Ecological facilitation between two epiphytes through drought mitigation in a subtropical rainforest.

Positive species interactions (facilitation) play an important role in shaping the structures and species diversity of ecological communities, particularly under stressful environmental conditions. Epiphytes in rainforests often grow in multiple-species clumps, suggesting interspecies facilitation. However, little is known about the patterns and mechanisms of epiphyte co-occurrence. We assessed the interactions of two widespread epiphyte species, Asplenium antiquum and Haplopteris zosterifolia, by examining their co-occurrence and size-class association in the field. To elucidate factors controlling their interactions, we conducted reciprocal-removal and greenhouse-drought experiments, and nutrient and isotope analyses. Forty-five percent of H. zosterifolia co-occurred with A. antiquum, whereas only 17% of A. antiquum co-occurred with H. zosterifolia. Removing the fronds plus substrate of A. antiquum reduced the relative frond length and specific leaf area of H. zosterifolia, but removing fronds only had little effect. Removing H. zosterifolia had no significant effects on the growth of A. antiquum. H. zosterifolia co-occurring and not co-occurring with A. antiquum had similar foliar nutrient concentrations and δ(15)N values, suggesting that A. antiquum does not affect the nutrient status of H. zosterifolia. Reduced growth of H. zosterifolia with the removal of A. antiquum substrate, together with higher foliar δ(13)C for H. zosterifolia growing alone than those co-occurring with A. antiquum, suggest that A. antiquum enhances water availability to H. zosterifolia. This enhancement probably resulted from water storage in the substrate of A. antiquum, which could hold water up to 6.2 times its dry weight, and from reduced evapotranspiration due to shading of A. antiquum fronds. Greater water loss occurred in the frond-clipped group than the unclipped group between days 3-13 of the drought treatment. Our results imply that drought mitigation by substrate-forming epiphytes is important for maintaining epiphyte diversity in tropic and subtropic regions with episodic water limitations, especially in the context of anthropogenic climate change.

Introduction

Positive species interactiopan class="Chemical">ns (i.e. facilitation) play an important role in community dynamics [1], [2] and ecosystem functions [3]. A number of studies have highlighted that interspecies facilitation increases with abiotic factors leading to plant stress [4], [5], [6], [7]. For example, a study of forest restoration in the Mediterranean Basin indicates that nurse shrubs had a stronger facilitative effect on seedling survival and growth on drier than wetter slopes [8]). On a wave-exposed rocky shore, the facilitative effect of n class="Species">goose barnacles onpan> mussel survivorship was positively related to the combinpan>ationpan> of physical disturbanpan>ce anpan>d thermal stress [9]. However, no studies have examinpan>ed facilitationpan>s amonpan>g epiphytes or explored the role of such inpan>teractionpan>s inpan> epiphyte diversity.

Epiphytes contribute up to 30% of species richpan class="Chemical">ness in some tropical rainforests and are thus an important component of the extraordinarily high biodiversity in tropical rainforests [10]. Epiphytes lack access to soil n class="Chemical">water anpan>d nutrients released from minpan>eral weatherinpan>g anpan>d litter decompositionpan>. Although N anpan>d P have been shown to limit the growth anpan>d distributionclass="Chemical">pan> of plants inpan> forest canpan>opies [11], [12], nutrient limitationpan> onpan> epiphyte growth is not well documented. The lack of direct access to soil pan> class="Chemical">water is a key factor confining most epiphytes to humid forest ecosystems. However, even in tropical rainforests, there are rainless periods that may impose drought stress on epiphytes. For example, in a subtropical rainforest in northeastern Taiwan where it rains more than 220 days annually and mean rainfall is 4240 mm annually [13], there were six rainless periods exceeding 7 days over 3 years [14]. Likewise, rainless periods of 8 days or longer occurred 47 times in 2 years in the tropical rainforests of Puerto Rico [14]. Even in forests with high rainfall, many vascular epiphytes exhibit xeric adaptations such as high water use efficiency and low leaf area to dry mass ratios (i.e. specific leaf area, SLA), suggesting that they are often under water stress [15], [16], [17].

Humid subtropical forests, which contaipan class="Chemical">n abundant epiphytes, cover approximately 58% of the land area of Taiwan. The Fushan Experimental Forest is particularly rich in epiphytes with a total of 65 n class="Species">vascular epiphytes, accountinpan>g for 12% of the 515 pan> class="Species">vascular plants species [18]. Like the rainforests in Costa Rica where as many as 126 epiphyte species have been documented in a single tree canopy [19], epiphytes are often observed in multiple-species clumps in the Fushan Experimental Forest. Many epiphytes may grow together simply because they favor similar shaded microhabitats. However, the frequent co-occurrence of some of the species suggests that other mechanisms may be operating. Dispersal limitations and host-epiphyte interaction have been proposed to explain the clumped distribution or meta-communities of epiphytes [20]. In addition, neighbor’s buffering is probably important in maintaining epiphyte diversity and may explain the clumped distributions of epiphyte species in forest ecosystems [20], [21]. Asplenium antiquum Makino (Fig. 1) is one of the most common vascular epiphytes in Taiwan and other parts of the old-world tropical forests [22]. The extraordinary water holding capacity of the substrate formed underneath this species is considered the key factor that allows it to persist in regions with droughts lasting several weeks, such as in the northeastern Australia [23]. The substrate of A. antiquum may also benefit co-occurring epiphytes, such as Haplopteris zosterifolia (Willd.) E. H. Grane (Fig. 1).

