Plant Soil DOI 10.1007/s11104-015-2574-6

REGULAR ARTICLE

The southern South American , coccineum exhibits intraspecific variation in growth and cluster-root formation depending on climatic and edaphic origins

Alejandra Zúñiga-Feest & Mabel Delgado & Angela Bustos-Salazar & Valeria Ochoa

Received: 12 December 2014 /Accepted: 22 June 2015 # Springer International Publishing Switzerland 2015

Abstract in from the north versus from the south. CR in Background and aims (CR) functioning seedlings from Curacautín (38°) were suppressed when has been studied mainly in Proteaceae species from P supply increased, though this was not seen in - (P)-deficient old soils. However, in southern South lings from Coyhaique (45°). America, six species occur in young P rich soils. The Conclusions Results suggest local root adaptation relat- aims were: i) to study the growth and CR formation of ed to both climatic and edaphic conditions. We hypoth- seedlings from populations esize that these features could favor Proteaceae persis- contrasting in edaphic and climatic conditions and, ii) tence in southern South American ecosystems. to study the effect of P availability on CR formation. Methods Seedlings were grown from collected Keywords Cluster roots . Phosphorus . Plasticity. from nine Chilean populations of E. coccineum (36° to Volcanic soils 45° S). After 9 months in a nursery, CR formation and growth were determined. Additionally, seedlings from the two populations at the extreme ends of the distribu- Introduction tion were maintained on sand and watered with nutrient solutions including or excluding P. Cluster roots (CR) have been described as dense Results All seedlings showed CR formation at 4 months clusters of fine rootlets found around a main axis old; however, CR allocation differed in that it was lower (Purnell 1960). These structures are often found in the Proteaceae family (Lambers et al. 2015; Responsible Editor: John Hammond. Lamont 1982; Skene 2000). Compared with non- cluster roots, CR have improved nutrient acquisi- * : : A. Zúñiga-Feest ( ) M. Delgado V. Ochoa tion; these structures increase the surface area Laboratorio de Biología Vegetal, Instituto de Ciencias Ambientales y Evolutivas, Facultad de Ciencias, Universidad available to absorb nutrients (Lambers et al. Austral de , Valdivia, Chile 2002, 2006) and exude large amounts of carbox- e-mail: [email protected] ylates, phosphatases (Gilbert et al. 1999;Neumann et al. 1999; Redel et al. 2008) and/or proteases A. Bustos-Salazar Escuela de Graduados, Facultad de Ciencias Forestales y (Paungfoo-Lonhienne et al. 2008; Schmidt et al. Recursos Naturales, Universidad Austral de Chile, Valdivia, 2003). The main factor inducing CR formation and Chile its concomitant carboxylate and acid phosphatase exudation is P deficiency (Shane and Lambers A. Bustos-Salazar Center for Climate and Resilience Research (CR), Santiago, 2005). This root adaptation is particularly impor- Chile tant in old climatically-buffered, infertile Soil landscapes (Hopper 2009) and also, in young vol- Donoso 2006) in which Olsen P ranges from 0.37 to canic soils. This latter type of environments, which 44 mg kg−1dry soil (Souto et al. 2009). Additionally, contain high total P but low P availability can be genetic differences among E. coccineum populations found in southern (Delgado et al. were reported by Souto and Premoli (2007) using iso- 2014; Lambers et al. 2012; Zúñiga-Feest et al. enzyme variation and morphology. These authors 2010). identified four groups: (i) north, (ii) central high eleva- The morphology, physiology, and ecological impor- tion, (iii) central low elevation and (iv) south. However, tance of CR have been studied mainly in Proteaceae to date, the root features of E. coccineum from various from southwestern and South Africa (Lambers regions have not been investigated nor have