High Rates of Endophytic Nitrogen Fixation and Rhizosphere Phosphatase Activity for Multiple Grass Species across Environmental Gradients in Serengeti National Park

Geofrey Soka^1*, Mark Ritchie², Ramesh Raina², Nancy Johnson³

¹Department of Wildlife Management, College of Forestry, Wildlife and Tourism, Sokoine University, Morogoro, Tanzania.
²Department of Biology, Syracuse University, Syracuse, New York, USA.
³School of Earth and Sustainability, Northern Arizona University, Flagstaff, USA.

DOI: 10.4236/oje.2026.163008                

Abstract

Nitrogen fixation in and phosphatase exudation to soil may be critical in mitigating nutrient limitation. However, little is known about these processes for different dominant herbaceous legume and grass species in natural grassland and savanna ecosystems. Here we report evidence of substantial, persistent root N₂-fixation associated with root and soil phosphatase activity for several dominant grass species and a common legume at five sites in the Serengeti ecosystem that differ in fire frequency, rainfall and soil N and P. N₂-fixation was measured with ¹⁵N₂ root incorporation assays for 225 plants in two different years. For 122 of these same plants, we also measured phosphatase activity in roots and rhizosphere soils. Community root biomass and per cent cover, as well as root and rhizosphere soil ¹⁵N for each species, were measured to allow area-specific estimates of N₂-fixation. Four dominant grass species and the legume exhibited comparable root diazotroph abundance and mass-specific N₂-fixation activity, but different species exhibited peak activity at different sites. Assayed rates were associated with greater root tissue ¹⁵N, indicating persistent seasonal N₂-fixation. Area-specific annual fixation from grass roots varied from 16 - 42 kgN^.ha^-1yr^-1, with the highest values at sites with the lower P, higher rainfall and/or greater fire frequency. Phosphatase activity in both roots and rhizosphere soil of all five species was significantly associated with root N₂-fixation. N₂-fixation in grasses may be a major, previously overlooked source of N for grasslands and savannas that may balance high mean annual N losses from grazing and fire. Fixed N may also stimulate phosphatase synthesis and exudation to mitigate P limitation.

Keywords

Nitrogen Fixation, Rhizosphere, Grass Species, Rainfall, Fire, Tropical Soil, Serengeti

1. Introduction

Native grassland and savanna plant communities on nutrient-poor soils can be surprisingly productive, exceeding 10⁴ kg^.ha^−1.yr⁻¹ even on soils with nearly undetectable levels of phosphorus (P < 10 ppm) and low soil nitrogen (N < 0.1%) [1]-[3]. Further, these environments often experience high annual N losses of 5 - 25 kg N.ha⁻¹yr⁻¹ and 3 - 5 kg P ha⁻¹ yr⁻¹ [4]-[7] due to frequent late-season fires, redistribution of N and P by herbivores, and/or intense livestock grazing [8]. Sources of the N and P to replace such losses are uncertain.

Fixation of atmospheric N₂ by diazotrophic bacteria in and around plant roots represents a major potential N input that may compensate for such N losses [9]-[12]. However, the magnitude of N₂-fixation in grasslands from free-living soil bacteria and herbaceous legumes is usually thought to be < 3 kg N ha⁻¹yr⁻¹ [13] [14], far less than the estimated losses of N and P. Leguminous trees and herbs that fix atmospheric N₂ are major contributors to productivity during tropical forest succession [15] [16] but can be sparse in savannas (< 5% cover) due to herbivory and mortality to fire [17]-[19] and their contribution to ecosystem N inputs is still largely unknown.

Even less well-understood is the contribution of grass root-associated N₂-fixation to grassland and savanna N budgets. Growing evidence suggests that wild grasses host abundant and active populations of “cryptic” root-endophytic N₂-fixing (diazotrophic) bacteria [13] [20]-[23] and recent measurements in roots and rhizosphere soil of different species of tropical C4 grasses suggest that such N-fixation may be substantial enough to mitigate N limitation [24] [25]. However, no studies of which we are aware have explored the prevalence of root-associated N₂-fixation among multiple grass species in grassland or savanna communities. Studies have also not addressed whether such grass root-associated N₂-fixation might interact with P limitation, and thus vary within and among species across soil N and P gradients. Legume-associated N₂-fixation is thought to be limited by trade-offs in the supply of carbon (C), as mediated by light and herbivory [13] [26], for the energy to support N₂-fixation and/or the supply of soil elements (P, Mo) that may limit synthesis of the N₂-fixing catalytic enzyme nitrogenase [10] [16] [17] [27]. However, grasses, and C4 grasses in particular, may exhibit a strong compensatory response to grazing that maintains leaf area and C assimilation [28] [29], potentially mitigating effects of herbivory on grass-associated diazotrophs. In addition, N₂-fixation might be favoured at lower P if fixed N is used to produce phosphatases that help extract P for plant uptake [30]. Water may also be important: higher soil water concentrations may reduce soil oxygen levels and inhibit nitrogenase activity, while also increasing net C assimilation and within-plant C availability, such that grass-associated N₂-fixation and diazotroph abundance may be greater at higher rainfall [27] [31]-[33].

