Gevuina Avellana and Lomatia Dentata, Two Proteaceae Species

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Gevuina avellana and Lomatia dentata, two Proteaceae species from evergreen temperate forests of South America exhibit contrasting physiological responses under nutrient deprivation

A. Zúñiga-Feest & F. Sepúlveda & M. Delgado & S. Valle & G. Muñoz & M. Pereira & M. Reyes-Diaz

Received: 9 December 2019 /Accepted: 13 July 2020 # Springer Nature Switzerland AG 2020

Abstract survival, physiological performance, and adjustment re- Aims Gevuina avellana and Lomatia dentata are garding cluster root (CR) formation and carboxylate Proteaceae species from evergreen temperate forests, exudation rate than L. dentata. but only G. avellana can colonize nutrient deprived Methods We evaluated relative growth rate, maximal volcanic depositions. We hypothesized that under nutri- photosynthetic rate, photochemical performance, nitro- ent deprivation, G. avellana would present higher gen (N) and phosphorus (P) leaf concentrations, specific leaf area, photosynthetic P and N use efficiency (PPUE Responsible Editor: Hans Lambers. and PNUE), CR formation and carboxylate exudation rate. Plants were grown in a greenhouse, using recent Electronic supplementary material The online version of this volcanic substrate and watered with different nutrient article (https://doi.org/10.1007/s11104-020-04640-y)contains supplementary material, which is available to authorized users. Hoagland (H) solutions: modified full Hoagland, without P, without N, or tap water. * : : : A. Zúñiga-Feest: ( ) F. Sepúlveda M. Delgado Results Both species showed high survival. G. avellana G. Muñoz M. Pereira exhibited higher growth rates (RGR), and higher CR Laboratorio de Biología Vegetal, Instituto de Ciencias number and biomass allocation under nutrient depriva- Ambientales y Evolutivas, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile tion, but lower carboxylate exudation rates than e-mail: [email protected] L. dentata. Malate, oxalate and succinate were detected : : in root exudates of G. avellana but only oxalate in A. Zúñiga-Feest F. Sepúlveda S. Valle L. dentata. However, PPUE and PNUE were higher in Centro de Investigaciones en Suelos Volcánicos, CISVo, Universidad Austral de Chile, Campus Isla Teja s/n, Valdivia, L. dentata than in G. avellana . Chile Conclusions Our hypothesis was not entirely accepted, : CR formation was more “constitutive” in G. avellana, M. Delgado M. Reyes-Diaz and composition of carboxylates was more diverse, with Departamento de Ciencias Químicas y Recursos Naturales, Facultad de Ingeniería y Ciencias, Universidad de La Frontera, lower exudation rates than L. dentata. Moreover, Temuco, Chile L. dentata showed higher PNUE and PPUE, partly : explained by thinner leaves. Different responses are M. Delgado M. Reyes-Diaz related with edaphic conditions, where both species Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus inhabit: more diverse to G. avellana and restricted to (BIOREN), Universidad de La Frontera, Temuco, Chile developed soils to L. dentata.