possible et al. 2006; Lamont 2003; Shane et al. 2004a). In con- correlations with P availability in soils or seeds been trast, knowledge about the ecophysiology and physiol- tested. ogy of CR from South American Proteaceae remains Recently, Delgado et al. (2014)proposedthatCR scarcely explored. However, there are some descriptions biomass allocation could be related to soil mineralogy. of CR for avellana Mol. (Grinbergs et al. 1987; Therefore, we sought to determine if CR size and bio- Ramírez et al. 1990) and recently the functioning and mass distribution in the root system of E. coccineum possible ecological role of CR for Embothrium seedlings from different populations, growing in the coccineum J. R. Forst.et G. Forst has been studied same conditions, exhibit equal or different responses to (Delgado et al. 2013, 2014; Lambers et al. 2012;Piper CR formation. Furthermore, we hypothesized that et al. 2013;Zúñiga-Feestetal.2010). E. coccineum seedlings from populations with low soil Unlike in southwestern Australia where there are P availability, growing under the same greenhouse con- ≥600 Protecaeae species (Pate et al. 2001), in Chile, ditions, would exhibit higher CR biomass than seedlings only six Proteaceae species occur: G. avellana, E. from populations with high soil P availability. In addi- coccineum, ferruginea Cav. R. Br., L. hirsuta tion, we hypothesized that E. coccineum seedlings from Lam., L. dentata Ruiz et Pavon R. Br. and populations with different genetic groups, growing un- myrtoidea Poepp. & Endl (Donoso 2006; Prance and der different nutritional conditions, would show differ- Plana 1998). Embothrium coccineum persists under di- ent responses in CR formation. The aims of this study verse environmental conditions and has a wide distribu- are (a) to evaluate the growth and CR formation in tion in South American temperate forests including recently emerged seedlings of E. coccineum growing seasonal forests, humid temperate rain forests, and Pat- under common nursery conditions, using seeds from agonian subantarctic forests in Chile and contrasting environments across different latitudes (Armesto et al. 1996;Pujana2007). Embothrium (36°–45°S) that differ in climate and edaphic conditions coccineum in Chile is found from 35° to 56° S and from (P soil availability, annual rainfall, and temperature), 0 to 1200 m (Rodriguez et al. 1983;Romero1986). This and (b) to study the effect of P supply on CR formation endemic small evergreen grows from the Mediter- using seedlings from distinct genetic groups inhabiting ranean region, with high temperatures in the summer, to different climatic and edaphic conditions. colder and drier areas in the Austral zone. The annual mean temperature and precipitation of the entire distri- bution range of E. coccineum is from 5 to 15 °C and Material and methods from 400 to >4000 mm per year, respectively (Donoso 2006; Pujana 2007). Embothrium coccineum has small Seeds and soil sampling seeds dispersed by wind; however, most seeds fall close to the maternal tree (Rovere and Premoli 2005) and Embothrium coccineum seeds were collected from Feb- germination rates are highest under full sun conditions ruary to March of both 2009 and 2013, from nine (Figueroa and Lusk 2001). Furthermore, E. coccineum populations along a latitudinal gradient in Chile shows a high colonizing ability especially in disturbed (36°71′–45°72′ S). These populations include the four areas such as volcanic deposits (ash and scoria), glacial genetic groups described by Souto and Premoli (2007): moraines, and roadsides (Alberdi and Donoso 2004; (i) north: Chillán, Concepción; (ii) central high eleva- Grubb et al. 2013; Veblen and Ashton 1978). It also tion: Curacautín; (iii) central low elevation: Loncoche, grows under diverse edaphic conditions (Alberdi 1995; Lanco, Puerto Montt, Chiloé; and (iv) south: Plant Soil

