DOI: 10.5061/dryad.9ghx3fftr
title: Endozoochory by black rhinoceroses enhances germination of a key arid savanna tree species

description: Megaherbivores are typically regarded as agents of top-down control,
 limiting woody encroachment through destructive foraging. Yet they also
 possess traits and engage in behaviours that facilitate plant success. For
 example, megaherbivores can act as effective endozoochorous seed
 dispersers. However, studies on facilitative roles are heavily biased
 toward the African savanna elephant (Loxodonta africana), with little
 attention paid to other species or to effects beyond germination across
 early ontogenic stages. The African black rhinoceros (Diceros bicornis),
 an obligate browser that exhibits frugivory and defecates in fixed dung
 middens, may offer ecologically distinct dispersal services. We conducted
 controlled experiments to investigate whether interactions between black
 rhinos and Vachellia erioloba, a leguminous tree of ecological
 importance in arid savannas, enhance germination, early seedling
 development, or seedling resilience to herbivory. Germination was compared
 among dung-derived seeds, untreated controls, and chemically scarified
 seeds. Seedling growth was assessed in dung versus sand, and under
 simulated black rhino herbivory. Dung-derived seeds germinated most
 steadily and produced the highest cumulative germination (+40 %) over the
 longest period (+13 days). Growth trials revealed that dung substrates did
 not enhance initial growth. Rather, seedlings being older conferred
 greater resilience to biomass loss than exposure to different substrate
 conditions. Our results provide the first experimental evidence of an
 apparent mutualism between black rhino and V. erioloba. This relationship
 is not driven by enhanced seedling development through legacy effects of
 gut passage, nor by dung conditions, as expected. Instead, it stems from
 the effects of gut passage on germination. In addition to increasing total
 germination, gut passage accelerates germination and extends the
 germination period, producing a seedling cohort with both older
 individuals and greater age variation — a population structure that may
 enhance persistence beyond the germination bottleneck. This research
 supports a more nuanced view of megaherbivores as both disturbance agents
 and mutualists in arid ecosystems.
description: <strong>Germination assays</strong>
 <em>Seed collection</em> Seeds
 were initially collected from black rhino dung to identify the study
 species, followed by mature pods. Dung samples were
 collected over two weeks each in April and July 2023, post-wet season
 (November to March). By this stage, all indehiscent pod-bearing species
 had produced mature pods, so their seeds should have been present in
 boluses if consumed by rhinos and able to withstand gut passage. The two
 collection periods were intended to contrast seeds with minimal (April) vs
 prolonged (July) environmental exposure. With assistance from field
 rangers, fresh black rhino dung (&lt;2 days) was located by
 backtracking from waterholes, and all intact seeds were removed from
 boluses. As rhinos have distinct territories, dung from a midden typically
 reflects the diet of only a few individuals. To maximize the number of
 individual rhinos sampled and capture variable gut passage effects on
 seeds, Dung seeds were collected across Tswalu. This also optimized the
 genetic diversity among seeds to account for potentially varied
 germination responses. Only <em>V.
 erioloba</em> seeds were found intact and in high densities (see
 SI). Accurate identification was confirmed by germinating and growing
 seedlings during the experiment, until distinctive characteristics
 emerged. In April, fallen pods were collected from
 beneath <em>V. erioloba</em> trees randomly distributed across
 Tswalu. Pods were diversely shaped and sized, particularly as distthe
 distance between trees increased (<em>pers. obs.</em>), which
 could be a morphological indication of genetic diversity, as found in
 <em>Vachellia caven</em> by Pometti et al. (2010).
 Seeds were processed immediately at the C.L.I.ME Laboratory,
 Stellenbosch University (Climate and Invasions: Mechanisms in Ectotherms;
 Department of Botany and Zoology). Pods were opened and seeds submerged in
 distilled water to check for bruchid beetle infestation and viability.
