variation in root system traits among african semi-arid savanna grasses: implications for drought...

Variation in root system traits among African semi-aridsavanna grasses: Implications for drought toleranceaec_2422 1..10

DAVID C. HARTNETT,1* GAIL W. T. WILSON,2 JACQUELINE P. OTT1 ANDMOFFAT SETSHOGO3

1Division of Biology, Kansas State University, 104 Ackert Hall, Manhattan, KS 66506, USA (Email:[email protected]), 2Department of Natural Resource Ecology and Management, Oklahoma StateUniversity, Stillwater, Oklahoma, USA; and 3Department of Biological Sciences, University ofBotswana, Gaborone, Botswana

Abstract In arid to semi-arid grasslands and savannas, plant growth, population dynamics, and productivity areconsistently and strongly limited by soil water and nutrient availability. Adaptive traits of the root systems of grassesin these ecosystems are crucial to their ability to cope with strong water and/or nutrient limitation and the increasingdrought stress associated with ecosystem degradation or projected climate change. We studied 18 grass species insemi-arid savanna of the Kalahari region of Botswana to quantify interspecific variation in three important rootsystem traits including root system architecture, rhizosheath thickness and mycorrhizal colonization. Drought-tolerant species and shorter-lived species showed greater rhizosheath thickness and fine root development but lowermycorrhizal colonization compared to later successional climax grasses and those characteristic of wetter sites. Inaddition, there was a significant positive correlation between root fibrousness index and rhizosheath thicknessamong species and a weak negative correlation between root fibrousness index and mycorrhizal colonization.Thesepatterns suggest that an extensive fine root system and rhizosheath development may be important complementarytraits of grasses coping with drought conditions, the former aiding in the acquisition of water by the grass plant andthe latter aiding in water uptake and retention, and reducing water loss in the rhizosphere. Within species, bothrhizosheath development and mycorrhizal colonization were significantly greater in a wet year than in a year withbelow-average precipitation.The observed patterns suggest that the primary benefit of rhizosheath development inAfrican savanna grasses is improved drought tolerance and that it is a plastic trait that can be adjusted annually tochanging environmental conditions. The functioning of mycorrhizal symbiosis is likely to be relatively moreimportant in infertile savannas where nutrient limitation is higher relative to water limitation.

Key words: grass, mycorrhiza, rhizosheath, root architecture, savanna.

INTRODUCTION

In the semi-arid savannas of southern Africa, declinesin grass production associated with vegetation andrangeland degradation have been driven by complexinteractions of changing land use and management,and climate. During recent decades, increased grazingpressure, in combination with drought, have resultedin declines in the cover of perennial grasses and anincrease in the cover of annual grasses and otherspecies of low economic and ecological value (VanVegten 1981; Skarpe 1986). Key to the conservationand sustainable management of these ecosystems is asound understanding of the ecology of their perennialgrasses and the mechanisms driving their responses toenvironmental change (Veenendaal 1991).

A key feature of semi-arid and arid savannas is thatplant growth, population dynamics and productivity

are consistently and strongly limited by water andmineral nutrients rather than light (Scholes 1997), incontrast with mesic grasslands that have higher canopydensity, are often light limited, and are characterizedby temporally and spatially shifting limiting resources(light, water, nitrogen) driven by complex interactionsof fire, grazing and climatic variability (Sinclair 1979;Deshmukh 1984; Knapp et al. 1998).

Several traits of the root systems of grasses arecrucial to their ability to cope with strong water and/ornutrient limitation. First, variation in grass plant archi-tecture has important implication for how grassesexploit their resources and respond to neighbours,grazers and disturbances (Briske 1991). The rootsystem architecture of grasses is key to acquisition oflimited water and mineral nutrients as it determinestheir total absorptive surface area and the volumeand depth of soil explored. Secondly, mycorrhizalsymbiosis is a key trait of savanna grasses that signifi-cantly enhances plant water and nutrient uptake andinfluences grass responses to fire, grazing or other

*Corresponding author.Accepted for publication May 2012.

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© 2012 The Authors doi:10.1111/j.1442-9993.2012.02422.xAustral Ecology © 2012 Ecological Society of Australia

disturbances (Veenendaal 1991; Hartnett et al. 2004).Mycorrhizal fungi can also inhibit soil degradation anderosion by exuding glycoproteins that enhance soilaggregate stability (Wilson et al. 2009). Most C4 grassspecies that dominate warm temperate or subtropicalgrasslands are obligate mycotrophs and are highlycolonized by arbuscular mycorrhizal fungi (AMF)(Hartnett &Wilson 2002).Thirdly, the development ofrhizosheaths is a trait frequently observed in grasses ofarid habitats. These relatively little understood com-plexes of sand particles, root hairs, mucilage, and oftenfungal and bacterial components are thought to func-tion in various ways to increase plant nutrient acqui-sition and/or water retention and uptake within thegrass rhizosphere. Rhizosheaths maintain higher mois-ture content, microbial densities and nitrogen concen-tration than surrounding soil and facilitate watermovement across the soil–root interface (Young 1995;North & Nobel 1997; Othman et al. 2004; Bhatnagar& Bhatnagar 2005; Bergmann et al. 2009).

