were unusually dry years. Fortunately the researchers adjusted, by refocusing their question on how plant communities respond to severe drought  (50% of intercepted rainfall), moderate drought (100%) and normal rainfall (150%).

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Herbaceous plant community being irrigated (notice the rainbow). Credit: Ellen Esch.

Esch and her colleagues set up their experiment at the San Diego State University Santa Margarita Ecological Reserve, which has a Mediterranean-type climate with mild, somewhat moist winters and hot dry summers.

EschD

Exotic grasses (here showing recently senesced Bromus madritensis) dominated the herbaceous sites. Credit: Ellen Esch

They wanted to know how climatic variability brought about by climate change would influence plant phenology (the timing of periodic ecological events), specifically green-up date (when plants begin turning green) and senescence date (when they turn brown and curtail photosynthesis). They expected that the native species, primarily sage-type shrubs, would be more drought-resistant than the exotic herbaceous vegetation, which was dominated by brome grass.  Climate change is predicted to increase climatic variability, which should increase the frequency and intensity of severe droughts (and also of unusually wet years).

An important measure of ecosystem functioning is its productivity – the amount of carbon taken up by an ecosystem, usually by photosynthesis.  More productive ecosystems have more energy available to feed consumers and decomposers.  More productive ecosystems also take up and store more carbon dioxide from the atmosphere, which can help reduce climate change. The researchers used a reflectance radiometer to calculate the Normalized Difference Vegetation Index (NDVI), which essentially calculates how green an area is, and is a good measure of productivity.  Esch and her colleagues hypothesized that drought would reduce overall ecosystem NDVI, but that native vegetation would be more buffered against the negative effects of drought than would the invasive exotic vegetation.

EschB

A student from a plant physiology class at San Diego State University measures NDVI. Credit: David Lipson

Each year from 2013 – 2016, the researchers set up 30 3X3 meter plots; 15 plots were dominated by exotic herbaceous species such as brome, and 15 plots had mostly native shrub species such as sage. Plots were treated the same, except for receiving either 50%, 100% or 150% of the fallen rain, which corresponded to severe drought, moderate drought and normal rainfall, respectively. Periodically, the researchers used a radiometer to measure NDVI for each plot.  They discovered that, as expected, drought reduced NDVI much more in the plots dominated by exotic herbaceous species (top graph below) than in the plots dominated by native shrubs (bottom graph).

EschFig1

NDVI on each measurement date for plots dominated by (top graph) exotic herbaceous species and (bottom graph) native shrub species. Red square = severe drought treatment, green circle = moderate drought, blue triangle = normal precipitation. Error bars = +/- 1 standard error.

What caused this difference in response to drought between exotic plant-dominated and native plant-dominated communities?  Mechanistically, the native shrubs have deeper roots than the exotic grasses, which may allow them to take up more water.  But how does this translate to differences in green-up date and senescence date?

EschE

A student measures stem elongation on a senescent native shrub, the black sage Salvia mellifera, near the very end of the growing season. Credit: Ellen Esch.

The researchers used two different NDVI measures to help answer this question.  Maximum NDVI is the greatest daily NDVI measure over the course of the growing season.  It is correlated with the maximum productivity of the plant community (at its greenest!).  In contrast seasonally integrated NDVI is a measure of productivity summed over the entire growing season.  Keeping those distinctions in mind, under extreme drought maximum NDVI was much lower in the exotic plots than the native plots.  But exotic plot performance increased with rainfall, so that under the wettest conditions (normal rainfall), exotic plot maximum NDVI was similar to native plot maximum NDVI (graph a below). However, when considered over the entire growing season, native plots were consistently more productive than exotic plots (graph c below).

EschFig2

Effect of rainfall on (a) maximum NDVI (top left), (c) seasonally integrated NDVI (top right), (b) green-up date (bottom left) and (d) senescence date (bottom right). Colors indicate dominant plot community composition (yellow = herbaceous, green = shrub) and point shape indicates growing season year (circle = 2013, square = 2014, diamond = 2015, triangle = 2016).

Phenology played an important role accounting for these differences in seasonally integrated NDVI.  At all rainfall levels, the native plant communities greened-up well before the exotic plant communities (graph b above). Exotic plants greened-up somewhat earlier as rainfall increased, while native plant green-up date was independent of rainfall. At all rainfall levels, native plots senesced about one month later than exotic plots, with increased rainfall delaying senescence in both native and exotic plant communities (graph d above).

