DOI: 10.5061/dryad.wpzgmsbwm
title: Elephant megacarcasses increase local nutrient pools in African savanna soils and plants

description: African elephants (Loxodonta africana) are the largest extant terrestrial
 mammals, with bodies containing enormous quantities of nutrients. Yet we
 know little about how these nutrients move through the ecosystem after an
 elephant dies. Here, we investigated the initial effects (1-26 months
 post-death) of elephant megacarcasses on savanna soil and plant nutrient
 pools in Kruger National Park, South Africa. We hypothesized that: (H1)
 elephant megacarcass decomposition would release nutrients into soil,
 resulting in higher concentrations of soil nitrogen (N), phosphorus (P),
 and cations near the center of carcass sites; (H2) carbon (C) inputs to
 the soil would stimulate microbial activity, resulting in increased soil
 respiration potential near the center of carcass sites; and (H3)
 carcass-derived nutrients would move from soil into plants, resulting in
 higher foliar nutrient concentrations near the center of carcass sites. To
 test our hypotheses, we identified 10 elephant carcass sites split evenly
 between nutrient-poor granitic and nutrient-rich basaltic soils. At each
 site, we ran transects in the four cardinal directions from the center of
 the carcass site, collecting soil and grass (Urochloa trichopus, formerly
 U. mosambicensis) samples at 0, 2.5, 5, 10, and 15 m. We then analyzed
 samples for CNP and cation concentrations and quantified soil microbial
 respiration potential. We found that concentrations of soil nitrate,
 ammonium, δ15N, phosphate, and sodium were elevated closer to the center
 of carcass sites (H1). Microbial respiration potentials were positively
 correlated with soil organic C, and both respiration and organic C
 decreased with distance from the carcass (H2). Finally, we found evidence
 that plants were readily absorbing carcass-derived nutrients from the
 soil, with foliar %N, δ15N, iron, potassium, magnesium, and sodium
 significantly elevated closer to the center of carcass sites (H3).
 Together, these results indicate that elephant megacarcasses release
 ecologically consequential pulses of nutrients into the soil that
 influence soil microbial activity and are absorbed by plants into the
 above-ground nutrient pools. These localized nutrient pulses may drive
 spatiotemporal heterogeneity in plant diversity, herbivore behavior, and
 ecosystem processes.
description: We performed this research in the southern part of the Kruger
 National Park (KNP), South Africa (24.996 S, 31.592 E, ~275m elevation).
 During the wet season in March 2023, we identified ten elephant carcass
 sites (1-26 months post-death), five on relatively nutrient-rich basaltic
 soil and five on nutrient-poor granitic soil. KNP section rangers provided
 precise GPS locations of where elephant carcasses had been found. These
 sites were recognizable <em>in situ </em>by a persistent
 bonefield, undigested gut contents, and an absence of herbaceous
 vegetation. At each site, we hammered a rebar post into the center of the
 megacarcass disturbance and ran 15 m transects out from the post in each
 of the four cardinal directions. We collected green leaf material from
 <em>Urochloa trichopus</em> (formerly <em>U.</em>
 mosambicensis), a common and abundant palatable grass species, and used an
 auger to collect soil samples to a depth of 10 cm at five points along
 each transect (0.5, 2.5, 5, 10, and 15 m). We pooled and homogenized the
 samples to yield one composite leaf and one composite soil sample per
 sampling distance from each carcass site. Soil samples were sieved in a
 5-mm metal sieve which was cleaned in between samples with 70% ethanol. On
 the day of collection, we used 5 g of each soil sample for soil
 respiration measurements (described below). The rest of each sample was
 stored plastic bags in a -20°C freezer until nutrient analyses. Leaf
 samples were stored in paper bags at room temperature until dried for
 analyses (see below). Soil samples were stored in a cooler during
 fieldwork. On the day they were collected, we used 5 g of each soil sample
 for soil respiration measurements (described below). The rest of each
 sample was stored in plastic bags in a -20°C freezer until nutrient
 analyses; they were stored in coolers with ice blocks during the
 transition from the freezer at the field site to the freezers at the
 labs. We tested our first hypothesis that elephant
 carcass decomposition would release nutrients into the soil by performing
 soil nutrient analyses. We sent 250 g of each soil sample to Eco-Analytica
 laboratory at the North-West University in Potchefstroom, South Africa for
 measurements of soil concentrations of ammonium
 [NH<sub>4</sub>]<sup>+</sup>, nitrate
 [NO<sub>3</sub>]<sup>-</sup>, phosphate
 [PO<sub>4</sub>]<sup>3-</sup>, and plant-available
 P. Samples were air-dried and sieved through &lt; 2mm mesh prior to
 chemical analysis. Plant-available P was extracted from 4 g of soil and 30
 ml extraction fluid (1:7.5 ratio) using an acid–ﬂuoride solution (P
 Bray-1), measured colorimetrically using a Systea EasyChem200 analyser,
 and expressed as mg/kg. The detection limit was 0.5 mg/kg, and plant
 available P measurements &lt;0.5 mg/kg were replaced with half the
 detection limit (0.25 mg/kg) (Croghan & Egeghy, 2003; Keenan
 & Beeler, 2023). Water-soluble nitrate and phosphate anions were
 extracted from volume on volume 100 ml soil and 200 ml deionized water,
 analyzed by ion chromatography on a Metrohm 930 Compact Flex System, and
 measured as mg/L. Ammonium (also 1:2 water extract) was analyzed
 colorimetrically using a Systea EasyChem200 analyzer and measured as mg/L.