 Only flawless, sinking seeds were selected as sample individuals. Dung
 seeds were rinsed with distilled water and processed similarly. By
 minimizing variability in initial seed quality, processing standardised
 the viability potential between seed treatment groups, facilitating a
 robust comparison. The first germination trial commenced within a week; it
 was delayed by 12 weeks lateto incubator constraints, during which seeds
 were stored at 22 ± 2 °C in paper bags.
 <strong>Microscope assessment</strong>
 Before the germination assays (Experiments 1 and 2), 10 seeds
 each from pods and dung were examined under a stereo microscope (LEICA
 M125, with MC190 HD camera), to check for visible differences in the
 external condition of the seed coats. Refer to Figure 1
 for a visual summary of experiments 1-4. Methodology is detailed
 below. Experiment 1: Disentangling the effects of gut
 passage on germination To assess whether gut passage
 enhanced germinability, our first germination trial compared dung seeds to
 pod seeds. Only April-collected seeds were used. 900
 pod seeds and 300 dung seeds were disinfected in  3% chlorine bleach (1:5)
 for two minutes. Pod seeds were divided into three groups (n=300
 <em>ea.</em>): untreated Control, HCl-treated, and HCl +
 GA-treated. To simulate acid scarification alone (disengaging from other
 digestive processes like mechanical abrasion and exposure to heat or
 enzymes), the second group was soaked in 0.000033 M HCl for 20 hours,
 followed by 0.00977 M HCl for another 20 hours. Protocols were designed
 based on digestive information in Clemens and Maloiy (1982) and
 Dierenfield (1993). The third group was soaked in gibberellic acid (GA₃;
 50 mg/L, Kimix, ≥90% purity) for 8 hours following HCl treatment. The
 control and GA groups served as minimum and maximum germination
 benchmarks, contextualising the Dung and HCl treatments. See SI for a
 detailed explanation and the underlying rationale of the HCl and GA
 treatments. The germination trial was run in a
 thermostatically controlled growth chamber (Panasonic MIR-254). Each
 treatment group (Dung, Control, HCl, and GA) comprised 20 replicate Petri
 dishes (9 cm), each lined with two layers of grade 1 Whatman filter paper,
 pre-moistened with 5 mL of distilled water, and containing 15 seeds. To
 prevent moisture loss, all Petri dishes were sealed with Parafilm M®. The
 growth chamber was equipped with warm white fluorescent tubes (KD21
 Striplight; Radiant, 16 W) and set to a fixed temperature regime of 16/32
 °C with a 12-hour thermoperiod and a matching light/dark photoperiod.
 Petri dishes were monitored daily for germinants (radicle ≥1 mm) over 86
 days (~11 weeks), well beyond the typical germination period for
 <em>V. erioloba</em> seeds (6–8 weeks; Palgrave, 1983).
 Moisture levels were maintained throughout. To avoid systematic effects
 related to positioning in the incubator, all dishes were re-randomized
 every two days, as in Yang et al. (1999). Germinants were transplanted to
 seedling trays every 14 days. Experiment 2: Assessing
 pod exposure effects on seed germinability This trial
 compared the germinability of dung seeds collected in April and July. Both
 sets were from the same cohort, but the July seeds had experienced several
 additional months of environmental exposure prior to ingestion by black
 rhinos. We hypothesised that July seeds would show reduced performance as
 a result, despite being visually intact. We used 600
 dung seeds from each month and 1,200 pod seeds (April-collected), divided
 into two control groups (n = 600 <em>ea.</em>). Each treatment
 (Dung April, Dung July, Control 1, Control 2) was sown in 40 replicate
 Petri dishes. Aside from the increased sample size, the experimental
 design replicated Experiment 1. The trial was terminated after 12 days due
 to time constraints linked to upcoming fieldwork.