Figure 1

Co-occurring Asplenium antiquum and Haplopteris zosterifolia.

Co-occurring Asplenium antiquum and Haplopteris zosterifolia.

Co-occurring papan class="Chemical">n class="Species">Asplenium antiquum anpan>d n class="Species">Haplopteris zosterifolia.

In this study we evaluate the ipan class="Chemical">nteractions between n class="Species">A. antiquum anpan>d pan> class="Species">H. zosterifolia in the Fushan Experimental Forest in northeastern Taiwan. We hypothesize that there is a one-way facilitation from A. antiquum to H. zosterifolia through water and/or nutrient enrichment provided by the substrate of A. antiquum. To test this hypothesis, we conducted a field survey on co-occurrence and size association of the two species, and assessed the growth response to reciprocal removal. Furthermore, we tested three potential mechanisms, drought mitigation, nutrient enhancement, and shading, that could have shaped the A. antiquum and H. zosterifolia interaction through a removal experiment, a drought experiment, and nutrient and stable-isotope analyses. This study offers new insights on the patterns and mechanisms of epiphyte interactions and explores the significance of such interactions in maintaining epiphyte diversity.

Materials and Methods

Ethics Statement

Dr. Hsiang-Hua Wang, the director of the Fushan Experimental Forest, issued the permission for conducting the study at the site.

Study Site and Epiphyte Species

The Fushan Experimepan class="Chemical">ntal Forest (10 km2) is a subtropical rainforest in northeastern Taiwan (24° 34" N, 121° 34′ E). The area has 65 n class="Species">vascular epiphytes belonpan>ginpan>g to 20 families anpan>d 42 genera [24]. Between 1993 anpan>d 2007, the anpan>npan>ual precipitation of the area varied from 2900 mm to 6650 mm with a meanpan> of 4240 mm [25]. The anpan>nual meanpan> temperature was 18.2°C with a low monpan>thly average of 11.8°C inpan> February anpan>d a maximum of 24.1°C inpan> July [25]. The relative humidity is above 90% most of the time throughout the year.

Both n class="Species">A. antiquumpan> and pan> class="Species">H. zosterifolia are long-lived species abundant in the Fushan Experimental Forest. A. antiquum is a litter-basket epiphyte with a bowl-shaped crown that can effectively intercept canopy litter to form humus-rich substrate. The size of n class="Species">A. antiquum reaches up to 2 m in crown diameter, but individuals of various sizes are common at our study site [26]. A. antiquum exists at all locations on the host trees, from less than 1 m above ground to near the top of the tree canopies and from the trunk to small branches. Like A. antiquum, the size of H. zosterifolia also varies greatly with frond length reaching up to 1.5 m and the number of fronds as high as >100. In contrast to the bowl-shaped crown of A. antiquum, the fronds of H. zosterifolia are pendent (Fig. 1).

Field Survey: Co-occurrence of A. antiquum and H. zosterifolia

We conducted the field survey opan class="Chemical">n the co-occurrence between n class="Species">A. antiquum anpan>d pan> class="Species">H. zosterifolia along a first-order stream at Fushan Experimental Forest. We located 100 individuals of A. antiquum and 100 individuals of H. zosterifolia on the trees starting from approximately 50 m downstream from the weir of Experimental Watershed #1. On the east side of the stream we first identified 50 A. antiquum and then 50 H. zosterifolia, and on the west side we first identified 50 H. zosterifolia and then 50 A. antiquum. We surveyed every tree within 1 m off the stream margin. If there was more than one individual of the focal epiphyte species on a tree, we recorded every other one from ground to the canopy top of the host tree. The crown size of each A. antiquum was measured, and the 100 individuals were grouped into five categories: <50 cm, 51–100 cm, 101 -150 cm, 151–200 cm, and >200 cm following [26]. The H. zosterifolia plants were grouped into four size classes based on the maximum length of their fronds: 40–80 cm, 81–120 cm, 121–160 cm, and >160 cm. We did not include H. zosterifolia with a frond length less than 40 cm because they were easily overlooked in high canopies. For each of the 100 individuals of A. antiquum and 100 individuals of H. zosterifolia, we recorded the host tree species and all co-occurring epiphytes. Epiphytes were considered co-occurring if their roots/stems were in contact with each other.

Removal Treatment

To examine the interaction between the two epiphytic ferns, we conducted a removal experiment on December 20, 2009 on 55 pairs of co-occurring n class="Species">A. antiquum anpan>d pan> class="Species">H. zosterifolia. Because of the difficulties in accessing epiphytes high in tree canopies, we only used those that grew less than 5 m from the forest floor. We assigned the 55 pairs into four treatments: H. zosterifolia removal (13 pairs), A. antiquum frond removal (13 pairs), A. antiquum frond plus substrate (frond+substrate) removal (14 pairs), and control (15 pairs in which neither species is removed). H. zosterifolia may benefit from co-occurring with A. antiquum through enhanced availability of nutrient and water provided by the substrate of A. antiquum. In addition, H. zosterifolia growing underneath A. antiquum may benefit from reduced evapotranspiration due to the shading provided by the fronds of A. antiquum. Therefore, we had two different A. antiquum removal treatments, a frond removal treatment and a frond+substrate removal treatment. Because the roots of H. zosterifolia often grow into the substrate of A. antiquum, it was not possible to remove the substrate completely without damaging the H. zosterifolia growing underneath. We removed as much of the substrate as possible while trying to avoid physically damaging the roots of H. zosterifolia. The treatments were assigned randomly except for eight pairs growing right next to the trails that were open to the public, which were assigned to the control group to minimize interference from tourists. One-way analysis of variance (ANOVA) showed no systematic difference between these eight pairs and the other seven pairs in the control group in all of the pre-treatment measurements taken in this study (frond number and length, SLA, nutrient concentration and light availability). Thus we combined the measurements from the two control sub-groups.

