Universidade Estadual De Campinas Instituto De

UNIVERSIDADE ESTADUAL DE CAMPINAS

INSTITUTO DE BIOLOGIA

ANNA ABRAHÃO

MINERAL NUTRITION IN TWO BIODIVERSITY HOTSPOTS: CAMPOS RUPESTRES AND KWONGAN

NUTRIÇÃO MINERAL EM DOIS HOTSPOTS DE BIODIVERSIDADE: CAMPOS RUPESTRES E KWONGAN

CAMPINAS

(2017)

ANNA ABRAHÃO

MINERAL NUTRITION IN TWO BIODIVERSITY HOTSPOTS: CAMPOS RUPESTRES AND KWONGAN

NUTRIÇÃO MINERAL EM DOIS HOTSPOTS DE BIODIVERSIDADE: CAMPOS RUPESTRES E KWONGAN

Thesis presented to the Institute of Biology of the University of Campinas in partial fulfilment of the requirements for the degree of Doctor in Ecology, in the scope of the Cotutelle Agreement between Unicamp and The University of Western Australia

Tese apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutora em Ecologia, no âmbito do Acordo de Cotutela firmado entre a Unicamp e a The University of Western Australia

ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA ANNA ABRAHAO, E ORIENTADA PELOS PROF. DR. RAFAEL SILVA OLIVEIRA, HANS LAMBERS E MEGAN HELEN RYAN

Orientador RAFAEL SILVA OLIVEIRA

Co-Orientadores: JOHANNES THIEO (HANS) LAMBERS, MEGAN HELEN RYAN

CAMPINAS

(2017)

Agência(s) de fomento e nº(s) de processo(s): CAPES, 88881.068071/2014 ORCID: 0000-0001-9295-2292

Ficha catalográfica Universidade Estadual de Campinas Biblioteca do Instituto de Biologia Mara Janaina de Oliveira - CRB 8/6972

Abrahão, Anna, 1988- Ab82m AbrMineral nutrition in two biodiversity hotspots : campos rupestres and kwongan / Anna Abrahão. – Campinas, SP : [s.n.], 2017.

AbrOrientadores: Rafael Silva Oliveira e Johannes Thieo Lambers. AbrCoorientador: Megan Helen Ryan. AbrTese (doutorado) – Universidade Estadual de Campinas, Instituto de Biologia. AbrEm cotutela com: The University of Western Australia.

Abr1. Plantas - Nutrição. 2. Solos. 3. Micorriza. 4. Nódulos radiculares de plantas. 5. Raizes (Botânica). I. Oliveira, Rafael Silva, 1974-. II. Lambers, Hans. III. Ryan, Megan. IV. Universidade Estadual de Campinas. Instituto de Biologia. VI. Título.

Informações para Biblioteca Digital

Título em outro idioma: Nutrição mineral em dois hotspots de biodiversidade : campos rupestres e kwongan Palavras-chave em inglês: Plants – Nutrition Soils Mycorrhizas Root nodules, Plant Roots (Botany) Área de concentração: Ecologia Titulação: Doutora em Ecologia Banca examinadora: Rafael Silva Oliveira [Orientador] Fernando Augusto de Oliveira e Silveira Ana Zangirólame Conçalves Soizig Anne Le Stradic Peter Groenendijk Data de defesa: 14-09-2017 Programa de Pós-Graduação: Ecologia

Campinas, 14 de setembro de 2017.

COMISSÃO EXAMINADORA

Prof. Dr. Rafael Silva Oliveira (Orientador) Prof. Dr. Johannes Thieo Lambers (Coorientador) Prof. Dr. Fernando Augusto de Oliveira e Silveira Dra. Ana Zangirólame Gonçalves Dra. Soizig Anne Le Stradic Prof. Dr. Peter Stoltenborg Groenendyk

Os membros da Comissão Examinadora acima assinaram a Ata de defesa, que se encontra no processo de vida acadêmica do aluno. AGRADECIMENTOS Eu gostaria de agradecer primeiramente ao Governo Federal Brasileiro por ter financiado o meu doutorado com bolsa e com financiamento para pesquisa. Sem o financiamento público não existiria universidade acessível para todos. Em segundo lugar, agradeço à minha mãe, que me estimulou a sempre buscar realizar os meus sonhos e por me ensinar a amar o cerrado. Agradeço também aos meus orientadores Rafael e Hans, que me deram todo o apoio necessário, e ao mesmo tempo me deram a liberdade para aprender a caminhar com as minhas próprias pernas pela estrada da pesquisa acadêmica. É muito bom ter orientadores tão empolgados com a ciência. Aprendi muito com eles parando para conversar sobre ideias de projeto sob o sol escaldante no meio do campo rupestre. Aprendi também que precisamos tentar tirar a ciência do nosso círculo acadêmico restrito e debate-la com a sociedade. A divulgação científica é um passo crucial para aproximar as pessoas do cerrado e dos campos rupestres. Para gostar é preciso conhecer. É preciso saber que sem as raízes, o solo não retém agua. Se o solo não absorver água, as nascentes não nascem e os rios secam. É preciso saber que não servem quaisquer raízes. O cerrado e os campos rupestres tem uma flora riquíssima e adaptada às condições de solo e clima, e ao fogo. Ao substituir as espécies nativas por outras com raízes mais rasas como a soja ou mais profundas como os eucaliptos, o ciclo dos elementos são modificados e os serviços ambientais se perdem. Agradeço então aos meus orientadores pelas ricas discussões sobre a importância dos ecossistemas nativos.

O trabalho todo durante o doutorado não seria possível sem o grupo incrível do qual tive a sorte de ser parte. Obrigada Lab de Ecologia Funcional da Unicamp. O meu maior agradecimento vai para a Pat, que me acompanhou em todos os campos e me ajudou muito com as análises em laboratório. A sua companhia foi incrível, Pat. Obrigada Grazi pelo carinho e pela competência enorme! O espírito colaborativo do lab foi muito importante para mim, e levo muitas amizades inesquecíveis como a Carol, as Fernandas, o Maurão, a Gabs e a Becky. Obrigada Andrés, Paulo, Chicken e Cleiton pelas ricas discussões de projeto. Obrigada à Isabela Fernandes, aluna de graduação que me acudiu com as amostras mesmo eu estando longe. Espero um dia ter um grupo de pesquisa tão legal quanto vocês novamente.

A estrada pela vida acadêmica não foi muito fácil. Eu agradeço então a todas as pessoas que conviveram comigo ao longo desses anos e me mantiveram feliz. Obrigada Zildinha, Talita e Zeca por me receberam de braços abertos na casa de vocês. Obrigada Lulu, Maíra e Su pelo cuidado e pela ternura. Obrigada Roni, Sophie e Bonitão pelos roques no meio da semana (e pela música dedicada a mim, voyage, voyage). Obrigada Thiara pelo

carinho, sensibilidade e dedicação a essa amizade tão gostosa. Obrigada ao André Rech, meu irmãozinho, grande inspiração. Obrigada Gugu Shimizu pelo companheirismo. Obrigada Johnny e Nathi por fazer cookies comigo até a Elisa chegar.

Durante o meu doutorado tive a sorte de poder participar do programa Ciência sem Fronteiras. Passei um ano na The University of Western Australia, como parte da cotutela de doutorado. Além de ter aprendido muito sobre cultivo de plantas, tive experiências maravilhosas e fiz muitas amizades deliciosas. Obrigada ao Hans que me fez me sentir em casa. Obrigada à minha orientadora Australiana, Megan por me acolher no seu laboratório, a sua dedicação com os seus alunos é incrível. Quando eu crescer quero ser igual a você. Obrigado Caio e Paulinha por me acolherem no apê de vocês. Obrigado Patty por me acolher no laboratório, fizemos um bom trabalho no nosso empreendimento cafeeiro! Obrigada Kenny por me ensinar como fazer um experimento com leguminosas nativas. Obrigada Wenli fofinha pelo carinho e pelas refeições chinesas deliciosas. Obrigada Seb, Maggie, Nathan e Albina pela viagem inesquecível a Shark Bay. Obrigada Adrien, Jorik e Guilhem pela amizade e apoio no experimento! Obrigada a todo o Lab Team da Megan, vocês são demais! Obrigada ao Conner, por ser meu companheiro nessa jornada. Por fim, obrigada Nana e Rodrigo pela energia contagiante. Adorei morar com vocês os últimos meses em Perth. Desejo muita saúde ao Baby que está a caminho.

Ao voltar para Campinas, tive outra experiência na mesma casa, que tinha virado a Fazendinha. O zelo que eu tinha pela casinha foi sustentado e desenvolvido pelas novas integrantes para tornar a Fazendinha o nosso lar. Obrigada Carolzinha, Anapê e Glacê por tornar a nossa casinha um ambiente tão agradável. Carol polentinha, sua sensibilidade é inacreditável. Glacê sua coragem é um exemplo incrível de superação, acredito muito no seu potencial. Anapê obrigada pelos conselhos sinceros! Amei passar o último ano com vocês e com as Megs. Obrigada a todos os agregados da Fazendinha!

Agradeço as pessoas que me dão apoio há mais tempo. Obrigada aos meus amigos de infância, Bellinha, Ia e Ana Flavia. Obrigada Marçalzinho, Leo, Darcy, Sylvia, Fernando, Norton, Tia Martinha e Tia Angela por torcerem por mim desde criança. Obrigada aos meus amigos da graduação, integrantes do condomínio do Yellow Submarine, que apesar de estarem espalhados geograficamente, estão sempre dando um jeito de se encontrar e se amar. Agradeço a todas as pessoas que me deram apoio de alguma forma durante os meus anos de doutorado. Por fim, agradeço a todos os membros da pré-banca e da banca que deram contribuições muito importantes para essa tese.

RESUMO

A distribuição de espécies ao longo de gradientes de disponibilidade de recursos depende da sua capacidade de obter e utilizar esses recursos de forma a sobreviver e se reproduzir em cada ambiente. Os nutrientes disponíveis no solo estão entre os recursos mais importantes na seleção das espécies capazes de se estabelecer em cada ambiente, e as estratégias de aquisição de nutrientes variam de acordo com sua disponibilidade. Em solos empobrecidos em fósforo (P), exsudatos radiculares do tipo carboxilatos são mais eficientes do que as micorrizas para a obtenção desse nutriente, enquanto que em disponibilidades intermediárias de P; as micorrizas têm um papel importante na aquisição de P. A fixação simbiótica de nitrogênio (N) representa um alto custo de P e é esperada quando o N é limitante para o crescimento. Cada estratégia representa custos importantes de carbono (C), criando uma demanda conflitante na alocação de C para a aquisição de P e N que varia de acordo com a disponibilidade desses nutrientes. Os carboxilatos mobilizam Mn do solo, que por sua vez se acumula nas folhas, enquanto que as micorrizas interceptam o Mn. Dessa forma, a abundância relativa das diferentes estratégias de aquisição de nutrientes pode ser avaliada por meio da concentração foliar de manganês (Mn). Uma vez absorvidos do solo, os nutrientes podem ser alocados para aumentar a eficiência fotossintética e remobilizados de tecidos senescentes. Nessa tese, eu investiguei as mudanças na aquisição e no uso de nutrientes ao longo de gradientes de fertilidade em solos muito antigos e inférteis nos campos rupestres no Brasil e no kwongan do sudoeste da Austrália. Um terço dos hotspots de biodiversidade ocorre em solos muito antigos e empobrecidos em P. Entender como plantas que ocorrem nesses ambientes adquirem e usam nutrientes é um passo importante para explicar os processos que geram e mantém essa alta biodiversidade em diferentes escalas.

No primeiro estudo, investiguei as estratégias de aquisição e uso de nutrientes ao longo de um gradiente de formação de solos, que também representa um gradiente de disponibilidade de fósforo. Eu hipotetizei que haveria uma maior proporção de espécies com micorrizas nos solos menos pobres em P e N nos campos rupestres, enquanto que nos solos mais pobres em P e N haveria uma maior proporção de espécies sem micorrizas, e com raízes nas quais a areia adere fortemente (raízes com bainha de areia). Eu também esperava que essas raízes liberassem altas taxas de carboxilatos para mobilizar P e que as espécies com essas raízes possuíssem alta [Mn] foliar. Finalmente, eu esperava que as espécies dos solos mais inférteis apresentassem maior eficiência fotossintética no uso de P e N (PPUE e PNUE, respectivamente). Eu observei uma clara diminuição no predomínio das espécies com

micorrizas, e um aumento da abundância de espécies com bainhas de areia nas raízes nos solos mais pobres em nutrientes. O [Mn] foliar foi alto independentemente da presença das bainhas de areia nas raízes, indicando que as bainhas não são essenciais para a liberação de carboxilatos. Eu observei valores semelhantes de PPUE e PNUE entre espécies dos diferentes solos do gradiente. Finalmente, a eficiência de remobilização de P foi muito alta em espécies ao longo de todo gradiente, enquanto que a remobilização de N foi moderada, o que reflete a maior limitação por P do que por N para o crescimento nesses ambientes.

No segundo estudo, investiguei as mudanças na aquisição e no uso de nutrientes em duas espécies de leguminosas ao longo de cronosequências de solo no kwongan, sudoeste da Austrália, e em casa de vegetação com diferentes suprimentos de P e N. Eu esperava que a proporção da raiz colonizada por fungos micorrízicos diminuísse com o aumento do suprimento de P, aumentando o [Mn] foliar, e que com a diminuição do suprimento de N, as plantas aumentariam a formação de nódulos, dado que P suficiente fosse suprido. Ao invés de investir em micorrizas e exsudação de carboxilatos quando submetidas a baixos suprimentos de P, as duas espécies investiram em alocação diferencial de biomassa para as raízes quando cultivadas na casa de vegetação. As duas espécies aumentaram a nodulação quando submetidas a baixos suprimentos de N. O suprimento adicional de P estimulou o crescimento das plantas, indicando limitação por P para o crescimento, mas não por N por causa da fixação simbiótica.

Em suma, a forte associação entre os atributos do solo e da vegetação que levaram à alta substituição de espécies na escala de comunidade e à plasticidade fenotípica intraespecífica na aquisição e no uso de nutrientes pode ajudar a explicar como os mecanismos subjacentes à distribuição de espécies operam em diferentes escalas.

ABSTRACT

Species distribution along gradients of resource availability depends on their capacity to acquire and use these resources to survive and reproduce in each environment. Soil nutrients are among the most important drivers of the selection of species capable of establishing in each environment, and nutrient-acquisition strategies vary according to their availability. In phosphorus- (P) impoverished soils, carboxylates are more efficient than mycorrhizas for P acquisition, while at intermediate P availability mycorrhizas play an important role. Symbiotic nitrogen (N) fixation has high P costs and is expected to occur when growth is N-limited. Each strategy represents a significant carbon (C) cost and creates a trade-off in C allocation for P and N acquisition which can change according to nutrient availability. The prevalence of the different P-acquisition strategies can be assessed through leaf [Mn], because carboxylates also mobilise soil Mn, which then accumulates in leaves, while mycorrhizas intercept Mn. Once taken up, nutrients can be allocated to increase photosynthetic nutrient-use efficiency and be remobilised from senescing tissues. In this thesis, I studied the shifts in nutrient- acquisition and -use strategies along fertility gradients in the nutrient-impoverished Brazilian campos rupestres and in the south-western Australian kwongan. One third of the biodiversity hotspots occur in old and P-impoverished soils. Understanding how species in these environments acquire and use nutrients is an important step to unveil the mechanisms that generate and maintain species diversity in different scales.

In the first study I investigated nutrient-acquisition and –use strategies of species occurring along a gradient of soil development that also represents a gradient of P availability. I hypothesised that the less P- and N-impoverished soils in campos rupestres would have a greater proportion of mycorrhizal species and that in the most impoverished soils a greater proportion of species would be non-mycorrhizal, with sand-binding roots (rhizosheaths). I also expected these specialisations to be associated with rapid rates of exudation of carboxylates and high leaf [Mn]. Finally, I expected the species from the most impoverished soils to exhibit greater photosynthetic P- and N-use efficiency (PPUE and PNUE) and greater P- and N-remobilisation efficiency. I observed a clear decrease in the prevalence of mycorrhizal species, and an increase in the abundance of species with rhizosheaths in the most impoverished soils. Leaf [Mn] was high, regardless of the presence of rhizosheaths, indicating rhizosheaths are not essential for carboxylate release. We observed similar PPUE and PNUE in species growing in the different soils and with different nutrient-acquisition strategies. Finally, P-remobilisation efficiency was very high in plants growing on all soil

types, and N-remobilisation efficiency was only moderate, reflecting the greater P- than N- limitation for growth in these habitats.

In the second study, I investigated shifts in nutrient-acquisition and –use strategies within two legume species along a soil chronosequence in the south-western Australian kwongan and grown in a glasshouse at varying P and N supply. I expected the proportion of the root length colonised by mycorrhizal fungi to decrease with increasing P supply, the amounts of rhizosphere carboxylates to increase at the lowest P supply, and leaf [Mn] and nodule formation to increase at low N supply, as long as enough P was available. Rather than investing in mycorrhizas or carboxylate exudation, the two species invested in differential biomass allocation to the roots at low-P when grown in the glasshouse. The two species increased nodulation at low N supply. Additional P supply stimulated plant growth, indicating growth was P limited, but they were not N-limited due to N-fixation.

Overall, the strong association I observed between plant and soil traits that led to variation in species abundance among plant communities and to the phenotypic plasticity in nutrient- acquisition and -use strategies within species may help explain how mechanisms underlying plant species distribution operate at different scales.

TABLE OF CONTENTS (SUMÁRIO)

CHAPTER 1. GENERAL INTRODUCTION ...... 14 1.1. Nutrient-acquisition strategies change with soil nutrient availability ...... 14 1.2. Nutrient-use efficiency in nutrient-impoverished soils ...... 18 1.3. Long-term soil development in nutrient-poor soils ...... 19 1.3.1. Study systems ...... 20 1.4. Significance of research ...... 24 1.4.1. Aims ...... 25 1.5. Thesis outline ...... 26 1.6. References ...... 27

CHAPTER 2. NUTRIENT-ACQUISITION AND -USE STRATEGIES IN A MOSAIC OF SOIL TYPES IN CAMPOS RUPESTRES AND CERRADO ...... 37 2.1. Abstract...... 37 2.2. Introduction ...... 38 2.3. Methods...... 43 2.3.1. Study area ...... 43 2.3.2. Soil sampling and analyses...... 44 2.3.3. Species selection ...... 45 2.3.4. Root sampling for nutrient-acquisition strategies ...... 46 2.3.5. Leaf collection for nutrient analyses ...... 47 2.3.6. Photosynthesis, photosynthetic nutrient-use efficiency and leaf mass per area ...... 48 2.3.7. Statistical analyses ...... 48 2.4. Results ...... 49 2.4.1. Soil data ...... 49 2.4.2. Nutrient-acquisition strategies ...... 50 2.4.3. Leaf P and N concentrations ...... 50 2.4.4. Photosynthetic nutrient-use efficiency ...... 51 2.5. Discussion ...... 51 2.5.1. Soil nutrient availabilities ...... 52 2.5.2. Nutrient-acquisition strategies ...... 52 2.5.3. Conservative nutrient use ...... 53 2.5.4. Photosynthesis, photosynthetic P- and N-use efficiency and functional traits ...... 54 2.5.5. Nutrient remobilisation efficiency and proficiency ...... 55 2.5.6. Conclusions ...... 56 2.6. Acknowledgments ...... 57 2.7. References ...... 57

CHAPTER 3. PHOSPHORUS- AND NITROGEN-ACQUISITION STRATEGIES IN TWO BOSSIAEA SPECIES (FABACEAE) ALONG TWO CHRONOSEQUENCES IN THE SOUTH-WESTERN AUSTRALIAN KWONGAN ...... 98 3.1. Abstract...... 98 3.2. Introduction ...... 99 3.3. Methods...... 102 3.3.1. Field collections ...... 102 3.3.2. Glasshouse experiment ...... 104 3.3.3. Statistical analyses ...... 107 3.4. Results ...... 108 3.4.1. Field data...... 108 3.4.2. Glasshouse experiment ...... 109 3.5. Discussion ...... 111 3.5.1. Leaf P and N concentrations ...... 111 3.5.2. Nutrient limitation, leaf N:P ratios and growth ...... 112 3.5.3. Phosphorus-acquisition strategies at different nutrient availabilities ...... 114 3.5.4. Leaf [Mn] and nutrient-acquisition strategies ...... 116 3.5.5. Nitrogen-acquisition strategies at different P and N availabilities ...... 117 3.5.6. Differences in nutrient-acquisition strategies between the two Bossiaea species ...... 118 3.5.7. Concluding remarks ...... 118 3.6. Acknowledgments ...... 119 3.7. References ...... 119

CHAPTER 4. GENERAL DISCUSSION ...... 151 4.1. Introduction ...... 151 4.2. Discussion ...... 152 4.2.1. Community level changes in nutrient-acquisition and -use strategies ...... 152 4.2.2. Within-species shifts in nutrient-acquisition and -use strategies ...... 154 4.3. Limitations, achievements and future direction...... 155 4.3.1. Limitations ...... 155 4.3.2. Achievements ...... 156 4.3.3. Future direction ...... 157 4.4. Conclusions ...... 157 4.5. References ...... 158

ANEXO I Documento referente a Bioética e/ou Biossegurança...... 162

ANEXO II Declaração direitos autorais ...... 163

...... 163 14

CHAPTER 1. GENERAL INTRODUCTION

The patterns of species distribution observed in ecological systems generally vary along a range of spatial, temporal, and organisational scales (Levin 1992). Unravelling the mechanisms that underlie the ecological patterns at different scales is paramount if we are to predict how these patterns are expected to change in response to environmental heterogeneity in resource distribution (Levin 2000) or to any changes in resource availability (Phoenix et al. 2006) or environment (e.g. climate change) (Brown et al. 2016; Bueno et al. 2017), but also to underpin restoration initiatives (Hopper 2009; Le Stradic et al. 2014). In order to understand patterns of plant species distribution along natural gradients of resource distribution, we have to understand resource-acquisition and -use mechanisms (Silvertown 2004). Since all plants require nutrients, soil nutrient availability is one of the most important drivers of plant species distribution (John et al. 2007; Perry et al. 2008). Plant species occur along belowground niche axes of nutrients, which can differ in depth and timing of uptake, chemical form and microbial mediation of nutrient uptake (Silvertown 2004), and also in how efficiently these nutrients are used by the plant (Lambers et al. 2010; Xu et al. 2012). Plant nutrient acquisition and use can be studied at different scales. At the community level, for example, one can investigate the relative abundance of species using different nutrient-acquisition and -use strategies along gradients of nutrient availability (Zemunik et al. 2015). At the species level, the reliance on different nutrient-acquisition and -use strategies can shift in response to nutrient availability (Pang et al. 2010; Pang et al. 2015; Albornoz et al. 2016; Png et al. 2017). At each scale, different patterns of plant mineral nutrition emerge.

1.1. Nutrient-acquisition strategies change with soil nutrient availability

Plants can acquire soil nutrients via two principal pathways: directly through the roots, or through a fungal symbiosis called mycorrhiza (Kaiser et al. 2015). Plants that acquire nutrients through arbuscular mycorrhizal fungi are advantaged through extension of the foraging zone beyond the roots due to the growth of thin fungal hyphae, which absorb soluble soil nutrients. Hence, plants in symbiosis with arbuscular mycorrhizal fungi can be called ‘scavengers’ (Lambers et al. 2008) as opposed to ‘miners’ (Fig. 1.1). Mining plants release highly localised root exudates that mobilise insoluble forms of nutrients (Lambers et al. 2008). The proportion of scavenging versus mining plants changes along gradients of nutrient availability (Zemunik et al. 2015). Natural gradients of soil nutrient availability can be found 15

along chronosequences, which are sequences of soils of varying age with the same parent material under the effect of similar climatic and biotic factors (Stevens and Walker 1970). Pedogenic processes cause phosphorus (P) loss with soil aging, while nitrogen (N) accumulates through biological fixation or atmospheric deposition (Walker and Syers 1976). However, very weathered soils are depleted in both P and N (Fig. 1.1). In these very old soils, the abundance of mining plants has been found to increase (Lambers et al. 2008; Oliveira et al. 2015; Zemunik et al. 2015) presumably due to their ability to acquire sparingly soluble forms of P; therefore the abundance of scavenging plants decreases proportionally (Lambers et al. 2008).

a b c

g q Scavenging

- strategy

e Mining

r t strategy

u N Total P

n Total N

t Young, poorly Old, nutrient-

developped soils impoverished soils

Soil age

Figure 1.1. Amounts of soil nutrients and shifts in plant phosphorus (P)-acquisition strategies during soil development. a. Younger soils are richer in phosphorus (P), where scavenging (mycorrhizal) plant species are more efficient in P acquisition and; b,c. Older soils are depleted in P and species with non-mycorrhizal mining strategies thrive. b. Rhizosheaths in Aulonemia effusa (Hack.) McClure (Poaceae); c. Long root hairs in Xyris sp. (Xyridaceae). 16

Adapted from Lambers et al. (2008).

Mycorrhizal fungi are a very diverse group and can form several types of symbiosis, such as ectomycorhizas, which cover the root system with intercellular hyphae (Hartig net), ericoid and orchid mycorrhizas, which occur specifically in Ericaceae and Orchidaceae and finally arbuscular mycorrhizas, which form intracellular arbuscules as an exchange interface between the plant and the fungus (Smith and Read 2008). The scavenging strategy occurs via symbiotic association with arbuscular mycorrhizal fungi only, because ecto- and ericoid mycorrhizas have access to additional insoluble organic forms of P and N (Lambers et al. 2008). Scavenging plants acquire soluble P, N (amino acids) and potassium (K) through the symbiosis; in exchange, plants supply the fungus with C as monosaccharides (Helber et al. 2011). The fungi germinates and penetrates the root cortex (Fig. 1.1a) without eliciting major plant defence responses (Kloppholz et al. 2011). They build absorptive hyphae into the soil and exploit soil patches unavailable to the roots (Bonfante and Genre 2010). The scavenging strategy can drain from 4 to 20% of the photosynthetic intake (Jakobsen and Rosendahl 1990). However, in mycorrhizal plants up to 50% of the P uptake by plants can be absorbed via mycorrhizal pathway (Li et al. 2006). When P is strongly bound to soil particles, the mining strategy is expected to be more efficient in P acquisition (Lambers et al. 2008).