 Testing early seedling growth and resilience
 Experiment 3: Monitoring early seedling development
 Seedlings from Experiment 1 were used to assess whether gut
 passage conferred legacy effects on early seedling development. We
 expected Dung seedlings to grow faster and taller than the Control
 seedlings. Germinated seeds were removed from Petri
 dishes every 14 days and transplanted into seedling trays filled with
 builder’s sand and perlite (1:1), a nutrient-deficient medium used to
 avoid confounding effects of substrate before the experiment. The trays
 were housed in a greenhouse with full-spectrum daylight supplementation
 and relatively stable temperatures (16/32 ± 5 °C). Each seedling received
 5 mL of distilled water weekly. After 28 days, seedlings were transplanted
 into 15 cm pots and moved to a climate-controlled room for monitoring.
 This staggered schedule created four batches, each spaced ~14 days apart,
 that were all introduced at ~42 days. Pots were filled
 with either Tswalu sand or fresh (1-2 day old) black rhino dung, collected
 in the field and sealed in heavy-duty bags for ≤2 weeks. Substrates were
 manually sifted to remove visible seeds. Each pot was layered with 5 cm of
 rockwool, 250 mL of perlite, and topped with either sand or
 dung. Seedlings from each germination treatment group
 (Control, Dung, HCl, and GA) were assigned as evenly as possible to each
 substrate. At large, five seedlings were planted per pot. However, when
 the number of seedlings in a batch was not divisible by five, one pot
 contained fewer individuals to accommodate the remainder. This variation
 was unavoidable, as batch size was determined by the total number of seeds
 that germinated during the 14-day window of the preceding germination
 trial. The variable <em>pot density</em> (i.e., the number of
 seedlings per pot) was recorded at planting and updated during biweekly
 monitoring to reflect any mortalities. Four additional pots (two per
 substrate), housed i-Buttons at 1 cm depth to monitor soil
 temperature. The room maintained a 16/32 °C cycle and
 12-hour photoperiod, lit by two full-spectrum, 600W LED plant ballasts
 (110 x 180 cm), delivering 500 ppm. A malfunction spiked daytime
 temperatures to a maximum of 41 °C for ~one week, with minimal effect on
 nighttime temperatures. This short-term fluctuation precisely replicated a
 Kalahari heat-wave, which occurs during the summer season we were
 simulating. Analysis revealed that seedling growth was
 unaffected. Pots received 150 mL of water twice weekly.
 Before watering, weekly soil moisture was recorded with an EC-5 probe
 (ProCheck logger). Pots were re-randomized biweekly. Plant growth
 measurements, namely plant length and basal stem diameter, were recorded
 every two weeks. Plant length was measured by tracing a string from the
 base of the cotyledons (where a scar remains after termination) to the
 apical bud and recording the string length. Stem diameter was measured
 using digital callipers below the first true leaves.
 Experiment 4: Clipping, to simulate black rhino
 herbivory To assess whether rhino dung promotes
 development and confers resilience to disturbance, seedlings were clipped
 to 2 cm, after which their recovery in either substrate was monitored. We
 expected seedlings grown in dung to recover most effectively, as they
 could rely on substrate-derived nutrients and moisture.
 Plants were cleanly clipped with pruning shears at 45 °,
 replicating the characteristic bite of a black rhino (Owen-Smith, 1988).
 Clipping prevents apical dominance and consequently induces branching, a
 common response to disturbance among <em>Acacias</em> in arid
 environments (Archibald & Bond, 2003). To accommodate this, each
 branch length was traced with straws and summed to calculate the total
 plant length. All other monitoring protocols matched those in Experiment
 3. All seedlings were clipped on the same day, but
 because batches were staggered by ~14 days, there was a ~51-day age gap
 between the oldest and youngest seedlings. This design allowed us to
 examine whether resilience increased with seedling maturity. The full
 monitoring period, including early growth and post-clipping recovery,
 lasted 93 days (±15 weeks). <strong>Data
 analysis</strong> All statistical analyses in
 this study were conducted using the R statistical environment, version
 4.4.2 (R Core Team, 2024). Experiments 1 and 2:
 Disentangling the effects of gut passage on germination and assessing the
 impact of field exposure on seed germinability We
 applied time-to-event models to analyse seed germination, as recommended
 by McNair et al. (2012) and Romano and Stevanato (2020), with
 methodological guidance from Onofri et al. (2010) and Scott et al. (1984).