An understanding of the patterns and functions ofrhizosheaths, mycorrhizas, root system architecture,and other root system traits is crucial, particularlygiven decreasing and more variable precipitation andmore intense and/or prolonged droughts predicted forsub-Saharan Africa and other grassland regions.However, there have been only a few studies examin-ing these root system traits in semi-arid or arid savannagrasses. High levels of mycorrhizal colonization insavannas of southern Africa (Veenendaal 1991;Veenendaal et al. 1992; Hartnett et al. 2004) and Aus-tralia (McGee 1989) suggest that this symbiosis maybe particularly important for grasses in savanna orgrassland habitats of poor nutrient and/or water status.The occurrence of rhizosheaths in South Africangrasses has been documented from herbarium speci-mens (Bailey & Scholes 1997), but their distribution,composition and function in southern African savan-nas have not been studied.

In an effort to better understand these traits ofsavanna grasses and their function in semi-arid eco-systems, we examined 18 perennial grass species insemi-arid savanna in the Kalahari region of Botswana.The specific objectives of our study were to: (i) assessinter-specific and inter-annual variation in root systemarchitecture, mycorrhizal fungal colonization andrhizosheath development; (ii) quantify patterns of cor-relation among these root system traits; and (iii)examine relationships between these traits and varia-tion in life history patterns and drought toleranceamong grass species.The general goal of our study wasto quantify the patterns of variation in root systemtraits and draw inferences from these patterns todevelop hypotheses concerning driving variables thatcan be robustly tested in future studies.

Based on previous studies suggesting that carbonallocation to mycorrhizal fungi versus allocation to fine

root development represent alternative strategies forsoil resource acquisition (Baylis 1975; Hetrick et al.1991), we hypothesized that this trade-off will beevident in a negative correlation between fine rootdevelopment and mycorrhizal colonization amongsavanna grasses. In addition, we hypothesized thatmycorrhizal symbiosis provides greater relative ben-efits to nutrient acquisition than to water status,whereas rhizosheaths provide greater benefits to plantwater status relative to nutrient acquisition. Thus wepredict that, among species and habitats rhizosheathsize will increase and mycorrhizal colonization willdecrease with increasing drought. We propose theadditional hypothesis (although not tested here) thatgrasses of relatively fertile but water-limited savannaswould show greater allocation to rhizosheath andfine root development whereas grasses of infertilesavannas would allocate more resources to mycorrhizaldevelopment.

METHODS

Study sites

Grass populations were sampled at three sites across theKalahari sandveld region of Botswana. The Kalahari savan-nas extend over 2.5 million square kilometres in the interiorof central southern Africa. This region is characterized bydeep, sandy arenosols (Moganane et al. 2000). The topogra-phy is characterized by wide plains, flat dunes, depressionsand pans. There is high local spatial variability and a strongseasonality to rainfall distribution in the Kalahari region.Thesummer-wet season typically occurs from October to April,with highest rainfall in January. The dry season typicallyoccurs from May through September, with the lowest rainfallin June and July. Over longer time scales, rainfall in Botswanashows a cyclical pattern, with clustering of wet and dry years(Tyson 1978). Mean annual precipitation decreases fromnortheast to southwest across Botswana, from >450 mm inthe northeast to <200 mm in southwest.The prominent landuses of semi-arid savanna areas in the Kalahari are commu-nal livestock grazing and wildlife conservation reserves, andalthough two of the sampling sites are currently managed aswildlife reserves, they have a previous history of severaldecades of heavy cattle grazing. Further description of theclimate, soils and vegetation of the region can be found inVeenendaal (1991) and Vossen (1988).

