Phosphorus-Acquisition and Phosphorus-Conservation Mechanisms of Plants Native to South-Western Australia Or to Brazilian Rupestrian Fields

Phosphorus-acquisition and phosphorus-conservation mechanisms of plants native to south-western Australia or to Brazilian rupestrian fields

Mariana Cruz Rodrigues de Campos MSc in Plant Biology

This thesis is presented for the degree of Doctor of Philosophy of Plant Biology of The University of Western Australia

The University of Western Australia Faculty of Natural and Agricultural Sciences School of Plant Biology 2011 2 ACKNOWLEDGEMENTS

I would like to thank my supervisors; Dr Hans Lambers, Dr Rafael Silva Oliveira and Dr Stuart James Pearse for all the advice, support and encouragement during my PhD.

The suggestions made by the thesis examiners, Dr Michelle Watt and Dr Rebecca Ostertag were extremely valuable and much appreciated. For helping me with key concepts and methodologies, I thank Dr Erik Veneklaas, Dr Mark Brundrett, Dr Michael Shane and Tamara Edmonds-Tibbett. I’d also like to acknowledge all administrative and support staff in the School of Plant Biology for the everyday work of keeping things running so I’d be allowed to focus on my thesis.

During my field work in Brazil, the supervision of Dr Rafael S. Oliveira allowed for the work to be much more efficient and targeted. His help and that of (unpaid) friends were absolutely essential and I truly appreciate and hope one day to repay Ana Luíza Muler, Maria Cecília Alvim Penteado, Hugo Galvão and Caio Guilherme Pereira.

I’d also like to thank the taxonomists who helped with the identification of the material collected in Brazil: Juliana Souza Silva (for the identification of Fabaceae), Marcelo Trovó (Eriocaulaceae), Maria das Graças Lapa Wanderley (Xyridaceae) and Dr Renato de Mello-Silva (Velloziaceae).

As my work progressed, I always had somebody giving me a good suggestion or helping me find solutions to my problems. The list of people who contributed in this way is much too long, but a special mention goes to the Plant Biology tea club members and to the glasshouse staff.

I extend my appreciation to the funding bodies that have financed me in my PhD research. Thanks to the University of Western Australia and the School of Plant Biology, I received a scholarship. The equipment, materials, travelling and attendance to conferences were made possible with the funding from Plant Biology Research Grant (2008-2011); ANZ Holsworth Wildlife Foundation Grant (2008, 2009, 2010); Brazilian National Research Council, CNPq (2008) and Mary Janet Lindsay of Yanchep Memorial Fund (2010).

My appreciation is extended to the directors and administrative staff of the field sites in which I worked in, both in Australia (Bold Park, Botanic Gardens & Parks Authority) and in Brazil (Santuário do Caraça, Propriedade Particular da Província Brasileira da

3 Congregação da Missão; Parque Nacional da Serra do Cipó; Parque Estadual do Rio Preto; and Parque Estadual da Serra do Cabral).

Finally, I thank my parents, family and friends in Brazil for supporting me in the decision of moving abroad; and for their continuous presence in my life, giving me comfort and love. Thanks also to those friends in Australia who have made me feel so welcome and happy and above all, to the never-ending support and encouragement of my very patient partner Troy.

4 5 SUMMARY

Phosphorus (P) is an essential macronutrient for life and it has been widely recognised as one of the most limiting nutrients for plant growth. The vast majority of P in the soil originates from the parent rock, and over prolonged periods of time, it is lost from the system due to weathering and leaching. This is the reason why P is particularly limiting in old soils, such as in the kwongan in the south-west of Australia; the fynbos in the Cape floristic region in South Africa, and the rupestrian fields in central Brazil. The study of P acquisition by native plants is of great importance, because these plants have evolved mechanisms and strategies to thrive under low availability of P; conditions in which crop species would not survive. Studying and understanding P-acquisition and P- conservation traits of native plants in their P-impoverished environments, is taking a step towards decreasing fertiliser use in crops and minimising its detrimental environmental consequences.

This thesis aims to deepen the understanding of P-acquisition and P-conservation mechanisms by addressing three aspects that were, until now, gaps in ecophysiology (chapter 1).

Through field work (chapter 2), I have compared and contrasted already well known P- impoverished environments to a much less studied and explored area: the rupestrian fields in central Brazil. Leaf nutrient resorption and root specialisations were the focus of the field work at nine sites in Brazil. I discovered that the species that occupy the rupestrian fields are taxonomically very distinct from those in the south-west of Australia, but that there is a functional convergence. I discuss the root specialisations found (root clusters and cluster-like, sand-binding roots, mycorrhizal associations and dense root hairs) and their relationship to soil fertility and the varying degrees of P resorption from senescing leaves.

Apart from this exploratory field work, I have also investigated how two different but co-occurring specialisations towards enhanced P-acquisition (cluster roots and mycorrhizal associations) are related to each other, to P supply and to the internal P concentration of an Australian native legume, Viminaria juncea (chapter 3). This waterlogging-tolerant species was found to be unique in that it maintains stable shoot P concentrations independent of P supply. As a consequence of the steady shoot P concentration, the suppression of mycorrhizal colonisation and cluster root formation was only marginal. 6 In addition, I have experimentally tested whether net P-uptake capacity is correlated with leaf P-resorption efficiency of species native to the Perth region, south-western Australia (chapter 4). My results indicate there is an inverse relationship between the capacity to down-regulate net P uptake and the P-resorption efficiency for Acacia truncata and A. xanthina (Fabaceae), as well as for Banksia attenuata, B. menziesii and Hakea prostrata (Proteaceae). Both Acacia species were able to down-regulate P-uptake capacity, but less efficient at remobilising P from senescing leaves. The three Proteaceae species were unable to down-regulate P-uptake capacity which led to P- toxicity symptoms, but on the other hand, they are extremely efficient and proficient at P-resorption.

P-impoverished environments are associated with a very large diversity of species and an equally large array of mechanisms to enhance P-acquisition and P-conservation. Although this was already known, the relationships between these traits and the variation across different locations have not been further explored.

The main conclusions of this thesis (chapter 5) include the discovery of specialised root structures in Brazilian rupestrian field specimens, including cluster roots in Proteaceae, cluster-like roots in Cactaceae and Cyperaceae, and sand-binding roots in Eriocaulaceae. The field work in Brazil also allowed us to discover a new species of Xyridaceae, and to conclude that the rupestrian fields are functionally equivalent to the fymbos and kwongan in terms of phosphorus nutrition. It made apparent how much there is yet to be learned from the vulnerable and diverse ecosystem that is the rupestrian fields. From one glasshouse experiment, I was also able to conclude that cluster root formation was positively correlated to mycorrhizal colonization in Viminaria juncea, which is a species that was able to maintain stable shoot phosphorus concentration across a wide range of treatments (0-50 mg P kg-1 dry soil). Probably due to the internal regulation of P, V. juncea did not supress the mycorrhizal colonization or the cluster root formation even at the highest treatments. An additional glasshouse experiment with four Australian native species provided us with an inverse correlation between P-uptake down-regulation and P resorption from senescing leaves. This means that the species which were able to remobilise P from senescing leaves more efficiently were less able to down-regulate their P-uptake, and ultimately suffered from P toxicity.

I believe that a broader, more generalised understanding of plant nutrition arises from studies where the interactions between characteristics are explored, and where different areas or different species are studied comparatively; this is what I aimed to achieve in

7 this thesis. The work I present in this thesis is important because it inter-connects knowledge of plant ecophysiology while adding new information and discussion; it has the practical application of being useful in the conservation and revegetation of the kwongan, fynbos and rupestrian fields, which are biodiversity hotspots; and finally, it adds to the early stages of the path of crop species manipulation for increased P-uptake and P-conservation which could result in less fertiliser use and decreased environmental damages from nutrient leaching into water bodies.

8 Statement of original contribution

The research presented in this thesis is an original contribution to the field of Plant Ecophysiology. The hypotheses and experiments presented and discussed in this thesis are my own original ideas and writing.

People who have made important contributions to this research in addition to those acknowledged in chapters 2, 3 and 4 are:

Hans Lambers, Stuart James Pearse and Rafael Silva Oliveira, who were the supervisors of my doctorate and who have guided me through the processes of forming hypotheses, designing experiments and writing the manuscripts.

Ricardo Brancalion, who provided technical support with ICP-OES analysis of leaf material included in chapter 2.

Michael Smirk, who provided technical support with ICP-OES analysis of leaf material included in chapters 3 and 4.

This thesis has been completed during the course of enrolment in a PhD degree at the University of Western Australia, and has not been previously used for a degree or diploma at any other institution.

Mariana Cruz Rodrigues de Campos

12th September 2011

9 10 INDEX

Acknowledgements...... 3

Summary...... 5

Statement of original contribution...... 9

Acronyms and equations used in this thesis...... 13

Chapter 1: General introduction and thesis outline

Phosphorus acquisition by roots and phosphorus conservation through efficient resorption from senescing leaves in plants native to south-western Australia or to Brazilian rupestrian fields...... 15

Chapter 2:

Phosphorus nutrition of Brazilian rupestrian field plant species in their old, nutrient- impoverished landscapes...... 39

Chapter 3:

Viminaria juncea (Schrad. & J.C. Wendl.) Hoffmanns does not vary its shoot phosphorus concentration and only marginally decreases its mycorrhizal colonisation and cluster-root dry weight under a wide range of phosphorus supplies...... 81

Chapter 4:

Down-regulation of net phosphorus uptake capacity is inversely related to leaf phosphorus resorption in four species from a phosphorus impoverished environment...... 101

Chapter 5: General discussion and conclusions

Linking the phosphorus acquisition and phosphorus conservation aspects studied in this thesis and adding Brazilian rupestrian fields to the group of worldwide phosphorus impoverished environments...... 123

11 12 Acronyms and equations used in this thesis

AM Arbuscular mycorrhizas

DL Detection limit

DW Dry weight

FW Fresh weight

LMA Leaf mass per area (g mg-1)

OM Organic matter

P Phosphorus (all other elements also follow periodic chart abbreviations)

Pi Inorganic phosphorus

Po Organic phosphorus

RWR Root weight ratio v/v volume/volume w/v weight/volume

[ ] Concentration (of element inside brackets)

Resorption equations

P resorption efficiency = ([P] in mature leaf – [P] in senesced leaf) / ([P] in mature leaf)

P resorption proficiency = ([P] in senesced leaf)

13 14 Chapter 1

General Introduction

Phosphorus acquisition by roots and phosphorus conservation through efficient resorption from senescing leaves in plants native to south-western Australia or to Brazilian rupestrian fields

The focus of this research is on specialisations that have evolved in plants naturally occurring environments impoverished of phosphorus (P), in particular root clusters, mycorrhizal associations and P conservation through resorption from senescing leaves. This is a particularly important topic because phosphorus is an essential nutrient for life and one that is commonly limiting for plant growth in natural and agricultural systems.

P-impoverished environments

Parental rocks that give origin to the soils are the main source of its P and, generally, very little P arrives via atmospheric deposition (Walker and Syers, 1976, Holford, 1997), e.g. dust (Okin et al., 2004, Tamatamah et al., 2005) or ash (Newman, 1995, Mahowald et al., 2005). Once a landscape is formed, and no geological events or major atmospheric deposition occur, the quantity of P in the soil declines over time, as has been observed in chronosequences in the United States of America (Lajtha and Schlesinger, 1988, Crews et al., 1995) and in New Zealand (Walker and Syers, 1976, Richardson et al., 2004).

An important factor that needs to be considered when studying P is that this nutrient is highly immobile in soil due to very little mass flow and slow diffusion (Lambers et al., 2006). Furthermore, P can be strongly sorbed to soil particles (Holford, 1997) which makes it hardly accessible to roots. Apart from the restricted quantity of phosphorus in solution, there is another challenge: plants are only able to take up inorganic phosphorus out of the many forms in which P can be found (Holford, 1997). This means that plants can only access a very small proportion of the P present in the soil.

A critical aspect of the Phosphorus cycle is fire. In a comprehensive review, Certini (2005) specifies that fire converts the organic form of P into the orthophosphate form, which has increased availability to plants, but may be easily bound to Al, Fe or Mn oxides, and that some P is lost through volatilisation, but not as substantially as is the

15 case with N. These are only some aspects in which fire influence the ecosystem. Some other very important aspects are the changes in pH after burning, influence on soil biota (Certini, 2005), and the overall influence on the individual and the species’ survival, seed germination or plant resprout, and therefore, on the overall evolution of any ecosystem.

In order to measure readily available P in the soil, a number of methods have been developed, such as the resin-extractable method, the Colwell method, the Bray-I and II methods, the Olsen method (Amer et al., 1955, Colwell, 1963, Bray and Kurtz, 1945, Olsen et al., 1954). The applicability of such methodologies depends on soil characteristics such as texture, mineralogy and pH (Holford, 1997, Mallarino and Atia, 2005). These tests provide only an indication of the readily available P to plants, as species native to regions where soil is tested as “deficient” still access soil P and thrive in such conditions (Handreck, 1997) due to specialised modes of P acquisition (detailed in “Root P-uptake” below).

A large number of studies have been performed around the world on nutrient limitation to plant growth focusing on nitrogen as the main limiting factor for plant productivity (as reviewed by Vitousek and Howarth, 1991, LeBauer and Treseder, 2008). More recently, the focus has shifted partly to the P limitation of soils (as reviewed by Vance et al., 2003, Lambers et al., 2008, Vitousek et al., 2010). This P limitation is especially pronounced in specific areas, which are highlighted below:

(a) South-western Australia

The south-west of Australia is a region formed in early-middle Tertiary age (Twidale and Campbell, 1988) that has been climatically buffered since the late Cretaceous (Hopper, 2009). This landscape has undergone intense weathering and leaching (Finkl, 1979, Wardell-Johnson and Horwitz, 1996), and has total levels of P usually below 150 mg P kg-1 soil (Beadle, 1966), often with undisputedly low values of readily available P under 1 mg kg-1 soil (Lambers et al., 2010).

South-western Australia has a very species-rich vegetation, and was classified as a world hotspot of biodiversity (Myers et al., 2000). Sandplain sclerophyllous shrubland vegetation, known locally as kwongan, occurs in south-western Australia (Hnatiuk and Hopkins, 1981). The kwongan flora evolved with phosphorus limitation in the soils and

16 its species present important physiological characteristics that allow them to live with restricted nutrients (Beadle, 1966).

Much literature has been devoted to plant adaptations to low P supply in south-western Australia (e.g. Bolan and Robson, 1987, Bougher et al., 1990, Lamont, 1982, Handreck, 1997, Adams et al., 2002, Shane et al., 2003a, Pearse et al., 2006a, Lambers et al., 2006). The occurrence of specialised modes of P-acquisition towards extremely P- impoverished soils is frequent (Skene, 1998, Brundrett, 2009, Lambers et al., 2010) in this region, particularly in the Proteaceae, Cyperaceae Restionaceae and Fabaceae, which are common taxa in the kwongan (Burbidge, 1960).

(b) Fynbos, South Africa

Fynbos represents a type of vegetation in the Cape floristic region in South Africa that holds several similarities to south-western Australia. As its Australian floristic counterpart, the Cape floristic province is also considered a hotspot of biodiversity (Myers et al., 2000). It originated from Gondwanaland, possesses a Mediterranean climate and is P-impoverished (Witkowski and Mitchell, 1987, Richards et al., 1997, Hopper, 2009).

P levels in the Cape floristic region show an average of 87 mg total P kg-1 soil which is comparable to the low values found in south-western Australia (Stock and Verboom, 2011). In the P-impoverished soils of the fynbos, many Proteaceae can be found, with root systems with specialised structures towards efficient P acquisition (Lamont, 1982, Lamont, 1983, Lambers et al., 2006), as is the case in the kwongan vegetation.

The rupestrian fields are a formation type that belongs to the cerrado biome (Eiten, 1972). They are characterised by the occurrence of rock outcrops (that give name to the formation) and sandy fields with shallow skeletal soils (Furley and Ratter, 1988). They generally originate from quartzites and less commonly granites or sandstones (Alves and Kolbek, 1994, Benites et al., 2007) and are believed to have been formed in the Pre- Cambrian era (Conceição et al., 2007).

The soil fertility in the cerrado has been previously explored in the literature (Goodland and Pollard, 1973, Lopes and Cox, 1977, Goedert, 1983, Furley and Ratter, 1988,

17 Marques et al., 2004), but little information is available for rupestrian fields (Conceição et al., 2007, Negreiros et al., 2008). Likewise, physiological ecology has not been a subject explored in the rupestrian fields – until now, studies of floristics and phytosociology have been the focus of scientific effort in this formation (Andrade et al., 1986, Giulietti et al., 1987, Alves and Kolbek, 1994, Romero and Nakajima, 1999, Amaral et al., 2006, Conceição et al., 2007).

The cerrado as a whole is a hotspot of biodiversity (Myers et al., 2000). High species richness (Andrade et al., 1986, Amaral et al., 2006, Conceição et al., 2007), high levels of endemism (Alves and Kolbek, 1994, Romero and Nakajima, 1999) and a large threat to habitats (Silva et al., 2007, Verola et al., 2007) are also the case for the rupestrian fields in particular. Since this formation has its origins in Gondwanaland and there are preliminary data that indicate that soil P levels are low, the rupestrian fields should be an ideal location to study root specialisations and plant nutritional status, and that can add to the global overview of plant specialisations in P-impoverished landscapes.

(d) Pantepuis, Guyana Shield

The pantepuis, higher regions of the tabletop mountains known as tepuis, are located along the border between Venezuela, Colombia, Guyana, French Guyana, Suriname and Brazil, and they are considered an old, climatically buffered, infertile landscape (Hopper, 2009) dating back to the Precambrian (Gibbs and Barron, 1983, Désamoré et al., 2010). The pantepuis have been recognised as nutrient-impoverished (Rull, 2004); although no data on soil P concentration ([P]) of the table mountains are available in the literature, an account for soils derived from the Guyana Shield in Venezuela showed <0.2 mg bicarbonate-extractable P kg-1 soil (Feeley, 2005). This ecosystem holds an incredibly rich flora, with ca. 15,000 taxa of vascular plants, of which a third are endemic (Berry and Riina, 2005). The most important families in the region are shared with the rupestrian fields in central Brazil (Berry and Riina, 2005, Alves et al., 2007) which could indicate that this is a potential area for the study of plant adaptations to P- impoverished environments.

The pantepuis are an area of pristine nature, mainly due to difficulty of access. The biggest threat to this ecosystem is global warming: if the temperature in the region increases by 2-4oC, between 10 and 30% of the habitats of endemic species will be lost (Rull et al., 2009). Very little ecophysiology has been studied in the pantepuis, and because of its origin, soil properties and biodiversity, there is indication that it would be 18 an area, which would complement the knowledge on P-acquisition and P-conservation mechanisms of plants.

Root P uptake

Inorganic phosphorus (Pi) in solution is taken up by the root via active transport, against an electrochemical potential gradient because the plasmalemma membrane potential is negative and there is a much higher concentration of Pi in the cytosol than in the soil (Schachtman et al., 1998). In conditions of low phosphorus supply, most plants can alter some of its root characteristics, and some species of plants have evolved specialised structures for enhanced P uptake. These characteristics and structures are discussed below:

(a) Transporter affinity expression

Kinetic and molecular data have shown that there are multiple transporters for inorganic P (Pi) of high- or low-affinity (Wykoff et al., 2007). Under P starvation, high-affinity transporters are expressed to a greater extent than in control situations (Burleigh and Harrison, 1999, Ragothama and Karthikeyan, 2005) in several species, including Medigaco trunculata, Arabdopsis thaliana and Glycine max. Increased expression of high-affinity transporters does not increase P uptake, but may improve internal P utilisation (Lambers et al., 2006). Therefore, increased expression plays a beneficial role in P-deficiency situations.

(b) Root architecture

Plants can modify their root architecture to allow for greater access to P in the soil (Ticconi and Abel, 2004). Increased root weight ratio has been observed in Eucalyptus grandis as a response to P limitation (Kirschbaum et al., 1992), and its consequences are the expansion of the root network in relation to aboveground biomass; that is, a change in the ratio between supply and demand for P. Other responses observed in P-limiting conditions are the increase of root length and of specific root length in Eucaliptus diversicolor (Brouwer, 1983), winter barley (Steingrobe et al., 2001), Capsicum annuum, Zea mays and Cucurbita pepo (Schroeder and Janos, 2005). Increased specific root length can have positive effects on P uptake by altering the ratio between root

19 surface and root mass, augmenting the absorptive surface in relation to the root volume. Another possibility of root architecture change in P-limiting situations is the allocation of more roots at the generally P-richer surface of the soil, as is the case of maize (Mollier and Pellerin, 1999) and bean (Lynch and Brown, 2001).

The formation of epidermal root hairs allows for greater surface area for water and nutrient absorption. When P is limiting, however, many plants produce an abundance of root hairs (Foehse and Jungk, 1983, Gahoonia and Nielsen, 1998, Schmidt, 2001) that increase the contact surface between the soil and the roots. The proliferation of hairs as a response to insufficient P has been observed in Arabidopsis (Bates and Lynch, 2001) as well as in crop varieties of wheat and barley (Gahoonia et al., 1997, Gahoonia and Nielsen, 2004) with the consequence of increased growth and nutritional status. High density of root hairs can also be promoted by factors other than low P, and has been observed as a response to increased moisture (Dittmer, 1949) and to fungal infection (Zangaro et al., 2005).

(d) Mycorrhizal associations

The association with mycorrhizal fungi is prevalent for vascular plants, and there are different types of mycorrhizal associations: arbuscular, orchid, ericoid and ectomycorrhizas (Brundrett, 2009). Of these groups, arbuscular mycorrhizas are the most commonly found in Angiosperms (74%), whereas all other groups combined add to 12% occurrence in Angiosperms (Brundrett, 2009). For this reason, our work focuses on arbuscular mycorrhizas.

Arbuscular mycorrhizas (AM) are incapable of growth without plants; so in exchange for carbon and a suitable habitat, they absorb nutrients from the soil and deliver it to the host plant (Brundrett, 2002).