S. Valle Instituto de Ingeniería Agraria y Suelos, Facultad de Ciencias Keywords Carboxylate exudation . Cluster roots . Agrarias, Universidad Austral de Chile, Valdivia, Chile Gevuina avellana . Lomatia dentata . Nutrient Plant Soil deprivation . South American Proteaceae . Volcanic structures that live around 20–30 days and exude car- depositions boxylates to the soil, effectively enhancing the acquisition of poorly available nutrients, especially mineral- bound inorganic and organic P (Lambers et al. 2006). Introduction The carboxylates released by CRs replace both organic and inorganic P adsorbed to soil particles, increasing the In an evolutionary context, South American Proteaceae concentration of P in the soil solution. Malate and citrate species have been considered outliers of the main cen- are the carboxylates most frequently released by CRs ters of distribution around the world (Prance and Plana (Delgado et al. 2014). Both have been reported in 1998). In fact, South Western Australia and South Afri- Proteaceae originating from ancient soils in South West- ca represent the highest diversity Proteaceae species ern Australia and South Africa (Shane et al. 2003; Shane sites worldwide. Currently eight genera of the and Lambers 2005), and in some southern South Amer- Proteaceae family inhabit South America. Euplassa, ican Proteaceae species such as Embothrium coccineum Panopsis and Roupala are the most widely distributed J.R. Forst. & G. Forst. and Orites myrtoidea (Delgado genera in South America, growing mainly in tropical et al. 2013, 2014; Ávila-Valdés et al. 2019). Moreover, areas. In contrast, Orites, Gevuina, Embothrium and CRs are induced by low P availability in several species Lomatia are distributed in southern South America in from different families such as Lupinus albus the cold, temperate areas of Patagonia from 30° S to 55° (Fabaceae), Myrica gale L. (Myricaceae), Hippophae S (Delgado et al. 2019). These genera grow mainly in rhamnoides L. (Elaeagnaceae), Grevillea robusta A. soils with higher total nutrient content than those from Cunn. Ex R. Br. and Hakea laurina R. Br. Central South America (de Britto et al. 2015;Hayes (Proteaceae) (Shane and Lambers 2005; Crocker and et al. 2018), except for Orites myrtoidea Poepp. & Endl., Schwintzer 1993). It is widely accepted that P limitation which grows exclusively in young skeletal-rocky soils induces CR formation, however the effect of N, an derived from volcanic depositions located in few popu- essential nutrient which is commonly less available in lations in the Andes Mountains (Pfanzelt et al. 2008; young volcanic soils (Gallardo et al. 2012; Piper et al. Hechenleitner et al. 2005). South American Proteaceae 2013), has not been studied under controlled conditions show contrasting ecophysiological traits compared to and neither considering the CR formation and function those of South Western Australia (Lambers et al. 2012; in southern South American Proteaceae species. In some Delgado et al. 2014; Hayes et al. 2018; Delgado et al. cases, a higher N supply under P limitation improves 2018). These differences include higher phosphorus (P) root growth and CR formation as found in Hippophae and nitrogen (N) concentration in leaves, lower nutrient rhamnoides (Shah et al. 2015), and a study of Crocker remobilization, lower photosynthetic use efficiency of P and Schwintzer (1993) showed that a specific chemical and N (Lambers et al. 2012; Delgado et al. 2018), and N source, urea more than nitrate, enhanced CR forma- thinner leaves (Torres 1998; Denton et al. 2007). How- tion in Myrica gale. In southern South America CR ever, it is unknown how southern South American formation was negatively correlated to soil N concen- Proteaceae could adjust these traits when grown under tration, but not to P concentration in austral differing nutrient availabilities. E. coccineum populations (Piper et al. 2013), suggesting All Proteaceae members, except for the genus that low N availability in soils could be a relevant factor Persoonia (Lamont 1982), have a root adaptation called for CR formation in this Proteaceae species. However, cluster roots (CRs), which is part of a mechanism to no experimental data sustaining the effect of N on CR improve mineral nutrition in soils with low nutrient formation have been provided yet for South American availability (Skene 1998;Lamont2003; Lambers et al. Proteaceae. 2008). In fact, these CRs were described for the first All southern South American Proteaceae species time, by Purnell in Purnell 1960, and later several au- have CR (Ramírez et al. 1990; Lambers et al. 2012; thors reported different aspects of their physiology and Zúñiga-Feest et al. 2010; Delgado et al. 2015;Ávila- ecology, focusing on the nutritional role of these root Valdés et al. 2019). These species inhabit a wide range structures in species that naturally grows on ancient soils of soil nutritional conditions but are commonly found in with low fertility (Lambers et al. 2011; Muler et al. soils of volcanic origin (Delgado et al. 2018). Volcanic 2014; Zemunik et al. 2015). The CRs are ephemeral soils (Andisols or andic Inceptisols) are characterized by Plant Soil a high total P content, but low availability (Borie and (Ramírez et al. 2004; Delgado et al. 2019). Additionally, Rubio 2003; Redel et al. 2008; Delgado et al. 2018) both species show contrasting individual CR size; compared to soils from south Western Australia Gevuina avellana has the largest CR recorded for south- (Lambers et al. 2012). In fact, some of these southern ern South American Proteaceae (Ramírez et al. 2004; South American Proteaceae are recognized as pioneers Zúñiga-Feest et al. 2014), whereas L. dentata has the in Chile and Argentina in sites with volcanic depositions smallest ones (Delgado et al. 2015). The carboxylate including Embothrium coccineum, Gevuina avellana composition and exudation rates of their CRs have not Mol., Lomatia hirsuta Lam. (Donoso 2006)andOrites yet been evaluated for either species. Moreover, it is myrtoidea (Ávila-Valdés et al. 2019). Lambers et al. unknown how these species adjust their CR formation (2012) proposed that southern South American and functioning when grown under different soil nutri- Proteaceae could act as “ecosystem engineers”,dueto ent availabilities. their low remobilization of nutrients from senescent We hypothesize that G. avellana will shows higher leaves, which increases nutrient content in the leaf litter, survival, relative growth rate, photochemical perfor- thereby increasing nutrient availability for other species. mance, photosynthetic nutrient use efficiency of N and Recently, Ávila-Valdés et al. 2019showed that P (PNUE and PPUE, respectively) and higher carbox- O. myrtoidea and E. coccineum can increase the P ylate exudation rates under nutritional deprivation treat- availability (Olsen-P) in their rhizosphere when grown ments than L. dentata, due to its capacity to colonize in pumice, a recent volcanic substrate, supporting in part recent volcanic depositions with low N and P availabil- the ecosystem engineer’s hypothesis of this Proteaceae ity for plants. Additionally, G. avellana will exhibit species but through a belowground nutrient enrichment. higher adjustment of CR formation in response to soil Additionally, Piper et al. (2019) partially agrees with nutrient availability and higher allocation to CR bio- this positive role of Proteaceae, because higher leaf P mass, especially when grown under low nutrient avail- concentration has been found in leaves of Acaena ability, than L. dentata.Therefore,theaimofthisstudy integerrima (Rosaceae) when grown with was to assess the effect of different nutrient availabilities E. coccineum seedlings in volcanic substrate from the on physiological performance, CR formation and car- Hudson volcano (Piper et al. 2019). However, their boxylate exudation of G. avellana and L. dentata grown ecosystem engineer role has been questioned with other under controlled conditions, and determine if this phys- studies, because remobilization of P and N in iological performance could be related to edaphic distri- E. coccineum leaves was higher compared with remo- bution of both species. bilization in leaves of coexisting species (Fajardo and Piper 2015). Gevuina avellana and Lomatia dentata are two Materials and methods southern South American species growing in similar latitudes in Chile, from 35° to 45° S and 32° to 42°S Seed collection, processing and maintenance of plant respectively, in Mediterranean (dry – humid) to temper- material ate climates and being part of evergreen temperate forests (Delgado et al. 2019). Gevuina avellana is a semi- Seedlings of G. avellana and L. dentata were produced shade tolerant species that sometimes acts as a pioneer from seeds collected in Valdivia, Region of Los Ríos, species, especially in recent volcanic depositions, in the Chile (39°38’ Sand73°5’ W), and grown under green- south of Chile (Donoso 2006). In contrast, L. dentata is house conditions in a mixture of volcanic sand and a shade-tolerant species that grows mainly in secondary perlite (1:1), from March to October of 2015, when temperate forests with rich soils and high humidity seedlings were transferred to 2 L pots with a substrate (Donoso 2006); and, as far as we know, it is absent on of volcanic sand. The sand was collected from the recent volcanic substrates. Specific information about Mocho Choshuenco volcano deposition (39°54’ S- chemical composition of the soils where both species 72°02’ W) in February 2015. This volcanic sand has naturally grows are scarce, being G. avellana associated an estimated age of 150 years old, considering the last with a wider range of nutrient variation (N from 14 to eruption in this area (Rawson et al. 2015). This volcanic 47 mg kg−1; P from ˂ 2 to 6.8 mg kg−1; organic matter material was chemically characterized in the laboratory content from 0.04% to 16%), comparing with L. dentata as follow: mineral N (mg kg−1): 23.8; P-Olsen Plant Soil

−1 (mg kg ); 2.1; pH (H2O): 6.8; total exchangeable bases CR/total plant biomass ratios were calculated. In addi- (cmol+ kg−1): 0.027; Al saturation (%) 6.94. tion, total number of CR in each seedlings for treatment and the frequency of plants with CR were calculated at Experimental conditions the end of the experiment.