The northernmost study site was the Khama Rhino Sanc-tuary (KRS), a 4300-ha reserve approximately 25 km northof Serowe Botswana (22°13′15″S, 26°41′44″E; elevation1230 m, mean annual precipitation = 425 mm). Prominentfeature of KRS include the Serwe Pan (a large grass-covereddepression with several natural water holes), a diverse com-munity of herbivores including white and black rhinoceros,and diverse plant communities including tree and shrubsavanna and open grasslands on the pans. The trees arepredominantly Terminalia sericea, Burkea africana, Peltopho-rum africanum, Croton gratissimus, Philoneptera nelsii, Combre-

2 D. C. HARTNETT ET AL.

© 2012 The Authorsdoi:10.1111/j.1442-9993.2012.02422.xAustral Ecology © 2012 Ecological Society of Australia

tum zeyheri, Combretum apiculatum, Ziziphus mucronata andvarious Acacia species such as Acacia erioloba, Acacia fleckiiand Acacia luederitzii. The shrub layer is mainly composedof Dichrostachys cinerea, Grewia flava, Grewia flavescens,Acacia mellifera, Bauhinia macranthera and Ximenia caffra.The grass cover includes Aristida meridionalis, Aristida con-gesta, Eragrostis pallens, Eragrostis superba, Pogonarthria squar-rosa, Heteropogon contortus, Digitaria eriantha and Cymbopogonpospischilii.

The second sample site was located in the 2500 km2

Khutse Game Reserve (KGR), approximately 170 km NWof Molepolole Botswana (23°26′11″S, 24°40′28″E; elevation1070 m, mean annual precipitation = 340 mm). The KGRadjoins the larger Central Kalahari Game Reserve. The veg-etation of KGR is predominantly shrub-savanna, dominatedby T. sericea, P. nelsii and A. erioloba (Bekker & DeWit 1991).The KGR supports a diverse fauna of large herbivores andlarge predators including lion, cheetah, brown hyena andwild dog.

The third sample site was located in the southern KalahariDune Bushveld (KDB) region in the southern Kalagadi Dis-trict (26°45′56″S, 20°38′58″E; elevation 873 m, meanannual precipitation <200 mm) a few km south of the Kala-gadiTransfrontier Park. Among the three study sites, this onenear the southern tip of Botswana is the most arid.This areais characterized by long sand dunes running in parallel rows,with crests rising up to 15–30 m above the dune valleysbetween them. The vegetation consist of scattered trees(Acacia erioloba, A. mellifera, Acacia luderitzii and Boscia albi-trunca) and dwarf shrubs (Rhigozum trichotomum, Lebeckialinearifolia), and the grass cover is dominated by Stipagrostisnamaquensis, Stipagrostis amavelius, Stipagrostis uniplumis,Schmidtia kalahariensis, Ar. meridionalis and Eragrostis leh-manniana (van Rooyen 2001).

Root trait measurements

We sampled a total of 18 species in 2007, 2008 and 2011(Table 1). Due to time and logistic constraints associatedwith each site visit, it was impossible to sample all 18 speciesfor all three sets of traits in all three years. Six species weresampled in multiple years and 12 species were sampled inonly one year (Table 1). All species are C4 caespitose grasses.We classified each species according to its life history andlongevity and its drought tolerance. Life histories vary fromruderal annual grasses adapted to highly disturbed sites tolong-lived climax perennial grasses. Seven of the species areannual or short-lived perennials (lifespan = 1–5 years), andthe remaining eleven are long-lived perennials.

Sampling in each year occurred in late March to mid-April when all of the grasses had reached maturity andwere near the end of their flowering period. At each sampletime, in each site we randomly selected 20 individual plants(genets) of each species. To minimize variation due toedaphic conditions, at each of the three locations studied,all species were sampled at the same local site and on well-drained sandy soils (avoiding pans, disturbed sites, etc.).For each plant, the basal crown and roots were carefullyexcavated to a depth of 15 cm, brushed free of soil and airdried. Samples were then placed in sealed plastic samplebags and transported to Kansas State University (USA) foranalysis.

Root system architecture was examined at KRS andKGR for 13 species in 2007 and seven species in 2008.Precipitation data obtained for the closest meteorologicalstations to the three sample location (Botswana Meteoro-logical Services) showed that total precipitation differedbetween the sampling years but the seasonal distribution ofrainfall was very similar. Precipitation at KRS during the

Table 1. Life history characteristics, drought tolerance and years sampled for savanna grass species in this study

Species Life history Drought tolerance Years sampled

Aristida congesta SP DT 2007a, 2008a, 2011aAristida diffusa LP na 2007aAristida junciformis LP NT 2007bAristida meridionalis LP DT 2008aAristida stipitata A/SP DT 2007aBrachiaria nigropedata LP DT 2007aCymbopogon popspichilli LP NT 2007a, 2008a, 2011aDigitaria eriantha LP NT 2007ab, 2008a, 2011aEragrostis cylindriflora A na 2007aEragrostis lehmanniana LP DT 2011aEragrostis rigidior SP DT 2007aHyperthelia dissoluta LP NT 2007a, 2008a, 2011aPennisetum macrourum LP NT 2007aPerotis patens A/SP DT 2007a, 2008aPogonarthria squarrosa SP na 2007a, 2008aSchmidtia kalahariensis A DT 2011cStipagrostis namaquensis LP DT 2011cStipagrostis uniplumis LP DT 2011a