Mycorrhizas can aid in plant nutrition by extending the volume of soil explored and by turning forms of P not accessible to plants into Pi through the release of exudates, such as organic acids and enzymes (Bolan, 1991, Brundrett, 2002, Barroso and Nahas, 2005). As mycorrhizas can utilise P in solution in the soil and they can also access organic P, they have been classified as scavenging structures (Lambers et al., 2008).

20 At very high or very low P supply, mycorrhizal infection could have a small effect, if any, on the P balance of the plants (Koide, 1991), and some results in the literature have indicated that mycorrhizas can be detrimental to the host plant when P supply is adequate (Peng et al., 1993, Siqueira and Saggin-Júnior, 2001, Aristizabal, 2008). Nevertheless, most research has found a positive effect of mycorrhizas on the P uptake of various species (Smith et al., 2011b), some of which are: onion (Mosse, 1973), subterranean clover (Pairunan et al., 1980, Bolan and Robson, 1987), ryegrass (Bolan and Robson, 1987), 14 Rosidae shrubs (Allsopp and Stock, 1995), 28 Brazilian native woody species (Siqueira and Saggin-Júnior, 2001) and Morella cerifera (Aristizabal, 2008).

(e) Exudates

Exudates can include a wide array of chemicals, such as amino acids, organic acids, sugars, vitamins, purines, enzymes and inorganic ions (Dakora and Phillips, 2002). Exudates play an important role in the solubilisation and availability of P to plants, either indirectly (through stimulation of microorganisms) or directly (through the conversion of organic P into inorganic P which the plants can take up) (Dakora and Phillips, 2002).

Phosphatase and phytase activity is increased when soil P levels are low (Dinkelaker and Marschner, 1992, Ozawa et al., 1995, Li et al., 1997) and there is evidence that what does, in fact, control their production and release is the internal plant [P] (Duff et al., 1994, Gilbert et al., 1999, Shane et al., 2003b). That these exudates have a positive effect in the plant P-uptake has been corroborated a number of times by experiments on the soils and exudates themselves (Jones and Brassington, 1998, Gerke et al., 2000), as well as on crop species (Ryan et al., 2001, Veneklaas et al., 2003)..Further research would help to understand the role of these exudates and their possible side effects (Marschner, 1998). Cluster roots can exude substances such as citrate, malate or malonate (collectively called carboxylates) that displace either organic or inorganic P from the soil particles, moving them into solution (Lambers et al., 2006). The exudation of carboxylates by cluster roots occurs in a burst (Shane and Lambers, 2005) and tends to be greater than that of species without root clusters (Grierson, 1992, Roelofs et al., 2001).

Exudates may also lower the pH of the soil where they are released (Haynes, 1990, Jones and Darrah, 1994), and where soil is alkaline, this is advantageous as well, as P 21 availability to the plant may increase (Dinkelaker et al., 1989). The role of exudates in acidic soils has not been largely explored, and it could be the case that the exudation of organic acids could result in toxic effects to the plants (Dakora and Phillips, 2002).

(f) Root clusters and cluster-like structures

Occurrence

Cluster roots, also known as proteoid roots, were described by Purnell (1960) as “dense clusters of rootlets of limited growth” from a study of over 40 Proteaceae species. In the genus Persoonia of Proteaceae no cluster roots are found in Australia (Lamont, 1982). In the Proteaceae of South Africa, all genera form cluster roots (Lamont, 1983). Cluster roots have also been reported in Chilean Proteaceae (Ramirez et al., 1990, Zúñiga-Feest et al., 2010), and in chapter 2 of this thesis (Campos et al., unpubl.), a first record of a cluster root in the Proteaceae Roupala montana in Brazil is presented. Since Purnell coined the term “proteoid roots” in 1960, very similar structures were found in other families, such as Fabaceae (Trinick, 1977, Lamont, 1972c, Gardner and Parbery, 1981), Casuarinaceae (Reddell et al., 1986, Racette et al., 1990, Khan, 1993), Myricaceae (Louis et al., 1990, Crocker and Schwintzer, 1993), Eleagnaceae (Skene et al., 1996) and Betulaceae (Hurd and Schwintzer, 1996). Due to their occurrence outside of the Proteaceae, a new term for such structures was proposed: cluster roots (Lamont, 1982).

Function

Cluster-root formation is triggered by low P supply (Lamont, 1972a, Crocker and Schwintzer, 1993, Crocker and Schwintzer, 1994, Shane and Lambers, 2005), and previous studies indicate that the internal plant [P] is what determines the production of root clusters (Marschner et al., 1987, Louis et al., 1990, Shane et al., 2003a). As much as P is a key nutrient in triggering the production of cluster roots, other factors can also affect cluster root formation (Dinkelaker et al., 1995), in particular iron and nitrogen deficiency (Shane and Lambers, 2005). The more species are studied, the more intricate cluster-root formation response appears to be (Watt and Evans, 1999).

Enhanced P uptake by cluster roots is observed due to a match between their enhanced morphology, anatomy, biochemistry and physiology (Shane and Lambers, 2005). Root clusters greatly increase the effective root surface and the volume of soil that can be explored (Lamont, 2003), and also exude large amounts of carboxylates in a burst,

22 which chemically mobilises nutrients in the soil (Neumann and Martinoia, 2002). Both proliferation of rootlets and exudation of reducing or chelating compounds can be observed independently in several species (Lambers et al., 2006), but the result of the combination of the exudation and the presence of dense clusters of rootlets has been shown to be advantageous (Hocking and Jeffery, 2004).

Other structures

Research with Cyperaceae (Davies et al., 1973, Lamont, 1974, Shane et al., 2005) has provided evidence for structures with similar function to cluster roots, but different appearance – these were named “dauciform” roots, due to their resemblance to a carrot.

In the Restionaceae, specialised structures were classified as “capillaroid” roots due to their thin diameter and capillary action that causes a high level of water retention and is possibly involved with nutrient uptake as well (Lamont, 1982).

The work presented in chapter 2 of this thesis also uses the term “cluster-like” for a species of Cyperaceae and a species of Cactaceae in rupestrian fields in central Brazil. These structures are not clearly structured as the cluster roots of Proteaceae, which were described by Lamont (1972b), but are, nonetheless, dense groupings of fine and short roots in a few regions of the root system. Despite their lack of strictly arranged architecture, these untidy-looking structures had soil particles firmly attached to them, which also implies the exudation of substances, and creates another similarity to cluster roots as defined above.

In two specimens of Syngonanthus niveus, Eriocaulaceae, a new root structure was observed. We called it cotton-like root due to its resemblance to a cotton ball. No exudates were obvious in these samples, and there were no other species with the same structures. Microscope observations indicated that these were roots, but further analyses were not performed, and the reference to this structure is best described by the images documented in Chapter 2 of this thesis.

(g) Sand-binding roots

The sand-binding roots, encountered in the Restionaceae, are roots covered by a sheath of sand agglutinated by an exudate released from the root which wrap each of the older roots in Lyginia barbata (Shane et al., 2009) and various species of Haemodoraceae (Smith et al., 2011a). Sand-binding roots have been demonstrated to be involved in

23 dormancy and water relations, and their role in nutrient acquisition remains to be investigated (Shane et al., 2009, Smith et al., 2011a).

P-conservation mechanisms in plants

In P-impoverished environments it is of the utmost importance that the limited P available gets used in the most efficient way possible, and that the least amount of P is lost from the plant through senescing tissues. Nutrient-conserving species are more likely to dominate nutrient-impoverished habitats (Wright and Westoby, 2003) and several mechanisms are involved in or related to P conservation. When P supply is scarce, plants are expected to exhibit slow growth, more scleromorphic leaves and long leaf longevity (Beadle, 1962, Beadle, 1966, Specht and Rundel, 1990, Lambers and Poorter, 1992, Wright et al., 2002, Wright and Westoby, 2003). Many species from P- impoverished environments also present high photosynthetic P-use efficiency (Wright et al., 2004a). The above mentioned traits increase the duration and efficiency of nutrient use, and are more important to species native to P-impoverished habitats than to fast- growing rates (Reich et al., 1997).

P-resorption from senescing leaves is a key mechanism for the conservation of this nutrient (Reich et al., 1995, Aerts, 1996, Wright and Westoby, 2003), as the plant avoids P losses to litter through resorption. Many studies have built knowledge towards finding the patterns of P resorption (Escudero et al., 1992, Aerts, 1996, Killingbeck, 1996, Reich et al., 1995, Wright and Westoby, 2003, Kobe et al., 2005, Freschet et al., 2010), but the data are still scarce, particularly for the Southern Hemisphere. There are two aspects of P-resorption: resorption efficiency (the percentage of nutrients, which is resorbed from senescing leaves related to mature green leaf [P]); and resorption proficiency (how much phosphorus is left in the senescing leaves). Both aspects are valid, and whereas resorption efficiency denotes how important P re-utilisation is for a particular species, resorption proficiency is of particular interest when evolutionary processes are concerned (Killingbeck, 1996).

Outline of thesis

This thesis aimed at exploring three specific points that remained open in the study of P- acquisition and P-conservation mechanisms of plants native to P-impoverished environments, in particular south-western Australia and Brazilian rupestrian fields. In this thesis, the following questions were addressed:

24 1. Are Brazilian rupestrian fields functionally equivalent to kwongan and fynbos in terms of (a) soil P availability, (b) plant nutrient status, (c) plant P-acquisition and (d) P-conservation mechanisms?

Answering these questions would allow for a generalisation of the knowledge on P- impoverished environments, and permit the inclusion of a non-Mediterranean environment with a very different species/family composition in the comparison. The hypothesis was that rupestrian fields would be functionally equivalent to kwongan and fynbos, and that root specialisations towards P-acquisition would be found in Proteaceae and in species in the most P-impoverished environments.

In order to get these answers, I have conducted field work at nine sites in rupestrian fields located in central Brazil, where soil samples were collected and analysed, as well as mature and senesced leaf samples and root samples of targeted families (Asteraceae, Eriocaulaceae, Xyridaceae, Velloziaceae, Cyperaceae, Fabaceae and Proteaceae). These data were analysed comparing the different families and sites in the studied area, and also compared in a wider context, with the inclusion of South African fynbos and south- western Australian kwongan data; the original hypothesis was confirmed.

2. Are cluster root formation and mycorrhizal colonisation in Viminaria juncea (Schrad. & J.C. Wendl.) Hoffmanns inversely correlated across a wide range of P supplies? How do these P-acquisition mechanisms and the supply of P relate to shoot [P]?

V. juncea is an unusual species in that is forms both mycorrhizal associations and cluster roots. This species might allow for a unique understanding of how the two P- acquisition strategies relate to each other, to a wide range of P treatments and to internal shoot [P]. Because multiple P-acquisition mechanisms are found concomitantly, a comparison without genetic or environmental bias is permitted. For this experiment, the hypothesis was that mycorrhizal colonisation would increase while cluster-root formation would decrease with greater provision of P until both mechanisms would be turned off at the highest P treatments.

This hypothesis was rejected through a glasshouse experiment with V. juncea individuals grown for 12 weeks in sealed pots with one of 21 P supplies (ranging from zero to 50 mg P kg-1 dry soil). At the end of the experiment, plants were harvested and their shoot [P] was measured, in addition to weighing shoots, roots, cluster roots and counting the number of cluster roots and mycorrhizal colonisation for each individual.

25 3. Is P-uptake down-regulation inversely correlated to leaf P-resorption efficiency in Proteaceae and Fabaceae species native to a P-impoverished environment in south-western Australia?

Although previous research has pointed towards a possible trade-off between P-uptake down-regulation and P-resorption efficiency, this correlation was yet to be confirmed experimentally. If the hypothesis that these are in fact inversely correlated is accepted, the implications of this finding may extend to an evolutionary link between the two; these findings would be of practical use when assessing the sensitivity of species to increased levels of P.

In a glasshouse experiment with Acacia truncata, A. xanthina, Banksia attenuata and B. menziesii grown at five P supplies over 10 weeks, the net P-uptake rate for each species was measured. Mature and senesced leaf samples of each species with the addition of Hakea prostrata were collected from the field to calculate the P-resorption efficiency of each, and the correlation between both traits was analysed, with the initial hypothesis corroborated.

Significance and contribution of the thesis

This work is important because it adds to the field of ecophysiology; not only by creating new knowledge, but inter-connecting it with what is already known and testing the relationships that exist between traits (P-uptake and P-resorption), between species (Acacia truncata, A. xanthina, Banksia attenuata and B. menziesii) and between ecosystems (kwongan, rupestrian fields and fynbos). Furthermore, there are two practical consequences of the study of plants in their P-impoverished environments. First, this study is essential in terms of conservation and revegetation of these biodiversity hotspots. Second, it is a step towards the manipulation of crop species for increased P-uptake and conservation and therefore, a decrease in fertiliser use and its negative environmental collateral effects.

All three chapters that address the above questions have produced novel and essential information towards a broader and more comprehensive understanding of P nutrition in plants. These three chapters are currently being prepared for publication in scientific journals in order to allow access to the wider scientific community to the important findings of this thesis.

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Chapter 2 Phosphorus nutrition of Brazilian rupestrian field plant species in their old, nutrient-impoverished landscapes

Mariana C. R. de Campos1, Rafael S. Oliveira1,2, Stuart J. Pearse1 and Hans Lambers1

1. School of Plant Biology, University of Western Australia. 35 Stirling Hwy, Crawley 6009. Australia.

2. Departamento de Biologia Vegetal, Universidade Estadual de Campinas. Rua Monteiro Lobato 255, Campinas 13083-862. Brazil.

This chapter will be prepared for submission to Annals of Botany in conjunction with an additional author who is collecting additional data in the rupestrian fields.

ABSTRACT

A field survey was conducted in order to generate basic knowledge of the soil properties of rupestrian fields in the cerrado, Brazil, and also on root characteristics and leaf nutrient status of key families in this area.

Nine sites in four conservation units were studied, and soil samples were taken from each for chemical and physical analyses. Mature and senesced leaves of over 30 species were collected for nutrient analyses and leaf mass per area (LMA) calculations, and their roots collected for arbuscular mycorrhizal colonisation count and observation of morphological modifications, such as cluster roots.

Soil results and an average leaf N:P ratio of 29 show that the rupestrian field soils are P- impoverished and that P is the limiting nutrient for plant growth. In accordance, we also found a wide range of root specialisations and high leaf P-resorption efficiency. The present results are compared with literature data for the South African Cape floristic region and for south-western Australia.

Our main findings include the discovery of the first specimen with a cluster root in the field in Brazil (Roupala montana, Proteaceae), sand-binding roots in five species of Eriocaulaceae, cluster-like roots and roots with abundant fine hairs. The average LMA was 167 g m-2 (range: 46-523 g m-2) which is lower than what is found in the Cape floristic region and in south-western Australia and may be partly explained by our

39 sample bias towards herbaceous species. The average P-resorption efficiency was 66% (range: 23-95%), a high value in comparison with a world dataset, but similar to values found in highly P-impoverished landscapes.

We conclude that plants in the rupestrian fields are functionally analogous to the even more P-impoverished Cape region and south-western Australia; however, much still remains to be discovered about this highly diverse and threatened ecosystem.

Keywords: campo rupestre, cerrado, cluster roots, leaf nutrients, mycorrhizas, N:P ratio, P nutrition, P-resorption efficiency, rupestrian field, soil analyses.

INTRODUCTION

The cerrado is a biome that originally covered 23% of the Brazilian territory (Furley and Ratter, 1988). It is located in the centre of the country and borders the Amazonian forest to the North-west; the semi-arid “caatinga” to the North-east; the Atlantic rainforest to the South-east, and the swamplands (“pantanal”) to the West. It is characterised by undulating topography and covered by a vegetation of grasses, woody plants, fire- resistant twisted trees with thick, corky bark, scleromorphic leaves, and vibrant flowers (Jepson, 2005). As it is a very large biome, spreading out in latitude and in contact with different ecosystems, not surprisingly the cerrado is a core area in terms of species richness (Furley, 1999). It has, in fact, the richest flora among the savannahs of the world (Klink and Machado, 2005), and is one of 25 global hotspots of biodiversity, greatly threatened by anthropogenic activities (Myers et al., 2000), mainly extensive cropping of soybean, maize, rice; and the widespread sowing of exotic grass species for pastures (Ratter et al., 1997). The estimates of how much of the cerrado remains in its natural state vary greatly, but even the most conservative affirm that half of the cerrado has been converted to crop or pasture (Jepson, 2005). Preservation by means of the establishment of reserves is scant for this biome, with only 2.2% of the cerrado legally protected (Klink and Machado, 2005).

The cerrado biome comprises several different formation types and various soils (Eiten, 1972). Amongst them are the “campos rupestres”, rupestrian fields, or rock outcrops that are characterised by shallow and skeletal soils (Furley and Ratter, 1988) with great predominance of quartzite (Alves and Kolbek, 1994, Benites et al., 2007). There are also the sand fields, or white-sand savannah, which are formations that resulted from the in situ erosion of quartzites (Alves and Kolbek, 1994). Soil depth in rupestrian fields

40 may reach over 100 cm in depth, but most of them are between 5 and 50 cm (Benites et al., 2007). Most soils in the cerrado are acidic with low fertility, especially in terms of readily available P (Goedert, 1983) which is partially accounted for by high levels of aluminium (Furley and Ratter, 1988).

Rupestrian fields are characterised by a marked seasonality in rainfall; the dry winters last typically for 3 or 4 months and have approximately 10 mm of rainfall monthly (May, June, July and August) of an annual average of 1300 mm (INMET, 1911-). Even though rainfall can be high in summer, the edaphic conditions of rupestrian fields do not allow for great water storage; hence the common occurrence of xeromorphic species in these areas (Alves and Kolbek, 1994).

Phosphate is the most immobile, inaccessible, and least available nutrient in soil (Holford, 1997). It is commonly a limiting nutrient for plant growth (Chapin III, 1980, Aerts and Chapin III, 2000), and the key limiting factor in ancient, highly weathered landscapes (Lambers et al., 2008). This macronutrient is essential for modern agriculture and food security, but the sources of rock phosphate used in crops may be depleted in 50-100 years (Cordell et al., 2009). Therefore, the study of phosphorus (P) nutrition is important, especially in terms of how native species prosper with limited supply of this nutrient.

Landscapes marked by very low soil P, such as south-western Australia (Holford, 1997) and the Cape Region in South Africa (Witkowski and Mitchell, 1987) are frequently hotspots of biodiversity (Myers et al., 2000). The species native to these regions are adapted to low levels of soil P through several mechanisms. A severely limiting P availability increases leaf scleromorphy (Wright et al., 2002, Specht and Rundel, 1990, Beadle, 1966) and is also associated with slow growth and longer leaf longevity (Lambers and Poorter, 1992, Wright and Westoby, 2003). Species from P-impoverished soils often present low leaf [P] (Aerts and Chapin III, 2000), high photosynthetic P-use efficiency (Wright et al., 2004a) and can exhibit high levels of P-resorption from senescing leaves (Kobe et al., 2005, Denton et al., 2007). These nutrient conservation mechanisms may co-occur with P-uptake specialisations, which include morphological responses, such as a root architecture that aids the exploration of the superficial layers of the soil, increased specific root length and increased root weight ratio or proliferation of root hairs (Lambers et al., 2006). Other specialisations are the development of root clusters, which increase the root surface area and exude compounds that release P from soil particles (Shane and Lambers, 2005); and the association with mycorrhizal fungi,

41 that extend the volume of soil that is explored and access nutrient patches (Tibbett, 2000).

While mycorrhizal associations are very common in land plants (Brundrett, 2009), they are proportionally less common in extremely P-impoverished landscapes where cluster roots are a major P-uptake specialisation (Brundrett, 2009, Lambers et al., 2010). Cluster roots are abundant on the most P-impoverished soils, but on less severely impoverished soils, cluster roots may be not as advantageous as mycorrhizas, due to higher carbon cost of cluster roots to the plant (Lynch and Ho, 2005).

The Brazilian cerrado originated from Gondwanaland, like South Africa and Australia, they also exhibit water deficit in the dry season and very low levels of soil P (Hopper, 2009). These similarities, as well as a high biodiversity in all three regions (Myers et al., 2000), have led us to propose that there are equivalent functional groups of species in the cerrado in Brazil and in the fynbos in South Africa and kwongan in south-western Australia. We aim to explore P-uptake strategies and assess aspects of plant P nutrition in the cerrado, in comparison with those in similar nutrient-impoverished landscapes. Although many studies have been carried out in the cerrado over the past 50 years, very little is known about root structures and the ecophysiology of species with regards to plant P nutrition. This is the first, though still preliminary investigation of root adaptations in P-impoverished habitats of the rupestrian fields and sandy areas of the cerrado, focused on representative taxa in the area (Xyridaceae, Eriocaulaceae, Cyperaceae, Velloziaceae and Asteraceae) as well as on taxa common in the fynbos and kwongan (Proteaceae, Fabaceae).

We expected to find many non-mycorrhizal root specialisations that may enhance P- uptake, such as high density of root hairs, evidence of root exudation and proteoid root clusters or dauciform roots. Our hypotheses were: (1) cluster roots would be found in Proteaceae and other specialisations, if found, would be present in the most abundant taxa; (2) a greater number of root specialisations would occur in the soils with least [P]; and (3) leaf P-resorption from senescing leaves would be associated with the presence of root specialisations.

42 MATERIAL AND METHODS

Field site characteristics

Our study was performed on nine field sites (Table 1) in the State of Minas Gerais, central Brazil. These sites belong to the Espinhaço Range and were chosen for their nutrient-impoverished soils and distribution across a latitudinal gradient. The climate is continental and for the region surveyed, minimum daily temperatures ranged between 10 and 15oC and maxima between 20 and 28oC (INMET, 1911-).

The field sites were all very patchy in terms of vegetation cover and soil characteristics, and for this reason, they were analysed individually instead of grouped by Reserve. A brief description of each site is provided below:

 Area of Philcoxia has white loose sand. No rocks were present in the sampled area, but there was a large (ca. 30 m high) rock wall approximately 300 m away. The vegetation was sparse with bushes clumped in groups and some herbs and cacti scattered in between.

