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Direct and indirect effects of Tillandsia recurvata on Prosopis laevigata in the Chihuahua desert scrubland of San Luis Potosi, Mexico
Journal of Arid Environments 104 (2014) 88e95
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Direct and indirect effects of Tillandsia recurvata on Prosopis laevigata in the Chihuahua desert scrubland of San Luis Potosi, Mexico Alejandro Flores-Palacios a, *, Cinthia Lorena Barbosa-Duchateau a, Susana Valencia-Díaz a, Ascención Capistrán-Barradas b, José G. García-Franco c a
Centro de Investigación en Biodiversidad y Conservación (CIByC), Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Col. Chamilpa, Cuernavaca 62209, Morelos, Mexico Facultad de Ciencias Biológicas y Agropecuarias, Universidad Veracruzana e Tuxpan, Carretera Tuxpan a Tampico Km. 7.5, Tuxpan, Veracruz, Mexico c Red de Ecología Funcional, Instituto de Ecología, A. C. Km 2.5 carretera antigua a Coatepec No. 351, Congregación El Haya, Xalapa 91070, Veracruz, Mexico b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 August 2012 Received in revised form 24 September 2013 Accepted 14 February 2014 Available online 15 March 2014
The function of epiphytes within ecosystems remains unclear; the high biomass and nutrient content of epiphytes suggests an indirect beneficial role, while other evidence indicates that epiphytes are directly harmful to their hosts. We studied the direct and indirect (associated with soil properties) effects of Tillandsia recurvata on Prosopis laevigata. Soils located below trees supporting large loads of T. recurvata differed in nutrient content from soils below trees carrying few T. recurvata and from bare soils. Greenhouse experiments showed that soils with added T. recurvata or P. laevigata litter increased seed germination and seedling survival of P. laevigata, but that the seedlings sown in soil with T. recurvata organic matter were shorter and of lower biomass. In vitro experiments showed that dichloromethanic and methanolic extracts of T. recurvata reduced the radicle size of P. laevigata seedlings. P. laevigata branches with >50% T. recurvata cover presented more dead and less live shoots than branches with <50% T. recurvata cover. Our data suggest that T. recurvata has a direct negative effect on P. laevigata. While T. recurvata does increase the organic matter and nutrient content of the scrubland soil, it also releases allelochemical compounds that act to reduce P. laevigata growth. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Allelopathy Bromeliaceae Epiphyte dead organic matter EDOM Structural parasitism
1. Introduction The function of vascular epiphytes in ecosystems remains poorly understood. It has been proposed that epiphytes are indirectly beneficial to their ecosystem (Nadkarni, 1986) because they capture nutrients from the atmosphere that are subsequently incorporated into the ecosystem via indirect paths (e.g. leachates, falling epiphytes) and to a lesser extent through direct paths, such as contact between the apogeotropic roots of the host and organic matter from dead epiphytes (Nadkarni, 1981). These pathways could be particularly important in forests where epiphytes are highly abundant and contain high concentrations of nutrients (Coxson and Nadkarni, 1995; Flores-Palacios and García-Franco, 2004); however, it has also been proposed that epiphytes have direct detrimental
* Corresponding author. Tel./fax: þ52 777 3297019. E-mail addresses: alejandro.fl[email protected], alefl[email protected] (A. Flores-Palacios), [email protected] (A. Capistrán-Barradas), jose.garcia.franco@ inecol.edu.mx (J.G. García-Franco). http://dx.doi.org/10.1016/j.jaridenv.2014.02.010 0140-1963/Ó 2014 Elsevier Ltd. All rights reserved.
effects on their hosts. Some authors have found evidence suggesting that epiphytes reduce the growth of their hosts and increase shoot and branch mortality (Benzing and Seemann, 1978; Caldiz and Fernández, 1995; Montaña et al., 1997). In scrubland areas dominated by Prosopis laevigata in the Southeastern Chihuahuan desert, the epiphytic bromeliad Tillandsia recurvata can reach a high biomass (up to 0.4 ton/ha) that is concentrated on the branches of P. laevigata (Flores-Palacios et al., unpublished data). The high abundance of T. recurvata in this scrubland suggests the potential role of this epiphyte as a source of organic material and nutrients for the ecosystem, contributing to the generation of fertility islands that influence the properties of the soil directly below the P. laevigata crown (Gerner and Steinberger, 1989; Olalde-Portugal et al., 2000). However, such high concentrations of T. recurvata on the branches could also affect P. laevigata growth. P. laevigata develops shoots, leaves and reproductive structures on its branches and the presence of T. recurvata in these quantities could therefore interfere with the fitness of the tree, as has been suggested in some other tree species colonized by this species (e.g. Montaña et al., 1997).
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In order to determine the possible indirect role of T. recurvata within the ecosystem, we: a) measured the nutrient content of T. recurvata, b) compared samples of soils ranging from bare soil to soil from directly below the P. laevigata trees supporting different loads of T. recurvata, and c) experimentally evaluated the effect of T. recurvata dry material and organic extracts on the germination of seeds, and growth and early seedling survival of P. laevigata. In order to explore the direct effects of T. recurvata on P. laevigata, we also: d) quantified the number of live and dead shoots on P. laevigata branches supporting different loads of T. recurvata, and e) determined the frequency of branches that support high T. recurvata loads. We hypothesized that T. recurvata is indirectly beneficial to the ecosystem because it probably contains many nutrients that, with the decomposition of fallen epiphytes, can promote the germination and growth of other plants. We also hypothesized that the direct negative effects of this epiphyte on P. laevigata would be minimal. 2. Materials and methods Field sampling and collection of plant material was conducted on the San Luis Potosi high plateau in the scrubland of the southern Chihuahuan desert, Mexico (Rzedowski, 1978). Mean annual precipitation at this site ranges from 300 to 500 mm, and mean annual temperature is 13e18 C. The scrubland vegetation of the area is composed of Larrea tridentata (Sessé & Moc. ex DC.) Coville (Zygophyllaceae), Flourensia cernua DC. (Asteraceae), Opuntia spp. (Cactaceae), Yucca spp. (Asparagaceae), Acacia spp. and P. laevigata (Humb. & Bonpl. ex Willd.) M.C. Johnst. (Fabaceae). Some areas are dominated by Agave spp. (Asparagaceae), Hechtia glomerata Zucc. (Bromeliaceae) and Dasylirium sp. (Liliaceae) (Rzedowski, 1978). At the sampling sites (Appendix A), the only true epiphyte species (holoepiphyte) is T. recurvata (L.) L. (Bromeliaceae), although six cactus species can occur as accidental epiphytes (Flores-Palacios et al., unpublished data). T. recurvata inhabits all of the study area scrub species over 1.7 m in height. Dry biomass of T. recurvata on P. laevigata can be predicted using tree height and a ball moss cover index that take a visual inspection of the epiphyte distribution in the tree into account (r2 ¼ 0.74, Flores-Palacios et al., unpublished data). Mean dry biomass of T. recurvata per tree is 0.35 0.80 kg (hereafter, mean S. D.), with a maximum of 9.4 kg per tree. The T. recurvata biomass is equally distributed between the inner and outer branches of P. laevigata, while the trunk presents little colonization (Flores-Palacios et al., unpublished data). 2.1. Nutrient content of the scrubland soil below P. laevigata trees with different T. recurvata loads From May to September 2006, in five locations chosen at random from a previous survey (Appendix A), soil samples representing three different soil conditions were taken: six soil samples from areas devoid of vegetation (bare soil), three from below the crown of three P. laevigata trees <3 m in height, and a further three from below P. laevigata trees >4 m in height. Each soil sample (12 samples per locality 5 localities) was collected from a plot 30 cm2 in area and 5 cm in depth. Soil samples were used to determine soil texture (percentage of sand, clay and silt), percentage of organic matter, pH and C, N, Pextractable, Ca, Mg, K and Na contents. For all samples, pH was recorded with a potentiometer (Barnard Mod. 20) after adding distilled water to 10 g of sample (1:10). Nitrogen was determined using the micro-Kjeldahl method (Etchevrs-Barra et al., 1997). Following digestion with HNO3, magnesium and calcium were determined by atomic absorption, sodium and potassium by flammometry, and extractable phosphorus by the vanadiume