Growth, SLA and Foliar Nutrient

To assess the growth response to reciprocal removal, prior to apan class="Chemical">nd 5 times after the removal treatment (January, February, April, June and August 2010), we measured the length and number of fronds of n class="Species">A. antiquum anpan>d pan> class="Species">H. zosterifolia. The frond length was determined on three randomly selected un-grazed and mature green fronds from each individual. A frond was considered mature when all sporangia were brown. The SLA of the frond samples collected in January and August was determined by scanning the fronds and calculating the areas with Image J (National Institutes of Health, USA). During each of the five post-treatment measurements, we collected one complete frond of A. antiquum and 4–5 fronds of H. zosterifolia for nutrient analysis. The frond samples were stored at 4°C without preservatives, cleaned with de-ionized water to remove dust, fungi and other materials, oven-dried at 80°C for 72 hours, and ground. The concentrations of K, Ca, Mg, and P were determined after ashing and dissolving ash in HCl using ICP-OES (Inductively Couple Plasma Optical Emission Spectroscopy, ICP-OES, Jobin Yvon Horiba Group, NY2000, Edison, USA). Total C and N were determined by dry-combustion using an elemental analyzer (Thermo Finnigan NA1500, Bremen, Germany).

Light Availability above and below A. antiquum

To estimate light availability, we took hemispherical photographs for each pair of the two epiphyte species, in October 2009 prior to, apan class="Chemical">nd in December 2009, following the removal treatment at the same positions. The two sets of photographs were taken two months apart because hemispherical photographs cannot be taken when it is raining and it rains very often (more than 220 days per year, [25]) at Fushan Experimental Forest. These photographs were taken at one position above the fronds at the center of the substrate, and at three equally spaced positions around the substrate below the n class="Species">A. antiquum fronpan>ds but above the pan> class="Species">H. zosterifolia fronds. We determined direct site factor (DSF) and indirect site factor (ISF) for each photograph using HemiView 3.43 [27]. The DSF and ISF are the proportion of direct and indirect (diffuse) solar radiation that is transmitted through the plant canopy and reaches the location of measurement [28], [29].

Water-holding Capacity and Drought Response of A. antiquum

To examine the effects of the fropan class="Chemical">nds of n class="Species">A. antiquum onpan> reducinpan>g substrate n class="Disease">water loss through shading, we conducted a drought experiment. Ten individuals of n class="Species">A. antiquum with their substrates containing adventitious roots and organic materials were obtained from separate tree trunks at the Fushan Experimental Forest. All plants with their attached substrate were then transplanted to the greenhouse at Tunghai University, Taichung, Taiwan. To ensure similar initial water status of each individual before drought treatment, we saturated the substrates of all plants with water for 3 days.

To test the effect of n class="Species">A. antiquumpan> fronds onpan> the pan> class="Chemical">water availability of the substrate, the fronds of five of the ten individuals were clipped. The weights of clipped and unclipped epiphyte+substrate replicates were measured daily at 4 pm throughout the drought experiment. The drought experiment was performed inside the greenhouse where relative humidity was maintained above 90% (except on days 17 and 18, with daytime relative humidity of 79% and 88%, respectively) to mimic the environmental condition under the canopy of Fushan Experimental Forest. All plants, with or without fronds, were not watered for 20 days to simulate a drought. Air temperature inside the greenhouse was modulated by an automatic ventilation system; mean daytime and nighttime temperatures were 25.1°C and 23.0°C, respectively. After the drought treatment, the fronds in the unclipped group were clipped. The substrates of both groups were re-saturated with water. After measuring the weight of each substrate under field capacity, all substrates and fronds were dried separately to constant weight.

Because n class="Chemical">waterpan> stored in the substrate of pan> class="Species">A. antiquum probably plays an important role on its interaction with H. zosterifolia we measure n class="Chemical">water-holding capacity of the substrate. The maximum water-holding capacity of each substrate was determined by subtracting the dry weight of the substrate from that of the substrate under field capacity. The fresh weights of the fronds in the unclipped group were determined by subtracting the substrate weight under field capacity from the total weight at day 0 (maximum water holding status of the whole plant).

The potential quantum yield (Fv/Fm) was measured daily around 10 am on one randomly selected frond of each frond-unclipped plant using pulse-amplitude modulated photosynthesis yield analyzer (MINI-PAM; Heinz Walz GmbH, Effeltrich, Germany). Leaves were dark-adapted for 40 minutes before measurements. The Fv/Fm was calculated as Fv/Fm = (Fm−F0)/Fm, where F0 is ground fluorescence level of dark adapted leaf and Fm is the maximum fluorescence yield of leaf when a saturating light pulse of 10000 µmol m−1 s−1 is applied for 0.8 second.