 Detection limits for soil ions were 0.01 mg/L, and soil ion concentrations
 measured as &lt;0.01 mg/L were replaced with half the detection limit
 (0.005 mg/L). To convert the nitrate, ammonium, and phosphate units from
 mg/L to mg/kg, we multiplied by 2, based on the 1:2 soil to water
 extraction ratio. To determine whether soil anions were
 distinct and elevated at the center of carcass sites relative to soil
 further from the center, concentrations of sodium (Na), magnesium (Mg),
 iron (Fe), calcium (Ca), potassium (K), and phosphorus (P) cations were
 measured using microwave-assisted digestion. Air-dried and sieved
 (&gt;2 mm) soil samples, weighed to 0.2 g, were microwaved in 9 ml 65%
 nitric acid (HNO<sub>3</sub>) and 3 ml 32% hydrochloric acid
 (HCl) according to EPA 3051b in a Milestone, Ethos microwave digester with
 UP, Maxi 44 rotor. A period of 20 minutes allowed the system to reach 1800
 MW at a temperature of 200 °C which was maintained for 15 minutes. After
 cooling, the samples were brought up to a final volume of 50 ml and
 analyzed on an Agilent 7500 CE ICP-MS fitted with CRC (Collision Reaction
 Cell) technology for interference removal. The instrument is optimized
 using a solution containing Li, Y, Ce, and Tl (1 ppb) for standard
 low-oxide/low interference levels (£ 1.5%) while maintaining high
 sensitivity across the mass range. The instrument was calibrated using
 ULTRASPEC® certified custom mixed multi-element stock standard solutions
 containing all the elements of interest (De Bruyn Spectroscopic Solutions,
 South Africa). Calibrations spanned the range of 0 – 30 ppm for the
 mineral elements Ca, Mg, Na, and K and 0 – 0.3 ppm for the rest of the
 trace elements. Elemental concentrations were expressed as
 mg/kg. Finally, to determine whether elevated N levels
 in soils were derived from the carcass, we sent 10 g of each sample to the
 BIOGRIP laboratory within the Central Analytical Facility at Stellenbosch
 University for measurements of soil %N and δ<sup>15</sup>N,
 obtained using a Vario Isotope Select Elemental Analyzer connected to a
 thermal conductivity detector and an Isoprime precisions isotope ratio
 mass spectrometer (IRMS). Samples were oven-dried at 60°C for 48 hours and
 milled to a fine powder using a Retsch MM400 mill (Germany). The powdered
 samples were weighed (2 – 60 mg) prior to combustion at 950°C. The gasses
 were reduced to N<sub>2</sub> (undiluted) in the reduction
 column, which was held at 600°C. A high organic carbon (HOC) soil standard
 (0.52 ± 0.02 %N), along with two international reference standards (USGS40
 (δ<sup>15</sup>N -4.52% AIR) and USGS41
 (δ<sup>15</sup>N +47.57% AIR)) were used for calibration. The
 N elemental content was expressed relative to atmospheric N as
 N<sub>2</sub> δ<sup>15</sup>NAIR (‰). The
 quantification limit for δ<sup>15</sup>N on the IRMS is 1 nA
 (nanoAmp), and the quantification limit for %N is 0.06%. The precision for
 %N was 0.02% and for δ<sup>15</sup>N is ±0.11%, determined
 using the HOC standard, which was run multiple times throughout the
 analysis. To test our second hypothesis that nutrient
 inputs to the soil would stimulate microbial activity, we measured soil
 organic C, water content, and microbial respiration potential. We sent 10
 g of each sample to the BIOGRIP laboratory for measurements of soil
 organic C using a Vario TOC Cube (Elementar, Germany). Samples (dried and
 milled as above) were weighed (10 – 60 mg), acidified using 10% HCl to
 remove the total inorganic C (carbonates), and dried overnight at 60°C.