A, annual; a, Khama Rhino Sanctuary; b, Khutse Game Reserve; c, Kalahari Dune Bushveld; DT, drought tolerant; LP,long-lived perennial (>5 years lifespan); na, not available; NT, non-tolerant (after van Oudtshoorn 1999); SP, short-livedperennial (2–5 years lifespan).

GRASS ROOT SYSTEM TRAITS AND DROUGHT TOLERANCE 3


2006–2007 growing season (7 months prior to 2007 sam-pling) was 386.7 mm which was 10% below the meanannual precipitation of 425 mm, whereas total precipitationduring the 2007–2008 season was 473.3 mm, 28% higherthan 2006–2007 and 11% higher than mean annual pre-cipitation. Four primary roots from each plant were analy-sed for physical root length, specific root length (metre rootlength/gram dry weight), and number of secondary and ter-tiary roots on each primary root. Each primary root wasstained in 1% Phloxin B for 2 min to improve resolution offine tertiary roots, then rinsed in water and spread on aplastic-coated grid and positioned under a plexiglass plate(Hetrick et al. 1991). The length of the primary root wasdetermined to the nearest mm. Specific length of the rootsystem was determined using the grid-line intersect methodof Tenant (1975). The number of intersections betweenroots and the grid lines, and the numbers of secondary andtertiary roots per primary root were counted (Rajapakse &Miller 1994). A root fibrousness index (RFI) was calculatedfollowing the methods of Hartnett et al. (2004) using theformula:

RFI

secondary tertiary branchescentimeter primary root=

+( )[] ×× specific root length

primary root diameter

The RFI is a measure directly related to the total length andsurface area of the grass root system.

Mycorrhizal colonization was measured for 13 species in2007 and seven species in 2008 at KRS and KGR. In addi-tion, two species at KDB were sampled in 2011. Roots werestained with trypan blue using the method of Koske andGemma (1989) to determine percent of root length colo-nized by AMF. Roots were scored for percentage colonizationusing the magnified gridline intersect method developed byMcGonigle et al. (1990).

Rhizosheath thickness was measured for seven species in2008 and six species in 2011 at KRS. Unlike 2008, whichhad above-average precipitation, 2011 was a very dry year.Precipitation at KRS in 2011 was 320 mm, 32% less than2008 and 25% less than the mean annual precipitation of425 mm. Eight primary roots were selected from each of thereplicate plants for determination of root diameter (cortexand stele), rhizosheath diameter, and rhizosheath thickness.Rhizosheath thickness was measured to the nearest 0.001 mmusing a Hirox KH-7700 digital microscope at 200 ¥magnification. Four random sites on each primary root weremeasured to obtain average root diameters. For measure-ment of the root diameter, the rhizosheath was broken awayfrom the cortex with tweezers.

Statistical analyses

The mean of each response variable (mycorrhizal coloniza-tion, RFI and rhizosheath thickness) was calculated usingwhole plants (genets) as replicates.The eighteen grass specieswere categorized as drought-tolerant or non-tolerant basedon information provided by Müller (1984), Gibbs Russellet al. (1990) and van Oudtshoorn (1999). The analysispresented was based principally on the classification ofvan Oudtshoorn (1999), although using species drought-tolerance information from the other sources did not changethe results.

A two-way treatment structure with year and species asfactors in a completely randomized design was used toanalyse RFI (PROC MIXED, SAS, 2008). Using the abso-lute value of the residuals of each combination of year andspecies for RFI, 14 groups of the year by species combina-tions with significantly different variances were identified.Denominator degrees of freedom were calculated usingKenward-Rogers approximation. A two-way treatmentstructure with year and species as factors in a completelyrandomized design with homogeneous variances was usedto analyse % mycorrhizal colonization for 2007 and 2008data (PROC GLM, SAS, 2008). Due to the missing treat-ment combinations, alternate main effects and interactioncontrasts according to Milliken and Johnson (2009) wereconsidered for both RFI and % mycorrhizal colonization.Only species collected in both 2007 and 2008 were used inthe contrast to test the year alternate main effect. Allspecies collected in 2007 and 2008 were included in thetest of species alternate main effect. In this contrast, specieswith more than one year of data were averaged across years.The year by species alternate interaction was tested usingthe six species that were collected in both years. A one-waytreatment structure with species as the factor in a com-pletely randomized design analysed % mycorrhizal coloni-zation of the two Kalahari grass species collected in 2011(Proc Mixed, SAS, 2008). A two-way treatment structurewith year and species as factors in a completely randomizeddesign with heterogeneous variances was used to analysesheath thickness (PROC MIXED, SAS, 2008). Five groupsof year by species combinations with significantly differentvariances were identified. The Kenward-Rogers approxima-tion was used to calculate the denominator degrees offreedom. Alternate main and interaction effects similar tothose used for RFI and % mycorrhizal colonization wereused to analyse sheath thickness.