Esch and her colleagues conclude that species composition (native shrub vs. exotic herbaceous plants) and drought both influence phenology and productivity in this important ecosystem. Climate change is predicted to increase the frequency of extreme droughts in this and other ecosystems.  Consequently, drought coupled with invasion by herbaceous species threatens to sharply reduce ecosystem productivity, which will decrease the food available for consumers and decomposers, and simultaneously reduce the amount of carbon dioxide taken up and stored by the ecosystem, thereby contributing to further climate change.

note: the paper that describes this research is from the journal Ecology. The reference is Esch, E. H.,  Lipson, D. A., and  Cleland, E. E.  2019.  Invasion and drought alter phenological sensitivity and synergistically lower ecosystem production. Ecology  100(10):e02802. 10.1002/ecy.2802. 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.

Turkey mullein trichomes gobble up protective pollen

I’m always amazed at how brilliant plants can be.  For example Billy Krimmel and Ian Pearse showed in 2013 that many plant species exude sticky substances that entrap small arthropods, thereby attracting predators, which then rid these plants of many herbivores that might otherwise consume their leaves or reproductive structures. Jennifer Van Wyk joined this research group (which included Laure Crova) in graduate school. They were hunting for predatory hemipterans (true bugs) for a different experiment, which involved looking for them on turkey mullein (Croton setiger). They found plenty of predators, but almost no prey.  This was puzzling; what were these predators eating?  Intrigued, the researchers swabbed the turkey mullein leaves for pollen and found relatively vast quantities of pollen trapped in the trichomes (hairlike protuberances) of the leaves. Much of the pollen was from other species, and the researchers suspected that the trichomes were removing pollen from pollinators (primarily bees) that came to visit. Presumably the predators, which included spiders, hemipterans and ants, were attracted to this highly nutritious pollen.

VanWykTurkeyMullein

Turkey mullein with predaceous hemipteran on lower right leaf. Credit: Billy Krimmel – http://www.miridae.com/our-team

Van Wyk and her colleagues wondered whether pollen capture benefitted turkey mullein. If turkey mullein used pollen to attract predators, and predators ate herbivores, pollen extraction by trichomes would be an adaptation that formed part of turkey mullein’s defense strategy.  If this is true, supplementing turkey mullein with additional pollen should increase visitation by predators, and decrease herbivore abundance.  With fewer herbivores, the researchers predicted less leaf damage.

Wykdamagedturkeymullein

Turkey mullein with herbivore-damaged leaves

Supplementing turkey mullein with additional pollen presents its own set of problems – most importantly coming up with enough pollen – in particular pollen that predators want to eat (Van Wyk and her colleagues collected a pound of oak pollen, only to find that predacious bugs were not interested in it).  The researchers grew sunflowers in greenhouses, secured squash pollen from friends’ gardens and used tuning forks to vibrate pollen from tarweed flowers.

The researchers then set up experiments using 60 turkey mullein plants from one population in 2013 and 80 plants from another population in 2014. Nearby plants were paired up, with one member of the pair receiving 150 mg of supplemental pollen each week from mid-August to mid-September.  They surveyed all arthropods visible to the naked eye, and categorized each species as predator or herbivore based on its primary diet (many of the arthropods were actually omnivorous).

In accordance with expectations, predator abundance was substantially greater in the supplemented populations in both years of the study. Spiders showed the most consistent increase, while Orius (the minute pirate bug) increased significantly in the 2014 population. The 2014 population had fewer arthropods of all species, possibly because it was immediately adjacent to an agricultural field.

WykFig1

Mean predator abundance per plant in 2013 (top) and 2014 (bottom). Geocoris is a Genus of big-eyed bugs, while Orius is the minute pirate bug. ** p < 0.01, † p < 0.1. Error bars are 1 standard error.

The results are less clear-cut with herbivore abundance.  Fleahoppers were 18% less abundant on supplemented plants in 2014, and slightly (not significantly) less abundant in supplemented plants in 2013.  Plants with a greater number of spiders had fewer fleahoppers, suggesting that spiders were eating them (or scaring them away). The researchers were unable to measure the abundance of an important herbivore, the grey hairstreak caterpillar, which forages primarily at night, and retreats into the soil during the heat of the day.