Coyhaique. Climatic and edaphic conditions of each 20 °C and the minimum temperature was 8 °C. location were extracted from Novoa et al. (1989). Soil The average photosynthetically active radiation samples were taken at each site, and chemical analyses fluctuated between 615 and 404 μmol photons were done in the laboratory of soil science of the Faculty m−2 s−1. of Agricultural Sciences, Universidad Austral de Chile (Table 1). Germination rate

Total P concentration in seeds The germination rate for each population was recorded over the course of 4 months. Mean germination rate per Because Proteaceae species frequently accumulate P in population and germination velocity index (IVG) were seeds when grown in P deficient soils (Groom and calculated as follows: IVG=P1/T1+P2/T2…….Pn/Tn, Lamont 2010), the P content in seeds of E. coccineum with Pi corresponding to the number of seeds germinat- from the different populations was determined. This was ed and Ti corresponding to the time interval (Latsague done colorimetrically using the phospho et al. 2010). antimonylmolybdenum blue complex method described by Drummond and Maher (1995), with some modifica- Cluster root (CR) formation and biomass distribution tions. Briefly, 0.5 g of leaf was ground, ashed at 550 °C in E. coccineum seedlings from different populations and digested three times using an acid mixture (H2O/ HCl/HNO3; 8/1/1). Subsequently, 10 mL of the acid Seedlings of each population were maintained at mixture was added to the ash, and allowed to stand for greenhouse conditions for 8 months from August 10 min. This mixture was then filtered and transferred to 2012 to March of 2013. These months correspond a volumetric flask filled to 50 mL of distilled water. to the seasonal growth period, which occurs in Then, 1 mL was blended with a mixed reagent: sulfuric spring and summer in Chile (Zúñiga-Feest et al. acid (5 M), ammonium molybdate tetrahydrate (0.038 2009). Pots containing seedlings were distributed M), potassium antimony tartrate (0.004 M) and a reduc- randomly in the greenhouse using a balanced ex- ing solution of ascorbic acid (0.3 M) in distilled water. perimental design. At the beginning and the end of The blue color formation of the complex was spectro- the experiment, 10 seedlings of each population photometrically measured at 700 nm, and Pi was were selected randomly to determine relative calculated. growth rate (in height and biomass) and biomass distribution. For this purpose, seedlings were har- Plant production and growth conditions vested and divided into parts containing only , stem, CR, and non-CR. Then, samples Four groups of 20 seeds per population were se- were dried at 60 °C using an oven (Venticell lected randomly to determine the fresh weight per MMM, Poland) for 48 h. Subsequently, the dried seed. The remaining seeds of each population were samples were weighed. The proportion of CR with maintained in gibberellic acid (250 mg L−1) solu- respect to total root weight and the ratio of CR/ tion for 24 h and subsequently washed with dis- total biomass were calculated; the number of CR tilled water and maintained in Petri dishes in a was also recorded. growth chamber at 20 °C for 2 weeks. Following this, all seeds were transferred to a 200 cm3 con- Nutritional supply tainer with composted pine bark. Then, the seeds were watered gently with distilled water to main- In order to study the effect of nutritional supply on tain field capacity. Seed germination under green- growth and CR formation, we focused on two geneti- house conditions was recorded over the course of cally distinct populations from contrasting soil origins 4 months. The conditions in the greenhouse and latitudinal distributions (Curacautín, 38°S and (39°73′ S) were monitored using a HOBO sensor Coyhaique, 45°S). For this purpose, 48 seedlings of (pendant temperature/Light Data Logger 64 k-UA- similar developmental stage (number of leaves) per 002-64), and the maximum recorded temperature population were selected randomly and were maintained from August to November was approximately on sand for 2 months under greenhouse conditions in a Plant Soil ) 1 − balanced experimental design. Then, four groups of kg

+ plants were separated (n=12) and watered weekly for 3 months (July to September) with different nutrient solutions as follows: full nutrient Hoagland solution (H), P deficient Hoagland solution (H-P), P excess Hoagland solution (H++P) and distilled water (W). The chemical composition of the Hoagland solution

(H) contained: Ca (NO3)2 (5 mM), KNO3 (5 mM), MgSO4 (2 mM), KH2PO4 (1 mM) and FeEDTA (1 mM). The H-P solution contained the same nutrients, d 2013. Soil samples were collected from except that it did not contain KH2PO4; K was incorpo- rated as KCL (1 mM). H++P was the same as the

) N mineral Extractable Al (cmol Hoagland solution but with the addition of 10 mM 1 − KH2PO4. The W group was watered with only distilled water without nutrients. At the end of the experiment, morphological parameters including height, total bio- mass, number of CR per plant, proportion of plants with CR, and CR allocation, were evaluated. )

1989 Statistical analysis

seeds were collected during 2010, 2011, an To assess differences among populations, one- and two- way ANOVA, Tukey tests, Contingency tables (chi- square), and Pearson correlation coefficients were ap-

(°C) Elevation (m) P Olsen (mg kg plied. Variables and parameters were as follows: P con- tent in seeds, height and biomass of seedlings, and ratios of shoot/root and cluster root/root. Two way ANOVA atic data from Novoa et al. ( and Tukey tests were used for determining germination Embothrium coccineum velocity kinetics (factors: population and days), the number of cluster roots, and the proportion of shoot / root and CR / root total (factors: population and nutrient solution). Contingency tables (chi-square) were used to compare the proportion of plants with CR from two contrasting populations (Curacautín and Coyhaique) t determined. Edaphoclim and from differing nutritional treatments. To assess the relationship between the variables of edaphic and cli- nfall (mm) Annual mean temperature matic conditions (annual mean temperature, annual rain- s from different populations where fall, P and N soil availability) and the P concentration in seeds, we calculated Pearson correlation coefficients.