 All samples were analyzed through combustion at 950°C. The released
 CO<sub>2 </sub>was measured by a non-dispersive infrared
 (NDIR) sensor. A high organic C (7.45 ± 0.14 %C) soil standard from
 Elemental Microanalysis Ltd (UK) was included during the analysis. The
 quantification limit for %C is 0.14%. The precision for the %C was 0.09%
 and was determined using the low organic C (LOC) standard (1.86 ± 0.14
 %C), which was run multiple times throughout the analysis.
 To quantify soil respiration and water content, we used an
 incubation method (Lemoine et al. 2023) in which 5 g (± 0.2 g) of each
 sample was placed into a 100 ml clear glass bottle, sealed, and flushed
 with CO<sub>2</sub>-free air. Following flushing, we incubated
 the bottles for one hour at 25°C. We then recorded
 CO<sub>2</sub> concentrations using an LI-850
 CO<sub>2</sub>/H<sub>2</sub>O infrared gas
 analyzer. After soil respiration measurements, we determined sample dry
 weight by drying each sample at 60°C for 24-48 hours until stable mass was
 achieved. We subtracted dry weight from starting weight to obtain soil
 water content. Finally, we used the dry weights and the Ideal Gas Law to
 standardize all respiration measurements to CO<sub>2</sub> μg
 h<sup>-1</sup>g dry soil<sup>-1</sup>.
 To test our third hypothesis that carcass-derived nutrients would
 be incorporated by plants, we measured foliar nutrient concentrations in
 <em>U. trichopus</em>. Two grams of each dried leaf sample was
 sent to the BIOGRIP laboratory for preparation and measurements of %N and
 δ<sup>15</sup>N via stable isotope analysis as described
 above. A Sorghum flour standard (1.47 ± 0.25 %N) from Elemental
 Microanalysis Ltd (UK) was used for calibration, along with two
 international reference standards (USGS40 and USGS41). The quantification
 limit for δ<sup>15</sup>N on the IRMS is 1 nA, and the
 quantification limit for %N is 1.3%. The precision for the %N was 0.02%
 and for δ<sup>15</sup>N is ±0.08‰. Limits were determined
 using the sorghum flour standard, which was run multiple times throughout
 the analysis. Additionally, we sent 5 g per sample to Cedara Analytical
 Services Laboratory to quantify micronutrients in grass tissue (P, Na, Mg,
 K, Ca, and Fe) using Inductively Coupled Plasma Optical Emission
 Spectroscopy (ICP-OES 5800, Agilent, USA). Samples were dried (110°C
 overnight) and milled to a fine powder. Subsamples (0.5 g) were ashed at
 450°C for 4 hours, and the ash was re-wet using 2 mL conc. HCl (32%).
 Samples were evaporated to dryness then re-suspended in 25 mL 1M HCl
 before filtering. Lastly, the filtrate was diluted with de-ionized water
 in a ratio of 5:20 filtrate to water. To calibrate the ICP-OES, solutions
 containing known amounts of each element were measured (10-20 ppm for Na
 and C, 200-1500 ppm for Fe, 0.5-3.75% for K, and 0.125-0.5% for P),
 prepared from 1000 ppm primary single standards. At three of the ten
 sites, we did not find sufficient plant material at the central point for
 analysis, resulting in a sample size of N = 7 for the center (distance =
 0.5m) measurement for leaf nutrient analyses.                  