WykFig2b

Mean number of fleahoppers  per plant in 2013 (left graph) and 2014 (right graph).  Blue bars indicated plants with supplemented pollen. * p < 0.05.

Lastly, supplemented plants suffered much less leaf damage than did unsupplemented plants.

WykFig2A

Mean number of damaged leaves per plant in 2013 (left graph) and 2014 (right graph).  Blue bars indicated plants with supplemented pollen. ** p < 0.01.

Taken together, these experiments indicate that turkey mullein uses its trichomes to capture pollen and attract a diverse army of predators, which reduce herbivore abundance and reduce damage to the plant.  It is possible that pollen supplementation could be used on a larger scale to reduce herbivore loads on agricultural crops.  More generally, it will be interesting to see whether other plants with sticky trichomes, such as the marijuana plant Cannabis sativa, also use their trichomes to attract predators and reduce herbivore abundance.

note: the paper that describes this research is from the journal Ecology. The reference is Van Wyk, J. I.,  Krimmel, B. A.,  Crova, L., and  Pearse, I. S..  2019.  Plants trap pollen to feed predatory arthropods as an indirect resistance against herbivory. Ecology  100( 11):e02867. 10.1002/ecy.2867. 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.

Birds and plants team up and trade off

For many years, ecologists have been puzzling over the question of why the world is so green.  Given the abundance of herbivores in the world, it seems, on the surface, that plants don’t stand a chance. The famous naturalist/ecologist Aldo Leopold was one of the first scientists to emphasize the role of predators, which provide service for plants by eating herbivores (his example was wolves eating deer, ultimately preserving the plant community growing on a hillside).  As it turns out there are many different predator species providing these services. Colleen Nell began her PhD program with Kailen Mooney with a keen interest on how insectivorous birds locate their prey, and how this could affect the plants that are being attacked by herbivorous insects.

COYE common yellowthroat simple

 A Common Yellowthroat perches on Encelia californica. Credit: Sandrine Biziaux.

Plants are not as poorly defended as you might expect (having sat on a prickly pear cactus I can  painfully attest to that).  In addition to thorns and other discouraging structures, many plants are armed with a variety of toxins that protect them against herbivores.  Thorns and toxins are examples of direct defenses.  But many plants use indirect defenses that involve attracting a predator to the site of attack.  Some plants emit volatile compounds that predators are attuned to; these compounds tell the predator that there is a yummy herbivore nearby.  Nell and Mooney recognized that plant morphology (shape and form) could also act as an indirect defense, making herbivorous insects more accessible to bird predators. They also recognized that we might expect a tradeoff between how much a plant invests in different types of defense.  For example, a plant that produces nasty thorns might not invest so much in a morphology attractive to predaceous birds.

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California Coastal Cactus Wren eating an orthopteran insect on a prickly pear cactus. Credit: Sandrine Biziaux.

What is a plant morphology that attracts birds?  The researchers hypothesized that birds might be attracted to a plant with simple branching patterns, so they could easily land on any branch that might be hosting a herbivorous insect (Encelia californica (first photo) has a simple or open branching pattern).  In contrast, birds might have a more difficult time foraging on insects that feed on structurally complex plants that host herbivorous insects which might be difficult to reach.

isocoma menziesii complex

Isocoma menziesii, a structurally complex plant. Credit: Colleen Nell.

The researchers chose nine common plant species from the coastal sage scrub ecosystem – a shrub-dominated ecosystem along the southern California coast. For each plant species they measured both its direct resistance and indirect resistance to herbivores.  Plants of each species were raised until they were four years old.  Then, for three months during bird breeding season, bird-protective mesh was placed over eight plants of each species, leaving five or six plants as unprotected controls.

IMG_3938

Kailen Mooney and Daniel Sheng lower bird-protective mesh over a plant. Credit: Colleen Nell.

After three months, the researchers vacuumed all of the arthropods from the plants, measured each arthropod, and classified it to Order or Family to evaluate whether the arthropod was herbaceous.

IMG_vaccum

Colleen Nell vacuums the arthropods from Artemisia californica. Credit: Colleen Nell.