Results

Edaphic parameters from different populations where Embothrium coccineum seeds were collected

Geographic and edaphic parameter The phosphorus availability (Olsen P) of the soil of all populations ranged from 2 to 27.4 mg kg−1 (Table 1). ChillánConcepciónCuracautín 36° S, 36°Loncoche 73° S, W 71° W 38°Lanco S, 71° W 1330 1383Valdivia 39° S, 72° W 2552Puerto MonttChiloé 2139 39° S,72° W 41° 39° S,72° S,73° W WCoyhaique 1942 1890 2532 45° 42° 13.2 S,72° S,73° 14.0 W W 9.7 1032 1942 12.5 10.4 10.9 12.2 10.4 7.2 4 985 728 196 9.8 ND 27.4 69 192 2 ND 190 300 6.8 3.4 3.8 18.9 21 2.1 10.5 4.4 ND 0.07 0.04 ND 0.04 70 ND ND 23.8 0.12 10.5 1.39 0.22 0.16 0.03 Populations Coordinates Annual rai the 20 cm upper soil profile, after litter layer. ND = No Table 1 Curacautin represented the population with the lowest Plant Soil values of P content in the soil. Mean annual tempera- value after 30 days (Fig. 1C). In contrast, seeds from tures of all populations ranged from 7.2 to 14 °C and Coyhaique started to germinate after 70 days and were correlated with latitude decreasing from the north reached the highest germination rate after 6 months to the south (r=−0.8, P<0.001). Annual rainfall ranged (data not shown). Germination velocity was significant from 1032 to 2552 mm with Coyhaique being the driest correlated with annual mean temperature (r=0.8, location and Curacautín being the wettest location. P<0.001) but not with annual rainfall (r=0.25, P= 0.18) or P seed concentration (r=0.26, P=0.20) Variation in weight, P concentration, and P content in seeds of E. coccineum from different populations Relative growth rate and CR formation of E. coccineum seedlings from different populations Seed weight was significantly different among populations. Chillán was the population with the After 6 months, significant differences in the rela- lowest seed weight and Puerto Montt had the tive growth rate in height (RGRh) and biomass highest seed weight. The phosphorus concentration (RGRb) of the plants were found. Values of RGRh ranged from 4.2 (Lanco) to 5.6 mg kg−1 ranged from 0.015 to 0.02 mm mm−1 day−1 and (Coyhaique; Table 2) and was weakly correlated RGRb ranged from 0.016 to 0.023 mg mg−1 day−1. with latitude (r=0.4, P=0.03), mean annual tem- Seedlings from Curacautín showed the lowest perature (r=−0.44, P=0.02) and annual rainfall values in both indices (Fig. 2). Both RGRh and (r=−0.4, P=0.04); seed weight and P concentra- RGRb were uncorrelated with P seed content (r= tion in seeds were not correlated with soil P avail- −0.03;P=0.85 and r=−0.1; P=0.55, respectively) ability (Olsen P) (r=0.02, P=0.88 and r=−0.1, P= and neither were correlated with seed weight (r= 0.65, respectively). 0.11; P=0.34 and r=0.001; P=0.99, respectively). Individual CR biomass decreased with latitude, Germination rate of E. coccineum seeds from different however this trend was not significant (r = populations −0.3; P=0.007). Two-month-old seedlings did not show significant Significant differences in germination rates of seeds differences in CR formation (Fig. 3A). However, from different populations were found to be correlated eight-month-old seedlings showed significant differ- with the latitudinal gradient (r=−0.6, P=0.001). In gen- ences in mean CR number per plant (20 to 45; eral, all populations started to germinate about 1 month Fig. 3C). All E. coccineum seedlings showed CR; how- after planting. Seed germination rate was approximately ever, the percentage of CR of total plant dry biomass 80 % after 4 months. Though, for the Coyhaique popu- differed significantly among populations and ranged lation (45°S) the germination rate was only 40 % after from 3.5 to 21 %, being significantly lower in northern 4months(Fig.1I). Seeds from Curacautín showed the populations (Concepción and Curacautín) than in south- highest germination velocity reaching the maximum ern populations. On the other hand, the dry weight of

Table 2 Seed weight and phosphorus (P) concentration of Embothrium coccineum from different populations. Each value represents the mean±SE (in parentheses), n=4. Different letters indicate significant differences among populations (P≤0.05). ND = Not determined

Populations Seed weight (mg) Seed P (mg g−1)Pcontent(μg P fresh seeds)

Chillan 10.5 (0.3) c ND ND Concepción 12.4 (0.1) ab 5.2 (0.0) ab 93.2 (2.7) a Curacautín 15.5 (0.7) b 4.5 (0.2) c 77.9 (4.0) ab Loncoche 14.0 (0.3) ab 4.4 (0.1) c 65.9 (4.1) b Lanco 13.2 (0.2) b 4.2 (0.2) c 67.7 (0.7) b Puerto Montt 16.9 (1.1) a 4.8 (0.1) bc 94.5 (8.3) a Chiloé 13.4 (0.6) b 4.7 (0.2) bc 83.2 (7.7) ab Coyhaique 16.1 (0.3) ab 5.6 (0.1) a 105.3 (2.5) a Plant Soil