description: # Elephant megacarcasses increase local nutrient pools in African savanna
 soils and plants
 [https://doi.org/10.5061/dryad.wpzgmsbwm](https://doi.org/10.5061/dryad.wpzgmsbwm) ## Datasets Dataset: Soil_Ions.csv This dataset contains soil ion measurements for 10 elephant carcass sites at distances of 0, 2.5, 5, 10, and 15m from the center of each carcass site. The columns contain identifying information for each site and sample, including the date the soil sample was collected ("Date_Collected"), the nickname identifier for the site ("Site"), the soil type ("Soil_Type"), the distance from the center of the carcass site in meters ("Distance_m"), and the age of the carcass ("Days_Post_Death"). The measurements taken at each site include phosphate ("PO4"), nitrate ("NO3"), and ammonium ("NH3") concentrations in mg/L and mg/kg, as well as the concentration of plant-available phosphorus ("P_Bray_I") in mg/kg. Dataset: Soil_Isotopes.csv This dataset contains soil isotope measurements for 10 elephant carcass sites at distances of 0, 2.5, 5, 10, and 15m from the center of each carcass site. The columns contain identifying information for each site and sample, including the date the soil sample was collected ("Date_Collected"), the nickname identifier for the site ("Site"), the soil type ("Soil_Type"), the distance from the center of the carcass site in meters ("Distance_m"), and the age of the carcass ("Days_Post_Death"). The measurements taken at each site include soil percent nitrogen ("N_per") and isotopic nitrogen-15 ("N15"). Dataset: Soil_Micronutrients.csv This dataset contains soil anion measurements for 10 elephant carcass sites at distances of 0, 2.5, 5, 10, and 15m from the center of each carcass site. The columns contain identifying information for each site and sample, including the date the soil sample was collected ("Date_Collected"), the nickname identifier for the site ("Site"), the soil type ("Soil_Type"), the distance from the center of the carcass site in meters ("Distance_m"), and the age of the carcass ("Days_Post_Death"). The anion measurements at each site include sodium ("Na_23"), magnesium ("Mg_24"), potassium ("K_39"), calcium ("Ca_43"), and iron ("Fe_57") concentrations in mg/kg. Dataset: Soil_Organic_Carbon.csv This dataset contains soil ion measurements for 10 elephant carcass sites at distances of 0, 2.5, 5, 10, and 15m from the center of each carcass site. The columns contain identifying information for each site and sample, including the date the soil sample was collected ("Date_Collected"), the nickname identifier for the site ("Site"), the soil type ("Soil_Type"), the distance from the center of the carcass site in meters ("Distance_m"), and the age of the carcass ("Days_Post_Death"). The measurements taken at each site include phosphate ("PO4"), nitrate ("NO3"), and ammonium ("NH3") concentrations in mg/L and mg/kg, as well as the concentration of plant-available phosphorus ("P_Bray_I") in mg/kg. Dataset: Soil_Respiration.csv This dataset contains soil respiration potential measurements for 10 elephant carcass sites at distances of 0, 2.5, 5, 10, and 15m from the center of each carcass site. The columns contain identifying information for each site and sample, including the date the soil sample was collected ("Date_Collected"), the nickname identifier for the site ("Site"), the soil type ("Soil_Type"), the distance from the center of the carcass site in meters ("Distance_m"), and the age of the carcass ("Days_Post_Death"). The measurements taken at each site include soil water content ("H2O_mmolpmol_obs") and soil respiration potential ("ugCO2_hr"). Dataset: Foliar_Isotopes.csv This dataset contains foliar (leaves from *Urochloa mosambicensis)* isotope measurements for 10 elephant carcass sites at distances of 0, 2.5, 5, 10, and 15m from the center of each carcass site. The columns contain identifying information for each site and sample, including the date the soil sample was collected ("Date_Collected"), the nickname identifier for the site ("Site"), the soil type ("Soil_Type"), the distance from the center of the carcass site in meters ("Distance_m"), and the age of the carcass ("Days_Post_Death"). The measurements taken at each site include leaf percent nitrogen ("N_per") and isotopic nitrogen-15 ("N15"). Three sites did not have sufficient grass for analysis at the distance of 0m, so the "N15" and "N_per" measurements are listed as "NA". Dataset: Foliar_Nutrients.csv This dataset contains foliar (leaves from *Urochloa trichopus)* isotope measurements for 10 elephant carcass sites at distances of 0, 2.5, 5, 10, and 15m from the center of each carcass site. The columns contain identifying information for each site and sample, including the date the soil sample was collected ("Date_Collected"), the nickname identifier for the site ("Site"), the soil type ("Soil_Type"), the distance from the center of the carcass site in meters ("Distance_m"), and the age of the carcass ("Days_Post_Death"). The micronutrient measurements at each site include sodium, magnesium, potassium, calcium, iron, and phosphorus in both percentages and mg/kg. Three sites did not have sufficient grass for analysis at the distance of 0m, so micronutrient measurements are listed as "NA". Note: all dates are recorded as M(M)/(D)D/YY (e.g., 3/15/23) ## Code Script: Megacarcass_Nutrients_Manuscript_Code.Rmd This is an annotated R Markdown file that can be used to generate the analyses and figures in the elephant megacarcass manuscript using the datasets listed above. The user needs to download necessary packages (listed in the markdown file) and set the file pathways for the above datasets. The script provides examples of each major analysis, which the use can then modify for any variable in the data. The code was originally run in R version 4.3.1.