Mining plants often have specialised root structures such as cluster roots (Neumann and Martinoia 2002), dauciform roots (Playsted et al. 2006), capillaroid roots (Lamont 1982) or very long root hairs that form rhizosheaths (Smith et al. 2011; Brown et al. 2017). Rhizosheaths are formed through the root exudation of mucilage that adheres soil particles very tightly (Fig. 1.1b) and tend to be larger in species with longer root hairs (Fig. 1.1c) (Brown et al. 2017). The cluster-roots found in many Proteaceae are known to release organic acids, or carboxylates plus protons, into the soil in exudative bursts (Shane et al. 2004; Delgado et al. 2014) and thereby mobilise P (Dinkelaker et al. 1995). However, the association between root specialisations and their role in mineral nutrition is not always clear (Brown et al. 2017; Güsewell and Schroth 2017). Some Carex species (Cyperaceae) with and without dauciform roots can equally access different forms of P; several Kennedia species (Fabaceae) produce high amounts of root exudates and appear relatively well able to access insoluble forms of P (Pang et al. 2015), but do not exhibit root morphological specialisations (Ryan et al. 2012). Therefore, mining plants are characterised by their ability to acquire insoluble forms of P and not, necessarily, by distinct root morphology. 17

The ability to mine P involves the release of root exudates capable of mobilising insoluble P (e.g., bound to oxides or hydroxides of Fe or Al). Carboxylates released into the soil solution can act as ligands; P mobilisation can occur by ligand-promoted mineral dissolution, where carboxylates are adsorbed to the soil surface and the metal-ligand complex is subsequently detached, releasing the previously bound P (Johnson and Loeppert 2006). Additionally, the organic anions of the carboxylates can replace P on ligand-exchange surfaces, releasing it for plant uptake (Gerke et al. 2000; Johnson and Loeppert 2006). As a result of fast rates of carboxylate release, micronutrients can also be mobilised and accumulate in leaves (Gardner and Boundy 1983). For example, leaf manganese (Mn) concentrations of carboxylate-releasing Proteaceae are greater than those of co-occurring species lacking this mining strategy (Hayes et al. 2014). In this context, Lambers et al. (2015) suggested that leaf [Mn] could be used as a proxy for carboxylate release when comparing co- occurring species in P-impoverished soils. Furthermore, arbuscular mycorrhizal colonisation of the roots can decrease leaf [Mn], either through direct interception of Mn by the fungi or indirectly through promotion of changes in the proportion of Mn-reducing to Mn-oxidising microbes in the rhizosphere (Bethlenfalvay and Franson 1989; Kothari et al. 1991; Nogueira et al. 2007). As a result, leaf [Mn] may be a good indicator for species that exhibit mining nutrient-acquisition strategies as opposed to those that exhibit scavenging.

At the community level, the relative abundance of mining species increases in the most P-impoverished soils (Oliveira et al. 2015; Zemunik et al. 2015). At the species level, each species can vary in the degree of investment in scavenging versus mining strategies according to nutrient supply, and there seems to be a trade-off between these two strategies (Ryan et al. 2012). For example, Kennedia species can invest carbon (C) in both arbuscular mycorrhizal fungi and carboxylate release, but investment in one appears to occur, at least to some degree, at the expense of investment in the other (Ryan et al. 2012). When inoculated with arbuscular mycorrhizal fungi, Kennedia species released less carboxylates then in the absence of inoculation (Ryan et al. 2012). Nutrient availability can also affect this investment pattern (Del-Saz et al. 2017). For example, increased P supply decreases carboxylate release in K. prostrata and K. prorepens (Pang et al. 2010), decreasing the relative importance of a mining strategy at high P supply. Phosphorus supply can also affect the investment in a scavenging strategy. Several Trifolium subterraneum cultivars present a greater proportion of the root length colonised by arbuscular mycorrhizal fungi at intermediate P levels, decreasing with increasing P supply and at the lowest P supply (Jeffery et al. 2017). A third nutrient- 18

acquisition strategy, symbiotic N fixation, requires further belowground carbon investment. Symbiotic N fixation through nodule formation also depends on plant C and P supply (Ryle et al. 1979) and is inhibited at sufficient N supply (Vitousek et al. 2010; Png 2016). Therefore, the changes in the relative importance of different nutrient-acquisition strategies observed along soil gradients at the community level can be observed at a smaller scale, within species grown at different nutrient supplies.

1.2. Nutrient-use efficiency in nutrient-impoverished soils

Nutrient-use efficiency is achieved through the use of nutrients for a balanced vegetative and reproductive growth, and this balance is highly depended on species habitats (Reich et al. 2014). Efficient nutrient use includes reducing nutrient demand (Aerts and Chapin 1999), increasing productivity per unit nutrient taken up, balancing allocation to vegetative and reproductive tissues (Veneklaas et al. 2012), and preventing nutrient loss through efficient nutrient remobilisation from senescing tissues (Yuan and Chen 2009; Vergutz et al. 2012). Efficient nutrient use does not manifest similarly in fertile and infertile habitats. In fertile habitats, plants are expected to have fast rates of resource acquisition (both water and nutrients), grow fast thereby increasing nutrient demand, and allocate nutrients to tissues with short lifespans (Lambers and Poorter 1992; Wright et al. 2004; Reich 2014) with low remobilisation efficiency (Kobe et al. 2005). Conversely, acquiring nutrients in nutrient- impoverished habitats is expensive and efficient nutrient use in infertile habitats is expressed as slow growth with lower nutrient demand, tissues with longer lifespans (Lambers and Poorter 1992; Nardoto et al. 2006; Bustamante et al. 2012), high productivity per nutrient taken up (Denton et al. 2007), and efficient nutrient remobilisation (Nardoto et al. 2006; Lambers et al. 2010).

Changes in nutrient-use efficiency can also be studied at different scales. At the community level, species occurring in more fertile soils tend to be less conservative in nutrient use than species occurring in nutrient-poor soils. However, nutrient-use efficiency can also be studied at the species level, since some traits related to nutrient-use efficiency can exhibit phenotypic plasticity to variable nutrient supply (Kramer-Walter and Laughlin 2017). For example, root nutrient concentration, relative growth rate and biomass allocation were highly variable, while root diameter and tissue density, specific root length, wood density and specific leaf area, were relatively constant in two angiosperms and two gymnosperms grown at different nutrient availability (Kramer-Walter and Laughlin 2017). Thus, plant traits can 19

differ in their plasticity to nutrient availability, but we can observe differences in nutrient-use efficiency at the species level.

Most studies that assessed changes in nutrient-use efficiency along natural gradients of nutrient availability were conducted in relatively fertile gradients. For example, the chronosequence in Hawaii extends over four million years of soil development (Crews et al. 1995), and the values of leaf [P] of plants growing in the oldest soils are relatively high on a global scale (Wright et al. 2004; Lambers et al. 2010), due to the import of P in dust from China (Chadwick et al. 1999). The semi-arid chronosequence in Arizona extends over six million years of soil development, and also presents relatively high soil [P] (Selmants and Hart 2010). However, the Franz Josef chronosequence in New Zealand extends over 120,000 years of soil development, and does reach similar low values of total soil [P] and leaf [P] to those amongst the most P-impoverished soils at the very oldest soils of the chronosequence (Richardson et al. 2004; Lambers et al. 2010). Even so, relatively little is known about how native species change nutrient-acquisition and -use strategies along severely nutrient- impoverished chronosequences occurring in seasonally-dry climates.

1.3. Long-term soil development in nutrient-poor soils

Nutrient-impoverished habitats can be found in soils exposed for long periods of time, with constant climate in the absence of soil rejuvenating processes such as loess deposition, volcanic activity, land slides or glaciations that carry important P contributions (Vitousek et al. 2010). Ultimately, long-term soil development leads to ecosystem retrogression. Ecosystem retrogression is defined as the depletion or reduction in access to these nutrients by plants and other organisms that slow ecosystem process rates such as net primary productivity, decomposition, and rates of nutrient cycling (Peltzer et al. 2010). Systems undergoing retrogression can be found, for example, in the kwongan vegetation in south-western Australia and in the very north of Australia, in the Cape Floristic Region in South Africa, in the Venezuelan Tepuis and in mountaintop vegetation called campos rupestres in Brazil (Hopper 2009; Silveira et al. 2016; Mucina 2017). Within these systems, we can find gradients of fertility (Oliveira et al. 2015), soil mosaics (de Carvalho et al. 2012) or chronosequences (Laliberté et al. 2012; Turner and Laliberté 2015), which offer unique model systems to understand how nutrient-acquisition and -use strategies change with soil (in)fertility at varying scales.

Because of pedogenic processes during soil development, old landscapes 20

undergoing ecosystem retrogression are more P-depleted than N-depleted (Walker and Syers 1976; Peltzer et al. 2010). As a result, plants growing in more developed soils tend to be P- limited for growth, while plants growing in younger soils tend to be N-limited for growth. Nutrient-limitation for growth can be accessed via foliar [N] to [P] ratios (N:P ratios) or through experimental nutrient addition (Aerts and Chapin 1999). Foliar N:P ratios under 10 indicate N-limitation, ratios between 10 and 20 indicate N-P co-limitation and ratios above 20 indicate P-limitation (Güsewell 2004). Although these cuts were based on global datasets, they are approximations based on experimental assessments. Liebig’s law of the minimum inspired the experimental assessments, whereby plants respond with growth when the scarcest resource is added, until another resource is limiting (Güsewell 2004). Therefore, P and N limitation can be assessed through experimental additions of these nutrients. The most limiting nutrient for growth depends on species ability to acquire these nutrients and is an important driver of species capacity to grow on nutrient-impoverished soils. As such, soil- nutrient limitation and mineral nutrition strategies strongly influence species distribution along (in)fertility gradients at the local scale.

Therefore, the focus of my thesis was to understand the changes in plant nutrient- acquisition and -use strategies at different scales in three long-term soil development gradients in Brazil and Australia, with contrasting climates. All sites are included in the list of world biodiversity hotspots, being priority areas for conservation (Myers et al. 2000). One soil development sequence was located in campos rupestres and the other two were located in the kwongan vegetation in south-western Australia.

1.3.1. Study systems

In Brazil, the campos rupestres I studied occur over quartzite outcrops in the southernmost section of the Espinhaço Range, at Serra do Cipó. The literal translation for campos rupestres is “rupestrian grasslands”. In 2005, the Espinhaço Range was designated a Biosphere Reserve by UNESCO (UNESCO 2005), and it now includes 16 protected areas (Le Stradic 2012), but campos rupestres are still highly threatened by mining activities, extensive cattle ranching, highly frequent fires and invasive species (Fernandes et al. 2014). They occur in highlands within the cerrado biome and are composed of a mosaic of soil types and vegetation (Alves et al. 2014). The soils in these environments are heterogeneous, ranging from thin soil layers between rock outcrops to deep sandy depositions in valleys and depressions (Benites et al. 2003). They are amongst the most P-impoverished soils in the 21

world (Oliveira et al. 2015; Silveira et al. 2016). The vegetation is dominated mainly by herbaceous and subshrub species within Poaceae, Cyperaceae, Xyridaceae, Velloziaceae, Cactaceae, Eriocaulaceae, Euphorbiaceae, Lamiaceae, Melastomataceae, Myrtaceae and Orchidaceae (Alves and Kolbek 1994; Rapini et al. 2008; Silveira et al. 2016). Campos rupestres are characterised by mild dry winters and wet summers, Cwb climate (dry-winter highland climate) in Köppen´s classification (Köppen 1900), with mean annual precipitation of ~1300 mm (Neves et al. 2005).

The soil development sequence I studied in the campos rupestres (Fig. 1.2a) was composed of three stages of soil development from the same parent material: 1) the least developed soils were rock outcrops (Fig. 1.2b), with a thin layer of organic soil between the rock layers (Benites et al. 2007); 2) the intermediate soils were gravel sands (Fig. 1.2c), and 3) the most developed and leached soil type were the white sands (Fig. 1.2d); these were also the most P- and N-impoverished. In addition to the three campos rupestres soil types, I included an adjacent cerrado (Fig. 1.2e) oxisol (Fig. 1.2f) which originated from a different parent material for comparison purposes. Total soil [P] varied sixfold along this fertility gradient; while total soil [N] varied threefold (Chapter 1). Previous studies have focused along this gradient. Le Stradic et al. (2015) investigated plant composition and structure of plants growing on gravel sands and white sands and found both soils are strongly acidic and nutrient-poor, but gravel sands are slightly less infertile, leading to the formation of different plant characteristic communities of each soil type. Negreiros et al. (2014) investigated how functional traits of plants growing on gravel sands and white sands positioned themselves along the competitive - stress-tolerant - ruderal framework developed by Grime (1977), using traits presented by Lambers and Poorter (1992). These authors found that campos rupestres plants all occur in the stress-tolerant corner of the triangle, but plants growing in gravel sands were positioned even further in the stress-tolerant corner, despite the slightly greater nutrient availability in these soils. de Carvalho et al. (2012) investigated the distribution of arbuscular mycorrhizal fungi along this gradient, including the rock outcrops and the cerrado soils. The density of spores and species richness of arbuscular mycorrhizal fungi was greatest for the intermediately-developed gravel sands, and did not differ among the other soil types. However, little is known about how these arbuscular mycorrhizal fungi colonise the roots of campos rupestres plants and contribute to their mineral nutrition. Also, the link between below- and aboveground plant traits has received little attention in campos rupestres (Oliveira et al. 2015; Oliveira et al. 2016; Brum et al. 2017). The aim of this study was to understand 22

how nutrient-acquisition and use shift along this gradient of nutrient-availability at the community level.

Figure 1.2. Soil development sequence in campos rupestres at Serra do Cipó, in Brazil. a. Landscape overview of campos rupestres; b. The least developed soil type, quartzite rock outcrops; c. The intermediately developed gravel sands; d. The most developed white sands; e. Adjacent cerrado savannah with f. oxisols.

In Australia, I studied two chronosequences with contrasting climates and vegetation. Both chronosequences originated from similar parent materials and comprise a series of parallel sand dunes deposited over two million years (Laliberté et al. 2012; Turner and Laliberté 2015; Turner et al. 2017). The younger calcareous dunes present alkaline pH and high total soil [P], while the old dunes are acidic and severely P-depleted (Turner and Laliberté 2015; Turner et al. 2017). The first south-western Australian chronosequence is 23

located along the Warren beach (Fig. 1.3a,b,c), 350 km south of Perth, and is cooler and wetter (~1200 mm annual precipitation, Csb - Mediterranean climate with very hot summers - in Köppen´s classification) than the second chronosequence. Total soil [P] varies ten-fold along this chronosequence while total soil [N] varies seven-fold (Turner et al. 2017). The vegetation in this chronosequence varies from an open scrubland to a closed Eucalyptus forest with an understorey kwongan vegetation (Turner et al. 2017).

The second kwongan south-western Australian chronosequence is located in Jurien Bay (Fig. 1.3b,d), 250 km north of Perth with a Csa climate in Köppen´s classification - Mediterranean climate with mild summers (~530 mm annual precipitation). Total soil [P] varies 40-fold along this chronosequence, while total [N] varies three-fold (Turner and Laliberté 2015). The vegetation in Jurien Bay varies from open woody scrubland on the youngest dunes to Banksia woodland on the oldest dunes (Laliberté et al. 2012; Hayes et al. 2014; Turner et al. 2017). Several aspects of plant mineral nutrition have been explored along the Jurien Bay chronosequence. Plants from the oldest, most nutrient-impoverished soils present a greater diversity of nutrient-acquisition strategies (Zemunik et al. 2015). At the community level, there is a very high species turnover (Zemunik et al. 2016) accompanied by changes in nutrient-acquisition (Zemunik et al. 2015) and -use in these species (Hayes et al. 2014). A smaller proportion of species growing on the oldest, most nutrient-impoverished soils rely on arbuscular mycorrhizas for nutrient-acquisition (Zemunik et al. 2015). Plant species occurring on the oldest soils have lower leaf [P] and [N], but greater leaf [Mn], and also greater P- and N-remobilisation efficiency than those growing in younger soils (Hayes et al. 2014). At the species level, little is known about how individual species switch nutrient- acquisition strategies along this chronosequence. Albornoz et al. (2016) grew two species capable of forming dual symbioses with ecto- or arbuscular mycorrhizas in soils collected along the Jurien Bay chronosequence and found a within-species shift from greater colonisation by arbuscular- to ectomycorrhizas with soil age. This shift is probably due to the ability of ectomycorrhizas to access organic P, present as a greater proportion of total P in older soils (Turner and Laliberté 2015; Albornoz et al. 2016). Png et al. (2017) found that legumes occurring in the oldest soils also invest more in organic P acquisition through the release of phosphomonoesterases than legumes occurring in younger soils. Finally, the investment of legumes in symbiotic N-fixing nodules and arbuscular mycorrhizal fungi in the older soils of the Jurien Bay chronosequence is also decreased due to the P limitation of N fixation and inefficiency of arbuscular mycorrhizas in acquiring P in the oldest soils. 24

Together, these results show that species occurring along the Jurien Bay chronosequence can shift their resource allocation to nutrient-acquisition strategies when nutrient availability is very low. These studies have focused on the investment in mycorrhizas and nodules, or nodules and root exudation, but not on the three strategies at the same time.

Figure 1.3. Stages of the dune chronosequences in south-western Australia. Warren chronosequence (a,b,c) and Jurien Bay chronosequences (d,e) are a series of dunes deposited along the south-western Australian coast along 2 million years. a. The youngest stage of the Warren chronosequences, dunes are relatively mobile; b. Stages 2 to 5 of the Warren chronosequence; the canopy is more closed and understorey community composed by woody shrubs; c. Last stage of the Warren chronosequence, open canopy, herbaceous understorey; d. Woody scrubs of stages 4 and 5 of the Jurien Bay chronosequence and e. Banksia woodland in stage 6 of the Jurien Bay chronosequence.

The soil development sequence in the campos rupestres, the chronosequence along the Warren beach and the Jurien Bay chronosequence offer unique model systems to understand how nutrient-acquisition and -use strategies change at different scales on some of the most nutrient-impoverished soils in the world.

1.4. Significance of research

Nutrient-acquisition and -use strategies are of significant ecological importance as they regulate the ability of plant species to occupy soils (Lambers et al. 2008). Plants can acquire nutrients directly from the soil or through microbial symbioses such as mycorrhizas and nitrogen-fixing microorganisms; how plants use these nutrients and return them to the soil 25

in the senesced tissues has implications for key ecosystem processes such as decomposition and nutrient cycling (Aerts and Chapin 1999). The relative importance of species with each nutrient-acquisition strategy and also the efficiency with which plants use nutrients changes along gradients of soil development that also represent gradients of soil fertility in campos rupestres and in the Jurien Bay chronosequence in south-western Australia (Lambers et al. 2008; Oliveira et al. 2015; Zemunik et al. 2015). At the species level, species can also shift their investment in different nutrient-acquisition strategies with changing P or N availability in these habitats (Abrahão et al. 2014; Albornoz et al. 2016; Png et al. 2017). However, the shifts in nutrient-acquisition strategy in campos rupestres were observed in soils formed from different parent materials (Oliveira et al. 2015), and we found no information on photosynthetic nutrient-use efficiency and nutrient remobilisation in these habitats. Additionally, although we now have evidence for within species shifts in the investment in mycorrhizas and in nodules along south-western Australian chronosequences (Albornoz et al. 2016; Png 2016), we do not know how species under variable P and N supply allocate resources to a third underground carbon sink, carboxylate release. The improved understanding of how species acquire and use nutrients at different scales (community and species level) in soils with contrasting nutrient availability will help unveil the factors that maintain species-rich plant communities in campos rupestres (Silveira et al. 2016) and in kwongan sand dunes in south-western Australia (Hopper and Gioia 2004).

1.4.1. Aims

Using the soil development sequence and the two 2-million years chronosequences in Warren and Jurien Bay as model systems, the overall aims of this research were to determine:

i) how the proportion of arbuscular mycorrhizal versus non-mycorrhizal species (scavenging versus mining strategies) changes at the community level along the soil development sequence in campos rupestres and cerrado and the implications of nutrient- acquisition strategies for nutrient-use efficiency; and, ii) how two legume species that occur along two chronosequences with contrasting climates in the kwongan vegetation in south-western Australia shift their investment in root arbuscular mycorrhizal colonisation, carboxylate release and nodule formation with varying P and N supply.

The specific aims of this research were to: 26

i) establish whether the relative proportion of mycorrhizal plants occurring in less developed and nutrient-impoverished soils is greater than that in plant growing in more nutrient-impoverished soils along a soil development gradient in campos rupestres; if the proportion of species with alternative root specialisations such as rhizosheaths is greater in the most nutrient-impoverished soils; and whether rhizosheaths are a good indicator of root function, using leaf [Mn] as a proxy for carboxylate release and arbuscular mycorrhizal colonisation in species growing in the different soil types in the campos rupestres; ii) understand if species occurring in more nutrient-impoverished soils of the campos rupestres have lower leaf [P] and [N], but greater remobilisation efficiency of these nutrients during senescence; and determine if species growing in more nutrient-impoverished soils of the campos rupestres have slower photosynthetic rates, but greater photosynthetic P- and N-use efficiency than those of species growing in less infertile soils. iii) determine whether two legume species native to south-western Australian kwongan vegetation switch from N to P limitation for growth along the long-term gradients of soil development; and if these two legume species vary leaf [P] and [N] according to P and N supply. We also aimed to test if they responded to nutrient addition with increased growth; iv) establish the effect of increasing N and P supply in the investment in underground C for carboxylate release, arbuscular mycorrhizal symbiosis and nodule formation in the same two legume species in a glasshouse experiment

1.5. Thesis outline

The two studies which I undertook to achieve the aims of my research are presented within the following three chapters:

Chapter 2

In my first study, conducted in the soil development gradient in campos rupestres, I tested the hypotheses that species occurring in more nutrient-impoverished soils would rely less on arbuscular mycorrhizas (scavenging strategy) for nutrient-acquisition, and would have alternative non-mycorrhizal root specialisations such as rhizosheaths (mining strategy) to acquire the insoluble forms of P. I expected that species with rhizosheaths would release large amounts of carboxylates into the rhizosphere, mobilising Mn and increasing leaf [Mn]. I also expected species growing on more nutrient-impoverished soils to present greater photosynthetic nutrient-use efficiency and greater nutrient-remobilisation efficiency. I compared these traits in ten species growing in each of the four soil types: for each soil type 27

the ten species belonged to the most dominant families and represented the different growth forms of each soil type.

Chapter 3

In my second study, I investigated the within-species shifts in nutrient-acquisition strategies of two legumes along two long-term soil development sequences in the south- western Australian kwongan. The study was conducted with Bossiaea linophylla R. Br. and B. eriocarpa Benth. for the Warren and the Jurien Bay chronosequences, respectively. I hypothesised that these species would switch from N- to P-limitation for growth along the chronosequence and that leaf [P] and [N] would increase with increasing P and N availability, both in the field and in a glasshouse experiment. I expected these species to increase the investment in carboxylate release and decrease the investment in arbuscular mycorrhizas at low P supply, thereby increasing leaf [Mn]. I also predicted that the two species would increase nodule formation with decreasing N supply, but only at sufficient P supply, due to the P costs of nodulation.

Chapter 4

The final chapter synthesises the findings of this research, discusses the limitations of the studies, and outlines potential areas for future research.

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CHAPTER 2. NUTRIENT-ACQUISITION AND -USE STRATEGIES IN A MOSAIC OF SOIL TYPES IN CAMPOS RUPESTRES AND CERRADO

Anna Abrahão, Patricia de Britto Costa, Hans Lambers, Sara Adrian, Alexandra Christine Helena Frankland Sawaya, Megan H. Ryan, Rafael S. Oliveira

2.1. Abstract

Nutrient acquisition is one of the most important processes influencing plant distribution along fertility gradients. The reliance on arbuscular mycorrhizal fungi decreases with decreasing P availability and the reliance on alternative non-mycorrhizal strategies increases accordingly. Campos rupestres soils in Brazil are among the most phosphorus (P)- impoverished soils in the world. We studied nutrient-acquisition strategies and nutrient-use efficiency of plants growing at three stages of soil development in campos rupestres soils and on less infertile cerrado soil. The three stages of campos rupestres soils were the rock outcrops, gravel sands and white sands, in increasing order of soil development. In each soil type we selected four individuals from each of 10 species that represented both the most dominant plant families and the diversity of plant functional types. We expected species from more nutrient-impoverished white sands would exhibit specialised mechanisms for nutrient acquisition such as prominent rhizosheaths and rapid rates of carboxylate exudation; the latter we expected would correspond to high leaf manganese (Mn) concentrations. We also expected these species to be more conservative in nutrient use, that is, to be more efficient in photosynthesis per unit P and nitrogen (N) (PPUE and PNUE, respectively) and in P and N remobilisation from senescing leaves. Total soil [P] and [N] was greater in the cerrado than in the campos rupestres soils, and more developed soils in campos rupestres were also increasingly more P and N-impoverished. Similar to what is known for other severely P- impoverished soils, campos rupestres species shifted nutrient-acquisition strategies along the soil development gradient. The proportion of the root length colonised by arbuscular mycorrhizal fungi decreased from 71% at the cerrado site to less than 1% at the most P- impoverished white sands. Conversely, the proportion of species with non-mycorrhizal nutrient-acquisition strategies such as rhizosheaths was greater on the most P-impoverished soils, but there were no differences in rhizosphere carboxylate amounts or leaf [Mn] among plants with these two nutrient-acquisition strategies, indicating that plants using both strategies released carboxylates into the rhizosphere. Leaf [P] and [N] were very low and 38

decreased with decreasing soil [P] and [N]. Leaf N:P ratios switched from indicating N-P co- limitation of plant productivity in the cerrado to P-limitation in the campos rupestres. Photosynthetic rates decreased along the soil developmental sequence, but PPUE was equally very high and PNUE moderately high at different soil developmental stages. Most species presented exceptionally high P-remobilisation efficiency during leaf senescence (>70%), and moderately high N-remobilisation efficiency. This suite of traits highlights the highly efficient P-use and moderate N-use efficiency in campos ruprestres and cerrado plants, likely reflecting that plant productivity was more strongly limited by P than by N in these nutrient- impoverished habitats. In conclusion, the set of below- and aboveground traits integrating nutrient-acquisition and -use strategies used in this study were key for the determination of the very high species turnover at a very small scale in campos rupestres.

Key-words: carboxylates, mycorrhizas, nutrient remobilisation, rhizosheaths, rupestrian grasslands, sand-binding roots

2.2. Introduction

Nutrient availability is one of the main drivers of plant species distribution (John et al. 2007; Perry et al. 2008). As sessile organisms, plants can only germinate, grow and reproduce where they are able to tolerate the environmental constraints. Therefore, efficient nutrient acquisition and -use is paramount for species performance in plant communities in nutrient-impoverished habitats. Nutrient-use efficiency is defined by the largest outcome at minimal nutrient cost (Reich et al. 2014). The largest outcome will depend on the species and its growth context. For crop species, the largest intended outcome is the largest yield in starch, proteins or oils (Reich et al. 2014). On the other hand, for native species, nutrient-use efficiency represents the use of “nutrients for an optimal and balanced vegetative and reproductive growth, which is most suitable to survive and compete in their respective habitat and niche” (Reich et al. 2014). Nutrient-use efficiency can be split into several components (Reich et al. 2014): nutrient demand (Aerts and Chapin 1999), productivity per unit nutrient taken up (Veneklaas et al. 2012; Xu et al. 2012), and efficient remobilisation (Vergutz et al. 2012), which minimises nutrient losses (Reich et al. 2014). In nutrient-impoverished habitats, plant species are under strong selection for specialised nutrient-acquisition strategies (Lamont 1982) as well as conservative nutrient use through tissues with low nutrient concentrations (Aerts and Chapin 1999), slow growth, long-lasting, well-defended tissues (Lambers and 39

Poorter 1992; Reich 2014), high productivity per unit of nutrient taken up (Lambers et al. 2011) and high remobilisation efficiency (Kobe et al. 2005). Species growing in nutrient- impoverished soils are therefore placed at the slow end of the ‘plant economics spectrum’ (Reich 2014). Because phosphorus (P) and nitrogen (N) are the most frequently limiting nutrients for plant productivity (Vitousek et al. 2010), our study focused on these two nutrients.