Fig. 1 Mean germination rates of Embothrium coccineum seeds± each graph capital letters show significant differences between standard errors. Seeds collected from different populations are as populations at the same time, lower case letters indicate significant follows, A Chillán, B Concepción, C Curacautín, D Loncoche, E differences during the germination period for each population (P≤ Lanco, F Valdivia, G Puerto Montt, H Chiloé, and I Coyhaique. In 0.05) individual CR ranged from 5 to 20.5 mg. However, both populations, a high frequency of plants with there were no significant differences among populations CR was observed though the proportion of plants although there was a tendency of lower values in south- with CR was different even within the same treat- ern populations (Table 3). ment; seedlings from Curacautín showed the highest In all populations, the frequency of plants with CR frequency under the W treatment (83 %), while showed an increase according to the age of the seed- Coyhaique seedlings had the highest frequency of lings: two-month-old seedlings showed frequencies of CR under the H-P treatment (83 %). In contrast, the 5–30 %, four-month-old seedlings had 80–100 % plants lowest frequency of seedlings with CR was observed with cluster roots, and all seedlings in all populations in Curacautín under H treatment (17 %), whereas had CR at 8 months (Fig. 3). seedlings from Coyhaique had slightly decreased cluster root formation under this treatment and had Effect of phosphorus on cluster root formation a high frequency of plants with CR even under the in seedlings from two populations H++P treatment (58 %) (Table 4). The shoot/root ratio of seedlings differed signif- Seedlings from Curacautín and Coyhaique showed icantly depending on the treatment and origin of significant differences in CR formation when they the seeds (Table 4). Curacautín had higher values were treated with different nutrient solutions. In than Coyhaique especially under H treatment. Plant Soil

Fig. 2 Relative growth rate in height±standard error (A)and Concepción, CUR: Curacautín, LON: Loncoche, LAN: Lanco, relative growth rate in biomass±standard error (B)ofEmbothrium VA: Valdivia, PM: Puerto Montt, Chi: Chiloé and COY: coccineum seedlings. Seedlings were produced using seeds col- Coyhaique. In each graph minor letters show significant differ- lected from different populations shown as follows, CO: ences between populations (P≤0.05)

Mean number of CR per plant was similar be- mortality when watered with excess P to the extent tween populations, being the lowest under the H that it was not possible to determine morphologi- (Curacautín) and the H++P (Coyhaique) treatments cal parameters (growth, shoot/root ratio, CR/root (Table 4). Seedlings from Curacautín showed high biomass) under this treatment (Table 4,NC).

Fig. 3 Mean cluster root (CR) number per plants±standard error PM: Puerto Montt, CHI: Chiloé and COY: Coyhaique. In each (A, B, C) and percentage of seedlings of Embothrium coccineum graph minor letters show significant differences between popula- having CR (D, E, F). Seedlings were produced using seeds collected tions (P≤0.05). Graphs A and D have different scales on the By^ from different populations shown as follows, CO: Concepción, axis CUR: Curacautín, LON: Loncoche, LAN: Lanco, VA: Valdivia, Plant Soil

Table 3 Cluster root* proportion of total plant dry biomass (%)±SE and mean cluster root biomass±SE in eight-month-old seedlings of Embothrium coccineum from different populations growing under same greenhouse conditions. SE in parenthesis. ield capacity, Minor letters shows significant differences among populations at the same development stage (P<0.05)

Populations % Cluster roots of total Dry weight of individual plant dry biomass Cluster roots (mg) nt dry biomass (CR/TDW) in Concepción 3.4 (0.6) c 20.5 (7.9) a Curacautín 11.2 (2.8) b 10.5 (4.6) a of P. NC = Not calculated owing to low Loncoche 21.7 (3.0) a 7.2 (2.1) a Lanco 11.3 (1.3) ab 19.4 (5.4) a Valdivia 18.4 (3.4) ab 11.7 (1.6) a

Puerto 15.8 (2.8) ab 6.2 (1.6) a ion as ratio of total pla Montt

Chiloé 12.5 (1.7) ab 5.1 (2.2) a g 3 months (July to September), watering at f Coyhaique 15.1 (2.6) ab 6.0 (1.7) a