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molybdenum method (Etchevrs-Barra et al., 1997), as has been carried out previously (Flores-Palacios and García-Franco, 2004). All soil and nutrient analyses were conducted in the laboratory of Functional Ecology and the Soil Biology Laboratory of the Instituto de Ecología, A. C. 2.2. Experiments of the role of T. recurvata dry material and organic extracts on P. laevigata early life stages We collected T. recurvata plants and soil samples from each study site. All T. recurvata specimens were growing on P. laevigata and, following collection, were cleaned to remove invertebrates, detritus, and dust, then oven-dried (FD 115-UL, Binder) at 30 C until reaching a constant dry weight. Dry T. recurvata material was ground to <3 mm in an electric mill (PULVEX S. A. de C.V. model Mini-100). The resulting dried ground material was stored in darkness at 15 C until use. A fraction of this dry material (200 g) was used for chemical analysis, while 800 g were used for extraction of organic extracts and the remaining 1.8 kg was utilized in the greenhouse experiments. From the fraction used for chemical analysis, we measured pH and C, N, P, Ca, Mg, K and N contents, as described previously. We conducted two experiments to test the effect of T. recurvata on seed germination, early growth and seedling survival in P. laevigata: one under greenhouse conditions and the other in vitro. In all germination experiments, seed coats were carefully cut to facilitate imbibition. In the greenhouse experiment, we sowed P. laevigata seeds in five soil mixtures: 1) soil only (soil collected form bare soil areas); 2) soil (89%) with T. recurvata organic matter (11%); 3) soil (89%) with P. laevigata litter (11%); 4) soil (89%) with T. recurvata (5.5%) and P. laevigata litter (5.5%); and 5) peat moss. All mixtures were prepared on a weight:weight basis. P. laevigata litter was obtained from 10 trees at the study area, ground, dried and analyzed for nutrient content as with the collected T. recurvata material. We used 125 seeds distributed in 25 0.5 L containers for each treatment, except for the peat moss, where only 6 containers were used. Seeds were sown at 1 cm depth. Containers were arranged at random in the greenhouse and irrigated daily. Greenhouse experiments were conducted from January 29 to June 25 2007. It was not possible to closely control the microclimatic conditions in the greenhouse, but we monitored the daily patterns of temperature and relative humidity with a datalogger (Onset HoBo ProSeries, model H08-32-IS) throughout the experiment. Because the experimental period covered three different seasons, we report the daily patterns of temperature and relative humidity for each season separately in the results. Seed germination was tested weekly for 28 days, according to seedling emergence. Seedling survival was monitored weekly for 20 weeks. To determine the germination capacity of the seed batch used in the experiment, 100 seeds were germinated in petri dishes (25 seeds per Petri dish) with filter paper as a substrate. For the in vitro experiment, we sowed P. laevigata seeds in organic extracts of T. recurvata of different polarities (hexanic, dichloromethanic and methanolic). Organic extracts were obtained from the 800 g of dried ground T. recurvata material. This material was consecutively macerated with three organic solvents (hexane, dichloromethane and methanol) at room temperature for three 72 h periods per solvent. Following maceration, extracts were filtered and concentrated under vacuum (Rota-evaporator Buchi R200) at 39 C for hexane and dichloromethane, and 46 C for methanol extraction. Extracts were stored at 15 C until use. We sowed the P. laevigata seeds under different concentrations (0, 0.01, 0.1, 1, 10, 100 and 1000 mg/ml) of the three organic extracts of T. recurvata (hexanic, dichloromethanic and methanolic). For each concentration within an extract, we sowed 10 seeds in each of three petri dishes using filter paper as a substrate. Petri dishes were placed
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in an environmental growth chamber (Scorpion Scientific A 50624, Mexico) with a photoperiod of 12 h light/12 h dark and temperature of 30 C for 15 days, and germination was recorded daily. In addition to seed germination, we also recorded the radicle length of each seed at the end of the experiment. 2.3. Relationship between T. recurvata load and the number of live and dead shoots on P. laevigata branches, and the frequency of branches with high T. recurvata loads To look for evidence of T. recurvata damage on P. laevigata branches, we randomly selected 50 trees in five locations that were >3 m in height (Appendix A). From each tree, we selected a pair of branches originating from the same node, each with a diameter of approximately 1 cm, but with contrasting T. recurvata cover. One branch had T. recurvata cover of <50% and the other >50%. For each branch, we measured basal diameter, length of the main axis, and number of live and dead shoots. In a different sample of 20 P. laevigata trees, we quantified the numbers of live and dead outer branches (diameter 1 cm) and, for each branch, we visually estimated the T. recurvata cover, which was categorized into cover classes (<25%, >25e50%, >50%).
component correlated positively with the organic matter (r ¼ 0.93), Ctotal (r ¼ 0.93), N (r ¼ 0.85) and Mg (r ¼ 0.64) content, while PC 2 explained 26.1% of the variation and correlated positively with sand (r ¼ 0.74) and P (r ¼ 0.69) content, but negatively with pH (r ¼ 0.61) and Ca content (r ¼ 0.61). The remaining soil variables were weakly correlated with the components (r < 0.60). In general, the PC ordination separated, within each sampling site, soil samples from below P. laevigata of height >4 m, samples from the bare soil and those from below P. laevigata of height <3 m (Fig. 1a); however, the effect of sampling location obscured the general pattern. With the exception of one site (Jagüey, Fig. 1d), at each location, soil samples from below P. laevigata of height >4 m were separated from the rest (Fig. 1b, c, e, f). In accordance with the PC analysis (especially with PC 1), the mean values of the soil properties suggest that, as T. recurvata load increases, the soils below contain more organic matter, Ctotal and N (Table 1) (all KruskalleWallis H > 12 and P < 0.05). Nutrient concentrations (Table 1) showed that P. laevigata leaves contain more nutrients than those of T. recurvata (ranging from 1.7 times more Ca to almost five times more P). This difference is significant when all the mean values of the nutrients are compared between P. laevigata and T. recurvata (Wilcoxon Signed rank test, z ¼ 2.47, P ¼ 0.013) (Table 1).
2.4. Data analysis We used principal components analysis (PCA) to test the association between soil condition (bare or from beneath different T. recurvata loads) and soil variables (Johnson and Wichern, 2002). We used a log-rank test (Kleinbaum and Klein, 2005) to determine whether seed germination and seedling survival differed among soil mixtures. We generated KaplareMeier curves for analysis of seedling survival and performed paired GehaneWilcoxon two-sample tests (Hollander and Wolfe, 1999) in order to identify groups where survival or germination differed from that of the others. To test whether the performance of P. laevigata seedlings differed between soil mixtures, we used analysis of covariance (Sokal and Rohlf, 1995). In this analysis, we used each P. laevigata seedling as a sampling unit, with soil treatment (four levels, excluding peat moss due to the accidental loss of these seedlings) as the factor and number of seedlings in each pot as a covariable. The response variables were seedling height, root length, aboveground biomass, belowground biomass and the aboveground: belowground biomass ratio. To test whether the T. recurvata organic extract concentrations influenced seed germination of P. laevigata, we performed a second order polynomial regression analysis (Sokal and Rohlf, 1995). In this analysis, the independent variable was the extract expressed as ln (extract concentration þ 0.001), while the response variable was seed germination (%). We used the same analysis to test whether extract concentration influenced radicle length in P. laevigata seedlings. To determine whether paired branches of P. laevigata with contrasting T. recurvata loads differed in diameter, length, T. recurvata biomass, total number of shoots, number of live shoots and number of dead shoots, we used a paired t test for each variable (Sokal and Rohlf, 1995). We performed a Wilcoxon signed rank test for those variables that did not fulfill the assumptions of normality (Hollander and Wolfe, 1999). 3. Results 3.1. Nutrient content of scrubland soils from below P. laevigata individuals with different T. recurvata loads The PCA explained 57.6% of the variation in two principal components (PC). PC 1 explained 31.5% of the variation; this
3.2. The effect of T. recurvata dry material and organic extracts on early life stages of P. laevigata Germination capacity of the seed batch used in the greenhouse experiment was 100%, and full germination took place within four days. During the greenhouse experiments, mean temperature was 19.5 C in winter (min 7.8; max 33.5), 22.3 C in spring (min 10.6; max 39.6), and 20.0 C in summer (min 10.6; max 35.2). During the course of the experiment, the mean daily pattern of temperature and relative humidity changed from winter to summer, with the highest temperatures recorded in spring and greatest humidity occurring in summer (Appendix A). Seed germination of P. laevigata occurred only in the first week of observation (Fig. 2a), and differed significantly between soil mixtures (c2 ¼ 88.4, d.f. ¼ 4, P < 0.0001) (Fig. 2a). Highest germination occurred in the peat moss and in the mixture of soil with litter of T. recurvata and P. laevigata. Germination was similar in the soil with litter of P. laevigata and in the soil with litter of T. recurvata, while germination in the latter soil/litter mixture did not differ significantly from germination in the soil with no added organic matter (Fig. 2a). Survival of P. laevigata seedlings differed between soil mixtures (c2 ¼ 24.2, d.f. ¼ 4, P < 0.0001) (Fig. 2b). There was a descending gradient of seedling survival from the peat moss to the soil mixed with litter of P. laevigata (Fig. 2b). Root length (F ¼ 0.15, d.f. ¼ 3, 65, P > 0.05) and the ratio of above- to belowground biomass (F ¼ 2.4, d.f. ¼ 3, 65, P > 0.05) in the seedlings were both similar between soil mixtures (Table 2), but seedlings differed significantly among soil mixtures in terms of height (F ¼ 5.0, d.f. ¼ 3, 65, P < 0.005), aboveground biomass (F ¼ 8.0, d.f. ¼ 3, 65, P < 0.0005), and belowground biomass (F ¼ 6.0, d.f. ¼ 3, 65, P < 0.0005). The general pattern for these variables was that the performance of P. laevigata seedlings was poorest in soil with T. recurvata litter (Table 2). There was no effect of the hexanic, methanolic and dichloromethanic extracts of T. recurvata on the germination of P. laevigata seeds (all regression F < 1.5, P > 0.05, r2 < 0.14). Hexanic extracts did not affect the radicle length of the P. laevigata seedlings (F ¼ 1.46, d.f. ¼ 2, 18, P ¼ 0.63, r2 ¼ 0.005), but the dichloromethanic (F ¼ 13.04, d.f. ¼ 2, 18, P < 0.0001, r2 ¼ 0.14) and methanolic (F ¼ 12.12, d.f. ¼ 2, 18, P < 0.0001, r2 ¼ 0.15) extracts had a smooth quadratic effect, causing reduced radicle length (Fig. 3).