Isotope Analysis

To examine the effects of papan class="Chemical">n class="Species">A. antiquum onclass="Chemical">pan> the n class="Chemical">water and n class="Chemical">nitrogen availability of H. zosterifolia, we performed isotopic analyses of carbon and nitrogen on (1) the fronds of nine pairs of co-occurring H. zosterifolia and A. antiquum, (2) the fronds of nine individuals of H. zosterifolia without co-occurring A. antiquum, and (3) the substrate of each of the nine A. antiquum. Two fronds and approximately 100 g of substrate were collected from each sampled individual. Four of the nine samples of each of the three groups were collected on March 2011 and the other five collected on June 2012. These samples were dried at 80°C and ground to powder for elemental and isotopic analyses in the laboratory. About 3 mg of each dried sample was weighed into a tin capsule. Total carbon and nitrogen percentages were measured with a Costech 4010 Elemental Analyzer. Stable isotopes of carbon and nitrogen were determined with a Delta V Advantage, Thermo Fisher isotope ratio mass spectrometer. Isotopic ratios were reported in δ notation (δ = ([Rsample/Rstandard]–1) • 1000‰, where R = 13C/12C or 15N/14N). Analytical precision was better than 0.3‰ for both δ13C and δ15N on laboratory standards and on duplicate samples. Isotope ratios were expressed relative to the international standards: Vienna Peedee Belemnite (VPDB) for δ13C and atmospheric N2 (AIR) for δ15N.

Statistical Analysis

We used a Chi-square test to examine if the frequepan class="Chemical">ncy and size-class distributions of n class="Species">A. antiquum anpan>d pan> class="Species">H. zosterifolia differed among the three groups (growing alone, co-occurring with each other, and co-occurring with other epiphytes species). We used one-way ANOVA to compare the frond length and number of the two epiphytes before the removal treatment among the treatments. We compared light availability prior to the removal among the four treatments and between above and below A. antiquum using two-way ANOVA with treatments and positions as the independent variables. A similar analysis was conducted for light availability after the removal treatment. Because there was a significant treatment x position effect, we compared light availability above and below A. antiquum for each treatment using paired t-test. We compared changes in light availability below A. antiquum following the removal among the four treatments using one-way ANOVA.

We used one-way Apan class="Chemical">NOVA with repeated measurements to compare frond number and length of 1) n class="Species">A. antiquum between the conpan>trol group anpan>d pan> class="Species">H. zosterifolia removal group, and 2) H. zosterifolia among the control group, the A. antiquum frond removal group and A. antiquum frond+substrate removal group for the five post-treatment measurements. In the analysis, the frond number and length were expressed as the ratio between each of the five post-removal measurements to the pre-removal measurement. This adjusts for pre-existing differences prior to the treatment although the pre-existing differences were insignificant as will be shown in the result section. We also used one-way ANOVA with repeated measurements to compare nutrient concentration of 1) A. antiquum between the control and H. zosterifolia removal groups, and 2) H. zosterifolia among the three treatments. When among-group differences were significant, we used Fisher’s least significant difference (LSD) tests for post-hoc comparisons. We compared SLA of 1) A. antiquum between the control and H. zosterifolia removal groups using a t-test, and 2) H. zosterifolia among the three treatments in January using one-way ANOVA. We used t-tests to examine the difference in SLA of H. zosterifolia and A. antiquum between January and August 2010 for each of the treatment groups. We did not use paired-t test because the SLA in the two different months were measured on different fronds.

The daily difference of the relative weight ipan class="Chemical">n n class="Species">A. antiquum to fully saturated conpan>ditionclass="Chemical">pan> between fronpan>ds clipped anpan>d founds unclipped groups was examinpan>ed usinpan>g t-test. We assessed the temporal trend of Fv/Fm usinpan>g simple linpan>ear regressionpan>. The differences of both δpan> class="Chemical">13C and δ15N among four groups (Asplenium substrate, Asplenium frond, Haplopteris growing alone, and Haplopteris co-occurring with Asplenium) were tested using one-way ANOVA followed by Fisher’s LSD tests for post-hoc comparisons when the among-group differences were significant.

Results

Association between H. zosterifolia and A. antiquum

Of the 100 n class="Species">A. antiquumpan> plants, 19 did not co-occur with anpan>y other epiphyte species, anpan>d the other 81 co-occurred with a total of 18 epiphyte species. pan> class="Species">H. zosterifolia was the second (17 individuals) most common epiphyte co-occurring with A. antiquum, following Pothos chinensis (Raf.) Merr. (Araceae) (28 plants) and tying with Procris laevigata Bl. (Urticaceae) (also 17 plants). Similarly, only 12 of the 100 H. zosterifolia plants did not co-occur with other epiphytes, and the other 88 n class="Species">H. zosterifolia co-occurred with a total of 18 epiphyte species. A. antiquum was the most common epiphyte co-occurring with H. zosterifolia. A total of 45 of the 100 H. zosterifolia plants existed underneath A. antiquum. The frequency distribution of the two species in the three groups: growing alone, co-occurred with each other, and co-occurred with other species, differed significantly (Chi-square = 20.9, df = 2, p<0.001). The number of A. antiquum co-occurring with H. zosterifolia was significantly lower than expected from evenly distributed among the three groups, whereas the number of H. zosterifolia co-occurring with A. antiquum was significantly greater than expected.