*Total cluster roots, including senescent cluster roots nd cluster roots allocat

Discussion

Intraspecific variation in germination rate of E. coccineum seeds from different populations land full nutrient solution; H++P: Hoagland with excess Being highly dormant, the seeds that originated from the population with the lowest mean annual temperature, annual rainfall, and P availability (Coyhaique) showed the lowest germination rate. As is widely known, dor- <0.05)

mancy varies in the long term (ecotypes or clines) or in P ngs were maintained under greenhouse conditions durin the short term (seed maturation environment) in several species (Fernández-Pascual et al. 2013). Considering that Coyhaique has the coldest environment and the oot ratio, mean number of CR per plant (No.CR), a most genetically isolated population (Bustos 2011; Ferrada 2009), the breaking of seed dormancy observed in the Coyhaique population grown under a common garden experiment (greenhouse 39°S) was the slowest as was expected. This pattern has been reported at broad geographical scales where seeds from higher altitude sites (cold environments) are positively correlated with cant differences among treatments ( higher dormancy in several species (Cavieres and Ar- royo 2000;Holm1994). Additionally, the Coyhaique atments as follows: W: Water; H-P: Hoagland without P; H: Hoag and Punta Arenas populations of E. coccineum have seedlings, from contrasting populations. Seedli increased rates of germination when they are exposed to winter stratification in both natural and greenhouse conditions (Piper F. and Ramírez F.; personal commu- nication). Recently, Fernández-Pascual et al. (2013) Percentage of plants with cluster roots (CR), shoot/r have suggested that local adaptation of an endemic WH-PHH++PWH-PHH++P 83 33 NC 17 75 83 58 75 1.1(0.2)b 3.2(0.5)a NC 4.3 (0.5)a 1.6(0.3)b 1.9 (0.2)b 2.3(0.3)ab 2.6 (0.2)a 2.2 (0.6)a 0.9 (0.5)b 0.2 (0.2)b NC 1.6 (0.4) 1.9 (0.5) 0.7 (0.2) 1.7 (0.4) 0.2 (0.1) 0.1 (0.1) 0.01 (0.1) NC 0.1 (0.2) 0.1 (0.1) 0.1 (0.0) 0.1 (0.0) Curacautín Coyhaique Nutrient treatment % plants with CR Shoot/Root No. CR CR/TDW survival. Minor letters shows signifi with different nutritional tre Embothrium coccineum mountain-inhabiting species from Spain can explain Table 4 Plant Soil dormancy differences among genetically isolated to differences in light intensity and temperature than that groups. Our results suggest that in cold environments previously reported by Delgado et al (2014). Both max- such as in Coyhaique, some seeds need winter stratifi- imum light intensity and temperature in our experiments cation to overcome dormancy in order to successfully were 500 μmol photons m2 s−1 and 20 °C. This is 1.5 grow. Timing of seed germination is critical for the times lower than that reported in the experiment carried survival of natural populations (Lambers et al. 2008). out during spring-summer in Australia 2012–2013 It is likely that dormancy plays a major role in the (Delgado et al. 2014). germination timing of the Coyhaique population in con- In all populations, the frequency of seedlings trast to northern populations where complete germina- with CR and the mean number of CR per plant tion can occur after 4 months. increased during the course of our experiment, such that all four-month-old seedlings had CR Intraspecific variation of E. coccineum seed weight (Fig. 4). From this result, we suggest that CR and P concentration formation is an early event in the life of seedlings and is probably key to plant Overall, the mean P concentration of seeds from all of establishment in natural conditions. Piper et al. the Chilean Proteaceae are significantly lower (3.2± (2013) reported a similar finding that small 0.4 mg g−1 DW) (Delgado et al. 2014) than the mean E. coccineum seedlings (≤1yearold)hadhigher P concentration often found in seeds from Proteaceae CR numbers than older seedlings (>1 year old) species of South Western Australia and South Africa under natural conditions. To date, it is yet un- (13.2±0.8 and 5.8±0.8 mg g−1 DW, respectively known how long CR formation occurs in (Groom and Lamont 2010)). E. coccineum; however, adult plants (around In addition, Proteaceae seeds from Chile are small 40 years old) growing under natural conditions in and have low P content. G. avellana, (Chilean young volcanic soils in southern Chile have been ) is one exception to this with higher values reported to have abundant CR formation (Personal of P content most likely due to its larger seed size in observation M. Delgado). comparison to other Chilean Proteaceae seeds (Delgado Our findings show differences in several features of et al. 2014). This finding indicates that recently emerged the root systems of seedlings that came from disparate seedlings, such as those found in Chile, have low P populations inhabiting various edaphic and climatic reserves at the time of germination in low P availability conditions; these responses included the frequency of soils compared with seeds from Proteaceae growing in CR production, CR allocation and CR mean number per extremely P poor soils, such as in Australia. plant. In general, Chilean Proteaceae such as E. coccineum, L. ferruginea and L. dentata, have small Intraspecific variation in growth and CR formation CR (≤ 1 cm) and also seem to have lower biomass of E. coccineum allocated to CR (5–10 %). In comparison, seedlings from other studies had much larger CR and CR biomass, Because E. coccineum has a wide latitudinal range in such as those grown under hydroponic conditions Chile, we initially hypothesized that CR formation and (Delgado et al. 2014; Zúñiga-Feest et al. 2014), and the proportion of CR to total plant dry biomass would be those of the southwestern Australian Proteacaeae higher in seedlings from populations with low soil P (SWA), such as prostrata R.Br. (Shane et al. availability. Our results agree in part with this idea, as 2004a, b)orGrevillea crithmifolia R.Br. (Shane and CR/plant biomass was significantly lower in seedlings Lambers 2006). In SWA species, cluster roots are re- from Concepción, which had the highest soil P avail- stricted to the upper layer of the soil, where nutrients are ability. In general, growth rates were similar between more abundant (Lamont 2003). In contrast, Chilean populations and were slightly lower than those reported Proteaceae growing on volcanic depositions in temper- by Delgado et al. (2014)forE. coccineum growing in ate regions frequently encounter abundant organic mat- hydroponic conditions and for grandis Willd. ter and thin litter layers in the soil profile (Bertrand and (Australian Proteaceae) growing in soil over a period of Fagel 2008; Tosso 1985). Because of this, it is possible 63 days (Barrow 1977). It is possible that the slow that the cluster roots of Chilean species can explore growth rates observed in our experiment could be due deeper horizons than SWA Proteaceae. In fact, Lambers Plant Soil

