DOI: 10.5061/dryad.8w9ghx3v3
title: Nitrogen and phosphorus availability alter tree-grass competition intensity in savannas

description: Plant essential macronutrients like nitrogen (N) and phosphorus (P) can
 limit savanna tree growth and are important determinants of savanna
 vegetation dynamics, along with rainfall, fire, and herbivory. How
 nitrogen and phosphorus shape tree-grass competition and their coexistence
 remain unclear, hindering our ability to predict how savannas may respond
 to altered nutrient cycling. Here, we evaluate (1) if trees and grasses
 respond differently to N vs. P availability, or (2) if grasses are more
 competitive in low nutrient environments while trees are more competitive
 in high nutrient environments. To do this, we grew saplings of 6 tree and
 1 grass species from Kruger National Park, South Africa, for 16 weeks
 under fully factorial nutrient and competition treatments (with/without
 competitors, low/high rate of N supply, and low/high rate of P supply)
 under a watering regime designed to mimic wet season rainfall in a mesic
 savanna. Trees and grasses foraged most aggressively for nitrogen and
 allocated biomass differently depending on nitrogen availability. Overall,
 tree growth decreased in competition with grass, even in high nutrient
 environments where they grew faster. Grasses were always better
 belowground competitors, utilizing aggressive nutrient foraging
 strategies, including high root phosphatase activity in response to
 nitrogen and large root biomass allocation. Synthesis: In low
 nutrient environments (e.g., on nutrient-poor sandy soils), nutrients may
 limit tree growth. Nutrient rich environments enable tree growth, but
 grasses continue to compete effectively with trees. Understanding what
 this means for ecosystem responses to nutrient availability is not
 trivial, especially in the context of fire and herbivory. However, it is
 clear that soil nutrients likely affect tree and grass growth and
 competition in savannas, which suggests that future changes in nutrient
 cycling, such as N deposition, may have important effects on savanna
 vegetation.
description: The primary goal of the experiment was to understand how trees
 and grasses respond to different nutrient and competition regimes. Six
 tree species and one grass species were selected based on phylogenetic
 diversity and nitrogen fixation ability for this experiment (Table 1). The
 grass species chosen for this experiment was <em>Melinis repens,
 </em>the only annual grass species common in Kruger National Park
 (Wigley-Coetsee and Staver 2020), one of the largest natural reserves that
 is situated in the low-lying savannas of South Africa.
 Plants were grown in a full-factorial nutrient-addition
 experiment with water availability held constant across treatments. Each
 pot was watered three times a day for a total of 400 mL per day, designed
 to mimic a wet growing season in the Pretoriuskop region of Kruger
 National Park, which receives about 750 mm of rainfall per year. The four
 nutrient treatments were (1) low N and P (), (2) high N and low
 P (), (3) low N and high P (), and (4) high N and P
 (). These nutrient treatments were applied factorially to trees
 grown with and without grasses and to grasses grown without trees for a
 total of 260 pots. Each nutrient and competition treatment was replicated
 five times in a randomized block design (except <em>Schotia
 brachypetala</em>, of which we had enough seedlings for only three
 replicates each). Grass and tree seeds were collected
 in Kruger National Park or purchased from Silverhill Seed Company in South
 Africa in 2019 and stored until germination. In July-August 2020, tree
 seeds were clipped and soaked in water for 24-72 hours before planted in
 potting soil. After sowing, seeds were watered and placed in growth
 chambers set at 28°C and 500 mmol/m<sup>2</sup>/s of light for
 the duration of germination. Grass seeds were germinated on watered
 potting soil in the same growth chambers. In September 2020, germinated
 tree seedlings and grasses were transplanted into 30-liter tree pots
 (Stuewe & Sons, Inc., 0.25 m diameter x 0.60 m height, sold as
 8-gallon pots) filled with turface (All-Season Turface, SiteOne
 Landscaping, New Haven CT). One tree sapling was planted per pot without
 grass competition, and two grass tufts were planted per pot without tree
 competition. For competition treatments, one tree sapling and two grass
 tufts were planted per pot (tree sapling in the middle, one tuft on either
 side). All pots were fertilized once a week for 16
 weeks at their assigned levels from October 2020-January 2021. Low
 nutrient treatments were designed to replicate nutrient limitation, while
 high nutrient treatments were designed to alleviate nutrient limitation
 while avoiding nitrogen or phosphorus toxicity. To determine the
 appropriate concentrations of N and P for each treatment, we derived
 monthly estimates of fertilizer from Tomlinson <em>et al.</em>
 (2019) along with field nutrient estimates from Ludwig <em>et al.
 </em>(2004). All  treatments received 100 mg N and
  treatments 10 mg P per pot per week to mimic field conditions
 via ammonium nitrate and potassium phosphate, with  treatments
 receiving 800 mg N and treatments 100 mg P per pot per week.