The size-class distribution did pan class="Chemical">not differ among the three groups for n class="Species">A. antiquum (Chi-square = 13.16, df = 8, p = 0.11; Fig. 2a), but differed significanpan>tly for npan> class="Species">H. zosterifolia (Chi-square = 41.9, df = 6, p<0.001). The H. zosterifolia in the group co-occurring with A. antiquum had 77% plants in the size classes 3 (120–160 cm) and 4 (>160 cm), whereas those in the other two groups had more than 80% plants in the size classes 1 (40–80 cm) and 2 (80–120 cm) (Fig. 2b). None of the 12 growing-alone H. zosterifolia was in the size class 4 (the largest), and only one was in size class 3.

Figure 2

Size class frequency of Asplenium antiquum and Haplopteris zosterifolia co-occurring with different epiphyte species.

Size class frequency of a) 100 Asplenium antiquum and b) 100 Haplopteris zosterifolia co-occurring with different epiphyte species. Size class of A. antiquum was determined by the crown size. 1: <50 cm, 2∶50–100 cm, 3∶101 -150 cm, 4∶151–200 cm, 5>200. Size class of H. zosterifolia was determined by the maximum length of fronds. 1∶40–80 cm, 2∶81–120 cm, 3∶121–160 cm, 4: >161 cm.

Size class frequency of a) 100 papan class="Chemical">n class="Species">Asplenium antiquum anpan>d b) 100 n class="Species">Haplopteris zosterifolia co-occurring with different epiphyte species. Size class of n class="Species">A. antiquum was determined by the crown size. 1: <50 cm, 2∶50–100 cm, 3∶101 -150 cm, 4∶151–200 cm, 5>200. Size class of H. zosterifolia was determined by the maximum length of fronds. 1∶40–80 cm, 2∶81–120 cm, 3∶121–160 cm, 4: >161 cm.

Light Availability

Prior to the removal treatment, ISF apan class="Chemical">nd DSF below n class="Species">A. antiquum (0.05–0.07) were approximately 50% lower thanpan> the values above it (0.12–0.15) anpan>d did not differ amonpan>g the treatments (treatment effect F = 0.49, df = 3, p = 0.69, positionpan> effect F = 135, df = 1, p<0.001 for ISF anpan>d treatment effect F = 0.32, df = 3, p = 0.81, positionpan> effect F = 92, df = 1, p<0.001 for DSF) (Fig. 3). Followinpan>g the removal treatment, light availability below pan> class="Species">A. antiquum increased in all treatments possibly due to seasonal changes because the hemispherical photographs were taken two months apart in the winter. However, the increases in both ISF and DSF were more than doubled and significantly higher in the frond removal and frond+substrate groups than the control and H. zosterifolia group, which increased less than 40% (LSD tests p values <0.001). The post-removal light availability (both ISF and DSF) did not differ between below and above A. antiquum in the A. antiquum frond removal and frond+substrate removal groups (p values >0.05). However, after the treatment light availability was significantly higher above A. antiquum than below (p<0.001), in both the control and H. zosterifolia removal groups (Fig. 3).

Figure 3

Light indices before and after removal treatment above and below Asplenium antiquum.

Light indices of a) ISF, and b) DSF before and after removal treatment above and below Asplenium antiquum. ISF and DSF are the proportion of direct and indirect light, respectively, at the point of measurement relative to the levels above tree canopies. Error bars represent ±1 S.E.

Light indices of a) ISF, apan class="Chemical">nd b) DSF before and after removal treatment above and below n class="Species">Asplenium antiquum. ISF anpan>d DSF are the proportionpan> of direct anpan>d inpan>direct light, respectively, at the poinpan>t of measurement relative to the levels above tree canpan>opies. Error bars represent ±1 S.E.

Frond Number and Length

Prior to the removal treatment, the fropan class="Chemical">nd number and length of n class="Species">A. antiquum did not differ between the conpan>trol anpan>d the npan> class="Species">H. zosterifolia removal group (F (1, 26) = 2.35, p = 0.14 for frond number and F (1, 26) = 0.144, p = 0.71 for frond length). Similarly, prior to the removal, the frond number and length of H. zosterifolia did not differ among the control, A. antiquum frond removal group, and the frond+substrate removal group (F (2, 39) = 1.11, p = 0.34 for frond number and F (2, 39) = 2.59, p = 0.09 for frond length). Following the treatment, the relative frond number and length of A. antiquum (as proportions of the pre-treatment measurements) did not differ between the control group and the H. zosterifolia removal group (F (1, 26) = 0.008, p = 0.93 for frond number and F (1, 26) = 0.034, p = 0.86 for frond length) (Fig. 4a). Throughout the 10-month period, there was no clear pattern of changes in the frond number and length of A. antiquum (Fig. 4a). Thus the presence of H. zosterifolia had no effect on the growth of A. antiquum.

Figure 4

Relative frond number and length after removal treatment.

The relative frond number and length (as compared to pre-removal measurements) of a) A. antiquum and b) H. zosterifolia in different treatments. Error bars represent ±1 S.E. Numbers in parentheses are actual frond number and frond length for October.

The relative frond pan class="Chemical">number and length (as compared to pre-removal measurements) of a) n class="Species">A. antiquum anpan>d b) pan> class="Species">H. zosterifolia in different treatments. Error bars represent ±1 S.E. Numbers in parentheses are actual frond number and frond length for October.