 Areial da Laje was an area with patches of sand and rock outcrops smaller than one metre in diameter scattered over 50% of the area. In the surroundings, there were patches of dark soil rich in organic matter and somewhat subject to waterlogging. The vegetation constituted of mainly herbs and short shrubs which had greater cover in the edge of the outcrops.

 Geraldo’s area is a site with large outcrops. The whole area sits close to the edge of the mountain, although the sampled area was not on the slope. The soil there was particularly scarce and shallow, and individuals were in close proximity to each other.

 Ranger base in an area with grey-brown soil in a flat area with small outcrops (< 1 m in diameter). Vegetation cover was of about 70%, and grasses, herbs and shrubs up to 2 m were frequent. The soil was not loose.

 Deco’s area is an infertile island of pale yellow loose sand herbs and small shrubs. It is close to a rock plateau with outcrops (only accessible by helicopter) and to rivers and creeks which are surrounded by riparian vegetation, including trees and plants that require high moisture, such as bromeliads.

 Heliport is a sandy site white, grey and black in colour, depicting the heterogeneity in the soil characteristics, particularly organic matter. The site is

43 used for helicopter landings, although extremely rare, and it a very flat area surrounded by small outcrops. The vegetation cover is constituted mainly of herbs and grass-like life forms, which are in greater density close to the rocks.

 Roadside is a site, as its name suggest, disting only 15 m from the road. It has large rock outcrops and yellow to orange loose crust soil. The main cover was of shrubs 0.5 to 2 m tall, with a less prominent herb cover.

 Campo de fora is a site with higher humidity and poor soil drainage in a very undulating relief. Eriocaulaceae was the dominant family in the sampled area, but the surroundings were very diverse, and also very stratified. Few grasses were present in the sampled area, which had small rock outcrops.

 Ponte de pedra is a site located on the top of a small mountain imbedded in a mountainous matrix. The region was full of large rock outcrops, and so was the sampled area. The soil was shallow but the most plastic, clayey and dark of all sampled sites. The area was quite humid and in close proximity to streams, rivers and waterfalls.

Table 1: Study sites with common name and site code assigned in this study. The reserve names refer to the conservation units where the sites are located in (Parque Nacional da Serra do Cabral; Parque Nacional da Serra do Cipó; Parque Estadual do Rio Preto and Reserva Privada do Santuário do Caraça). All of the sites are located in the State of Minas Gerais, Brazil. The last column divides the sites into three categories: Sand, small outcrops (up to 1 m3) and large outcrops (more than 1 m3). In all landscapes in Cabral, Cipó and Rio Preto, base material is quartzite and arenite. In Caraça, outcrops were granitic.

Common site name Reserve Site code GPS location Landscape Area of Philcoxia Cabral CB-1 S17°42' W44°11' Sand Areial da Laje Cabral CB-2 S17°41' W44°17' Small outcrops Geraldo's area Cipó CP-1 S19°16' W43°35' Large outcrops Ranger base Cipó CP-2 S19°14' W43°31' Small outcrops Deco's area Rio Preto RP-1 S18°05' W43°20' Sand Heliport Rio Preto RP-2 S18°05' W43°20' Sand Roadside Rio Preto RP-3 S18°06' W43°20' Large outcrops Campo de fora Caraça CR-1 S20°07' W43°31' Small outcrops Ponte de pedra Caraça CR-2 S20°06' W43°28' Large outcrops

Some areas are particularly sandy (Figure 1a), while others are a mixture of sand and exposed rock (Figure 1b, c and d) and some are mainly rocky (Figure 1e and f). The species of interest in our study were never found growing directly on a rock outcrop, but

44 invariably on the soil patches in between. The soil depth was not measured objectively, but while digging for the root samples, soils in the outcrop areas were mostly between 15 and 50 cm in depth.

The field trip was performed in February and March 2009, at the end of the wet summer season.

Figure 1: Examples of plants and sites in the cerrado of central Brazil. Two locations in the Parque Nacional da Serra do Cabral are shown (a), CB-1 and (b), CB-2. In (c), Reserva Privada do Santuário do Caraça, location CR-1 is shown with a detail of an Eriocaulaceae species. Parque Nacional da Serra do Cipó areas CP-1 (d) and CP-2 (e) and an area adjacent to RP-2 in the Parque Estadual do Rio Preto (f).

Soil sampling

Soil samples that represent the substrate where the target species grow were collected at each study site. A single sample for each point was made at a depth of 0-10 cm. The number of samples collected varied with the size of the surveyed area at the study site and also with the number of plant species collected in each area. A larger number of soil samples were collected where more plant species were sampled.

All 21 soil samples collected were taken back to the Department of Soil Science at the University of Sao Paulo. The samples were sifted, homogenised and analysed following the standardised methods of the Institute of Agriculture of Campinas (IAC). The methods and detection limits (DL) for the analyses performed are: pH measured in 10 45 mM CaCl2; readily available P by the ion-exchange resin method (Amer et al., 1955), DL 2 mg kg-1 (due to calibration curve); readily available P by the Bray-II method (Bray and Kurtz, 1945), DL 2 mg kg-1 (due to calibration curve); organic matter by colourimetry, DL 1 mg kg-1; K by atomic emission spectrophotometry with ion- exchange resin extraction, DL 0.1 mmolc kg-1; Ca by atomic emission spectrophotometry with ion-exchange resin extraction, DL 1 mmolc kg-1; Mg by atomic emission spectrophotometry with ion-exchange resin extraction, DL 0.3 mmolc kg-1; Al by colourimetry with potassium chloride extraction, DL 0.8 mmolc kg-1; S by turbidimetry with calcium phosphate extraction, DL 1 mg kg-1; B by colourimetry with warm water extraction, DL 0.14 mg kg-1; Cu, Zn, Mn and Fe by atomic absorption spectrophotometry with diethylene triamine pentaacetic acid (DTPA) extraction, DL, respectively, 0.1 mg kg-1; 0.1 mg kg-1; 0.2 mg kg-l; and 0.7 mg kg-1.

Plant sampling

Plant material was collected according to the availability of target species at each site. We aimed to collect three species (n=3 for each species) of the families Xyridaceae, Eriocaulaceae and Fabaceae, in addition to three replicates of all species of Cyperaceae, Velloziaceae and Proteaceae. Three replicates of Lychnophora sp. (Asteraceae) were also collected as a control, as this was the one species that occurred at every site and was not expected to present root specialisations. The low sample number for each species (3) reflects the difficulty in finding individuals of the same species in the same area in such a biodiverse region. It was also chosen in order to allow for the collection of a larger number of species, which suits the purpose of an initial screening project.

From targeted individuals, we collected fully expanded, mature, green leaves with no apparent damage, as well as senesced leaves still attached to the plant or from the top layer of litter, when attached senesced leaves were unavailable. The roots of the same individuals were excavated and field observations of cluster roots or other visible specialisations were recorded. Root samples were collected and kept in 70% (v/v) ethanol. The whole root system was collected when possible, and when it was not possible, a subsample of the terminal portion of the roots was taken instead.

Leaf mass per unit leaf area (LMA) was calculated using a flat scanner with the software ImageJ (National Institute of Health 2009) and a balance with three digit precision. Mature and senesced leaves were dried with a lamp oven in the field and in a convection oven at 60oC for three days upon return from the field. 46 Dried leaves were ground with mortar and pestle, digested with nitric and perchloric acid and analysed for nutrients through inductively coupled plasma mass spectrometry (ICP-OES, model Optima 7300 DV, Perkin Elmer, Massachusetts, USA). The carbon and nitrogen results were obtained through combustion under continuous helium flux in an elemental analyser (Carlo Erba, model CHN-1110, Milan, Italy).

With the green and senesced leaf nutrient analyses it was possible to calculate the resorption efficiency, which equals the difference of the concentration of nutrients in green and senesced leaves divided by the concentration of nutrients in the green leaves. Resorption proficiency values are presented, and they indicate the amount of nutrients left in the senesced leaves.

Root samples kept in ethanol were prepared following the protocol used by Brundrett et al. (Brundrett et al., 1996). Roots were cleared (diaphanised) with 10% (w/v) KOH in an autoclave for 20 minutes at 121oC and then stained with 0.05% (w/v) Trypan Blue and preserved in lactoglycerol. Arbuscular mycorrhizal colonisation counts were performed under a stereomicroscope and recorded as a percentage of colonisation. The colonisation count method (Brundrett et al., 1996) is performed using a Petri dish with a square grid, in which the total number of points where roots cross the grid are counted, as well as the number of points with mycorrhizal evidence cross the grid. The division of the latter by the former gives a root colonisation percentage. Arbuscules, vesicles and hyphae were included in the counts of this studty. During this process, any structural root modifications were also recorded.

Statistical analyses

Analyses of variance and HSD (Honestly Significant Difference) tests, also known as Tukey tests, were performed using the software R (version 2.13.0. The R Foundation for Statistical Computing, 2011).

47 RESULTS

Soil analyses

The soil pHCaCl2 of all sites was acidic: between 3.4 and 4.3 (Table 2), and not significantly different among sites at p<0.05 (Table 3). Also not significantly different were the results for concentrations of Ca, Mg, Zn, and B between all sites (Table 3). Extremely low values of Cu were found at seven of the sites, with only two locations (RP-3 and CR-2) showing concentrations above the detection limit (Table 2).

Organic matter (OM) averages ranged from 12.3 g OM kg-1 soil in CB-1 to 48 g OM kg- 1 soil in CR-2 (Table 2). Interestingly, at each site, there was a lower and a higher value encountered which resulted in no significant differences among sites (Table 3).

Analysis of the texture of the soil showed low percentages of clay (3 to 5.3%) for six of our sites. CP-2, RP-3 and CR-2 were the exceptions, with 13-15% clay. Both areas in the Cipó reserve (CP-1 and CP-2) exhibited the highest silt percentages (Table 2).

The most variable soil parameter in our analyses was Fe, with results between 6 and 298 mg Fe kg-1 soil. Al was also highly variable, ranging from 4 to 32 mg Al kg-1 soil. Results of the concentration of K differed by an order of magnitude; values for soil S showed a maximum difference of only 40% (Table 2).

Two distinct P analyses were performed. The results of the Bray-II P analysis were not significantly different among sites, ranging from 6 to 17 mg P kg-1 soil, whereas the resin extraction method produced values ranging from undetectable to 10 mg P kg-1 soil (Table 2) and these were significantly different between sites.

48 Table 2: Chemical and physical analyses of the soil samples collected at 0-10 cm depth. Units displayed as mg kg-1 symbolise mg of the element analysed per kg of dry soil. The number of soil samples is shown in the second column (n). Results are presented as averages with range in brackets. OM is organic matter; UDL means under detection limit; * is for one sample removed for being UDL and ‡ for two samples removed for being UDL. Ca and Mg data are not shown in the table as all samples had a result of 1.0 mg kg-1.

Site n pH OM Sand Silt Clay Resin P Bray-II P K Al S Cu Zn Mn Fe B -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 code g kg %%%mg kg mg kg mg kg mg kg mg kg mg kg mg kg mg kg mg kg mg kg CB-1 3 4.0 (3.8-4.3)) 12 (11-13) 95 (92-96 2 (1-5) 3 (-) UDL 6 (-) 0.1 (-) 4 (3-7) 3 (-) UDL 0.1 (-)* 0.4 (-) ‡ 7 (6-9) 0.2 (-) ‡ CB-2 3 3.8 (3.4-4.1) 21 (15-28) 94 (91-96) 2 (1-3) 4 (3-8) 3 (2-4)) 11 (6-19) 0.4 (0.1-0.8) 7 (5-10) 4 (3-6) UDL 0.2 (0.1-0.4) * 0.7 (0.3-1.8) 60 (9-102) 0.3 (0.2-0.5) CP-1 2 3.8 (3.7-3.9) 22 (16-28) 76 (72-80) 19 (15-23) 5 (-) 3 * 8 (7-8) 0.3 (0.2-0.4) 7 (-) 3 (-) UDL 0.3 (0.1-0.4) 0.5 (0.4-0.5) 135 (78-192) 0.4 (0.3-0.4) CP-2 2 3.6 (3.5-3.6) 37 (31-42) 61 (58-64) 26 (-) 13 (10-16) 5 (4-5) 10 (-) 0.4 (-) 17 (14-19) 5 (4-5) UDL 2.0 (0.2-3.8) 0.8 (0.5-1.1) 218 (210-225) 0.6 (0.5-0.7) RP-1 2 4.0 (3.6-4.3) 15 (14-16) 95 (93-96) 3 (1-4) 3 (-) UDL 7 (-) 0.1 (-) 4 (3-5) 3 (-) UDL 1.0 (-)* 0.6 (0.2-1.0) 8 (6-9) 0.3 (-) * RP-2 2 3.8 (3.7-3.8)) 22 (-) 94 (92-95) 4 (2-5) 3 (-) 3 (-) 7 (6-8) 0.3 (-) 8 (-) 3 (-) UDL 1.3 (-)* 0.4 (0.3-0.4) 155 (134-175) 0.5 (0.4-0.5) RP-3 1 3.8 32 79 6 15 4 9 1.7 12 4 0.7 0.2 1.9 249 0.4 CR-1 3 3.8 (3.7-4.0) 18 (12-28) 91 (84-95) 3 (2-6) 5 (3-10) 3 (2-4) * 7 (5-10) 0.4 (0.2-0.6) 9 (8-11) 4 (3-6) 0.1 (-) ‡ 0.4 (0.3-0.4) 1.1 (0.7-1.3) 131 (47-298) 0.3 (0.2-0.5) CR-2 1 3.4 48 73 12 15 10 17 1.3 32 8 0.3 0.7 1.7 242 0.6

Table 3: Results of the HSD (Honestly Significant Difference) Test. For each column, a different letter implies a significant difference. By convention, “a” is always the highest value. In the second column, “n” refers to the number of soil samples in each location.

Site code n pH OM Sand Silt Clay resin P Bray-II P K Ca Mg Al S Cu Zn Mn Fe B CB-13acabbb a baacbc abba CB-2 3 a abc ab b ab ab a ab a a bc ab c a ab ab a CP-1 2 a abc c ab ab b a ab a a bc b c a ab ab a CP-2 2 a ab d a ab ab a ab a a ab ab c a ab a a RP-12a bca abb b a b a a c b c aabba RP-2 2 a abc ab ab b ab a ab a a bc b c a ab ab a RP-3 1 a abc bc ab a ab a a a a abc ab a a a a a CR-1 3 a abc ab ab ab b a ab a a bc ab bc a ab ab a CR-2 1 a a c ab a a a a a a a a ab a a a a

49 Leaf nutrient analyses

The results of the 189 leaf nutrient analyses were grouped in three ways: by species (Table 4), by family (Table 5) and by site (Table 6).

Mature leaf [P] values were invariably higher than values for senesced leaves, showing that P was resorbed prior to abscission. Amongst the sampled species, mature leaf [P] ranged from 128 to 1173 µg P g-1 leaf DW with an average of 536 µg P g-1 leaf DW. For senesced leaves, the [P] and resorption proficiency was at an average of 187 µg P g- 1 leaf DW (ranged from 27 to 657 µg P g-1 leaf DW). The average P-resorption efficiency (that is, what percentage of nutrients were resorbed before abscission) at the species level ranged from 23% for Mimosa misera to 95% for Stylosanthes guianensis (Figure 2). Syngonanthus niveus had the highest [P] in mature leaves with 1173 µg P g-1 leaf DW; 71% of which was remobilised prior to senescence. Chamaecrista cf. desvauxii var. langsdorfii also appeared to show luxury consumption of P, with a total of 1031 µg P g-1 leaf DW but had very low P-resorption efficiency (36%) and low P- resorption proficiency (how little nutrients were left in the abscised leaf): its senesced leaves contained an astonishing 657 µg P g-1 leaf DW, higher than most species showed in their mature green leaves (Figure 2). The average P-resorption efficiency for all of the species studied was 66%. At the family level, the P-resorption efficiencies were 53% (Asteraceae); 83% (Cyperaceae); 76% (Eriocaulaceae); 52% (Fabaceae); 44% (Proteaceae); 63% (Velloziaceae); and 64% (Xyridaceae).

Trace elements, Cu, Mn and particularly Zn, showed great variation between species. The results of mature leaf analyses ranged between undetectable values and 17 µg Cu g- 1 leaf DW; 11 and 543 µg Mn g-1 leaf DW; and 7 and 124 µg Zn g-1 leaf DW. The analyses of [Ni] in leaves ranged from 0.1 to 2.7 µg Ni g-1 leaf DW; and [Si] ranged from 15 to 93 µg Si g-1 leaf DW.

The ratio of nitrogen to phosphorus (N:P) was high, with a species minimum of 14, maximum of 61 and average of 29. The variation was greater between species in a family than between families. No clear patterns were found when grouping sites for N:P values (data not shown), but the spread of families across the N:P values is weakly grouped, even though with an overlap (Figure 3). All N:P ratios and LMA results are presented organised by study site (Table 6) and by families and species (Table 7).

50 71 1200 36

82 47 85 85 83 73 33 23 95 60 54 68 leaf DM)leaf DM) 74 56 41 77 72 -1-1 600 63 82 69 44 62 75 Leaf [P] [P] Leaf Leaf 75 70 (µg P gg (µg P(µg P 0 n 0n 0 n 0n 0 n 0n 0 n +n + n +n + clcl 0 0 clcl 0 0 clcl 0 0 clcl + + clcl 0 0 clcl 0 0 clcl + + clcl + + clcl 0 0 clcl + + clcl + + clcl + + clcl + + clcl + + rcrc + + sbsb + + sbsb + + sbsb + + sbsb + + . . nodnod + + nodnod + + sb/clsb/cl 0 0 spsp sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. Cyper Cyper menolopio menolopio desvauxiidesvauxii Vellozia Vellozia Vellozia XyrisXyris tenella tenella MimosaMimosa cf.cf. cf. cf. Xyris asperula asperula Xyris Xyris Mimosa misera misera Mimosa Mimosa Vellozia albiflora albiflora Vellozia Vellozia Lychnophora Lychnophora VelloziaVellozia resinosa resinosa “new species 80”80” species species “new “new 98”98” species species “new “new XyrisXyris subsetigera subsetigera Xyris obcordata obcordata obcordata Xyris Xyris Roupala montana montana montana Roupala Roupala Xyris obtusiuscula obtusiuscula Xyris Xyris Actinocephalus Actinocephalus Actinocephalus Andira Andira Syngonanthus niveus niveus niveus Syngonanthus Syngonanthus Xyris Xyris Xyris Xyris Xyris StylosanthesStylosanthes guianensis guianensis Paepalanthus elongatus elongatus Paepalanthus Paepalanthus Syngonanthus bisulcatus bisulcatus Syngonanthus Syngonanthus Actinocephalus bongardii bongardii bongardii Actinocephalus Actinocephalus Syngonanthus verticillatus verticillatus verticillatus Syngonanthus Syngonanthus PaepalanthusPaepalanthus geniculatus geniculatus Actinocephalus polianthus polianthus polianthus Actinocephalus Actinocephalus Actinocephalus cabralensis cabralensis cabralensis Actinocephalus Actinocephalus Chamaecrista Chamaecrista Chamaecrista

Figure 2: Phosphorus (P) concentrations in mature leaves (total bar length) and its remobilised fraction (dark). The values on top of the bar represent the P- remobilisation efficiency (%), and the light bars at the bottom correspond to the P-remobilisation proficiencies of each species, that is the amount of P that is not remobilised from the senescing leaves. Species with only one sample analysed were not shown in this graph. Codes after species names are: sb (sand-binding); cl (cluster-like); n (none); nod (nodules); rc (root cluster); + (mycorrhizal); 0 (not mycorrhizal, or not confirmed).

51 Table 4: Leaf analyses results grouped by species. The column “sample” describes the type of material (“sen” is senesced, and “mat” is mature), and number of samples in brackets. Results are in µg of nutrient g-1 leaf dry weight. N/a is not analysed and n/d is not detected.