 Along with N and P fertilization, each treatment received the same
 quantity of N- and P-free Hoagland’s micronutrient solution, which
 included all essential micronutrients: potassium, calcium, sulfur,
 magnesium, chloride, sodium, manganese, zinc, copper, molybdenum, boron
 and iron. Pots were located in a single greenhouse bay at Yale Marsh
 Botanical Gardens in a block-randomized design, kept at 28°C for the
 duration of the experiment, and supplemented with grow lights for 12 hours
 a day to replicate a sub-tropical growing season. Soil moisture was
 measured throughout the experiment to ensure that soil moisture remained
 constant across pots. <em>Plant
 Responses</em> At the time of transplanting, 10
 individuals of each tree and grass species were harvested for initial size
 and biomass estimates. For tree seedlings, we recorded starting biomass,
 stem height, basal diameter, diameter at 10 cm height, root diameter, root
 length, wet and dry leaf stem mass, and fine (sorted into two diameter
 classes: 0-1 mm and 1-2 mm) and coarse root (&gt; 2 mm) dry mass. For
 grasses, we recorded basal circumference, maximum culm and sward height,
 root diameter, and wet and dry weight of aboveground and belowground grass
 biomass. Throughout the experiment, measurements were
 taken every four weeks. Tree measurements consisted of stem height, basal
 diameter, and diameter at 10 cm. Grass measurements consisted of basal
 circumference and sward height. Along with tree and grass measurements,
 light availability was measured using an fPAR meter (fraction of incident
 photosynthetically active radiation, 400-700 nm) at three different
 heights (tree base, tree canopy height, and grass height). At the final
 harvest in January 2021, we repeated all routine measurements and measured
 wet and dry weights of each plant tissue type. Time to harvest did not
 differ among species or blocks nor with respect to any experimental
 treatment (<em>n</em> = 260). To calculate
 mass gain, we subtracted the mean total dry weight of the 10 tree
 seedlings or grasses harvested at the start of the experiment from the
 final total dry weights at the end of the experiment. We used allometric
 relationships to evaluate whether competition and nutrient treatments
 influenced the growth form of trees. To do this, we calculated the
 allometric exponent <em>b</em>  as follows (Feldpausch et al.
 2011): for both shoot and root systems. For shoots, we used stem height
 and basal diameter (mm). For roots, we used taproot length for ‘height’
 and root diameter (mm). We refer to the traits represented by these
 allometric exponents as ‘shoot elongation’ and ‘root elongation’,
 respectively.  Competition intensity was calculated by dividing the
 average mass gain of a tree or grass grown with competition by the average
 mass gain of a tree or grass grown out of competition and subtracted by
 one for each nutrient treatment within one species
 (<em>i.e.</em>, only individuals of the same species were
 compared). Negative values (&lt; 0) indicate competitive effects while
 positive values (&gt; 0) indicate facilitative effects (Carlyle et al.
 2010, Jabot and Pottier 2012). To quantify root
 phosphatase activity, we used a method adapted from Turner et al. (2001),
 using para-nitrophenyl (pNPP) as an analogue for phosphomonoesterase, a
 root phosphatase that hydrolyzes simple phosphate monoesters to release a
 phosphate ion for plant uptake (Browman and Tabatabai 1978, Tabatabai
 1994). For each plant within a pot, we collected three subsamples of fine
 roots for measuring phosphomonoesterase activity and one for measuring
 color production without substrate added. For each plant, 500 mg of fresh
 root was added to a glass vial with 9 mL of 50 mM sodium acetate-acetic
 acid buffer (pH 5.0) and shaken in a water bath at 28°C for five minutes
 to simulate wet season surface soil temperatures at Kruger National Park.
 To initiate the reaction, we added 1.0 mL of 50 mM pNPP (5.0 mM final
 concentration). The reaction was terminated by mixing 0.5 mL of
 buffer/substrate solution with 4.5 mL of 0.11 M NaOH, which was then
 vortexed and measured for absorbance at 405 nm against paranitrophenol
 (pNP) standards. Two sodium acetate-acetic acid buffer and substrate
 blanks were run for every assay. Roots were removed from the vials and
 dried at 65°C for 3 days to express the activity as μmol pNP
 g<sup>-1</sup> dry root mass
 h<sup>-1</sup>.