The duration of the experiment was 6 months during the growing season of the southern hemisphere, from Oc- Photosynthesis and maximum quantum yield tober 15th of 2015 to April 15th of 2016, in the green- of photosystem II (Fv/fm) house of the Universidad Austral de Chile, Valdivia. Temperatures were recorded using a HOBO Pendant Before the harvest of the seedlings, maximum photo-

Temperature/Light Data Logger, and ranged from 8 °C synthetic rate (Amax) and maximum quantum yield of to 30 °C, with an average temperature of 20.8 °C. The photosystem II (Fv/fm) were determined. To determine average daily light intensity was 300 μmol photons Amax, we used an infrared gas analyzer (ADC-LCA4 m−2 s−1, fluctuated between 530 and 200 μmol photons Analytical Development Co., Hoddesdon, UK). These −2 −1 m s . Treatments consisted of 4 different nutrient determinations were made at ambient CO2 concentra- availability conditions: a) modified full Hoagland nutrition (≈360 ppm), outdoor temperature (16–20 °C) and ent solution (H), b) modified Hoagland solution without 250 μmol photons m−2 s−1. For this, a fully expanded phosphorus (H-P), c) modified Hoagland solution with- leaf of 5 seedlings were measured for each nutritional out nitrogen (H-N) and tap water (W). For each treat- treatment and species. Before these measurements, we ment, 9 seedlings of each species were randomly select- determined light saturation curves for photosynthesis for ed. Modified full Hoagland nutrient solution (H), was both species where, the maximum photosynthetic re- −2 −1 prepared as follow (μM): 400 Ca (NO3)2,200K2SO4, sponse was around 250 μmol photons m s . 54 MgSO4,100KH2PO4, 10 Fe-EDTA, 0.3 Na2MoO4, For photochemical performance we measured Fv/ 0.1 ZnSO4,0.02CuSO4,2.4H3BO3, 20 KCl. For mod- fm using a pulse-amplitude modulated fluorimeter ified Hoagland without P (H-P), KH2PO4 was not (FMS 2, Hansatech Instruments Ltd., U.K.). In brief, added, and for modified Hoagland without N (H-N), minimal fluorescence (Fo) was measured in attached Ca (NO3)2 was not added (Hoagland and Arnon 1950; leaves, dark-adapted for 25 min before measure- Zúñiga-Feest et al. 2015). The pH of the nutrient solu- ments, of four seedlings per nutritional treatment tions was ≈ 5.5 and substrate pH ranged from 5.1 to 5.8 per species. After that, the leaves were illuminated during the experiment. The solutions were applied twice by a short pulse (0.8 s) of 8000 μmol s−1 m−2 light per week to each pot as needed; plants were additionally to obtain the maximum fluorescence (Fm). The Fv/ watered with tap water to maintain water availability. fm ratio was then calculated (where Fv =variable fluorescence (Fv = Fm − Fo)andFm =maximum Biomass and growth rate fluorescence). This parameter is widely used as an indicator of environmental stress in plants. An opti-

Relative growth rate for height (RGRh) was calculated mal value is around 0.83, and lower values indicate from plant height measurements at the beginning and damage of photosystem II (Maxwell and Johnson end of the experiment. Relative growth rate for biomass 2000; Souza et al. 2004).

(RGRb) was determined for ten seedlings per species. The RGRb was calculated using the difference of log10 of dry biomass at the end and beginning of the experi- Specific leaf area (SLA) determinations ment, using the formula described by Barrow (1977). At the end of the experiment the seedlings were harvested The specific leaf area (SLA) was determined using the and separated into stems, leaves, non-cluster and cluster same fully expanded leaves where Amax was measured roots, and eachorgan was dried in an oven at 60 °C before. The area of each leaf was calculated using a LI- (MMM Medcenter, Einrichtungen GmbH Venticell, COR Model LI-3100 Area Meter, and dry weight was Germany) for 48 h or until constant dry weight. Then, determined as previously described. The values of SLA samples were weighed using an analytical balance were calculated as the ratio of leaf area to leaf biomass (RADWAG AS220-C2, Poland). The shoot/root and for each treatment and species (n =6). Plant Soil

Chemical analysis of leaves, N/P ratio Statistical analysis and photosynthetic nutrient use efficiency calculations To determine significant differences among species The mature leaves from each nutritional treatments and nutritional treatments, and possible interaction and species were washed with distilled water and among factors, we used two-way ANOVA and dried at 60 °C, for 48 h. The dried samples were Tukey test (p < 0.05) with the program ground and used to analyze N and P concentrations. STATGRAPHICS Centurion XVI.I. Correlations The P concentration was determined by spectropho- were made between specific leaf area (SLA) and tometry using the vanadophosphomolybdate meth- PPUE; also between SLA and PNUE values, for od, previously samples calcination and N concentra- each species and treatments. tionwasdeterminedbyaciddigestionandKjeldahl distillation. All methods are described by Sadzawka et al. (2004). The nitrogen (PNUE) and phosphorus Results (PPUE) photosynthetic use efficiency per unit leaf were calculated using the measurements of Amax, Survival and growth rate SLA and N and P concentration in mature leaves as described by Delgado et al. (2018). The ratio of N/P Survival rate was 90% to 100% for both species under concentrations in leaves was used as an indicator of all nutritional treatments. The highest RGRb was N or P limitation, where values below 14 are indic- reached with the modified Hoagland solution (H) and ative of N limitation and values over 16 indicate P H-P for G. avellana (5.3 to 5.1 mg g−1 day−1 respec- limitation. Values of N/P ratio between 14 and 16 tively) and with modified Hoagland solution for indicate that plant growth is equally limited by N L. dentata (6.5 mg g−1 day−1), being these values statis- and P (Güsewell 2004; Koerselman and Meuleman tically different between species (p < 0.05) (Table 1). 1996). Additionally, L. dentata showed significant differences

in RGRb values among treatments, being around three Collection and identification of root exudates times higher under full nutrient treatment (H) compared with W, and around two times higher than H-N and H-P. The collection of root exudates was conducted using However, G. avellana only showed significant differ- intact, whole root systems of transpiring plants for each ences in RGRb when grown with H-N and W compared nutritional treatment and species. Before the harvest, to H and H-P, and this reduction was smaller than the root system of each seedling were carefully separated reduction observed in L. dentata (Table 1). Values of from the substrate, gently washed using running water RGRh were in general similar between species, but and then were submerged in 0.25 mM of CaSO4 solu- significant differences under H treatment were ob- tion (18–20 °C as above) and shacked softly for 2.5 h. served, being RGRh higher to L. dentata than After this period, solutions were filtered through a G. avellana. When both species were watered without