Nutrient-acquisition efficiency is influenced by root architecture with morphological and physiological specialisations for acquisition from the soil (Lynch and Brown 2001; Haling et al. 2013), uptake kinetics (Xu et al. 2012), and radial transport inside the roots (Baligar et al. 2001). Plants can acquire nutrients directly from the soil or through mycorrhizal symbiosis (Kaiser et al. 2015). Mycorrhizal associations are the dominant nutrient-acquisition strategy in terrestrial plants, especially for P acquisition, which can be greatly enhanced through the actions of mycorrhizal fungi (Smith and Read 2008; Brundrett 2009). However, in habitats where P is severely limiting for productivity, the carbon costs of maintaining a mycorrhizal symbiosis can overcome the benefits of the association (Lynch and Ho 2005; Lambers et al. 2010; Ryan et al. 2012). In severely P-impoverished soils, plants present alternative non-mycorrhizal root specialisations such as cluster roots (Lamont 1972; Shane and Lambers 2005), dauciform roots (Lamont 1974; Playsted et al. 2006) and capillaroid roots (Lamont 1982; Shane et al. 2011). Additionally, plants occurring in nutrient- impoverished soils can present a root specialisation that occurs throughout the angiosperms called rhizosheaths (Brown et al. 2017; Pang et al. 2017). Rhizosheaths are sheaths of soil particles strongly adhered to the root surface formed through the exudation of mucilage (McCully 1999), with an increasingly recognised role in nutrient acquisition (North and Nobel 1997; Schmidt et al. 2003; Smith et al. 2011; Abrahão et al. 2014; Brown et al. 2017). Rhizosheath development depends on the length of the root hairs, but also on soil texture and water content (Haling et al. 2014). Root specialisations such as rhizosheaths are expected to locally release large amounts of root exudates that mobilise forms of P otherwise unavailable to plants (Dinkelaker et al. 1995; Skene 1998; Oburger et al. 2009; Playsted et al. 2010; Abrahão et al. 2014) and can also influence local redox potential and activity of microbial populations and convert soil N forms, leaving them available to plants (Xu et al. 2012).

The link between root specialisations and exudation is not straightforward. Güsewell and Schroth (2017) investigated dauciform root formation and exudation in Carex species (Cyperaceae) and found that species with dauciform roots had equal access to 40

different forms of P to species without dauciform roots. Additionally, mycorrhizal species can also release carboxylates, although there is a trade-off for belowground C investment. Therefore, caution must be taken when linking root form and function (Shipley et al. 2016), and direct evidence of root exudation is needed (Güsewell and Schroth 2017). As a result of the release of organic anions or carboxylates by the roots, soil manganese (Mn) is mobilised (Gardner et al. 1982; Grierson and Attiwill 1989) and can accumulate in leaves (Gardner and Boundy 1983). Therefore, leaf [Mn] has been suggested as a proxy for carboxylate release in P-poor soils (Lambers et al. 2015b), but there are caveats since fast transpiration rates can also increase leaf [Mn] through mass-flow in sandy soils (Huang et al. 2017). In contrast to carboxylate-releasing roots, arbuscular mycorrhizal fungi intercept Mn or change its solubility and lower leaf [Mn] in mycorrhizal plants (Bethlenfalvay and Franson 1989; Kothari et al. 1991; Nogueira et al. 2007). Consequently, leaf [Mn] is expected to be a good indicator of nutrient-acquisition strategies in plants occurring in the same habitat.

The first component of nutrient-use efficiency is conservative nutrient use. In nutrient-impoverished habitats, plants invest in slow growth with leaves with low nutrient concentrations (Aerts and Chapin 1999) of well-defended tissues (Reich 2014) which increases the residence time of the nutrient in the plant (Berendse and Aerts 1987; Westoby et al. 2002). Plants accumulate fibres, build thick cell walls, sclerenchyma and secondary plant compounds, all of which increase the lifespan and defence against herbivores and abiotic stress (Berendse and Aerts 1987; Wright and Cannon 2001). Long-lasting leaves have a greater leaf mass per area (LMA) (Lambers and Poorter 1992; Reich 2014), which further reduces leaf nutrient concentration on a mass basis (Lambers et al. 2011). These traits can be integrated into Grime’s stress-tolerant corner of the competitive – stress-tolerant – ruderal triangle (C-S-R) (Grime 1977; Negreiros et al. 2014). Therefore, efficient nutrient use does not manifest as fast rates of biomass accumulation in nutrient-limited habitats, but instead is expressed as slow growth of long-lasting tissues with low nutrient concentrations.

The second component of nutrient-use efficiency is productivity per unit nutrient taken up (Veneklaas et al. 2012; Xu et al. 2012). Species at the slow end of the plant economic spectrum present slow photosynthetic rates on a mass basis, slow growth rates, low leaf [N] and [P] and high LMA (Lambers and Poorter 1992; Wright et al. 2004; Reich 2014). Productivity per unit nutrient taken up represents the instantaneous rate of carbon fixation per unit of nutrient in the plant (Berendse and Aerts 1987). High LMA reduces photosynthetic N- use efficiency (PNUE) either because a larger fraction of the leaf N is allocated to cell-wall 41

material or defence compounds and does not contribute directly to photosynthetic rates (Berendse and Aerts 1987; Poorter and Evans 1998), or because of N overinvestment in Rubisco without a sink (Pons et al. 1993). However, high photosynthetic P-use efficiency (PPUE) is not necessarily correlated with low LMA (Veneklaas et al. 2012; Lambers et al. 2015a), since many species achieve fast photosynthetic rates at high LMA (Denton et al. 2007; Lambers et al. 2010; Lambers et al. 2015a). High PPUE and PNUE can be achieved through reducing leaf [P] and [N] of several P and N fractions. For example, young leaves of Proteaceae from severely P-poor soils replace phospholipids in cell membranes by sulfolipids or galactolipids (Lambers et al. 2012). These species also function at very low ribosomal RNA levels in leaves and hence decrease P and N demand (Sulpice et al. 2014). Therefore, plants from nutrient-impoverished environments can achieve high PPUE and PNUE through several mechanisms.

The third component of nutrient-use efficiency is efficient remobilisation and reallocation from senescing tissues. Since nutrient acquisition from the soil represents a cost to the plants, efficient plants are expected to withdraw a large fraction of the nutrients prior to senesced tissue shedding, thereby reducing nutrient losses. The remobilised nutrients can be used to build new tissues. Nutrient-remobilisation efficiency is greater in plants with decreased nutrient status (Vergutz et al. 2012). Therefore, the remobilisation efficiency is expected to be higher in plants growing in more nutrient-impoverished soils (Kobe et al. 2005; Hayes et al. 2014). Another important aspect of the remobilisation process is how much nutrient is left in senesced leaves. Killingbeck (1996) introduced the concept of nutrient- remobilisation proficiency, which is the final nutrient concentration in fully-senesced leaves. The litter nutrient concentration is vital for plants growing on nutrient-poor soils, because these plants are primarily dependent on soil litter for nutrient uptake. Nutrient-remobilisation proficiency plays a fundamental role in nutrient cycling and plant-soil feedbacks through litter decomposition (Killingbeck 1996; Hobbie 2015), a key plant trait that plays a significant role in ecosystem functioning (Lavorel et al. 2011; Lavorel 2013). Low nutrient-content litter with high concentrations of secondary plant metabolism compounds takes longer to be decomposed and for nutrients to become available again (Benites et al. 2007; Cornwell et al. 2008). Other plant parts under fast turnover rates such as cluster roots can also be under strong selection for high remobilisation efficiency (Shane et al. 2004). Hence, nutrient- remobilisation efficiency and proficiency is paramount for nutrient conservation and efficient use in plants. 42

The strategies to accomplish high nutrient-acquisition and -use efficiency change along gradients of nutrient availability (Chadwick et al. 1999; Hidaka and Kitayama 2009; Hayes et al. 2014; Zemunik et al. 2015). Along chronosequences, that is, long-term soil development sequences from the same parent material with a constant climate (Stevens and Walker 1970), soil nutrient concentrations change with increasing soil age (Walker and Syers 1976; Chadwick et al. 1999; Turner and Laliberté 2015). Phosphorus is continuously lost through erosion and leaching from the parental material, while N can accumulate due to biological fixation or atmospheric deposition, but is also lost from very old soils (Walker and Syers 1976; Peltzer et al. 2010). Consequently, plant growth on old soils tends to be P-limited rather than N-limited (Richardson et al. 2004; Parfitt et al. 2005; Lambers et al. 2010; Hayes et al. 2014). Plants occurring on oldest soils are expected to be more nutrient-acquisition and - use efficient than plants occurring on younger soils. Hopper et al. (2016) pointed out that twelve out of thirty five global biodiversity hotspots are old, P-impoverished landscapes (Old, Climatically Buffered, Infertile Landscapes, OCBILs). Similar patterns of nutrient-acquisition strategies are starting to emerge in old habitats in South America, South Africa and Australia (Lamont 1982; Abrahão et al. 2014; Hayes et al. 2014; Oliveira et al. 2015; Zemunik et al. 2015; Hopper et al. 2016; Silveira et al. 2016; Mucina 2017). We expect that understanding mineral nutrition in OCBILs will help unveiling the mechanisms underlying the generation and maintenance of the high biodiversity in nutrient-impoverished landscapes.

In South-America, campos rupestres are an old-growth grassland ecosystem (Veldman et al. 2015) with sparse shrubs and trees; campos rupestres occurs on mountaintops within the Brazilian savannah, the cerrado (Eiten 1972; Alves et al. 2014). Campos rupestres harbours an extremely high species richness (Giulietti 1987; Silveira et al. 2016) of lineages originating from Gondwanaland (Mello-Silva et al. 2011). Campos rupestres soils are shallow, acidic and are amongst the most P-impoverished soils in the world (Oliveira et al. 2015; Oliveira et al. 2016; Silveira et al. 2016) and frequently influenced by fires (Le Stradic et al. 2016). They are composed of a mosaic of soil types that form a gradient of N and P availability at the low end of the fertility gradient (de Carvalho et al. 2012; Le Stradic et al. 2015; Oliveira et al. 2015). We compared species nutrient-acquisition and –use strategies along gradient of soil development within the campos rupestres and it an adjacent oxisol covered with cerrado sensu stricto (Ribeiro and Walter 2008). We expected the plants from the different soils within more P-impoverished campos rupestres to use different strategies to achieve high efficient in nutrient acquisition and use than those of the cerrado. 43

Given the above, we tested five hypotheses. 1) Cerrado plants would be highly dependent on arbuscular mycorrhizal fungi for nutrient uptake and hence be highly colonised, while campos rupestres species would present a decreasing proportion of the root colonised by arbuscular mycorrhizal fungi along the soil development gradient, and increase the frequency of root specialisations such as rhizosheaths and high carboxylate release due to the inefficiency of arbuscular mycorrhizas in extremely P-poor soils. 2) Non-mycorrhizal species with carboxylate-releasing strategies would present greater leaf [Mn] than mycorrhizal species, 3) Plants growing in the more P-impoverished campos rupestres soils would show evidence of more conservative nutrient-use, that is, leaves with lower [P] and [N] (greater leaf N:P ratios) and more scleromorphic leaves (high LMA) and with mycorrhizal species showing greater leaf [P] and [N] than non-mycorrhizal species due to their occurrence on less nutrient-impoverished soils; 4) Photosynthetic rates would decrease with decreasing soil [P], but PPUE would be greater in the most P-impoverished soils, while PNUE would be less responsive to soil [N]; and 5) P- and N-remobilisation efficiency and proficiency from senescing leaves would increase with decreasing P and N soil availability. We expected that the integration of these below- and aboveground traits would help explain the very high species turnover at a very small spatial scale observed by Le Stradic et al. (2015).

2.3. Methods

2.3.1. Study area

The field site is located at Reserva Vellozia, Serra do Cipó at 900 to 1200 m above sea level, at the southern end of the Espinhaço Range, in the state of Minas Gerais, south-eastern Brazil (19° 16' 56'' S, 43° 35' 38'' W). The predominant vegetation type is campo rupestre. Campos rupestres are a montane, nutrient-poor, grassy-shrubby, fire-prone vegetation mosaic with rock outcrops (Alves et al. 2014; Silveira et al. 2016; Mucina 2017). The campos rupestres of Serra do Cipó occur over quartzite outcrops (Saadi 1995). However, present within the campos rupestres are also cerrado savannahs (Eiten 1972) over oxisol soil patches (de Carvalho et al. 2012). In this mosaic of campos rupestres soils, we chose three soil types occurring nearby, and a cerrado soil for comparison (Fig. 2.2.1a). All the soil types occur within a 700 m transect. Within the campos rupestres the thin soil layers within rock outcrops (Fig. 2.1b) are pedogenically the less developed soils, gravel sands are the intermediate stage (Fig. 2.1c) and seasonally water-saturated white sands are the most weathered soils (Fig. 2.1d). During pedogenesis, P erodes away, while N is expected to slowly 44

accumulate due to biological N-fixation, but to be lost in the oldest soils (Walker and Syers 1976). As a result, both soil [P] and soil [N] are lower in the white sands (most weathered) than in the gravel sands (intermediately weathered soils), especially during the dry season (Le Stradic et al. 2015), but Mehlich-1 soil [P] is similarly low in the rock outcrops and the cerrado soils (2-3 mg P kg-1 soil). We used the same sites as studied by de Carvalho et al. (2012). Our gravel sands are described as quartz gravel field, and our white sands as sandy bogs in de Carvalho et al. (2012).

The cerrado soils originate from a different parent material than do the campos rupestres. The cerrado soils are a clay loam, while the three campos rupestres soils are sandy loams (de Carvalho et al. 2012). The campos rupestres soils originate from quartzite rocks (Neves et al. 2005), which are naturally P-poor (Porder and Ramachandran 2013; Silveira et al. 2016). Depending on the local topography, campos rupestres soils can range from thin soil layers between rock outcrops to deep sandy depositions in valleys (Benites et al. 2007). Some of the soils in the valleys can be seasonally water-saturated and accumulate non-decomposed organic matter (Benites et al. 2003; Benites et al. 2005), such as our white sands. Seasonally- low temperatures, nutrient-poor soils and aluminium saturation contribute to slow soil decomposition of soil organic matter by soil microbes (Benites et al. 2007).

The regional climate is classified as Cwb, according to Köppen´s system (Köppen 1900), with marked seasonality, a dry cool winter and a rainy warm summer. Mean minimum monthly temperature recorded from 1961 to 2016 by a meteorological station at Conceição do Mato Dentro (19° 1' 12'' S; 43° 25' 48'' W), 30 km north of the study site, ranged from 10°C in July to 19°C in January, and maximum monthly temperatures ranged from 25°C in July to 30°C in February (http://www.inmet.gov.br). Mean annual precipitation is 1313 mm (http://www.inmet.gov.br).

2.3.2. Soil sampling and analyses

We collected three soil samples of 500 g each from each soil type. Each soil sample was a compound sample from ten soil cores at 0-10 cm collected within ten square meters of the same soil type. Samples from the same soil type were collected at least 100 m apart at the beginning of the rainy season in November 2015 (Fig. 2.2). We analysed total soil [P], the proportion of organic [P], total soil [N] and exchangeable cations to better understand the nutrient dynamics during the pedogenesis process. Total [P] was determined by ignition

(550°C, 1 h) and extraction in 1 M H2SO4 (16 h, 1:50 soil to solution ratio), with P detection 45

by automated neutralisation and molybdate colorimetry on a Lachat Quikchem 8500 (Hach Ltd, Loveland, CO, USA). Readily-exchangeable P (Resin [P]) was determined by extraction with anion-exchange membranes as described in Turner and Laliberté (2015). Organic and non-occluded inorganic P were determined by the NaOH-EDTA extraction method (Turner and Laliberté 2015). Total soil [N] was determined by combustion and gas chromatography on a Thermo Flash 1112 analyser (CE Elantech, Lakewood, NJ, USA). Soil pH was determined in a 1:2 soil-to-solution ratio in both water and 10 mM CaCl2 using a glass electrode. Exchangeable cations were determined by extraction in 0.1 M BaCl2 with detection by ICP-OES (Optima 7300DV; Perkin Elmer Inc, Wellesley, MA, USA). Total C and N were determined by automated combustion and gas chromatography with thermal conductivity detection using a Thermo Flash 1112 analyzer (CE Elantech, Lakewood, New Jersey). Soil was shaken in 1 M KCl, and the solution was filtered through a Whatman No. 42 filter paper for the determination of ammonium and nitrate. Ammonium was determined by automated colorimetry at 660 nm following reaction with phenolate, while nitrate was determined at 520 nm following cadmium-catalyzed reduction to nitrite and reaction with sulfanilamide at pH 8.5. Both ammonium and nitrate were determined using a flow injection analyzer (Lachat Quikchem 8500, Lachat Instruments, Loveland, CO, USA). All soil analyses were carried out at the Soils Laboratory of the Smithsonian Tropical Research Institute, Panama.

2.3.3. Species selection

Previously, a strong association was established between species composition and soil attributes for the gravel sands and white sands in the campos rupestres at Serra do Cipó (Le Stradic et al. 2015). We observed that an association existed for the other two soil types: rock outcrops and the cerrado oxisol. We selected representatives of the most dominant plant families (Le Stradic et al. 2015) on each soil type that also represented the diversity of plant functional types for each soil type (graminoids, herbs, woody shrubs, rosettes and trees). In each soil type we selected 10 species and four individuals per species (Table 2.1 Sup. Info). Although the chosen species are only a very small fraction of the ~1600 plant species pool(Giulietti 1987), we expected them to represent the different life forms in campos rupestres. 46

2.3.4. Root sampling for nutrient-acquisition strategies

We assessed three categories of nutrient-acquisition strategy: 1) presence of a rhizosheath, 2) colonisation by arbuscular mycorrhizal fungi and carboxylates associated with root tips. In the middle of the growing (rainy) season (February 2015) we collected at least 30 cm of fresh fine roots from four individuals per species and immediately stored them in 50% (v/v) ethanol so we could later assess mycorrhizal colonisation. Prior to mycorrhizal assessment, all the roots were examined for the presence of rhizosheaths with a stereomicroscope (Leica M80, Heerbrugg, Switzerland) and photographed with a Leica DFC295 3.0 MP digital camera with focus stacking using Leica Application Suite 3.8.0 (Leica, Wetzlar, Germany). The presence of rhizosheaths was recorded if the roots still had soil attached even after shaking the roots in the vials and scrubbing with a paint brush.

For mycorrhizal assessment, the roots were cleared in 10% (w/v) KOH and kept in a water bath at 90°C for 3-6 h until the roots were clear, replacing the KOH when necessary. If the roots were still dark, they were transferred them for 10-50 min to 3% (v/v)

H2O2 mixed with 20% (v/v) NH4OH (10:1) (Koske and Gemma 1989). After clearing, the roots were rinsed, acidified in 1% (v/v) HCl overnight, and stained them with acidified glycerol (glycerol, deionised water and 1% (v/v) HCl, 10:9:1) containing 0.05% (w/v) Trypan blue (Koske and Gemma 1989) in a water bath at 90°C for 1-3 h. The stained roots were stored in acidified glycerol until assessment of mycorrhizal colonisation with the slide method in 30 cm of roots (Giovanetti and Mosse 1980) at 200 x magnification with a compound microscope (Olympus BX51, Tokyo, Japan) mounted with a Leica DFC 295 digital camera (Leica, Wetzlar, Germany). We observed several fungal structures such as sclerotia and hyphae from dark septate fungi in the roots of cerrado and campos rupestres species, however we only recorded the proportion of roots colonised by arbuscular mycorrhizal fungi because the identity and the role of other fungi in plant nutrition are unknown. We did not observe ecto- or ericoid mycorrhizas in the roots inspected in our study. Therefore, roots were recorded as colonised by arbuscular mycorrhizal (AM) fungi if we observed Arum- or Paris- type structures (Dickson 2004) such as arbuscules, vesicles or hyphal coils.

Carboxylates are mainly released at the root tips (Lambers et al. 2006), and Ryan et al. (1995) found similar carboxylate efflux in excised root tips of Triticum aestivum when compared with the efflux measured in the roots of intact seedlings; therefore, we used excised root tips for carboxylate collection. To collect the carboxylates in the field, we excavated and excised three root tips per plant, measuring 1-3 cm with the rhizosphere soil attached. The 47

rhizosphere soil was defined as the soil remaining after gently shaking the roots (Veneklaas et al. 2003). Rhizosheath-forming species had more soil attached than species without rhizosheaths. Thus the carboxylates measured in rhizosheath-forming species had a greater contribution from the soil than those of species without rhizosheaths. The measured carboxylates were those released from the root and the rhizosphere carboxylates, both microbial- and plant-released. We immersed the root tips including the cut end in 1 ml 0.1% (v/v) formic acid for 5 min, to avoid further microbial breakdown of the organic acids. This trap solution was immediately filtered through a 0.22 μm syringe filter and the samples kept on ice for a maximum of 2 h before freezing at -20°C until analysis. Each root tip was weighed in order to express rhizosphere carboxylate amounts per unit root fresh weight. The samples were analysed with an ultra-high performance liquid chromatography system with a triple quadrupole mass spectrometer (UPLC-MS) and an electrospray ionisation source, Acquity UPLC-TQD (Waters, Milford, MA, USA) as described in Abrahão et al. (2014).

2.3.5. Leaf collection for nutrient analyses

We collected fully-expanded sun-exposed mature leaves at the end of the rainy season in April 2014 for nutrient analyses from the same individuals we collected roots. If the individual did not have enough leaves for nutrient analyses, we marked and collected neighbouring individuals (<2 m apart). For at least three leaves per individual, we positioned leaf traps made of 100% polyamide tulle meshes held to the stems with galvanised steel wires. For herbaceous species that kept the leaves after senescence (e.g., Poaceae, Cyperaceae, Xyridaceae), we marked mature leaves with crochet cotton thread with a plastic label. At the beginning, and in the middle, of the dry season (end of May and beginning of August 2014) we checked the tulle leaf traps and collected senesced leaves from all the species. All the leaves per individual were bulked. The leaves were oven-dried at 60°C for seven days and ground with a Geno/Grinder 2010 (SPEX SamplePrep, Metuchen, NJ, USA) using stainless steel bearings for 6 min at 950 rpm. A subsample of mature and senesced leaves was digested in concentrated HNO3: HClO4 (4: 1) (Sarruge and Haag 1974) for P and micronutrient analyses. Phosphorus was determined colourimetrically with the ascorbic acid method (Braga and Defelipo 1974). Manganese was determined by atomic absorption spectrometry (Varian Spectra AA 220FS, Varian, Mulgrave, Australia). Another subsample was digested with the Kjeldahl digestion method (Bradstreet 1965) and N determined by atomic absorption spectrometry as above. Leaf nutrient analyses were done at Laboratório de análise de plantas of Universidade Federal de Viçosa, Brazil. Phosphorus- and nitrogen-remobilisation 48

efficiency from senesced leaves was calculated as 100% × (1 − [Nutrient]senesced

/[Nutrient]mature).

2.3.6. Photosynthesis, photosynthetic nutrient-use efficiency and leaf mass per area

Photosynthesis was measured in the field with a Licor LI 6400 XT (Licor, -6 3 -3 Lincoln, NE, USA) with a CO2 mixer to provide stable CO2 supply at 380 x 10 m CO2 m air on three leaves per individual and four individuals per species. To set the time interval and photosynthetically active radiation (PAR, 400-700 nm) of photosynthesis measurements, we made diurnal and light-response curves for Eriocaulaceae sp. 3 (herb), Barbacenia flava (woody rosette), Lagenocarpus tenuifolius (graminoid) and Trembleya laniflora (woody shrub); species choice was designed to reflect the diversity of functional types in the study (Table 2.1. Sup Info). The light-response curves started at 1000 µmol photons m–2 s–1, and gradually increased to 2500 µmol photons m–2 s–1. We started logging the photosynthetic rates at 2500 µmol photons m–2 s–1 when stable and recorded photosynthetic rates at each PAR interval, decreasing by 200 µmol photons m–2 s–1 at each step until 500 µmol photons m–2 s–1 (e.g., 2500, 2300, 2100 µmol photons m–2 s–1, etc.). Because PAR above 1700 µmol photons –2 –1 m s decreased CO2-assimilation rates of B. flava, and plants started to close their stomata after 14.00 h, we recorded the photosynthetic rates of all the species in the study from 8.30 h to 13.30 h with PAR set to 1700 µmol photons m–2 s–1. Each measurement was logged after photosynthetic rates were stable for at least one minute.

To calculate photosynthetic P- and N-use efficiency (PPUE and PNUE, respectively), we divided photosynthetic rates by leaf [P] and [N] reported on an area basis. We used leaf mass per area to convert leaf [P] and [N] from mass to area basis. To calculate leaf mass per area, we collected at least three leaves per individual for three individuals per species, kept them wrapped in moist paper towels and scanned the leaves the same day. The leaves were oven-dried at 60°C for a week and weighed. Leaf area was calculated with imageJ (Schindelin et al. 2015).

2.3.7. Statistical analyses

All statistical analyses involved model selection using corrected Akaike Information Criterion (AICc) (Zuur et al. 2009) in the R environment (R Development Core Team 2017). For the soil data, one-way ANOVAs were used to compare soil attributes among soil types. A model with intercept only (null model) was compared with a model with soil type as an independent variable. We checked model assumptions graphically (Zuur et al. 49

2009), and if they were not met, variance was modelled using generalised least square models (gls) with the nlme package (Pinheiro et al. 2017). For the plant data, we used linear mixed models with species as a random factor using the nlme package (Pinheiro et al. 2017). If the models presented residual heteroscedasticity or non-normality, plant attributes were modelled using generalised linear mixed models (glmm) with gamma distribution with nlme or glmmTMB (Magnusson et al. 2017). Because mycorrhizal colonisation and nutrient remobilisation were a percentage, we modelled them using glmm with beta distributions with the glmmADMB package (Skaug et al. 2016) adding 10-11 to all the values, because zeros cannot be included in the model. We modelled the presence or absence of arbuscular mycorrhizal colonisation and rhizosheaths in each species with generalised linear models (glm) with a binomial distribution. We selected random terms in gls and fixed terms in all the models using the corrected Akaike Information Criterion (AICc) (Zuur et al. 2009). If the differences in AICc between models were less than two units, we chose the most parsimonious model (Arnold 2010). If any term was in the selected model, Tukey post-hoc comparisons were performed with the lsmeans package (Lenth 2016).

2.4. Results

2.4.1. Soil data

Total soil [P] by ignition was very variable among soil types, being greater in the cerrado oxisol and lowest in the more developed white sands (Fig 3a, Table 2.2 Sup. Info). Total [P] in the cerrado oxisol was 171 ± 46 mg P kg-1 soil (least square means ± standard error), 41 ± 4 mg P kg-1 soil at the less developed rock outcrops, 59 ± 8 mg P kg-1 soil in the intermediately developed gravel sand soils and 28 ± 2 mg P kg-1 soil in the more developed white sands. The resin-extractable [P] was much lower in the cerrado than in the campos rupestres (Fig 3b, Table 2.2 Sup. Info). Resin [P] in the cerrado oxisol was 0.4 ± 0.4 mg P kg- 1 soil, 2.8 ± 0.4 mg P kg-1 soil at the rock outcrops, 2.5 ± 0.4 mg P kg-1 soil in the gravel sands and 1.8 ± 0.4 mg P kg-1 soil in the white sands. Most of the P was in organic forms in all the soil types (Fig 3c, Table 2.2 Sup. Info). The proportion of organic [P] out of total P extracted with NaOH was 60% for the cerrado, 73% for the rock outcrops, 58% for the gravel sands and 63% for the white sands. Total soil [N] was greater for the cerrado soils and the gravel sands (Fig 3d, Table 2.2 Sup. Info). In the cerrado soils, total [N] was 1.4 ± 0.2 4 g N kg-1 soil, 0.9 ± 0.1 g N kg-1 soil at the rock outcrops, 1.2 ± 0.1 at the gravel sand and 0.4 ± 0.1 g N kg-1 soil at the white sands. 50

2.4.2. Nutrient-acquisition strategies

We found the following structures characteristic to arbuscular mycorrhizas: arbuscules (Fig. 2.4a,b), coils (Fig. 2.4a,b) and vesicles (Fig. 2.4c) in 27 out of the 40 studied species. The proportion of AM species varied from 100% in the cerrado to 50-60% in the campos rupestres soils, especially at the more developed gravel sand and white sand (Table 2.3 Sup. Info, Fig. 2.5a). The proportion of the root colonised by arbuscular mycorrhizal fungi decreased from 71% at the cerrado site to less than 1% at the most nutrient-impoverished white sands site (Table 2.3 Sup. Info, Fig. 2.5b). The proportion of species with rhizosheaths (Fig. 2.6a,b,c) increased from 10% at the cerrado site to 100% at the white sands (Table 2.3 Sup. Info, Fig. 2.5c).