description: # Nitrogen and phosphorus availability alter tree-grass competition
 intensity in savannas ##
 Description of the data and file structure There are two files:
 treedata.csv and grassdata.csv. The tree data file contains all data for
 tree species in the experiment while the grass data contains all data for
 grass species. ## Tree Data Column Names Pot = pot number in experiment
 Species = Species Code Block = Block in the randomized block design NTrt =
 nutrient treatment. C = low N + low P, N = high N + low P, P = low N +
 high P, NP = high N + high P GTrt = grass treatment. G- = without grass,
 G+ = with grass Sample_ID = sample ID used for phosphatase enzyme assays
 RSDiam = Basal diameter of tree (in cm)\ Diam10cm = tree diameter at 10 cm
 (if tall enough) in cm StemHt = stem height in cm RootLn = root length in
 cm RootDiam = root diameter in cm FPARBase = FPAR (nm of light) at the
 base of the tree FPARTree = FPAR at tree canopy height FPARGrass = FPAR at
 grass sward height FPARDate = date FPAR measurement was taken FPARWeather
 = weather description (how it could potentially impact FPAR measurement)
 FPARTime = time of day the measurement was taken Notes = any notes taken
 at harvest (eg was tree dead at harvest) BagStem = bag weight of stem (g)
 StemWet = weight of wet stem + bag (g) StemDry = weight of dry stem + bag
 (g) BagLeaf = bag weight of leaf (g) LeafWet = weight of wet leaf + bag
 (g) LeafDry = weight of dry leaf + bag (g) BagCroot = bag weight of coarse
 root (g) CrootWet = weight of wet coarse root + bag (g) CrootDry = weight
 of dry coarse root + bag (g) BagFine12 = bag weight of fine root (1-2mm)
 (g) Fine12Wet = weight of wet fine root (1-2mm) + bag (g) Fine12Dry =
 weight of dry fine root (1-2 mm) + bag (g) BagFine01 = bag weight of fine
 root (0-1 mm) (g) Fine01Wet = weight of wet fine root (0-1 mm) + bag (g)
 Fine01Dry = weight of dry fine root (0-1 mm) + bag (g) HarvestDate.x =
 date the tree was harvested Fresh_root_g = weight of fresh root taken for
 phosphatase measurement (g) Dry_root_g = weight of dry root taken for
 phosphatase measurement (g) Total_fresh_fine01root_g = total fresh weight
 of fine (0-1 mm) root to include all fine roots (0-1mm) including those
 taken for phosphatase measurements - in g Total_dry_fine01root_g = total
 dry weight of fine (0-1 mm) root to include all fine roots (0-1mm)
 including those taken for phosphatase measurements - in g
 Total_fresh_fine02root_g = total fresh weight of fine (0-2 mm) root to
 include all fine roots (0-2mm) including those taken for phosphatase
 measurements - in g Total_dry_fine02root_g = total dry weight of fine (0-2
 mm) root to include all fine roots (0-2mm) including those taken for
 phosphatase measurements - in g\ Total_fresh_root_g = total fresh weight
 of all roots, including all those taken for phosphatase measurements - in
 g Total_dry_root_g = total dry weight of all roots, including those taken
 for phosphatase measurements - in g Total_fresh_stem_g = total wet stem
 weight (bag weight subtracted- in g) Total_dry_stem_g = total dry stem
 weight (bag weight subtracted- in g) Total_fresh_leaf_g = total wet leaf
 weight (bag weight subtracted- in g) Total_dry_leaf_g = total dry leaf
 weight (bag weight subtracted- in g) Total_fresh_AGB_g = total wet
 aboveground weight (bag weight subtracted - in g) Total_dry_AGB_g = total
 dry aboveground weight (bag weight subtracted - in g) SDryF = total dry
 stem weight (bag weight subtracted- in g)\ LDryF = total dry leaf weight
 (bag weight subtracted - in g) CRDryF = total dry coarse root weight (bag
 weight subtracted - in g) FR1DryF = total dry fine root (1-2 mm) weight
 (bag weight subtracted - in g) FR0DryF = total dry fine root (0-1mm)
 weight (bag weight subtracted - in g) RSRatio = root shoot ratio DryTotal
 = all biomass dry weights added in g (stem + leaf + coarse root + fine
 root (1-2mm) + fine root (0-1mm) FineRootDry = all dry fine root biomass
 in g (fine root 1-2mm + fine 0-1mm) DryRootTotal = all dry root biomass in
 g (coarse root + fine root 1-2mm + fine 0-1mm) Treatment = nutrient
 treatment. C = low N + low P, N = high N + low P, P = low N + high P, NP =
 high N + high P RSDmm = basal diameter converted to mm Nitrogen = nitrogen
 treatment. N+ = high N, N- = low N Phosphorus = phosphorus treatment. P+ =
 high P, P- = low P Competition = competition treatment. G+ = with grass,
 G- = without grass Speciesy = full species names BNFAbility = fixing or
