Nodulation Alleviates Water Stress in Vachellia Sieberiana

14 Species in the genus Vachellia (Fabaceae) have a global tropical and sub-tropical distribution. 15 Numerous Vachellia species are currently observed to be expanding their indigenous ranges and 16 increasing in dominance globally, suggesting an overarching driver. Most Vachellia species enhance 17 nitrogen uptake mutualistically via specialized root nodule structures. Nodules contain N 2 -fixing 18 rhizobia that consume host supplied carbon to catalyse atmospheric N 2 into a plant useable form, a 19 key element in plant growth. The rhizobial mutualism of some Vachellia species may be vital to 20 understanding changing patterns of ecological success observed across the savanna precipitation 21 gradient. 22 Here, we investigated how the seedling root development and physiology of two dominant savanna 23 woody species, the arid-adapted Vachellia erioloba and the mesic-adapted Vachellia sieberiana , 24 responded to simulated drought events. Seedlings of both species were grown at 4%, 8% and 16% 25 soil moisture content (SMC) for four months. Seedling growth and allometry of arid-adapted V. 26 erioloba was unresponsive to water stress treatments, and no nodulation was observed, reflecting a 27 fixed higher relative investment in belowground biomass. In contrast , V. sieberiana roots were 28 nodulated, but developed the highest nodule biomass and growth rate when grown at the lowest 29 soil moisture (4% SMC). These patterns suggest that effective life history strategies for the arid- 30 adapted species precludes the need for rhizobial mutualism, possibly due to more “open” N cycling 31 and lower competitive interactions in arid systems, while the more “closed” N cycling in mesi c 32 savannas, and higher competitive stress, may favour nodulation, especially under low water supply 33 that limits root access to soil nitrogen, and signals a more competitive environment and an 34 advantage from N 2 -fixing.


Introduction 37
Savannas account for a fifth of the earth's land surface across four continents, including half of Africa 38 (Sankaran et al., 2005;Scholes & Archer, 1997), and are "open ecosystems" (Bond, 2019), 39 characterised by discontinuous woody cover and a continuous herbaceous ground layer (House et  for encroachment (Liu et al., 2013). 51 In Africa, the majority of encroaching woody species are legumes, belonging to the genera Vachellia 52 and Senegalia (Stevens et al., 2017). Many of these species have the capacity to fix atmospheric 53 nitrogen (N2) via a rhizobial mutualism (Scholes & Archer, 1997;Sprent, 1995), a trait that possibly 54 influences their regional dominance ( Seymour, 2008;Sprent & Gehlot, 2010). It has also been suggested that if N is not a limit on plant 63 growth, the ability to nodulate is of limited advantage (Sprent & Gehlot, 2010), as substantial energy 64 must be invested in traits that facilitate survival in water limited conditions. Arid savannas leaf N 15 65 reflects an "open" N cycle (Aranibar et al., 2004;Midgley, Aranibar, Mantlana, & Macko, 2004) . 66 Nodulating legumes, such as Vachellia sieberiana, are more common in mesic savannas (~800-67 1000mm MAP) (Figure 1.c). Mesic savannas are typically N limited; due to high rates of soil nutrient 68 leaching (Zahran, 1999), competition with grasses (due to increased productivity associated with 69 higher annual rainfall) ( (Bond & Midgley, 2003). Plant 75 survival is determined by interacting factors that vary in their relative importance along productivity 76 gradients including water stress, herbivory, and fire (Kraaij & Ward, 2006;Sankaran et al., 2005). 77 Hence, understanding seedling growth and recruitment of Vachellia species could help understand 78 the key traits that underlie varying savanna vegetation dynamics across rainfall gradients. A seldom 79 considered aspect of savanna seedling success is how water stress (Sankaran, 2019) alters the 80 functionality of the legume-rhizobium symbiosis (Serraj, 2003). Previously it has been proposed that 81 water stress can reduce N2-fixation by reducing carbon nodule metabolism, introducing oxygen 82 limitation, thus causing reduction of N2-fixation product transport (Serraj, 2003). Understanding 83 interactions between water stress and nodule production related to plant growth will help 84 determine the functional role of N2-fixation in seedling success (Krug, 2017 Here we investigated how soil moisture relates to the growth and nodulation response of two 90 woody Vachellia species, one arid and one mesic, both considered encroachers (Hauwanga,91 per water treatment, water treatments were applied of: no water, 0.020 m 3 /m 3 , 0.080 m 3 /m 3 , 0.100 158 m 3 /m 3 , 0.120 m 3 /m 3 , 0.180 m 3 /m 3 , 0.220 m 3 /m 3 , 0.280 m 3 /m 3 , 0.330 m 3 /m 3 and 0.380 m 3 /m 3 . After 159 three weeks V. exuvialis seedlings were removed, and the soil from each pot weighed and oven 160 dried at 105°C to determine appropriate SMC for watering treatment using the following equation: Height (mm) of each seedling was measured weekly. Ten seedlings of each species of each 175 treatment were harvested at three points after commencing the water treatments. Harvests were at 176 one (Harvest 1), two (Harvest 2) and three months (Harvest 3) post water treatment. Seedlings were 177 separated into above and belowground biomass and roots were carefully washed to maintain fine 178 root mass. Nodules were removed from the roots using forceps and cleaned using a paint brush. For 179 each harvested plant, the final plant height, dry aboveground and belowground biomass (g), and 180 nodule dry biomass (g) were recorded. All plant material was oven dried at 65°C for 36 hours 181 (Kambatuku et al., 2013).