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Fig. 1. PCA ordination of soil samples collected from below three different canopy conditions in five locations in the scrublands of the Chihuahuan desert on the San Luis Potosi high plateau, Mexico. Canopy conditions reflect increasing abundance of the epiphyte T. recurvata; bare soil (empty circles), canopy <3 m in height (asterisks) and canopy >4 m in height (solid squares). On each axis, the explained variance and associated soil properties are shown.
3.3. Relationship between T. recurvata load and number of live and dead shoots on branches on P. laevigata and the frequency of branches with high T. recurvata loads Branches of P. laevigata with T. recurvata cover of <50% were similar to branches with a cover >50%, in terms of diameter (W ¼ 286, P > 0.05), length (t ¼ 1.78, d.f. ¼ 49, P > 0.05) and total number of shoots (W ¼ 141, P > 0.05) (Table 3). Branches with T. recurvata cover of <50% presented more live shoots than branches with >50% cover (W ¼ 502, P < 0.01); while branches with >50% cover featured a higher T. recurvata biomass (t ¼ 9.52,
d.f. ¼ 49, P < 0.001) and a greater number of dead shoots (t ¼ 6.11, d.f. ¼ 49, P < 0.001). In the P. laevigata sampled, the majority of the branches (499) had <25% T. recurvata cover, 46 branches with coverage of between 26 and 50%, and 20 branches with >50%, presenting a distribution that was significantly biased (c2 ¼ 770, d.f. ¼ 2, P < 0.0001) toward branches with low coverage of T. recurvata. We found an association (c2 ¼ 22, d.f. ¼ 2, P < 0.0001) between T. recurvata cover and the frequency of dead branches. The proportion of dead branches was 0.04 in the cover category of <25% (22 dead branches from a total of 499 branches observed), but increased to 0.15 in the 26e50% cover
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Table 1 Chemical properties of soil samples, and Tillandsia recurvata and Prosopis laevigata leaves. Soil samples come from below three different canopy conditions. All samples were collected in five locations in the scrublands of the Chihuahuan desert, on the San Luis Potosi high plateau, Mexico. Canopy conditions reflect increasing abundance of the epiphyte T. recurvata, from bare soil to soil below a canopy >4 m in height. Mean values (1 standard deviation) are presented for each variable. Different letters between soil samples indicate significant differences between median values, according to a KruskalleWallis test, followed by the Nemenyi multiple comparison procedure (P < 0.05). Variable
Bare soil (n ¼ 30)
Clay (%) Sand (%) Silt (%) pH Organic matter (%) Ctotal (%) Ca (cmol/kg) K (cmol/kg) Mg (cmol/kg) Na (cmol/kg) Ntotal (ppm) Pextractable (ppm)
29.55 36.89 33.85 6.92 1.50 0.87 25.51 2.10 1.68 0.47 0.16 41.61
Soil under P. laeviagata of canopy height: <3 m (n ¼ 15)
4.05 7.11 5.32 0.84 1.05b 0.61b 11.53n.s. 0.68n.s. 0.66n.s. 0.15n.s. 0.07n.s. 40.62b
31.52 37.34 31.12 6.47 2.46 1.43 25.82 2.29 1.68 0.48 0.18 43.84
3.45 6.07 5.30 0.89 1.54a 0.89a 14.08 0.97 0.70 0.14 0.06 43.12b
>4 m (n ¼ 15) 32.72 37.23 30.05 6.45 4.62 2.68 25.41 2.78 2.51 0.52 0.29 49.58
category (7 dead branches from a total of 46 branches) and to 0.25 in the >50% cover category (5 dead branches from a total of 20 branches). 4. Discussion Epiphytes play an important role in the nutrient dynamics of forest and woodlands because they reach high biomass in the trees and can capture atmospheric nutrients that can be subsequently released to the ecosystem through stemflow, apogeotropic roots and when the epiphytes fall to the forest floor (Coxson and Nadkarni, 1995). In the study area, T. recurvata is the only holoepiphyte; it can present a high biomass on P. laevigata (up to 0.4 ton/ha) and has nitrogen-fixing bacteria on its leaves (Puente and Bashan, 1994). This feature enables T. recurvata to contribute both organic matter and nutrients to the soil below. However, our results suggest that the nutrient content of T. recurvata is poor compared to both P. laevigata leaves and the nutrient contents of other epiphytic communities. The P and N content of T. recurvata is lower than that observed in leaves of Tillandsia capillaris in a dry woodland in Argentina, which was reported to be 3.1 mg/g for P and 24.4 mg/g for N (Abril and Bucher, 2009). It also was lower than the nutrient content found in humid environments. For example, the P and N content of T. recurvata was lower than the P content of the debris captured by bromeliads (0.19 mg/g) and in the bromeliad leaves (0.48 mg/g) in a humid tropical forest in Puerto Rico (Richardson et al., 2000). In Neotropical montane cloud forests, the P content of epiphyte dead organic matter (EDOM) ranged from 0.54 to 0.66 mg/g in Colombia (Hofstede et al., 1993), from 2.19 mg/g to 3.50 mg/g in Mexico (Flores-Palacios and García-Franco, 2004) and from 1.25 mg/g to 1.49 mg/g in American coniferous forest (Lang et al., 1980; Nadkarni, 1984). The N content of T. recurvata was lower than that found in the bromeliad leaves of a humid tropical forest in Puerto Rico (7.20 mg/ge7.50 mg/g; Richardson et al., 2000), and was lower than the N content of debris captured by bromeliads (14.50 mg/ge18.10 mg/g) in the same study, and in the EDOM of Neotropical montane cloud forest in Colombia (8.12 mg/ge 8.53 mg/g) (Hofstede et al., 1993) and Mexico (17.96 mg/ge 19.15 mg/g) (Flores-Palacios and García-Franco, 2004). Perhaps the low nutrient content of T. recurvata relative to those studies evaluating the EDOM nutrient content could be due to the fact that EDOM contains nutrients coming both from the epiphytes themselves and from another sources (e.g. invertebrates, detritus and dust). However, T. recurvata is an atmospheric Tillandsia with a
4.07 6.53 4.76 1.01 2.16a 1.25a 10.49 1.23 1.17 0.20 0.11 45.83a
Tillandsia recurvata (n ¼ 4)
4.98 90.54 52.52 41.84 9.75 4.44 5.77 0.73 41.82
013 1.24 072 1.02 0.24 1.18 0.47 0.06 4.43
Prosopis laevigata (n ¼ 2)
5.26 93.01 53.95 71.25 27.36 14.54 14.47 2.44 201.99
0.12 0.08 0.05 0.76 0.03 1.12 1.62 0.12 5.46
Ratio Prosopis/ Tillandsia
1.0 1.0 1.7 2.8 3.3 2.5 3.3 4.8
limited capacity for retaining EDOM in its roots and between its leaves. Being of low nutrient content, T. recurvata is likely to contribute only slightly to the creation of fertility islands in the studied scrubland; however, principal component analysis separated the soil samples collected from below the crown of P. laevigata >4 m in height, where the T. recurvata load is high, from the rest of the soil samples. The importance of the contribution of T. recurvata to the soil composition under the taller individuals of P. laevigata should not be discounted, since T. recurvata provides both organic matter and nutrients. Nonetheless, the contribution made by the nutrient rich leaves of the tree is likely to be of relatively greater importance. The greenhouse experiments give us a better understanding of the role of T. recurvata when incorporated into the soil; the increased organic matter clearly promoted seed germination and seedling survival in P. laevigata, although this could have been the result of increased water retention rather than the increase in nutrient availability. Surprisingly, the greenhouse and in vitro experiments also showed that T. recurvata could negatively affect the performance of P. laevigata seedlings. Greenhouse experiments showed that seedlings of P. laevigata were shorter and presented lower above- and belowground biomass when grown in soils with added T. recurvata material. Perhaps the short height and low aboveground biomass were caused by a reduction in the development of root biomass, especially since the in vitro experiments showed that dichloromethanic and methanolic extracts of T. recurvata reduced root development in P. laevigata. T. recurvata contains various compounds that could be allopathic (Valencia-Díaz et al., 2012; de Queiroga et al., 2004) and organic extracts of T. recurvata have been shown to reduce the germination of sympatric epiphytic species (Valencia-Díaz et al., 2012). Among these allelopathic compounds, diterpene phytol is known as a root growth inhibitor and is present in dichloromethanic extracts (Valencia-Díaz et al., 2012). Our results suggest that, although T. recurvata is a source of organic matter and nutrients for the scrubland soil, it could also exert an allelopathic effect on the seedlings of its host. In addition to the probable allelopathic effect of T. recurvata on scrubland soil, we found that P. laevigata branches with >50% T. recurvata cover had more dead shoots and less live shoots than branches with <50% cover. The same effect of T. recurvata has previously been reported in the tree Parkinsonia praecox (Fabaceae) in a scrubland in the semiarid region of Tehuacan, Mexico (Montaña et al., 1997). Our evidence suggests that T. recurvata directly reduced the growth of P. laevigata. Perhaps the damage that
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Fig. 2. Germination (a) and seedling survival (b) of Prosopis laevigata seeds/seedlings sown in different soil mixtures. Soil (empty circles); soil with T. recurvata litter (asterisks); soil with P. laevigata litter (solid triangles); soil with litter of T. recurvata and P. laevigata (empty squares); and peat moss (solid circles). Different letters indicate significant differences between proportions (GehaneWilcoxon test, P < 0.05).