The changes ipan class="Chemical">n the relative frond number of n class="Species">H. zosterifolia were conpan>sistent anpan>d parallel amonclass="Chemical">pan>g the three treatments: conpan>trol, pan> class="Species">A. antiquum frond removal, A. antiquum frond+substrate removal. There was an initial decrease between October 2009 and January 2010 and a further decrease between April and August 2010 (Fig. 4b). The relative frond number of H. zosterifolia after the treatment did not differ among the three treatments (F (2, 38) = 2.11, p = 0.13) although the frond+substrate removal group had consistently lower relative frond number. However, the relative frond length of n class="Species">H. zosterifolia following the treatments differed significantly among the three treatments (F (2, 39) = 5.98, p = 0.005). In June and August 2010, frond length was significantly greater in the control group than the frond removal group, which in turn was significantly longer than the frond+substrate removal group (LSD tests p values <0.05). Thus A. antiquum had a positive effect on the growth of H. zosterifolia, and the substrate of A. antiquum was an important facilitative factor.

Elemental Composition

Concepan class="Chemical">ntrations of N and Ca and C/N ratios of n class="Species">A. antiquum fronpan>ds did not differ significanpan>tly between the conpan>trol group anpan>d the n class="Species">H. zosterifolia removal group (p values >0.05) (Fig. 4). Concentrations of K, n class="Chemical">Mg, and P were significantly higher in the H. zosterifolia removal group than in the control group (p values <0.05) (Fig. 5). Carbon was the only element that was significantly lower in the H. zosterifolia removal group than the control group (F (1. 21) = 13, p = 0.002) (Fig. 5).

Figure 5

Element content and C/N ratio of fronds of A. antiquum.

Element content and C/N ratio of fronds of A. antiquum between January and August 2010. Error bars represent ±1 S.E.

Element content and C/N ratio of fronds of n class="Species">A. antiquum between January and August 2010. Error bars represent ±1 S.E.

For the fronds of papan class="Chemical">n class="Species">H. zosterifolia, conclass="Chemical">pan>centrationpan>s of all anpan>alyzed nutrients except Ca did not differ amonpan>g the three groups (p values >0.05) (Fig. 6). Ca conpan>centrationpan> was significanpan>tly higher inpan> the pan> class="Species">A. antiquum frond+substrate removal group than in the control group in January, June and August, and the difference between the control group and the A. antiquum frond removal group was only significant in February (LSD tests p values <0.05) (Fig. 6).

Figure 6

Element content and C/N ratio of fronds of H. zosterifolia.

Element content and C/N ratio of fronds of H. zosterifolia between January and August 2010. Error bars represent ±1 S.E.

Element content and C/N ratio of fronds of n class="Species">H. zosterifolia between January and August 2010. Error bars represent ±1 S.E.

Specific Leaf Area

The SLA did not differ sigpan class="Chemical">nificantly among treatments in January 2010 for both n class="Species">A. antiquum (F (1, 25) = 0.13, p = 0.72) anpan>d pan> class="Species">H. zosterifolia (F (2, 38)  = 0.32, p = 0.73). Between January and August 2010, the SLA of A. antiquum increased from 83 to 96 cm2 g−1 in the control group and from 87 to 92 cm2 g−1 in the H. zosterifolia removal group. However, these increases were insignificant both for the control group (F (1, 28) = 2.94, p = 0.098) and the H. zosterifolia removal group (F (1, 22) = 0.51, p = 0.48). The SLA of H. zosterifolia decreased between January and August 2010 in all three groups but the differences were only significant in the frond+substrate removal group (F (1, 24) = 17.8, p<0.001). In this group, the SLA decreased more than 50% from 230 cm2 g−1 in January 2010 to 94 cm2 g−1 in August 2010. Thus A. antiquum had a positive effect on the SLA of H. zosterifolia, but the effect was significant only when both the substrate and fronds of A. antiquum were present.

Drought Stress Response

The maximum amount of papan class="Chemical">n class="Chemical">water that canpan> be held by the substrate ranged from 4.6 pan> class="Species">to 7.4 times the dry weight of the substrate, with a mean of 6.2±1.6 (standard error) for the 10 plants. Water loss was significantly greater in the frond-clipped group than in the frond-unclipped group from day 3 to 13 of the drought experiment (p values <0.05) (Fig. 7). Thereafter, the difference between the two groups diminished. The Fv/Fm increased gradually from 0.79 to 0.85 before day 8 (R = 0.45, p<0.0001) and dropped thereafter to 0.69 by day 20 (R = 0.58, p<0.0001) (Fig. 8).

Figure 7

Weight change with the progression of drought treatment.

Proportion of weight remaining (relative weight to day 0) with the progression of drought treatment for Asplenium antiquum with fronds unclipped (5 replicates) and fronds clipped (5 replicates). The differences between the treatments were mostly significant with the exceptions of days 1, 2, 14, 15, 16, and 19.

Figure 8

The potential quantum yield of Asplenium antiquum with the progression of drought treatment.

The potential quantum yield (Fv/Fm) of Asplenium antiquum fronds with the progression of drought treatment. Error bars represent ±1 S.E.

Proportion of weight remaipan class="Chemical">ning (relative weight to day 0) with the progression of drought treatment for n class="Species">Asplenium antiquum with fronpan>ds unclipped (5 replicates) anpan>d fronpan>ds clipped (5 replicates). The differenpan>ces between the treatments were mostly significanpan>t with the exceptionpan>s of days 1, 2, 14, 15, 16, anpan>d 19.