Nell and Mooney evaluated the herbivore resistance of each plant species by measuring herbivore density in the bird-exclusion plants.  Relatively few herbivorous arthropods in plants that were protected from birds would indicate that these plants had strong direct defenses against herbivores.  The researchers also evaluated indirect defenses as the ratio of herbivore density on bird exclusion plants in comparison to controls (technically the ln[exclusion density/control density]).  A density of herbivores on plants protected from birds that is much greater than the density of herbivores on plants that allowed birds would indicate that birds are eating many herbivores. Finally, Nell and Mooney estimated plant complexity by counting the number of times a branch intersected an axis placed through the center of the plant at three different angles.  More intersecting branches indicated a more complex plant.

The researchers expected a tradeoff between direct and indirect defenses.  As predicted, as herbivore resistance (direct defense) increased, indirect defenses from birds decreased among the nine plant species.

NellFiga

Tradeoff between direct herbivore resistance and indirect defense by predaceous birds, for nine common plant species in the coastal sage scrub ecosystem.

The researchers also expected that more structurally complex plants would be less accessible to birds because complex branching would interfere with bird perching and foraging.  Thus Nell and Mooney predicted that structurally more complex plants would have weaker indirect defenses from birds, which is precisely what they discovered.

NellFigc

Indirect defenses (from birds) in relation to plant structural complexity .

Given that structurally complex plants received little benefit from birds, you might expect that they had greater direct defenses in the form of herbivore resistance.  Once again the data support this prediction.

NellFigb

Direct defenses (herbivore resistance) in relation to plant  structural complexity.

Initially, Nell was uncertain about whether increased plant complexity would deter insectivorous birds.  She points out that the top predators in this ecosystem are birds of prey that circle overhead in search of vulnerable birds to eat.  Structurally complex plants might provide refuge for insectivorous birds, which could result in them spending more time foraging in complex plants.  But the research showed the opposite trend. Plant complexity reduced the foraging efficiency of these small insectivorous birds, who prefer foraging on plants with relatively simple structure, which are easier to access and tend to host more prey.

note: the paper that describes this research is from the journal Ecology. The reference is Nell, C. S., and  Mooney, K. A..  2019.  Plant structural complexity mediates trade‐off in direct and indirect plant defense by birds. Ecology  100( 10):e02853. 10.1002/ecy.2853.  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.

Tadpoles shun trout across time

At a young school child (so long ago I can’t recall exactly when) I was exposed to Ernst Haeckel’s dictum that “ontogeny recapitulates phylogeny.”  More interested in language than biology at the time, I thought “cool – three words that I’m clueless about.” Though biological thinking about ontogeny – the processes of growth and development – has changed since Haeckel’s time, interest has, if anything, grown more intense across disciplines. Tiffany Garcia has explored her lifelong fascination with ontogeny by focusing her research on amphibians, which are famous for their distinct stages of development, each with unique habitats and ecological requirements. Working with eggs and tadpoles of the Pacific chorus frog (Pseudacris regilla), Garcia and her colleagues investigated whether stress associated with the presence of predators during one developmental stage (for example an egg) would carry over to influence behavior or development of subsequent stages.

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The Pacific chorus frog (Pseudacris regilla). Credit Brett Hanshew.

A tadpole’s anti-predator strategy can be influenced by other factors besides carry-over from earlier developmental stages.  For example, we might expect that tadpoles whose ancestors lived in association with predators for many generations might have evolved a different anti-predator strategy than did tadpoles whose ancestors lived in a less threatening environment (this would be an adaptive effect). Tadpoles may also show very short-term changes in behavior or development (this is termed plasticity) if exposed to a cue that indicated a possible predation threat.

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Collecting newly laid eggs at Three Creeks Lake. Credit: Lindsey Thurman

These three processes operate over very different time scales (long term – adaptive; intermediate term – carry-over; short term – plastic).  Garcia and her colleagues designed an experiment to explore how these processes might interact to influence a tadpole’s anti-predator strategy.  To investigate long term adaptive effects, the researchers collected newly laid (fertilized) eggs from lakes with and without rainbow trout (Oncorhynchus mykiss). They investigated carry-over effects by conditioning these eggs with four different environments during development: (1) trout odor, (2) cues from injured tadpoles (alarm cues), (3) trout odor paired with alarm cues, and (4) a water control (no odors nor cues).  The researchers created alarm cues by grinding up four juvenile tadpoles in 150 ml of water, and trout odor by housing 30 juvenile rainbow trout in a 200 L tank filled with well water.  They then conducted behavioral and developmental assays on tadpoles to see how adaptive, carry-over and plastic effects influenced tadpole growth, development and behavior.