Given the general lack of knowledge about grass root-associated N₂-fixation and its role in mediating N and/or P limitation under herbivory and fire, here we expand upon a previous study [13] to explore N₂-fixation in six different dominant grass species plus a common legume in the Serengeti ecosystem, Tanzania. We (1) test whether these grass species exhibit significant root-associated N₂-fixation, (2) assess the magnitude of fixation across sites that differ in soil N and P, rainfall, and fire history, (3) evaluate the association between such fixation and synthesis and exudation of phosphatase enzymes, and (4) estimate plant cover, root biomass and tissue ¹⁵N to scale up mass-specific rates to area-specific and seasonal estimates of N₂-fixation.

2. Materials and Methods

2.1. Study Sites

Figure 1. Map showing the locations of the five study sites (black circles) across Serengeti National Park, Tanzania. Each site is labelled with a three-letter code that corresponds to Table 1.

Measurements were made in each of two years (2022-2024) at five sites in Serengeti National Park, Tanzania (Figure 1), selected from a set of eight sites in a Long-Term Grazing Exclosure (LTGE) experiment [29] [34]-[36] established in 2001. These five sites exhibit both considerable variation in soil N, P, plant species composition, and above- and belowground biomass [31]. We focused on unfenced areas subject to herbivory to be able to scale N₂-fixation activity assays to area-specific measurements that might apply across the study area.

2.2. Sampling Design

N₂-fixation measures were made on randomly selected individuals of the target species in the 4 m × 4 m unfenced plots of the LTGE experiment at each site. All other soil and plant community measures were from these same plots. The target grass (Poaceae) species were Digitaria macroblephara, Eragrostis tenuifolia, Panicum maximum, Pennisetum mezianum, Sporobolus fimbreatus, and Themeda triandra. All six genera have broad distributions in the tropics, and T. triandra is common in regions of 600-1000 mm rainfall in Africa, Indonesia and Australia [37]. The six grass species chosen exhibited mean cover > 5% at least one of the five sites, and four species (D. macroblephara, P. maximum, P. mezianum and T. triandra) exceeded that cover in 3 of the 5 sites. In addition, we assayed a ubiquitous legume (Fabaceae), Indigofera volkensii, which had the highest cover compared to other legume species (1% - 2% of total cover) by a factor of > 3 at each of the five sites.

2.3. Plot Level Measurements

Root biomass was estimated from 3 pooled 10 cm × 40 cm diameter cores within each plot using the flotation method to separate live roots from dead and soil detritus [13] in June 2022 (at the end of the wet season). Cover of each species was estimated visually in April 2023 (during the peak of the wet season) in each of four 2 m × 2 m quadrats within each 4 m × 4 m plot and then averaged [31] [32]. Plant and total soil element concentrations were measured for plant leaves and soils sieved from root cores, respectively, with benchtop methods at the Soil Analysis Laboratory at Sokoine University of Agriculture in Morogoro, Tanzania [31]. Total plant and soil N were analyzed using the Kjeldahl method, while plant and soil P were analyzed with standard persulfate digestion. These methods in this laboratory have contributed significant prior published data and reflect similar differences among samples as samples analyzed with combustion and infrared methods that are otherwise not available in Tanzania [31] [38].

2.4. Root and Soil Phosphatase Activity Measurements

We used the para-nitrophenyl phosphate (pNPP) colourimetric assay, originally developed by [39], as the standard protocol used to measure both root and soil phosphatase activity. This method measures the enzymatic hydrolysis of pNPP, which releases the yellow-colored compound p-nitrophenol (pNP). The intensity of the yellow colour, proportional to the enzyme activity, was measured using a spectrophotometer at a wavelength of 400 nm - 420 nm.