Total rhizosphere carboxylate amount was very low (<1 μmol g-1 root FW) and did not differ among soil types, between the AM and NM species (Fig. 2.7a) or between species with and without rhizosheaths (Fig. 2.7b). Mature leaf [Mn] did not differ among soil types and on each soil type, mature leaf [Mn] did not differ between AM and NM species (Fig. 2.8a) or between species with or without rhizosheaths (Fig. 2.8b). The lowest mature leaf [Mn] was in Stryphnodendron adstringens (Fabaceae, 13 mg Mn kg-1 DW) growing in the cerrado soil and the greatest mature leaf [Mn] was in “Lychnophora jolyana sp. Ined.” (Asteraceae, 478 mg Mn kg-1 DW) growing on the rock outcrops.

2.4.3. Leaf P and N concentrations

Mature leaf [P] was greatest in the cerrado soils and less in the campos rupestres soils (Table 2.3 Sup. Info, Fig. 2.9a). Senesced leaf [P] was similar among soil types and leaf P remobilisation was similarly high among soil types, between 75 and 88% (Table 2.3 Sup. Info, Fig. 2.9a,b). The species with the greatest mature leaf [P] was Neea theifera (Nyctaginaceae, 1.1 mg P g-1 DW) from rock outcrops and the species with the lowest mature leaf [P] was Xyris pilosa (Xyridaceae, 0.14 mg P g-1 DW) from the gravel sand. Mature leaf [P] did not differ among AM and NM species (Fig. 2.9c) or species with and without rhizosheaths (Table 2.3 Sup. Info, Fig. 2.9d).

Mature leaf [N] was lowest in the gravel sands and white sands soils and greatest in the cerrado soils (Fig. 2.10a). Senesced leaf [N] was similar among soils and leaf N remobilisation was greatest in the white sands (59%) and lowest in the gravel sands (35%, Fig. 2.10a,b). Mature leaf [N] did not differ among AM and NM species (Fig. 2.10c), but was greater in species with rhizosheaths than species without rhizosheaths (Table 2.3 Sup. Info, 51

Fig. 2.10d). The species with the greatest mature leaf [N] was also N. theifera (32 mg N g-1 DW) from rock outcrops, and the species with the lowest mature leaf [N] was Bulbostylis paradoxa (Cyperaceae, 5 mg N g-1 DW) from the gravel sand. Leaf N: P ratios of mature leaves were lowest in the cerrado soils and varied from 11 in a Poaceae growing in the cerrado soils to 46 in an Eriocaulaceae growing on the rock outcrops (Fig. 2.11).

2.4.4. Photosynthetic nutrient-use efficiency

CO2-assimilation rates were lower for the species growing on the white sands -2 -1 (Table 2.3 Sup. Info, Fig. 2.12). CO2-assimilation rates varied from 8 ± 1 μmol CO2 m s on -2 -1 the more nutrient-impoverished white sands to 13 ± 1 μmol CO2 m s at the least nutrient- impoverished cerrado site. Only two species showed assimilation rates faster than 20 μmol -2 -1 CO2 m s : “L. jolyana sp. Ined.” growing on the rock outcrops and Marcetia taxifolia (Melastomataceae) growing on gravel sands. Photosynthetic P-use efficiency did not differ -1 among species in different soil types (Table 2.3 Sup. Info), and varied from 106 μmol CO2 g P s-1 on the cerrado soil to 189 on the white sands. Photosynthetic P-use efficiency did not differ between AM and NM species (Fig. 2.13a) or species with or without rhizosheaths (Fig. 2.13b). Photosynthetic N-use efficiency also did not differ among soil types and varied from 5 -1 -1 -1 -1 μmol CO2 g N s on the rock outcrops to 9 μmol CO2 g N s on the gravel sand. Photosynthetic N-use efficiency was greater for AM than for NM species due to higher leaf [N] on an area basis in NM species, not faster photosynthetic rates in AM species (Fig. 2.13c), but PNUE did not differ between species with and without rhizosheaths (Fig. 2.13d).

2.5. Discussion

Campos rupestres soils are among the most P-impoverished soils in the world (Oliveira et al. 2015; Silveira et al. 2016). We investigated several plant traits related to P- and N-acquisition and -use efficiency in campos rupestres and cerrado plants in four nutrient- impoverished soils that form an (in)fertility gradient. Our results emphasise the shifts in nutrient-acquisition strategies and the very high P-use and moderate N-use efficiency in campos rupestres and cerrado plants, probably reflecting the stronger P- than N-limitation of plant productivity in these habitats. We found very nutrient-conservative traits in species growing on the mosaic of soil types reflecting the slow-growth strategy of campos rupestres and cerrado plants (Lambers and Poorter 1992; Bustamante et al. 2012a; Negreiros et al. 2014; Reich 2014). The turnover o below- and aboveground nutrient-acquisition and –use 52

strategies are very likely to be important drivers of the very high species turnover observed in different soil types.

2.5.1. Soil nutrient availabilities

Total soil [P] was greater in the cerrado than in the campos rupestres soils, while soil P-availability (resin P) indicated lower P availability in the cerrado. These lower values are associated with the inability of the anion-exchange resin to extract P bound to Fe and Al oxides in the cerrado soils (Fink et al. 2016; Schmitt et al. 2017). However, leaf [P] was greater in plants growing on the cerrado soils, indicating that the plants growing on these soils are capable of taking up P bound to Al and Fe oxides, also facilitated by the less acidic pH in the cerrado soils (Table 2.4 Sup. Info). Total [P] was slightly greater in gravel sands than in rock outcrops, probably due to plant-soil nutrient feedbacks during soil formation, which also increased total [C]. Total soil [N] in the cerrado was within the expected range (1.5 mg g-1) for these soils (Bustamante et al. 2004), and the very low soil [N] of the campos rupestres was also similar to that in other quartzitic campos rupestres (Messias et al. 2013; Oliveira et al. 2015). In summary, the soil nutrient data showed that the less developed rock outcrops and the most developed white sands were the most impoverished in P and the white sand soils were more impoverished in N .

2.5.2. Nutrient-acquisition strategies

Along gradients of ecosystem development, plants are expected to switch from being highly dependent on arbuscular mycorrhizas for P acquisition in young and P-richer soils to non-mycorrhizal in old, severely P-impoverished soils (Lynch and Ho 2005; Miller 2005; Lambers et al. 2008). Our first hypothesis was that the same shift would occur in our soil development mosaic. In accordance with our expectations, we observed a clear shift in the proportion of mycorrhizal species, and also in the proportion of the root length colonised by arbuscular mycorrhizal fungi, from > 50% in the less P-impoverished cerrado and rock outcrops to <1% in in the more P-impoverished white sands. Moreover, we observed a major increase in the proportion of species forming rhizosheaths with P-impoverishment, supporting our first hypothesis. There seems to be a trade-off between C use to support the mycorrhizal symbiosis and C use for rhizosheath formation and maintenance (i.e. root hair production, mucilage release), and function (i.e. carboxylate release). The dominance of non-mycorrhizal root specialisations in the most nutrient-impoverished soils demonstrated their ability to access sparingly available forms of P in strongly P-depleted soil such as in campos rupestres. 53

All the rhizosphere carboxylate amounts we measured in the field were extremely low (<1 μmol g-1 root FW). A hydroponically-grown non-mycorrhizal cactus from campos rupestres showed up to 200 μmol g-1 root FW of total carboxylate release, possibly indicating that our field-based measurements only detected a small portion of the exudates. In Hakea prostrata and Embothrium coccineum (Proteaceae), the organic anions are released in an exudative burst (Shane et al. 2004; Delgado et al. 2014). Even though we collected fresh roots in the growing season, it is unlikely that we collected all the roots at the time of the peak carboxylate release. Moreover, carboxylates are rapidly degraded by soil microbiota (Evans 1998; Hashimoto 2007) or adsorbed onto soil particles (Oburger et al. 2009). Therefore, our field-based rhizosphere carboxylate collections likely underestimated the actual amount of carboxylates released by the root.

Our second hypothesis was that leaf [Mn] would be a good indicator of the amount of carboxylates released, because of their solubilisation effect on soil Mn (Lambers et al. 2015b). Additionally, arbuscular mycorrhizal fungi can significantly decrease the amount of Mn transferred to the host plants (Bethlenfalvay and Franson 1989; Kothari et al. 1991; Nogueira et al. 2007). However, leaf [Mn] did not differ between AM and NM species and also in species with and without rhizosheaths. Therefore, we rejected our second hypothesis that non-mycorrhizal and rhizosheath-bearing species would present greater leaf [Mn] than mycorrhizal species. The lack of differences in mature leaf [Mn] between mycorrhizal and non-mycorrizal species can be due to the fact that mycorrhizal species can also release carboxylates (Ryan et al. 2012). Concurrently, species with and without rhizosheaths can also release carboxylates (Güsewell and Schroth 2017). We only observed a tendency for rhizosheath-bearing species to present greater leaf [Mn]; this could eventually be attributed to a change in root exudation of carboxylates (Lambers et al. 2015b) which was not observed in all species, or in the microbial composition of the rhizosphere (Kothari et al. 1991). Therefore, although we did not find a strong link between rhizosheath formation, carboxylate release and leaf [Mn], the strong increase in the proportion of species with long root hairs (rhizosheaths) demonstrates the important role of root hairs in severely P-impoverished soils (Lynch 2011).

2.5.3. Conservative nutrient use

Our third hypothesis was that plants growing in the more nutrient-impoverished soils would present more nutrient-conservative leaves. We expected plants from P- impoverished soils to have a low P and N demand for biomass production producing leaves 54

with lower nutrient concentrations, greater leaf N: P ratios and greater LMA. We also expected non-mycorrhizal species to have lower leaf [P] and [N] than mycorrhizal species. In fact, mature leaf [P] was half for plants growing in the most P-impoverished campos rupestres soils compared with that of plants in the less P-impoverished cerrado soils, but it did not change between species growing on different campos rupestres soils and did not reflect the nutrient-acquisition strategies. Mature leaf [N] was also greater in the cerrado plants than in the campos rupestres plants, especially at the intermediate stage of soil development, the gravel sand, but did not completely reflect total soil [N], because soil total [N] was greatest in the cerrado and gravel sand, and leaf [N] was lowest in the gravel sand. Mature leaf [N] was also greater in species with rhizosheaths than in those without rhizosheaths. It is possible that the greater leaf [N] in species with rhizosheaths was due to increased N uptake through the release of proteases for organic N acquisition (Schmidt et al. 2003; Paungfoo-Lonhienne et al. 2008). Leaf N: P ratios were lowest in the cerrado plants, with a mean value of 18, indicating N-P co-limitation for growth (Güsewell 2004) as previously observed in cerrado trees (Nardoto et al. 2006) and grasses (Lannes et al. 2012; Lannes et al. 2016). The leaf N:P values of campos rupestres plant species were above 20, indicating P limitation for growth, as similarly observed by Oliveira et al. (2015). Finally, leaf mass per area was similarly high on all soil types, representing the construction of well-defended leaves across our soil gradient (Lambers and Poorter 1992). The very high leaf mass per area of the campos rupestres species, together with high leaf dry matter content of the scleromorphic leaves places the campos rupestres species at the stress-tolerant corner of the C-S-R triangular scheme (Grime 1977; Lambers and Poorter 1992; Grime et al. 1997; Negreiros et al. 2014). Overall, we accepted our third hypothesis and both the cerrado and campos rupestres species presented very low leaf [P], moderate leaf [N] and high LMA, reflecting their very conservative P-use strategy (Aerts and Chapin 1999; Bustamante et al. 2012b).

2.5.4. Photosynthesis, photosynthetic P- and N-use efficiency and functional traits

Our fourth hypothesis was that photosynthetic rates would decrease while PPUE and PNUE would increase along the soil development gradient, from less P-impoverished cerrado to strongly P-impoverished campos rupestres. Photosynthetic rates varied from 13 to -2 -1 8 μmol CO2 m s from the cerrado (oxisol) and rock outcrops (less developed campo rupestre soil) to the white sands (most developed, P- and N-impoverished campo rupestre soil), supporting our hypothesis. Similarly, photosynthetic rates of dominant species occurring along a chronosequence in New Zealand decreased with decreasing soil P and N availabilities 55

(Whitehead et al. 2005). The moderately low values for photosynthetic rate for campos rupestres species, when compared with the global values (Wright et al. 2004), reflect the conservative use of nutrients of plants at the “slow” end of the leaf economics spectrum (Reich 2014). Plants at this end of the continuum have high leaf tissue density, long leaf life spans and slow rates of resource acquisition and photosynthesis (Reich 2014), as also observed in our study. Due to the very high irradiance in the campos rupestres, it seems that a set of anatomical and physiological strategies has been selected to protect the photosynthetic apparatus (Pereira et al. 2017). For example, Vochysia thyrsoidea presents a hypodermis in the leaves that can protect against dehydration of the chlorenchyma and thus photoinhibition (Pereira et al. 2017) and Coccoloba cereifera leaves contain anthocyanins (Milanez 1951) that can provide photoprotection (Steyn et al. 2002; Pereira et al. 2017).

Despite the slow photosynthetic rates, species growing on different soils achieved similarly very high photosynthetic P-use efficiencies, similar to the extremely efficient Banksia and Hakea species (Proteaceae) growing in similarly P-impoverished habitats in south-western Australia (Denton et al. 2007; Sulpice et al. 2014). The global mean PPUE calculated from the dataset used in Wright et al. (2004) by Lambers et al. (2010) is 103 µmol -1 -1 CO2 g P s , similar to the values observed for the cerrado plants, but half of the values observed in plants growing in the white sands, although they were not significantly different due to the large variation of PPUE in each soil type. Mean PNUE in our study was very -1 -1 similar to the global mean value of 6.3 µmol CO2 g N s (Lambers et al. 2010) in all soil types, and slightly greater in AM when compared with NM species in our study. This difference is due to greater leaf [N] on an area basis in NM species, due to a possible moderating effect of mycorrhizas intercepting N as they do with P (Smith and Read 2008; Nazeri et al. 2014). Photosynthetic N-use efficiencies were not as high as those of PPUE when compared with global values, possibly due to the stronger P-limitation than N-limitation of plant productivity as shown by the leaf N: P ratios. Photosynthetic nutrient-use efficiency is expected to increase in infertile soils (Hidaka and Kitayama 2009), but since all the soils in our study are infertile, species growing in all the soils presented very high PPUE, but moderate PNUE, rejecting our hypothesis of increased PPUE and PNUE along the soil development gradient.

2.5.5. Nutrient remobilisation efficiency and proficiency

Species from infertile habitats are expected to be more efficient in nutrient remobilisation and thus avoid the costs of nutrient uptake (Aerts and Chapin 1999; Kobe et al. 56

2005; Yuan and Chen 2009; Vergutz et al. 2012; Hayes et al. 2014). Therefore, our fifth hypothesis was that species from soils with lower P and N availabilities (i.e. the white sands) would remobilise more P and N. Species from all soil types remobilised more than 75% of P during leaf senescence, reaching >90% P remobilisation in Velloziaceae and Poaceae; however, there were no differences among soil types. Remobilisation of P and N from senescing leaves of the cerrado species was very similar to the values previously reported by Nardoto et al. (2006), Miatto et al. (2016), and Kozovits et al. (2007) but we have not found published values of P and N remobilisation efficiency in campos rupestres plants. Species from all soils presented very low senesced leaf [P] (<0.06 mg g-1 in the white sands), one tenth than global estimates (Vergutz et al. 2012), representing a great remobilisation proficiency (Killingbeck 1996). Leaf N-remobilisation efficiency was greatest (60%) for the white sands, where total soil [N] was lowest, supporting our hypothesis on N-remobilisation. The ability of plants to remobilise N usually increases with latitude, while the ability to remobilise P increases towards the equator due to the patterns of N and P limitation of plant productivity (Yuan and Chen 2009). Therefore, a greater capacity to remobilise P than N was expected in our study, due to the greater P limitation for growth in the severely leached campos rupestres and cerrado soils. The shedding of very low-nutrient leaves into the litter contributes to the slow decomposition rates and slow nutrient cycling in the system, maintaining low soil nutrient concentrations (Aerts and Chapin 1999; Benites et al. 2007; Kozovits et al. 2007; Jacobson et al. 2011). Nutrient-remobilisation proficiency is a clear example of plant traits that shape the ecosystem processes at the “slow” end of the plant economics spectrum (Lavorel et al. 2011; Reich 2014). In summary, we found support for the hypothesis that N remobilisation would be greater in N-poor soils, but P remobilisation was equally high for all four soil types.

2.5.6. Conclusions

Functional traits of plant species growing in campos rupestres reflect the harsh conditions of these environments (Negreiros et al. 2014) with very efficient nutrient use. The shift from mycorrhizal nutrient-acquisition strategies in soils with greater P availability to non-mycorrhizal strategies in severely P-impoverished soils indicates that the benefits of nutrient acquisition through mycorrhizas do not overcome the costs associated with mycorrhizal maintenance at very low P availability (Lambers et al. 2008; Lambers et al. 2010). More than 60% of the studied species presented rhizosheaths, validating the prevalence of non-mycorrhizal root specialisations with long root hairs in the most P-impoverished soils 57

(Lamont 1982; Miller 2005; Shane et al. 2006; Abrahão et al. 2014). Field-based carboxylate collections did not provide reliable measurements due to the ephemeral nature of carboxylates in the rhizosphere, which makes it difficult to detect the pulse of release in the rhizosphere. Differences of leaf [Mn] in species acquiring nutrients through different strategies were less than the differences within strategies, probably indicating that regardless of mycorrhizal colonisation and the presence of rhizosheaths, most species have the ability to release carboxylates for P-acquisition. The very low leaf [P] and [N] of mature and senesced leaves, high LMA, intermediate photosynthetic rates with high PPUE and high P-remobilisation efficiencies reflect the efficient use of P in these species (Aerts and Chapin 1999; Wright et al. 2004; Veneklaas et al. 2012). Based on the leaf N: P ratios, P was more strongly limiting for plant growth than N was; therefore, strategies to increase P-use efficiency are likely under stronger selection than those conferring N-use efficiency, leading to relatively low values of PNUE compared with global averages, as opposed to very high PPUE values, relative to global averages. Taken together, the high turnover of below- and aboveground nutrient- acquisition and –use strategies contribute to explain the very high species turnover in different soil types observed at a very small spatial scale.

2.6. Acknowledgments

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes, PhD scholarship to Anna Abrahão and Project Grant PVE 88881.068071/2014-01). We thank the owner of the private reserve Reserva Vellozia (Dr. Geraldo Wilson Fernandes), Instituto Estadual de Florestas of Minas Gerais (license 056/2014) and Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio, license 43511-1) for allowing us to work in the field sites. We thank Dr. Juliana L. S. Mayer for allowing us to work at the Plant Anatomy lab at Unicamp. We also thank Isabela Fernandes for the lab work support.

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Zuur A, Ieno E, Walker N, Saveliev A, Smith G (2009) Mixed effects models and extensions in ecology with R. Gail M, Krickeberg K, Samet JM, Tsiatis A, Wong W, editors. New York, NY: Spring Science and Business Media.

a b

c d

Figure 2.1. Mosaic of soil types in mountaintop campos rupestres of Serra do Cipó, Minas Gerais, Brazil. a. Oxisols covered with cerrado savannah vegetation showing the leaf trap on a branch of Myrsine guianensis (Myrsinaceae), b. Rock outcrops with thin soil layers in the background and gravel sands in the front, c. Details of the gravel sands, with exposed quartz gravel d. Detail of the white sands, dominated by grasses and sedges.

N➤ IImage © 2017 CNES // Aiirrbus © 2017 Googlle 100 m Image © 2017 CNES / Airbus © 2017 Google Figure 2.2. Map of the study site in campos rupestres at Serra do Cipó, Minas Gerais Brazil, with a mosaic of different soil types: cerrado oxisol in orange, rock outcrops in black, gravel sands in grey and white sands in white. The road on the top right corner is MG-010 km 111.

a b 4

)

g 3 200 k

(

]

(

] P 2

[

100 s l 1

o T 0

o p d d o p d d d ro n n d ro n n rra tc sa sa rra tc sa sa e u el e e u el e C k o v hit C k o v hit c ra W c ra W Ro G Ro G Soil type Soil type

)

1 c - d g Pi Po

)

1 1.5

( 100 -

]

[

(

o 75

] s 1.0

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a 50

t 0.5

o x 25

−

O 0

a 0.0

o p d d o p d d

l d o n n d o n n ra cr sa sa ra cr sa sa a r ut l r ut l t e e e e C o ve hit C o ve hit o k a k a oc r W oc r W T R G R G Soil type Soil type

Figure 2.3. Soil phosphorus (P) and nitrogen (N) concentrations in a mosaic of soil types in mountaintop campos rupestres at Serra do Cipó, Espinhaço range, Brazil. a. Total soil [P] by ignition, b. Resin-extractable P, c. NaOH-extractable inorganic (Pi) and organic (Po) phosphorus, d. Total soil [N]. Points are least-square means and bars are 95% confidence intervals.

a b c

Figure 2.4. Arbuscular mycorrhizal structures observed in roots of cerrado and campos rupestres plants from mountaintop habitats at Serra do Cipó, Espinhaço range, Brazil. a. Arbuscules (arrow head) and coils (arrow) in roots of Lagenocarpus rigidus (Cyperaceae) growing in rock outcrops, scale bar = 50 μm, b. arbuscules (arrow head) and coils (arrow) in roots of Kielmeyera coriacea (Clusiaceae) growing in the cerrado, scale bar = 100 μm and c. vesicles in roots of a grass species (Poaceae 1) growing in the cerrado, scale bar = 100 μm.

a b c Mycorrhizal status AM NM 100 No rhizosheath Rhizosheath

)

(

)

(

% 100

(

o 75

p 75 n 80

a 50

r z

50 i

y 25 i 40

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o p d d o p d d P o p d d d ro n n d ro n n d ro n n rra tc sa sa rra tc sa sa rra tc sa sa e u el e e u el e e u el e C k o v hit C k o v hit C k o v hit c ra W c ra W c ra W Ro G Ro G Ro G Soil type Soil type Soil type

Figure 2.5. Nutrient-acquisition strategies of 10 species per soil type in a mosaic of soil types in mountaintop campos rupestres at Serra do Cipó, Espinhaço range, Brazil. a. Proportion of mycorrhizal species (AM) and non-mycorrhizal species (NM), b. Proportion of the root length colonised by arbuscular mycorrhizal fungi, c. Proportion of species with rhizosheaths. Points are least-square means and bars are 95% confidence intervals.

Figure 2.6. Rhizosheaths observed in roots campos rupestres plants from mountaintop habitats at Serra do Cipó, Espinhaço range, Brazil. a. Dauciform roots of Bulbostylis paradoxa (Cyperaceae) growing in rock outcrops, scale bar = 2 mm, b. rhizosheaths of a bamboo species Aulonemia effusa (Poaceae) growing in rock outcrops, scale bar = 500 μm and c. roots of Barbacenia flava (Velloziaceae) growing in rock outcrops, scale bar = 2 mm.

a b

) AM AM NM No rhizosheaths Rhizosheaths

t 1.00 1.00

o 0.75 0.75

(

a 0.50 0.50

c 0.25 0.25

p s 0.00 0.00

i h do op nd nd do op nd nd

R r r rra tc sa sa rra tc sa sa e u el e e u el e C k o v hit C k o v hit c ra W c ra W Ro G Ro G Soil type Soil type

Figure 2.7. Carboxylate content in the rhizosphere of 10 species per soil type in a mosaic of soil types in mountaintop campos rupestres at Serra do Cipó, Espinhaço range, Brazil. a. Rhizosphere carboxylate amounts of species colonised by arbuscular mycorrhizal fungi (AM) and non-mycorrhizal species (NM), b. Rhizosphere carboxylate amounts of species with and without rhizosheaths. Points are least-square means and bars are 95% confidence intervals.

a b AM AM NM No rhizosheaths Rhizosheaths

100 300

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e 25

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0 −100 o p d d o p d d d ro n n d ro n n rra tc sa sa rra tc sa sa e u el e e u el e C k o v hit C k o v hit c ra W c ra W Ro G Ro G Soil type Soil type

Figure 2.8. Mature leaf manganese (Mn) concentration from 10 species per soil type in a mosaic of soil types in mountaintop campos rupestres at Serra do Cipó, Espinhaço range, Brazil. a. Leaf [Mn] of species colonised by arbuscular mycorrhizal fungi (AM) and non- mycorrhizal species (NM), b. Leaf [Mn] of species with and without rhizosheaths. Points are least-square means and bars are 95% confidence intervals.

a b Mature Senesced

)

% 90

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)

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[ [

f f

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e 0.25 e

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o p d d o p d d d ro n n d ro n n rra tc sa sa rra tc sa sa e u el e e u el e C k o v hit C k o v hit c ra W c ra W Ro G Ro G Soil type Soil type

Figure 2.9. Leaf phosphorus (P) concentrations in leaves from 10 species per soil type in a mosaic of soil types in mountaintop campos rupestres at Serra do Cipó, Espinhaço range, Brazil. a. Leaf [P] from mature (circles) and senesced (triangles) leaves, b. Percentage of P remobilised from senesced leaves, c. Mature leaf [P] of species colonised by arbuscular mycorrhizal fungi (AM, circles) and non-mycorrhizal species (NM, triangles), d. Mature leaf [P] of species with (triangles) or without rhizosheaths (circles). Points are least-square means and bars are 95% confidence intervals.

a b Mature Senesced 100

15 )

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n 75

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i 10

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f f

a a 5

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o p d d o p d d d ro n n d ro n n rra tc sa sa rra tc sa sa e u el e e u el e C k o v hit C k o v hit c ra W c ra W Ro G Ro G Soil type Soil type

Figure 2.10. Leaf nitrogen (N) concentrations in leaves from 10 species per soil type in a mosaic of soil types in mountaintop campos rupestres at Serra do Cipó, Espinhaço range, Brazil. a. Leaf [N] from mature (circles) and senesced (triangles) leaves, b. Percentage of N remobilised from senesced leaves, c. Mature leaf [N] of species colonised by arbuscular mycorrhizal fungi (AM, circles) and non-mycorrhizal (NM, triangles) species, d. Mature leaf [N] of species with (triangles) or without rhizosheaths (circles). Points are least-square means and bars are 95% confidence intervals

P : 20

e L 10

o p d d d ro n n rra tc sa sa e u el e C k o v hit c ra W Ro G Soil type

Figure 2.11. Leaf N:P ratios of leaves from 10 species per soil type in a mosaic of soil types in mountaintop campos rupestres at Serra do Cipó, Espinhaço range, Brazil. Points are least- square means and bars are 95% confidence intervals.