T. recurvata exerts on branches of P. laevigata is physical, since evidence in the same study area shows that P. laevigata branches that support T. recurvata can present anatomical changes in the wood; however, the hydraulic conductivity of these branches must be studied in order to determine whether such anatomical changes affect tissue function (Rodríguez et al., 2007). Among the branch population, only 3.5% of the branches presented a T. recurvata cover greater than 50%, suggesting that, overall, the direct negative effects of T. recurvata may be slight, even though the proportion of dead branches was highest in this T. recurvata cover category. In view of the fact that epiphytes can be highly abundant in scrublands and forests and can contain large amounts of nutrients,
it is hypothesized that they can exert an indirect positive influence on the ecosystem. Conversely, because epiphytes can be present in high concentrations on their hosts, it has also been suggested that they may have a direct harmful effect upon them. Our results suggest that, contrary to expectations, the only holoepiphyte in the study area could have a negative allelopathic effect on the soil in addition to the expected direct negative effect on the branches of P. laevigata. In the studied area, this could also be occurring in the sympatric Acacia trees that can contain T. recurvata loads as large as those of P. laevigata, but further study is required in order to confirm this. T. recurvata is a widely distributed vascular epiphyte (Smith and Downs, 1977) and is the dominant epiphyte in some
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Table 2 Mean values (1 standard deviation) of variables associated with Prosopis laevigata seedling performance following 20 weeks growth in different soil mixtures. Different letters indicate significant differences between mean values (Tukey test, P < 0.05). n.s. ¼ non-significant differences between means. Variable
Soil (n ¼ 8)
Soil with T. recurvata litter (n ¼ 27)
Soil with P. laevigata litter (n ¼ 12)
Soil with litter of T. recurvata and P. laevigata (n ¼ 22)
Seedling height (cm) Root length (cm) Aboveground biomass (g) Belowground biomass (g) Above/below ground biomass ratio
12.46ab 24.84n.s. 0.31a 0.20ab 2.42n.s.
4.68b 19.57 0.05b 0.05b 1.10
12.81a 25.93 0.32a 0.27a 1.18
10.60a 23.42 0.19ab 0.16a 1.09
(3.59) (7.99) (0.31) (0.24) (3.12)
(1.21) (12.06) (0.03) (0.03) (1.33)
(4.83) (9.75) (0.19) (0.16) (0.27)
(6.35) (10.16) (0.18) (0.11) (0.45)
Fig. 3. Relationship between the radicle length of Prosopis laevigata seedlings growing on organic extracts of the epiphyte Tillandsia recurvata, and organic extract concentration. n.s. ¼ non-significant relationship.
tropical forests (Vergara-Torres et al., 2010). In addition to constituting nutrient sources, epiphyte communities dominated by T. recurvata could in fact be sources of allelopathic compounds in the decaying litter, a phenomenon suggested in tree litter in previous studies (Lodhi, 1978; Mazzoleni et al., 2007). Perhaps a synergistic negative effect of T. recurvata on the soil and on the branches of their host can cause decay in some trees that are highly
Table 3 Mean values (1 standard deviation) of variables measured in Prosopis laevigata branches (diameter 1 cm) with different categories of Tillandsia recurvata cover. Different letters indicate significant differences between mean values (t or Wilcoxon Paired test, P < 0.05, see text). n.s. ¼ non-significant differences between means. Variable
T. recurvata cover
Branch diameter (mm) Branch length (cm) T. recurvata biomass (g) Total number of shoots Number of live shoots Number of dead shoots
13.1n.s. 64.0n.s. 6.8b 15.2n.s. 11.5a 3.7b
<50%
>50%
12.0 18.8 7.5 8.0 6.9 3.1
11.9 59.1 21.0a 16.6 9.5b 7.1a
2.3 18.0 12.0 10.4 8.1 4.8
infested by epiphytes (e.g. Benzing and Seemann, 1978). Experimental research focusing on both components of the ecosystem (soil and canopy) is required in order to broaden our understanding of this aspect of the interaction between these species. Acknowledgments Samuel Arechaga helped during fieldwork. The comments and critique of K. MacMillan and two anonymous reviewers improved the manuscript. This research was supported by a grant from the Oficina de Sanidad Forestal, Comisión Nacional Forestal (Project 10140) to JGGF, a PROMEP grant to AFP (PROMEP/103.5/05/1901) and INECOL (20030-10144). An earlier version, circumscribed to the greenhouse experiments only, was presented by CLBD as a Bachelors thesis at the Faculty of Biological Sciences, Universidad Autónoma del Estado de Morelos, Mexico. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jaridenv.2014.02.010.
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References Abril, A.B., Bucher, E.H., 2009. A comparison of nutrient sources of the epiphyte Tillandsia capillaris attached to trees and cables in Cordoba, Argentina. J. Arid Environ. 73, 393e395. Benzing, D.H., Seemann, J., 1978. Nutritional piracy and host decline: a new perspective on the epiphyte-host relationship. Selbyana 2, 133e148. Caldiz, D.O., Fernández, L.V., 1995. The role of the epiphytic weeds Tillandsia recurvata and T. aëranthos in native rural and urban forest. Int. J. Ecol. Environ. Sci. 21, 177e197. Coxson, D.S., Nadkarni, N.M., 1995. Ecological roles of epiphytes in nutrient cycles of forest ecosystems. In: Lowman, M.D., Nadkarni, N.M. (Eds.), Forest canopies. Academic Press, San Diego, pp. 495e543. de Queiroga, M.A., de Andrade, L.M., Chagas, K.F., de Fátima, M., Sobral da Silva, M., Barbosa-Filho, J.M., Vasconcelos, E., 2004. Chemical constituents from Tillandsia recurvata. Fitoterapia 75, 423e425. Etchevrs-Barra, J., Goijberg, E.G., López-Romer, R.M., Padilla-Cuevas, J., AlvaradoLópez, J., Hidalgo-Moreno, C., Cruz-Huerta, M., Guerrero-Peña, A., Gutiérrez, B., Miranda-Cruz, E., 1997. Manual de procedimientos analíticos para análisis de suelos y plantas del laboratorio de fertilidad de suelos. IRENAT, Colegio de Posgraduados, Sociedad Mexicana de la Ciencia del Suelo A. C, México, D.F. Flores-Palacios, A., García-Franco, J.G., 2004. Effect of isolation on the structure and nutrient budget of oak epiphyte communities. Plant Ecol. 173, 259e269. Gerner, W., Steinberger, Y., 1989. A proposal mechanism for the formation of fertile islands in the desert ecosystem. J. Arid Environ. 16, 257e262. Hofstede, R.G.M., Wolf, J.D.H., Benzing, D.H., 1993. Epiphytic biomass and nutrient status of a Colombian upper Montane rain forest. Selbyana 14, 37e45. Hollander, M., Wolfe, D.A., 1999. Nonparametric Statistical Methods. John Wiley & Sons, Danvers. Johnson, R.A., Wichern, D.W., 2002. Applied Multivariate Statistical Analysis. Prentice Hall, Upper Saddle River. Kleinbaum, D.G., Klein, M., 2005. Survival Analysis: a Self-learning Text. SpringerVerlag, New York. Lang, G.E., Reiners, W.A., Pike, L.H., 1980. Structure and biomass dynamics of epiphytic lichen communities of balsam fir forest in New Hampshire. Ecology 61, 541e550. Lodhi, M.A.K., 1978. Allelophatic effects of decaying litter of dominant trees and their associated soil in a lowland forest community. Am. J. Bot. 65, 340e344.