Additional contrasts examining drought tolerance, and dif-ferences between years for individual species were includedin the analyses of RFI, % mycorrhizal colonization, andrhizosheath thickness and comparison-wide error rate wascontrolled using Fisher’s Least Significant Difference. Pat-terns associated with phylogeny were examined by additionalsimilar contrasts among grass sub-families, but an insuffi-cient number of species were studied to examine patterns atfiner taxonomic levels (e.g. among tribes or sub-tribes).

Using the variables of mean RFI, % mycorrhizal coloniza-tion, and rhizosheath thickness of the seven species col-lected in 2008, a negative monotonic association betweenRFI and % mycorrhizal colonization, a positive monotonicassociation between RFI and rhizosheath thickness, anda general monotonic association between % mycorrhizalcolonization and sheath thickness were evaluated withKendall’s Tau-b using exact permutation tests (Proc Freq,SAS, 2008).

RESULTS

Root system architecture, as measured by mean RFI,ranged from approximately 1.0 to >30.0 amongspecies. RFI varied more than 10 fold among allspecies sampled (F13,88.7 = 66.4, P < 0.0001) and



among the grass species sampled only in 2007(F12,69.3 = 49.6, P < 0.0001, Fig. 1). Mean primary rootdiameter also differed significantly among speciesbut showed considerably less inter-specific variation,ranging from 1.05 mm to 1.99 mm (F6,19 = 40.3,P < 0.0001, data not shown). Several species, includ-ing Aristida diffusa, Aristida junciformis, Brachiarianigropedata, Hyperthelia dissoluta and Pennisetum mac-rourum had relatively coarse, unbranched root systemswith mean RFI values �2.0. By contrast, Perotis patensand Po. squarrosa had very fine root systems, withmean RFI values of 33.0 and 25.0 respectively. Theremaining species showed intermediate RFI values,varying from 5.0 to 15.0 (Fig. 1). Those grass speciesclassified as drought tolerant (Table 1) had signifi-cantly greater RFI than drought-intolerant species(Table 2).

Root system architecture also differed significantlybetween years (F1,55.8 = 11.1, P = 0.0016), and anova

showed a significant species ¥ year interaction forRFI (F5,58.8 = 12.5, P < 0.0001). Among the six speciesmeasured in both 2007 and 2008, three species

(Ar. congesta, Pe. patens and D. eriantha) showed sig-nificantly higher RFI (P = 0.04) in the dry year (2007)than in a wet year (2008) while one species(H. dissoluta) had significantly lower RFI in 2007 thanin 2008 (P < 0.0001).

Mycorrhizal colonization of roots ranged from<30% to >80% and also varied significantly among allgrass species (F13,298 = 92.2, P < 0.0001) and amongspecies only sampled in 2007 (F12,289 = 43.0,P < 0.0001, Fig. 2). The two species sampled onlyfrom the most arid site (KDB) also differed signifi-cantly in mycorrhizal colonization, S. namaquensisshowing more than double the colonization levelsthan Sc. kalahariensis (F1,37 = 395.1, P < 0.0001,Fig. 2).

There was a consistent pattern among grass speciesof different life history. In the 2007 sampling, all of theannual or short-lived perennial species (Ar. congesta,Aristida stipitata, Eragrostis cylindriflora, Eragrostisrigidior, Pe. patens and Po. squarrosa) showed averageroot colonization levels of <44%, whereas all of thelong-lived perennial species had colonization levels of>47%. Mycorrhizal colonization was 13% lower inspecies classified as drought tolerant than non-droughttolerant species (Table 2).

Mycorrhizal colonization levels also differedbetween years (F1,298 = 407.0, P < 0.0001). All of thesix species measured in both 2007 and 2008 showedsignificantly higher colonization in the wet year (2008)than in the dry year (2007) (t298 � 4.1, P < 0.0001 forall six contrasts).