)

- 20

2 15

m 10

(

i t 5

s s 0

A o p d d d ro n n rra tc sa sa e u el e C k o v hit c ra W Ro G Soil type

Figure 2.12. Carbon assimilation from 10 species per soil type in a mosaic of soil types in mountaintop campos rupestres at Serra do Cipó, Espinhaço range, Brazil. Points are least- square means and bars are 95% confidence intervals.

a b AM NM No rhizosheaths Rhizosheaths

)

- 300 300

200 200

o 100 100

(

E 0 0

P o p d d o p d d d ro n n d ro n n rra tc sa sa rra tc sa sa e u el e e u el e C k o v hit C k o v hit c ra W c ra W Ro G Ro G Soil type Soil type

c d AM NM No rhizosheaths Rhizosheaths

)

- 15 15

2 10 10

m 5 5

(

N 0 0

P o p d d o p d d d ro n n d ro n n rra tc sa sa rra tc sa sa e u el e e u el e C k o v hit C k o v hit c ra W c ra W Ro G Ro G Soil type Soil type

Figure 2.13. Phosphorus and nitrogen photosynthetic-use efficiency (PPUE and (PNUE, respectively) from mycorrhizal (AM) and non-mycorrhizal (NM) species and species with and without rhizosheaths from 10 species per soil type in a mosaic of soil types in mountaintop campos rupestres at Serra do Cipó, Espinhaço range, Brazil. a, c PPUE and PNUE as dependent on the presence of the colonisation by arbuscular mycorrhizal fungi, respectively, b,d. PPUE and PNUE as dependent on the presence of rhizosheaths, respectively. Points are least-square means and bars are 95% confidence intervals.

Table 2.1 Sup. Info. Species studied in a mosaic of soil types in mountaintop campos rupestres at Serra do Cipó, Espinhaço range, Brazil. Leaf phosphorus (P), nitrogen (N) and manganese (Mn) concentrations; carbon assimilation, percentage of root colonised by arbuscular mycorrhizal fungi (AM col), leaf mass per area (LMA), mycorrhizal status (AM, presence of arbuscules or vesicles) and presence of rhizosheaths (R, 0 for absence and 1 for presence)

Plant habit A Leaf Leaf Leaf (μmol [P] [N] [Mn] P N CO2 AM Soil mg g- mg g- mg kg- remob. remob. m-2 s- col LMA type Species Family 1 1 1 (%) (%) 1) (%) (g m2) AM R

Byrsonima tree Cerrado verbascifolia Malpighiaceae 0.6 14.3 21.3 69.3 65.5 14.3 55.5 176.2 AM 0

Stryphnodendro tree Cerrado n adstringens Fabaceae 0.6 16.6 13.4 75.0 37.8 11.0 70.8 162.1 AM 1

Roupala tree Cerrado montana Proteaceae 0.5 9.1 34.3 71.8 24.3 10.3 72.1 234.4 AM 0

Dalbergia tree Cerrado miscolobium Fabaceae 0.7 19.1 63.7 82.2 43.2 13.6 75.2 194.1 AM 0

Kielmeyera tree Cerrado coriacea Clusiaceae 0.8 11.6 19.5 81.2 57.5 15.7 36.9 209.6 AM 0

Myrsine tree Cerrado guianensis Myrsinaceae 0.6 9.0 30.6 74.0 51.0 11.7 64.1 183.1 AM 0

Erythroxylum Erythroxylacea tree Cerrado suberosum e 0.8 13.4 16.3 82.0 44.2 14.2 66.5 217.0 AM 0 85

Cerrado Poaceae sp. 1 Poaceae graminoid 0.8 8.3 29.2 92.4 68.0 12.6 93.2 151.1 AM 0

Echinolaena graminoid Cerrado inflexa Poaceae 0.7 12.1 146.1 74.9 69.5 9.7 92.7 59.0 AM 0

Cerrado Poaceae sp. 2 Poaceae graminoid 0.5 5.9 19.4 80.6 48.5 16.6 82.6 238.8 AM 0

Rock Barbacenia woody outcrop flava Velloziaceae rosette 0.5 11.1 340.7 92.2 46.8 13.8 9.6 289.3 NM 1

Rock Vellozia herbaceous outcrop caruncularis Velloziaceae rosette 0.6 10.7 137.5 91.4 40.7 7.9 8.8 208.8 NM 0

Rock Aulonemia herb outcrop effusa Poaceae 0.4 10.6 29.8 95.3 66.1 13.6 60.7 242.3 AM 1

“Lychnophora treelet Rock jolyana sp. outcrop Ined” Asteraceae 0.3 8.5 477.7 78.8 56.4 20.2 55.2 293.4 AM 1

Rock treelet outcrop Neea theifera Nyctaginaceae 1.1 32.8 57.4 69.9 -4.2 11.6 75.0 195.3 AM 1

Rock Trembleya Melastomatace shrub outcrop laniflora ae 0.2 6.6 82.7 62.2 41.8 15.7 81.8 357.6 AM 1

Rock Lavoisiera Melastomatace shrub outcrop cordata ae 0.3 9.5 50.5 76.6 63.6 14.1 59.3 188.0 AM 0

Rock Vochysia tree outcrop thyrsoidea Vochysiaceae 0.4 8.6 97.4 64.1 20.9 15.6 39.9 422.0 AM 0

Rock Eriocaulaceae Eriocaulaceae rosette 0.2 8.0 34.3 82.6 36.1 5.3 75.3 151.1 AM 0 86

outcrop sp. 1

Rock Lagenocarpus graminoid outcrop rigidus Cyperaceae 0.3 8.2 61.0 93.7 73.2 10.8 36.6 219.0 AM 0

Gravel Bulbostylis woody sand paradoxa Cyperaceae rosette 0.4 5.5 25.5 84.0 14.9 12.7 4.3 88.4 NM 1

Gravel Vochysia shrub sand pygmea Vochysiaceae 0.3 7.1 111.7 44.6 -12.0 11.5 37.4 402.2 AM 0

Gravel Melastomatace shrub sand Marcetia sp. 1 ae 0.3 9.7 359.7 12.5 NA 12.3 22.6 92.7 AM 1

Gravel Lagenocarpus graminoid sand tenuifolius Cyperaceae 0.3 6.0 76.0 91.1 66.2 9.2 46.0 246.3 AM 1

Gravel Byrsonima sp. shrub sand 1 Malpighiaceae 0.4 10.2 41.8 75.4 46.9 16.9 35.5 312.6 AM 1

Gravel Cryptangium graminoid sand verticillatum Cyperaceae 0.4 6.6 60.3 85.4 44.3 3.5 2.5 154.2 NM 1

Gravel herb sand Xyris pilosa Xyridaceae 0.1 5.6 34.6 82.3 36.9 9.6 4.4 141.0 NM 1

Gravel Coccoloba shrub sand cereifera Polygonaceae 0.5 9.5 110.4 78.2 29.4 11.8 2.9 450.8 NM 1

Gravel Marcetia Melastomatace shrub sand taxifolia ae 0.4 11.9 393.4 36.7 30.1 22.5 3.7 97.7 NM 1

Gravel Trachypogon Poaceae graminoid 0.4 7.3 25.2 89.7 62.3 11.1 1.2 245.5 NM 1 87

sand sp.

White Actinocephalus herbaceous sand geniculatus Eriocaulaceae rosette 0.3 9.9 100.5 89.4 NA 5.5 5.3 137.9 NM 1

Xyris herb itatiayensis or White Xyris sand longiscapa Xyridaceae 0.2 6.2 54.0 94.0 66.1 4.7 1.3 269.9 NM 1

White Lagenocarpus herb sand tenuifolius Cyperaceae 0.3 7.2 103.9 92.5 76.0 9.3 8.7 151.5 AM 1

White Lagenocarpus graminoid sand tenuifolius Cyperaceae 0.3 7.0 65.4 93.7 71.8 8.9 1.5 235.1 NM 1

White Rynchospora graminoid sand ciliolata Cyperaceae 0.3 7.5 102.6 80.0 61.9 5.2 4.9 119.2 NM 1

White Cephalostemon herb sand riedelianus Rapateaceae 0.4 8.0 31.6 92.2 71.7 7.0 25.4 232.2 AM 1

White Eriocaulaceae herbaceous sand sp. 3 Eriocaulaceae rosette 0.2 8.7 26.4 87.0 47.3 8.2 5.3 178.6 AM 1

White graminoid sand Apochloa sp. Poaceae 0.3 8.5 55.4 95.6 67.3 5.1 2.8 309.7 AM 1

Xyris nubigena herb or X. White pterygoblephar sand a Xyridaceae 0.2 6.8 76.4 84.4 51.9 4.6 6.2 276.3 AM 1 88

White Richterago herb sand arenaria Asteraceae 0.3 8.4 121.1 84.7 47.3 7.5 3.8 412.4 NM 1 89

Table 2.2 Sup Info. Model selection for soil attributes. Explanatory variables were compared through the corrected Akaike Information Criterion (AICc), ~1 represents the null model (intercept only). The delta AICc (dAICc) represents the difference between the model with the lowest AICc (best model) and the other models. The ΔAICc of the best model is therefore zero. df represents the degrees of freedom of each model and Akaike weights represent the relative likelihood of each model.

Dependent Explanatory Best model AICc ΔAICc df Weights variable variables

Total soil [P] gls(Total_P_mg_P_kg~ Description, ~1 143.1 22.8 3 < 0.001 weights= varPower(form= ~ fitted(.))) ~ Soil type 120.3 0 6 1

Total soil [N] ~1 -35.9 3.6 2 0.14 aov(Total_N_mg_g~ Description) ~ Soil type -39.5 0 5 0.86

Resin [P] ~1 41.3 2.4 2 0.23 aov(Resin_P_mg_P_kg~ Description) ~ Soil type 38.9 0 5 0.77

Oxalate [P] ~1 46.9 0 2 0.959 aov(P_mg_kg_oxal~1) ~ Soil type 53.2 6.3 5 0.041

Olsen [P] ~1 32.6 0.6 2 0.42 aov(NaHCO3_P_mg_P_kg~1) ~ Soil type 32 0 5 0.58

NaOH Pi aov(NaOH_Pi_mg_p_kg~ Description) ~1 97.1 9.6 2 0.0083 90

~ Soil type 87.5 0 5 0.9917

NaOH Po ~1 105 10.9 2 0.0043 aov(NaOH_Po_mg_p_kg~ Description) ~ Soil type 94.1 0 5 0.9957

NaOH Pt ~1 117.3 13.9 2 < 0.001 aov(NaOH_Pt_mg_p_kg~ Description) ~ Soil type 103.5 0 5 1

Table 2.3 Sup. Info. Model selection for plant traits between soil types and nutrient-acquisition strategies. Abbreviations as in Table 2.1.

Dependent variable Best model Independent variables AICc ΔAICc df weight

Mature leaf [P] lme(fixed = P ~ Phys, random = | ~1 -314.4 17.3 6 < 0.001 Species, weights = varIdent(form = | Phys) ~Soil type -331.7 0 9 1

Senesced leaf [P] lme(fixed = Psen ~ Phys, random = | ~1 -507 0.4 6 0.45 Species, weights = varIdent(form = | Phys)) ~Soil type -507.4 0 9 0.55

P remobilisation (%) glmmadmb(RemobP~ ~1 -314 0.9 3 0.39 Phys+(1|Species), family="beta", link="logit") ~Soil type -314.9 0 6 0.61

Mature leaf [P] ~1 -314.4 17.3 6 < 0.001

lme(fixed = P ~ Phys, random = | ~ Soil type + AM -330.7 1 10 0.38 Species, weights = varIdent(form = | Phys)) ~ Soil type -331.7 0 9 0.62

AM -312.5 19.2 7 < 0.001

Mature leaf [P] lme(fixed = P ~ Phys, random = | ~1 -314.4 17.3 6 < 0.001 Species, weights = varIdent(form = | ~Soil type + -330 1.7 10 0.3012 Phys)) Rhizosheath

~Soil type -331.7 0 9 0.6971

~Rhizosheath -319.6 12.1 7 0.0016 92

Mature leaf [N] lme(fixed = N ~ Phys, random = | ~1 747.9 2.4 6 0.23 Species, weights = varIdent(form = | Phys) ~Soil type 745.5 0 9 0.77

Senesced leaf [N] lme(fixed = Nsen ~ 1, random = | ~1 632 0 6 0.88 Species, weights = varIdent(form = | Phys) ~Soil type 636 4 9 0.12

N remobilisation (%) lme(fixed = RemobN ~ Phys, ~1 -5.9 2.5 6 0.22 random = | Species, weights = varIdent(form = | Phys) ~Soil type -8.4 0 9 0.78

Mature leaf [N] ~1 795.3 2.2 6 0.184

lme(fixed = N ~ Phys, random = | ~ Soil type + AM 795.3 2.2 10 0.185 Species, weights = varIdent(form = | Phys)) ~ Soil type 793.1 0 9 0.556

AM 797.1 4 7 0.075

Mature leaf [N] ~1 795.3 4.2 6 0.08

~Soil type + lme(fixed = N ~ Phys + special, 791.2 0 10 0.645 random = | Species, weights = Rhizosheath varIdent(form = | Phys)) ~Soil type 793.1 2 9 0.242

~Rhizosheath 797.1 6 7 0.033

Proportion of glm(AM~Phys, mycorrhizal species ~1 52.6 6.1 1 0.046 family=binomial(link = "logit")) (%) 93

~Soil type 46.5 0 4 0.954

Proportion of root lme(fixed=Col_percent~Phys, ~1 1407.4 51.9 6 < 0.001 colonised by AM (%) random= ~1|Species, weights=varIdent(form=~1|Phys)) ~Soil type 1355.5 0 9 1

Proportion of species ~1 55 19 1 < 0.001 with rhizosheaths (%) glm(special~Phys, family=binomial(link = "logit")) ~Soil type 36 0 4 1

Total rhizosphere ~1 42.8 0 3 0.72 carboxylates lme(fixed = Total_carbox ~ 1, random = | Species) ~Soil type 44.7 1.9 6 0.28

Mature leaf [Mn] glmer(Mn~Phys+(1|Species), ~1 1545.3 6.8 3 0.033 family= Gamma(link = "inverse")) ~Soil type 1538.5 0 6 0.967

Mature leaf [Mn] ~1 1483.5 6.3 3 0.022

lme(fixed = Mn ~ Phys, random = | ~Soil type + AM 1477.4 0.3 7 0.449 Species, weights = varIdent(form = | Phys)) ~Soil type 1477.1 0 6 0.518

~AM 1484.9 7.7 4 0.011

Mature leaf [Mn] lme(fixed = Mn ~ 1, random = | ~1 1672.7 0.6 6 0.29 Species, weights = varIdent(form = | ~Soil type + Phys)) 1674.1 2.1 10 0.14 Rhizosheath 94

~Soil type 1673.8 1.8 9 0.16

~Rhizosheath 1672 0 7 0.4

Total rhizosphere ~1 42.8 0.8 3 0.317 carboxylates

lme(fixed = Total_carbox ~ 1, ~Soil type + AM 45.4 3.4 7 0.087 random = | Species) ~Soil type 44.7 2.7 6 0.124

~AM 42 0 4 0.472

Total rhizosphere ~1 42.8 0 3 0.51 carboxylates

~Soil type + lme(fixed = Total_carbox ~ 1, 46.3 3.5 7 0.09 random = | Species,) Rhizosheath ~Soil type 44.7 1.9 6 0.2

~Rhizosheath 44.7 1.9 4 0.2

Proportion of root ~1 1346 49.1 6 < 0.001 colonised by AM (%)

lme(fixed = Col_percent ~ Phys, ~Soil type + random = | Species, weights = 1299.2 2.3 10 0.24 Rhizosheath varIdent(form = | Phys) ~Soil type 1297 0 9 0.76

~Rhizosheath 1330.3 33.4 7 < 0.001

PPUE lme(fixed = PhotoP ~ 1, random = | ~1 1751.5 0 6 0.84 Species, weights = varIdent(form = | Phys)) ~Soil type 1754.8 3.3 9 0.16

PPUE ~1 1751.5 0 6 0.58

lme(fixed = PhotoP ~ 1, random = | ~Soil type + AM 1755.5 4 10 0.08 Species, weights = varIdent(form = | Phys)) ~Soil type 1754.8 3.3 9 0.11

~AM 1753.4 1.9 7 0.23

PPUE ~1 1751.5 0.5 6 0.391

~Soil type + lme(fixed = PhotoP ~ special, 1756.8 5.8 10 0.027 random = | Species, weights = Rhizosheath varIdent(form = | Phys)) ~Soil type 1754.8 3.8 9 0.076

~Rhizosheath 1751 0 7 0.506

PNUE lme(fixed = PhotoN ~ 1, random = | ~1 798.5 0 6 0.57 Species, weights = varIdent(form = | Phys)) ~Soil type 799.1 0.6 9 0.43

PNUE ~1 798.5 0.2 6 0.29

lme(fixed = PhotoN ~ 1, random = | ~Soil type + AM 799.5 1.1 10 0.18 Species, weights = varIdent(form = | Phys)) ~Soil type 799.1 0.8 9 0.21

~AM 798.4 0 7 0.31 96

PNUE ~1 798.5 0 6 0.43

~Soil type + lme(fixed = PhotoN ~ 1, random = | 801.4 2.9 10 0.1 Species, weights = varIdent(form = | Rhizosheath Phys)) ~Soil type 799.1 0.6 9 0.32

~Rhizosheath 800.6 2.1 7 0.15

Table 2.4. Sup Info. Mean soil chemical attributes of soils collected in mountaintop campos rupestres at Serra do Cipó, Espinhaço Range, Brazil. Each value represents the mean of three soil samples. Comb. means automated combustion.

Al Fe Ca K Mg Mn Na NH4 NO3 Total C Total N Soil type pH pH (mg (mg (mg (mg (mg (mg (mg C: N (mg (mg (%) (%) kg-1) kg-1) kg-1) kg-1) kg-1) kg-1) kg-1) kg-1) kg-1)

Method Water CaCl2 BaCl2 BaCl2 BaCl2 BaCl2 BaCl2 BaCl2 BaCl2 Comb. Comb. KCl KCl

Cerrado 6.5 5.8 3.03 1.83 1596.13 18.27 8.17 0.80 2.07 2.84 0.14 20.10 17.40 6.73

Rock outcrop 4.1 3.8 58.10 15.47 53.00 24.20 2.43 0.87 5.10 1.69 0.09 17.83 10.17 7.07

Gravel sand 4.5 3.8 83.87 14.30 55.20 24.97 0.50 0.77 1.83 1.97 0.12 16.33 14.07 4.30

White sand 4.4 3.8 34.80 8.77 4.90 3.63 0.10 0.13 2.10 0.61 0.04 15.67 3.77 1.80

CHAPTER 3. PHOSPHORUS- AND NITROGEN-ACQUISITION STRATEGIES IN TWO BOSSIAEA SPECIES (FABACEAE) ALONG TWO CHRONOSEQUENCES IN THE SOUTH-WESTERN AUSTRALIAN KWONGAN

Anna Abrahão, Rafael S. Oliveira, Megan H. Ryan, Etienne Laliberté, Hans Lambers

3.1. Abstract

The relative abundance of species with different nutrient-acquisition strategy shifts in response to soil nutrient availability during long-term ecosystem development, but little is known about shifts in nutrient-acquisition strategies within single species occurring along soils in distinct stages of long-term ecosystem development. In old and severely phosphorus (P)-impoverished soils, mycorrhizal plant species decrease in relative importance, whereas plants with non-mycorrhizal root specialisations for P-acquisition (e.g., carboxylate release) become more abundant. In order to assess nutrient-acquisition strategies used by plants, leaf manganese (Mn) concentrations have been suggested as a proxy, increasing with carboxylate release and decreasing in mycorrhizal plant species. Additionally, nodulation in legumes is expected to occur at low nitrogen (N) availability, but only when enough P is available. We investigated whether two congeneric legume species (Bossiaea linophylla and B. eriocarpa) growing along two chronosequences with contrasting climates on the south-western Australian coast altered nutrient-acquisition strategies with changes in N and P availability. To assess nutrient limitation and nutrient-acquisition strategies in young plants we grew them in a glasshouse at varying N (10, 30 and 50 mg N kg-1 soil) and P (0, 0.5, 5 and 50 mg P kg-1 soil) supply. Leaf N: P ratios and the responses to nutrient addition suggested that seedlings of both species exhibited P-limitation in all treatments. This apparent P-limitation may be due to the very high leaf [N] of legumes afforded by symbiotic N fixation. Arbuscular mycorrhizal colonisation did not vary in the field for either species, while it decreased with increasing N and P supply in the glasshouse. Surprisingly, root exudation of carboxylates increased with increasing P and N supply for B. linophylla and decreased with increasing N supply for B. eriocarpa. At low P supply, both species invested in morphological rather than physiological adaptations for P uptake. Leaf [Mn] of the glasshouse plants was not related to carboxylate release. Nodule production declined with increasing N supply for both species, as expected. The two congeneric species show different shifts in nutrient-acquisition strategies with 99

nutrient supply. We conclude that the within-species response to increasing P supply is not the same as that previously observed at the community level.

Key-words: Carboxylates, Mycorrhiza, Nodule, OCBIL, Root

3.2. Introduction

Nutrient limitation is an agronomic concept adopted in ecology, and hard to test in native ecosystems without disrupting the systems. Nutrient-limited crop plants respond positively to the addition of growth-limiting nutrients with an increased production of biomass (Vitousek et al. 2010; Meharg and Marschner 2012). In contrast, native plants in nutrient-impoverished ecosystems often hardly respond to nutrient addition, and may even experience nutrient toxicity (Specht 1963; Chapin et al. 1986), suggesting that their adaptation to nutrient impoverishment, for instance, inherently slow growth, may restrict their ability to respond to addition of nutrients. Understanding the response of native plants to changes in nutrient availability will be crucial if we are to understand the factors controlling their distribution and abundance in landscapes where soil nutrient levels vary greatly, and eventually in landscapes subjected to eutrophication, for example increased atmospheric nitrogen (N) deposition (Phoenix et al. 2006; Bustamante et al. 2012) or P arriving in run-off, fire retardants or dust (Lambers et al. 2013). Since nutrient availability is one of the main drivers of plant species distribution (John et al. 2007; Perry et al. 2008), plants can only germinate, grow and reproduce where they are able to tolerate local environmental conditions. Therefore, the ability to acquire nutrients in oligotrophic habitats is crucial for plant establishment.

Nutrient-acquisition strategies at the community level vary with soil age and associated changes in growth-limiting soil nutrients (Walker and Syers 1976; Wardle et al. 2008; Turner and Condron 2013; Hayes et al. 2014; Zemunik et al. 2015). Nutrient limitation can be assessed through nutrient addition experiments (Lannes et al. 2016), or in natural systems called chronosequences. A chronosequence is a sequence of soils developed on similar parent materials and relief under the effect of constant climatic and biotic factors (Stevens and Walker 1970). Pedogenic processes cause phosphorus (P) loss from the soil, while N is absent from most parent materials and is slowly accumulated by biological fixation, so that plant growth on old soils tends to be P-limited rather than N-limited (Walker and Syers 1976). Nutrient limitation can also be indirectly assessed through foliar nutrient ratios, such as N: P, once controlled for phylogenetic and environmental differences (Redfield 100

1958; Güsewell 2004; Richardson et al. 2004; Wardle et al. 2004; Vitousek et al. 2010). Therefore, assessing nutrient-limitation and nutrient-acquisition strategies along gradients of nutrient availability can help explain species niche partitioning and species distributions (Laliberté et al. 2013; Laliberté et al. 2014).

Nutrient limitation for plant productivity influences the relative abundance of species with different nutrient-acquisition strategies. In a well-studied chronosequence in the south-western Australian kwongan vegetation, mycorrhizal plants are more prominent in younger, more fertile soils, while non-mycorrhizal cluster-rooted plants are more abundant in older, more P-impoverished soils (Zemunik et al. 2015). Each strategy represents carbon (C) costs, which must be balanced against benefits in terms of acquisition of growth-limiting nutrients (Lynch and Ho 2005; Kaiser et al. 2015). Root specialisations like cluster roots exude a large amount of carboxylates that can cost over half of daily photosynthates if we include root growth and respiration (Lambers et al. 2006); however, the highly-localised exudation of organic anions can desorb strongly-bound forms of P (Jones 1998; Ryan et al. 2001; Lambers et al. 2002; Oburger et al. 2009). As a result of rapid rates of carboxylate release, soil manganese (Mn) is mobilised (Gardner et al. 1982; Grierson and Attiwill 1989; Dinkelaker et al. 1995) and can accumulate in leaves (Gardner and Boundy 1983; Shane and Lambers 2005; Muler et al. 2014). Mycorrhizal fungi also require a large carbon supply from the plant to build their hyphae, but can extend the P-acquisition volume beyond the root hair zone (Bitterlich and Franken 2016; Kikuchi et al. 2016). Mycorrhizal colonisation can significantly decrease the amount of Mn transferred to the host plant (Bethlenfalvay and Franson 1989; Kothari et al. 1991; Nogueira et al. 2007; Nazeri et al. 2014) or influence the proportion of oxidising to reducing microbiota, also leading to decreased Mn availability (Kothari et al. 1991). Therefore, leaf [Mn] may be used as a proxy to understand if plants rely on carboxylate release or mycorrhizal associations for P-acquisition in P-poor soils (Lambers et al. 2015). The shifts in relative abundance of mycorrhizal and non-mycorrhizal plants with changes in P availability are well studied (Oliveira et al. 2015; Zemunik et al. 2015), while the within-species shifts in P-acquisition strategies with changes in P availability are poorly known (Pang et al. 2010; de Campos et al. 2013; Albornoz et al. 2016; Png et al. 2017).

The investment in N-acquisition strategies also shifts with varying soil N and P availability. Nodule formation is inhibited by sufficient N supply (Streeter and Wong 1988), but stimulated by high P supply (Albornoz et al. 2016), due to plant growth stimulation increasing N demand (Robson et al. 1981). Nodule formation represents large C and P costs 101

(Ryle et al. 1979), but the additional N supply compensates this C investment by allowing faster rates of photosynthesis (Tjepkema and Winship 1980). Some species use more than one nutrient-acquisition strategy, but some legumes like Kennedia (Fabaceae, Papillionoideae) species, native to the south-western Australian kwongan, release carboxylates, form mycorrhizal associations and nodules (Adams et al. 2002; Ryan et al. 2012). It is likely that other co-occurring legumes also present all three strategies. Very few studies have tested the effects of nutrient addition on how species are positioned along the trade-off axis between C investment in mycorrhizas versus carboxylates (Ryan et al. 2012), or between mycorrhizas and nodules (Larimer et al. 2014). Here, we test the effect of increasing N and P supplies on three nutrient-acquisition strategies (i.e. mycorrhizas, carboxylate release and nodule formation).