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Mazzoleni, S.G., Bonanomi, G., Giannino, F., Rietkerk, M., Dekker, S.C., Zucconi, F., 2007. Is plant biodiversity driven by decomposition processes? An emerging new theory on plant diversity. Community Ecol. 8, 103e109. Montaña, C., Dirzo, R., Flores, A., 1997. Structural parasitism of an epiphytic bromeliad upon Cercidium praecox in an intertropical semiarid ecosystem. Biotropica 29, 517e521. Nadkarni, N.M., 1984. Epiphyte biomass and nutrient capital of a neotropical elfin forest. Biotropica 16, 249e256. Nadkarni, N.M., 1981. Canopy Roots: convergent evolution in rainforest nutrient cycles. Science 214, 1023e1235. Nadkarni, N.M., 1986. The nutritional effects of epiphytes on host trees with special reference to alteration of precipitation chemistry. Selbyana 9, 44e51. Olalde-Portugal, V., Frías-Hernández, J.T., Aguilar-Ledezma, A.L., Pescador, N., Aguilera-G, L.I., 2000. Caracterización Microbiólogica de Suelos de Isla de Fertilidad de Mezquite [Prosopis laevigata (Humb. & Bonpl. Ex. Wild) M. C. Johnst.]. In: Hernández, J.T.F., Portugal, V.O., Carter, J.V. (Eds.), El Mezquite árbol de usos múltiples, estado actual del conocimiento en México. Universidad de Guanajuato, México, pp. 95e107. Puente, M.E., Bashan, Y., 1994. The desert epiphyte Tillandsia recurvata harbours the nitrogen-fixing bacterium Pseudomonas stutzeri. Can. J. Bot. 72, 406e408. Richardson, B., Richardson, M.J., Scatena, F.N., McDowell, W.H., 2000. Effects of nutrient availability and other elevational changes on bromeliad populations and their invertebrate communities in a humid tropical forest in Puerto Rico. J. Trop. Ecol. 16, 167e188. Rodríguez, S.A., Terrazas, T., León, E.A., Salas, M.E.H., 2007. Modificaciones de la corteza de Prosopis laevigata por el establecimiento de Tillandsia recurvata. Bol. Soc. Bot. México 81, 27e35. Rzedowski, J., 1978. La Vegetación de México. Libreros Mexicanos Unidos, S. A. (LIMUSA), D.F. Smith, L.B., Downs, R.J., 1977. Tillandsioideae (Bromeliaceae). Flora Neotrop. Monogr. 14, 663e1492. Sokal, R.R., Rohlf, F.J., 1995. Biometry. Freeman and Company, New York. Valencia-Díaz, S., Flores-Palacios, A., Rodríguez-López, V., Jiménez-Aparicio, A.R., 2012. Effects of Tillandsia recurvata extracts on the seed germination of Tillandsia spp. Allelopathy J. 29, 125e136. Vergara-Torres, C.A., Pacheco-Álvarez, M.C., Flores-Palacios, A., 2010. Host preference and host limitation of vascular epiphytes in a tropical dry forest of central Mexico. J. Trop. Ecol. 26, 563e570.
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Journal of Arid Environments journal homepage: www.elsevier.com/locate/jaridenv
Direct and indirect effects of Tillandsia recurvata on Prosopis laevigata in the Chihuahua desert scrubland of San Luis Potosi, Mexico Alejandro Flores-Palacios a, *, Cinthia Lorena Barbosa-Duchateau a, Susana Valencia-Díaz a, Ascención Capistrán-Barradas b, José G. García-Franco c a
Centro de Investigación en Biodiversidad y Conservación (CIByC), Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Col. Chamilpa, Cuernavaca 62209, Morelos, Mexico Facultad de Ciencias Biológicas y Agropecuarias, Universidad Veracruzana e Tuxpan, Carretera Tuxpan a Tampico Km. 7.5, Tuxpan, Veracruz, Mexico c Red de Ecología Funcional, Instituto de Ecología, A. C. Km 2.5 carretera antigua a Coatepec No. 351, Congregación El Haya, Xalapa 91070, Veracruz, Mexico b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 August 2012 Received in revised form 24 September 2013 Accepted 14 February 2014 Available online 15 March 2014
The function of epiphytes within ecosystems remains unclear; the high biomass and nutrient content of epiphytes suggests an indirect beneficial role, while other evidence indicates that epiphytes are directly harmful to their hosts. We studied the direct and indirect (associated with soil properties) effects of Tillandsia recurvata on Prosopis laevigata. Soils located below trees supporting large loads of T. recurvata differed in nutrient content from soils below trees carrying few T. recurvata and from bare soils. Greenhouse experiments showed that soils with added T. recurvata or P. laevigata litter increased seed germination and seedling survival of P. laevigata, but that the seedlings sown in soil with T. recurvata organic matter were shorter and of lower biomass. In vitro experiments showed that dichloromethanic and methanolic extracts of T. recurvata reduced the radicle size of P. laevigata seedlings. P. laevigata branches with >50% T. recurvata cover presented more dead and less live shoots than branches with <50% T. recurvata cover. Our data suggest that T. recurvata has a direct negative effect on P. laevigata. While T. recurvata does increase the organic matter and nutrient content of the scrubland soil, it also releases allelochemical compounds that act to reduce P. laevigata growth. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Allelopathy Bromeliaceae Epiphyte dead organic matter EDOM Structural parasitism
1. Introduction The function of vascular epiphytes in ecosystems remains poorly understood. It has been proposed that epiphytes are indirectly beneficial to their ecosystem (Nadkarni, 1986) because they capture nutrients from the atmosphere that are subsequently incorporated into the ecosystem via indirect paths (e.g. leachates, falling epiphytes) and to a lesser extent through direct paths, such as contact between the apogeotropic roots of the host and organic matter from dead epiphytes (Nadkarni, 1981). These pathways could be particularly important in forests where epiphytes are highly abundant and contain high concentrations of nutrients (Coxson and Nadkarni, 1995; Flores-Palacios and García-Franco, 2004); however, it has also been proposed that epiphytes have direct detrimental
* Corresponding author. Tel./fax: þ52 777 3297019. E-mail addresses: alejandro.fl[email protected], alefl[email protected] (A. Flores-Palacios), [email protected] (A. Capistrán-Barradas), jose.garcia.franco@ inecol.edu.mx (J.G. García-Franco). http://dx.doi.org/10.1016/j.jaridenv.2014.02.010 0140-1963/Ó 2014 Elsevier Ltd. All rights reserved.
effects on their hosts. Some authors have found evidence suggesting that epiphytes reduce the growth of their hosts and increase shoot and branch mortality (Benzing and Seemann, 1978; Caldiz and Fernández, 1995; Montaña et al., 1997). In scrubland areas dominated by Prosopis laevigata in the Southeastern Chihuahuan desert, the epiphytic bromeliad Tillandsia recurvata can reach a high biomass (up to 0.4 ton/ha) that is concentrated on the branches of P. laevigata (Flores-Palacios et al., unpublished data). The high abundance of T. recurvata in this scrubland suggests the potential role of this epiphyte as a source of organic material and nutrients for the ecosystem, contributing to the generation of fertility islands that influence the properties of the soil directly below the P. laevigata crown (Gerner and Steinberger, 1989; Olalde-Portugal et al., 2000). However, such high concentrations of T. recurvata on the branches could also affect P. laevigata growth. P. laevigata develops shoots, leaves and reproductive structures on its branches and the presence of T. recurvata in these quantities could therefore interfere with the fitness of the tree, as has been suggested in some other tree species colonized by this species (e.g. Montaña et al., 1997).