Fig. 4 Representative pictures of recently emerged seedlings produced from seeds collected in Curacautín (a) and Coyhaique (b). Plants were maintained under greenhouse conditions, growing in sand and watered with distilled water as is described in BMaterial and methods^.Theback scale bar represents 1 cm, both seedlings shows cluster roots indicated by red arrows

et al. (2012) found that cluster roots of mature concentrated mainly on the east side of the Andes in G. avellana can reach up to 1.5 m in depth. Argentina (Naranjo and Stern 1998). It is possible that volcanic eruptions differentially impact southern South Effect of phosphorus on cluster root formation America and have influenced soil development on re- in E. coccineum seedlings from two origins gional scales. This in turn could allow for root adapta- tion in Proteaceae species. However, these hypotheses In our experiment, seedlings from Curacautín seeds have not yet been evaluated systematically but they suppressed CR formation under H; however, seedlings could inspire future research. from Coyhaique seeds maintained CR formation The observed suppression of CR formation under H (Table 4). In addition, the shoot/root ratio of seedlings treatment in seedlings from Curacautín could be an from Curacautín was higher than that of seedlings from indication of local adaptation related to the minimization Coyhaique when both were exposed to full nutrient of carbon costs involved in CR formation and exuda- treatments. Plants of E. coccineum from Coyhaique tion. The differences in sensitivity to P excess could be form part of the South genetic group described by related to genetic differences of these populations and/or Souto and Premoli (2007) which is characterized by could be due to the differences in climatic and edaphic being more geographically and genetically isolated than conditions of each area. However, further evaluation is other populations. In fact, using chloroplast and nuclear needed to determine the extent to which E. coccineum is markers (Bustos 2011;Ferrada2009), some studies locally adapted to soils with differences in nutrient have found that this South group is the most genetically availability. differentiated population. Low soil nutrient availability has been reported as an Coyhaique has a lower annual mean temperature and evolutionary driver in Proteaceae from Mediterranean annual rainfall than Curacautín. Furthermore, these two environments. The low nutrient availability in soils is locations differ in terms of their volcanic input. thought to influence P absorption mechanisms (Shane Curacautín is located between two of the most active et al. 2004b). As such it has been shown in some volcanoes, Villarrica and Llaima, both of which have species (e.g. H. prostrata) which evolved in very P erupted several times per decade in recent history poor soils have a low capacity to down regulate P (Dzierma and Wehrmann 2010). In contrast, Coyhaique uptake, and experience P toxicity even at low P soil is a region with a lower density of volcanoes. Conse- levels (Shane et al. 2004b). In contrast, other Proteaceae quently, this region historically had a lower nutrient species, such as crithmifolia, that grow in input from volcanic ash and also ash deposits have been relatively P richer soils in SW Australia (region Plant Soil characterized by extremely P impoverished soils) show ancestors are thought to have originated during the early a strong capacity to down-regulate P uptake (Shane and Paleocene (65–56 Mya) (Johnson and Briggs 1975). Lambers 2006). Then Embothrium coccineum continued to evolve dur- In our experiment, seedlings from Curacautín grow- ing the Eocene (45 Myr) (Barker et al. 2007) and expe- ing under the H++P treatment exhibited symptoms of P rienced the uplift of the Andes Mountains around 23 toxicity with the consequent death of seedlings. This is Mya. Following this, E. coccineum endured the intense most likely because the P concentration used in the volcanic activity during the Pliocene and experienced experiment was too high for this tree species (1 mM the glacial conditions of the Pleistocene (Heusser 1990; P). Delgado et