Methods
Study Site - Fieldwork was conducted from September 2016 to September 2018 at Ol Pejeta Conservancy (“OPC”; 0°0’52.62”N, 36°51’58.64”E, 1800 m above sea level). This 360 km2conservancy receives ca. 250-300 mm of rainfall in wet seasons (March to May; October to December, and intervening periods are typically dry and hot with monthly rainfall of ca . 30-50 mm (Ol Pejeta Conservancy Dept. of Wildlife Conservation, unpublished data). The OPC elephant population (ca . 130-300 depending on forage availability, OPC records) disproportionately imposes heavy damage onA. drepanolobium in areas where P. megacephala has invaded (Riginos et al. 2015; Palmer et al. 2020). Ground-dwellingP. megacephala ants have expanded from human habitation areas on OPC into black-cotton savannas for the past ca . two decades (Riginos et al. 2015), where they occupy trees and soil. During this study, P. megacephala extended each monitored invasion front by ca . 50 m/yr (invading ca . 40-50 trees per 50 x 50 m area; Pietrek et al. in press)
Survey Regime - We measured leaf gas exchange (photosynthesis and transpiration) in concurrent Before-After-Control-Impact (BACI) and factorial experiments (Fig. S3) during rainy and dry seasons in 2017 and 2018. Both experimental designs are described below, followed by specific details of our plant physiology surveys. For each surveyed tree, we measured leaf water potential at mid-day and before dawn to 1) confirm assumptions that all sites had similar soil water status within each ca. 2-week survey period, 2) to confirm that our designations of “wet” and “dry” seasons were appropriate relative to studies of other East African acacias (Gebrehiwot et al. 2005; Gebrekirstos et al. 2006), and 3) to calculate leaf water potential range, which can be compared with leaf gas exchange rates to indicate changes in water management by the plant.
Before-After-Control-Impact Experiment – To assess short-term impacts of P. megacephala invasion, we measured gas exchange rates and leaf water potential on the same trees before and after invasion, and compared those to concurrent measurements on uninvaded trees that were protected by native C. mimosae . We surveyed trees in plots near the invasion front (“Transition” sites) before and after invasion, and also surveyed non-manipulated trees <1 km from each Transition site (“Control” sites) that remained unaffected byP. megacephala range expansion over the course of the study. All sites were accessible to large herbivores. In the July 2017 dry season and November 2017 wet season, we surveyed 20-24 adult trees (1.5-2 meters tall) at each Transition (pre-invasion) and Control site.Pheidole megacephala workers expanded into Transition sites in December 2017, and we repeated surveys at each site in the May 2018 wet season and September 2018 dry season. Five trees were destroyed (evidently by elephants) between December 2017 and May 2018 and were excluded from analyses.
Factorial experiment comparing longer-term (>5 years) impact of invasion In the factorial experiment, we tested direct and indirect effects of invasive P. megacephala , nativeC. mimosae, and vertebrate herbivores on leaf and canopy gas exchange, and also compared gas exchange between longer-term invaded (ca. 5 years) and uninvaded tree stands. We measured leaf water potential and gas exchange rates in two dry (July 2017 and September 2018) and two wet seasons (November 2017 and May 2018). Treatment factors were large herbivores (present vs. excluded) and ants (present vs. excluded), resulting in four treatments (Fig. S3). We conducted our experiment in three sites where acacias had been invaded for ca.5 years (“Invasion”; estimated from surveys of P. megacephalaabundance from 2013-2015 and rates of expansion of nearby invasion fronts), and in 3 neighboring (< 2 km away) uninvaded sites with comparable tree density (“Uninvaded” sites). We constructed an electric fence exclosure at each site to exclude large herbivores (>20 kg) from a 50 x 50 m plot (0.25-ha) containingca . 40 adult trees (1.5-2 meters tall). We marked 40 adult trees (1.5-2 m tall) in a plot of similar area and tree density ca. 200 m from each fenced plot to serve as the herbivore-present treatment. Each site comprised two plots, with a total of ca . 80 marked trees at each site. We fogged canopies of 20 trees in each plot with 0.6% alpha-cypermethrin (2-3 days in full sunlight, World Health Organization, 2013), to remove ants at the start of the experiment in 2016. To prevent the reestablishment of ant colonies, we applied sticky barriers (Tanglefoot ® Insect Barrier, Contech Enterprises, Victoria, BC, Canada) to the trunks of those same trees (e.g., see Stanton and Palmer 2011). To maintain ant exclusion treatments, we reapplied sticky barriers as needed, and injected insecticide into domatia if they were colonized by foundress ant queens (recognizable by domatia holes sealed with carton material).