1. Soil phosphatase activity protocol

Fresh soil was typically used, passed through a 2 mm sieve, and, in some cases, air-dried and stored at 4˚C. One g of soil was mixed with 4 mL of Modified Universal Buffer (MUB), which sets the pH (e.g., pH 6.5 for acid phosphatase, pH 11 for alkaline phosphatase), and 1 mL of 0.115 M pNPP substrate. The mixture was incubated for 1 hour at 37˚C. The reaction was stopped by adding 1 mL of 0.5 M CaCl₂ and 4 mL of 0.5 M NaOH. The soil suspension was centrifuged, and the supernatant was filtered, and the absorbance was read at 405 nm. Activity was expressed as 𝜇mol pNP released per gram of soil per hour (𝜇mol g⁻¹h⁻¹).

2. Root phosphatase activity protocol

Fine roots (≤ 1 mm or first three orders) were carefully washed to remove adhering soil. Between 0.3 - 1.0 g of roots were placed in a vial with 9 mL of 50 mM buffer (e.g., sodium acetate for acid phosphatase, pH 5.0) and 1 mL of 50 mM pNPP. The roots were shaken for 1 hour at 27˚C. 0.5 mL of the sample solution was added to 4.5 mL of 0.11 M NaOH. Absorbance was read at 405 nm and compared against a standard curve prepared from pNP. Activity was expressed per unit of root dry mass (𝜇mol pNP g⁻¹_rooth⁻¹).

2.5. N₂-Fixation Activity Assays

Labelled N₂ uptake was measured using an in situ incubation method [40], modified for whole roots of I. volkensii and T. triandra and other grasses [13]. Assays were performed during periods following at least 100 mm of rain in the previous month: in November 2022 and March 2023. In June 2022, roots of 5 plants of each of six target grass and one legume species were rinsed thoroughly of soil with distilled water and assayed for N₂-fixation at each of the five sites where we encountered plants with 0.02% and thin 50 m of the permanent plots of the LTGE experiment (Table 1). This resulted in measurements of 25 D. macroblephara, P. mezianum, T. triandra and the legume I. volkensii, plus 15 measurements for P. maximum, 10 for S. fimbreatus, and 5 for E. tenuifolia (which was only found at the MSB site). Following these measurements, in March 2023, we re-measured N₂-fixation in the roots of four species for which N₂-fixation was detected (mean N fixed significantly different from zero) in 2022 (D. macroblephara, P. mezianum, T. triandra and the legume I. volkensii) for five new individual plants at each of the five LTGE sites.

For each assay, whole roots of target individual plants of each species were excavated, rinsed of visible soil and divided into control and enriched subsamples of approximately 20 g fresh mass (3 g dry) each in 60 ml chambers (syringes). An additional 3 root samples from each of the seven species were additionally surface-sterilized following [41]: sequential immersion in 99% alcohol, CaOCl (2%), chloramine T (2%) with 2 - 3 drops of Tween^® and an antibiotic solution containing streptomycin sulphate, gentamicin sulphate (0.01%). Surface-sterilized roots and non-sterilized roots differ primarily in the presence of microbial life on their exterior, which affects both microbial analysis and plant behaviour. Surface sterilization removed epiphytic microbes (surface dwellers) to allow for the study of endophytes. This was a critical, yet potentially destructive, step in investigating plant-microbe interactions, as the process must kill surface bacteria without harming the internal endophytes or the root tissue itself. Measurements were then compared to roots receiving only rinsing to determine if N₂-fixation was due to root surface associated bacteria or endophytes (living inside root tissue). The enriched sample received 50% ¹⁵N-enriched atmosphere at 1-atm pressure by injecting 20 ml of 99% ¹⁵N₂ gas. Both control and enriched samples were incubated at 5 cm soil depth for 30 min, after which samples were cooled to < 0˚C in a portable solar-powered box freezer (−20˚C) to stop the reaction. Assayed roots were dried, ground and analyzed for δ¹⁵N to 0.01 ^o/_oo (UC Davis Stable Isotope Laboratory). N₂ uptake (μg N ^.g^−1.hr⁻¹) was estimated as [N] × [(δ¹⁵N_enriched − δ¹⁵N_control) × 2]/(50% × 30 min x g sample), where [N] = μg N/g of roots or soil, δ¹⁵N is the difference between sample and atmospheric ¹⁵N, 50% is the gas enrichment, 30 is the assay time (min) and the factor 2 converts rate 30 min⁻¹ to rate hr⁻¹. Mass-specific rates were obtained by wet-dry mass conversions and the wet mass of roots assayed. To include δ ¹⁵N sampling error in the estimates of δ¹⁵N of root and soil tissues, we did not correct the negative δ¹⁵N_enriched − δ¹⁵N_control to zero and evaluated whether mean δ¹⁵N_enriched − δ¹⁵N_control > 0, including any negative values in the distribution for each site x species combination.