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In order to determine the possible indirect role of T. recurvata within the ecosystem, we: a) measured the nutrient content of T. recurvata, b) compared samples of soils ranging from bare soil to soil from directly below the P. laevigata trees supporting different loads of T. recurvata, and c) experimentally evaluated the effect of T. recurvata dry material and organic extracts on the germination of seeds, and growth and early seedling survival of P. laevigata. In order to explore the direct effects of T. recurvata on P. laevigata, we also: d) quantified the number of live and dead shoots on P. laevigata branches supporting different loads of T. recurvata, and e) determined the frequency of branches that support high T. recurvata loads. We hypothesized that T. recurvata is indirectly beneficial to the ecosystem because it probably contains many nutrients that, with the decomposition of fallen epiphytes, can promote the germination and growth of other plants. We also hypothesized that the direct negative effects of this epiphyte on P. laevigata would be minimal. 2. Materials and methods Field sampling and collection of plant material was conducted on the San Luis Potosi high plateau in the scrubland of the southern Chihuahuan desert, Mexico (Rzedowski, 1978). Mean annual precipitation at this site ranges from 300 to 500 mm, and mean annual temperature is 13e18 C. The scrubland vegetation of the area is composed of Larrea tridentata (Sessé & Moc. ex DC.) Coville (Zygophyllaceae), Flourensia cernua DC. (Asteraceae), Opuntia spp. (Cactaceae), Yucca spp. (Asparagaceae), Acacia spp. and P. laevigata (Humb. & Bonpl. ex Willd.) M.C. Johnst. (Fabaceae). Some areas are dominated by Agave spp. (Asparagaceae), Hechtia glomerata Zucc. (Bromeliaceae) and Dasylirium sp. (Liliaceae) (Rzedowski, 1978). At the sampling sites (Appendix A), the only true epiphyte species (holoepiphyte) is T. recurvata (L.) L. (Bromeliaceae), although six cactus species can occur as accidental epiphytes (Flores-Palacios et al., unpublished data). T. recurvata inhabits all of the study area scrub species over 1.7 m in height. Dry biomass of T. recurvata on P. laevigata can be predicted using tree height and a ball moss cover index that take a visual inspection of the epiphyte distribution in the tree into account (r2 ¼ 0.74, Flores-Palacios et al., unpublished data). Mean dry biomass of T. recurvata per tree is 0.35 0.80 kg (hereafter, mean S. D.), with a maximum of 9.4 kg per tree. The T. recurvata biomass is equally distributed between the inner and outer branches of P. laevigata, while the trunk presents little colonization (Flores-Palacios et al., unpublished data). 2.1. Nutrient content of the scrubland soil below P. laevigata trees with different T. recurvata loads From May to September 2006, in five locations chosen at random from a previous survey (Appendix A), soil samples representing three different soil conditions were taken: six soil samples from areas devoid of vegetation (bare soil), three from below the crown of three P. laevigata trees <3 m in height, and a further three from below P. laevigata trees >4 m in height. Each soil sample (12 samples per locality 5 localities) was collected from a plot 30 cm2 in area and 5 cm in depth. Soil samples were used to determine soil texture (percentage of sand, clay and silt), percentage of organic matter, pH and C, N, Pextractable, Ca, Mg, K and Na contents. For all samples, pH was recorded with a potentiometer (Barnard Mod. 20) after adding distilled water to 10 g of sample (1:10). Nitrogen was determined using the micro-Kjeldahl method (Etchevrs-Barra et al., 1997). Following digestion with HNO3, magnesium and calcium were determined by atomic absorption, sodium and potassium by flammometry, and extractable phosphorus by the vanadiume
89
molybdenum method (Etchevrs-Barra et al., 1997), as has been carried out previously (Flores-Palacios and García-Franco, 2004). All soil and nutrient analyses were conducted in the laboratory of Functional Ecology and the Soil Biology Laboratory of the Instituto de Ecología, A. C. 2.2. Experiments of the role of T. recurvata dry material and organic extracts on P. laevigata early life stages We collected T. recurvata plants and soil samples from each study site. All T. recurvata specimens were growing on P. laevigata and, following collection, were cleaned to remove invertebrates, detritus, and dust, then oven-dried (FD 115-UL, Binder) at 30 C until reaching a constant dry weight. Dry T. recurvata material was ground to <3 mm in an electric mill (PULVEX S. A. de C.V. model Mini-100). The resulting dried ground material was stored in darkness at 15 C until use. A fraction of this dry material (200 g) was used for chemical analysis, while 800 g were used for extraction of organic extracts and the remaining 1.8 kg was utilized in the greenhouse experiments. From the fraction used for chemical analysis, we measured pH and C, N, P, Ca, Mg, K and N contents, as described previously. We conducted two experiments to test the effect of T. recurvata on seed germination, early growth and seedling survival in P. laevigata: one under greenhouse conditions and the other in vitro. In all germination experiments, seed coats were carefully cut to facilitate imbibition. In the greenhouse experiment, we sowed P. laevigata seeds in five soil mixtures: 1) soil only (soil collected form bare soil areas); 2) soil (89%) with T. recurvata organic matter (11%); 3) soil (89%) with P. laevigata litter (11%); 4) soil (89%) with T. recurvata (5.5%) and P. laevigata litter (5.5%); and 5) peat moss. All mixtures were prepared on a weight:weight basis. P. laevigata litter was obtained from 10 trees at the study area, ground, dried and analyzed for nutrient content as with the collected T. recurvata material. We used 125 seeds distributed in 25 0.5 L containers for each treatment, except for the peat moss, where only 6 containers were used. Seeds were sown at 1 cm depth. Containers were arranged at random in the greenhouse and irrigated daily. Greenhouse experiments were conducted from January 29 to June 25 2007. It was not possible to closely control the microclimatic conditions in the greenhouse, but we monitored the daily patterns of temperature and relative humidity with a datalogger (Onset HoBo ProSeries, model H08-32-IS) throughout the experiment. Because the experimental period covered three different seasons, we report the daily patterns of temperature and relative humidity for each season separately in the results. Seed germination was tested weekly for 28 days, according to seedling emergence. Seedling survival was monitored weekly for 20 weeks. To determine the germination capacity of the seed batch used in the experiment, 100 seeds were germinated in petri dishes (25 seeds per Petri dish) with filter paper as a substrate. For the in vitro experiment, we sowed P. laevigata seeds in organic extracts of T. recurvata of different polarities (hexanic, dichloromethanic and methanolic). Organic extracts were obtained from the 800 g of dried ground T. recurvata material. This material was consecutively macerated with three organic solvents (hexane, dichloromethane and methanol) at room temperature for three 72 h periods per solvent. Following maceration, extracts were filtered and concentrated under vacuum (Rota-evaporator Buchi R200) at 39 C for hexane and dichloromethane, and 46 C for methanol extraction. Extracts were stored at 15 C until use. We sowed the P. laevigata seeds under different concentrations (0, 0.01, 0.1, 1, 10, 100 and 1000 mg/ml) of the three organic extracts of T. recurvata (hexanic, dichloromethanic and methanolic). For each concentration within an extract, we sowed 10 seeds in each of three petri dishes using filter paper as a substrate. Petri dishes were placed
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in an environmental growth chamber (Scorpion Scientific A 50624, Mexico) with a photoperiod of 12 h light/12 h dark and temperature of 30 C for 15 days, and germination was recorded daily. In addition to seed germination, we also recorded the radicle length of each seed at the end of the experiment. 2.3. Relationship between T. recurvata load and the number of live and dead shoots on P. laevigata branches, and the frequency of branches with high T. recurvata loads To look for evidence of T. recurvata damage on P. laevigata branches, we randomly selected 50 trees in five locations that were >3 m in height (Appendix A). From each tree, we selected a pair of branches originating from the same node, each with a diameter of approximately 1 cm, but with contrasting T. recurvata cover. One branch had T. recurvata cover of <50% and the other >50%. For each branch, we measured basal diameter, length of the main axis, and number of live and dead shoots. In a different sample of 20 P. laevigata trees, we quantified the numbers of live and dead outer branches (diameter 1 cm) and, for each branch, we visually estimated the T. recurvata cover, which was categorized into cover classes (<25%, >25e50%, >50%).
component correlated positively with the organic matter (r ¼ 0.93), Ctotal (r ¼ 0.93), N (r ¼ 0.85) and Mg (r ¼ 0.64) content, while PC 2 explained 26.1% of the variation and correlated positively with sand (r ¼ 0.74) and P (r ¼ 0.69) content, but negatively with pH (r ¼ 0.61) and Ca content (r ¼ 0.61). The remaining soil variables were weakly correlated with the components (r < 0.60). In general, the PC ordination separated, within each sampling site, soil samples from below P. laevigata of height >4 m, samples from the bare soil and those from below P. laevigata of height <3 m (Fig. 1a); however, the effect of sampling location obscured the general pattern. With the exception of one site (Jagüey, Fig. 1d), at each location, soil samples from below P. laevigata of height >4 m were separated from the rest (Fig. 1b, c, e, f). In accordance with the PC analysis (especially with PC 1), the mean values of the soil properties suggest that, as T. recurvata load increases, the soils below contain more organic matter, Ctotal and N (Table 1) (all KruskalleWallis H > 12 and P < 0.05). Nutrient concentrations (Table 1) showed that P. laevigata leaves contain more nutrients than those of T. recurvata (ranging from 1.7 times more Ca to almost five times more P). This difference is significant when all the mean values of the nutrients are compared between P. laevigata and T. recurvata (Wilcoxon Signed rank test, z ¼ 2.47, P ¼ 0.013) (Table 1).