All species sampled in 2008 and 2011 possessedrhizosheaths but they varied by an order of magnitudein mean rhizosheath thickness, ranging from 75 mm to820 mm (Fig. 3). The rhizosheaths appeared to becomposed of sand particles and both root hairsand fungal hyphae, but the specific composition ofthe sheaths is unknown and is currently underinvestigation. Rhizosheath thickness varied signifi-cantly among all grass species (F10,144 = 76.1,P < 0.0001, Fig. 3) and among species sampled onlyin 2008 (F6,109 = 50.7, P < 0.0001). Rhizosheath thick-ness was significantly larger in drought-tolerantspecies than drought-intolerant species (Table 2). In2011, the perennial species from the most arid site(S. namaquensis) had the largest rhizosheaths of almost

Fig. 1. Root fibrous index (RFI) (see text for description)of grasses of semi-arid Kalahari savanna. Error barsare �1 SE. Open circles = April 2008 sampling; closedcircles = April 2007 sampling.

Table 2. Comparison of means of root system traits between drought-tolerant and non drought-tolerant grass species

Rhizosheath thickness (mm) Mycorrhizal colonization (%) RFI

Non drought-tolerant grasses 279.8 62.1 3.97Drought-tolerant grasses 463.2 49.2 9.82F-statistic F1,138 = 113.3 F1,298 = 160.8 F1,25.1 = 72.1Probability <0.0001 <0.0001 <0.0001

RFI, root fibrousness index.



3 mm in diameter, whereas the annual species fromboth KDB and KRS had among the thinnestrhizosheaths. The only exception was the perennialD. eriantha, which in both years showed the thinnestrhizosheaths among all species (Fig. 3).

Rhizosheath thickness was significantly differentbetween years (F1,84.9 = 160.8, P < 0.0001). All fourspecies that were sampled at KRS in both 2008 and2011 (Ar. congesta, D. eriantha, H. dissoluta, and Cym-bopogon pospischilli) produced significantly thicker

rhizosheaths in the wet year (2008) than in the dry year(2011) (P < 0.005). However, their respective differ-ences between years were not similar as indicatedby the significant year by species interaction(F3,86.8 = 16.3, P < 0.0001).

Although it was not a primary objective of our study,an additional analysis revealed an association betweenroot system traits and the phylogenetic relationships ofthe grasses studied. All three root system traits exam-ined differed significantly among grass sub-families(Table 3). In particular, grasses in the sub-familyChloridoideae showed the highest RFI (approximatelythree times higher than other species) and the lowestmycorrhizal colonization, whereas grasses in the sub-family Panicoideae showed the lowest RFI and highestmycorrhizal colonization (Table 3). Grasses of thesub-family Arundinoideae were intermediate for bothRFI and mycorrhizal colonization, but they showedthe highest rhizosheath development of all species(Table 3).

Among grass species, there was a significant negativeassociation between mean RFI and mean mycorrhizalcolonization, but no significant positive associationbetween RFI and rhizosheath thickness nor a signifi-cant association between mycorrhizal colonization andrhizosheath thickness (Table 4).

DISCUSSION

Mycorrhizal symbiosis is widespread among grassesand grasslands (Allen 1996; Hartnett & Wilson2002). Root colonization by AMF results in numer-ous potential benefits to the host plant, includingincreased acquisition of mineral nutrients, enhancedphotosynthesis and growth, improved plant waterrelations, and enhanced resistance to pathogens.Recent evidence suggests that AMF also play keyroles in ecosystem processes such as enhancedcarbon sequestration and improved soil aggregatestabilization and reduction in soil erosion (Wilsonet al. 2009). In the present study, all grass speciesexamined were highly colonized by AMF, with colo-nization levels varying among grass species, fromapproximately 30% to 80% of root length colonized.These AMF colonization levels are similar to those ofother semi-arid and relatively nutrient poor savannas(e.g. O’Connor et al. 2002; Hartnett et al. 2004).However, in a similar study, Wilson and Hartnett(1998) sampled mature grasses in a more productivemesic temperate grassland of similar low fertility andfound notably lower AMF colonization levels. Thesedifferences suggest that mycorrhizal symbiosis is par-ticularly important to plant and ecosystem function-ing in arid and semi-arid grasslands where light isusually abundant but soil resources are strongly lim-

Fig. 2. Percent mycorrhizal colonization of roots of semi-arid Kalahari savanna grasses. Error bars are �1 SE. Closedcircles = April 2007 sampling; open circles = April 2008sampling; triangles = April 2011 sampling.