2.6. Soil and Plant Tissue Stable Isotope Analysis

To assess whether fixed N contributes significantly to plant tissue N, and therefore whether assayed N₂-fixation is a persistent process in the species tested, we also collected and dried at 45˚C for five days 50 g rhizosphere soil (within 1 cm of roots). Soils were analyzed for δ¹⁵N at the UC Davis Stable Isotope Laboratory (Davis, CA, USA). δ¹⁵N was obtained for control plant roots from those analyzed for the ¹⁵N₂- plant roots atmospheric ¹⁵N₂. Greater proportions of atmospheric N in plant tissue are associated with a greater difference between tissue ¹⁵N and that of soils, which are typically higher in ¹⁵N than the atmosphere [40].

2.7. nifH Gene Copy Number

As a measure of N₂-fixation potential, we measured copy number for the nifH gene coding for the Fe-protein in the enzyme nitrogenase in the ¹⁵N₂ uptake assayed roots using a customized DNA extraction followed by quantitative PCR [13]. Copy number of nifH genes was measured from standard molecular methods of DNA extraction [42] followed by assessment with standard real-time PCR methods [43] of the number per g sample of the nitrogenase genes, nifH. Frozen soil and roots were transported in sealed plastic bags at −20˚C by express courier, and DNA was extracted in duplicate from soil and plant samples essentially as described previously [42] with an optimized extraction [13]. To conduct real-time PCR, we designed nifH gene-specific degenerate primers following standard methods [43] to maximise the amplification of most of the nifH genes in different prokaryotic taxa [44] [45] (forward primer: CSATCAACTTCCTBGARGA, reverse primer: GCCATCATBTCRCCGGA).

2.8. Area-Specific N₂-Fixation (NF_A)

Community weighted mean (+ s.e.m.) area-specific N₂-fixation, NF_A (µg N^.m^−2.hr⁻¹) at each site was estimated as the sum of directly measured mean mass-specific N₂-fixation of plant species i (NF_{M, i}, μg^.g^−1.hr⁻¹) multiplied by estimated total community root biomass (RB, g/m²) and proportional cover of species i, COVER_i [13] (Equation 1). This assumes that root biomass for a species is proportional to its aboveground cover. Annual area-specific N₂-fixation, ANF_A,i for each species was estimated by further multiplying NF_A,i by the mean number of “wet days” (WD) (Table 1) where soil moisture exceeded a 10% threshold associated with root microbial activity [29] and by 12 hr^.day⁻¹ (as N₂-fixation can occur at night) and by appropriate area and mass conversions. WD represents the number of “wet days” (days with suitable moisture for microbial activity) over a specific period, which drives nitrogen fixation in that area. Soil moisture was measured using the Gravimetric Method [46], which measures water mass relative to dry soil mass (Wet Weight − Dry Weight)/Dry Weight).

$N{F}_{A,i} =N{F}_{M,i}\times RB\times COVE{R}_i$ (1)

$AN{F}_{A,i}=N{F}_{A,i}\times WD\times {10}^{-9}kg/\mu g\times {10}^4{m}^2/ha\times 12hr/day$ (2)

Total NF_A is estimated by summing ANF_A,I for the three diazotrophic grass species and I. volkensii.

2.9. Statistics

All analyses were performed with SPSS 27 (IBM Corp., Released 2020. IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY) Generalized Linear Models using maximum likelihood estimates. The Likelihood Ratio (LR) test statistic was used to ensure optimal statistical reproducibility. Calculated N₂-fixation activity, which was approximately lognormally distributed but featured some negative values due to sampling error in tissue ¹⁵N, was performed on an adjusted logarithmic transformation$N{F}_{\log ,}=\ln \left(1+Y-\min \left(Y\right)\right)$ . Copy numbers of nifH transcripts were log-transformed before analysis as well.