To understand the investment in mycorrhizal colonisation, carboxylate exudation and nodule formation with increasing P and N supply, we studied two legumes, native to the south-western Australian kwongan. The legumes belong to the genus Bossiaea, are mycorrhizal, and associate with Bradyrhizobium sp. within nodules to fix nitrogen (Lange 1959; Brundrett and Abbott 1991; Thrall et al. 2011; Parker 2015). The species occur along the Jurien Bay or Warren chronosequences, on sand-dunes covering 2-million years of pedogenesis (Turner et al. 2017). Total soil P concentration varies 40-fold along the Jurien Bay and 10-fold along the Warren chronosequence (Turner and Laliberté 2015; Turner et al. 2017). We also grew these species in a glasshouse in order to remove possible confounding factors driving nutrient-acquisition strategies in the field. Bossiaea linophylla R.Br. occurs along all the stages of the Warren chronosequence, while B. eriocarpa Benth. only occurs at the three oldest stages of the Jurien Bay chronosequence. We hypothesised plants of both Bossiaea species to a) increase their leaf nutrient concentrations with increasing N and P supply, switching from N to P-limitation along the chronosequences in the field, but that both species would have nutrient-conserving traits such as slow growth and little growth-response to nutrient addition in the glasshouse. We expected b) low P supply, regardless of N supply, to stimulate carboxylate release and increase leaf [Mn] and to inhibit arbuscular mycorrhizal colonisation. Finally, we hypothesised c) high N supply at low P supply to inhibit nodulation in the glasshouse, and high P supply to enhance nodulation, because of the high P requirements of nodules. 102

3.3. Methods

3.3.1. Field collections

3.3.1.1. Study area

We conducted the study along two chronosequences on the south-western Australian coast, each consisting of a series of parallel sand dunes deposited over more than 2 million years (Turner et al. 2017). There are ten 10 by 10 m permanent plots installed at each chronosequence stage at the Jurien Bay chronosequence and five 20 by 20 m plots per stage at the Warren chronosequence (Laliberté et al. 2014; Turner et al. 2017). The Jurien Bay chronosequence (30.29° S, 115.04° E) has a warmer (mean annual temperature, MAT 19°C) and drier (mean annual precipitation, MAP 533.2 mm) climate than the Warren chronosequence (34.62° S, 115.89° E; MAT 15°C, MAP 1184 mm) (Turner et al. 2017). Changes in soil N and P availability across both sequences are consistent with predictions of long-term soil and ecosystem development (Walker and Syers 1976; Turner and Condron 2013). Total soil [P] is highest at the youngest stages and is lowest in the oldest stages; total soil [N] is greatest in intermediate stages (Turner et al. 2017). However, the total soil P concentration of the parent material is much higher at the Jurien Bay chronosequence (384 g m-2, 1 m depth) than at Warren (35.6 g m-2). Both chronosequences reach the same levels of total soil [P] at the oldest stages (2-4 g m-2) (Turner et al. 2017). The proportion of P as organic P is higher along the Warren than at the Jurien Bay chronosequence (Turner et al. 2017). The pedogenic processes that accompany the dune system development include loss of carbonates and a pH decline from 8 to 4 at both chronosequences. Complete soil and vegetation descriptions can be found in Turner et al. (2017). An interesting feature of these landscapes is that they present high vascular plant species diversity and high endemism at very low nutrient availability (Hopper and Gioia 2004; Hopper 2009; Hopper et al. 2016).

3.3.1.2. Species selection

We selected two congeneric native legume species that occur along one of the chronosequences, and each present at least two nutrient-acquisition strategies. This allowed us to test if the species switched nutrient-acquisition strategies according to N or P limitation along the chronosequences, and also in a glasshouse experiment. Bossiaea linophylla is an erect shrub from 0.4 to 2.2 m tall that grows mainly in sandy soils, coastal limestone, dunes and granite rocks of the South-West Province of Australia, from Bunbury to Albany (FloraBase 2017). Bossiaea linophylla occurs at all the stages of the Warren chronosequence 103

(pers. obs.). Bossiaea eriocarpa is an erect or spreading shrub to 1 m high that grows on sandy soils of the South-West Province from Geraldton to Albany (FloraBase 2017) and grows at stages four to six of the Jurien Bay chronosequence (Zemunik et al. 2016).

Both B. linophylla and B. eriocarpa are legumes and species within this genus associate with N2-fixing Bradyrhizobium spp. and form nodules (Lange 1959; Thrall et al. 2011; Parker 2015). Bossiaea is also colonised by arbuscular mycorrhizal fungi (Brundrett and Abbott 1991; Zemunik et al. 2015). Some south-western Australian herbaceous legumes release large amounts of carboxylates (Ryan et al. 2012), and we expected Bossiaea to do the same, because of the relatively high leaf [Mn] of B. eriocarpa along the Jurien Bay chronosequence (G. Zemunik, unpublished data).

3.3.1.3. Plant material

We collected mature, fully-expanded leaves of B. linophylla from three individuals per plot within 30 m of five plots per chronosequence stage (Table 3.1 Sup. mat.). Bossiaea linophylla occurred at five stages (1-4 and 6). No plots were set up in the stage 5 described in Turner et al. (2017). Since B. eriocarpa does not occur along the entire chronosequence, we collected leaves from four individuals per plot in a total of 10 plots from three stages in September 2016 (Table 3.1 Sup. mat.). B. eriocarpa only occurred in one plot of stage 5. Leaves were bulked per plot, oven-dried for 96 h at 70°C and ground with a Geno/Grinder 2010 (SPEX SamplePrep, Metuchen, NJ, USA) using ceramic bearings for 3 min at 1500 rpm. Leaf nutrient analyses were conducted at ChemCentre, Perth, Australia. A subsample was digested in concentrated HNO3: HClO4 (3: 1 v/v) and analysed for Ca, Cu, Fe, K, Mg, Mn, Mo, P, S and Zn concentrations in an Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES). All digests were first analysed using a simultaneous Varian Vista Pro (Varian Australia Pty Ltd, Australia), radially configured ICP-OES equipment fitted with an Auto Sampler (CETAC ASX-510, USA). A second subsample was analysed for total leaf [N]. Samples were digested using a the Kjeldahl method with a mixture of salicylic and sulfuric acid with hydrogen peroxide (Bradstreet 1965), diluted and analysed by the Berthelot colourimetric determination (Varley 1966; Searle 1984) using a two-channel autoanalyser (Technicon AAII).

3.3.1.4. Field mycorrhizal colonisation

To measure mycorrhizal colonisation, we collected at least 30 cm of young roots from the same plants from which we collected leaves and immediately stored them in ethanol 104

(50% v/v). In the laboratory, we cleared the roots in KOH (10 % w/v) at room temperature for 7 days and further heated them in a water bath at 90°C for 1-3 h. We acidified the roots in HCl (1% v/v) overnight and transferred them to Parker blue ink in white vinegar (5% v/v) for three hours at room temperature (Vierheilig et al. 1998). We thoroughly rinsed the samples to remove excess ink and stored them in acidified glycerol (50% v/v with 5% HCl). We assessed mycorrhizal colonisation by counting the number of arbuscular mycorrhizal hyphae, arbuscules and vesicles in 30 cm of root using the slide method at 200x magnification (Giovannetti and Mosse 1980).

3.3.2. Glasshouse experiment

We set up a factorial design with four P treatments (0, 0.5, 5 and 50 mg P kg-1 soil) and three N treatments (10, 30 and 50 mg N kg-1 soil) with eight replicates for each treatment and two plants per pot to allow measurement of carboxylate release on one plant and mycorrhizal colonisation on the other (total number of pots per species: 4 x 3 x 8 = 96). We also had two non-planted pots per treatment to measure microbial carboxylates.

3.3.2.1. Germination

We purchased seeds of B. linophylla and B. eriocarpa from Nindethana Seed Service, Western Australia. To break dormancy, we boiled the seeds for 60 s (Bell et al. 1993) and then soaked them in sterile deionised (DI) water with aeration overnight. We surface- sterilised the seeds in 70% (v/v) ethanol for 10 s, rinsed three times in sterile DI water, submerged seeds for 10 s in 2% (v/v) sodium hypochlorite and then rinsed them in DI water a further three times. We spread the seeds on two 18.5 cm diameter filter papers in a 20 cm diameter glass Petri dish and placed them in a controlled-temperature room in the dark at 15°C and sprayed daily with DI water (Bell et al. 1995). After the radicle emerged (21 to 48 days), seeds were transferred into steam-pasteurised washed river sand in seed trays (cell dimensions: 36 x 38 x 60 mm) and kept in the dark until cotyledons emerged. As soon as the cotyledons emerged, the seed trays were transferred to a glasshouse with a shade cloth reducing the light intensity to 30% in January 2016. To keep the seedlings moist, seed trays were covered with a plastic cover. Between February and April 2016 the glasshouse temperatures varied between 13 and 34°C during the day and between 13 and 24°C during the night. The relative humidity ranged from 31 to 75% and peak irradiance varied from 700 to 2000 μmol m-2 s-1. The seedlings were watered every second day with DI water. 105

3.3.2.2. Plant growth

We lined pots of 8.5 cm × 8.5 cm × 18 cm depth with plastic bags and filled with 1.08 kg of washed, air-dried, double steam-pasteurised, coarse river sand. We fertilised river sand with a P- and N-free basal nutrient solution containing: K 62 mg kg-1, S 43 mg kg-1, Ca 27 mg kg-1, Fe 14 mg kg-1, Cl 35 mg kg-1, Mg 7.9 mg kg-1, Cu 1.3 mg kg-1, Zn 2.3 mg kg-1, -1 -1 -1 Mn 9.4 mg kg , B 0.12 mg kg , Mo 0.09 mg kg . We added N as potassium nitrate (KNO3) -1 to 10, 30 or 50 mg N kg soil (N10, N30, N50) and P as monopotassium phosphate (KH2PO4) to 0, 0.5, 5 or 50 mg P kg-1 soil (P0, P0.5, P5, P50). We did not include a zero N treatment, as native legumes showed poor establishment in such a treatment in previous experiments (G.K. Png unpublished data).

On top of the sand was added 120 g of field soil, which was intended to act as inoculum for arbuscular mycorrhizal fungi and rhizobia. The field soil was a 1:1 mix of soil collected under B. eriocarpa and B. aquifolium. We collected the soil under B. eriocarpa from 0-10 cm in a sandy soil in bushland at Harrisdale in the Perth metropolitan area (32°10’ S, 115°92’ E), Australia. We collected the soil under B. aquifolium from 0-10 cm in a lateritic soil in bushland at Waroona, ~100 km south of Perth, Australia. We used the field soil mix to represent the diversity of habitats of occurrence of Bossiaea in the field. We did not have access to soil under B. linophylla at the time of the experimental set-up.

Chemical analyses of the soils were conducted at ChemCentre, Perth, Australia (Table 3.1). Total N was measured after digestion with sulfuric acid, in the presence of a catalyst. Using a Technicon AA11 segmented flow auto-analyser, the digest was diluted, and N was determined by reaction with chlorine and salicylic acid to form a blue compound (Berthelot reaction). Total P was determined using the same digest colourimetrically as the phosphomolybdenum blue complex (Murphy and Riley 1962) with a Shimadzu UV-Vis spectrophotometer (Columbia, Maryland, United States). Other soil nutrients were measured using the Mehlich-3 extraction followed by ICP-OES analysis in a Arian Vista axial ICP-OES (Rayment and Lyons 2011).

Three months after the first seeds germinated (April 2016 - spring), seedlings were transplanted to the pots prepared with nutrients and soil inoculum. After transplanting, the soil surface in each pot was covered with 10 g of polyethylene beads to minimise soil evaporation. Over the following three months, we watered the plants with DI water to 90% 106

field capacity three times a week, and then to 70% once a week from June 2016. We recorded pot weight before each watering. The position of the pots was re-randomised fortnightly. Between April and August, the glasshouse temperatures varied between 10 and 27°C during the day, and between 10 and 22°C during the night. The relative humidity ranged from 28 to 82% and peak irradiance varied from 900 to 1500 μmol m-2 s-1.

3.3.2.3. Harvest

Plants were harvested in August 2016. We removed and separated the two plants per pot for either carboxylate collection (plant A) or mycorrhizal colonisation analysis (plant B). For plant A, the soil that remained attached to the roots after gently tapping the roots was considered rhizosphere soil (Veneklaas et al. 2003). To collect the rhizosphere carboxylates, we gently immersed the roots covered with rhizosphere soil of plant A in a known volume

(~20 ml) of 0.2 mM CaCl2 for 1 min. We filtered 1 ml of the rhizosphere extract with a 22- µm syringe filter into 25 µl of concentrated orthophosphoric acid and froze the samples at - 20°C. Carboxylate concentrations were determined using HPLC, following Cawthray (2003). Working standards of tartaric, formic, malic, malonic, lactic, acetic, maleic, citric, succinic, oxalic, fumaric, cis-aconitic and trans-aconitic acids were used. To report carboxylate amount per gram of rhizosphere soil, we collected the rhizosphere soil in a paper filter and dried it at 105°C during four days prior to weighing. Soil moisture also influences carboxylate amount, so we also collected a sample of the bulk soil from the pots, both top soil (field soil used as inoculum) and bottom soil (washed river sand) and calculated soil water content. After carboxylate collection, we rinsed the plant in DI water, removed the excess water with a paper towel, and weighed the whole plant, the roots, the stem and the leaves separately. To test if leaf mass per area was affected by nutrient supply, leaves were scanned and leaf area later calculated using imageJ (Schindelin et al. 2015). Leaves and stems were dried at 70°C for four days. Roots were wrapped in damp paper towels in a zip lock bag for a maximum of 48 h at 4°C. Nodules were then counted and removed and roots floated in a transparent tray in water and scanned, and analysed for root length, root surface area and average diameter with WinRhizo (WinRhizo system, Regent Instruments, Quebec, Canada). Roots were then dried at 70°C for 4 days.

3.3.2.4. Mycorrhizal analysis

To measure mycorrhizal colonisation, roots of plant B were rinsed, treated as the field samples, except for staining. We treated the stem and leaves as described above for plant 107

A. Glasshouse root samples were stained in Trypan Blue (0.05% w/v) for an hour in a water bath at 90°C, rinsed and stored as described for field samples. We assessed mycorrhizal colonisation using the intersect method by counting the number of arbuscular mycorrhizal hyphae, arbuscules and vesicles at each intersect (Giovannetti and Mosse 1980) for at least 180 intersects. Note that the assessment method used for the field samples can result in a slightly higher estimate of the level of colonisation than the method we used for the glasshouse samples (Giovannetti and Mosse 1980).

3.3.2.5. Leaf nutrient concentrations

Two leaf samples per pot were bulked for nutrient analyses. Dry leaves were ground as described above, a subsample was digested using concentrated HNO3: HClO4 (3:1) and analysed for Ca, Cu, Fe, K, Mg, Mn, Mo, P, S and Zn concentrations in an Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES, Perkin Elmer 5300DV, Waltham, USA). A second subsample was used for total N concentration using the combustion method (Elementar Vario Macro, Germany). All the glasshouse nutrient analyses were performed at Earth and Environment Analysis Laboratory (EEAL), The University of Western Australia. To estimate the control of nutrient uptake, we calculated coefficients of variation (CV) of leaf nutrient concentrations as the standard deviation of leaf nutrient concentration divided by the mean of leaf nutrient concentration.

3.3.3. Statistical analyses

All statistical analyses for the field data and glasshouse experiment involved model selection using corrected Akaike Information Criterion (AICc) (Zuur et al. 2009). For the field data, one-way ANOVAs were used to compare plant attributes among stages. A model with intercept only (null model) was compared with a model with chronosequence stage as an independent variable. We checked model assumptions graphically (Zuur et al. 2009), and if they were not met, variance was modelled using generalised least square models (gls) with the nlme package (Pinheiro et al. 2017). If the models presented residual heteroscedasticity or non-normality, nutrient concentrations were modelled using generalised linear models (glm) with gamma distribution. Because mycorrhizal colonisation is a percentage, we modelled it using beta regressions with the betareg package (Cribari-Neto and Zeileis 2010), adding 10-11 to all the values, because zeros cannot be included in betareg. We selected random terms in gls and fixed terms in all the models using the corrected Akaike Information Criterion (AICc) (Zuur et al. 2009). If the differences in AICc between models 108

were less than two units, we chose the most parsimonious model (Arnold 2010). If the term chronosequence stage was in the selected model, Tukey post-hoc comparisons were performed with the lsmeans package (Lenth 2016).

For the glasshouse experiment, two-way ANOVAs were used to model differences between the plant attributes measured in N and P supply treatments. We compared a null model with models with P and N supply, with and without interactions, as independent variables, and with only P or only N supply as independent variables. To meet model assumptions gls, glm and betareg were used as described above. Post-hoc comparisons were undertaken as described above. All statistics were undertaken in R 3.3.2 (R Development Core Team 2017). Only significant effects were graphed using ggplot2.

3.4. Results

3.4.1. Field data

Leaf [P] of B. linophylla followed available soil [P] along the high-rainfall Warren chronosequence, being lowest in younger and older soils (Fig. 3.1a, CV of leaf [P] = 32%, Table 3.2 Sup. mat.). In contrast, B. eriocarpa along the low-rainfall Jurien Bay chronosequence maintained a similar, relatively low, leaf [P] at stages 4-6 where it occurred (Fig. 3.1b, Table 3.2 Sup. mat., CV = 19%). Leaf [N] of B. linophylla was greatest at stage 3, being lower on younger and older soils (Fig. 3.1c, Table 3.2 Sup. mat., CV= 16%). Again, B. eriocarpa maintained a similar leaf [N] at stages 4-6 (Fig. 3.1d, Table 3.2 Sup. mat., CV=11%). Leaf N: P ratios of B. linophylla were similar among stages 1-4, and higher at stage 6 (Fig. 3.1e, Table 3.2 Sup. mat., CV=109%)). Leaf N: P ratios of B. eriocarpa were similar among stages 4-6 and greater than those of B. linophylla (Fig. 3.1f, CV= 11%).

Leaf [Mn] varied greatly among chronosequence stages (Fig. 3.2a-b). In B. linophylla, the leaf [Mn] started at 11 ± 1 mg Mn kg-1 DW at stage 1, and reached a very high 568 ± 77 mg Mn kg-1 DW at stage 6 (Fig. 3.2a, Table 3.2 Sup. mat., CV=150%). In B. eriocarpa, leaf [Mn] was constant around 27 ± 10 mg Mn kg-1 DW at stages 4 and 5, but increased to 98 ± 9 mg Mn kg-1 DW at stage 6 (Fig. 3.2b, Table 3.2 Sup. mat.).

The level of mycorrhizal colonisation was similar at all stages for both species (Fig. 3.2c-d). In B. linophylla, mycorrhizal colonisation varied from 35 ± 5% at the first stages to 58 ± 7% at stage 4 (Fig. 3.2c, Table 3.2 Sup. mat.). In B. eriocarpa, mycorrhizal 109

colonisation varied between 41 ± 6% at stage 4 and 8 ± 6% at stage 5 (Fig. 3.2d, Table 3.2 Sup. mat.).

3.4.2. Glasshouse experiment

Leaf [P] increased with increasing N and P supply for both species (Fig. 3.3a, b note P-N interaction only for B. eriocarpa). In both species leaf [P] approximately doubled from P0-P50 (Fig. 3.3b, CV of leaf [P] of B. linophylla = 52%, B. eriocarpa =39%). Leaf [N] also varied with increasing N and P supplies in both species (Fig. 3.3c, d, Table 3.3 Sup. mat., no interaction between N and P supply). In B. linophylla, leaf [N] increased approximately 30 % between P0-P50 treatments and 15% between N10-N50 treatments (CV= 17%). In B. eriocarpa, maximum leaf [N] variation was 26% between N10-N50 treatments (CV= 18%). Leaf N: P ratios decreased with increasing P supply, but increased with increasing N supply for both species (Fig. 3.3e, f, Table 3.3 Sup. mat., no N-P interaction). In B. linophylla N: P ranged from 25 in P50/N10 to 52 in P0/N50 (CV= 27%). In B. eriocarpa N: P ranged from 32 to 63 in the same treatments (CV= 23%).

Total dry biomass was very similar for the two species, and increased more than three-fold between P0 and P50 (Fig. 3.4, Table 3.3 Sup. mat.), but did not vary with increasing N supply (Table 3.3 Sup. mat.). Changes in the total biomass were due to increases in leaf, stem and root biomass in both species (Fig. 3.4). However, root length, root volume and root surface area increased with increasing P supply in B. linophylla only (data not shown, Table 3.3 Sup. mat.). Root: mass ratio decreased with P supply in B. linophylla and decreased with increasing P supply, but increased with increasing N supply in B. eriocarpa (data not shown, Table 3.3 Sup. mat.).

While increasing N supply did not affect biomass in B. linophylla, specific root length (root length/root dry weight) decreased with increasing P supply and increased with increasing N supply in the P0 and P0.5 treatments, but decreased with increasing N supply in the P5 and P50 supplies (data not shown, Table 3.3 Sup. mat.). Specific root length did not vary with P and N supply in B. eriocarpa (data not shown, Table 3.3 Sup. mat.). Leaf mass per area was 49 to 60 g m-2 throughout treatments for both species (data not shown, Table 3.3 Sup. mat.).

We detected citrate (59-98% of total amount of carboxylates for B. linophylla and 0-77% for B. eriocarpa), malate (0-38% for B. linophylla and 7-74% for B. eriocarpa), malonate (0-1% for B. linophylla and 0-23% in B. eriocarpa) and lactate (undetected for B. 110

linophylla and 0-20% in B. eriocarpa) in decreasing order of amounts (Fig. 3.5). We also detected acetate, maleate and fumarate, but in much lower amounts. Total carboxylate release in B. linophylla was positively affected by both increasing P and N supply (P-N interaction), but we did not find differences in pairwise comparisons (Fig. 3.6a, Table 3.3 Sup. mat.). Total carboxylates in the rhizosphere varied from 2.4 ± 0.6 μmol g-1 root DW at P0/N10 to 13.5 ± 15.7 μmol g-1 root DW at P0/N50 in B. linophylla. In the P50 treatment, B. linophylla showed 5.8 ± 2.2 μmol g-1 root DW at N10 and 22.9 ± 16.2 μmol g-1 root DW at N50. The total amount of carboxylates in the rhizosphere of B. eriocarpa was one order of magnitude less than that in B. linophylla (Fig. 3.6b, Table 3.3 Sup. mat.). Total carboxylates decreased with increasing N supply, with no pairwise differences, from 0.39 ± 0.13 μmol g-1 root DW at N10 to 0.07 ± 0.04 μmol g-1 root DW at N50. The very high confidence intervals of total carboxylate in the N50 treatments in both species is because of the small sample size in this treatment, due to the high mortality rate.

Mycorrhizal colonisation decreased with increasing N supply in B. linophylla, but independent of P supply (Fig. 3.7a, Table 3.3 Sup. mat.). In B. eriocarpa, mycorrhizal colonisation decreased with increasing P supply, being highest at intermediate N supply (N30) (30% colonisation) and lowest in P0.5 (4% colonisation, Fig. 3.7b, Table 3.3 Sup. mat.). Numbers of nodules per plant decreased with increasing N supply for both species, but increased with increasing P supply in B. linophylla (Fig. 3.7c,d, Table 3.3 Sup. mat.; note no P-N interaction). In the P0 treatment, B. linophylla presented 11 nodules at N10, but only 1 nodule at N50, and in the P50 treatment, 24 nodules at N10 and 14 at N50. In B. eriocarpa, only N supply affected nodule formation. Numbers of nodules of B. eriocarpa decreased from 10 in N10 to 5 in N50 (Fig. 3.7d, Table 3.3 Sup. mat.).

Leaf [Mn] of B. linophylla decreased with increasing P supply, but increased with increasing N supply (Fig. 3.8a, no N-P interaction). In B. linophylla, leaf [Mn] halved from P0 to P50, but increased 30% between N10-N30 (CV= 26%). However, total leaf Mn content (leaf [Mn] multiplied by leaf mass), increased with increasing P supply (Fig. 3.8b so the decrease in leaf [Mn] with P supply was due to a dilution effect. In B. eriocarpa leaf [Mn] decreased 30% with increasing P supply from P0 to P50, being constant among N treatments (Fig. 3.8c, CV= 24%) and total leaf Mn of B. eriocarpa content did not change with increasing N or P supply (Fig. 3.8d).

Mortality rate increased greatly with increasing N supply in both species and was unaffected by P supply (Fig. 3.9, Table 3.3 Sup. mat.). In B. linophylla, mortality rates 111

increased from 29% at N10 to 75% at N50 (Fig. 3.9a). In B. eriocarpa, mortality rates increased from 23% at N10 to 53% at N50 (Fig. 3.9b).

3.5. Discussion

We investigated whether P and N availability affected P- and N-acquisition strategies of two legume species native to the south-western Australian kwongan by investigating foliar [P], [N] and [Mn] and root mycorrhizal colonisation along two strong gradients of soil age with distinct P and N availabilities and climates (Turner et al. 2017). Additionally, we investigated how varying P and N supply affected foliar [P] and [N], biomass production and allocation, carboxylate release, mycorrhizal colonisation and nodulation in a glasshouse experiment. Our results show that growth of the two Bossiaea species is usually P-limited due to the extra N source of the N-fixation, and the two species presented different shifts in nutrient-acquisition strategies with changes in nutrient- availability.

3.5.1. Leaf P and N concentrations

Leaf [N] and [P] of the glasshouse-grown Bossiaea plants were of the same order of magnitude as those of plants in the field. Leaf [P] in the field (0.2-1.3 mg P g-1 DW) and in the glasshouse (0.3-0.9 mg P g-1 DW) were below the global average leaf [P] (1.43 mg P g-1 DW) estimated by Vergutz et al. (2012), while leaf [N] of both field (15-21 mg N g-1 DW) and glasshouse-grown Bossiaea plants (15-28 mg N g-1 DW) were equal to, or even higher than the global average (18.4 mg N g-1 DW) (Vergutz et al. 2012). The high [N] may be due to the inherently high N and P demand of legumes (Sprent 1999; Vitousek et al. 2002).

Our first hypothesis was that B. linophylla and B. eriocarpa would change leaf [N] and [P] in response to increasing supply. Leaf [N] and [P] of both field- and glasshouse- grown Bossiaea plants increased with increasing N and P supplies. Leaf [N] and [P] of B. eriocarpa in the field were unchanged along the last three stages where it occurs likely due to the similar soil availabilities of these nutrients. Leaf [P] and [N] of B. eriocarpa were similar to those of the nitrogen-fixing species, but higher than the concentrations of species with other nutrient-acquisition strategies at the same chronosequence stages of Jurien Bay (Hayes et al. 2014). Leaf [P] of B. eriocarpa in the field was very similar to those of co-occurring legumes from stages 4 to 6, and lower than leaf [P] and [N] of legumes from the younger stages where B. eriocarpa does not occur (Png et al. 2017). Gompholobium tomentosum, which acquires a greater proportion of N from nitrogen fixation presented the same leaf [P] (0.6 mg P g-1 DW) 112

as that of a species that acquires no N through fixation, Labichea cassioides at stage 4 (Png et al. 2017), and only a slightly higher leaf [P] than that of B. eriocarpa (0.4 mg P g-1 DW). At stage 6, where Acacia pulchella, Jacksonia floribunda and J. hakeoides all acquire a small portion of their N through fixation, leaf [P] is also very similar to that measured in B. eriocarpa (Png et al. 2017). Leaf [N] of B. eriocarpa was also similar to leaf [N] of G. tomentosum (stage 4) and A. pulchella (stage 6), but higher than that of most other species assessed by Png et al. (2017). Therefore, the species’ dependence on N-fixation was not related to leaf [P] or [N] along the oldest stages of the Jurien Bay chronosequence.

When compared with co-occurring glasshouse-grown legumes provided with 50 mg P g-1 soil and 25 mg N g-1 soil, leaf [P] of glasshouse-grown B. eriocarpa was one-fifth of that of A. pulchella and one-fourteenth of that of J. floribunda, while leaf [N] of these species was very similar to that of B. eriocarpa (Png 2016). These differences led to much lower N: P ratios of A. pulchella and J. floribunda than of B. eriocarpa at similar nutrient supplies. These two species did not produce any nodules in any treatment (Png 2016). The low investment in N acquisition through N-fixation might account for the stronger N-limitation of these species when compared with B. eriocarpa.

Bossiaea plants did not vary in leaf [N] to a great extent and seemed to down- regulate N uptake with increasing N availability. However, the high mortality rate of B. linophylla (75%) and B. eriocarpa (50%) observed at the highest N supply (50 mg kg-1), especially at low P supply (0 and 0.5 mg P kg-1 soil), might indicate a threshold beyond which N-uptake cannot be down-regulated. The mortality may therefore be due to a stoichiometric imbalance (Schulze 1989), rather than simply N-toxicity, because mortality decreased at higher P supplies. Lower mortality at higher P supplies might also be due to the dilution of the N when biomass increased. Also, the total N supplied to the plants was readily available, in the form of potassium nitrate. In the field, total soil [N] can be higher, but is likely in a less available organic form (Turner and Laliberté 2015), and, thus, less likely to cause toxicity. Therefore, we accepted our hypothesis that leaf [N] and [P] increased with increasing N and P supply.