2.4. Data analysis We used principal components analysis (PCA) to test the association between soil condition (bare or from beneath different T. recurvata loads) and soil variables (Johnson and Wichern, 2002). We used a log-rank test (Kleinbaum and Klein, 2005) to determine whether seed germination and seedling survival differed among soil mixtures. We generated KaplareMeier curves for analysis of seedling survival and performed paired GehaneWilcoxon two-sample tests (Hollander and Wolfe, 1999) in order to identify groups where survival or germination differed from that of the others. To test whether the performance of P. laevigata seedlings differed between soil mixtures, we used analysis of covariance (Sokal and Rohlf, 1995). In this analysis, we used each P. laevigata seedling as a sampling unit, with soil treatment (four levels, excluding peat moss due to the accidental loss of these seedlings) as the factor and number of seedlings in each pot as a covariable. The response variables were seedling height, root length, aboveground biomass, belowground biomass and the aboveground: belowground biomass ratio. To test whether the T. recurvata organic extract concentrations influenced seed germination of P. laevigata, we performed a second order polynomial regression analysis (Sokal and Rohlf, 1995). In this analysis, the independent variable was the extract expressed as ln (extract concentration þ 0.001), while the response variable was seed germination (%). We used the same analysis to test whether extract concentration influenced radicle length in P. laevigata seedlings. To determine whether paired branches of P. laevigata with contrasting T. recurvata loads differed in diameter, length, T. recurvata biomass, total number of shoots, number of live shoots and number of dead shoots, we used a paired t test for each variable (Sokal and Rohlf, 1995). We performed a Wilcoxon signed rank test for those variables that did not fulfill the assumptions of normality (Hollander and Wolfe, 1999). 3. Results 3.1. Nutrient content of scrubland soils from below P. laevigata individuals with different T. recurvata loads The PCA explained 57.6% of the variation in two principal components (PC). PC 1 explained 31.5% of the variation; this
3.2. The effect of T. recurvata dry material and organic extracts on early life stages of P. laevigata Germination capacity of the seed batch used in the greenhouse experiment was 100%, and full germination took place within four days. During the greenhouse experiments, mean temperature was 19.5 C in winter (min 7.8; max 33.5), 22.3 C in spring (min 10.6; max 39.6), and 20.0 C in summer (min 10.6; max 35.2). During the course of the experiment, the mean daily pattern of temperature and relative humidity changed from winter to summer, with the highest temperatures recorded in spring and greatest humidity occurring in summer (Appendix A). Seed germination of P. laevigata occurred only in the first week of observation (Fig. 2a), and differed significantly between soil mixtures (c2 ¼ 88.4, d.f. ¼ 4, P < 0.0001) (Fig. 2a). Highest germination occurred in the peat moss and in the mixture of soil with litter of T. recurvata and P. laevigata. Germination was similar in the soil with litter of P. laevigata and in the soil with litter of T. recurvata, while germination in the latter soil/litter mixture did not differ significantly from germination in the soil with no added organic matter (Fig. 2a). Survival of P. laevigata seedlings differed between soil mixtures (c2 ¼ 24.2, d.f. ¼ 4, P < 0.0001) (Fig. 2b). There was a descending gradient of seedling survival from the peat moss to the soil mixed with litter of P. laevigata (Fig. 2b). Root length (F ¼ 0.15, d.f. ¼ 3, 65, P > 0.05) and the ratio of above- to belowground biomass (F ¼ 2.4, d.f. ¼ 3, 65, P > 0.05) in the seedlings were both similar between soil mixtures (Table 2), but seedlings differed significantly among soil mixtures in terms of height (F ¼ 5.0, d.f. ¼ 3, 65, P < 0.005), aboveground biomass (F ¼ 8.0, d.f. ¼ 3, 65, P < 0.0005), and belowground biomass (F ¼ 6.0, d.f. ¼ 3, 65, P < 0.0005). The general pattern for these variables was that the performance of P. laevigata seedlings was poorest in soil with T. recurvata litter (Table 2). There was no effect of the hexanic, methanolic and dichloromethanic extracts of T. recurvata on the germination of P. laevigata seeds (all regression F < 1.5, P > 0.05, r2 < 0.14). Hexanic extracts did not affect the radicle length of the P. laevigata seedlings (F ¼ 1.46, d.f. ¼ 2, 18, P ¼ 0.63, r2 ¼ 0.005), but the dichloromethanic (F ¼ 13.04, d.f. ¼ 2, 18, P < 0.0001, r2 ¼ 0.14) and methanolic (F ¼ 12.12, d.f. ¼ 2, 18, P < 0.0001, r2 ¼ 0.15) extracts had a smooth quadratic effect, causing reduced radicle length (Fig. 3).
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Fig. 1. PCA ordination of soil samples collected from below three different canopy conditions in five locations in the scrublands of the Chihuahuan desert on the San Luis Potosi high plateau, Mexico. Canopy conditions reflect increasing abundance of the epiphyte T. recurvata; bare soil (empty circles), canopy <3 m in height (asterisks) and canopy >4 m in height (solid squares). On each axis, the explained variance and associated soil properties are shown.
3.3. Relationship between T. recurvata load and number of live and dead shoots on branches on P. laevigata and the frequency of branches with high T. recurvata loads Branches of P. laevigata with T. recurvata cover of <50% were similar to branches with a cover >50%, in terms of diameter (W ¼ 286, P > 0.05), length (t ¼ 1.78, d.f. ¼ 49, P > 0.05) and total number of shoots (W ¼ 141, P > 0.05) (Table 3). Branches with T. recurvata cover of <50% presented more live shoots than branches with >50% cover (W ¼ 502, P < 0.01); while branches with >50% cover featured a higher T. recurvata biomass (t ¼ 9.52,
d.f. ¼ 49, P < 0.001) and a greater number of dead shoots (t ¼ 6.11, d.f. ¼ 49, P < 0.001). In the P. laevigata sampled, the majority of the branches (499) had <25% T. recurvata cover, 46 branches with coverage of between 26 and 50%, and 20 branches with >50%, presenting a distribution that was significantly biased (c2 ¼ 770, d.f. ¼ 2, P < 0.0001) toward branches with low coverage of T. recurvata. We found an association (c2 ¼ 22, d.f. ¼ 2, P < 0.0001) between T. recurvata cover and the frequency of dead branches. The proportion of dead branches was 0.04 in the cover category of <25% (22 dead branches from a total of 499 branches observed), but increased to 0.15 in the 26e50% cover
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Table 1 Chemical properties of soil samples, and Tillandsia recurvata and Prosopis laevigata leaves. Soil samples come from below three different canopy conditions. All samples were collected in five locations in the scrublands of the Chihuahuan desert, on the San Luis Potosi high plateau, Mexico. Canopy conditions reflect increasing abundance of the epiphyte T. recurvata, from bare soil to soil below a canopy >4 m in height. Mean values (1 standard deviation) are presented for each variable. Different letters between soil samples indicate significant differences between median values, according to a KruskalleWallis test, followed by the Nemenyi multiple comparison procedure (P < 0.05). Variable
Bare soil (n ¼ 30)
Clay (%) Sand (%) Silt (%) pH Organic matter (%) Ctotal (%) Ca (cmol/kg) K (cmol/kg) Mg (cmol/kg) Na (cmol/kg) Ntotal (ppm) Pextractable (ppm)
29.55 36.89 33.85 6.92 1.50 0.87 25.51 2.10 1.68 0.47 0.16 41.61
Soil under P. laeviagata of canopy height: <3 m (n ¼ 15)
4.05 7.11 5.32 0.84 1.05b 0.61b 11.53n.s. 0.68n.s. 0.66n.s. 0.15n.s. 0.07n.s. 40.62b
31.52 37.34 31.12 6.47 2.46 1.43 25.82 2.29 1.68 0.48 0.18 43.84
3.45 6.07 5.30 0.89 1.54a 0.89a 14.08 0.97 0.70 0.14 0.06 43.12b
>4 m (n ¼ 15) 32.72 37.23 30.05 6.45 4.62 2.68 25.41 2.78 2.51 0.52 0.29 49.58
category (7 dead branches from a total of 46 branches) and to 0.25 in the >50% cover category (5 dead branches from a total of 20 branches). 4. Discussion Epiphytes play an important role in the nutrient dynamics of forest and woodlands because they reach high biomass in the trees and can capture atmospheric nutrients that can be subsequently released to the ecosystem through stemflow, apogeotropic roots and when the epiphytes fall to the forest floor (Coxson and Nadkarni, 1995). In the study area, T. recurvata is the only holoepiphyte; it can present a high biomass on P. laevigata (up to 0.4 ton/ha) and has nitrogen-fixing bacteria on its leaves (Puente and Bashan, 1994). This feature enables T. recurvata to contribute both organic matter and nutrients to the soil below. However, our results suggest that the nutrient content of T. recurvata is poor compared to both P. laevigata leaves and the nutrient contents of other epiphytic communities. The P and N content of T. recurvata is lower than that observed in leaves of Tillandsia capillaris in a dry woodland in Argentina, which was reported to be 3.1 mg/g for P and 24.4 mg/g for N (Abril and Bucher, 2009). It also was lower than the nutrient content found in humid environments. For example, the P and N content of T. recurvata was lower than the P content of the debris captured by bromeliads (0.19 mg/g) and in the bromeliad leaves (0.48 mg/g) in a humid tropical forest in Puerto Rico (Richardson et al., 2000). In Neotropical montane cloud forests, the P content of epiphyte dead organic matter (EDOM) ranged from 0.54 to 0.66 mg/g in Colombia (Hofstede et al., 1993), from 2.19 mg/g to 3.50 mg/g in Mexico (Flores-Palacios and García-Franco, 2004) and from 1.25 mg/g to 1.49 mg/g in American coniferous forest (Lang et al., 1980; Nadkarni, 1984). The N content of T. recurvata was lower than that found in the bromeliad leaves of a humid tropical forest in Puerto Rico (7.20 mg/ge7.50 mg/g; Richardson et al., 2000), and was lower than the N content of debris captured by bromeliads (14.50 mg/ge18.10 mg/g) in the same study, and in the EDOM of Neotropical montane cloud forest in Colombia (8.12 mg/ge 8.53 mg/g) (Hofstede et al., 1993) and Mexico (17.96 mg/ge 19.15 mg/g) (Flores-Palacios and García-Franco, 2004). Perhaps the low nutrient content of T. recurvata relative to those studies evaluating the EDOM nutrient content could be due to the fact that EDOM contains nutrients coming both from the epiphytes themselves and from another sources (e.g. invertebrates, detritus and dust). However, T. recurvata is an atmospheric Tillandsia with a