Tree physiological measurements In the BACI and factorial experiments, we conducted all plant physiology measurements on fully-expanded leaves growing from non-lignified shoots in the unshaded sections of the upper canopy. Leaf-level light-saturated photosynthetic and transpiration rates [henceforth “leaf-level photosynthesis” (Amax-leaf ) and “leaf-level transpiration” (Eleaf )] were measured using a LI-6400XT Portable Photosynthesis System (Li-Cor Biosciences, Lincoln, NB) during sunny or partly cloudy days from 07:30-11:30. In longer-term Invaded and Uninvaded sites, we counted all mature leaves and measured leaf area of 5 randomly-selected mature leaves for a random subset of 7-17 acacias per treatment in our factorial experiment in 2018 ( N = 102 in wet season, N = 61 in dry season; means ± SEM in Table S1) to estimate total leaf area. We then multiplied total leaf area by leaf-level photosynthesis and transpiration to estimate idealized light-saturated whole-canopy photosynthesis and transpiration capacities [henceforth “canopy-level photosynthesis” (Amax-canopy ) and “canopy-level transpiration” (Ecanopy )].
Amax and E are calculated from gas exchange rates measured in the ideal environment within a controlled cuvette, and likely are higher than net photosynthesis and transpiration of a tree in naturally variable conditions (McGarvey et al. 2004). We therefore compare these approximations at the leaf- (-leaf ) and canopy-level (-canopy ) to estimate relative differences in gas exchange for trees in our field experiments, but they do not estimate the absolute effect of invasion on carbon fixation.
We measured pre-dawn (ψPD ) and mid-day leaf water potential (ψMD) on the same day as the gas exchange measurements for each study site using a Model 610 Plant Pressure Chamber (PMS Instruments, Corvallis, OR). Treatment means (± SEM) of ψPD andψMD are in Tables S2 and S3. Wet seasonψPD ranged from ca . -1.0 to -1.5 MPa and dry season ψPD ranged fromca . -1.9 to -2.1 MPa; studies of related tree species in the region recorded ψPD of ca . -2.0 MPa in dry conditions (Gebrekirstos et al. 2006).
For each tree we calculated diurnal leaf water potential range (∆ψ) as the difference between pre-dawn and mid-day leaf water potentials (∆ψ= ψPDψMD). ∆ψ demonstrates the range of viable water conditions that a leaf will experience (Gebrehiwot et al. 2005; Gebrekirstos et al.2006): that range (negative with an upper limit of zero) is fundamentally created by stomatal water loss (Henry et al. 2019) and made more negative by loss of vascular hydraulic conductivity (Lambers et al. 2008; Scoffoni et al. 2017). Plants will often remain within a species-specific ∆ψ (e.g., Gebrekirstos et al. 2006), while photosynthesis and transpiration can vary without affecting ∆ψ as a result of osmotic or stomatal adjustments (Inoueet al. 2017; Martínez‐Vilalta & Garcia‐Forner 2017; Hochberget al. 2018; Zhang et al. 2019). Further details on our methods for measuring tree physiology parameters can be found in Note S1.
Statistical Analysis - We used generalized linear mixed models (GLMMs) to analyze data in the BACI and factorial experiments. For the BACI experiment, we constructed individual GLMMs for each season (wet/dry) for Amax-leaf ,Eleaf , and ∆ψ. In the BACI GLMMs, sampling year (2017, 2018) and site type (Transition, Control) and their interaction term were fixed effects. We included site as a random effect in all GLMMs. The BACI analysis produces 3 terms: effects of 1) Site and 2) Year, which indicate significant underlying differences between Transition and Control sites and interannual differences for all trees between survey years (respectively), and 3) an interaction term, which indicates if changes that occurred for leaf physiological traits between the 2017 and 2018 surveys were different for Transition trees (which were invaded at the end of 2017) and for Control trees. Only the interaction terms are discussed in the results section, while Site and Year (i.e. , underlying differences) are reported in the supplement (Note S3). For the factorial experiment, we constructed separate GLMMs for each season (wet/dry) forAmax-canopy ,Ecanopy ,Amax-leaf ,Eleaf , and ∆ψ. In the factorial experiment GLMMs, invasion status (longer-term Invaded or Uninvaded) was a fixed effect, the exclusion of herbivores and ant occupants were fixed effects nested within invasion status, and we pooled data for the two dry seasons and for the two wet seasons.
Analyses were conducted using JMP Pro 15.1.0 (SAS Institute, Cary, North Carolina, USA). Further details on GLMMs are in Note S2.