Fig. 3. Rhizosheath thickness of semi-arid Kalaharisavanna grasses. Error bars are �1 SE. Closed circles = April2008 sampling; open circles = April 2011 sampling.



iting. In these ecosystems the symbiosis providessignificant benefits in terms of enhanced acquisitionof limiting soil resources, while the high light andrates of photosynthesis enable grasses to allocatesignificant carbohydrates to support their fungalsymbionts.

Among the species studied, annual or short-livedperennial grasses showed lower AMF colonizationthan long-lived perennial species. This is consistentwith a general trend noted in other ecosystems oflower colonization and mycorrhizal dependencyamong annual and ruderal plant species adapted tohighly disturbed sites (Miller 1987; Trappe 1987;Wilson & Hartnett 1998). Because highly disturbedsoils often lack mycorrhizal hyphal networks orhave limited propagule (spore) densities, theestablishment success of ruderal species is likelyenhanced if they have little or no dependency onmycorrhizal symbiosis. Mycorrhizal colonizationwas also lower among highly drought-tolerantgrass species than in drought-intolerant species. Thisis consistent with a pattern noted in experimentalburn plots in savannas at Kruger National Parkwhere mycorrhizal colonization was significantlylower in grass populations in more water-limitedannually burned plots than in infrequently burnedplots where water was less limiting (Hartnett et al.2004).

Root system architecture, as measured by RFI,also varied significantly among grass species, butthe patterns were exactly the opposite of thoseobserved for mycorrhizal colonization. The mostdrought-tolerant grass species showed much greaterroot branching and fine root development than theless drought-tolerant species, and there was anegative association between RFI and mycorrhizalcolonization among species. This supports thegeneral hypothesis that there is a trade-off betweencarbon allocation to root growth and allocation tosymbiotic fungi and that the development of anextensive fine root system and support of mycorrhizalsymbiosis represent alternative strategies for theacquisition of limiting soil resources in grasses(Hetrick et al. 1990; Wilson & Hartnett 1998). Thedifferent patterns observed in drought-tolerant versusdrought-intolerant grass species indicate that, understrongly water-limited conditions the benefitsof fine root development are higher relative to theircarbon costs than are the benefits of mycorrhizalsymbiosis. These observed patterns lead to ourgeneral prediction that mycorrhizal symbiosis is rela-tively more important to grasses of infertile savannas,whereas high RFI is more important in fertile savan-nas were water is more strongly limiting relative tonutrients.

Of the three traits examined in this study, least isknown about the patterns, composition, and ecologi-cal role of rhizosheaths. Rhizosheaths are cylindricalcoatings of grass roots composed of a complex ofroot hairs, sand particles, and possibly fungal hyphaeand/or bacteria, all cemented together by mucilageand perhaps other products exuded from the roots orhyphae. Both AMF and non-AMF hyphae have beenobserved in some rhizosheaths, but the relative abun-dance and roles of root hairs and different fungalcomponents in the enmeshment of sand particles inrhizosheaths is not clear (Goodchild & Myers 1987;Degens et al. 1996; Othman et al. 2004; Moreno-Espindola et al. 2007). Several studies have suggestedthat rhizosheaths play an important role in increasingnutrient and/or water acquisition by grasses, and the

Table 3. Comparison of means of root system traits among grass sub-families

Rhizosheath thickness (mm) Mycorrhizal colonization (%) RFI

Arundinoideae 528.6C 52.0B 5.57B

Panicoideae 279.8A 65.0C 3.96A

Chloridoideae 469.4B 38.0A 15.1C

F-statistic F2,121 = 89.76 F2,298 = 275.88 F2,26.4 = 98.14Probability <0.0001 <0.0001 <0.001

For each trait, different superscript letters indicate significant differences between sub-families at the P < 0.05 level. RFI, rootfibrousness index.

Table 4. Kendall’s Tau-b correlations between root systemtraits among semi-arid savanna grass species

tb P

Root fibrousness index vs. mycorrhizalcolonization

-0.714 0.01

Root fibrousness index vs. rhizosheaththickness

0.048 ns

Rhizosheath thickness vs. mycorrhizalcolonization

0.333 ns

Probability values of the exact test for Kendall’s Tau-bwere obtained in sas using the Jonckheere-Terpstra test. ns,not significant.



largest and most coherent rhizosheaths are typicallyassociated with grasses in sandy soils and underdry conditions (Watt et al. 1994). Plants withrhizosheaths have a greatly increased root surface incontact with a considerable volume of soil, facilitat-ing water and ion uptake in dry soils. Plants withlarger rhizosheaths have been shown to achievegreater total P uptake (Delhaize et al. 2009). Somerhizosheaths harbour a higher density of bacteriathan the surrounding soil and may also facilitatenitrogen fixation (Wullstein 1980; Buckley 1982;Othman et al. 2004; Bergmann et al. 2009).Rhizosheaths may also play an important role indrought-tolerance of grasses in arid environments.They may facilitate water movement across the root-soil interface by eliminating or greatly reducing theroot–soil air gaps (North & Nobel 1997). In addi-tion, rhizosheath soil can be significantly wetter thanbulk soil, and it has been suggested that exudateswithin the rhizosheath may increase the waterholding capacity of sandy soil (Young 1995). Thisenhancement of water retention and uptake maybe a crucial factor maintaining root function and lim-iting root mortality in grasses coping with droughtconditions.