Family/Species sample P N K Ca Mg Al S Cu Zn Mn Fe B Ni Si Asteraceae Lychnophora sp. sen (6) 211 7266 2607 3623 1014 715 638 8.1 23 161 800 86 1.7 55 mat (6) 453 9929 11326 3591 1418 170 878 10.3 28 182 278 84 2.7 34 Cyperaceae Cyperus sp. sen (4) 111 5659 606 2699 683 95 623 2.3 19 528 135 78 1.5 136 mat (4) 669 18405 3920 3443 1287 61 1357 0.9 23 412 52 66 1.5 93 Eriocaulaceae Actinocephalus cabralensis (Silveira) Sano sen (2) 110 4281 2147 379 383 512 376 2.7 6 93 2150 39 0.3 48 mat (2) 301 11035 9218 637 464 318 776 5.5 9 106 1260 31 0.8 59 Actinocephalus polianthus (Bong.) Sano sen (3) 128 6899 761 711 440 215 561 5.4 8 62 195 57 0.8 55 mat (3) 487 17141 15057 1787 1154 69 1108 3.8 17 160 103 59 1.3 27 Actinocephalus sp. sen (4) 101 5239 644 939 556 75 410 0.7 12 93 125 44 0.4 63 mat (3) 434 8067 6968 1155 2592 19 743 1.9 8 178 35 48 0.4 33 Actinocephalus bongardii (A.St-Hil.) Sano sen (3) 95 5265 433 1290 418 317 534 3.2 50 399 844 39 1.6 54 mat (3) 627 13848 10241 1199 1211 49 1113 5.4 36 543 133 55 1.7 31 Paepalanthus argyropus Silveira sen (1) 120 3626 801 486 122 165 311 2.4 9 27 213 66 0.6 26 mat (1) 698 10064 12401 84 195 30 982 4.0 14 11 40 66 0.3 23 Paepalanthus elongatus (Bong.) Koern sen (3) 56 5047 854 366 127 744 491 5.0 15 98 1303 42 1.6 84 mat (3) 308 12308 13330 917 794 220 1140 4.2 34 450 429 50 1.5 40 Paepalanthus erectifolius Silveira sen (1) 78 7338 310 603 143 403 637 3.4 9 52 503 71 1.0 32 mat (1) 289 17687 22253 963 1165 118 1234 4.4 22 318 107 63 1.5 15 Paepalanthus geniculatus (Bong.) Koern sen (3) 54 4776 839 458 180 808 660 2.8 7 54 1266 36 0.9 48 mat (3) 215 10438 16045 654 1011 288 1642 5.6 12 235 318 61 0.9 34 Syngonanthus bisulcatus (Koern) Ruhland sen (4) 126 7759 1475 1281 481 785 631 4.4 16 159 893 64 1.4 106 mat (4) 491 9306 5681 1150 1139 117 1100 4.5 30 325 168 44 1.9 42

52 Table 4, continued

Family/Species sample P N K Ca Mg Al S Cu Zn Mn Fe B Ni Si Syngonanthus niveus (Bong.) Ruhland sen (2) 338 n/a 844 1525 194 156 587 n/d 61 191 132 88 0.8 85 mat (1) 1173 n/a 2544 885 487 138 772 n/d 92 175 61 60 0.7 78 Syngonanthus verticillatus (Bong.) Ruhland sen (2) 95 3215 609 166 187 219 298 0.3 8 13 268 49 0.1 43 mat (3) 643 11063 11744 643 1218 55 1074 2.0 37 59 79 51 0.3 28 Fabaceae Andira cf. menolopio sen (3) 187 10675 239 2325 300 64 660 2.3 6 85 102 37 0.5 41 mat (3) 700 16401 1794 3232 1465 26 1203 2.7 7 284 61 57 0.7 59 Chamaecrista cf. desvauxii var. langsdorfii (Kunth ex Vogel) H.S. Irwin & Barneby sen (1) 657 n/a 4477 4109 784 80 741 5.3 18 131 75 98 1.6 94 mat (5) 1031 28486 6540 6062 1718 103 1502 17.1 29 398 261 74 2.0 28 Mimosa misera Benth. sen (3) 486 12859 1321 2338 663 170 696 13.0 8 273 165 55 0.4 52 mat (5) 630 13496 2234 1240 809 99 788 7.1 11 204 118 37 0.3 31 Mimosa sp. sen (2) 451 13316 2238 1397 707 102 701 5.1 11 58 174 62 0.4 39 mat (3) 673 17458 3196 1544 1325 25 980 3.1 15 70 59 46 0.7 35 Stylosanthes guianensis (Aubl.) Sw. sen (1) 27 6328 395 1422 277 208 676 3.4 13 189 260 54 2.0 17 mat (3) 603 19239 12104 5253 2855 150 1789 9.6 53 484 150 88 1.8 22 Proteaceae Roupala montana Aubl. sen (12) 281 6517 1843 3248 669 638 678 3.6 6 156 558 45 2.0 43 mat (15) 506 9073 4555 2928 1369 298 856 3.7 8 153 137 54 1.3 30 Velloziaceae Vellozia albiflora Pohl sen (1) 246 5513 813 8004 1015 98 645 2.7 19 58 117 70 1.2 40 mat (2) 621 11656 2668 4404 1335 84 886 6.5 36 112 87 29 0.3 33 Vellozia resinosa Mart. sen (2) 131 6069 370 1851 160 81 497 1.7 16 120 94 56 0.2 22 mat (2) 724 13795 5617 2422 902 10 943 5.4 30 209 28 42 1.0 31 Vellozia sp. sen (2) 381 9673 1759 9941 846 56 989 3.1 76 77 64 52 0.6 32 mat (1) 715 16623 4551 8101 1524 14 1206 7.2 124 49 37 55 1.3 31

53 Table 4, continued

Family/Species sample P N K Ca Mg Al S Cu Zn Mn Fe B Ni Si Xyridaceae Xyridaceae "new species 56" sen (1) 192 4762 363 2266 262 111 450 4.0 14 37 151 49 1.1 18 mat (1) 657 9264 2819 1446 933 21 960 3.9 16 85 42 55 0.3 22 Xyris asperula Mart. sen (4) 88 6841 1206 951 565 1061 349 3.7 9 67 1663 50 1.3 81 mat (4) 288 10506 10088 1049 849 179 798 11.7 14 118 266 69 1.4 59 Xyris obcordata Kral e Wanderley sen (3) 283 5674 1933 1081 423 132 448 2.7 12 60 154 64 0.6 51 mat (4) 615 9043 6446 1055 605 30 663 5.2 16 65 54 64 0.2 38 Xyris obtusiuscula L.A. Nilsson sen (2) 38 5974 377 1065 165 279 391 2.6 8 56 284 54 0.5 39 mat (2) 128 5540 7626 502 303 67 726 2.7 8 58 55 55 0.1 35 Xyris "new species 80" sen (3) 187 6187 894 1161 246 44 462 1.7 8 119 69 42 0.1 31 mat (3) 579 10601 4294 921 445 62 739 3.9 15 119 85 40 0.5 32 Xyris "new species 98" sen (3) 164 6133 1779 906 321 295 500 1.7 8 137 305 45 0.1 42 mat (3) 295 8738 6271 1358 566 99 669 4.2 13 259 150 77 0.1 45 Xyris subsetigera Malme sen (3) 116 5720 1082 1332 236 1331 570 7.5 17 80 1657 84 1.4 126 mat (3) 304 8023 6526 2480 631 302 889 11.8 29 142 380 80 2.0 38 Xyris tenella Kunth sen (3) 58 4227 999 595 79 393 430 1.5 7 42 642 78 0.6 76 mat (3) 231 10674 9185 1587 669 66 932 4.0 14 224 84 78 0.1 44

54 Table 5: Leaf analyses results grouped by families. The column “sample” describes the type of material (“sen” is senesced, and “mat” is mature), and number of samples in brackets. Results are in µg of nutrient g-1 leaf dry weight.

Family sample P N K Ca Mg Al S Cu Zn Mn Fe B Ni Si Asteraceae sen (6) 211 7266 2607 3623 1014 715 638 8.1 23 161 800 86 1.7 55 mat (6) 453 9929 11326 3591 1418 170 878 10.3 28 182 278 84 2.7 34 Cyperaceae sen (4) 111 5659 606 2699 683 95 623 2.3 19 528 135 78 1.5 136 mat (4) 669 18405 3920 3443 1287 61 1357 0.9 23 412 52 66 1.5 93 Eriocaulaceae sen (28) 114 5446 909 806 337 430 513 2.9 19 126 740 52 0.9 65 mat (27) 477 11645 11056 995 1158 129 1089 3.9 26 255 248 52 1.1 36 Fabaceae sen (10) 360 11393 1403 2231 536 120 689 6.5 9 151 148 55 0.7 47 mat (19) 749 19432 5008 3505 1556 85 1230 8.8 22 291 142 59 1.1 34 Proteaceae sen (12) 281 6517 1843 3248 669 638 678 3.6 6 156 558 45 2.0 43 mat (15) 506 9073 4555 2928 1369 298 856 3.7 8 153 137 54 1.3 30 Velloziaceae sen (5) 254 7399 1014 6318 605 74 724 2.5 41 90 87 57 0.6 30 mat (5) 681 13505 4224 4350 1200 40 973 6.2 51 138 53 39 0.8 32 Xyridaceae sen (21) 138 5743 1182 1065 308 523 448 3.1 10 79 721 59 0.7 63 mat (23) 380 9184 7088 1300 621 112 780 6.4 16 138 153 66 0.7 42

55 leaf DW) leaf DW) -1-1 N (µg gg N (µgN (µg

-1 P (µg g leaf DW)

Figure 3: Leaf [N] plotted against leaf [P]. Colours relate to botanical family: Fabaceae in dark blue; Cyperaceae in red; Velloziaceae in pink; Eriocaulaceae in green; Proteaceae in light blue; Xyridaceae in yellow; Asteraceae in black.

Table 6: Leaf analyses results grouped by sampling site. The column “sample” indicates the type of material (“sen” is senesced, and “mat” is mature), and number of samples in brackets. The unit is µg of nutrient g-1 leaf dry weight; and for LMA (Leaf Mass per Area), g m-2. LMA was not calculated for sites CR-1 and CR-2.

Site sample P N K Ca Mg Al S Cu Zn Mn Fe B Ni Si N:P LMA CB-1 sen (16) 232 8285 648 2516 501 136 629 4.3 20 240 151 62 1.0 72 mat (17) 651 13343 2768 2688 1213 88 1005 4.1 25 276 89 47 1.0 51 22 181 CB-2 sen (27) 201 6443 1549 1565 545 613 489 3.6 12 75 873 55 1.0 64 mat (30) 531 10983 7901 1501 1131 136 879 5.8 18 97 221 58 1.0 38 23 152 CP-1 sen (6) 56 4501 919 527 130 600 545 2.1 7 48 954 57 0.7 62 mat (8) 295 14215 10898 2644 999 162 1387 8.4 16 284 272 71 1.0 34 48 163 CP-2 sen (5) 73 6218 331 1968 361 311 549 2.5 8 88 290 58 0.9 34 mat (9) 406 12179 9181 3068 1717 156 1213 4.4 25 226 87 68 1.0 24 35 145 RP-1 sen (9) 315 8473 1419 3741 465 73 648 3.4 26 140 80 54 0.4 39 mat (10) 894 18583 5279 3287 1105 62 1035 8.7 33 214 80 54 1.0 32 21 241 RP-2 sen (6) 146 6593 1270 809 380 255 530 3.5 8 100 250 51 0.5 48 mat (6) 391 12940 10664 1572 860 84 888 4.0 15 210 126 68 0.7 36 33 94 RP-3 sen (6) 198 5725 3745 2751 943 363 562 2.8 8 149 155 73 2.7 28 mat (6) 354 8616 8990 2637 1445 232 686 4.3 8 141 81 70 2.6 28 25 259 CR-1 sen (9) 89 5344 790 996 260 797 532 5.2 28 192 1268 55 1.6 88 mat (9) 413 11393 10032 1532 878 190 1047 7.1 33 379 314 62 1.7 36 30 CR-2 sen (3) 214 6519 472 3976 550 671 919 7.6 11 366 928 35 1.3 64 mat (3) 480 8550 3653 3179 1085 350 1017 7.5 9 445 365 35 0.8 45 18

57 Table 7: Average results of nitrogen to phosphorus ratio on a mass basis (N:P) and leaf mass per area (LMA; g m-2) for families (in bold) and species. The second column (n) shows the number of samples analysed and n/a stands for not analysed.

Family/Species n N:P LMA Asteraceae 6 25 197 Lychnophora sp. 625197 Cyperaceae 4 24 71 Cyperus sp. 42471 Eriocaulaceae 27 32 98 Actinocephalus cabralensis 236n/a Actinocephalus polianthus 33597 Actinocephalus sp. 31979 Actinocephalus bongardii 325n/a Paepalanthus argyropus 114n/a Paepalanthus elongatus 340n/a Paepalanthus erectifolius 161120 Paepalanthus geniculatus 34998 Syngonanthus bisulcatus 420n/a Syngonanthus niveus 1 n/a 149 Syngonanthus verticillatus 31746 Fabaceae 19 30 135 Andira cf. menolopio 324201 Chamaecrista cf. desvauxii var. langsdorfii 535126 Mimosa misera 524138 Mimosa sp. 326117 Stylosanthes guianensis 33992 Proteaceae 15 18 250 Roupala montana 15 18 250 Velloziaceae 5 21 435 Vellozia albiflora 221523 Vellozia resinosa 219283 Vellozia sp. 123497 Xyridaceae 23 29 132 Xyridaceae "new species 56" 1 14 128 Xyris asperula 437205 Xyris obcordata 41668 Xyris obtusiuscula 243110 Xyris "new species 80" 32097 Xyris "new species 98" 33091 Xyris subsetigera 326n/a Xyris tenella 346225

Roots

In our samples, we found some roots covered by a layer of sand, i.e. sand-binding roots, in five species of Eriocaulaceae (Actinocephalus cabralensis, A. polianthus, A. bongardii, Paepalanthus erectifolius and P. geniculatus, Figure 4a). The sand-binding sheath covered approximately three quarters of the roots, from the tip to the base.

Unlike the above mentioned species of Eriocaulaceae, some other species presented much less extensive sand-binding (Figure 4b). Other roots, such as those of

58 Syngonanthus verticillatus (Figure 4c) were covered in very fine root hairs, not only at the tip of the root, but over their entire length. A total of 40% of the collected species showed full root hair cover, most of them in the Eriocaulaceae and Xyridaceae, but also in the Cyperaceae and Asteraceae families.

Cluster-like structures were found in some specimens of Cactaceae, Xyridaceae and Eriocaulaceae (Figures 4d and 4e), but they cannot be considered “true” cluster roots as described in the literature. Only one cluster root was observed in the field (Figure 4f). It was recorded in one individual of Roupala montana (Proteaceae).

The root samples indicated that amongst the species sampled, only 37% presented some arbuscular mycorrhizal colonisation, and 15% of these had a level of colonisation under 5%. The extent to which all of the roots were colonised varied between 0 and 62% (Table 8), with great variation between samples of the same species, and also some variation between the sites. The colonisation of mycorrhizas was not correlated with soil P concentration (Figure 5).

Figure 4: Root modifications found in the rock outcrops and sand plains of the cerrado in central Brazil, June 2009. Abundance of fine hairs in Xyris asperula (a) and in Syngonanthus verticillatus (b); agglomerated sand in the roots of a Cactaceae (c) and the pseudo-clusters found after washing these roots (d); cotton-like structure found in Syngonanthus niveus (e); sand- binding roots of Actinocephalus polianthus in the field (f) and a cross-section of the same roots (g); root cluster of Roupala montana (h); and sand granules adhered to roots of Chamaecrista cf. desvauxii var. Langsdorfii (i). Scale bars correspond to 2 cm (a, c); 2 mm (b, g, i); 5 cm (f); 1 cm (d, e, h). 59 Table 8: Arbuscular mycorrhizal colonisation (%) across different species and sites. Samples with inconclusive observations or that had inadequate material were excluded. Results are ungrouped; each value represents one individual root count. CB-1 is Area of Phicoxia, CB-2 is Areal da Laje, CP-1 is Geraldo’s area, CP-2 is Ranger Base, RP-1 is Deco’s area, RP-2 is Heliport, RP-3 is Roadside, CR-1 is Campo de Fora and CR-2 is Ponte de Pedra.

Family/Species CB-1 CB-2 CP-1 CP-2 RP-1 RP-2 RP-3 CR-1 CR-2 Asteraceae Lychnophora sp. 4 Lychnophora sp. 0 Lychnophora sp. 0 Lychnophora sp. 0 Lychnophora sp. 0 Lychnophora sp. 4 Cyperaceae Cyperus sp. 0 Cyperus sp. 0 Cyperus sp. 0 Eriocaulaceae Actinocephalus cabralensis 14 Actinocephalus cabralensis 0 Actinocephalus polianthus 24 Actinocephalus polianthus 1 Actinocephalus sp. 0 Actinocephalus sp. 0 Actionocephalus bongardii 12 Actionocephalus bongardii 17 Actionocephalus bongardii 19 Paepalanthus argyropus 0 Paepalanthus elongatus 32 Paepalanthus elongatus 43 Paepalanthus elongatus 41 Paepalanthus erectifolius 4 Paepalanthus erectifolius 5 Paepalanthus geniculatus 5 Paepalanthus geniculatus 23 Paepalanthus geniculatus 20 Syngonanthus aciphyllus 0 Syngonanthus aciphyllus 0 Syngonanthus aciphyllus 0 Syngonanthus bisulcatus 0 Syngonanthus bisulcatus 0 Syngonanthus bisulcatus 2 Syngonanthus niveus 0 Syngonanthus verticillatus 0 Syngonanthus verticillatus 0 Syngonanthus verticillatus 0 Fabaceae Andira cf. menolopio 0 Andira cf. menolopio 62 Andira cf. menolopio 0

Table 8, continued

Family/Species CB-1 CB-2 CP-1 CP-2 RP-1 RP-2 RP-3 CR-1 CR-2 Chamaecrista cf. desvauxii var. langsdorfii 1 Chamaecrista cf. desvauxii var. langsdorfii 7 Chamaecrista cf. desvauxii var. langsdorfii 8 Mimosa misera 0 Mimosa misera 0 Mimosa misera 0 Mimosa misera 0 Mimosa misera 0 Mimosa sp. 0 Mimosa sp. 4 Mimosa sp. 0 Stylosanthes guianensis 0 Proteaceae Roupala montana 1 Roupala montana 0 Roupala montana 11 Roupala montana 20 Roupala montana 47 Roupala montana 36 Roupala montana 4 Roupala montana 0 Velloziaceae Vellozia albiflora 0 Vellozia albiflora 0 Vellozia resinosa 0 Vellozia resinosa 0 Vellozia sp. 0 Vellozia sp. 0 Xyridaceae Xyris asperula 0 Xyris obcordata 0 Xyris obcordata 0 Xyris obcordata 0 Xyris sp. 0 Xyris "new species 80" 0 Xyris "new species 80" 0 Xyris "new species 98" 1 Xyris "new species 98" 0 Xyris "new species 98" 0 Xyris subsetigera 0 Xyris subsetigera 28 Xyris subsetigera 0 Xyris tenella 0

61 Mycorrhizal colonisationMycorrhizal (%)

-1 Soil Bray-II P (mg kg soil)

Figure 5: Mycorrhizal colonisation (%) as related to Bray-II P results for soils where the root samples were collected. Each point represents one plant sample and the colours relate to the site where it was collected: CB-1 in black; CB-2 in dark grey; CP-1 in red; CP-2 in green; RP-1 in pink; RP-2 in yellow; RP-3 in grey; CR-1 in dark blue; CR2 in light blue.

Out of our targeted families, no mycorrhizas were found in any specimens of Velloziaceae (n=6) or Cyperaceae (n=3); 14% of Xyridaceae had some colonisation (n=14); 33% of both Fabaceae (n=15) and Asteraceae (n=6) were colonised; as well as 54% of Eriocaulaceae (n=28); and the highest value of colonisation, 75%, was encountered for Proteaceae (n=8), represented in this study solely by Roupala montana. There was also a wide range of colonisation values observed across study sites. Of the collected specimens in CB-1, 18% were colonised (n=11); 21% in CB-2 (n=24); 83% in CP-1 (n=6); 60% in CP-2 (n=5); 7% in RP-1 (n=15); 60% in RP-2 (n=5); 75% in RP-3 (n=4); 78% in CR-1 (n=9) and 100% in CR-2 (n=1).

From the results collected, it was not possible to determine whether there is an association between root specialisations and P resorption.

62 DISCUSSION

Our findings demonstrate that the soils in the cerrado present low or sometimes undetectable levels of readily available P (below 2 mg P kg-1 soil). In addition, we also measured high levels of Al and Fe, and a pH in a very acidic range that accounts for the low levels of readily available P. The sandy soils with such properties in an environment that has a marked dry season could be challenging for plant growth. However, the native species we have studied in these locations exhibit several traits that allow for the occupation of such an environment. We have discovered root clusters, profusely haired roots, sand-binding roots and mycorrhizal associations in the studied taxa. We also measured leaf nutrient concentrations and found some of the highest ever- recorded values of P-resorption efficiency.

Fire is a driving factor of cerrado ecology, and therefore, of rupestrian fields ecology. In this work, we have not gathered evidence of fire history or included it as a nominal factor. Instead, we accept fire as part of the system and its influences on the nutrient cycles and study the resulting conditions.

This is the first, albeit preliminary, survey of cerrado plants with a focus on plant nutrition. Our findings allow for a comparison with other nutrient-impoverished ecosystems.

Soil analyses

We encountered heterogeneity of the physical and chemical properties of the soils sampled. This was expected, since numerous studies report spatial heterogeneity regardless of the scale studied (Facelli and Facelli, 2002). We did not, however, find a gradient of soil fertility between conservation units – in fact, readily available P was not significantly different between most sites.

Sandy soils with low clay content and organic matter, such as found at the majority of our sites, are representative of rupestrian fields (Benites et al., 2007). Coarser soil texture increases soil drainage and consequently affects nutrient availability and nutrient uptake by the roots. The low pH, ranging between 3.4 and 4.3, is commonly associated with a very low availability of P, Ca, Mg and K; it can also cause Al and Mn toxicity effects (Marschner, 1991). The low availability of P in rupestrian field soils can be partly explained by the high levels of Al and Fe, as well as the low pH (Holford, 1997).

63 Readily available soil P was measured with the resin method, which is considered to be the most appropriate method when soil P values are very low and has been used in the cerrado by other researchers (Raij et al., 1994). Readily available soil P was also measured by the Bray-II method, suitable for the acidic soils of the rupestrian fields; this is an alternative technique that allows for comparison with soil data from other regions, such as South Africa. The results of the two analyses did not correspond perfectly: the resin-extractable P results showed a significant difference amongst some sites, whereas no significant difference between sites was detected by the Bray-II method. Independent of which set of results is analysed, it is possible to affirm that, although not as extremely low as south-western Australian kwongan or Cape fynbos, the available P in the rupestrian fields we have studied is low. The low results encountered in rupestrian fields agree with the prediction that white sandy soils resulting from in situ erosion of quartzite in an ancient geological formation dating between 950 and 650 million years ago (Alves and Kolbek, 1994) would be P impoverished.

The present results cannot be considered representative for the entire cerrado biome, which comprises several formations (Eiten, 1972), and extends over two million km2 (Klink and Machado, 2005). It is also a biome that is located over several different geomorphic surfaces and types of soil (Marques et al., 2004). Our results of extractable P and percentage of clay are lower than those found by other researchers in different formation types of cerrado (Cruz Ruggiero et al., 2002), as well as different areas of rupestrian fields (Conceição et al., 2007). However, a study in the Cipó Reserve has also found values of resin-extractable P below 1 mg kg-1 soil (Negreiros et al., 2008) and between 2 and 3 mg kg-1 soil (Benites et al., 2007), corroborating our results that these are particularly P-impoverished areas.