3. Results

The five LTGE sites used in this study varied substantially in soil nutrients and mean annual rainfall (Table 1), with data assembled from previous studies [29], [47]. Mean annual rainfall ranged from 515 - 903 mm/yr, soil N varied by a factor of 2 (0.11 - 0.22%) and total soil P varied by a factor of 10 (138 - 1172 ppm). Soil pH (5.71 - 7.07) and texture (e.g., 6.2 - 35.9 % clay) also varied substantially.

Table 1. Mean (s.e.m.) rainfall, soil characteristics (N = 6 at each site), and fire frequencies at the five study sites in Serengeti National Park.

Site	Rainfall (mm/yr, N = 17)	Soil N (%)	Soil C (%)	Soil P ppm	pH	Silt (%)	Clay (%)	Bulk Density (g/cm3)	Fires 2012-2022
KCW	801 (78)	0.22 (0.03)	1.77 (0.09)	138.8 (78.7)	5.71 (0.11)	35.5 (3.3)	23.9 (2.4)	1.07 (0.17)	7
SWRC	655 (97)	0.25 (0.06)	2.67 (0.08)	423.0 (105.8)	6.15 (0.17)	52.0 (1.8)	12.5 (2.8)	0.96 (0.04)	3
MSB	903 (73)	0.14 (0.07)	2.20 (0.21)	752.2 (82.5)	6.00 (0.26)	31.2 (3.6)	35.9 (2.9)	0.90 (0.10)	7
SOT	515 (51)	0.11 (0.02)	1.91 (0.14)	1172.6 (109.7)	7.07 (0.12)	55.4 (4.1)	12.5 (2.4)	0.84 (0.08)	1
TOG	683 (77)	0.15 (0.06)	1.85 (0.07)	609.8 (26.5)	5.96 (0.14)	28.0 (1.5)	6.2 (1.3)	1.22 (0.17)	6

3.1. Nitrogen Fixation

Figure 2. N₂-fixation sampled across two wet seasons in roots of six grass species (Dig mac, Digitaria macroblephara; Era ten, Eragrostis tenuifolia; Pan max, Panicum maximum; Pen mez, Pennisetum mezianum; Spo fim, Sporobolus fimbreatus and The tri, Themeda triandra across five sites (ordered by increasing mean annual rainfall in Serengeti National Park, Tanzania). A. Mean (+ s.e.m.) copy number (PCR units) per g root of the nifH gene for the nitrogenase enzyme for each species across all sites. B. Mean (+ s.e.m.) root mass-specific uptake of N by each species.

Mass-specific copy numbers of nifH genes varied significantly, and by over four orders of magnitude, among sites, species and site x species combinations (Table 2, Figure 2(A)). Across all sites combined, mean root mass-specific nifH varied significantly among species (Table 2), with two species (D. macroblephara and P. mezianum) exhibiting 100 - 150 times higher copy numbers than three grass species (E. tenuifolia, S. fimbreatus, P. maximum). The legume I. volkensii and the grass T. triandra contained similar mean copy numbers to these three grass species but exhibited a much greater range (Figure 2(A)). Mean copy numbers for these species also varied significantly among sites (Table 2), and the sites with the highest copy numbers differed among species (Site x Species Interaction) (Table 2, Figure 3(A)). Variation in nifH copy number was very weakly correlated with mass-specific N₂-fixation (R² = 0.02, N = 215, P = 0.04).

Figure 3. Box and whisker representations of mass-specific N₂-fixation for A. a legume and B-D three grass species across five sites in Serengeti National Park, Tanzania. Asterisks indicate means for species x site combinations that are significantly different from zero.

Table 2. Outcomes of Generalized Linear Models to assess variation in nitrogenase gene copy number, root mass-specific N₂-fixation and phosphatase activity by site, species, and their interaction.

	Site			Species			Species x Site
	Χ2	df	P	Χ2	df	P	Χ2	df	P
ln(nifH copy number)	82.129	4	<0.001	57.318	6	<0.001	31.593	15	0.007
ln(Mass-Specific N2-Fixation)	3.904	4	0.419	19.483	6	0.003	28.861	15	0.017
Root Phosphatase Activity	3.291	4	0.510	13.256	6	0.039	9.671	14	0.786
Soil Phosphatase Activity	3.869	4	0.424	17.807	6	0.007	8.367	14	0.869