0.2 μm membrane filter, and stored at −20 °C. Then, N (H-N and W), RGRh was significantly reduced the samples were lyophilized and dissolved in 600– (Table 1). Shoot/root ratio was higher in L. dentata than 800 μL of deionized-sterile water and filtered again G. avellana comparing under the same treatment and (0.22 μm) for HPLC injection. The chromatographic significant different values among treatments for both separation was performed in an HPLC system (Jasco species were observed (Table 1). The highest value was LC-Net II/ADC) equipped with a photodiode array de- observed in seedlings of L. dentata watered with H-P tector (DAD) (Jasco MD 2015 Plus) and a Sphere (3.08 ± 0.2) and the lowest in G. avellana grown in W Column Heater according to Meier et al. (2012). The (0.63 ± 0.08). flow rate was 1 mL min−1 and injected samples were detected at a wavelength of 210 nm. Identification of Specific leaf area, photosynthesis rate and fluorescence organic anions (oxalate, malate, citrate, iso-citrate and of PSII succinate) was determined by comparing retention times and by comparison with standards for each organic acid Leaves of G. avellana showed specific leaf area (SLA) anion. values around half that of L. dentata, independent of Plant Soil

Table 1 Relative growth rates of biomass (RGRb) and height differences (p < 0.05) between species; lowercase letters indicate (RGRh), shoot/root ratio and frequency of plant with CR for both significant differences (p < 0.05) among nutritional treatments. species and nutritional treatments. Values of means ± standard Values replaced with (*) are less than 0.01 error (SE) are shown. Capital letters indicate significant

−1 −1 −1 −1 Species Nutritional treatment RGRb (mg g day )RGRh (mm mm day ) Shoot/Root Ratio Plants with CR %

Gevuina avellana H5.3±*Ba 0.2 ± * Ba 1.69 ± 0.1 Ba 100 H-N 4.0 ± * Ab 0.1 ± * Ab 0.83 ± 0.4 Bb 100 H-P 5.1 ± * Aa 0.2 ± * Aa 1.61 ± 0.2 Ba 100 W4.2±*Aab 0.1 ± * Bc 0.63 ± 0.1 Bb 100 Lomatia dentata H6.5±*Aa 0.3 ± * Aa 2.46 ± 0.4 Ab 22 H-N 2.5 ± * Bb 0.1 ± * Ac 1.44 ± 0.3 Ac 75 H-P 2.6 ± * Bb 0.2 ± * Ab 3.08 ± 0.2 Aa 100 W2.6±*Bb 0.1 ± * Ac 2.84 ± 0.3 Aa 88

nutritional treatment (Table 2). Leaves of L. dentata maintained with H (Table 2). The maximum quantum showed differences among treatments, where H-P yield of PSII (Fv/fm) had no significant differences showed the highest value, and the lowest one under H among species or nutritional treatments, with an average treatment (143 ± 11 and 103 ± 21 cm2 g−1,respectively). value of 0.7 (Table 2). However, there were no differences for SLA of G. avellana seedlings between nutritional treatments CR formation, biomass allocation and individual CR (p >0.05). dry weight The response of maximal photosynthetic rate (Amax) to nutritional treatments was different among species Seedlings of G. avellana formed CR in all plants under (Table 2). Maximal photosynthetic rate was higher for all nutritional treatments (100%). However, L. dentata both G. avellana and L. dentata when watered with H, showed a high frequency of plants with CR in treat- and this variable was only affected (p < 0.05) by nutrient ments without nutrients (100–75%) and a low frequency limitation (H-P, H-N, W) for G. avellana. For under full Hoagland treatment (22%) (Table 1). Differ- G. avellana Amax was around four times lower in seed- ences among seedlings of both species were observed in lings maintained with W treatment than seedlings the total number of CR per plant and the CR/total

Table 2 Maximum photosynthetic rate (Amax), maximum quan- differences (p < 0.05) between species; lowercase letters indicate tum yield of photosystem II (Fv/fm) and specific leaf area (SLA) significant differences (p < 0.05) among nutritional treatments. of both species under nutritional treatments. Values of means ± Values replaced with (*) are less than 0.01 standard error (SE) are shown. Capital letters indicate significant

−2 −1 2 −1 Species Nutritional treatment Amax μmol CO2 m s Fv/fm SLA cm g

Gevuina avellana H 3.7 ± 1.3 Aa 0.75 ± 0.0 Aa 62 ± 6 Ba H-N 2.0 ± 0.6 Bb 0.67 ± 0.9 Aa 54 ± 4 Ba H-P 3.2 ± 0.4 Ba 0.75 ± 0.1 Aa 66 ± 4 Ba W 1.0 ± 0.4 Bb 0.79 ± * Aa 53 ± 3 Ba Lomatia dentata H 4.1 ± 0.7 Aa 0.76 ± * Aa 103 ± 21 Ab H-N 3.3 ± 0.2 Aa 0.76 ± * Aa 121 ± 6 Aab H-P 4.0 ± 0.7 Aa 0.76 ± * Aa 143 ± 11 Aa W 3.9 ± 0.9 Aa 0.62 ± 0.1 Aa 126 ± 10 Aab Plant Soil