3.5.2. Nutrient limitation, leaf N:P ratios and growth

Nutrient limitation is diagnosed by how primary productivity and other biological processes respond to added nutrients (Vitousek et al. 2010). Nutrient limitation can be assessed through direct measurements such as rate of biomass production, or alternatively 113

through indirect measurements of nutrient ratios in leaves. Nitrogen to phosphorus ratios above 16 represent P-limitation of plant growth (Redfield 1958; Wardle et al. 2004). We expected that the growth of Bossiaea would switch from N to P limitation along the chronosequences, in accordance with the ecosystem development model of Walker and Syers (1976). Along the Warren chronosequence, N: P ratios of B. linophylla ranged between 17 and 19 along the first four stages, and reached 28 at the last stage, indicating P-limitation at all stages. Since B. eriocarpa only occurred at the last three stages of the Jurien Bay chronosequence, all of which have low P availability, growth of all plants in the field was P- limited, with N: P ratios between 43 and 48, in agreement with the long-term soil development model (Walker and Syers 1976). The apparent P limitation of plant productivity in Bossiaea can be due to the high leaf [N], supported by N-fixation in nodules. Therefore, our results did not support our hypothesis that Bossiaea would switch from N to P limitation along the chronosequences, due to their high leaf [N].

We assessed the nutrient limitation of young Bossiaea plants measuring variation in growth with varying N and P supply in a glasshouse experiment. We expected that species that occur in P-impoverished soils would present slow growth rates in order to reduce nutrient-demand (Lambers and Poorter 1992). The two Bossiaea species in our study accumulated less than one gram of dry weight in five months, even at the highest nutrient supply. Co-occurring legumes from the same stages, Acacia pulchella and Jacksonia floribunda, accumulated up to 5 g at similar nutrient supplies (50 mg P kg-1 soil, 25 mg N kg-1 soil) and up to 10 g at higher N supplies (300 mg N kg-1 soil) over a six-month period (Png 2016). The slow growth of Bossiaea reveals a very conservative strategy in nutrient use for growth. Despite the slow biomass accumulation, total biomass of both species more than tripled from P0 to P50, but it did not respond to increasing N supply, probably due to the tight regulation of N-uptake. The range of P supplies was chosen to represent the maximum variation of total soil [P] found in the field along the two chronosequences (Turner et al. 2017). Thus, our glasshouse results suggest that the growth of B. linophylla and B. eriocarpa seedlings in the field might be P-limited, rather than N-limited. The very high N: P ratios of glasshouse-grown plants support this contention (>25 in all treatments). Liebig´s law of the minimum proposes that growth is controlled by the scarcest nutrient (Güsewell 2004). Since nitrogen fixation provides the plants with enough N, it is possible that increased N supply also increased P demand, leading to this apparent P-limitation. Therefore, we accepted our 114

hypothesis that Bossiaea would show conservative nutrient-use traits such as slow growth, but they did respond to P addition in a modest way.

3.5.3. Phosphorus-acquisition strategies at different nutrient availabilities

The changes in leaf [N] and [P] with nutrient availabilities allowed us to test if nutrient-acquisition strategies changed as observed at the community-level by Zemunik et al. (2015). We tested if the lowest P supplies stimulated carboxylate release, as observed in several native and crop species (Keerthisinghe et al. 1998; Shane et al. 2003; Pearse et al. 2006). In contrast, we found that for B. linophylla increasing P supply stimulated the release of carboxylates into the rhizosphere (Fig. 3.6a). A similar result was observed by (D’Angioli et al. 2017) for Zea mays and Huang et al. (2017) for Agonis flexuosa (Myrtaceae), which also grows along the entire Warren chronosequence. When grown in a glasshouse, rhizosphere carboxylates of A. flexuosa increased two-fold between 0 and 30 mg P kg-1 soil treatments. The increased amount of carboxylates in the rhizosphere of A. flexuosa occurred in the treatments with lowest root: shoot ratios and highest photosynthetic rates, possibly indicating release of excess carbon by the roots. The amount of rhizosphere carboxylates of B. linophylla was of the same order of magnitude as those of several Kennedia species inoculated with arbuscular mycorrhizal fungi (Ryan et al. 2012) and those measured on Cullen australasicum, Bituminaria bituminosa, Medicago sativa and Trifolium subterraneum (Nazeri et al. (2014), but much less than those presented for Cicer arietinum and Lupinus albus by Veneklaas et al. (2003) and K. nigricans by Suriyagoda et al. (2012). At the lowest P supplies, B. linophylla invested in morphological adaptations such as greater specific root length, rather than in carboxylate release. The carboxylate amounts in the rhizosphere of B. eriocarpa were one order of magnitude less than those in B. linophylla, and they decreased with increasing N rather than increasing P supply. Despite the lower carboxylate release in B. eriocarpa, the leaf [P] and total biomass were similar to those of B. linophylla, possibly because B. eriocarpa invested in alternative P-acquisition strategies, such as the release of phosphatases into the soil as observed in other legumes occurring along the Jurien Bay chronosequence. Bossiaea eriocarpa also invested in greater biomass allocation as shown by the greater root mass ratio at low P. We rejected our hypothesis that low P supply would stimulate carboxylate exudation, but we observed a greater investment in morphological modifications for P uptake.

The proportion of mycorrhizal plant species decreases along the Jurien Bay chronosequence (Zemunik et al. 2015), so we expected that the proportion of the root length colonised by mycorrhizal fungi would decrease also along the chronosequence within a single 115

species. We expected that the proportion of the root colonised by arbuscular mycorrhizal fungi would be low at a very low P supply, high at intermediate P supply and also be low at high P supply. In the field, mycorrhizal colonisation was constant along the chronosequences for both species. At the oldest stages, the costs of maintaining a mycorrhizal symbiosis are expected to be greater than the benefits (Lambers et al. 2008b). At extremely low soil [P], where B. eriocarpa occurs, arbuscular mycorrhizal fungi might have a different role. Rather than increase the uptake of P, arbuscular mycorrhizal fungi may provide protection against soil pathogens (Laliberté et al. 2015; Albornoz et al. 2017). Because the total soil [P] at the youngest stages of the Warren chronosequence is much lower than that at the youngest stages at Jurien Bay (Turner et al. 2017), it is possible that the role of arbuscular mycorrhizal fungi in pathogen defence dominates much earlier during pedogenesis. This might explain why mycorrhizal colonisation was constant, but high along the two chronosequences. We rejected our hypothesis that mycorrhizal colonisation would vary with P supply in the field.

In the glasshouse experiment, increasing P supply did not affect mycorrhizal colonisation in the roots of B. linophylla, similar to that of Viminaria juncea (de Campos et al. 2013). Instead, root colonisation declined with increasing N supply. Arbuscular mycorrhizas have traditionally been associated with P uptake. However, studies over the past 30 years show increasing evidence of inorganic (Govindarajulu et al. 2005) and organic (Leigh et al. 2009) N uptake and transfer to the host through arbuscular mycorrhizas (George et al. 1995). Despite the evidence for N transfer, evidence of enhanced growth and N uptake is hard to find (Reynolds et al. 2005). Nitrogen fertilisation inhibits arbuscular mycorrhizal colonisation in grasslands (Johnson et al. 2003), but these grasslands are N-limited, rather than P-limited as the systems in our study. The decrease in mycorrhizal colonisation at high N supply can be explained by an increased allocation to photosynthesis, leading to the inhibition of resource allocation to roots, and particularly C-transfer to root symbionts (Marschner et al. 1996; Johnson 2010). This pattern is expected when enough P is supplied to the plant. Additionally, the decrease in mycorrhizal colonisation at high N supply might be due to the greater carboxylate release, creating a trade-off in carbon investment in carboxylate release and in mycorrhizal fungi (Ryan et al. 2012). The mycorrhizal response to nutrient addition in B. eriocarpa differed from that in B. linophylla. Mycorrhizal colonisation decreased with increasing P supplies and was greatest at intermediate N supplies. Thus, we partially rejected our hypothesis of changes in mycorrhizal colonisation with P supply in the glasshouse. Therefore, the shift in the prevalence of species that acquire nutrients through arbuscular 116

mycorrhizas along the chronosequence observed at the community level in Jurien Bay (Zemunik et al. 2015) did not happen within Bossiaea species, either in the glasshouse or in the field. The changes in the proportion of nutrient-acquisition through mycorrhizas is due to changes in species composition (Laliberté et al. 2013; Zemunik et al. 2016), rather than within-species changes in nutrient-acquisition strategies.

3.5.4. Leaf [Mn] and nutrient-acquisition strategies

Leaf [Mn] cannot be compared between chronosequence stages due to the large differences in soil pH, which affect Mn availability; therefore we only present within-stage comparisons with co-occurring species. We expected that leaf [Mn] would increase with increased carboxylate release and decrease with increased mycorrhizal colonisation. If Bossiaea released more carboxylates than co-occurring species at each stage, we would expect them to have greater leaf [Mn] (Lambers et al. 2015). In B. linophylla, leaf [Mn] was very similar to those of co-occurring species at every stage of Warren (Huang et al. 2017). Co-occurring species, including Proteaceae, along the Warren chronosequence (Huang et al. 2017) presented similar leaf [Mn] to those of B. linophylla; this may indicate facilitation by carboxylate-releasing neighbouring plants, as demonstrated before (Muler et al. 2014). The similarity between leaf [Mn] of these species may be due to the root entangling observed in the field, where we rarely found Bossiaea roots separated from cluster roots. However, any role of facilitation needs to be confirmed using targeted glasshouse experiments. Leaf [Mn] of non-mycorrhizal species had slightly greater leaf [Mn] than that of B. eriocarpa, but leaf [Mn] of mycorrhizal plant species along the Jurien Bay chronosequence were also similar to those of B. eriocarpa at stages 4 and 5 (Hayes et al. 2014). Manganese acquisition of B. eriocarpa may also have been facilitated by Proteaceae, especially at stage 6, a Banksia woodland. Additionally, rhizosphere microorganisms may have played a role in increasing Mn availability in the oldest soils (Kothari et al. 1991). We surmise the increase in leaf [Mn], relative to that of other species at the same location, along the chronosequences is a combination of increased carboxylate-release, increased availability through mobilisation by rhizosphere microbiota and facilitation, thus only partly accepting our hypothesis based on field data.

Both B. linophylla and B. eriocarpa behaved as Mn accumulators in the glasshouse experiment, presenting leaf [Mn] greater than 500 mg kg-1 in every treatment. In the field, only B. linophylla reached similar values at the last stage of the Warren chronosequence. We expected increasing carboxylate release to increase rhizosphere Mn 117

availability and hence leaf [Mn] (Lambers et al. 2015). However, there was no relationship between leaf [Mn] and total carboxylate release, or total leaf Mn content and carboxylate release in either species. The carboxylate collection method employed in this study only measures instantaneous rhizosphere carboxylates, which can be rapidly consumed by soil microbiota or adsorbed onto soil particles (Evans 1998; Hashimoto 2007). The amount of carboxylates measured in one collection might not reflect the amount released along plant development. Therefore, carboxylates released may still have contributed to the very high leaf [Mn] of both species in the glasshouse. Differences in leaf [Mn] could also be due to mycorrhizal fungi intercepting the excess Mn. The mycorrhizal colonisation did not differ among treatments, but mycorrhizal fungal interception of soil Mn depends on extraradical hyphal length, which we did not measure. We suggest that greater hyphal length at high P supply and lower extraradical hyphal length at higher N supply might intercept more Mn, decreasing Mn transfer to the host. Therefore we rejected our hypothesis on leaf [Mn] based on the glasshouse data.

3.5.5. Nitrogen-acquisition strategies at different P and N availabilities

We expected nitrogen limitation to stimulate nitrogen fixation, given enough P was available (Sprent 1999). In the glasshouse experiment, we found that high N supply effectively decreased nodule formation in both species as expected (Streeter and Wong 1988). Nitrate application reduces nitrogenase activity and induces nodule senescence through several mechanisms, but most recently, down-regulation of genes involved in ATP generation in the mitochondria of Medicago truncatula was shown to be the mechanism behind the nitrate effect on nodulation (Cabeza et al. 2014). In B. linophylla, nodule formation was also stimulated at high P supply, as expected due to the high P-cost of nodulation (Pang et al. 2011; Raven 2012). The range of leaf [P] was greater among treatments in B. linophylla than in B. eriocarpa. This might explain why nodule formation in B. eriocarpa responded only to increasing N, but not to increasing P supply. Therefore, we observed that N and P availabilities can drive changes in N-acquisition strategies. We confirmed our third hypothesis that increased N supply reduces nodulation; however, increased P supply only stimulated nodulation in B. linophylla, due to the more variable leaf [P] and therefore, less tight control of P uptake. 118

3.5.6. Differences in nutrient-acquisition strategies between the two Bossiaea species

We observed very distinct responses to nutrient availability in both species. We emphasise that the responses to multiple nutrients are more complex than that of single nutrient addition experiments due to possible interactions and stoichiometric effects. Bossiaea linophylla occurred at all the stages of the Warren chronosequence. The presence at all stages requires the ability to extract nutrients from a wide array of soils, from alkaline to acidic. The availability of many nutrients varies with soil pH (Lambers et al. 2008a); therefore, their acquisition requires different strategies. Bossiaea eriocarpa was restricted to the older P- impoverished soils. The three stages where it occurred presented similar N and P availabilities and pH, demonstrating its limited capacity to grow on alkaline soils. Bossiaea eriocarpa also presented the same nutrient-acquisition strategies at all the stages where it occurred, probably reflecting the constant leaf [N] and [P] at different stages. When grown in the glasshouse, B. eriocarpa grew well with the wide range of N and P supplies with acidic pH. Therefore, it is likely that its occurrence in the field is due to its low capacity to acquire nutrients at high soil pH. The restricted capacity of B. eriocarpa to acquire nutrients in alkaline soils may indicate a specialisation, as opposed to the generalist capacity of B. linophylla to occupy a wider range of soil pHs.

3.5.7. Concluding remarks

We conclude that the shifts in nutrient-limitation and in nutrient-acquisition strategies observed at the species level are not the same as observed at the community level. We also emphasise that the responses to multiple nutrients are more complex than that of single-nutrient addition experiments, but are important to understand shifts in nutrient- acquisition strategies, and the ability of plants to occur along natural gradients of soil fertility. The release of rhizosphere carboxylate release was not up-regulated at low P supply, but the species invested more in morphological adaptations for P uptake. Mycorrhizal colonisation of Bossiaea did not change along the chronosequences, possibly due to a relatively unimportant role for the fungi in nutrient uptake at very low P availability, and to their provision of defence against soil pathogens in exchange for plant carbon. Nodule formation was down- regulated by increased N supply, as expected. We conclude that although the two species belong to the same genus, they have distinct nutrient demands and responses to nutrient additions, probably reflecting their different capacities of occupying different soils in the kwongan vegetation mosaic. 119

3.6. Acknowledgments

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes, PhD scholarship to Anna Abrahão and Project Grant PVE 88881.068071/2014-01) and by the School of Plant Biology of the University of Western Australia. We thank the Western Australian Department of Parks and Wildlife for the licenses to work in Jurien Bay (License SW017907 and Regulation 4 Authority Permit CE005400). We also would like to thank Adrien Frémont, Jorik Smits, Guilhem Reboul, Patrick Hayes, Daniel Kidd, Tim Lardner, Greg Cawthray, Rob Creasy, Bill Piasini, Francis Nge, Rodrigo Pires and Elzbieta Halladin for the practical support at the University of Western Australia and in the field.

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129

Table 3.1. Soil chemical analyses of the substrates used in the glasshouse experiment. Nutrient and aluminium (Al) concentrations are reported in mg kg-1. M3 refers to Mehlich-3 extractions. Total nitrogen (N) and phosphorus (P) were determined after extraction with sulfuric acid. Percentages under soil names refer to the proportion of each soil used in the pots.

pH pH N P Al Ca Cu Fe K Mg Mn Zn

(H2O) (CaCl2) (total) (total) (M3) (M3) (M3) (M3) (M3) (M3) (M3) (M3)

Washed river sand (90%) 6.6 6.1 <0.05 31 72 78 0.2 50 9 38 7.4 2.4 Harrisdale sand 5.1 4 0.43 15 46 250 0.1 15 6 31 1.3 0.3 Bossiaea eriocarpa (5%) Waroona lateritic soil B. aquifolium 6.1 5.1 1.78 250 550 200 0.5 37 92 53 12 <0.1 (5%)

130

a B. linophylla b B. eriocarpa 2.0 2.0

) 1.5 1.5

( 1.0 1.0

L 0.5 0.5

0.0 0.0 1 2 3 4 6 4 5 6 c d 30 30

)

1 - 20 20

(

a 10 10

0 0 1 2 3 4 6 4 5 6 e f 80 80

60 60

P P

: :

N N

f 40 f 40

a a

e e

L L

20 20

0 0 1 2 3 4 6 4 5 6 Chronosequence stage Chronosequence stage

Figure 3.1. Leaf phosphorus (P) and nitrogen (N) concentrations (in mg g-1 dry weight), and N: P ratios of Bossiaea linophylla along the Warren chronosequence (a, c, e) and B. eriocarpa along the Jurien Bay chronosequence (b, d, f). Points represent least-square means and bars, 95% confidence intervals. 131

a B. linophylla b B. eriocarpa 1000 1000

)

1 750 750

m 500 500

(

a 250 250

0 0

1 2 3 4 6 4 5 6

c d 100 100

)

(

o 75 75

o l 50 50

h 25 25

M 0 0

1 2 3 4 6 4 5 6 Chronosequence stage Chronosequence stage

Figure 3.2. Leaf manganese concentrations (mg Mn kg-1 dry weight of Bossiaea linophylla along the Warren chronosequence (a) and B. eriocarpa along the Jurien Bay chronosequence (b). Percentage of root colonised by arbuscular mycorrhizal fungi in roots of B. linophylla along the Warren chronosequence (c) and B. eriocarpa along the Jurien Bay chronosequence (d). Points represent least-square means and bars, 95% confidence intervals. Chronosequence age increases with stage.

132

Figure 3.3. Leaf nutrient concentrations of Bossiaea linophylla and B. eriocarpa grown in a glasshouse at different nitrogen (N) and phosphorus (P) supplies. a) and b) P concentrations , c) and d) N concentrations (mg g-1 dry weight), e) and f) leaf N:P ratios. Points represent least-square means and bars, 95% confidence intervals. 133

a B. linophylla b B. eriocarpa 0.8 0.8 Leaf Leaf

) Stem Stem

( 0.6 Root 0.6 Root

0.4 0.4

l 0.2 0.2

0.0 0.0 0 0.5 5 50 0 0.5 5 50 P supply (mg kg-1) P supply (mg kg-1)

Figure 3.4. Total plant biomass divided in leaves, roots and stems of Bossiaea linophylla (a) and B. eriocarpa (b) grown in a glasshouse at different nitrogen and phosphorus (P) supplies. Least-square means and 95% confidence intervals are shown. There was no effect of N supply, so the tissue dry weights were average across N treatments, and no interaction of P supply with N supply, except for stem dry weight of B. eriocarpa where P and N supply affected stem biomass (no interaction between N and P).

134

)

W B. linophylla B. eriocarpa

o P0 P0.5 P5 P50 P0 P0.5 P5 P50

1 100 100

m 75 75

m Acetic Acetic

( Citric Citric

t Fumaric Fumaric

a 50 50 l Lactic Lactic

x Maleic Maleic

b r 25 Malic 25 Malic

c Malonic Malonic

h 0 0

s 103050 103050 103050 103050 103050 103050 103050 103050

i -1 -1

h N supply (mg kg ) N supply (mg kg )

Figure 3.5. Proportion of each carboxylate in the rhizosphere of Bossiaea linophylla (a) and B. eriocarpa (b) grown in a glasshouse at different nitrogen (N) and phosphorus (P) supplies. The values are mean per treatment.

135

B. linophylla ) B. eriocarpa

)

-1 D 10 30 50 60

W N supply (mgkg )

1.00

- o 60

g 0.75

l - 0.50

g 40

m l 0.25

40 (

m 0.00

(

t 10 30 50

y 20

20 x

0 r

e 0

−20 i

l i −20

a 0 0.5 5 50 10 30 50

o -1 -1 T P supply (mg kg soil) N supply (mg kg soil)

Figure 3.6. Total carboxylates in the rhizosphere of Bossiaea linophylla (a) and B. eriocarpa (b) grown in a glasshouse at different nitrogen (N) and phosphorus (P) supplies. The insert in b shows the same data with a different range of y axis values to represent the variation in rhizosphere carboxylates of B. eriocarpa at a smaller scale. Points represent least-square means and bars, 95% confidence intervals.

136

a B. linophylla b B. eriocarpa

50 -1 10 30 50

) N supply (mg kg )

(

)

40 % 50

(

i 40

n 30

o n 30

20 o

i l 20

o 10

c r 10

c 0 y 0

M 10 30 50 0 0.5 5 50 N supply (mg kg-1 soil) P supply (mg kg-1 soil)

c d

N supply (mg kg-1) 10 30 50 30

s 30

e l 20

o 20

e 10

m u 0 N 0

0 0.5 5 50 10 30 50 P supply (mg kg-1 soil) N supply (mg kg-1 soil)

Figure 3.7. Percentage of root mycorrhizal colonisation (a, b) and number of nodules of Bossiaea linophylla (a, c) and B. eriocarpa (b, d) grown in a glasshouse at different nitrogen (N) and phosphorus (P) supplies. Points represent least-square means and bars, 95% confidence intervals.

137

a B. linophylla b B. linophylla N supply (mg kg-1) 10 30 50 0.3

1500

)

m 0.2

(

1000 M

(

] a 0.1

500 L

[

0 0.0 0 0.5 5 50 0 0.5 5 50

c B. eriocarpa d B. eriocarpa 1500 0.3

)

(

g 1000 0.2

(

]

500 f 0.1

[

L 0 0.0 0 0.5 5 50 0 0.5 5 50 P supply (mg kg-1 soil) P supply (mg kg-1 soil)

Figure 3.8. Leaf manganese concentrations of Bossiaea linophylla (a) and B. eriocarpa (c) grown in a glasshouse at different nitrogen (N) and phosphorus (P) supplies and total leaf manganese content of B. linophylla (b) and B. eriocarpa (d). Points represent least-square means and bars, 95% confidence intervals. Nitrogen is only shown in the figures when included in the best model.

138

a B. linophylla b B. eriocarpa 1.00 1.00

a 0.75 t 0.75

0.50 0.50

b 0.25 0.25

0.00 0.00 10 30 50 10 30 50 N supply (mg kg-1 soil) N supply (mg kg-1 soil)

Figure 3.9. Proportion of mortality of Bossiaea linophylla (a) and B. eriocarpa (b) grown in a glasshouse at different nitrogen (N) and phosphorus (P) supplies. Points represent least-square means and bars, 95% confidence intervals. Phosphorus was not significant in the model and therefore was not added to the figure.

139

Table 3.1. Sup. mat. Study plot locations within the dune systems of the high-rainfall Warren and low-rainfall Jurien Bay chronosequences. Five plots were positioned within each stage: the location of each plot’s centre point is listed. Stages were originally described by Laliberté et al. (2012); (Turner and Laliberté 2015)

Site Dune system Plot code Stage Latitude Longitude Warren Meerup unstable sand WAR-QY-A 1 34° 37' 31.9" S 115° 52' 2.6" E WAR-QY-B 1 34° 37' 30.4" S 115° 52' 6.5" E WAR-QY-C 1 34° 37' 29" S 115° 52' 7.9" E WAR-QY-D 1 34° 37' 32.9" S 115° 51' 58.2" E WAR-QY-E 1 34° 37' 28" S 115° 52' 12" E Meerup leached calcareous WAR-QM-A 2 34° 37' 14.5" S 115° 53' 15.4" E sand WAR-QM-B 2 34° 37' 14.6" S 115° 53' 9.8" E WAR-QM-C 2 34° 37' 15.5" S 115° 53' 11.9" E WAR-QM-D 2 34° 37' 15.4" S 115° 53' 14.3" E WAR-QM-E 2 34° 37' 13.9" S 115° 53' 21.6" E WAR-QM-F 2 34° 37' 16.7" S 115° 53' 7.2" E Meerup podzols over WAR-QO-A 3 34° 37' 17.1" S 115° 53' 34.5" E calcareous sands WAR-QO-B 3 34° 37' 17.2" S 115° 53' 38.3" E WAR-QO-C 3 34° 37' 17.9" S 115° 53' 42" E WAR-QO-D 3 34° 37' 19.9" S 115° 53' 43.2" E WAR-QO-E 3 34° 37' 17" S 115° 53' 30.5" E Meerup podzols in WAR-SY-A 4 34° 36' 44.3" S 115° 54' 25.2" E siliceous sands WAR-SY-B 4 34° 36' 46.6" S 115° 54' 26.7" E 140

WAR-SY-C 4 34° 36' 48.9" S 115° 54' 23.9" E WAR-SY-D 4 34° 36' 51" S 115° 54' 20.9" E WAR-SY-E 4 34° 36' 42.4" S 115° 54' 22.9" E Cleave (Northcliffe) WAR-BAS-A 6 34° 35' 37" S 115° 54' 56.2" E WAR-BAS-B 6 34° 35' 40.3" S 115° 54' 55.7" E WAR-BAS-C 6 34° 35' 37.6" S 115° 54' 51.5" E WAR-BAS-D 6 34° 35' 33.9" S 115° 54' 52.5" E WAR-BAS-E 6 34° 35' 35" S 115° 54' 56.4" E Jurien Bay West Spearwood S.W.8 4 30° 14' 7.0" S 115° 4' 14.4" E S.W.36 4 30° 11' 29.3" S 115° 4' 27.4" E S.W.34 4 30° 1' 39.3" S 115° 2' 20.0" E S.W.3 4 30° 8' 35.6" S 115° 3' 43.6" E Deep-sand Spearwood S.DS.3 5 30° 15' 32.1" S 115° 6' 25.7" E Bassendean B.HR.5 6 30° 17' 39.2" S 115° 11' 5.7" E B.HR.2 6 30° 17' 42.3" S 115° 11' 15.5" E B.L.9 6 30° 10' 5.5" S 115° 6' 20.5" E B.L.14 6 30° 10' 51.1" S 115° 7' 43.5" E B.L.5 6 30° 9' 51.6" S 115° 7' 21.5" E

141

Table 3.2. Sup. mat. Model selection of the fixed factors in the field-collected nutrient concentrations and mycorrhizal colonisation of Bossiaea linophylla and B. eriocarpa along the Warren and Jurien Bay chronosequences, respectively. Indp var are the independent variables in the model compared through the corrected Akaike Information Criterion (AICc), ~1 represents the null model (intercept only). The delta AICc (dAICc) represents the difference between the model with the lowest AICc (best model) and the other models. The dAICc of the best model is therefore zero. df represents the degrees of freedom of each model and Akaike weights represent the relative likelihood of each model.