3.2. Mass-Specific N₂-Fixation (Diazotroph Activity)

As suggested by the pattern for nitrogenase gene copy number, over all sites, plant species varied significantly in mean root mass-specific N₂-fixation (diazotroph activity) (Figure 2(B), Table 2). Four species’ 95% confidence intervals did not overlap zero, indicating evidence for consistent N₂-fixation across individuals in these species, which included three grasses, D. macroblephara, P. mezianum, T. triandra and the legume I. volkensii (Figure 2(B)). Mean N₂-fixation in these three grass species and the legume ranged from 10 - 25 μg^.g^−1.hr⁻¹, well above the means for each of the three inactive grass species (−0.5 to 0.02 μg^.g^−1.hr⁻¹), whose 95% CI each included zero. The nifH gene copy number was not correlated with measured N₂-fixation at the level of individual root samples (r = 0.04, N = 221, P = 0.550), but mean N₂-fixation rate for a species was correlated with its mean nifH gene copy number (r = 0.744, N = 7, P = 0.022, one-tailed test for an expected increasing relationship).

Figure 4. Persistent mass-specific N₂-fixation (diazotroph activity) inferred from the difference between plant root and soil ¹⁵N for three grasses, D. macroblephara, P. mezianum, T. triandra and the legume I. volkensii across 5 sites in Serengeti National Park.

For the four species that occurred within the LTGE experiment permanent plots, we found that, as in the previous analysis with all seven species, the four species differed significantly in mean mass-specific N₂-fixation (X²_4,200 = 18.42, P = 0.013), but the mean across species did not differ among sites (X²_4,200 = 3.83, P = 0.53). There was a significant interaction between site and species (X²_16,200 = 29.07, P = 0.023), which reflected the fact that different species’ peak mass-specific N₂-fixation occurred at different sites (Figure 4) and the confidence interval for the peak rates for each species did not include zero. D. macroblephara roots exhibited peak diazotroph activity at the driest site with the lowest soil N (SOT) and at a site with higher rainfall but the lowest P (KCW). P. mezianum roots exhibited peak activity also at the drier, low N site (SOT) but were largely inactive at the other sites. In contrast, legume I. volkensii roots displayed peak activity at a site with the highest soil N but intermediate rainfall and soil P, while T. triandra roots exhibited peak activity at the lowest soil P site (Figure 4(D)).

Figure 5. For all seven plant species tested, across all sites, relationship between mean short-term N₂-fixation activity and the difference in leaf versus soil ¹⁵N, an indicator of persistent N₂-fixation through the season.

Across all seven species, N₂-fixation was strongly correlated with the difference between root tissue ¹⁵N and rhizosphere soil ¹⁵N (X²_1,223 = 61.59, P < 0.001) (Figure 5), suggesting persistent N₂-fixation in species and at sites with high measured N₂-fixation activity. This correlation was strong for species with high mean diazotroph activity, such as the legume I. volkensii, D. macroblephara and P. mezianum, but weaker for species with lower activity and nifH copy number.

3.3. Phosphatase Activity and N₂-Fixation

Soil (rhizosphere) and root phosphatase activity was associated with each other (Figure 6(A)), and phosphatase activity on each substrate was associated significantly (P < 0.0001) with that plant’s N₂-fixation activity (Figure 6(B), Figure 6(C)) across all four N₂-fixing species.

Figure 6. Relationship between A. phosphatase activity in roots versus soil, B. Root phosphatase activity and N₂-fixation activity, and C. soil phosphatase activity and N₂-fixation activity for all four N₂-fixing plant species across all sites in Serengeti National Park.

3.4. Area-Specific Nitrogen Fixation

Area-specific N₂-fixation was greatest at low soil N:P, consistent with a response to N-limitation, and at high soil N:P, where its association with higher rhizosphere soil phosphatases are consistent with a response to P-limitation. Cover of our target species also varied substantially across sites (Table 3), as did total aboveground and root biomass.

Table 3. Mean (s.e.m.) plant above- and belowground biomass, cover for each species and for all N₂-fixing species at each of the five study sites.