1a). Similar number of CR per plant were found among 50 treatments for G. avellana, except in H treatment, which (a) Aa Aa was slightly lower. However, L. dentata showed signif- 40 Aa Aa icantly higher average CR number per plant in seedlings Ab 30 maintained with W, compared to the other treatments, and under H treatment the lowest number of CR per 20 Bb Bb plant were detected, being observed only in two seed- 10 lings. The CR/total plant biomass ratio ranged from 20 Total CR per plant to 40% in G. avellana, being the lowest for H and H-P 0 * HH-NH-PW and the highest for W and H-N (Fig. 1b). Lomatia Lomatia dentata Gevuina avellana dentata showed a very small variation in this ratio, ranging from 1 to 8%, being the highest in seedlings 0.6 maintained with W treatment. Additionally, G. avellana (b) Aa tio

a showed higher individual CR dry weight compared with Aa L. dentata in all treatments (24 to 71 mg and 0.33 to 0.4 0.5 mg, respectively, Fig. 1c). Ab Ab 0.2 Ba Carboxylate exudation: Rate and composition /Total biomass r Bb CR * Bb 0.0 Significant differences for carboxylate composition and HH-NH-PW exudation rates were observed among species and treat- Lomatia dentata Gevuina avellana ments (Fig. 2). Oxalic, malic and succinic acids were detected in the root system of G. avellana and only 100 Aa ) (c) oxalic acid in that of L. dentata. The rate of carboxylate -1 80 exudation was higher in L. dentata than G. avellana, Ab 60 especially when seedlings were watered with H-N or H- Ab 40 Ab P. Seedlings of L. dentata showed the highest carbox-

ass (mg CR 20 ylate exudation rate when were watered with H-P

om −1 −1 i 1.0 μ Ba Ba Ba (205.8 mol g h oxalate). G. avellana seedlings 0.5 showed more similar carboxylate exudation rates among CR b 0.0 * nutritional treatments, being succinate exuded at the HH-NH-PW μ −1 −1 Lomatia dentata Gevuina avellana highest rate (27 mol g h ), and then oxalate (5 μmol g−1 h−1). Malate was also detected when these seedlings were watered without N, but its value did not − − Fig. 1 a Total cluster root (CR) number per plant, b CR/total exceed 1.4 μmol g 1 h 1 (Fig. 2). biomass ratio and (c) individual biomass CR biomass in seedlings of Gevuina avellana and Lomatia dentata growing in different nutritional treatments. Biomass values correspond to dry weigh Leaf nutrient concentrations and photosynthetic nutrient (DW). Values shown the mean ± standard error (SE) (n =9).In use efficiency L. dentata in H treatment (*) value represent the mean CR number from only two seedlings that showed CRs, and was not considered Both species showed differences (p <0.05)forleafP in the ANOVA. Capital letters indicate significant differences (p < 0.05) between species; lowercase letters indicate significant concentration among treatments. Phosphorus concentra- −1 differences (p < 0.05) among nutritional treatments for each specie tion in leaves ranged from 0.2 to 0.5 mg P g DW in L. dentata and 0.4 to 0.6 mg P g−1 DW in G. avellana (Table 3). In L. dentata, the highest values were found in biomass ratio (Fig. 1). Gevuina avellana showed higher H treatment, being statistically different from other treat- numbers (p < 0.05) of mean CR per plant than ments (p < 0.05). However, G. avellana seedlings L. dentata, except under W treatment. The mean CR showed the highest leaf P concentration in seedlings per plant in G. avellana ranged from 24 to 35, whereas watered with H-N. lomatia dentata and G. avellana in L. dentata it ranged from 2 to 27 CR per plant (Fig. showed different leaf N concentration for each Plant Soil

Fig. 2 Carboxylate exudation rates on FW basis of Gevuina 250 avellana and Lomatia dentata Aa seedlings grown with different 200 nutritional treatments. Average

) 50

values ± standard error (SE) are -1 shown (n = 6). Capital letters in- Ba

udation rate 40 dicate significant differences x FW h A A (p < 0.05) among treatment for -1 30 each organic acid within each species; lowercase letters indicate 20 B significant differences (p <0.05) (molg Ca between species for each organic C 10 Cb Bb Ab acid and nutritional treatments Da Organic acids e Db 0 HH-NH-PW HH-NH-PW Gevuina avellana Lomatia dentata

Oxalic Malic Succinic

nutritional treatment (Table 3).Thevaluesrangedfrom in L. dentata (Table 3). Lomatia dentata showed the 1.5 to 6.7 mg N g−1 DW in L. dentata and from 4.5 to highest values when seedlings were watered with H-N 10.5 mg N g−1 DW in G. avellana.ValuesofleafN and H-P (PPUE) and the lowest in H, being the highest concentration for L. dentata were about three times PNUE value under H-N. (Table 3). lower (p < 0.05) in seedlings watered without N and W compared to seedlings watered with H treatment; and N/P ratio under different nutrient treatments around two times lower for G. avellana under similar conditions. PPUE and PNUE were higher (p < 0.05) in Differences (p < 0.05) were observed among species L. dentata than G. avellana, in all nutritional treatments. and treatments for N/P ratio. When both species were PPUE in G. avellana was around four times lower in W watered with H-N, the N/P ratios were ≤ 10 and when treatment respective to H treatment (Table 3). The mean was watered with H-P, the N/P ratio values were ≥ 24, − − values of PPUE ranged from 12 to 60 μmol C g 1 Ps 1 indicating N and P limitation, respectively (Fig. 3). In − − in G. avellana and from 65.7 to 121.6 μmol C g 1 Ps 1 seedlings maintained with the full nutrient treatment,

Table 3 Phosphorus (P) and nitrogen (N) concentrations in (SE) are shown. Capital letters indicate significant differences leaves. Photosynthetic phosphorus use efficiency (PPUE) and (p < 0.05) between species; lowercase letters indicate significant photosynthetic nitrogen use efficiency (PNUE) of both species differences (p < 0.05) among nutritional treatments. Values re- under nutritional treatments. Values of means ± standard error placed with (*) are less than 0.01

Species Nutritional [N] leaf mg N g−1 [P] leaf mg P g−1 DW PPUE μmol C g−1 Ps−1 PNUE μmol C g−1 Ns−1 treatment DW