 Unlike germination traditional methods (indices, ANOVAs, or linear
 regressions), time-to-event approaches model the distribution of
 individual germination events, rather than cumulative germination
 proportions, offering greater statistical power. We considered three main
 types of models: Kaplan-Meier (non-parametric), Cox proportional-hazards
 (semi-parametric), and Accelerated Failure Time (parametric) models. We
 formatted germination data from either trial in binary format. After
 inspecting survival curves and log-log plots, we found the data exhibited
 a slightly leptokurtic distribution, which violated parametric
 assumptions, particularly in the second trial. Consequently, the
 non-parametric (Kaplan-Meier) and semi-parametric (Cox
 proportional-hazards) approaches were chosen for reporting in both
 analyses. Models were run using the
 <em>survival</em> package (Therneau, 2023; version 3.8-1) and
 visualised with <em>survminer</em> (Kassambara et al., 2021;
 version 0.4.9). Statistical significance was assessed using log-rank
 (Kaplan-Meier) and Wald (Cox) tests, with p-values reported and 𝛼=0.05.
 The Kaplan-Meier log-rank test models the survival function 𝑆(𝑡), or the
 probability of a seed not germinating at time 𝑡; this is the inverse of
 the cumulative incidence 1−𝑆(𝑡)/𝐹(𝑡), representing the instantaneous
 probability of germination. The Cox model estimates the hazard function
 𝐻(𝑡), or the cumulative "risk" of germination.
 Experiments 3 and 4: Monitoring early seedling development and
 clipping, to simulate black rhino herbivory Seedling
 growth in sand and dung substrates, pre- and post-clipping, was analysed
 by means of Generalized Linear Mixed Models (GLMMs), using the
 <em>glmer</em> function (<em>Lme4</em> version
 1.1-35.4; Bates et al., 2015). Basal stem diameter,
 converted to stem area (πr²),  was selected as the main response variable
 because this measure demonstrated the best model fit. This aligns with
 findings that stem diameter scales with leaf biomass across a wide variety
 of tree species (Enquist & Niklas, 2002; Sun et al., 2019). Plant
 growth data were separated into two datasets (pre- and post-clipping) that
 were treated as separate experiments, so analysed independently but using
 the same statistical framework. Data from the first 18 days were excluded
 from Experiment 3 because seedlings only had a radicle and cotyledons
 protruding from the seed, making stem-related measurements unfeasible.
 Monitoring thus spanned 4 weeks for Experiment 3 (pre-clipping) and 9
 weeks for Experiment 4 (post-clipping). Before the main
 analysis, substrate differences in soil moisture and temperature were
 tested using Welch’s t-tests. Collinearity among predictors was evaluated
 via Pearson’s correlation matrices (<em>chart.Correlation</em>
 function; <em>PerformanceAnalytics</em> version 2.0.4;
 Peterson & Carl, 2020) and variables with a coefcoefficient75 were
 excluded from joint models. Model distributions were selected based on
 residual plots and fir diagnostics (<em>fitdistrplus</em>
 version 1.2-1; Delignette-Muller & Dutang, 2015). Although the
 data were normally distributed, heteroscedasticity necessitated a
 semi-parametric approach. The Gamma distribution provided the best fit for
 both datasets and was theoretically applicable, since it accommodates
 positive, skewed, and continuous data, and is commonly used for repeated
 measures (Eric et al., 2021). Unlike the normal distribution (mean, SD),
 the Gamma distribution uses shape and scale parameters, which offer more
 flexibility in variance (Eric et al., 2021). We applied a log link, which
 also accommodates positive-only data with positively-skewed errors and can
 represent an underlying multiplicative process, which is common in
 ecology. Candidate model selection was conducted to
 determine optimal model structure (Tables S1 and S2). While the
 experimental treatments were retained in all candidate models, the
 selection process considered the inclusion of additional biologically