When worldwide data are included in the comparison, soils from rupestrian fields in central Brazil are still low in [P], but they are not the most P-impoverished. Soils in Australia are remarkably low in P (Beadle, 1966) and in south-western Australia, topsoils are particularly P-impoverished; in WA sandplains, readily-available P values are commonly between 1 and 3 mg P kg-1 soil (Jeschke and Pate, 1995, Samadi and Gilkes, 1998, Fisher et al., 2006), but can be found under 1 mg P kg-1 soil in many sites (Lambers et al., 2010, Lambers et al., 2006).

Fynbos soils in the Cape floristic region are also impoverished in P, and could be placed between those levels found in Australian kwongan and in the rupestrian fields in Brazil.

64 In different studies in the fynbos, soil P ranges between 0.4 and 2.5 mg resin- extractable P kg-1 soil (Mitchell et al., 1984); between 0.1 and 3.2 mg resin-extractable P kg-1 soil for fynbos areas (Witkowski and Mitchell, 1987); and between 61 and 95 mg total P kg-1 soil (Richards et al., 1997). A recent review using a large database found an average of 87 mg total P kg-1 soil for the fynbos in the Cape (Stock and Verboom, 2011). To allow for comparison, we consider total P as 20-fold readily-available P (Lambers et al. 2010).

With these values, it is possible to consider that the kwongan in south-western Australia, the fynbos in South Africa and the rupestrian fields in Brazil share similar environments in terms of soil conditions, but with a slightly increasing level of soil P fertility (Table 9).

Leaf N and P concentrations

With regards to leaf N concentration ([N]), our survey revealed a range of 5.5 to 28.5 mg N g-1 leaf DW, and an average of 12.7 mg N g-1 leaf DW. We would like to point out that most species in our survey are herbaceous, and the leaf nutrient concentrations are affected by growth forms (Wright et al., 2004b, Poorter et al., 2009). Fabaceae were above our average of rupestrian field species; which is expected since many of them are potentially N2-fixing species. Cyperaceae also showed relatively high leaf [N], whereas Asteraceae, Proteaceae and all Xyridaceae showed leaf [N] values below average. In a global comparison, the leaf [N] of the rupestrian field species included in this study is just below the world average of 12.9 mg N g-1 leaf DW; and markedly higher than south-western Australian and Cape flora values (Lambers et al., 2010, Stock and Verboom, 2011). Such high leaf [N] values can be partly explained by the constant presence of potentially N2-fixing legumes in rupestrian fields. Depending on the site, this family can vary from only a minor representative of the flora (Conceição et al., 2007) to one of the families with the largest number of species (Amaral et al., 2006, Giulietti et al., 1987).

65 Table 9: Comparison of soil [P], leaf [P], leaf N:P ratio and leaf LMA between the rupestrian fields, Brazil; south-western Australia and the Cape floristic region, South Africa. Values presented are averages with the range in brackets. Two groups of soil data from rupestrian fields are presented; according to analysis methodology (see Material and Methods). The symbol * defines data originally presented as total P and converted to readily available P by dividing by 20 (Lambers et al. 2010), and ** signifies that value presented here is an average of multiple sources within the cited paper.

Readily available soil P Leaf [P] Leaf N:P ratio LMA (mg P kg-1 dry soil) (mg P g-1 leaf DW) (g m-2) 3.4 (UDL-10.0); resin 0.54 (0.13-1.17) 29 (14-61) 167 (46-523) Rupestrian fields, Brazil (this study) 9.1 (6.0-17.0), Bray-II (Lambers et al. 2010) (0.4-6.0) 0.26 ** 22.4 ** 409 ** South-western Australia (Stock & Verboom 2011) 4.5 (0.7-27.0)* 0.5 (0.1-3.8) 16.6 (1.0-82.1) (Lambers et al. 2010) 0.35 ** 24.6 ** 260 ** Cape region, South Africa (Stock & Verboom 2011) 4.4 (0.9-15.5)* 0.3 (0.1-1.1) 22 (10-80)

66 The P concentration in mature leaves in this survey ranged from 0.13 to 1.17 mg P g-1 leaf DW, with an average of 0.54 mg P g-1 leaf DW. All collected species of Cyperaceae, Fabaceae and Velloziaceae showed values above the average. Roupala montana, the only species in which a cluster root was found, had lower than average leaf [P] (0.50 mg P g-1 leaf DW). Our results showed a lower range than those of woody cerrado species (0.3 – 1.5 mg P g-1 leaf DW) (Araújo and Haridasan, 2007). The results presented in our survey are somewhat higher than those for plants in south-western Australia (0.26 mg P g-1 leaf DW) and Cape flora, South Africa (0.34 mg P g-1 leaf DW), but are almost half of the world average of 1.02 mg P g-1 leaf DW (Lambers et al., 2010). When the review of Stock and Verboom (2011) is considered, however, the values change in order, and 0.5 mg g-1 is the leaf [P] averaged for Western Australia and a lower value of 0.3 mg P g-1 leaf DW is the average for the Cape. The discrepancy between the reviews of Lambers et al. (2010) and of Stock and Verboom (2011) could have been caused by the inclusion of different datasets, the extent of the area considered in each case or by how this data are averaged (individuals or species). Regardless of the review used for comparison, the values found in Brazilian rupestrian fields are higher than those of Western Australia and the Cape floristic region (Table 9).

In terms of P-resorption efficiency, there are no reviews on large groups of species from different biomes around the world. Rather, values of specific families and regions are presented in individual publications, which make the comparison very biased. Nonetheless, data from this survey show a great range of P-resorption efficiencies, ranging from 23 to 95%, which is not abnormal, considering P resorption efficiencies of forest species also present great variation (McGroddy et al., 2004). The maximum value obtained in our research is an extremely high value, since the highest P-resorption efficiency value in the literature is 89% (Wright and Westoby, 2003).

The P-resorption efficiency average in this survey was 66%. The average value in the present study is very similar to that found in Australian nutrient-poor, water-limited sites: 63% P-resorption efficiency (Wright and Westoby, 2003), but higher than the 50% average found in a large dataset with focus on North American and European sites (Aerts, 1996). Although the P-resorption efficiency values in the present study must not be taken as a representative value for the region, they are a first step towards the knowledge of P resorption in the rupestrian fields.

Proficiency values found for the sampled species in the campo rupestre had an enormous variation: from 27 to 657 µg P g-1 leaf DW. Half of these values fall under

67 130 µg P g-1 leaf DW and are comparable to those found for Banksia species (Denton et al., 2007) which live in the extremely P-impoverished environment of south-western Australia. The average of all species collected in this study was of 187 µg P g-1 leaf DW, which is very similar to the Australian species average (Wright and Westoby, 2003) and much lower than the world average resorption proficiency of 450 µg P g-1 leaf DW (Killingbeck, 1996). The highest values of residual P in senesced leaves in this survey were not restricted to a particular family. In fact, congeneric species in this study presented very different P-resorption proficiency results. Although our measurements are of realised and not potential resorption proficiency (Killingbeck, 1996), they differ enough to suggest that P-resorption proficiency is not likely to be phylogenetically related.

Nitrogen to phosphorus ratio

In the species collected in the rupestrian fields, the range of nitrogen to phosphorus ratio (N:P) was 14 to 61; with a species average of 29. The variation of results between species and genera was pronounced (e.g., Eriocaulaceae and Xyridaceae).

With the exception of two species whose ratio was 14 (Paepalanthus argyropus and Xyridaceae “new species 56”), all other 28 species had a N:P ratio of 16 or above, indicating that P is the limiting nutrient for plant growth in the rupestrian fields (Koerselman and Meuleman, 1996). This agrees with the low soil [P] data.

Leaf N and P are known to correlate strongly across species and functional classifications, as leaf P is stoichiometrically related to leaf N (Watanabe et al., 2007), but at vegetation level, broad generalisations can be drawn – N:P ratios lower than 10 and higher than 20 correspond to N or P limitations, respectively (Gusewell, 2004). When rupestrian field species are put in a global perspective, we observe that the average N:P ratio for the selected species in this study (29) is higher than the averages in other P-impoverished environments (Table 9). For south-western Australia, the calculated average N:P ratios were 24 (Lambers et al., 2010) and 17; with a range of 1 to 82 (Stock and Verboom, 2011). For the Cape floristic region, the calculated values were 25 (Lambers et al., 2010) and 22; with a range of 10 to 80 (Stock and Verboom, 2011).

Leaf mass per area index

68 Leaf mass per area (LMA) is a factor that is correlated to some other characteristics of plants. Habit, for instance, influences LMA, in that herbs and graminoids tend to have lower LMAs than evergreen species, for instance (Poorter et al., 2009). There are several other factors that influence LMA, such as radiation, atmospheric composition, water, temperature, salinity, soil compaction and also nutrients (Poorter et al., 2009).

Rupestrian field species are commonly xeromorphic (Alves and Kolbek, 1994) and possess a high LMA. The LMA values presented here ranged between 46 and 523 g m-2, and it must be pointed out that most of the species we collected were herbaceous, with the exception of Lychnophora sp. and Roupala montana – these would tend to decrease the LMA values found, which was not the case. The two highest values (497 and 523 g m-2) are for two Vellozia species, which are very characteristic genus of rupestrian fields (Conceição et al., 2007), especially in the Espinhaço Range (Franceschinelli et al., 2006).

As with N:P data, LMA is not necessarily a consistent characteristic of a family. In the families with larger numbers of species samples in this study, such as Fabaceae, Eriocaulaceae and Xyridaceae, the variation is evident.

There are records of LMA between 14 and 1500 g m-2 worldwide (Wright et al., 2004b), with a global average of 144 g m-2 (Lambers et al., 2010). In dry, P-impoverished landscapes, associated with higher leaf N:P ratios, high LMA values are expected. In the Cape floristic region, the average LMA is 241 g m-2; in Australia, it is 268 g m-2, whereas in south-western Australia it is 411 g m-2 (Lambers et al., 2010). In such a context, the present average LMA of 167 g m-2 in the rupestrian fields is relatively low (Table 9). Amongst the possible reasons for this low average LMA are that: (1) despite a pronounced dry season, rainfall in rupestrian fields is still higher than in the Cape or south-western Australia, with 1300 mm rainfall per year; and (2) most of the species we have collected are herbaceous, and the growth form affects LMA (Poorter et al., 2009). Araújo and Haridasan (2007) encountered LMA values between 91 and 333 g m-2 for 15 woody cerrado species. A more extensive screening of the species that occur in the rupestrian fields is needed in order to assess whether our data are biased by sampling.

69 Root specialisations

Mycorrhizas

The two families in this study that exhibited no arbuscular mycorrhizal associations were Cyperaceae and Velloziaceae. In the families Eriocaulaceae, Fabaceae and Xyridaceae, some, but not all species had individuals colonised by arbuscular mycorrhizal fungi. Asteraceae and Proteaceae, both with just one species targeted, had individuals positive and negative for mycorrhizal infection. It is important to point out that the absence of mycorrhizas in a sample does not mean the species from which that sample was collected is invariably non-mycorrhizal.

Mycorrhizas are a prevalent symbiosis, occurring worldwide in 82% of all vascular plants, and arbuscular mycorrhizas (AM) are the most common, occurring in 73% of all taxa (Brundrett, 2009). However, in particular environments, the dominance of a certain group of species or soil conditions may affect this proportion. For Western Australia and the Cape region, the proportion of plants with AM is smaller and there is an increase in the non-mycorrhizal fraction of species (Brundrett, 2009, Lambers et al., 2010).

No extensive research on mycorrhizal colonisation has been done in the plant species of rupestrian fields. In our work, limited to a few families, we have observed AM in only 38% of individuals analysed, and in at least one individual of 47% of the species. Most interestingly, the two locations where there was no detectable resin-extractable P (CB-1 and RP-1) were also the two sites with the least occurrence of AM. This observation reinforced previous experimental observations that arbuscular mycorrhizal associations are favoured in intermediate P levels (Siqueira and Saggin-Júnior, 2001, Reddell et al., 1997) (Lambers et al., 2008)

In the Cipó region, where our resin-extractable P results show moderate values of readily available P (3 to 4.5 mg P kg-1 soil), our counts showed much higher levels of mycorrhizal colonisation: 83 and 60% of individuals in the two sites. The spores in the soil of the Cipó have previously been studied, and an astounding 49 species of AM were found there, which represents 23% of the AM species described worldwide (Carvalho, 2010). We believe that a screening approach is necessary to reveal what role arbuscular mycorrhizas play in the rupestrian fields in general; including not only the most representative species, but all species in an area.

70 In terms of Proteaceae, in particular, we have found AM associations in five out of seven Roupala montana specimens collected. Colonisation was up to 47% in RP-3 site. This is remarkable, because Proteaceae are acknowledged as a non-mycorrhizal family (Brundrett and Abbott, 1991, Skene, 1998); other studies in Brazil have also observed mycorrhizas in R. montana (Dettman et al., 2007) and in R. luscens (Thomazini, 1974).

Sand-binding roots

Sand-binding roots were found in Eriocaulaceae species in the rupestrian fields, with a very similar structure detailed for south-western Australian Restionaceae Lyginia barbata (Shane et al., 2009) and several species of Haemodoraceae (Smith et al., 2011). Amongst the species sampled, five out of 12 Eriocaulaceae species possessed sand- binding roots. These were most pronounced for Actinocephalus bongardii, A. cabralensis and A. polianthus, but also clear in Paepalanthus erectifolius and P. geniculatus. When the specimens in the herbarium of the University of Campinas, Brazil, were consulted, we found many pressed individuals of the same species with sand-binding roots.

Water stress and potentially nutrient deficiency (Smith et al., 2011, Shane et al., 2009) have been raised as evolutionary forces for the development of sand-binding roots. We have not explored whether this is the case for the Eriocaulaceae species, and only with further ecophysiological research will it be possible to unravel if the rupestrian field species are functionally analogous to the south-western Australian species.

In a number of other species, such as Xyris obcordata, roots were found with sand firmly attached to them that resisted washing and even scraping. However, these did not form a sheath, and are most likely the result of local exudation. Up to date, there have been no studies on root exudates of rupestrian field species. Given the low nutritional status of the soils and the circumstantial evidence of root exudation through the samples collected, we believe this is common in these regions, and that a study of exudates would assist in further understanding of rupestrian species’ nutrition.

Root clusters

This survey shows the first record of a root cluster in field conditions in Brazil. Clusters were found in only one individual of Roupala montana (Proteaceae). The occurrence of root clusters in R. montana was supported by the germination of seeds of this species in

71 washed sand. After ca. 3 months, most of the seedlings had small root clusters (Pedro Brancalion, unpubl.).

Different to the simple and compound root clusters of South African and Western Australian Proteaceae (Shane and Lambers, 2005), the root clusters found in R. montana are inconspicuous, with less density of hairs and in a marginally organised structure. They were found ca. 5 cm deep in the soil, unlike the Proteaceae and Viminaria juncea which commonly form a mat of root clusters under the leaf litter (Lamont, 2003). The root clusters of R. montana are visually much more similar to the clusters of Fabaceae species, such as Lupinus albus (Dinkelaker et al., 1995). We were able to bring to light that root clusters also occur in Proteaceae in the rupestrian fields. This implies that the level of P in the soil was not high enough to suppress the formation of such a structure. It is essential to note, however, that Proteaceae are not as significant a family in the rupestrian fields as they are in south-western Australia or in the Cape region in South Africa. Since R. montana is also a species that forms associations with mycorrhizas, we bring up the question of how these two adaptations interact with each other and what is their proportional relevance to the P nutrition of this species.

Other unusual root structures

In our excavations, we have found other interesting structures. We cannot affirm that these structures are related to P nutrition, but given the low availability of P in the soil, it is a possibility. In our root samples, there were also numerous samples with an extensive cover of very fine hairs. In fact, more than half of the species (17 of 31) were covered over the whole root with fine clear hairs. These often had sand granules firmly attached to them which did not come loose when rinsed with water, suggesting these hairs may be releasing exudates.

In a specimen of Cactaceae (unidentified species) and in Cyper sp., cluster-like structures were found. The structures observed (Figure 5c and 5d), when excavated, have blocks of sand attached to the roots in particular clusters, but after being washed, look as normal roots, not arranged in a particular way but clustered in sections of the primary root axis.

Our understanding of such structures is that there is not a boundary between a “normal” root and a root cluster, but a large array of strategies that look like a gradient: from the non-specialised, straight root with root hairs only at the root cap to fully haired roots, to pseudo-clusters (or cluster-like), to simple clusters, and finally, compound cluster roots (Table 10), and also different levels of exudation in these root clusters or cluster-like 72 structures. They may be very different in their ontogeny, but they appear to conform functionally to the gradient of soil P-impoverishment.

An association between roots specialisations and P resorption could not be drawn from the data collected here. The presence of structural root specialisation, such as nodules, root clusters or cluster-like structures and sand-binding roots was prevalent in the species sampled, and not enough species without specialisations were collected for a correlation to be drawn. The comparison to mycorrhizal colonisation was also not possible, since mycorrhizal colonisation data varied largely between samples of the same species (Table 8), whereas P resorption efficiency was calculated as an average for each species. This question remains open and could be explored by future research based on the preliminary results delivered here.

One of the most intriguing and unusual structures found was the cotton-like structure of Syngonanthus niveus (Figure 5e). This was observed in two specimens, but we could not further analyse these structures, as the material had to be used in other analyses and would be insufficient for an anatomical description.

73 Table 10: Types of structural roots, example of species of occurence and its image.

Type of root Species Picture

“Normal” Xyris tenella

Prolific hairs Syngonanthus verticillatus

Sand-binding Paepalanthus erectifolius

Cluster-like or Cactaceae sp. Pseudo-cluster

Simple cluster Roupala montana

Compound Banksia menziesii cluster

74 CONCLUSIONS

Our survey in the rupestrian fields unveiled many novel findings: the first record of a cluster root in natural field conditions in Brazil; sand-binding structures in Eriocaulaceae species; “cluster-like roots”; and the cotton-like roots yet to be described. Considering the results of soil resin-extractable P and knowledge from other nutrient- impoverished locations worldwide, we propose that these structures are very likely to be linked to P nutrition. Based on our data, it was not possible to draw a correlation between root specialisations and P resorption; and we suggest the preliminary information discovered in this study is used as basis for a more thorough and extensive study on the matter.

The nine study sites in four conservation units in the Espinhaço Range in Minas Gerais have higher levels of readily extractable P in the soils than south-western Australia or the Cape floristic region, but they are still P-impoverished in global terms. The low levels of soil P are reflected not only in the root specialisations, but also in the species’ very high N:P ratios and high P-resorption efficiency from senescing leaves.

We have positioned the white sand areas of the rupestrian fields as functionally equivalent to the kwongan in south-western Australia and the fynbos in the Cape floristic region in South Africa, despite the fact that the species composition is markedly different.

The rupestrian fields are remarkably intricate ecosystems and still very unexplored by science. In doing this preliminary ecophysiological work in the field of plant P- nutrition, we hope to draw attention to this threatened ecosystem that is incredibly rich in species and full of adaptative mechanisms yet to be discovered.

ACKNOWLEDGEMENTS

Thanks to the University of Western Australia for the IPRS/SIRF PhD scholarship for the first author; and to the School of Plant Biology for the funding and infrastructure provided. This research was supported by the Australian Research Council and additional grants were given by the Brazilian National Research Council (CNPq), ANZ Holsworth Wildlife Foundation and The Mary Janet Lindsay of Yanchep Memorial Fund. We also thank the volunteers who have helped with field work and sample analyses: Ana Luíza Muler, Maria Cecília Alvim Penteado, Hugo Galvão, Caio Guilherme Pereira and Ricardo Viani; and the taxonomist who helped with the

75 specimens identification: Juliana Souza Silva, Marcelo Trovó, Maria das Graças Lapa Wanderley and Dr. Renato de Mello-Silva. We also thank the Parks and Reserves which have allowed us to work in their area: Bold Park (Botanic Gardens and Parks Authority), Parque Nacional da Serra do Cipó, Parque Estadual do Rio Preto, Parque Estadual da Serra do Cabral and Santuário do Caraça (Reserva Particular) and to the Forestry Insitute of Minas Gerais (IEF-MG).

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79 Chapter 3

Mariana C. R. de Campos1, Stuart J. Pearse1, Rafael S. Oliveira1,2 and Hans Lambers1

1. School of Plant Biology, University of Western Australia. 35 Stirling Hwy, Crawley 6009. Australia.

2. Departamento de Biologia Vegetal, Universidade Estadual de Campinas. Rua Monteiro Lobato 255, Campinas 13083-862. Brazil.

This chapter is currently under preparation as an article

for submission to New Phytologist.

ABSTRACT

The south-western Australian legume species Viminaria juncea (Schrad. & J.C.Wendl.) Hoffmanns is unusual in that it forms both cluster roots and mycorrhizal associations. Our aim was to identify if these root specialisations are expressed at differential supplies of phosphorus (P) and at different shoot P concentrations [P].

Seedlings were planted in sealed pots and provided 0.5 g of mycorrhizal inoculum and basal nutrients plus one of 21 P treatments (n=7), ranging from 0 to 50 mg P kg-1 dry soil. Plants were harvested at 12 weeks after planting, and roots, shoots and cluster roots were measured for length, fresh and dry weight. The number of cluster roots and the percentage of mycorrhizal colonisation were also counted; and shoot [P] was calculated. Shoot biomass accumulation increased with increasing P supply until a shoot DW of 3 g was reached at a P supply of 27.5 mg P kg-1 dry soil. Mycorrhizal colonisation was highest (ca. 30%) at 2.5-7.5 mg P kg-1 dry soil, and then decreased with increasing P supply without disappearing completely. The number of cluster roots per plant was, on average, 28; remarkably, it dropped only marginally with increasing P supply. Most intriguingly, shoot [P] was not significantly different across treatments (p=0.17), with an average of 1.4 mg P kg-1 shoot dry weight.