Gevuina H 10.5 ± 0.7 Aa 0.4 ± 0.0 Aab 49.0 ± 6.7 Ba 1.9 ± 0.2 Bb avellana H-N 4.5 ± 0.6 Ab 0.6 ± 0.1 Aa 21.3 ± 3.6 Bb 2.7 ± 0.4 Ba H-P 9.4 ± 1.7 Aa 0.4 ± * Abc 60.4 ± 9.0 Ba 2.9 ± 0.7 Ba W5.4±0.7Ab 0.5 ± * Ab 12.1 ± 2.3 Bc 1.2 ± 0.3 Bc Lomatia dentata H6.7±1.3Ba 0.5 ± 0.1 Aa 65.7 ± 5.6 Ac 5.5 ± 0.8 Ab H-N 1.5 ± 0.1 Bb 0.2 ± * Bb 121.6 ± 4.9 Aa 13.5 ± 2.3 Aa H-P 7.5 ± 0.3 Ba 0.3 ± * Bb 118.4 ± 0.9 Aa 3.9 ± 0.4 Ab W2.4±*Bb 0.3 ± * Bb 80.0 ± 3.4 Ab 9.3 ± 3.6 Aab Plant Soil

et al. 2014), as well as a high content of protein (Medel 2001). Therefore, seed nutrient provision can help to sustain the growth of G. avellana seedlings, thus explaining the higher leaf N and P concentrations in G. avellana watered with nutrient deficient solutions (Table 3). Moreover, the features of G. avellana seeds mentioned before can also support the colonizing capacity of this species in recent volcanic substrates. There are no information about G. avellana seeds nutrient content collected from trees growing on different edaphic conditions, however size variation have been reported comparing populations (Donoso 1978), and in all cases, seed size is higher than L. dentata seeds size. Fig. 3 Mean N/P ratio calculated from nitrogen and phosphorus In contrast, L. dentata seedlings are found frequently concentration of the leaves of seedlings of Lomatia dentata (black in secondary evergreen forests with more developed circles) and Gevuina avellana (grey squares) maintained in differ- soils and high organic matter content (Donoso 2006) ent nutritional treatments. Value corresponds to mean ± standard error (SE) (n = 10) Horizontal lines indicate limitation by N and, as far as we know, the species is absent from (values <14) or limitation by P (values >16), or equal limitation recent volcanic substrates. Other explanation could by both N and P (values 14–16), for detailssee Materials and be that G. avellana seedlings had higher root absorp- Methods. Capital letters indicate significant differences (p <0.05) tion capacity than L. dentata, because their CR rep- between species; lowercase letters indicate significant differences (p < 0.05) among nutritional treatments within each species resent higher surface of interaction with the substrate and/or higher absorption capacity per root biomass. The volcanic sand used in our experiment had low G. avellana had values >16, indicative of P limitation, nutrient content; then the nutrients added by and L. dentata around 14, indicative of equal limitation watering, represented the main provision to seed- by N or P. When seedlings were watered with W treat- lings. Additionally, the volcanic sand used in our ment, the N/P ratios were lower than 14 for both species, experiment has around three times lower plant avail- indicative of N limitation (Fig. 4). able water capacity than an organic load develop soil (Table 1, Supplementary material). Probably, G. avellana’s root system had higher efficiency to Discussion acquire nutrients than L. dentata, considering the high drainage of the substrate used. Gevuina avellana and Lomatia dentata showed high Considering that G. avellana can live under full survival rates in all treatments, as well as relatively high sun light conditions, and L. dentata is a shade toler- vitality as inferred from Fv/fm values, suggesting that ant species (Donoso 2006), contrary to our expecta- both species seedling can survive in nutrient poor vol- tions, values of Amax were lower in G. avellana than 2 −1 canic substrates. However, significant differences in in L. dentata (2.4 and 3.8 μmol CO2 m s , respec- relative growth rates were observed, where L. dentata tively) while leaf P and N concentrations were in was more strongly affected by nutrient limitation than general higher in G. avellana. Thus, the calculated G. avellana. In agreement with part of our hypothesis, PPUE and PNUE values were lower for G. avellana G. avellana showed higher relative growth rate of bio- than L. dentata. The PPUE and PNUE found in mass under nutritional limitation (H-N, H-P and W) than seedlings of G. avellana and L. dentata were lower L. dentata;butL. dentata showed significantly higher than those reported for adult trees of G. avellana and biomass growth rates than G. avellana under H treat- L. dentata growing under natural conditions ment. These different responses in growth between spe- (Delgado et al. 2018). This result was probably due cies can be related to their initial nutrient seed provision. to reduced light intensity in the greenhouse. In fact, In fact, G. avellana has the largest seed size among the in our experiment, the light saturation point was southern South America Proteaceae species and has fifty lower than values reported by Castro-Arévalo et al. times higher seed P content than L. dentata (Delgado (2008) and by Delgado et al. (2018)forbothspecies Plant Soil a) b) c)

d) e) f)

h) g)

Fig. 4 Pictures of Gevuina avellana and Lomatia dentata. a) G. avellana seedling. e) Microscopic cross section of L. dentata seedling showing entire root system. b) G. avellana G. avellana leaf. g) L. dentata leaf, different colored bars shows sapling growing on recent volcanic deposition close to Osorno width (yellow), palisade parenchyma (white) and epidermis with volcano, south of Chile (see white arrow in the center of the cuticle (red), scale left bottom side 50 μm. f) cluster root of picture). c) G. avellana root system, showing different cluster root L. dentata and h) cluster root of G. avellana showing “claviform” development stages, red line highlight mature cluster root. d) structures of each rootlet, f and h have the same scale

−1 −1 growing under full sun light. Both species showed H-N or H-P, being 122 and 118 μmol CO2 g Ps , significant differences in PPUE and PNUE among respectively. nutritional treatments, however L. dentata, showing Differences in the leaf anatomy between the studied higher values of PPUE when seedlings were watered species here could explain this low value of PPUE and Plant Soil