Site	Root Biomass g/m2		D. macroblephara		P. mezianum		T. triandra		I. volkensii		Total
KCW	374.0	(50.0)	0.3	(0.3)	0.2	(0.2)	31.7	(10.4)	1.8	(1.6)	34.0	(6.2)
MSB	502.5	(59.3)	5.0	(2.3)	5.0	(2.5)	30.7	(12.7)	1.3	(0.9)	42.0	(9.3)
SOT	571.6	(61.4)	13.3	(6.0)	26.7	(4.4)	10.0	(2.4)	1.8	(1.6)	51.8	(7.2)
SWRC	470.7	(30.7)	7.0	(4.1)	6.2	(3.6)	5.5	(2.1)	2.0	(0.6)	20.7	(5.2)
TOG	671.6	(78.3)	7.7	(1.4)	0.0	(0)	25.2	(6.4)	1.4	(1.3)	34.2	(4.6)

4. Discussion

To our knowledge, these mass-specific measurements for multiple grass species in natural ecosystems across multiple species, years, and soil and rainfall conditions are unprecedented [10] [13]. Mass-specific rates for grasses in certain combinations of soil and rainfall conditions approached those of the ubiquitous legume I. volkensii. The correlation between the difference in root and soil ¹⁵N and N₂-fixation activity suggests that N_2-fixation occurred consistently over the growing season [5] [10] (Figure 2(D)), since differences in tissue ¹⁵N from soil likely reflect consistent acquisition of atmospheric versus soil N in developing tissue. These ¹⁵N data support the veracity of our extrapolations of short-term N₂-fixation over time in calculating annual area-specific N fixation (Figure 5). Overall, these data suggest that grasses fix atmospheric N at much higher rates than previously thought, and the biomass dominance of such grasses translates into larger-than-expected short-term and seasonal estimates of fixed N at the ecosystem level on extremely N- and P-poor soils across the Serengeti ecosystem.

Rates of N₂-fixation were equivalent in surface-sterilized versus surface-rinsed roots, supporting the hypothesis that the majority of N₂-fixing activity occurred inside the root by endophytic diazotrophs. Such endophytes inhabit internal plant tissues, which may provide more efficient, direct, and protected nitrogen fixation than rhizosphere-associated bacteria. Although rhizosphere bacteria may contribute to N₂-fixation, our results suggest that the protected environment of the plant is a critical, often superior, site for diazotrophic activity. Soil nutrient availability in the Serengeti differs significantly, characterized by a distinct gradient of high-nutrient soils in the southeast, driven by volcanic ash, and lower-nutrient, more acidic soils in the northwest. Our comparisons of grass root-associated N₂-fixation across sites may provide, to our knowledge, the first test of different hypotheses about environmental drivers of ecosystem N₂-fixation in tropical grasslands or savannas [9] [10]. The high N₂-fixation rates measured at low soil soil N (Figure 3(A)) are consistent with the hypothesis that N₂-fixation is favoured under conditions of strong N-limitation. However, high rates at high soil N and low extractable P were unexpected, as they imply higher fixation under low P availability when typically N-fixation in agricultural legumes is stimulated by the addition of P and thus is presumed P-limited [9] [10]. The association between N₂-fixation and rhizosphere phosphatase concentrations (Figure 3(B)), as has been found for some trees [11] [30], supports the hypothesis that N₂-fixation in grasses may mitigate P limitation in P-poor soils [30]. Higher activity of phosphatases, which require substantial N to synthesize and solubilize phosphate from organic matter and mineral particles in soil [30], was expected at low soil P. However, our results suggested that increased phosphatase activity association with N₂-fixation occurred at both low N and low P sites. Higher phosphatase activity at low soil P would be consistent with a hypothesis that phosphatase synthesis subsidized by atmospheric fixed N may enhance P-acquisition in P-limited conditions. Mechanisms to explain elevated phosphatase activity at low soil N (Figure 3(C)) are less clear. One hypothesis is that allocation of fixed N to phosphatase synthesis might compensate for, and thus be inversely correlated with, lower benefits of arbuscular mycorrhizal (AM) fungi at low soil N, where AM fungi might be more likely to be parasitic [15] [17] [30]. However, without data on AM fungal production, this hypothesis awaits future investigation.

Annual fixed N estimates from grass-associated N₂-fixation at the plot level ranged from 20-52 kgN ha⁻¹ yr⁻¹ (Table 3). These estimates were conservative for the ecosystem as a whole, as they did not account for possible N₂-fixation by other grass species we have not yet tested or by leguminous woody plants. Nevertheless, they represent some of the first-ever estimates of area-specific N₂-fixation by grass endophytes. They are much higher than rates previously assumed or measured from “cryptic” N₂-fixation [9] [10], even on soils with intermediate soil N and/or P. These annual estimates strongly suggest that these N₂-fixing grasses provide substantial “biofertilization” on either N- or P-poor soils, which in Serengeti correspond to soils < 0.1% total N and/or < 40 ppm total P.