The virtually constant shoot [P] in V. juncea over the very wide range of P supplies is unprecedented. To maintain these stable values, this species down-regulates its growth

80 rate when no P is supplied; conversely, it down-regulates its P-uptake capacity very actively at the highest P supplies, when its maximum growth rate has been reached. We propose that the persistence of cluster roots and mycorrhizal colonisation up to the highest P treatments is a consequence of its carefully controlled shoot [P]. The presence of both P-acquiring adaptations and a strong down-regulating mechanism of P-uptake capacity demonstrate atypical ecophysiology. We speculate that this may be related to the habitat of this N2-fixing species, a relatively nutrient-rich micro-habitat in an extremely P-impoverished landscape.

Keywords: cluster roots, legume, shoot phosphorus concentration, mycorrhiza, phosphorus supply, Viminaria juncea.

INTRODUCTION

Soils impoverished of phosphorus (P) are very common in ancient, highly weathered landscapes (Lambers et al., 2008). South-western Australia is one of the most P- impoverished regions on Earth (Handreck, 1997), but also harbours one of the world’s hotspots of biodiversity (Myers et al., 2000). Many soils in south-western Australia typically have less than 1 mg of readily available P kg-1 dry soil (Singh and Gilkes, 1991, Lambers et al., 2006, Lambers et al., 2010).

Native species in south-western Australia often exhibit a variety of P-conservation mechanisms, including long leaf longevity, high degree of sclerophylly, high photosynthetic P-use efficiency and high P-resorption efficiency (Lambers et al., 2010). Mechanisms that enhance P uptake, such as mycorrhizal associations and cluster roots (Lamont, 1982) are present in the most abundant taxa in south-western Australia, and these root modifications that enhance P uptake can be divided into two groups of strategies: the scavenging mycorrhizal associations and the mining cluster roots (Lambers et al., 2008).

Cluster roots (also known as proteoid roots, after the family in which they were first discovered) are dense clusters of rootlets of limited growth (Purnell, 1960). They increase the root surface area and exude carboxylates, which allow the plant to mine small patches of P from soil in which P is not readily available to most plants. Cluster roots can be simple or compound and vary greatly in size and longevity (Lamont, 1983, Dinkelaker et al., 1995, Shane and Lambers, 2005). Cluster roots have been reported in a wide range of species, genera and families (Lambers et al., 2006), but have been

81 researched most extensively in Proteaceae native to Australia and South Africa and in Lupinus (Fabaceae), particularly Lupinus albus L. (Gardner and Parbery, 1981, Marschner et al., 2002, Shane et al., 2003b). Cluster roots vary in their appearance, and cluster roots of V. juncea are classified as simple cluster roots, as only one order of rootlets emerges from the axis (Lamont, 1972c). The cluster roots of V. juncea observed in our experiment ranged from 3 to 20 mm.

Increasing P supply decreases the formation of root clusters in the field or in glasshouse experiments (Lamont, 1972a, Crocker and Schwintzer, 1993, Crocker and Schwintzer, 1994, Shane and Lambers, 2005, Zúñiga-Feest et al., 2009) for a number of species from different families on different continents. In their 1994 work, Crocker and Schwintzer found no relationship between the leaf [P] of Myrica (now Morella) gale L. and the occurrence of cluster roots, but this finding was contradicted by further research on other species (Louis et al., 1990, Shane et al., 2003a, Dinkelaker et al., 1995) using foliar application of P or split-root systems.

Arbuscular mycorrhizas form a very common symbiosis with terrestrial plants; these associations between fungi and plants are present in ca. 73% of all terrestrial plant species (Brundrett, 2009). When it comes to P acquisition, mycorrhizal plants are considered scavengers (Lambers et al., 2008). The hyphae effectively increase the nutrient acquiring surface of the root in return for plant-derived carbohydrates (Marschner, 1998, Smith et al., 2011b); they allow for faster and more comprehensive exploitation of the soil, accessing pools of nutrients that roots would be otherwise unable to access (Tibbett, 2000).

Although both mycorrhizal and cluster-root strategies increase P uptake by the plants, there is often a prevalence of one or the other, according to the level of P in the soil. Extremely P-impoverished soils in south-western Australia are most commonly inhabited by non-mycorrhizal, cluster-root bearing species, such as Proteaceae and Cyperaceae (Lambers et al., 2010). As the P levels increase slightly, the ecology changes and mycorrhizal species such as Myrtaceae become more abundant (Lambers et al., 2006).

Some cluster-root forming species have been reported to have mycorrhizal associations, (Skene, 1998), but there is very limited information on the ecophysiology of species capable of a mycorrhizal symbiosis as well as cluster-root formation. These species provide the opportunity to explore the relationship between P supply, tissue-P dynamics and P acquisition of both root specialisations within the same species, as dependent on P

82 supply. Casuarina cunninghamiana Miq., a cluster-root and mycorrhizal species has been previously studied (Reddell et al., 1997) and from an experiment with six levels of P (0.1 to 250 mg P kg-1 soil), it was observed that both mycorrhizal colonisation and the number of cluster roots decreased with an increase in P supply from 0.1 to 10 mg P kg-1 soil, but from that point on, both colonisation and cluster-root formation decreased.

Viminaria juncea, an Australian native species of Fabaceae forms both cluster roots (Lamont, 1972b) and is colonised by arbuscular mycorrhizas (Brundrett and Abbott, 1991). Due to its multiple P-acquisition root specialisations, V. juncea was chosen as our experimental species to investigate whether cluster roots were expressed at the very lowest P supply, to be replaced by mycorrhizal associations at slightly higher soil P levels. Both P-acquisition mechanisms are expected to be suppressed at even higher P supply, as generally found for mycorrhizal associations (Pairunan et al., 1980, Bougher et al., 1990, Bolan, 1991, Aggangan et al., 1996, Reddell et al., 1997, Valentine et al., 2001) and cluster roots (Lamont, 1972a, Crocker and Schwintzer, 1993, Crocker and Schwintzer, 1994, Shane and Lambers, 2005, Zúñiga-Feest et al., 2009).

We hypothesise that as P supply in soil is increased, shoot [P] will increase, providing the signal to produce fewer cluster roots and greater mycorrhizal colonisation. By providing a much finer range of soil P supplies than used for Casuarina cunninghamina (Reddell et al., 1997), we aimed to determine a ‘turning point’ where the mining cluster- root formation strategy is replaced by the strategy of scavenging mycorrhizal association.

MATERIAL AND METHODS

Species characterisation

Viminaria juncea (Schrad. & J.C. Wendl.) Hoffmanns is an erect, often weeping shrub that may reach up to six metres in height, and occurs in forest ecosystems, open woodlands and heathlands near lakes and swamps, river banks and winter-wet depressions (Walker and Pate, 1986, Herbarium, 1998-). It is a species endemic to Australia (Walsh and Entwisle, 1996). In its early development, V. juncea seedlings lose their leaves and the modified petioles (phyllodes) replace them as the photosynthesising structures in the adult plants. The germination of its seeds is facilitated by heat shock, which concurs with the fact that this is a species native to fire-prone environments (Bell, 1999). It is also a species commonly found in disturbed areas, at a lower level in the

83 landscape (Figure 1a), suggesting it may have higher nutrient requirements and/or tolerance than, for instance, P-sensitive Banksia species (Figure 1b).

a b

Figure 1: Two locations at Yule Brook Reserve (Goble-Garratt et al., 1981). Panel (a), lower land where Viminaria juncea is abundant and where invasive species occur. Note Banksia stand in the background. Panel (b), area where Banksia attenuata and B. menziesii occur, as well as several other native species, but with no V. juncea specimens. Area ‘b’ is elevated ca. 2 metres higher than area ‘a’.

Glasshouse work

Seeds of Viminaria juncea were purchased from a local supplier (Nindethana Australian Seeds). Their provenance is not known, but the phenotypic variation in the grown seedlings was large and we assume they were genetically distinct. The seeds had their dormancy broken through hot-water treatment prior to germination. The hot-water treatment consisted of placing the seeds in a sieve and dipping them in boiling water for ten seconds. They were distributed on Petri dishes with filter paper immediately afterwards, and placed in a 15˚C chamber in May 2010. The Petri dishes were kept moist until germination.

84 In order to expose the young experimental seedlings to mycorrhizal fungal spores found in their natural habitat, the germinated seeds were planted into seedling trays filled with field soil. This soil was collected from Yule Brook reserve, adjacent to the roots of naturally growing V. juncea individuals (Goble-Garratt et al., 1981).

After 12 weeks, most of the seedlings had reached 4 cm in height and had at least 4 phyllodes. At this stage (1 October 2009) they were removed from the seedlings trays. The roots were shaken to remove excess field soil, but not washed, and the seedlings were transplanted into individual 2-litre pots. These sealed pots were lined with clear plastic to prevent any leaching and filled with 1.5 kg of sterilised, washed river sand.

Each pot received an addition of 1 g of Glomus sp. inoculum at the tip of the root; and a basal nutrient solution without phosphorus containing (per kilogram of soil): 100 mg

CaCl2.2H2O; 140 mg K2SO4; 70 mg NH4NO3; 80 mg MgSO4.7H2O; 15 mg

MnSO4.4H2O; 10 mg ZnSO4.7H2O; 5 mg CuSO4.5H2O; 0.7 mg H3BO3; 0.5 mg

CoSO4.7H2O; 0.4 mg Na2MoO4.2H2O; 20 mg FeNaEDTA. Additional doses of

NH4NO3 (70 mg) were supplied after 4, 6, 8 and 10 weeks. Plants were exposed to 21 P treatments ranging from zero to 50 mg P per kg-1 dry soil in increments of 2.5 mg P per kg-1 dry soil. Each treatment had seven replicates, and the nutrients were added by preparing a solution with deionised water and applying it to the pots.

The pots were kept in a root-cooling tank and watered with deionised water when required for the 12 week duration of P treatment. The temperature in the glasshouse was buffered by evaporative air-conditioning, and varied between 22.5 and 28.5oC during the day and between 9.5 and 21.0oC at night. The relative humidity was 30 to 65% during the day and 46 to 73% at night. The glasshouse allowed ca. 70% of the ambient light to penetrate in, and the daily peak of light intensity at plant level varied between 1450 and 2010 µmol m-2 s-1. Plants were harvested on 5 February 2010, after 12 weeks of treatment.

Harvest

All plants were harvested on the same day (5 February 2010) and had the length of their longest phyllodes measured. The plant material was separated into shoots, non-cluster roots and cluster roots. The number of cluster roots per individual was recorded, and the fresh weight (FW) of cluster roots, roots and shoots was also measured. Nodules were not taken into account in this study.

85 A root sample was removed from each individual and placed in ethanol for the mycorrhizal colonisation count. The remaining material was placed in a 60oC oven for three days, after which the dry weight (DW) of cluster roots, roots and shoots was also recorded.

In order to obtain shoot [P] results, the dried shoots were ground with mortar and pestle, digested with nitric and perchloric acid and phosphate concentrations were determined using the malachite green method (Motomizu et al., 1983) with a spectrophotometer (Multiskan Spectrum, Thermo Fischer Scientific, Massachusetts, USA).

Colonisation counts

The roots set aside for mycorrhizal analyses were prepared following the protocol used by Brundrett et al. (1996). Roots were cleared (diaphanised) with 10% (w/v) KOH in an autoclave for 20 minutes at 121oC and then stained with 0.05% (w/v) Trypan Blue and preserved in lactoglycerol.

Arbuscular mycorrhizal colonisation counts were performed under a stereomicroscope (SteREO DiscoveryV8, Carl Zeiss MicroImaging, Göttingen, Germany) with a 5x objective lens and an 8:1 zoom. A Petri dish marked with a quarter-inch grid was used to count the root intersections and the colonised root intersections. Colonisation was considered as the number of colonised roots that intersected the grid divided by the total number of roots that intersected the grid. Hyphae, arbuscules and vesicles were all considered as evidence of colonisation and were all counted together.

Statistical analyses

Analyses of variance were performed, considering the P treatment as the factor and each of the other measurements as the variables, all in one-way analyses of variance (ANOVA), using Genstat 12th Edition, 2009 (VSN International Ltd). Data with non- homogenous residual plots were log-transformed. Trendlines were checked against the data for their fit using an ANOVA on the linear regression between the original data and the predicted values of the trendline.

86 RESULTS

Growth response to P supply

Plant growth responded positively to increased P addition to the soil. Shoot DW was significantly higher (P<0.001) for the plants with greater P supply (Figure 2a). Average values for the different treatments ranged from 0.5 g to 3.5 g of shoot DW. Figure 2a shows a quasi-linear increase in shoot DW up to a treatment of 27.5 mg P kg-1 soil, when the values stabilised at around 3 g.

87 a 800 b ab ab ab ab ab ab ab ab ab ab ab ab ab ab 600 ab ab ab ab ab 400 a

200

Shoot length (mm) length Shoot 0

c 4.5 shoots b bc bc bc c roots bc bc abc bc cluster roots abc bc abc abc 3.0 abc abc abc abc abc

1.5 abc ab a Dry weight (g) weight Dry 0 0.6 a c ab abc abc abc abc abc abc abc abc 0.4 abc abc abc bc abc c abc abc abc abc abc

0.2 Root Mass Ratio 0 0 5 10 15 20 25 30 35 40 45 50 Phosphorus treatment (mg P kg-1 soil)

Figure 2: Growth of Viminaria juncea as dependent on P supply; (a) Average of total shoot length according to P treatments; (b) Root Weight Ratio as dependent on P supply; (c) shoot dry weight (DW) (green) root DW (yellow) and cluster-root DW (brown). Stacked columns give total dry weight. No root results are significantly different (roots p=0.06 and cluster roots p=0.23) between P treatments. Bars indicate standard error (n=7) and letters above columns indicate significant differences observed in the HSD Tukey test with a significance value of 0.05.

88 The root DW did not vary significantly between treatments (P=0.061) (Figure 2a). Values of root DW varied from an average of 0.5 g to 1.8 mg, and represented between 25 and 50% of the total plant weight. Significantly different values of root weight ratio (RWR, root DW divided by plant total DW) (p<0.001) were found between treatments, but the trend was weak, as RMR decreased significantly until a P supply of 15 mg P kg- 1 soil, but beyond that values were not significantly different from the no-P treatment (Figure 2b).

The lengths of the longest phyllode at harvest were significantly different (p< 0.05) for only two of the 21 P treatments. There was an increase in shoot length from ca. 291 mm in the no-P treatment to a maximum of 700 mm, in the 37.5 mg P kg-1 soil treatment. The HSD Tukey Test revealed that, with the exception of the 37.5 mg P kg-1 soil treatment, all results were not significantly different from each other, and the additional P did not lead to a greater length of the longest phyllode (Figure 2c).

Shoot phosphorus

Plants of all treatments showed remarkably little variation in their shoot phosphorus concentration, despite the very wide range of P supply; the average [P] was 1.4 mg P kg-1 shoot DW, and the range was from 1.19 to 1.68 mg P kg-1 shoot DW (Figure 3). The best-fitted model was a linear curve with a negative slope, which would indicate that the plants being supplied the highest P levels are the ones with the lowest shoot [P]. However, these results are not statistically different from each other (p=0.173), and the shoot [P] must, therefore, be considered constant, which is a remarkable result, that we have not seen reported in the literature before.

The total plant P content increased with increasing P supply (Figure 3), due to the fact that the plants accumulated more biomass with increasing P supply. The logarithmic curve fitted to the model illustrates how total plant P content increased at low to moderate P supplies, and then, from moderate to high supplies, the increase in total P saturated(Figure 2B), in agreement with the P saturation of plant growth.

89 6 a 6 a a ab a aa ab ab ab ab ab ab leaf DW) 4 ab ab ab 4 -1 ab ab ab ab 2 2

b shootTotal P (mg) Shoot [P] (mg P g Shoot [P] 0 0 0 5 10 15 20 25 30 35 40 45 50

Phosphorus treatment (mg P kg-1 soil)

Figure 3: Shoot P concentration (dark bars) and total shoot P content (light bars) as dependent on P supply. P concentration: y=-0.01x+1.53; R2=0.35; p=0.17; P content: y=1.14ln(x)+0.65; R2=0.68, curve fit p<0.005. Bars indicate standard error (n=7). Letters above bars indicate the results of a Tukey Test on total shoot P content (light blue). Shoot P concentration showed no significant variation (p=0.05).

Mycorrhizal colonisation and cluster root formation

Plants in 17 out of 21 treatments presented some form of vesicular-arbuscular mycorrhizal colonisation. Vesicles and few hyphae were observed in the specimens, but no arbuscules. Other fungi (most likely saprophytic) were also present in the roots of V. juncea.

There was an inverse correlation of -0.32 between the mycorrhizal colonisation and the level of P treatment; that is, more colonisation occurred in the lower P treatments. A significant decreasing linear trend (R2= 0.28; p<0.001) was observed with increasing P supply (Figure 4A). Similarly, there was a significant linear trend for decrease in the number of cluster roots per plant with an increase in P supply (R2= 0.14; p<0.05) (Figure 4A). However, the effect of P supply on both colonisation and cluster-root formation was marginal, compared with what has been found in the literature for other species.

No significant difference was found in the dry weight of cluster roots per plant, as dependent on P supply (Figure 2B); the values ranged from 28 to 153 mg, without any pattern or trend. Not even in the initial levels of P treatment was any pattern observed. However, there were significant differences between the ratio of cluster roots to total root dry weight. This ratio followed a logarithmic downward trend (R2=0.50; p<0.001),

90 with similar ratios for most treatments above 10 mg P g-1 soil (Figure 4B). The average size of cluster root per individual plant (data not shown) was also not significantly different between treatments (p=0.12). In our samples, no senesced cluster roots were observed.

60 30 a

40 20

20 10 % mycorrhizal colonisation mycorrhizal % Number of clusters per plant

0 0 20 b 15

Cluster to total root ratio (DW) 0 0 5 10 15 20 25 30 35 40 45 50

-1 Phosphorus treatment (mg P.kg soil)

Figure 4: Number of cluster roots per plant in red (y=-0.59x+34.07; R2=0.14; p<0.05) and percentage of mycorrhizal colonisation in blue (y=-0.45x+9.72; R2=0.28; p<0.001) as dependent on P supply (a). In (b), the ratio of cluster root weight to total root dry weight was y=- 2.86Ln(x)+12.85; R2=0.50; p<0.001. Bars indicate standard error (n = 7).

DISCUSSION

Viminaria juncea, an Australian Fabaceae species, showed very little response to an increasing P supply. Unlike most species, it did not stop the production of cluster roots at high P levels (up to 50 mg P kg-1 dry soil), neither did its mycorrhizal associations cease. Even more remarkably, increasing supply of P did not affect this species’ level of shoot [P]. We discuss some of the possible reasons and mechanisms for these unusual ecophysiological responses.

91 P supply affects growth, but does not affect shoot [P]

Viminaria juncea growth responded to additional P-supply until saturation was reached at around 27.5 mg P kg-1 dry soil; from this growth curve we infer that the range of phosphorus treatments applied was adequate and that the experiment ran for the necessary length. The differences in growth were observed solely in the biomass of shoots, not in the biomass of roots or root clusters. The most typical plant response to an increase in P supply is to show greater accumulation of biomass (de Groot et al., 2001, Müller et al., 2000, Cassman et al., 1980), but in some cases the effect on root growth is inconspicuous, as with Lupinus albus (Pearse et al., 2006b). A pronounced differential growth between shoots and roots would be reflected in the root weight ratio (RWR), such as the case with Triticum aestivum, Brassica napus and Pisum sativum (Pearse et al., 2007), but our results showed only a slight increment in shoot growth and a non- significant increase in root weight. The consequence was an apparent decrease of RWR with increasing P supply, but that was not, for most treatments, significant. RWR is, in fact, not altered with different P supplies in a number of species (Pearse et al., 2006a).

In terms of its shoot [P] V. juncea behaved in an extraordinary manner. Other species have been reported to increase leaf or shoot [P] in response to increasing P supply (Pearse et al., 2006a, Peng et al., 1993, de Groot et al., 2003). The very strong control V. juncea possesses over shoot [P] is unprecedented and implies an incredibly fine adjustment of plant growth when P supply is scarce, and on the other end of the spectrum, a powerful down-regulation of P-uptake capacity at the highest levels of P. We propose that the reason for such a fine tuning of leaf [P] is that V. juncea inhabits areas that are subject to waterlogging and could, therefore, be exposed to sporadic flushes of nutrient availability. V. juncea is a legume whose degree of nodulation decreases sharply with decreasing P supply; moreover, when it depends on symbiotic

N2 fixation as its source of N, investment in cluster roots is suppressed when investment in nodules is increased (Walker and Pate, 1986). Taken together, this might be a situation where preferential allocation of P to nodules, rather than shoots, and down- regulation of P-uptake capacity is favoured. An explanation of how this control of P uptake and leaf [P] is achieved by V. juncea is yet to be produced.

92 Root adaptations for P uptake decrease marginally with increasing P supply, and remain present even at the highest treatments.

Several studies have shown that mycorrhizal colonisation favours P uptake of plants (Koide, 1991, Aristizabal, 2008, Jakobsen et al., 1992) and when P supply increases, the percentage of root colonisation decreases (Pairunan et al., 1980, Bougher et al., 1990, Reddell et al., 1997, Valentine et al., 2001).

In our experiment with V. juncea, an increasing P supply did affect mycorrhizal colonisation, but not as expected. The decrease in colonisation was observed particularly between the P treatments of 2.5 to 12.5 mg P kg-1 dry soil; but after this point, the level of colonisation varied with no particular direction, resulting in a minute decreasing overall trend. Most importantly, at the highest levels of P, mycorrhizal colonisations of ca. 8% were observed, instead of the expected lack of infection. We argue that this lack of suppression of mycorrhizal colonisation is due to an internal control of shoot [P]. As no sharp increase in shoot [P] was observed, a control from the plant must not have been exerted or required on the mycorrhizal infection.

In the case of cluster roots, there was also a very slight decreasing trend in the number of root clusters per plant, and no significant difference in cluster root DW was observed dependent on P treatment. Similar to mycorrhizal infection, cluster roots were not suppressed at the highest levels of P, and our interpretation of this is that the stable values of shoot [P] explain why there was no suppression of the production of root clusters across P treatments.