PNUE. In fact, G. avellana have slightly thicker leaves world (Elser et al. 2007; Güsewell 2004;vonOheimb than L. dentata (Fig. 4), which could reduce carbon et al. 2010). We used this N/P ratio as a proxy to study diffusion and assimilation rate and consequently lower nutrient limitation effect in our experiment, using seed- photosynthetic use efficiency of both nutrients. Under lings without nutrient provision. According to the N/P our experimental conditions, specific leaf area values ratios established by Koerselman and Meuleman (1996), were higher than those reported by Delgado et al. (2018) L. dentata was more frequently limited by N than and by Lusk (2002). In this regard, L. dentata watered G. avellana, even when it was watered with full with different nutrient solutions exhibited higher SLA Hoagland solution. To better understand nutrient limita- plasticity than G. avellana,specificallyL. dentata seed- tion in our seedlings, we used reaction norm of growth lings showed 40% higher SLA under H-P treatment, rate under different types of nutrient limitation (N or P), compared with H (Table 2). or co-limitation describe by Harpole et al. (2011). These Low photosynthetic rates have been explained by results shows that L. dentata was co-limitated by N and low leaf N concentrations (Wright et al. 2004), however P, in opposition to G. avellana that showed a “serial in our experiment, even under N deprivation, photosyn- limitation” by N (Supplementary material, Fig. 1). thetic rates for L. dentata were similar to seedlings G. avellana showed a positive response on RGRb when watered with full nutrient solution (Table 2). Under was supplemented by N, under H and H-P treatments; nutrient deprivation, L. dentata maintained a very low however L. dentata had a positive response in RGRb growth rate with few expanded leaves (Tables 1 and 2, only when was maintain in H (both N ad P provision) respectively), probably as a strategy to maintain enough (Supplementary Material, Fig. 1).Basedinourresults, N for photosynthetic rate by unit of leaf surface and to Lomatia dentata has multiple nutrient requirements, in sustain a positive carbon balance at plant level and comparison with G. avellana, and this features could survive. In this context, Lusk (2002)reportedthatsev- explain their predominance in more developed soils, eral evergreen tree species from southern South Amer- where L. dentata can maximize their growth to compete ica produce thin leaves (with high SLA) that help to in more diverse plant communities (Harpole et al. 2017), increase net carbon gain at the leaf and whole-plant level but as far as we know, L. dentata is absent from recent as part of a general strategy to maintain survival in volcanic substrates. In contrast, G. avellana can grow and shaded environments, conditions in which L. dentata colonize recent volcanic depositions, where probably a is normally present. minimum of N could be provided by mosses and lichens, Low SLA values have been considered part of an as pioneering species (Gallardo et al. 2012), but also can ecological strategy of Proteaceae species from South grow in more developed soils. When G. avellana grows Western Australia, involving a very slow leaf decompo- in more fertile soils probably suppress their CR forma- sition and release of nutrients to the soil, predominantly tion, this are in agreement with recent evidence of CR occurring during fires (Lambers et al. 2012). In contrast, suppression in seedlings of G. avellana,growinginsim- in temperate rain forests of southern South America, ilar greenhouse conditions, when P Olsen value was Proteaceae species have higher SLA (Lusk 2002; ≥12 ppm (Velasco 2019). Delgado et al. 2018), and leaves have higher nutrient Our experimental conditions were probably extreme- concentrations than South Western Australia Proteaceae ly poor, especially in N, because N/P ratio was ˂15 to species (Lambers et al. 2012; Delgado et al. 2018). Leaf L. dentata compared to values of 22 reported by decomposition rates of Proteaceae also seems to be Delgado et al. (2018) from leaves of adult’s trees of this faster in southern South America than in South Australia species, growing in the Coastal Mountain range. Other (Lambers et al. 2012; Vivanco and Austin 2008; values of N/P reported to temperate forest species shows Cárdenas et al. 2018). Gevuina avellana shares some frequently limitation of P in Lomatia hirsuta saplings ancestral features with Proteaceae from South Western growing in young substrates in Llaima volcano Australia such as coriaceous leaves (Jordan et al. 2008) chrosequence (N/P = 17,3) but equal limitation of N with low SLA, but L. dentata has thinner leaves as do and P to L. dentata (N/P = 14,6) (Gallardo et al. 2012), many other Angiosperms species from temperate forests being in agreement with co-limitation observed in our of Southern south America (Lusk 2002). experiment. Additionally, Diehl et al. (2003)showed The N/P ratio has been used in plant communities to that L. hirsuta was the species more P limited (N/P = study nutrient limitation in several ecosystems around the 20), compared with several Nothofagus species (N/P Plant Soil from 13 to 17), when are growing in north Patagonian by Ramírez et al. (1990), from seedlings growing in forest in the south of Argentina. The association with similar greenhouse conditions (31% of total plant bio- mycorrhiza in the case of Nothofagus species, which mass) and volcanic sand substrate from the south of help with P acquisition, have been related with low Chile. values of N/P (Hevia et al. 1999). However, values of Gevuina avellana showed a higher carboxylate exu- N/P reported by Delgado et al. (2018)inadultstreesto dation rate than L. dentata, when watered with modified the south of Chile, showed that all Proteaceae species Hoagland solution. However, L. dentata showed a very evaluated (Lomatia hirsuta, L. ferruginea, L. dentata, O. high oxalate exudation rate when watered without P, myrtoidea, G. avellana and E. coccineum)weremore these rate was four times higher than the citrate exuda- limited by P than N. Probably in this case, related with tion rate reported by Delgado et al. (2014), from high P retention in this soils, with high content of E. coccineum seedlings. Because CRs represent a rela- allophan (Borie and Rubio 2003); and probably by a tively low portion of the biomass of the root system of higher demand for growth, at their development stage. L. dentata, we can argue that this high exudation rate Very limited information of N/P ratio have been was mainly attributable to non-CR tissues. It is well reported in seedlings, to compare appropriately our re- known that both non-CR and CR can exude carboxyl- sults. However, recently Fajardo and Piper (2019)ina ates (Roelofs et al. 2001) but exudation rates are typi- similar pot experiment, showed that G. avellana seed- cally higher in CR than in non-CR. Future studies lings had very low shoot N/P values (˂ 10), when was should collect exudates from different roots structures, growing in tephra or nursery substrate. This information in order to determine how L. dentata can sustain the are in agreement with our results that showed N limita- carboxylate exudation rate detected here. tion to G. avellana, probably due high capacity of this Differences in carboxylate composition were also species, to acquire P from low available pools through found among the studied species. Gevuina avellana CR functioning, become limiting N. exuded three different carboxylates: succinic, oxalic Contrary to our hypothesis, we found that CR forma- and malic acid, whereas for L. dentata only oxalic acid tion seems to be more constitutive in G. avellana than in was detected. It has been suggested