In our study, there was considerable interspecificvariation in rhizosheath size among Kalahari grassspecies, similar to patterns observed among SouthAfrican grasses by Bailey and Scholes (1997). Ourobservation that rhizosheath thickness was signifi-cantly greater for drought-tolerant species than lesstolerant species provides further support for thehypothesis that the primary ecological function ofrhizosheaths in these semi-arid ecosystems isimproved plant water relations. Thus, rhizosheathsmay play an increasingly important role in grasslandecosystem function under climate change, particu-larly under predicted increasing intensity and/orduration of droughts. The increased root hairdevelopment and mucilage production and exu-dation that will be required to develop largerrhizosheaths will represent an increased carbon costto the grass plant. However, increasing drought con-ditions will only increase the limitation of soilresources relative to light limitation for photosynthe-sis in savannas and grasslands, increasing the benefitof rhizosheath development relative to its carboncosts.

The interspecific differences in these root systemtraits (RFI, rhizosheath development and mycorrhizalcolonization) and the clear differences in traitsamong grass sub-families indicate that these traits areat least partially under phylogenetic control. Furtherstudy of additional species representing differenttribes and sub-tribes will further elucidate these pat-terns. The grass sub-family Chloridoideae is generallyconcentrated in more arid habitats in the tropics

relative to other sub-families. The high RFI andlower mycorrhizal colonization among grasses inthe Chloridoideae in this study (as well as the highRFI and low mycorrhizal colonization amongdrought-tolerant species) suggests that RFI is moreimportant to drought tolerance than is mycorrhizalsymbiosis.

Significant inter-annual variation in these traitsindicates that they are also partially under environ-mental control. Both mycorrhizal colonization andrhizosheath development varied between years withinspecies. Whereas our comparisons among speciesindicated that more drought-tolerant grasses havelarger rhizosheaths, comparisons within speciesbetween years showed greater rhizosheath develop-ment and higher mycorrhizal colonization during awet year (2008) than during a dry year (2007 or2011). Thus, these two traits show high plasticityand can be readily adjusted from year to yeardepending upon moisture availability. Greaterrhizosheath development during a favourable grow-ing season is expected because root exudate produc-tion, growth of root hairs and hyphal growth are alldependent on the amount of plant carbon gainduring its growth season. Greater rhizosheath devel-opment during a favourable growing season thenlikely increases grass survivorship during the subse-quent dry season or a subsequent dry year. This plas-ticity may also be important for savanna grassescoping with increasing precipitation variability asso-ciated with climate change, as it allows them to capi-talize on periods of high water availability anddevelop root system traits (e.g. thick rhizosheaths andmycorrhizal fungal growth) that will enhance subse-quent survivorship during periods of low resourceavailability.

Overall, the higher RFI and rhizosheath size butlower mycorrhizal colonization among drought-tolerant grasses and the significant negative correla-tion between RFI and mycorrhizal colonizationamong species together support the hypothesis thatthe relative importance of these different root systemtraits varies spatially and temporally with shifts in therelative limitation of different plant resources. Thedevelopment of a highly branched fine root systemand the development of rhizosheaths may be impor-tant complementary traits of grasses coping withdrought conditions, the former aiding in the acquisi-tion of limiting soil water by the grass plant and thelatter aiding in water retention and reducing the rateof water loss in the rhizosphere. The fact that grassspecies with high RFI and larger rhizosheaths alsoshowed lower mycorrhizal colonization, and thatmycorrhizal colonization was low in drought-tolerantgrasses, together support the general hypothesis thatthe primary benefit of arbuscular mycorrhizas sym-biosis in these semi-arid savannas is improved



nutrient acquisition by the host plant rather thanimproved water status.

ACKNOWLEDGEMENTS

We thank Mbaki Muzila of the University of BotswanaHerbarium for assistance with grass identificationsand Beth Gastineau and Ben VanderWeide for assis-tance with the fieldwork. This research was partiallysupported by the National Science FoundationLong-Term Ecological Research Program (USA) andthe Institute for Grassland Studies, Kansas StateUniversity.

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