These annual estimates may balance substantial N losses that can be experienced in tropical grasslands and savannas. Up to 7 kgN ha⁻¹ yr⁻¹ in aboveground plant tissue can be transported by herbivores to small portions of the landscape beneath trees, near water, or to livestock corrals [7] [8], and losses from fire [4] [6] can be as high as 16 - 25 kgN ha⁻¹ yr⁻¹. Consequently, our high measured N₂-fixation rates may result from N-limitation imposed by herbivory and fire. These losses, coupled with possible extra demand for N to synthesize phosphatases on very P-poor soils, might explain why the highest annual N₂-fixation rates occurred at the KCW site (Figure 5). Thus, sustaining high abundances of active diazotrophic endophytes in grass roots may be an important functional adaptation to multiple nutrient stresses. However, such high rates of associative nitrogen fixation in grass species likely require significant amounts of photosynthetically derived carbon to fuel nitrogenase activity in roots and rhizosphere. The reliance of tropical grasses on associative or associative-symbiotic bacteriameans they cannot “manage” the nitrogen fixation as efficiently, often resulting in higher carbon costs per unit of nitrogen fixed compared to fixation in legume nodules. Studies suggest that for every 1 kg of nitrogen (N) fixed, 3 to 10 kg of C per kg of N are required depending on the efficiency of the bacteria-root association. Based on a fixation rate of 20 - 52 kgN ha⁻¹ yr⁻¹, the estimated physiological carbon costs include a total annual carbon requirement of roughly 60 to 520 kgC ha⁻¹ yr⁻¹. This figure is well below estimated C assimilation of 720 - 950 kg C ha⁻¹ yr⁻¹ [48]. Therefore, such high N₂-fixation rates measured in this study are feasible.

Our results likely apply beyond the Serengeti, as we sampled across a broad range of soils that include typical tropical sandy loam P-deficient soils [1] [49], and more N-poor soils characteristic of volcanic regions in Africa [47] and temperate grasslands [50] [51]. The pan-tropical distribution of each of the three N₂-fixing grass species implies application beyond the Serengeti as well (see Methods). Some temperate species, such as Panicum virgatum, contain high copy numbers of nifH genes, putative N-fixing taxa, and evidence of active N₂-fixation [20] [52], so N₂-fixation by temperate grasses may be more important than currently realized. The weak correlation between nifH gene copy numbers and phenotypic N₂-fixation rates suggests that gene presence reflects potential, not realized function, which may be more tightly controlled by immediate environmental factors such as moisture, temperature, N- or P-demand, and cellular regulation [53] [54].

Spatial variability in nutrients, herbivore intensity, plant species composition and diversity, fire, and climate, which all vary substantially across the Serengeti landscape and co-influence each other [55], may impose additional influence on N₂-fixation. High plant species richness and robust vegetation cover significantly enhance soil nutrient dynamics, driving increased nitrogen, phosphorus, and potassium levels through complex ecological feedbacks. Regular, annual burning in the grasslands affects soil P levels, with fire often being the primary source of P replenishment rather than soil weathering. However, exploring these influences was beyond the scope of this paper. Given the unprecedented measurement of N₂-fixation in individual plant species across multiple sites, our study now may anticipate studies that explore such factors.

Our study demonstrates that multiple species of grasses in the Serengeti ecosystem fix atmospheric N through endophytic bacterial activity, but this fixation varies substantially among species across sites. Nitrogen fixation was consistently high in some species (D. macroblephara) but high only at certain sites for the other three species. Nitrogen fixation activity measures were strongly correlated with indicators of persistent season-long N fixation, and this persistence, coupled with the local biomass dominance of the key species, suggests high rates of nitrogen fixation formerly assumed to occur only in legume-dominated systems. N losses can be substantial in tropical grasslands subject to grazing and fire, so high nitrogen fixation rates are not only feasible but may be required to achieve N balance. Future research has many further questions to address, such as how soil ratios of N to P, spatial variation in herbivory intensity, and fire might affect such N fixation. Finally, the question remains as to how commonly high rates of N₂-fixation occur across the global distribution of tropical grasslands and savannas characterized by high rainfall and low P soils.

Acknowledgements

The study was supported by NSF grants DEB 0842230, and 1557085. We thank Emilian Mayemba for field assistance and Doug Frank, Jason Fridley, Katie Becklin, and Jamie Lamit for comments.

Conflicts of Interest

The authors declare that they have no competing interests.

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