GarciaFig2

Overview of the experimental design.

Garcia and her colleagues discovered that early exposure to trout odor had very little effect on growth and development, with body size and stage of development equivalent to that of controls.  In contrast exposure of eggs to tadpole alarm cues or to alarm cues + trout odor resulted in smaller, less developed fish (see table below).  In addition there was no effect of evolutionary history – eggs from lakes with and without trout showed similar patterns of growth and development.

GarciaTable1

Tadpole size and development in response to the four conditioning  treatments.  Higher Gosner stage numbers indicated more developed tadpoles. A tadpole hatches at Gosner stage 21 and begins metamorphosis at Gosner stage 42.

The next question is how do tadpoles respond behaviorally from exposure to different environments over the long, intermediate and short time scale?  To test tadpole anti-predator behavior, the researchers placed an individual tadpole into a tub that had a 6 X 8 cm piece of corrugated black plastic, which the tadpole could use as a refuge.  The researchers added to each tub one of the following: water (as a control (C)), tadpole alarm cues (AC), trout odor (TO), or alarm cues + trout odor (AC+TO).  After an acclimation period, a researcher noted the position of the tadpole (under the refuge or out in the open) every 20 minutes over a 3-hour time period.

There were no effects of evolutionary history on refuge use.  Tadpoles from lakes with and without trout showed similar patterns of refuge use.  However, embryonic conditioning to alarm cues and trout odor had a large effect on refuge use.  The left graph below shows the response of tadpoles from all four conditioning groups (C, AC, TO and TO+AC) to the addition of water.  As you can see, tadpoles that hatched from eggs that were conditioned with AC+TO were most likely to use refuges, while tadpoles from AC only or TO only eggs were somewhat more likely to use refuges. The pattern repeats itself when tadpole alarm cues are added to the water (second graph from left).  However when trout odor is added to the water, the responses are much more extreme, but follow the same pattern (third graph).  Lastly, when confronted with alarm cues and trout odor, tadpoles increase refuge use dramatically, but again show the same pattern, with tadpoles from control eggs using refuges the least, and tadpoles from eggs conditioned with alarm cues and trout odor using refuges the most (right graph).

GarciaFig6

Refuge use by tadpoles in response to embryonic conditioning and experimental exposure. C = water control, AC = tadpole alarm cue, TO = trout odor, and AC+TO = tadpole alarm cue and trout odor. Blue bars are means and gray bars are 95% confidence intervals.

There are two processes going on here.  First, over the short term, tadpoles are more responsive to the strongest cues, increasing refuge use when exposed to both tadpole alarm cues and trout odor.  Second, over the intermediate term, there is solid evidence for carry over effects.  Tadpoles that hatched from eggs conditioned with alarm cues and/or trout odor showed markedly increased refuge use than did tadpoles that hatched from control eggs.

These predator-induced responses impose a cost to the tadpoles.  Tadpoles exposed to alarm cues and trout odor while still in the egg were smaller and less developed, and probably metamorphosed into smaller frogs.  Many studies have shown that smaller frogs have reduced reproductive success.  The researchers recommend further studies to explore these trade-offs between survivorship, growth rate, development rate and size at metamorphosis. These studies are particularly essential, because rainbow trout are a non-native predator to these lakes.  Studies such as these allow conservation ecologists to understand the evolution and development of predator-prey interactions when novel species are introduced into an ecosystem.

note: the paper that describes this research is from the journal Ecology. The reference is Garcia, T. S.,  Bredeweg, E. M.,  Urbina, J., and  Ferrari, M. C. O..  2019.  Evaluating adaptive, carry‐over, and plastic antipredator responses across a temporal gradient in Pacific chorus frogs. Ecology  100( 11):e02825. 10.1002/ecy.2825.  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.

 

 

 

 

 

 

 

 

 

 

 

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.

StantonCanopy

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.

Stantoncrane

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.

Stantonstockings

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.

Stantonfixationlab

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.

StantonFig1A

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.

StantonFig1B

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.

StantonFig2B

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.

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.