Dependent variable Species Best model Indp var. AICc ΔAICc df weight

Leaf [P] B. linophylla aov(P_mg_g~Stage, data=war) ~1 14.9 10.5 2 0.0053

Stage 4.4 0 6 0.9947

B. eriocarpa aov(P_mg_g~1, data=jur) ~1 -17.1 0 2 0.987

Stage -8.4 8.7 4 0.013

Leaf [N] B. linophylla aov(N_mg_g~Stage, data=war) ~1 151.4 18 2 < 0.001

Stage 133.3 0 6 1

B. eriocarpa aov(N_mg_g~1, data=jur) ~1 47.5 0 2 0.9918

Stage 57.1 9.6 4 0.0082

Leaf N:P B. linophylla aov(NP~Stage, data=war) ~1 172.3 24.9 2 < 0.001

Stage 147.4 0 6 1

B. eriocarpa aov(NP~1, data=war) ~1 66.2 0 2 0.984

Stage 74.4 8.2 4 0.016

Leaf [Mn] B. linophylla aov(Mn_mg_kg~Stage, data=war) ~1 317.8 52.7 6 < 0.001

Stage 265.2 0 10 1 142

B. eriocarpa aov(Mn_mg_kg~Stage, data=jur) ~1 107.9 7.5 2 0.023

Stage 100.4 0 4 0.977

Mycorrhizal B. linophylla betareg(Mean_Percent_col~1,data=war) ~1 -17 0 2 0.86 colonisation

Stage -13.4 3.6 6 0.14

B. eriocarpa betareg(Mean_Percent_col~1,data=jur) ~1 -4.1 0 2 0.7

Stage -2.4 1.7 4 0.3

143

Table 3.3. Sup. mat. Model selection of the fixed factors in a glasshouse experiment where we grew Bossiaea linophylla and B. eriocarpa at variable N and P supplies. Abbreviations as in Table 3.3.

Dependent variable Species Best model Indp var. AICc ΔAICc df weight Total dry weight B. linophylla ~1 -258.3 18 2 < 0.001 P* N -272.6 3.7 13 0.12 glm(Total dry weight ~P,family=Gamma(link = P+N -272.5 3.8 7 0.11 inverse)) P -276.3 0 5 0.77 N -254.7 21.6 4 < 0.001 B. eriocarpa ~1 -41.2 26.6 2 < 0.001 P* N -66.1 1.8 13 0.19 glm(Total dry weight ~P, family= Gamma(link = P+N -67.9 0 7 0.46 inverse)) P -67.3 0.5 5 0.35 N -42.7 25.2 4 < 0.001 Root dry weight B. linophylla ~1 -258.3 18 2 < 0.001 P* N -272.6 3.7 13 0.12 glm(Root dry weight ~P, family= Gamma(link = P+N -272.5 3.8 7 0.11 inverse)) P -276.3 0 5 0.77 N -254.7 21.6 4 < 0.001 B. eriocarpa ~1 -171.9 14 2 < 0.001 P* N -180.3 5.6 13 0.038 glm(Root dry weight ~P, family= Gamma(link = P+N -184.8 1.1 7 0.35 inverse)) P -185.9 0 5 0.611 N -172.4 13.5 4 < 0.001

Stem dry weight B. linophylla glm(Stem dry weight ~P, ~1 -280.9 40 2 < 0.001 family= Gamma(link = P* N -320.9 0 13 0.495 144

inverse)) P+N -317 4 7 0.069 P -320.7 0.3 5 0.436 N -277.3 43.6 4 < 0.001 B. eriocarpa ~1 -272.8 44.5 2 < 0.001 P* N -317.3 0 13 0.526 glm(Stem dry weight ~P+N, family= Gamma(link = P+N -316.8 0.5 7 0.412 inverse)) P -313 4.3 5 0.062 N -275.8 41.5 4 < 0.001 Leaf dry weight B. linophylla ~1 -180.1 18.7 2 < 0.001 P* N -193.9 4.9 13 0.068 glm(Leaf dry weight ~P, family= Gamma(link = P+N -195.5 3.3 7 0.153 inverse)) P -198.8 0 5 0.779 N -176.8 22 4 < 0.001 B. eriocarpa ~1 -145.1 31.8 2 < 0.001 P* N -176.9 0.1 13 0.39 glm(Leaf dry weight ~P, family= Gamma(link = P+N -177 0 7 0.41 inverse)) P -175.6 1.3 5 0.21 N -147.1 29.8 4 < 0.001 Leaf [P] B. linophylla ~1 33.5 144.1 3 < 0.001 P* N -110 0.5 14 0.43 gls(Leaf [P]~P+N, weights= P+N -110.6 0 8 0.57 varPower(form= ~ fitted(.)) P -94.8 15.8 6 < 0.001 N 27.9 138.5 5 < 0.001

B. eriocarpa gls(Leaf [P]~P*N, weights= ~1 -32.7 49.1 3 < 0.001 varPower(form= ~ fitted(.)) P* N -79.9 1.9 14 0.271 145

P+N -81.9 0 8 0.718 P -73.5 8.4 6 0.011 N -38.1 43.8 5 < 0.001 Leaf [N] B. linophylla ~1 342.1 51.8 2 < 0.001 P* N 302.1 11.8 12 0.0027 aov(Leaf [N]~P+N) P+N 290.3 0 7 0.9925 P 300.9 10.6 5 0.0048 N 330.9 40.7 4 < 0.001 B. eriocarpa ~1 298 10.7 3 0.0041

gls(Leaf [N]~P+N,, P+N 287.3 0 8 0.858 weights=varExp(form=~Pcont) P 291.4 4.1 6 0.1092 N 294.1 6.8 5 0.0287 Leaf N:P B. linophylla ~1 477.5 64.7 2 < 0.001 P* N 419.7 6.8 12 0.0315 aov(Leaf N:P~P+N) P+N 412.8 0 7 0.9623 P 422.9 10.1 5 0.0062 N 480.6 67.8 4 < 0.001 B. eriocarpa ~1 423.8 36.6 2 < 0.001 P* N 397.9 10.7 11 0.0048 aov(Leaf N:P~P+N) P+N 387.2 0 7 0.9946 P 402 14.8 5 < 0.001 N 426 38.8 4 < 0.001 Leaf [Mn] B. linophylla ~1 937.2 51.8 2 < 0.001 aov(Leaf [Mn]~P+N) P* N 889.9 4.5 13 0.095 P+N 885.4 0 7 0.905 146

P 914.8 29.4 5 < 0.001 N 919.7 34.2 4 < 0.001 B. eriocarpa ~1 808 13 2 < 0.001 P* N 799.1 4 13 0.072 aov(Leaf [Mn]~P) P+N 795.7 0.6 7 0.393 P 795.1 0 5 0.534 N 810.3 15.3 4 < 0.001 Root length B. linophylla ~1 1190.5 7 3 0.0207 P* N 1189 5.5 14 0.0441 gls(Root length ~P+N, weights= P+N 1185.6 2.1 8 0.2394 varPower(form= ~ fitted(.)) P 1183.5 0 6 0.6926 N 1194.2 10.8 5 0.0032 B. eriocarpa ~1 859.3 17.3 3 < 0.001

gls(Root length ~P, weights= P+N 843.3 1.3 8 0.3373 varPower(form= ~ fitted(.)) P 842 0 6 0.6578 N 851.9 9.9 5 0.0047 Specific root length B. linophylla ~1 550.7 14.2 2 < 0.001 P* N 536.5 0 13 0.9304 aov(Specific root length~P*N) P+N 542 5.5 7 0.0587 P 547.1 10.6 5 0.0047 N 546.8 10.3 4 0.0055 B. eriocarpa ~1 438.9 0 3 0.8375 P+N 450 11.1 8 0.0033 aov(Specific root length~1) P 445 6.1 6 0.0393 N 442.8 3.9 5 0.1199 147

Root surface area B. linophylla ~1 1177.9 11 3 0.0033 P* N 1171.2 4.4 14 0.0894 gls(Root surface area ~P, weights= varPower(form= ~ P+N 1170.7 3.8 8 0.1176 fitted(.)) P 1166.9 0 6 0.7894 N 1181.9 15 5 < 0.001 B. eriocarpa ~1 858.5 19.9 3 < 0.001 gls(Root surface area ~P+N, P+N 838.6 0 8 0.8019 weights= varPower(form= ~ fitted(.)) P 841.5 2.9 6 0.1894 N 847.7 9.1 5 0.0086 Root volume B. linophylla ~1 859.8 15.2 3 < 0.001 P* N 846.8 2.2 14 0.24 gls(Root volume ~P, weights= P+N 849.5 4.9 8 0.06 varPower(form= ~ fitted(.)) P 844.6 0 6 0.7 N 860 15.4 5 < 0.001 B. eriocarpa ~1 640.9 22.7 3 < 0.001 gls(Root volume ~P+N, P+N 618.1 0 8 0.941 weights= varPower(form= ~ fitted(.)) P 624.4 6.3 6 0.041 N 626 7.9 5 0.018 Root average diameter B. linophylla ~1 47.3 0 2 0.3709 P* N 57.9 10.6 13 0.0019 aov (Root average diameter ~1) P+N 49.6 2.3 7 0.1188 P 47.4 0.1 5 0.3646 N 49.2 1.9 4 0.1437 B. eriocarpa ~1 40.5 0 2 0.508 aov(Root average diameter ~1) P* N 58.5 18 12 < 0.001 148

P+N 47.4 6.9 7 0.016 P 45.9 5.4 5 0.035 N 40.8 0.3 4 0.441 Number of nodules B. linophylla ~1 443.8 56 3 < 0.001 P* N 395.7 7.9 14 0.019 gls(Number of nodules ~P+N, weights= varPower(form= ~ P+N 387.8 0 8 0.981 fitted(.)) P 414.6 26.8 6 < 0.001 N 440.7 52.9 5 < 0.001 B. eriocarpa ~1 295 0.5 3 0.387 gls(Number of nodules ~N, P+N 297.7 3.3 8 0.097 weights= varPower(form= ~ fitted(.)) P 300.6 6.1 6 0.023 N 294.5 0 5 0.493 Mycorrhizal B. linophylla -199 13.3 2 < 0.001 colonisation ~1 P* N -211.7 0.6 13 0.38 betareg(Mycorrhizal colonisation~N) P+N -209.1 3.2 7 0.1 P -199.5 12.8 5 < 0.001 N -212.3 0 4 0.51 B. eriocarpa ~1 -105 9.2 2 0.007 P* N -109.4 4.8 13 0.063 betareg(Mycorrhizal P+N -114.2 0 7 0.689 colonisation ~P+N) P -111.4 2.8 5 0.168 N -109.7 4.5 4 0.074 Total carboxylates B. linophylla ~1 433.1 37.6 3 < 0.001 gls(Total carboxylates ~P*N, weights= varPower(form= ~ P* N 395.5 0 14 1 fitted(.))) P+N 410.5 15 8 < 0.001 149

P 414.4 18.9 6 < 0.001 N 421.6 26.1 5 < 0.001 B. eriocarpa ~1 242.6 5.8 2 0.0517 P* N 248.2 11.4 12 0.0031 glm(Total carboxylates ~N, family= Gamma(link = P+N 244.7 7.9 7 0.0176 inverse)) P 248.2 11.3 5 0.0032 N 236.8 0 4 0.9244 Leaf mass per area B. linophylla ~1 394.5 0 2 0.653 P* N 408.7 14.3 13 < 0.001 aov(Leaf mass per area ~1) P+N 400.9 6.5 7 0.026 P 398.5 4 5 0.088 N 396.5 2.1 4 0.233 B. eriocarpa ~1 448.4 0 2 0.4014 P* N 456.4 8 13 0.0074 aov(Leaf mass per area ~1) P+N 451.6 3.1 7 0.0831 P 448.5 0.1 5 0.3787 N 450.7 2.3 4 0.1294 Mortality B. linophylla ~1 276.2 23.2 1 < 0.001 P* N 261.3 8.3 12 0.013 glm(Mortality ~N, P+N 256.5 3.6 6 0.143 family=binomial(link = logit)) P 280 27.1 4 < 0.001 N 252.9 0 3 0.844 B. eriocarpa ~1 259.3 9.5 1 0.0081 glm(Mortality ~N, P* N 262 12.2 12 0.0021 family=binomial(link = logit)) P+N 254.8 5 6 0.0767 150

P 264.2 14.4 4 < 0.001 N 249.8 0 3 0.9124 Root: mass ratio B. linophylla -195.9 25.7 2 < 0.001 ~1 glm(RootMass~P, family= P* N -212.2 9.5 13 0.0053 Gamma(link= inverse)) P+N -221.7 0.0 7 0.6040

P -220.8 0.9 5 0.3907 N -195.3 26.3 4 < 0.001 B. eriocarpa ~1 -135.0 10.1 2 0.0048 aov(RootMass~P+N) P* N -135.3 9.8 13 0.0056 P -145.1 0.0 7 0.7350 N -142.9 2.2 5 0.2506 151

CHAPTER 4. GENERAL DISCUSSION

4.1. Introduction

In view of changing soil nutrient availability (Phoenix et al. 2006) and increasing ecosystem threats (Lambers et al. 2013; Fernandes et al. 2014), it is important to understand the mechanisms underlying the patterns of native plant distribution in order to underpin restoration efforts. Old and severely nutrient-impoverished habitats, particularly those occurring on sandy soils, are extremely sensitive to disturbance, and restoration is very slow and expensive (Hopper 2009; Le Stradic et al. 2014). Therefore, understanding how species acquire and use nutrients from soils differing in nutrient availability can help us establish species plasticity and limitations regarding mineral nutrition (Kramer-Walter and Laughlin 2017), and offer important information for restoring degraded landscapes (Silveira et al. 2016). Understanding nutrient-acquisition and -use strategies in soil mosaics of varying nutrient availability will also help to unravel the mechanisms underlying the patterns of species diversification and distribution (Laliberté et al. 2013; Silveira et al. 2016; Zemunik et al. 2016).

The relative proportion of species with different nutrient-acquisition strategies, and also the within-species investment in different nutrient-acquisition strategies, change with nutrient availability (Lambers et al. 2008; Pang et al. 2010; de Campos et al. 2013; Oliveira et al. 2015; Zemunik et al. 2015). Long-term soil development sequences provide excellent models to understand these changes, and how they relate to species distributions along these gradients. In this thesis, I investigated how nutrient-acquisition and -use strategies occur at the community and species level in three long-term soil-development sequences. The first sequence was in mountaintop vegetation called campos rupestres in Brazil (Chapter 2) and the other two were in sand dunes along the south-western Australian coast (Chapter 3). All soil-development sequences presented increasing P-impoverishment, leading to increased P- limitation for plant growth. The shifts observed in nutrient-acquisition strategies at the community scale in the campos rupestres were the same as those observed at the community scale for the long-term soil development sequence in south-western Australia (Zemunik et al. 2015). For example, the proportion of mycorrhizal species decreased, and that of non- mycorrhizal species increased along the soil sequence in campos rupestres. However, at the species level, the species occurring along the two chronosequences in south-western Australia did not show the same pattern as observed at the community level (Png 2016). Therefore, the 152

shifts in nutrient-acquisition strategies observed at the community level are due to the turnover of species with different nutrient-acquisition strategies along the long-term soil development gradients (Zemunik et al. 2016), and not to within species changes in the investment in particular nutrient-acquisition strategies.

In this short general discussion, I synthesise the main findings of my thesis in two sections: changes in nutrient-acquisition and -use strategies at the community level along the soil-development gradient in campos rupestres and within-species changes in nutrient- acquisition and -use strategies in two Bossiaea species along the Warren and Jurien Bay chronosequences. I also discuss the limitations and achievements of my project and finish with suggestions for future key areas for research on mineral nutrition in nutrient- impoverished habitats.

4.2. Discussion

4.2.1. Community level changes in nutrient-acquisition and -use strategies

I expected the investment in arbuscular mycorrhizas (scavenging strategy) to decrease along the chronosequence. At the community scale, I found the same decrease in the proportion of mycorrhizal species along the gradient of soil development in the campos rupestres as observed in the well-studied Jurien Bay chronosequence (Zemunik et al. 2015). Interestingly, I also found the proportion of the root length colonised by arbuscular mycorrhizal fungi to decrease with increasing age along the soil-development sequence. This decrease might be due to a lower efficiency of arbuscular mycorrhizas to acquire sparingly forms of P in the oldest and most-impoverished soils (Parfitt 1979; Lambers et al. 2008). The few arbuscular mycorrhizal species found in the oldest, most nutrient-impoverished soils, might provide a benefit for their host based on a protective role of arbuscular mycorrhizas against soil pathogens (Laliberté et al. 2015) as observed for plants growing along the Jurien Bay chronosequence (Albornoz et al. 2017). In summary, the relative abundance of species relying on a scavenging strategy through arbuscular mycorrhizal fungi for P-acquisition was greatest at the less-developed, less infertile soils.

A great proportion of the non-mycorrhizal species in the most P-impoverished soils had roots with rhizosheaths. I hypothesised these rhizosheaths would indicate the presence of large amounts of carboxylates into the rhizosphere. However, I found only low amounts of carboxylates in the rhizosphere of all plants. It is not possible to say whether this indicates that all species were little reliant on carboxylates for P uptake (and hence not P- 153

miners) or whether my methodology was not suited to address this issue. The latter is possible as the presence of rhizosphere carboxylates is difficult to measure in the field due to the ephemeral nature of carboxylates release, their sorption to soil particles (Evans 1998; Hashimoto 2007) and, perhaps, other issues such as the disturbance involved in excavating roots. I used leaf [Mn] as an indicator of carboxylate-releasing mining strategies for P- acquisition. However, I did not find greater leaf [Mn] in rhizosheath-forming species, probably because species without rhizosheaths can also release carboxylates. Although root hair length is an important morphological adaptation for P acquisition in P-impoverished soils (Lynch 2011), there is no direct link between rhizosheath formation and capacity to take up different forms of P, as recently pointed out by Güsewell and Schroth (2017) for dauciform roots. According to Güsewell and Schroth (2017), species with and without dauciform roots have equal access to different forms of P. My results indicate that species release carboxylates independently of the presence of rhizosheaths. However, the greater relative abundance of rhizosheath forming species in the soils with the lowest P availability reinforces the importance of long root hairs for P-acquisition, regardless of carboxylate release. More detailed within-species studies are necessary to confirm each species’ mining strategy for P- acquisition in campos rupestres soils, but we do not discard the importance of long root hairs for P uptake.

Campos rupestres species achieved very high nutrient-use efficiency through reducing nutrient demand, presenting leaves with very low [P] and [N], very high remobilisation efficiency and proficiency from senescing leaves; and very high photosynthetic P- and N-use efficiency at high leaf mass per area. Leaf N: P ratios indicated N-P co- limitation for growth in the cerrado plants and strong P-limitation in the campos rupestres. Therefore, we found similarly efficient nutrient-use strategies for aboveground traits, but very distinct belowground nutrient-acquisition strategies in campos rupestres plants. Together, the species distribution along the trait axes of nutrient-acquisition and -use strategies reflect their ability to grow in different soil types within the soil mosaic of severely nutrient-impoverished campos rupestres soils.

Species occupying severely nutrient-impoverished soils such as campos rupestres present traits that allow them to acquire nutrients at very limited availabilities. Restoration efforts are based on the paradigm that degraded soils need to be fertilised in order to re- establish the vegetation (Holl et al. 2000). However, fertilisation of plants from nutrient- impoverished habitats can lead to nutrient-toxicity rather than successful plant establishment 154

(Le Stradic 2012; Lambers et al. 2013). As such, restoration of campos rupestres is a very slow and expensive process and further understanding of the species dependence on the soil microbiota is needed to foster successful restoration efforts. Although a low proportion of the root system is colonised by arbuscular mycorrhizal fungi, the role of the symbiosis is not limited to mineral nutrition and their role in plant establishment and nutrient cycling needs further attention (Delavaux et al. 2017). Campos rupestres sites are also very important for water percolation and aquifer formation (Silveira et al. 2016). Conservation of campos rupestres remnants is therefore important if we want to preserve the ecosystem services provided by plants and soil in these biodiversity hotspots.

4.2.2. Within-species shifts in nutrient-acquisition and -use strategies

Leaf N: P ratios of Bossiaea linophylla occurring along all the stages of the Warren chronosequence indicated shifts from N- to P-limitation for growth along the chronosequence. Bossiaea eriocarpa, occurring only in the three oldest stages of the Jurien Bay chronosequence also presented strong-indications of P-limitation, as expected in the pedogenesis model (Walker and Syers 1976). These ratios could represent a real P-limitation for growth or only an apparent P-limitation due to the stoichiometric imbalance caused by the extra N supply of symbiotic N fixation. The two species presented extremely slow growth rates in the glasshouse, and only responded to P supply (not to N) with increased growth, indicating a real P-limitation for growth in the young seedlings grown in sand in a glasshouse.

The shifts from N to P-limitation were not accompanied by a decrease in investment in arbuscular mycorrhizal symbiosis by B. linophylla and B. eriocarpa along the chronosequences. The similarly high proportions of root colonised by arbuscular mycorrhizal fungi along the two chronosequences might be due to the protective role of the fungi against soil pathogens (Albornoz et al. 2017). I expected the two Bossiaea species to increase the investment in the P-mining strategy at low P through increased carboxylate release. However, B. linophylla had decreasing amounts of carboxylates to the rhizosphere with decreasing P supply. This means that, the main P-acquisition strategy of B. linophylla under P limitation is not via carboxylate release. Hence, morphological adaptations such as greater specific root length in B. linophylla (Lynch 2007), and greater root mass ratio in B. eriocarpa may account for P uptake at low P supply (Freschet et al. 2015). Since the growth of Bossiaea in the glasshouse experiment was extremely slow, we also believe they might rely on facilitation from neighbouring species for nutrient-acquisition in the field (Callaway and Walker 1997), but further evidence is needed to confirm this hypothesis. Finally, nodule formation was 155

inhibited when enough N was supplied in both species, a common response in legumes (Streeter and Wong 1988; Houlton et al. 2008). In conclusion, P uptake was possibly increased through root morphological adaptation when P was limiting for growth, and N was acquired symbiotically, through N-fixation and/or arbuscular mycorrhizas. The shifts in reliance on arbuscular mycorrhizal fungi with nutrient supply observed at the community level were not observed at the species level. Therefore, the species turnover along the chronosequence is determining the shifts in the relative abundance of each nutrient- acquisition strategy.

4.3. Limitations, achievements and future direction

4.3.1. Limitations i) Carboxylate-collection methods

Measuring carboxylate release into the rhizosphere can be done in several ways (Oburger et al. 2013). I tried to collect rhizosphere carboxylates released from root tips and present in their rhizosphere in the field in the campos rupestres, but the measurements may not have provided a good representation of the actual amounts released and hence the P- aquisition strategy of the host plant . Under more controlled conditions, I collected rhizosphere carboxylates from B. linophylla and B. eriocarpa, and only the former contained significant amounts, maybe due to grater consumption by the rhizosphere microbiota in B. eriocarpa (Martin et al. 2016). The measured carboxylates amounts could not be correlated with leaf [Mn] because we only collected a snapshot of carboxylates, at the end of the experiment and it probably did not reflect the total amount released during the experiment. Therefore, in order to have carboxylate measurements that reflect the species’ reliance on the P mining strategy, we would need continuous measurements such as non-destructive collections with micro-suction cups, or continuous in-situ collection methods (Oburger et al. 2013). Due to the large number of replicates in my glasshouse experiment, and the limited amount of time I had at the University of Western Australia, I preferred the more traditional methods of carboxylate collection, at the expense of potentially more informative results. ii) Diversity of fungal species in root-collected field samples

In roots of both campos rupestres plant species and Bossiaea species collected in the field, I observed very diverse fungal structures that I did not classify as arbuscular mycorrhizal fungi due to the lack of vesicles, coils or arbuscules. Some roots presented highly melanised hyphae, dark-septate hyphae, sclerotides, and several other unidentified structures. 156

Due to the large number of samples and the very time-consuming slide method of root colonisation assessment, I chose not to try to identify root-associated non-mycorrhizal fungi based on root morphology. However, I believe some of these fungi could have and important role in mineral nutrition, particularly the dark-septate fungi (Mandyam and Jumpponen 2005) and they deserve further attention. iii) Nitrogen dynamics in the campos rupestres

I investigated several aspects of P-acquisition in the P-impoverished campos rupestres, but they are also very N-poor, and very little is known about how N enters and is cycled in the system. Legumes are not very abundant in campos rupestres (Le Stradic et al. 2015); therefore, symbiotic N fixation in legumes would not be a large contribution to N input into the system. Carnivorous plants also contribute to N-entrance into the system, but are not very abundant either (Le Stradic et al. 2015). Nitrogen fixation can be conducted by epilithic and endolithic cyanobacteria and lichens (Jacobi et al. 2007; Alves et al. 2014). Symbiotic bacteria in the gut of termites are also capable of fixing N (French et al. 1976), and since termites are among the dominant fauna in tropical soils (Black and Okwakol 1997; da Cunha et al. 2006); they may represent and important N input source, as well as participate in N cycling in campos rupestres (Black and Okwakol 1997). Free-living soil (cyano)bacteria can also fix N (Bellenger et al. 2014; Mirza et al. 2014), but nothing is known about their role in severely N-poor soils of campos rupestres. Therefore, stronger quantitative data are needed to understand the extent to which each of these sources contributes to N input in campos rupestres.

4.3.2. Achievements

Despite the limitations listed above, during the course of my PhD I achieved most of my project goals. I successfully examined the extent to which plants invest C on arbuscular mycorrhizas along a gradient of soil (in)fertility in campos rupestres and I found that campos rupestres species are highly efficient in P and N use for photosynthesis, and also in P and N remobilisation. At the species level, I found that Bossiaea switch their investment into morphological root adaptations, but not in arbuscular mycorrhizal colonisation, in response varying P supply. In these species, carboxylate release was not the strategy used for P acquisition at low P. Therefore, I found different patterns for mineral nutrition at the community level in campos rupestres from that at the species level in kwongan, revealing the importance of studying mineral nutrition and underlying mechanisms at different scales. 157

4.3.3. Future direction

In view of the limitations of my project discussed above, and further questions that appeared during my PhD that were not included in my goals, I suggest that further work is needed to understand:

 How do plant roots release carboxylates and what are the best sampling methods that give a definite value for the plant reliance on carboxylate-associated P mining under field conditions?  What are the non-mycorrhizal fungi colonising roots of native plant species: are they saprophytic, pathogenic, parasitic, and do they contribute to nutrient-acquisition?  What is the contribution of different sources of N input in campos rupestres?  What is the relationship between rhizosheath formation and nutrient- acquisition?  What is the role of plant-associated and free-living microorganisms in ecosystem functioning in campos rupestres, including those inside the rhizosheaths?  How do plant-soil feedbacks influence plant community assembly in campos rupestres?

4.4. Conclusions

I found an important shift in nutrient-acquisition strategies both at the community and species level along long-term soil development in retrogressive systems. I observed a convergent shift in the decrease of the proportion of mycorrhizal species with increasing P- impoverishment between campos rupestres in Brazil and kwongan vegetation in south- western Australia (Chapter 2). Campos rupestres species presented very efficient nutrient- remobilisation and photosynthetic nutrient-use efficiency, especially for P, in accordance with the stronger P- than N-limitation for productivity in these systems. At the species level, I found that at low P availability, root morphological adaptations of B. linophylla and B. eriocarpa appeared to be more important than symbiotic strategies for P uptake. The extra N- source from nitrogen fixation increased leaf [N] relative to leaf [P], leading to an apparent P limitation for growth in the studied legumes. Taken together, the findings on the strong association between plant and soil traits, leading either to the observed patterns of species turnover at the community scale (Le Stradic et al. 2015; Zemunik et al. 2015; 2016), or to phenotypic plasticity in nutrient-acquisition and -use traits at the species level provide 158

important support to understand how different mechanisms underlying plant species distribution operate at different scales along soil gradients.

4.5. References

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ANEXO I Documento referente a Bioética e/ou Biossegurança

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ANEXO II Declaração direitos autorais