 non-fixing Family = phylogenetic family Encroacher = encroacher status
 Stmm = stem diameter converted to mm lgStmm = stem diameter logged after
 converting to mm shootelongation = shootelongation RTLmm = root length in
 mm RTDmm = root diameter in mm lgRTLmm = log of root length in mm lgRTLmm
 = log of root length in mm RTbeta1 = root elongation\ propfine = fine root
 mass fraction propcoarse = coarse root mass fraction propleaf = leaf mass
 fraction prop01fine = fine root (0-1mm) mass fraction prop12fine = fine
 root (1-2mm) mass fraction proproot = total root mass fraction propstem =
 stem massfraction propsum = checking that all prop parts add to 1 propabg
 = proportion aboveground biomass PlantID = treeID SowedDate = date sowed
 TransplateDate = date transplanted to pots HarvestDate.y = date of Harvest
 DaysGrownSinceTransplant = number of days the tree grew since transplant
 DaysGrownSinceSowed = number of days grown since seeds were planted
 mean_initialbiom = initial biomass of seedlings RGR = relative growth rate
 finalminusinitial = log(TotalDry) - log(mean_initialbiomass) massgain =
 TotalDry - mean_initalbiomass (in g) mean_phos = phosphatase activity for
 the tree (averaged across 3 replicates per tree) ## Grass Data Columns Pot
 = pot number GrassSpecies = Melinis Repens, grass species Species =
 species of tree the grasses were grown with Block = block in the
 randomized block design NTrt = nutrient treatment. C = low N, low P, N =
 high N, low P, P = low N, high P, NP = high N, high P GTrt = grass
 treatment (all G+ since they are grasses) Sample_ID = phosphatase enzyme
 assay sample ID BasalC1 = basal diameter of grass 1 in pot (cm)
 MaxSwardHt1 = maximum sward height of grass 1 in pot (cm) MaxCulmHt1 =
 maximum culm height of grass 1 in pot (cm) BasalC = basal diameter of
 grass 2 in pot (cm) MaxSwartHt = maximum swart height of grass 2 in pot
 (cm) MaxCulmHt = maximum culm height of grass 2 in pot (cm) BagGrass1 =
 bag weight of grass 1 (g) Grass1Wet = total wet weight of aboveground
 grass 1 + bag (g) Grass1Dry = total dry weight of aboveground grass 1 +
 bag (g) BagGrass2 = bag weight of grass 2 (g) Grass2Wet = total wet weight
 of aboveground grass 2 + bag (g) Grass2Dry = total dry weight of
 aboveground grass 2 + bag (g) BagGrassRoot = bag weight of root biomass
 bag (g) GrassRootWet = total wet weight of belowground biomass grass 1 + 2
 (g) GrassRootDry = total dry weight of belowground biomass grass 1 + 2 (g)
 HarvestDate = date pot was harvested Fresh_root_g = fresh root (g) taken
 for phosphatase Dry_root_g = dry root (g) taken for phosphatase
 Total_fresh_root_g = total fresh root (g) of grass + root taken for
 phosphatase Total_dry_root_g = total dry root (g) of grass + roots taken
 for phoshpatase Total_fresh_ABG_g = total fresh aboveground biomass of
 grass (bag weight subtracted, grasses added together) Total_dry_ABG_g =
 total dry aboveground biomass of grass (bag weight subtracted, grasses
 added together) GDry1F = aboveground biomass of grass 1 (g - bag
 subtracted) GDry2F = aboveground biomass of grass 2 (g - bag subtracted)
 GDryRootF = belowground biomass of grasses (g - bag subtracted) RSRatio =
 root shoot ratio DryTotal = total dry weight in the pot AbvTot = total dry
 weight aboveground biomass in pot Treatment = nutrient treatment
 Competition = competition treatment, T- = without tree, T+ = with tree
 Nitrogen = nitrogen treatment, N+=high N, N- = low N Phosphorus =
 phosphorus treatment, P+ = high N, P- = low N Speciesy = full species name
 of tree grown with grass BNFAbility = nitrogen fixation ability of the
 tree grown with grass Family = phylogenetic family of tree grown with
 grass Encroacher = encroacher category of tree grown with grass PlantID =
 unique plant ID SowedDate = date grass seeds were sowed TransplantDate =
 date grasses were transplanted HarvestDate.y = date grasses were harvested
 DaysGrownSinceTransplant = days grown since grass transplant
 DaysGrownSinceSowed = days grown since grass seeds were planted
 Grass.Treatment = tree species and grass treatment mean_initialbiom = mean
 initial biomass (grasses harvested at transplant) of grasses RGR =
 relative growth rate of grasses finalminusinitial = log(TotalDry) -
 log(massgain) massgain = TotalDry - mean_initialbiom mean_phos =
 phosphatase activity (averaged 3 replicates per pot) ## Code/Software All
 analyses were run in R using packages: lme4, emmeans, lsmeans, and
 ggplot2.