al. (2014), showed that 6 months old Romero 1986;Villagrán2001). Under this evolutionary E. coccineum seedlings (produced from seeds collected scenario, species with high plasticity such as at 40° S) can shed leaves when they are exposed to high E. coccineum, could be highly successful and could Plevels(10–250 μM nutrient solutions). After shedding persist under diverse and dynamic conditions produced their leaves, these plants were able to survive through by natural perturbations. In contrast, in southwestern their down-regulated P uptake by producing new leaves Australia and South Africa, where the highest with low P concentration. However, in our experiments Proteaceae species diversity is found, the soils are seedlings from Curacautín died when they were watered weathered and infertile because these regions have had with excess P, and then they were not able to regrow. It is geological and climatic buffered conditions (Hopper probable that these small-sized seedlings had little car- 2009; Lambers et al. 2010). Also, these environments bon reserves for re-growth after being exposed to these with Mediterranean climate have been warmer and drier high P level causing irreversible damage. An additional than regions in the central-south of Chile. These differ- explanation for the contrasting results between both ences in both soil and climate could explain why experiments could be that in our case, the seedlings were Proteaceae species in different regions of the world grown from July to September (winter), however differ in diversity and plasticity. In contrast to that seen Delgado et al. (2014) measured their seedlings from in southwestern Australia and South Africa, in southern November to January (spring to summer). Larger differ- South America there are few species of Proteaceae but encesingrowthratehavebeenreportedinE. coccineum some of them have high plasticity. In summary, this in warmer than in cooler seasons (Escobar et al. 2006; study provides the first evidence of intraspecific varia- Zúñiga-Feest et al. 2009). tion in CR formation of E. coccineum, aspecieswhich evolved in highly disturbed environments along broad Plasticity in the South American Proteaceae geographic, climatic and edaphic conditions.

The high colonizing capacity of several Proteaceae spe- Acknowledgments The authors would like to thank the Chilean cies in southern South America, such as E. coccineum Science Council FONDECYT grants N° 1130440 and 11080162 (Zúñiga-Feest A.) for support this research. Besides, we give and L. hirsuta (Alberdi et al. 2009;Segura-Uauy1999) thanks to FONDECYT Postdoctoral research N°3150187 and has been discussed mainly in relation to photosynthetic CONICYT N° 21140737 (PhD scholarship)/CONICYT/ features; however, few studies have focused on root FONDAP 15110009 for funding these two young researchers: adaptations. Our results reveal that: i) E. coccineum Delgado M. and Bustos-Salazar A., respectively. Also, we would like to thank Dr. Hans Lambers and Dr. Peterson who provided seedlings from different populations growing at the important comments to improve this manuscript. Finally, we thank same latitude (39° 73′ S) showed differences in CR Viveros Bosques del Sur, Instituto de Ciencias Ambientales y allocation, size and formation and ii) seedlings from Evolutivas for greenhouse facilities, Alejandro Vera, Dra. Frida populations of contrasting latitude and temperature Piper, and Dr. Luis Corcuera for seed collection facilities in the field. (Curacautín and Coyhaique) showed different responses to nutritional treatments. These results confirm that E. coccineum root growth could be related to genetic identity and this could in turn indicate that some degree References of local adaptation is occurring at the root level. The high plasticity found in E. coccineum popula- Alberdi M (1995) Ecofisiología de especies leñosas de los tions is potentially due to the ancestral history of the bosques hidrófilos templados de Chile:Resistencia a la Proteaceae family in South America where the clade sequía y bajas temperaturas. In: J Armesto, C Villagrán, Plant Soil

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