Our findings differ from those for every other species known in the literature, including the work on cluster-root and mycorrhizal species Casuarina cunninghamiana (Reddell et al., 1997). There is a strong difference between the cluster-root formation and mycorrhizal colonisation encountered by Reddell (1997) and in the present study; as in the C. cunninghamiana work, the number of cluster roots and percentage of colonisation declined in a clear linear trend. Unfortunately, Reddell (1997) did not present data on leaf [P] which precludes us to extend our interpretation that leaf [P] would be the main control of cluster-root formation and mycorrhizal infection in C. cunninghamiana.

The role of cluster roots in V. juncea has not been studied extensively. The specimens grown for 12 weeks in the glasshouse had very small and inconspicuous clusters, which resemble those of Lupinus albus, also a legume. The resemblance extends to the fact that root clusters of L. albus are largely but not completely suppressed by the addition 93 of phosphorus in solution cultures (Shane et al., 2003b). We bring up the possibility that, as is the case of some Lupinus species (Pearse et al., 2006a), the root clusters in V. juncea could be more or less active due to their level of exudation; and that the constant presence of cluster roots could act as a mechanism to quickly respond to demand and availability of P.

Viminaria juncea is unique in its shoot [P] control and an exception to Australian native species.

A control of shoot [P] such as found for V. juncea in this experiment is not paralleled by any other species studied so far. The unaltered shoot [P] prevents the suppression of cluster roots and mycorrhizal associations, and this combination of factors could be considered as evidence of adaptation of V. juncea to its ecological niche in terms of N and P nutrition. Our reasoning is that this legume occupies areas that are P impoverished, but are subject to waterlogging and consequently a potential peak of nutrients leached from the surrounding areas. This is a species that should not be affected by P toxicity, but that would also be equipped for the periods of N and P impoverishment.

The discovery of the present precise control of shoot [P] in this legume opens up many possibilities in the study of physiological ecology, in particular the modes of regulation of shoot [P] and P uptake and allocation, and the persistence, longevity and exudation of cluster roots when P is not in short supply. The present findings are further evidence of the incredible diversity of physiological mechanisms that can be encountered. With their control of growth and P uptake, they provide an alternative to “shutting down” mycorrhizal associations and cluster-root formation when P supply is no longer restricted.

CONCLUSIONS

Cluster-root formation and mycorrhizal colonisation were positively correlated for V. juncea in a range of P treatments; and therefore, we have rejected our initial hypothesis. What we have found was that both mycorrhizal colonisation and the number of cluster roots per plant only marginally decreased and were not fully suppressed at even the highest P supply (50 mg P kg-1 dry soil).

94 We have discovered that V. juncea is unique in the fact that is has stable shoot [P] in the P treatment range of 0 to 50 mg P kg-1 soil. We have inferred that a very fine control is in place for this species, with an exceptional capacity for down-regulation of P-uptake. Furthermore, we have deduced that the constant shoot [P] is the reason why the cluster roots and mycorrhizas are not fully suppressed in the high-P treatments. All that is related to a constant shoot [P]: down-regulation of P-uptake capacity and maintenance of structures of enhanced P acquisition allow V. juncea to be ready for both situations of high or low levels of phosphorus. However, a new intriguing question is raised: if shoot [P] is constant, what parameters or mechanisms are involved in the down-regulation of P-uptake capacity?

ACKNOWLEDGEMENTS

Thanks to the University of Western Australia for the IPRS and SIRF scholarships and the School of Plant Biology for the research funding and infra-structure; to ANZ’s Holsworth Wildlife Foundation Grant and The Mary Janet Lindsay of Yanchep Memorial Fund for additional grants. This work was supported by the Australian Research Council (ARC). We are very thankful for the help and advice from Prof Erik Veneklaas and Ana Luíza Muler in germinating and cultivating the seedlings for the initial phase of the experiment.

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98 99 Chapter 4

Down-regulation of net phosphorus uptake capacity is inversely related to leaf phosphorus resorption in four species from a phosphorus impoverished environment

Mariana C. R. de Campos1, Stuart J. Pearse1, Rafael S. Oliveira1,2 and Hans Lambers1

1. School of Plant Biology, University of Western Australia. 35 Stirling Hwy, Crawley 6009. Australia.

2. Departamento de Biologia Vegetal, Universidade Estadual de Campinas. Rua Monteiro Lobato 255, Campinas 13083-862. Brazil.

This chapter is being prepared for submission to Plant, Cell & Environment. Resorption data for three additional species which was not able to be processed by the ICP-OES technicians before the deadline of this thesis will be included in the publication.

ABSTRACT

Previous research suggested a trade-off between the capacity of plants to down-regulate phosphorus (P) uptake and the efficiency of P-resorption from senescent leaves by species from P-impoverished environments. To further investigate this, four Australian native species (Banksia attenuata R.Br., B. menziesii R.Br., Acacia truncata (Burm.f.) Hoffmanns and A. xanthina Benth.) were selected and grown for 10 weeks under hydroponic conditions. Plants were supplied with a basal nutrient solution (excluding P) and one of five P treatments. Acacia plants received between 0 and 500 µM P; while Banksia plants received between 0 and 10 µM P, to avoid major P-toxicity symptoms in these highly P-sensitive species.

For both Acacia species the net P-uptake rates measured at 10 µM P decreased logarithmically with increasing P supply during growth. In contrast, B. attenuata linearly increased its net P-uptake rate from a solution with 10 µM P with increasing P supply during growth. B. menziesii showed no significant response. Leaf [P] of the four species supported this finding, with A. truncata and A. xanthina showing an increase up to a saturation value of 29 and 32 mg P g-1 leaf dry mass, respectively (at 500 µM P), whereas B. attenuata and B. menziesii both exhibited a linear increase in leaf [P], reaching 16 and 20 mg P g-1 leaf dry mass, respectively, without approaching a

100 saturation point. The Banksia specimens grown at 10 µM P showed mild symptoms of P toxicity, i.e. yellow spots in some leaves and drying and curling of the tips of the leaves.

Leaf P-resorption efficiency was 69% (B. attenuata), 73% (B. menziesii), 34% (A. truncata) and 36% (A. xanthina). Their P-resorption proficiency values were 0.08 mg P g-1 leaf dry mass (B. attenuata and B. menziesii); 0.32 mg P g-1 leaf dry mass (A. truncata); and 0.36 mg P g-1 leaf dry mass (A. xanthina). Our results support the hypothesis that the ability to resorb P from senescing leaves is inversely related to the capacity to down-regulate net P uptake. We demonstrate that species that are adapted to extremely P-impoverished soils, such as many south-western Australian Proteaceae, have developed extremely high P-resorption efficiencies, but lost their capacity to down-regulate net P-uptake mechanisms.

Keywords: down-regulation, net P uptake, nutrient-poor soils, phosphorus toxicity, remobilisation, resorption.

INTRODUCTION

Australian soils are generally characterised as containing low concentrations of phosphate with levels decreasing from east to west across the country (Beadle, 1966). In south-western Australia, bicarbonate-extractable phosphorus (P) values (“plant- available” or readily-extractable P) range from 0.9 to 47 mg kg-1 (Singh and Gilkes, 1991). The readily-extractable P content of these soils is extremely low because they have developed from low-P content parental material (such as sandstones and beach sand) and because these landscapes have been climatically buffered since the Jurassic (Hopper, 2009), without glaciations for millions of years (Lambers et al., 2008). The native species living on soils low in P must present special strategies to survive and remain competitive in these environments. These include maximising P-acquisition, P- use efficiency, and conservation of P within the plant. Specific strategies can include association with mycorrhizas, development of cluster roots, exudation of chemicals that ‘unlock’ the P from soil particles, high photosynthesis per unit of P, sclerophylly, high leaf longevity, and highly efficient P-resorption from senescing leaves (Lambers et al., 2010, Lamont, 1982, Marschner, 1998).

As a nutrient that can be limiting to growth, plants usually respond to added P with a positive response in biomass or leaf [P] (Elser et al., 2007, Ostertag, 2010). However, when in excess, P-uptake can be down-regulated as demonstrated by the addition of

101 inorganic phosphorus (Pi) or by mycorrhizal colonisation (Burleigh and Harrison, 1999). The species less able to down-regulate their P-uptake (adapted to soils with low P contents) can develop symptoms of P-toxicity when supplied with P above the normal levels in soil (Ozanne and Specht, 1981). An example arises from a study on P-uptake rates and down-regulation of Australian native species and crop plants, where extreme P-toxicity was found in Hakea prostrata R.Br. (Shane et al., 2004b, Shane and Lambers, 2006, Shane et al., 2004a).

Plants may resorb nutrients from leaves prior to their senescence, and these nutrients are mobilised into more juvenile photosynthetic structures (Foulds, 1993). There is still debate as to whether P-resorption from senescing leaves is a key nutrient-conservation mechanism (Chapin III, 1980, Aerts, 1996) or if it is not that important when compared with leaf longevity (Aerts, 1996, Reich et al., 1995, Escudero et al., 1992). In Western Australian species, both high resorption efficiency (Wright and Westoby, 2003) and high leaf longevity are often observed (Wright et al., 2004), suggesting that in ecosystems where P is extremely scarce, a combination of these mechanisms contribute to the success of the plant species that inhabit them.

When studying nutrient resorption from senescing tissues, there are two complementary parameters that can be calculated: nutrient-resorption efficiency (the percentage of nutrients that a plant can remove from its senescing leaves compared to how much is in its adult leaves), and nutrient-resorption proficiency (how little nutrient is left, in absolute terms, in the senesced leaves). Killingbeck (1996) postulated that efficiency values are best suited for resolving issues related to the conservation of nutrients and, therefore, reduction of subsequent nutrient uptake. Proficiency values, on the other hand, appear to be a more objective measure of the degree to which selection has acted to minimise nutrient loss. Attempts have been made to correlate resorption efficiencies and proficiencies with other parameters, such as soil fertility and growth forms, sometimes arriving at conflicting conclusions (Killingbeck, 1996, Aerts, 1996). There are indications, therefore, that the P-resorption efficiency (and potentially the proficiency) both influences nutrient uptake and is influenced by it.

The Proteaceae are a conspicuous family in the south-western Australia (Pate et al. 2001), and they are typically non-mycorrhizal species that are dominant on soils lowest in P content (Lambers et al., 2006). The vast majority of the species in this family make cluster roots, which release exudates to make sorbed phosphorus available for uptake (Shane and Lambers, 2005). The Fabaceae are also a conspicuous and diverse family in

102 south-western Australia, and some species are also able to form cluster roots; most are mycorrhizal and a large proportion forms root nodules. Fabaceae tend to be more tolerant of higher levels of soil P than Proteaceae (Handreck, 1997), presumably due to their ability to down-regulate their P-uptake capacity. In the present study we hypothesised that there is an inverse relationship between a species capacity to down- regulate its P-uptake system and its P-resorption efficiency and proficiency. Our hypothesis is that Banksia attenuata and B. menziesii (Proteaceae) will not be capable of down-regulation of their P-uptake capacity, and will accumulate P in their leaves until they show symptoms of P-toxicity. On the other hand, they are expected to have a very high P-resorption efficiency and withdraw P from senescing leaves to an extremely low level (high proficiency). In contrast, we expect Acacia truncata and A. xanthina (Fabaceae) to be able to down-regulate their P-uptake capacity, but to be less efficient and proficient at P-resorption. To test our hypothesis, we measured P-uptake at a standard P concentration for plants of four species grown at a range of P supplies in a glasshouse. We also measured the P concentrations in fully mature and recently senesced leaves on plants of the same species growing in their natural habitat.

MATERIAL AND METHODS

Glasshouse plant cultivation

Acacia truncata (Burm.f.) Hoffmanns, A. xanthina Benth. (Fabaceae) and Banksia attenuata R.Br. and B. menziesii R.Br. (Proteaceae) were chosen for this experiment because they are local to Perth, Western Australia and because they belong to families which are very abundant and species-rich in Australia. The choice of these four species allowed for comparisons between the families and between the genera, as well as comparisons between all individual species.

Seeds were purchased from a local nursery (Nindethana Australian Seeds) and germinated in Petri dishes with moist filter paper at 15oC. When the radicles emerged, the seedlings were transferred to trays containing sterilised, washed sand placed in a glasshouse. When they had reached the size of 3 cm (11 weeks), they were removed from the sand, gently washed and transplanted to individual pots in an aerated hydroponic system in a glasshouse on the 29th of June (beginning of winter).

The glasshouse was equipped with root-cooling tanks that kept the nutrient solutions at 16oC. The relative humidity of the glasshouse varied between 50 and 70% at night and

103 decreased to 30 to 50% during the day. The temperature ranged from 5 to 15oC at night and from 18 to 26oC during the day for the duration of the experiment. The area the plants were located in received around 70% of external sunlight, varying from a daily peak of 800 to 1650 µmol m-2 s-1. Each species grew for ten weeks under five phosphorus treatments with six replicates per treatment.

The nutrient solution contained (in µM): 200 CaNO3, 100 K2SO4, 54 MgSO4, 20 KCl, 2

Fe-EDTA, 2.4 H3BO3, 0.3 Na2MoO4, 0.24 MnSo4, 0.1 ZnSO4, and 0.02 CuSO4, made up with deionised water and with a pH of 5.8 (Shane and Lambers, 2006), but the addition of KH2PO4 for the P treatments differed with a range of [P] supplied to the plants for 10 weeks. Based on preliminary experiments, we used a range from zero to 10 µM P for the Banksia species; and for the Acacia species, expected to be more tolerant to P, we used a range from zero to 500 µM P. The P-sensitive B. attenuata and B. menziesii were given the following treatments: 0; 0.1; 1; 5 and 10 µM P; the more P- tolerant A. xanthina and A. truncata were given 0; 0.5; 5; 50 and 500 µM P (all in individual 2 litre pots). Solutions were replaced daily.

Net P-uptake determination

After ten weeks, all plants were transferred to a no phosphorus basal nutrient solution for 20 hours. On the day of harvest, they were all supplied with a [P] of 10 µM (in a total volume of 200 ml). The optimal concentration and volume of solution to measure P uptake were based on preliminary experiments and published experiments (Shane et al., 2004a).

To measure P-uptake of each individual plant, samples (1 ml) of each solution were taken every half hour from 10.30 am until 12.30 pm, and then hourly until 4.30 pm. These nutrient solution samples were stored at 5oC until analysed using the malachite green method (Motomizu et al., 1983) in a microplate spectrophotometer (MultiSkan, Thermo Scientific, Massachusetts, USA). Only the values in the linear phase (between 10.30 am and 12.30 pm) were used to calculate net P-uptake. In order to compare P- uptake rates, the P-uptake of each individual plant was divided by its root fresh weight.

All individual plants were harvested and had their stems and roots separated. Shoots and roots were weighed for fresh weight (FW). They were then dried in a 60˚C oven for three days and weighed to determine their dry weight (DW).

104 Leaf P concentration of experimental plants

To calculate the leaf P concentration, all individuals had their leaves dried, ground with mortar and pestle and homogenised. They were then subjected to a nitric-perchloric acid digestion, diluted and analysed using the malachite green method (Motomizu et al. 1983).

Resorption

To calculate the P-resorption proficiency of the same four species, fully expanded leaves (green, mature and not visibly damaged) and recently senesced leaves (either completely dry but still on the plant or on the top layer of litter) of B. attenuata, B. menziesii, A. truncata and A. xanthina were collected in Bold Park (S31.95, E115.77), a native bushland in the Perth metropolitan area. Samples from field specimens had to be collected for these analyses because these species are slow-growing and experimental plants had no senescing leaves at the end of the study. In addition to those four species, samples of Hakea prostrata were also collected. Three replicates of both mature and senesced leaves were taken for each species (each replicate was from a separate shrub or tree no closer than five metres from one another). Each replicate consisted of leaves taken from various points of the same plant. Leaves were gently brushed to remove sand and dust and then dried in an oven over three days at 60oC. The dried material was ground with mortar and pestle, digested with nitric and perchloric acid and then analysed by inductively coupled plasma mass spectrometry (ICP-OES, model Optima 7300 DV, Perkin Elmer, Massachussetts, USA).

The P-resorption efficiency was calculated as the difference between mature and senesced leaf [P] divided by mature leaf [P]. P-resorption proficiency, or the amount of P present in the senesced leaves, was also determined. All [P] values in this study are expressed on a leaf dry mass basis.

Statistics

Single-factor analyses of variance considering the P treatments as factors were conducted for all of the data, species by species and then grouped by genus; and finally with all species together. All weight measurements were log-transformed prior to the ANOVAs, since they did not have equal variances, and the residuals “fanned out”. These analyses were performed with Genstat 12th Edition, 2009 (VSN International Ltd). Means are presented with standard errors.

105

RESULTS

Effect of P treatments on growth

Acacia truncata, A. xanthina and B. menziesii exhibited a positive growth response to the addition of P to an intermediate treatment level, and then showed no further accumulation or decrease in biomass with higher levels of P (Figure 1). A. xanthina accumulated the most biomass of all species (5.04 g shoot FW and 4.08 g root FW), and A. truncata the least (1.45 g shoot FW and 1.04 g root FW). B. menziesii reached values (4.63 g shoot FW and 3.93 g root FW) approximately twice those of B. attenuata (2.80 g shoot FW and 1.61 g root FW). With the exception of B. attenuata, all species had a significant difference in dry weight between treatments (p<0.05). The highest biomass values mentioned above were not found at the highest P-treatment, but at an intermediate one. For A. truncata, it was the 50 µM P treatment that resulted in the most root and shoot weight. For A. xanthina, 5 µM and for B. menziesii, 1 µM P was optimal for growth. Fresh and dry weight analyses presented the same trends, and plant water content was on average 78%.

3 (a) Acacia truncata 4 (c) Banksia attenuata c 3 aaaa a 2 bc c 2 1 b a 1 0 0 0 0.5 5 50 500 00.11510

(b) Acacia xanthina (d) Banksia menziesii 6 c 6

Fresh weight (g) weight Fresh bc c bc bc 4 4 b ab b a

2 a 2

0 0 0 0.5 5 50 500 00.11510

[P] supplied to plants during growth (µM)

Figure 1: Fresh weight of (a) Acacia truncata, (b) A. xanthina, (c) Banksia attenuata, and (d) B. menziesii grown at five P concentration in nutrient solution (0; 0,5; 5; 50 and 500 µM P for Acacia, and 0; 0.1; 1; 5 and 10 µM P for Banksia). Dark columns represent root fresh weight (FW) and light columns represent shoot FW. Bars indicate standard errors, n=6. Letters above the bars indicate the pairwise comparison of means (p=0.05) and show that root and shoot reach maximum fresh weight at an intermediate P concentration for three species. B. attenuata was the

106 exception, showing no growth response to P supply. Note that the scales on the Y-axes differ between panels.

The root weight ratio (RWR); or root FW to total plant FW, also differed between treatments for all species except B. attenuata (Figure 2), but only presented a significant linear decrease trend for B. menziesii. RWR (g root FW g-1 plant FW) varied between 41 and 51 for A. truncata; 44 and 66 for A. xanthina; 33 and 39 for B. attenuata and 36 and 51 for B. menziesii.

Acacia truncata Acacia xanthina Banksia attenuata Banksia menziesii a 0.5 ) -1 ab a ab ab bc bc 0.4 b ab c a a ab ab 0.3 a a a a b 0.2 a

0.1 Root weightratio (g g

0.0 0 0.5 5 50 500 0 0.5 5 50 500 0 0.1 1 5 10 0 0.1 1 5 10

[P] supplied to plants during growth (µM)

Figure 2: Root weight ratio (total root dry weight as a fraction of total plant dry weight) as dependent on P concentration in the nutrient solution for Acacia truncata, A. xanthina, Banksia attenuata and B. menziesii grown at five P concentrations in nutrient solution (0; 0,5; 5; 50 and 500 µM P for Acacia, and 0; 0.1; 1; 5 and 10 µM P for Banksia). The root weight ratio of B. menziesii was the only one with a significant decrease along P treatments (p<0.001, n=6). Letters above the bars indicate significant difference in a HSD tukey test.

Leaf P concentration

Acacia species did not increase leaf [P] linearly with increasing P supply (Figure 3a), but reached a saturation point of approximately 16 mg P g-1 leaf DW. There was a linear trend for the three lowest P concentrations (0, 0.5 and 5 µM), but when the data obtained for the higher P concentrations during growth are included, the trend that would fit the data best would be a saturation curve, or logarithmic curve. There is a point, approximately 10 µM P, when accumulation of P in the leaves becomes only slight regardless of the nutrient solution concentration (Figure 3a); from that point on,

107 A. truncata increased it leaf [P] from 11 to 15 mg P g-1 leaf DW and A. xanthina, from 12 to 17 mg P g-1 leaf DW. On the other hand, both Banksia species leaf [P] increased linearly with increasing P supply during growth (Figure 3b). B. menziesii accumulated 1.3 times more P than B. attenuata in response to 10 µM P supply (Figure 3b).

8 a

leaf DW) 0

-1 050 500

Leaf [P] (mg g 8

4 b 0 01 5 10

[P] supplied to plants during growth (µM)

Figure 3: Leaf [P] plotted against the concentration of P at which the plants (n=6) were grown (n=6). On panel (a), leaf [P] in Acacia truncata (solid circles) and A. xanthina (open squares) increased linearly when P supply is up to 10 µM but then reached a plateau as plants got saturated. On panel (b), the response of Banksia attenuata (solid losangles) and B. menziesii (open triangles) was linear, and its [P] increased to a lesser extent than those of Acacia species. The equations for the fitted trendlines, squared R and significance levels are: Acacia truncata (y=1.04Ln(x)+8.38; R2=0.76; p<0.001); Acacia xanthina (y=1.28Ln(x)+9.34; R2=0.69; p<0.001); Banksia attenuata (y=0.79x+1.82; R2=0.98; p<0.001); Banksia menziesii (y=1.27x+0.96; R2=0.98; p<0.001).