4.07 6.53 4.76 1.01 2.16a 1.25a 10.49 1.23 1.17 0.20 0.11 45.83a
Tillandsia recurvata (n ¼ 4)
4.98 90.54 52.52 41.84 9.75 4.44 5.77 0.73 41.82
013 1.24 072 1.02 0.24 1.18 0.47 0.06 4.43
Prosopis laevigata (n ¼ 2)
5.26 93.01 53.95 71.25 27.36 14.54 14.47 2.44 201.99
0.12 0.08 0.05 0.76 0.03 1.12 1.62 0.12 5.46
Ratio Prosopis/ Tillandsia
1.0 1.0 1.7 2.8 3.3 2.5 3.3 4.8
limited capacity for retaining EDOM in its roots and between its leaves. Being of low nutrient content, T. recurvata is likely to contribute only slightly to the creation of fertility islands in the studied scrubland; however, principal component analysis separated the soil samples collected from below the crown of P. laevigata >4 m in height, where the T. recurvata load is high, from the rest of the soil samples. The importance of the contribution of T. recurvata to the soil composition under the taller individuals of P. laevigata should not be discounted, since T. recurvata provides both organic matter and nutrients. Nonetheless, the contribution made by the nutrient rich leaves of the tree is likely to be of relatively greater importance. The greenhouse experiments give us a better understanding of the role of T. recurvata when incorporated into the soil; the increased organic matter clearly promoted seed germination and seedling survival in P. laevigata, although this could have been the result of increased water retention rather than the increase in nutrient availability. Surprisingly, the greenhouse and in vitro experiments also showed that T. recurvata could negatively affect the performance of P. laevigata seedlings. Greenhouse experiments showed that seedlings of P. laevigata were shorter and presented lower above- and belowground biomass when grown in soils with added T. recurvata material. Perhaps the short height and low aboveground biomass were caused by a reduction in the development of root biomass, especially since the in vitro experiments showed that dichloromethanic and methanolic extracts of T. recurvata reduced root development in P. laevigata. T. recurvata contains various compounds that could be allopathic (Valencia-Díaz et al., 2012; de Queiroga et al., 2004) and organic extracts of T. recurvata have been shown to reduce the germination of sympatric epiphytic species (Valencia-Díaz et al., 2012). Among these allelopathic compounds, diterpene phytol is known as a root growth inhibitor and is present in dichloromethanic extracts (Valencia-Díaz et al., 2012). Our results suggest that, although T. recurvata is a source of organic matter and nutrients for the scrubland soil, it could also exert an allelopathic effect on the seedlings of its host. In addition to the probable allelopathic effect of T. recurvata on scrubland soil, we found that P. laevigata branches with >50% T. recurvata cover had more dead shoots and less live shoots than branches with <50% cover. The same effect of T. recurvata has previously been reported in the tree Parkinsonia praecox (Fabaceae) in a scrubland in the semiarid region of Tehuacan, Mexico (Montaña et al., 1997). Our evidence suggests that T. recurvata directly reduced the growth of P. laevigata. Perhaps the damage that
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Fig. 2. Germination (a) and seedling survival (b) of Prosopis laevigata seeds/seedlings sown in different soil mixtures. Soil (empty circles); soil with T. recurvata litter (asterisks); soil with P. laevigata litter (solid triangles); soil with litter of T. recurvata and P. laevigata (empty squares); and peat moss (solid circles). Different letters indicate significant differences between proportions (GehaneWilcoxon test, P < 0.05).
T. recurvata exerts on branches of P. laevigata is physical, since evidence in the same study area shows that P. laevigata branches that support T. recurvata can present anatomical changes in the wood; however, the hydraulic conductivity of these branches must be studied in order to determine whether such anatomical changes affect tissue function (Rodríguez et al., 2007). Among the branch population, only 3.5% of the branches presented a T. recurvata cover greater than 50%, suggesting that, overall, the direct negative effects of T. recurvata may be slight, even though the proportion of dead branches was highest in this T. recurvata cover category. In view of the fact that epiphytes can be highly abundant in scrublands and forests and can contain large amounts of nutrients,
it is hypothesized that they can exert an indirect positive influence on the ecosystem. Conversely, because epiphytes can be present in high concentrations on their hosts, it has also been suggested that they may have a direct harmful effect upon them. Our results suggest that, contrary to expectations, the only holoepiphyte in the study area could have a negative allelopathic effect on the soil in addition to the expected direct negative effect on the branches of P. laevigata. In the studied area, this could also be occurring in the sympatric Acacia trees that can contain T. recurvata loads as large as those of P. laevigata, but further study is required in order to confirm this. T. recurvata is a widely distributed vascular epiphyte (Smith and Downs, 1977) and is the dominant epiphyte in some
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Table 2 Mean values (1 standard deviation) of variables associated with Prosopis laevigata seedling performance following 20 weeks growth in different soil mixtures. Different letters indicate significant differences between mean values (Tukey test, P < 0.05). n.s. ¼ non-significant differences between means. Variable
Soil (n ¼ 8)
Soil with T. recurvata litter (n ¼ 27)
Soil with P. laevigata litter (n ¼ 12)
Soil with litter of T. recurvata and P. laevigata (n ¼ 22)
Seedling height (cm) Root length (cm) Aboveground biomass (g) Belowground biomass (g) Above/below ground biomass ratio
12.46ab 24.84n.s. 0.31a 0.20ab 2.42n.s.
4.68b 19.57 0.05b 0.05b 1.10
12.81a 25.93 0.32a 0.27a 1.18
10.60a 23.42 0.19ab 0.16a 1.09
(3.59) (7.99) (0.31) (0.24) (3.12)
(1.21) (12.06) (0.03) (0.03) (1.33)
(4.83) (9.75) (0.19) (0.16) (0.27)
(6.35) (10.16) (0.18) (0.11) (0.45)
Fig. 3. Relationship between the radicle length of Prosopis laevigata seedlings growing on organic extracts of the epiphyte Tillandsia recurvata, and organic extract concentration. n.s. ¼ non-significant relationship.
tropical forests (Vergara-Torres et al., 2010). In addition to constituting nutrient sources, epiphyte communities dominated by T. recurvata could in fact be sources of allelopathic compounds in the decaying litter, a phenomenon suggested in tree litter in previous studies (Lodhi, 1978; Mazzoleni et al., 2007). Perhaps a synergistic negative effect of T. recurvata on the soil and on the branches of their host can cause decay in some trees that are highly
Table 3 Mean values (1 standard deviation) of variables measured in Prosopis laevigata branches (diameter 1 cm) with different categories of Tillandsia recurvata cover. Different letters indicate significant differences between mean values (t or Wilcoxon Paired test, P < 0.05, see text). n.s. ¼ non-significant differences between means. Variable
T. recurvata cover
Branch diameter (mm) Branch length (cm) T. recurvata biomass (g) Total number of shoots Number of live shoots Number of dead shoots
13.1n.s. 64.0n.s. 6.8b 15.2n.s. 11.5a 3.7b
<50%
>50%
12.0 18.8 7.5 8.0 6.9 3.1
11.9 59.1 21.0a 16.6 9.5b 7.1a
2.3 18.0 12.0 10.4 8.1 4.8
infested by epiphytes (e.g. Benzing and Seemann, 1978). Experimental research focusing on both components of the ecosystem (soil and canopy) is required in order to broaden our understanding of this aspect of the interaction between these species. Acknowledgments Samuel Arechaga helped during fieldwork. The comments and critique of K. MacMillan and two anonymous reviewers improved the manuscript. This research was supported by a grant from the Oficina de Sanidad Forestal, Comisión Nacional Forestal (Project 10140) to JGGF, a PROMEP grant to AFP (PROMEP/103.5/05/1901) and INECOL (20030-10144). An earlier version, circumscribed to the greenhouse experiments only, was presented by CLBD as a Bachelors thesis at the Faculty of Biological Sciences, Universidad Autónoma del Estado de Morelos, Mexico. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jaridenv.2014.02.010.
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