that the exuded L. dentata. However, G. avellana showed larger CR size carboxylate spectra could reflect adaptations to local than L. dentata, with significant variation in their indi- soil conditions. Citrate and malate are the most abundant vidual CR biomass depending on nutritional treatment. carboxylates in the rhizosphere, and they can effectively Instead, L. dentata had a greater number of CRs when mobilize retained P from soil (Roelofs et al. 2001). subjected to nutrient deprivation, compared with H Oxalic acid exudation was reported as the predominant treatment. Moreover, and as expected, higher values of carboxylate exuded in the South American Proteaceae CR/total plant biomass were observed in G. avellana Euplassa cantareirae Sleumer, a species able to grow in than L. dentata seedlings, reaching the highest values the acidic and seasonally flooded soils in Restinga forest when both species were grown without nutrients. Low in Brazil (de Britto et al. 2015). For this species, the role CR biomass allocation has been explained as an adap- of oxalic acid could be similar to citrate in Aluminium tation of southern South America Proteaceae species (Al)-rich soils, as Gerke et al. (2000) reported before. when grown in fertile soils. This occurs in Embothrium Therefore, it is possible to suggest that oxalate exuded coccineum, in which only 5–10% of the total plant by L. dentata could avoid Al toxicity, because iron (Fe) biomass goes to CR biomass when was watered with and Al have been reported as predominant P complex full nutrients (Zúñiga-Feest et al. 2014), or high P con- forms in the southern Chile soils (Borie and Rubio centration in hydroponic conditions (Delgado et al. 2003). In this regard, it is important to note that 2014). Comparing our results of CR biomass allocation G. avellana and L. dentata are found to grow in soils to other Proteaceae species, G. avellana showed values with high Al saturation (63%) in the Coastal Mountain similar to Banksia or Hakea, both South Western Aus- range in the South of Chile (Delgado et al. 2018)and tralian Proteaceae, for whom CR biomass could reach both species have been reported as Al- about 40–65% of total root biomass (Dinkelaker et al. hyperaccumulators species (Delgado et al. 2019). Addi- 1995;Lamont1982). In our experiment, CR biomass of tionally, oxalate exudation has been related to Al alle- G. avellana was on average 40% of total plant biomass viation, as occurs for Tithonia diversiflora (Asteraceae), (W treatment), being slightly lower the values reported where increased Al availability induces oxalate Plant Soil exudation by the entire root system (Olivares et al. volcanic originated soils. In opposition, G. avellana 2002). Carboxylates, such as oxalate and citrate, have maintains more constitutive CR formation in all nutri- been reported to increase P availability in limestone soil, tional conditions, with a higher biomass investment and in which some Australian Proteaceae species grow carboxylate diverse, but lower exudation rate. In addi- (Ström et al. 1994; Jones and Darrah 1994). tion, G. avellana seedlings sustain higher growth rate Succinic and oxalic acid exudation rates showed under nutrient deprivation, and as an adults produce big significant variations among treatments in G. avellana, seeds with higher nutrient content. These features of the highest rates reached when seedlings were watered both species, as different adaptations could be related with H and without P treatments. Succinic acid has a to their capacity to specialization for live in different high affinity for iron (Fe) (Cmuk et al. 2009), which environments, G. avellana can colonize and grow in a could cause the release of P adsorbed to Fe compounds wider geographical distribution, including in recent vol- in the soil. In L. dentata, a higher carboxylate exudation canic depositions where L. dentata is absent. However, rate was detected when watered without P, but also L. dentata showed co-limitation of N and P, that could without N, than when grown with full Hoagland solu- be related with their capacity to grow almost exclusively tion. A potential role of the exuded carboxylates could in temperate forest and more develop soils. Considering influence microbial communities in their rhizosphere. It the small proportion of the world covered by southern is well known that different species of free-living bac- South American Proteaceae and the low number of these teria can use these carboxylic acids as a carbon source species, the differences reported here are notable. (Bacilio-Jimenez et al. 2003;Baisetal.2006). A specific diversity of microbial functional groups was recently detected for E. coccineum (Sanchez-Salazar et al., Conclusions manuscript in preparation), as well as an increase of oxalotrophic bacteria in the rhizosphere of Our initial hypothesis was not entirely accepted, how- E. coccineum growing on pumice, a recent volcanic ever under nutrient deprivation Gevuina avellana substrate, when the seedlings exude only oxalate showed a higher relative growth rate, CR number and (Muñoz et al., submitted manuscript). However, further CR biomass allocation than L. dentata. Gevuina research is need it to evaluate if carboxylate exudation avellana showed CR formation in all treatments evalu- through CR could select specific microbial groups. ated; however, L. dentata suppressed it when grown Southern South American plants have evolved under under full nutrient solution. Three carboxylates were frequent volcanic activity and glaciations (Kooyman detected in G. avellana with a low exudation rate, while et al. 2014), and therefore some plants could experience only oxalate with a high exudation rate was found for episodic periods of nutrient enrichment, low P availabil- L. dentata.Finally,L. dentata showed higher values of ity and high Al saturation. The Lomatia genus is an PNUE and PPUE related to their thinner leaves. Growth ancient member of the Proteaceae (Barker et al. 2007) responses analyzed by norm of reaction, shows that and in southern South America, there are three species G. avellana was N limited and L. dentata was co- (L. dentata, L. hirsuta and L. ferruginea)thatgrowin limited by N and P. All their differences described here, contrasting environments. Lomatia dentata is the spe- are associated with the environments in which both cies with a narrowest geographic distribution compared species are currently found, G. avellana could to other Lomatia species (Donoso 2006). The genus inhabiting more diverse edaphic conditions, even as a Lomatia have three extinct species in southern South pioneer species in young volcanic substrates, and America (L. occidentalis, L. patagonica and L. dentata are more restricted to grows on developed L. preferruginea)(Pujana2007) in opposition to soils and the shade of other species in temperate forests. Gevuina, that is a monotypic genus and evolutionarily younger (Lambers et al. 2012). Thus, it is possible that Acknowledgements Financial support was provided by Fondo CR formation observed only during nutrient deprivation Nacional de Desarrollo Científico y Tecnológico de Chile in L. dentata reflects a suppression of these structures to (FONDECYT) 1130440 and 1180699 regular grants. MD thanks avoid carbon costs, over the course of their evolution, FONDECYT initiation project N° 11170368. Technical assistance from Felipe Ramírez, Ruben Mondaca in greenhouse experiments and can alternatively exude carboxylates from the entire and Ruth Espinoza on soil nutrient analysis. Also, give thanks Ms. root system to alleviate high Aluminum content in their Caroline Dallstream and Ms. Daniela Gallardo by English edition. Plant Soil

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