P uptake

Both Acacia species significantly decreased P-uptake rates from a 10 µm P solution when grown at higher P concentrations (Figure 4a). A. truncata decreased its uptake

108 from 0.26 to 0.03 nmol P g-1 root FW s-1; and A. xanthina from 0.08 to -0.03 nmol P g-1 root FW s-1. Interestingly, six individuals of Acacia (three of each species) grown at the highest [P] (500 µM) did not take up any P during the measurement period; in fact, they showed P efflux from the root system into the solution during the measurement period, observed in the negative average of P-uptake rate by A. xanthina. In contrast to the response of the Acacia species, B. attenuata and B. menziesii did not show reduction of their P-uptake in relation to the P supply during growth (Figure 4b): B. attenuata grown with no P addition had a P-uptake of 0.023 nmol P g-1 root FW s-1 and grown at the greatest P treatment (10 µM P), an uptake of 0.036 nmol P g-1 root FW s-1. Similarly, B. menziesii had P-uptake rates of 0.026 (no P) and 0.027 nmol P g-1 root FW s-1 (10 µM P).

109

0.25 a

) 0.15 -1

0.05 root FW s FW root -1 0500 50 -0.05 500

0.06 b

0.04

0.02 Net P uptake rate (nmolrate uptake P Net g P

0 01 6 10

[P] supplied to plants during growth (µM)

Figure 4: Net P-uptake rates calculated from P-depletion of a 10 µM P solution (n=6). The X- axis shows the [P] at which plants were grown for 10 weeks. On panel (a), net P-uptake rates of Acacia truncata (solid circles) and A. xanthina (open squares) decreased significantly following a logarithmic trend. On panel (b), uptake of Banksia attenuata (solid losangles) increased and B. menziesii (open triangles) was not significantly related to the P supply during growth. The negative values in A. xanthina indicate that plants leached P into the nutrient solution. The difference between the two genera is statistically significant (significance value of 0.05), but not between the two species of the same genus. The equations for the fitted trendlines, squared R and significance levels are as follows: Acacia truncata (y=-0.02Ln(x)+0.13; R2=0.96; p=0.02); Acacia xanthina (y=-0.01Ln(x)+0.04; R2=0.79; p<0.01); Banksia attenuata (y=0.001x+0.03; R2=0.75; p=0.05); Banksia menziesii (y=0.0003x+0.02; R2=0.13; p=0.55). Note the difference in Y-axis scale.

110 Resorption of leaf P

The [P] in green, fully expanded, mature leaves and in recently senesced leaves of plants collected from plants growing in their natural habitat is shown in Figure 5. Leaf [P] of mature and senesced leaves were compared between species. The average mature and senesced leaf [P] values found for A. truncata were of 0.54 and 0.32 mg P g-1 leaf, respectively. A. xanthina had 0.57 and 0.36 mg P g-1 leaf in its mature and senesced leaves, respectively. The mature leaf [P] values are not significantly different (significance level of 5%) between both Acacia species, and neither are the senesced leaf [P] values. A lack of difference was also found for the two Banksia species, with mature leaf [P] of 0.26 (B. attenuata) and 0.29 mg P g-1 leaf (B. menziesii) and senesced leaf [P] of 0.08 mg P g-1 leaf for both species.

0.8 69% 73% 84% 36% 41%

0.6

0.4

0.2 Leaf [P] (mg P g-1 leaf DW) 0.0 Banksia Banksia Hakea Acacia Acacia attenuata menziesii prostrata xanthina truncata

Figure 5: Leaf P concentrations for mature and senesced leaves for each species. Dark columns represent averages (n=3) of mature leaves and light columns, averages (n=3) of senesced leaves; whiskers represent standard errors. Samples were collected from Bold Park, Perth (S31.95, E115.77). The percentage value shown above the bars represents the remobilisation efficiency rate.

P-resorption efficiency was not significantly different (significance level of 5%) between species of the same genus, but was remarkably different between Acacia (34% and 41%) and Banksia (69% and 73%). The mature leaf [P] of Banksia was lower than Acacia, as they were able to resorb most P from the leaves during leaf senescence before leaf shedding. The P-resorption proficiency of Banksia was high, with an average of only 0.08 mg P.g-1 dry leaf left in the senescing leaves for either species.

111 On average, B. attenuata drew its leaf [P] down to 0.08 mg P g-1 dry leaf - the same value as for B. menziesii, and just under that of H. prostrata (0.085 mg P g-1 dry leaf). The senesced leaves of A. xanthina; however, retained 0.36 mg P g-1 dry leaf, and Acacia truncata; 0.32 mg P g-1 dry leaf, showing a 4-fold variation in P-resorption proficiency between Acacia on one hand and Banksia and Hakea on the other.

A correlation was calculated between the slope of the curve of net P uptake and P resorption efficiency, and the correlation value encountered was 0.79. This correlation included the four species of this study (A. xanthina, A. truncata, B. attenuata and B. menziesii) and Hakea prostrata, for which the net P uptake curve slope was obtained from Shane et al. (2004b).

DISCUSSION

An increase in P supply to B. attenuata, B. menziesii, A. truncata and A. xanthina resulted in an increase in biomass only when the P supply was relatively low, but at higher P supply a decrease in biomass was observed and symptoms of P toxicity became apparent for some species, as previously observed for a range of Australian species from severely nutrient-impoverished environments (Specht and Groves, 1966, Ozanne and Specht, 1981, Grundon, 1972, Specht, 1963, Groves and Keraitis, 1976, Handreck, 1997). The level of external [P] at which this happens varies among species (Groves and Keraitis, 1976), as they have different P demands and different capacities to down-regulate their P-uptake.

P-resorption efficiencies and proficiencies in situ

B. attenuata and B. menziesii both have very low mature leaf [P], about half the average leaf [P] for Australian plants (0.49 mg g-1) and a quarter of the world average (1.02 mg g-1) (Lambers et al., 2010). Nevertheless, they resorbed most of their leaf P during leaf senescence, with P-resorption efficiencies (relative to their initial P content) of 69% and 73% respectively. H. prostrata exhibited a similar pattern to B. attenuata and B. menziesii: its P-resorption efficiency was 84%. A. xanthina and A. truncata exhibited a mature leaf [P] of 0.55 mg g-1, twice as much as the Banksia species, but their P- resorption efficiencies were much lower, 36% and 41%, respectively. In a study of 73 Australian evergreen taxa from nutrient-poor, water-limited sites the mean P-resorption efficiency was 63% (Wright and Westoby, 2003), which is higher than the present

112 results for A. truncata and A. xanthina, whereas the data for B. attenuata, B. menziesii and H. prostrata showed greater efficiency. Resorption data in the literature and data collected here should be considered as measured values, and are not necessarily the definitive values. In this study, for instance, the P-resorption efficiency for B. attenuata was 69%, whereas in a different study with collections in 2005, the value was only 27% (Denton et al., 2007). The differences between resorption efficiencies can be explained by various reasons, such as water availability, timing of abscission or shade (Killingbeck, 1996). Interestingly, the same study included B. menziesii with a P resorption efficiency of 72%, similar to the value found in the present study.

The two genera studied here also differed in their P-resorption proficiency (absolute measure of how little is left in the senesced leaves); B. attenuata and B. menziesii being more proficient at resorbing P from senescing leaves than the two Acacia species studied. The differences in proficiency between species of the same genus were not statistically significant, but they were significantly different between genera. The proficiency values found for B. attenuata and B. menziesii in this study (80 µg P g-1 DW) fall within the known range for Banksia species: 29 to 128 µg P g-1 DW (Denton et al., 2007). They are very similar to the values found for H. prostrata, of 85 µg P g-1 DW, also a slow-growing Proteaceae which is sensitive to P toxicity, like B. attenuata and B. menziesii. A. xanthina and A. truncata showed P-resorption proficiencies of 360 and 320 µg P g-1 DW, respectively, and were four times less proficient at resorbing P than the two studied Banksia species. They were still proficient in a global context (450 µg P.g-1 DW) (Killingbeck, 1996), but less proficient than the average 180 µg P.g-1 DW for Australian evergreen species (Wright and Westoby, 2003).

Unlike the P-sensitive B. attenuata and B. menziesii, A. truncata and A. xanthina had greater tolerance of higher levels of P, which may be one of the traits that allow these legumes to be successful at early stages following fires (Bell and Koch, 1980) and also allows them to occupy niches in the landscape with slightly higher nutrient concentrations, such as drainage areas and disturbed edges of the vegetation formation. On the other hand, A. xanthina and A. truncata were not as proficient in P-resorption, leaving more of this growth limiting nutrient in its senesced leaves.

A previous study with a large number of species and considering environmental fertility variation had results which suggested that mature leaf nutrient concentration is an important determinant of senesced leaf [P], and P-resorption efficiency declines with increasing green leaf [P] (Kobe et al., 2005). In agreement, both Acacia species in our

113 study accumulated P in their leaves to a higher degree than the Banksia species did, and also showed a lower P-resorption proficiency and efficiency.

Effects of P supply on plant growth

A. truncata, A. xanthina and B. menziesii plants produced most biomass at an intermediate P concentration. The highest P concentrations did not result in additional biomass production or inhibited growth for these species and led to toxicity symptoms in Banksia leaves. B. attenuata did not produce any additional biomass with increased P supply. The seedlings of B. attenuata have a very high P content in their cotyledons (Denton et al., 2007) and it is a very slow growing plant, which may explain why no increase in biomass was observed. High cotyledon P content and slow growth rate; however, are also characteristics of B. menziesii. Therefore, this does not seem to be the explanation as to why only B. attenuata did not respond to P supply. Despite the fact that the growth of B. attenuata was similar amongst treatments, a clear increase was observed for leaf [P] with increasing P supply. Possibly the outcome of this experiment could be different if the experiment was conducted over a longer period of growth.

A decrease in biomass allocation to roots with increasing P supply is a common response among many plant species, and even though differences may be relatively small, they have been found to be consistent (Kirschbaum et al., 1992). This is not the case in this study, where the root weight ratio (RWR) did not vary according to treatments as expected, except for B. menziesii, with a significance value smaller than 0.01 for a linear regression of RWR plotted against P treatment. The absence of variation in RWR for B. attenuata, A. truncata and A. xanthina are not, however, a novel finding. White lupin also showed no response in RWR to P treatments (Pearse et al., 2006), and we raise the possibility that cluster-root bearing species may not always have a RWR decline with increasing P supply. A less pronounced RWR difference as dependent on P treatment in species that form root clusters would make sense ecologically, as clusters greatly increase P-uptake with a very little additional increase in root weight.

Effect of P supply on leaf [P]

After 10 weeks of supply of P treatments Banksia leaves in the 10 µM P treatment had dried and curled leaf tips, and some presented dark spots on the leaves (Figure 6). This

114 effect was more pronounced on the oldest leaves, but not exclusive to them. It is relatively common for Australian species subjected to increased P supply to present leaf symptoms of P toxicity, although these occur at varying levels of P, as reviewed by Shane et al. (2004a). The leaves of Acacia, unlike those of Banksia, were not visually damaged in any way, even at the highest P concentration, which was considerably higher than that used for Banksia. The results on leaf [P] provide evidence that A. truncata and A. xanthina were able to down-regulate their P-uptake in the present experiment, whereas B. attenuata and B. menziesii showed a very low capacity to down- regulate their P-uptake system.

Figure 6: Symptoms of toxicity due to excess phosphorus. (a) Banksia attenuata with the leaf tips dried and curled up; (b) B. menziesii with dark spots on the leaves; and (c) B. menziesii with pigment loss and dried leaf margins.

115 Net P-uptake rates

Both Acacia species studied were able to down-regulate their P-uptake rates. A. truncata grown without P for 10 weeks had an average uptake value (n=6) of 0.26 nmol P g-1 root FW s-1, and this decreased to 0.16; 0.07; 0.07; and 0.03 nmol P g-1 root FW s-1 (plants grown at 0.5; 5; 50 and 500 µM P, respectively). A. xanthina presented down- regulation of P-uptake as well. However, plants grown in the no-P solution showed net P-uptake rates of 0.08 nmol P g-1 root FW s-1, much lower than those for A. truncata. Its P-uptake values decreased with increasing P supply to 0.07; 0.04; 0.03 and -0.03 (plants grown at 0.5; 5; 50 and 500 µM P, respectively), ending in a negative value, which suggests that the plants grown at 500 µM released P into the solution. In contrast B. attenuata increased (significance level of 5%) its P-uptake rate with increasing P supply. The B. attenuata individuals grown at the highest P supply for 10 weeks had a P-uptake rate ca. 60% greater (0.036 nmol P g-1 root FW s-1) than the plants grown with no P (0.023 nmol P g-1 root FW s-1).

B. menziesii, like B. attenuata, did not down-regulate its P-uptake, and the difference between treatments was not statistically significant. The P-uptake rates of B. menziesii ranged between 0.02 and 0.027 nmol P g-1 root FW s-1. Shane et al. (2004a) studied the P-uptake rates and P toxicity in H. prostrata (also native to south-western Australia) and determined the uptake rates at 5 µM P. Those plants were grown at eight P concentrations, ranging from 0 to 100 µM P. Although H. prostrata almost doubled its uptake rate when comparing plants grown at 0 and 0.2 µM P, its uptake rate then stabilised at 0.05 nmol P g-1 FW s-1 in the 0.2 to 1 µM P range. Interestingly, the uptake rate then decreased by half at the highest P concentrations (10; 50 and 100 µM P), coinciding with a suppression of cluster-root formation. Such a decrease in uptake was not observed in our experiment with B. attenuata and B. menziesii, which could be a difference specific to species, but could also be related to the developmental stage of the individuals (Shane et al., 2004b used older plants in their experiment). No studies have been performed to date in order to assess how the net P-uptake rates vary with plant age, and this information would be very insightful.

Linking P-uptake and P-resorption

As hypothesised, a strong inverse correlation (79%) was encountered between the down-regulation of P-uptake and the P resorption efficiency. This is the first attempt at such a correlation, and even though only five species were included in the analysis, they 116 represent three genera (Acacia, Banksia and Hakea) of two very important species in the Australian kwongan.

The genes that control P down-regulation are likely to be basal and its loss, secondary, as there is more ecological significance in down-regulating P-uptake to avoid P toxicity than in up-regulating, as the root kinetics benefits are limited by soil processes (Lambers et al., 2006, Lambers et al., 2011). High P-resorption efficiency and proficiency, on the other hand, appear to be a trait acquired from recent selection (Killingbeck, 1996). There is little evidence that the nutritional status of the plant controls P-resorption (Aerts, 1996) and little evidence of consistent phylogenetic influence on it (Killingbeck, 1996). A biochemical explanation for P-resorption proficiencies or efficiencies is yet to be proposed (Lambers et al., 2011).

We suggest that there is a link between the genetic attributes that allow for the plant to possess extreme P-remobilisation efficiency and those responsible for down-regulation of P uptake. This hypothesis arises from the inverse correlation found between the capacity to down-regulate P-uptake (at increasing P supply) and the resorption of P from senescing leaves in five south-western Australian species: B. attenuata, B. menziesii and H. prostrata (Proteaceae) and A. truncata and A. xanthina.

CONCLUSIONS

As hypothesised, we observed an inverse correlation between the P-resorption efficiency and the capacity to down-regulate P-uptake in the five species studied here. The species included in this study fall into two groups: one with highly specialised physiology aiding survival in P scarce soil conditions (H. prostrata, B. attenuata and B. menziesii); and the second with greater tolerance to P supply but with a less efficient P- conservation mechanism (A. truncata and A. xanthina).

The species we have studied are representative of two contrasting survival strategies within nutrient-impoverished landscapes. One strategy where plants possess a low capacity for P-resorption from senescing leaves but control their uptake (avoiding P toxicity); and another, where plants are extremely efficient in P-resorption, but perhaps as a consequence they cannot down-regulate their P-uptake.

The species used in this study co-occur in some areas in south-western Australia; however, they are not found side by side, and we believe that this is due to the complex mosaic of soils providing different niches. Phosphorus uptake down-regulation and P-

117 resorption efficiency and proficiency can, in part, explain some of the niches occupied by different species in these nutrient-poor habitats. By studying traits relating to P uptake and utilisation efficiency as well as other ecophysiological traits, we can begin to understand intricate structure of the complex and incredibly species-rich region of the south-west of Western Australia.

ACKNOWLEDGEMENTS

Thanks to The University of Western Australia for a doctorate scholarship (IPRS/SIRF) and the School of Plant Biology for the funding and infra-structure. This research was supported by the Australian Research Council (ARC) and additional grants were provided by ANZ Holsworth Wildlife Foundation and The Mary Janet Lindsay of Yanchep Memorial Fund. We would also like to thank the colleagues who helped with the experiment and made valuable suggestions.

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120 121 Chapter 5

General discussion and conclusions

Linking the phosphorus acquisition and phosphorus conservation aspects studied in this thesis and adding Brazilian rupestrian fields to the group of worldwide phosphorus impoverished environments

In each chapter of this thesis, a previously unexplored facet of P-acquisition or P- conservation mechanism was studied. In this chapter, I present the main conclusions of each work and tie these findings together, stating the significance and contribution of my thesis.

Through work in Brazilian rupestrian fields, I was able to sufficiently characterise the soils of such formations with regards to chemical and physical properties, and I also provided a novel account of the nutrient status of both mature and senesced leaves for over 30 species. With regards to the study of roots, I collected the first records of sand- binding roots in the cerrado in Brazil (in Eriocaulaceae), as well as the first record of cluster roots in a plant grown in its natural environment in the same habitat. Roupala montana, a Proteaceae in which cluster roots were found, also had mycorrhizal associations. This study also provided the finding of new root structures that are cluster- like but are as yet undescribed in Cactaceae and Cyperaceae, as well as the “cotton-like” structure in an Eriocaulaceae species. All of these first-time findings, as well as the discovery of three species of Xyridaceae new to science demonstrate just how little rupestrian fields have been studied, and how much there is to learn from this threatened environment.

Rupestrian fields were characterised as functionally equivalent to the kwongan in south- western Australia and the fynbos in the Cape floristic region, South Africa. I arrived at this conclusion through the analysis of soil paired with the observation of structures presumably specialised for P-acquisition and the measurement of plant P-resorption efficiency, proficiency and N:P ratios. The three regions referred to above do not only share their origin and a similarly high biodiversity, but also the mechanisms with which their plants have adapted to the P-impoverished environment. Fire, although not examined in this study, is also a major factor in all three ecosystems. It seems like the differences in climate (Mediterranean in the Cape and south-western Australia and

122 tropical in the rupestrian fields) and in species composition did not counteract the similarities in soil nutrient availability.

In a glasshouse experiment with the Australian native legume Viminaria juncea, I had the opportunity to come across a species with an unheard-of control of shoot [P]. V. juncea did not vary its shoot [P] as dependent on a wide range of P supply. The presumed implications of this internal [P] control are that this species suppresses its growth when P supply is low, and strongly down-regulates its P-uptake capacity when provided with additional P. Another set of interesting consequences of the constant shoot [P] is that mycorrhizal colonisation and cluster-root formation were not suppressed, even at the highest level of P treatment (50 mg P kg-1 soil). With the results of this experiment, the initial hypothesis was rejected: cluster-root formation and mycorrhizal colonisation were not inversely correlated across P treatments for V. juncea which turned out to be an unsuitable species to address this question due to its unique shoot [P] stability.

Another experiment was performed as part of this thesis. The comparison of the capacity for net P-uptake rate and leaf P-resorption across five species from south- western Australia corroborated our prediction that these would be inversely correlated. For Acacia xanthina, A. truncata, Banksia attenuata, B. menziesii and Hakea prostrata, down-regulation of P-uptake capacity is inversely correlated to P-resorption efficiency. The existence of such inverse correlation suggests evolutionary processes that probably took place in P-impoverished environments, where some species became highly adapted to extremely low soil [P] (B. attenuata, B. menziesii and Hakea prostrata), but lost the ability to down-regulate their P-uptake capacity. The two strategies found between the Fabaceae and the Proteaceae species studied here need to be expanded through further research to other species and regions, but if the correlation is sustained, leaf analyses could become an easy and simple method to assess whether species are prone to P- toxicity, and aid in the management and conservation of land.

My thesis has explored the similarity between different P-impoverished environments, detailing some of the differences and similarities between south-western Australia, the Cape floristic region and the rupestrian fields, as well the variability of characteristic in the latter. I have also analysed the parallel occurrence of two modes of specialised root P-uptake in V. juncea, which eventuated to be a unique species in the maintenance of a constant shoot [P]. Finally, I also tied together root uptake and P-conservation mechanisms in two Fabaceae and three Proteaceae species of south-western Australia.

123 All of the observed findings and patterns have fulfilled my aim to contribute to a broader, more generalised understanding of plant nutrition in P-impoverished environments.

This thesis contributes to knowledge in the field of ecophysiology, and should also have significant impact due to the potential application of some of its discoveries. I believe that the results of my research are a significant step towards the understanding of the intricate mechanisms which allow plants to live in P-impoverished ecosystems. It is also a step towards the future goal of finding the basis of P-acquisition and P-conservation mechanisms in order to, potentially, select these traits or introduce them to crops. This could diminish the use of phosphate fertiliser, that has major detrimental side-effects to the environment, and which is becoming harder to mine.

I end my thesis suggesting that future research should include areas in the world that have been less explored, such as more areas of rupestrian fields or the pantepuis in northern South America. Research in South America is still young, with most of the universities founded in the mid-1900s; and funding in developing countries is often very restrictive. International collaboration, such as established in this PhD project is highly beneficial to both parties, and allows for the development of knowledge that is needed for a more generalised understanding of plant ecophysiology. I also suggest the inclusion of carboxylate studies as well in the field surveys, which we were not able to perform, but that would most certainly reveal exciting findings. Further studies in physiology and molecular biology of V. juncea are also highly commendable, as it would be of great interest to unveil the mechanism behind its strict shoot [P] control. The way in which V. juncea down-regulates its P-uptake capacity to maintain a constant shoot [P] is another very interesting unexplored point. Finally, there is the suggestion of the inclusion of a broader range of species and regions to test whether the inverse correlation found for down-regulation of P-uptake capacity and P-resorption efficiency is held. By answering three questions in this thesis, I have come across many others questions, big and small; I hope that my thesis and publications help entice other researchers to the beautiful and curious study of P-impoverished environments.

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