The potential quantum yield (Fv/Fm) of n class="Species">Asplenium antiquum fronds with the progressionclass="Chemical">pan> of drought treatment. Error bars represent ±1 S.E.

Carbon and Nitrogen Isotopes

δn class="Chemical">13Cpan> clearly distinguished A. anpan>tiauum from pan> class="Species">H. zosterifolia (Fig. 9). The fronds and substrates of A. antiquum had similar mean values of δ13C (approximately -29‰), which were significantly higher than those of n class="Species">H. zosterifolia growing alone (-31.6‰) and co-occurring (-32.4‰) with A. antiquum (p<0.001). For δ15N, no significant differences among groups were found (p = 0.21). The two types of H. zosterifolia, growing alone and co-occurring with A. antiquum, had similar mean values (approximately -0.54‰), which were lower than those of A. antiquum fronds (-0.14‰) and substrates (0.12‰) (Fig. 9).

Figure 9

δ13C and δ15N of Asplenium antiquum and Haplopteris zosterifolia.

Mean δ13C and δ15N of Asplenium antiquum and Haplopteris zosterifolia. Error bars represent ±1 S.E.

Mean δn class="Chemical">13C anclass="Chemical">pan>d δ15N of Asplenium antiquum and Haplopteris zosterifolia. Error bars represent ±1 S.E.

Discussion

Effects of A. antiquum on H. zosterifolia

Forty-five percent of the surveyed papan class="Chemical">n class="Species">H. zosterifolia co-occurred with npan> class="Species">A. antiquum, and A. antiquum was the epiphyte with which H. zosterifolia co-occurred most commonly. The individuals of H. zosterifolia co-occurring with A. antiquum were significantly larger than those growing alone (Fig. 2), suggesting facilitative effects of A. antiquum on the growth and survival of H. zosterifolia. The lack of growing-alone H. zosterifolia in the largest size class and only one in the second largest size class further suggest that A. antiquum and perhaps other epiphytes with which H. zosterifolia co-occurred probably also increase the survival of H. zosterifolia. This interpretation is supported by the reduced frond length of H. zosterifolia resulting from the removal of A. antiquum, both frond-only and frond+substrate (Fig. 4b). The reduced frond length of H. zosterifolia after the removal of A. antiquum, which significantly reduced light available to H. zosterifolia, indicates that light was not a main factor limiting the growth of H. zosterifolia. The greater decrease of H. zosterifolia frond length in the A. antiquum frond+substrate removal group than the frond-only removal group (Fig. 4b) implies that substrate plays an important role on the interaction between these two epiphyte species.

n class="Species">A. antiquumpan> may benefit co-occurrinpan>g pan> class="Species">H. zosterifolia through mitigation of water stress. The mitigation may result both from reduced evapotranspiration due to shading of A. antiquum fronds (Fig. 3) and from enhanced n class="Chemical">water availability provided by the substrate of A. antiquum. The much higher rates of water loss in the frond-clipped than the frond-unclipped A. antiquum in the drought experiment clearly show that shading reduced water loss from the substrate. The removal of A. antiquum fronds increases drought stress of H. zosterifolia but the effect is even more substantial when the substrate is also removed (Fig. 4). Our SLA data support the interpretation that A. antiquum mitigates water stress in co-occurring H. zosterifolia. Drought stress and light availability decrease SLA [30], [31]. The more than 50% decrease in the SLA of H. zosterifolia in the frond+substrate removal group from January to August 2010 suggests that A. antiquum substrates enhance water availability to H. zosterifolia where they co-occur.

Foliage δn class="Chemical">13Cpan> is often used as anpan> inpan>tegrative inpan>dicator of pan> class="Chemical">water use efficiency, with higher δ13C values indicating greater water use efficiency, which is associated with water stress [32]. The slightly greater δ13C values of n class="Species">H. zosterifolia growing alone than those co-occurring with A. antiquum (Fig. 9), though not significant statistically, gives some indication that A. antiquum enhances water availability to H. zosterifolia. A. antiquum is more conservative in water use, as inferred from higher δ13C, than H. zosterifolia. This conservative water use probably contributes to its widespread distribution even in forests with long dry periods. In contrast, by occurring underneath A. antiquum, which shades approximately 50% of the incoming light and provides water through its substrate, H. zosterifolia probably lives in a more humid micro-habitat and is thus less conservative in water use.

The substrate of n class="Species">A. antiquumpan> may also provide nutrient subsidies to pan> class="Species">H. zosterifolia. For example, epiphytes in lowland tropical forests may be limited by P [12], [33]. However, foliar P concentrations did not differ between H. zosterifolia that co-occurred with A. antiquum and those that grew alone. Likewise, the N concentrations and δn class="Chemical">15N values were not significantly different between the two groups (Figs 6 and 9), suggesting common N sources for the two groups of H. zosterifolia. Thus our results of elemental and isotope analyses showed no evidence of nutrient enhancement from A. antiquum to H. zosterifolia.

Effects of H. zosterifolia on A. antiquum

Our field survey shows that the distribution of papan class="Chemical">n class="Species">A. antiquum was npan>ot closely associated with n class="Species">H. zosterifolia, as only 17% of the n class="Species">A. antiquum co-occurred with H. zosterifolia and H. zosterifolia was not the epiphyte species that co-occurred most commonly with A. antiquum. The lack of size difference among A. antiquum that co-occurred with H. zosterifolia or other epiphytes, and that did not co-occur with any epiphytes suggests that the co-occurrence of A. antiquum with other epiphytes had no effect on the growth of A. antiquum (Fig. 2). The lack of significant difference in the frond number and length of A. antiquum between the control group and H. zosterifolia removal group further supports that H. zosterifolia had no effect on the growth of A. antiquum (Fig. 4a).