Statistical analyses 184
All analyses was conducted using R 3 5.1 (R Core Team, 2020). The weekly measurements of seedling 185 height were used to create a linear mixed model, using the lme4 package ( Table 2).
The growth and allocation patterns of the arid V. erioloba did not vary across water treatment (df= 207 2, F=1.45, p>0.050) (Figure 3.a, Figure 3.b & Table 2). Relative to V. sieberiana, V. erioloba seedlings 208 were shorter (df= 2, p>0.050) ( Table 3 & 4). There was an 800% increase in the number of individuals 215 that nodulated between Harvest 1 (two months old) and Harvest 2 (three months old) (Table 4). 216 There was an 11% increase in the number of individuals that nodulated between Harvest 2 (three 217 months old) and Harvest 3 (four months old) (Table 4). 218 Nodule biomass and count was affected by water treatment (df=2, F=4.40, p<0.050) (Figure 4.a & 219 Table 3). These patterns fluctuated across the experiment with three seedlings grown in 8% SMC 220 developing nodules at around two months old, seedlings grown in 16% SMC collectively producing 221 the highest nodule count across all three harvests. However, seedlings grown in 4% SMC developed 222 nodules with the highest nodule biomass in Harvest 3 (four years old) (Table 4) (1) How does water availability affect seedling growth rates? 228 Contrary to expectation we found that the growth of mesic V. sieberiana seedlings increased with a 229 decline in water availability. However, links between reduced water availability and increased growth/success have been seen in other Vachellia species: rapid above and belowground growth in 231 Vachellia tortilis and Vachellia raddiana has been found to occur during the dry season in Southern 232 Israel (Winters et al., 2018). These two species are also known nodulators (Sprent, 2009)  . 258 Alternatively, V. erioloba extends into most arid parts of the African desert; more than any other 259 tree species (R. D. Barnes, Fagg, & Milton, 1997). Therefore, perhaps it was unaffected by water 260 treatment because the seedlings were not drought stressed. We suggest that future experiments 261 using V. erioloba incorporate a more severe drought treatment to better understand their growth 262 traits relating to their arid niche. 263 Rooyen, 2020). In this experiment, to allow seedlings the opportunity to establish they were exposed 297 to four weeks of continuous watering before drought treatments were imposed. We suggest that 298 future experiments could identify the minimum watering period for seedling establishment to 299 understand how seedlings will adapt to these climatic changes. 300 301

Conclusion 302
Drought has a wide range of effects on closely related Vachellia species relative to their 303 environmental niche and associated growth traits. A high growth rate is not necessarily an indicator 304 of plant success (Adams et al., 2016); it must be considered alongside the ability of a plant to survive and reproduce under a range of environmental conditions. For V. sieberiana to be successful in the 306 mesic environment it requires extended periods of growth to escape fire and compete with grass. 307 Nodulation enables a flexible N supply to enhance growth over such time periods. In this experiment 308 V. sieberiana's increased nodulation triggered by drought stress suggests that nodulation assists the 309 withstanding of water stress within its environmental niche. The growth traits of V. erioloba, 310 remained unaffected by drought stress, potentially attributable to its high tolerance to aridity (R. D. 311 Barnes et al., 1997). Perhaps, the measure of success for V. erioloba is not rapid growth but being 312 able survive in a water limited environment via methods of below ground investment. These 313 patterns suggest that effective life history strategies for the arid-adapted species precludes the 314 requirement for rhizobial mutualism, due to more "open" N cycling and lower competitive 315 interactions in arid systems (Aranibar et al., 2004). Whereas in the mesic savanna, the more "closed" 316 N cycling and higher competitive stress, may favour nodulation, especially under low water supply 317 that limits root access to soil nitrogen, and signals a more competitive environment and an 318 advantage from N2-fixing ( Table 1 The output of a mixed model for the effect of water treatment on the growth (height) of V. 598 erioloba (VE) and V. sieberiana (VS) seedlings. 4%, 8% and 16% correspond the soil moisture content 599 the seedlings were grown in. These measurements were taken three weeks following germination for 600 15 weeks. Soil moisture treatments were applied at week two. The individual seedling was tested as 601 a random effect. The standard deviation is represented in brackets. Significance is indicated as follows: 602 *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. 603 Vachellia erioloba Height (mm)  Table 2 The coefficients (β), lower (CI 2.5%) and upper (CI 97.5%) confidence intervals from the whole 605 plant dry biomass (grams), below ground biomass (grams) and the root: shoot ratio of seedlings 606 harvest at four months old (during Harvest 3). Treatment refers to the water availability treatment. 607

Coefficients
Species refers to V. erioloba (VE) and V. sieberiana (VS) and The output from 2 way ANOVA for testing 608 the effect of water availability treatment and differences between species in the whole plant biomass 609 (grams), below ground biomass (grams) and above: below ground ratio of V. erioloba (VE) and V. 610 sieberiana (VS) seedlings that were harvested at four months old (during Harvest 3). Treatment refers 611 to the water availability treatment. Significance is indicated as follows: *p<0.05, **p<0.01, 612 ***p<0.001, ****p<0.0001. 613 Below ground biomass (g)     The height of V. erioloba and V. sieberiana measured at weekly intervals over a four-month period. Treatment 4% (orange), 8% (purple) and 16% (blue) correspond the soil moisture content the seedlings were grown in. These measurements were taken three weeks following germination. Soil moisture treatments were applied at week 2 (Wickham, 2016).