 motivated covariates like soil moisture, temperature, and pot density, as
 well as feasible interaction terms. Candidate models were ranked using the
 second-order Akaike Information Criterion (AICc), computed via
 the <em>AICc</em> function (<em>AICcmodavg</em>
 version 2.3-3; Mazerolle, 2023). AICc was preferred over AIC due to small
 size, since it includes a correction for low numbers of observations
 relative to model parameters (Hurvich & Tsai, 1989). Model
 rankings were based on ΔAICc values and cumulative Akaike weights (aictab
 function). Following Burnham & Anderson (2002), models with ΔAICc
 ≤ 2 were considered equally plausible; those with ΔAICc between 2–7 had
 less support, and those with ΔAICc &gt; 7 were excluded from
 interpretation. If ΔAICc ≤ 2 between the top models, biological relevance
 was considered before selecting the final model.  Multicollinearity among
 covariates was assessed using variance inflation factors (VIFs), or
 generalised VIFs (GVIFs) for models with interaction terms, via the
 <em>vif</em> function (<em>car</em> version 3.1-2;
 Fox et al., 2019). We checked for overdispersion using the
 <em>dispersion_glmer</em> function
 (<em>blmeco</em> version 1.4; Korner-Nievergelt et al., 2015).
 Marginal and conditional R<sup>2</sup> values were calculated
 via <em>r.squaredGLMM</em> (<em>MuMIn</em> version
 1.48.4; Bartoń, 2024).
description: # Endozoochory by black rhinoceroses enhances germination of a key arid
 savanna tree species
 [https://doi.org/10.5061/dryad.9ghx3fftr](https://doi.org/10.5061/dryad.9ghx3fftr) ## Description of the data and file structure This dataset was collected to investigate the role of black rhinoceroses (*Diceros bicornis*) in facilitating the recruitment of *Vachellia erioloba*, a keystone tree species in arid savannas. The study aimed to evaluate the whether a mutualism exists, through the study of i) whether gut passage significantly enhances germination, ii)  whether seed viability is diminished by the duration between pod shedding and seed dormancy breakage (alleviated by early consumption), iii) whether legacy effects of rhino gut passage enhance the initial growth of seedlings and iv) whether seedlings grown in dung recover better after simulated herbivory (clipping) than those grown in sand. We conducted a series of experiments to test each of these hypotheses. Surviving individuals from Experiment 1, testing germination, were integrated into successive experiments (Experiments 3 and 4), testing growth and recovery. Only Experiment 2 used a unique seed set. These findings contribute to understanding how black rhino presence influences *Vachellia erioloba* recruitment dynamics at early ontogenic stages of development. ### Files and variables #### Experiment 1 **Description:** Data file [Experiment_1.csv 112.35 KB] consists of germination rates of seeds collected from black rhino dung and pods, as well as two chemically treated pod seed groupings  ##### Variables * Treatment  Seeds were treated with hydrochloric acid (HCl), or HCl and then gibberellic acid (GA), or were untreated and consisted of seeds from pods, or were seeds untreated but collected from dung, so had undergone black rhino gut passage.  * petri_dish: 300 seeds per treatment group were germinated within petri dishes, with 15 seeds per petri dish and dishes labelled A-Z. r petri dish 26, AA, BB, and so on....  * start_date: Beginning date of germination trial  * end_date: Termination date of germination trial * time_days: A count of how many days over which the trial was conducted  * germinated_yn: Count ofthe  number of germinations over the days #### Experiment 2 **Description:** Data file [Experiment_2.csv 208.58 KB]  consists of germination rates of seeds collected from black rhino dung and pods, from two different time periods in the year (i.e., with different degrees of environmental exposure).  ##### Variables * Treatment: Two sets of "treatment" seed groups were either untreated and consisted of seeds from pods (Control 1 and Control 2). The other two seeds were also untreated but collected from dung, so had undergone black rhino gut passage, in April (Dung A) or June (Dung J) * petri_dish: 300 seeds per treatment were germinated within petri dishes, with 15 seeds per petri dish and dishes labelled A-Z, then after petri dish 26, AA, BB, and so on....  * start_date: Beginning date of germination trial  * end_date: Termination date of germination trial * time_days: A count of how many days over which the trial was conducted  * germinated_yn: Count of the number of germinations over the days #### Files: Experiments 3 and 4 **Description Summary Stats:** This subsection prefaces Experiments 3 and 4. Summary statistics regarding the differences in a) temperature and b) soil moisture between pots containing Dung and Sand are included, in data files a) Summary_Stats_soil_moisture.csv 22.54 KB and b) Summary_Stats_temperature.csv 276.92 KB **\ \ Description Exp 3:** Data file [Experiment_3.csv 26.05 KB] consists of seedling growth measurements post-germination under different substrate conditions (sand and dung) **Description Exp 4:** Data file [Experiment_4.csv 64.4 KB] consists of further growth metrics following simulated black rhino herbivory (clipping). ##### Variables * ID: Individual seeds were named according to their treatment, petri dish number, and order of germination out of the petri dish (1-15) - same seedlings as in experiment 1.  * Treatment: Same as in experiment 1.  * batch: Seeds were removed from petri dishes, held in seedling trays, and introduced into the climate-controlled room in batches every 14 days, resulting in subgroupings of seedlings, A-D.  * pot_number: Numbering system for the pot that each seedling was planted in.  * Substrate: The pots were filled with black rhino dung or sand from the study site * pot_density: Number of plants planted in a single pot * stem_width_mm: Repeat measures of the width of each seedling stem between the scar made by the cotyledons and the first set of true leaves. A fine measure of plant growth.  * stem_area: Calculated from stem width, using the formula for the area of a circle.  * SM*_*AVG*: s*oil moisture values, calculated from* EC*5 soil moisture probes, connected to a Procheck logger * T_Median: median SOIL temperature, calculated from 15-minute measures of temp from i-buttons buried 1cm into both the sand and dung substrates.  * T_Max: maximum SOIL temperature, calculated from 15-minute measures of temp from i-buttons buried 1cm into both the sand and dung substrates.  *NOTE: Correlation matrices were assessed in the same R scripts but using modified files, Experiment_3_corr_matrix.csv 16.39 KB and Experiment_4_corr_matrix.csv 37.25 KB #### Software files hosted by Zenodo All .csv files were analysed in R Studio, within the R scripts listed below, whose names all correspond intuitively with the datasets utilised in each one.  * Summary_Stats_-_soil_moisture.R 8.59 KB * Summary_Stats_-_temperature.R 21.21 KB * Experiment_1.R 45.72 KB * Experiment_2.R 44.46 KB * Experiment_3.R 65.02 KB * Experiment_4.R 57.83 KB #### Supplemental files hosted by Zenodo Figures 1 to 7 are all the figures included within the published paper:  * Figure_1_-_FULL_overview.svg 2.42 MB * Figure_2_-_Stereo_microscope.svg 2.54 MB * Figure_3A_-_Experiment_1.svg 84.36 KB * Figure_3B_-_Experiment_1.svg 152.76 KB * Figure_4A_-_Experiment_2.svg 76.33 KB * Figure_4B_-_Experiment_2.svg 142.03 KB * Figure_5A_-_Summary_Stats_soil_moisture.svg 462.27 KB * Figure_5B_-_Summary_Stats_Temp.svg 3.02 MB * Figure_6_-_Experiment_3.svg 152.68 KB * Figure_7_-_Experiment_4.svg 220.34 KB Figures S1 to S2 are all the figures included within the supplementary information, which is publicly available:  published paper:  * Figure_S1A_-_Experiment_1.svg 144.69 KB * Figure_S1B_-_Experiment_1.svg 125.33 KB * Figure_S2A_-_Experiment_2.svg 190.3 KB * Figure_S2B_-_Experiment_2.svg 124.76 KB ## Access information Other publicly accessible locations of the data: * None Data was derived from the following sources: * None