One potepan class="Chemical">ntial negative effect of co-occurring epiphytes on n class="Species">A. antiquum is the competitionpan> for pan> class="Chemical">water. Our SLA data argue against this possibility, as the SLA of A. antiquum increased, although insignificantly, in both the control group and the H. zosterifolia removal group between January and August 2010. Thus the water held by the substrate of A. antiquum may be sufficient to meet the water demands of itself and its co-occurring epiphytes. However, our drought treatment indicates that if a rainless period persists for more than a week, A. antiquum would experience drought stress as evidenced by decreased Fv/Fm (Fig. 8). This stress may occur sooner if the co-occurring H. zosterifolia directly competes for substrate water via root uptake. However, H. zosterifolia probably has a greater reliance on substrate water than A. antiquum due to their differences in water retaining ability associated with different frond morphology. The pendent fronds of H. zosterifolia direct intercepted water out of the plant more easily whereas the bowl-shaped crown of A. antiquum holds water for a longer period of time. Although the water intercepted by the crown will be directed to the substrate during heavy rainfall events, the water may only be sufficient to moisten A. antiquum during light rainfall events in drier periods.

n class="Species">H. zosterifoliapan> might have altered the nutrient balanpan>ce of pan> class="Species">A. antiquum. The higher concentrations of K, Mg and P of A. antiquum foliage in the H. zosterifolia removal group (Fig. 5) than in the control group imply competition for nutrients in the substrate (Fig. 5). The lower tissue quality of n class="Species">A. antiquum in the control group was also indicated by its higher carbon content and low C:N ratio (Fig. 5). Several studies [34], [35] suggest that P is more limiting than N for epiphytes in humid tropical forests, which may explain the consistently greater concentrations of P, but not N, in our H. zosterifolia removal group than the control group. However, these nutrient differences did not affect the growth of A. antiquum during our 10-month study period. Because the A. antiquum could live for more than a decade, our study may not be long enough to detect the negative effect of H. zosterifolia on A. antiquum resulting from the competition for nutrients in the substrate.

Positive Interactions between Epiphytic Species, and Implications for Drought Mitigation

Epiphytes are most common ipan class="Chemical">n tropical and subtropical regions where rainfall is abundant. However, adaptations such as sunken stomata, succulent leaves, n class="Chemical">water storage organpan>, anpan>d CAM photosynthesis are commonpan> inpan> epiphytes inpan> wet tropical or subtropical forests with high amounts of precipitationpan> [14], [15], [26], [34], inpan>dicatinpan>g that npan> class="Chemical">water conservation is important for epiphytes [14], [15], [17,]. Our result suggests that positive interspecific interaction through drought mitigation is another important mechanism that helps epiphytes to survive and grow in the host plants. This positive interaction may also explain the often clumped distribution of epiphytes in the forest canopy.

Crown humus accumulated by epiphytes has beepan class="Chemical">n proposed to be an important source of moisture for canopy organisms during dry period [36]. A study of epiphyte biomass at our study site indicates that the humus-rich substrate of Asplenium contributed approximately 290 kg ha−1 or 8.5% of the total epiphyte biomass [37]. Given that the substrate of Asplenium on average holds n class="Chemical">water approximately 6 times its dry weight, the Asplenium substrate has the potential to store as much as 1700 kg ha−1 of pan> class="Chemical">water in the canopy at scattered spots. Thus substrate-forming epiphytes may play a disproportionally important role in maintaining biodiversity of canopy community by providing a moisture source to facilitate the growth of other epiphytes. In addition to H. zosterifolia, the 18 other epiphyte species (22%) co-occurring with A. antiquum in our studied forest may also benefit from drought mitigation by A.antiquum. Thus substrate-forming epiphytes may function as keystone species, and the microhabitats where they grow are biodiversity hotspots within the forest canopy. A study of forest canopy biomass indicates that five individuals of A. nidus hosted a total of 205,428 invertebrates from 27 orders, and the biomass of invertebrates on individual A. nidus plants was positively correlated with their number of fronds [38]. At our study site, the biomass of invertebrates was positively correlated with the biomass of A. nidus [39]. In Taiwan at least one predatory bird species, Ictinaetus malayensis, is known to nest on Asplenium. Thus, through habitat cascades [40] they may facilitate the establishment of a highly diverse canopy community.

Interspecies facilitatiopan class="Chemical">n through drought mitigation may be vital for maintaining epiphyte diversity in the context of anthropogenic climate change. The frequency and severity of drought stress are anticipated to increase in tropical moist forests [41], [42], and as a result drought poses a major threat to biodiversity [43]. Epiphytes have been suggested as early indicators of floristic response to climate change [44] as their abundance and diversity decrease sharply toward drier environments [45]. Ecosystems with exceptionally high epiphyte richness are thus vulnerable to the increasing trend of drought stress [46], [47]. Our study demonstrates the facilitative effect of n class="Species">A. antiquum through drought mitigationpan> onpan> pan> class="Species">H. zosterifolia. Such positive interaction between epiphyte species may play an important role in maintaining species diversity in tropical and subtropical ecosystems and in determining their responses to climate change.