Tropical Terrestrial And Epiphytic Ferns Have Different Leaf Stoichiometry With Ecological Implications.

doi:10.21203/rs.3.rs-709718/v1

Terrestrial and epiphytic herbaceous forest species have different ecology and leaf stoichiometry. In tropical regions, a great component of herbaceous forest species is represented by ferns with different lifeforms. However, little is known about the differences in leaf stoichiometry between the lifeforms. We account for the concentrations of leaf elements (N, P, K, Ca and Mg) between terrestrial and epiphyte lifeforms and evolutionary clades. The fern species were sampled from the forest of Brunei Darussalam. Five leaves were collected from 5 individuals from 16 terrestrial and 4 epiphytic ferns. The leaves were then acid-digested and analyzed. Epiphytic species had higher concentration of most of the leaf elements. The N:P ratio showed that the epiphytic species being much more nutrient-limited, relying on stochastic events, compared to the terrestrial species which have a constant availability of soil elements. Epiphytes showed a higher concentration of P, which could be explained by their luxury consumption. Epiphytes accumulate elements in a higher concentration than is needed by their normal metabolic activity. Furthermore, epiphyte species have a significantly higher concentration of Ca which could be interpreted as necessity of coping with severe habitat conditions with schlerophyll leaves. The results bring in more information on the poorly studied stoichiometry of tropical Asian fern species. Important in understanding the eco-physiology of terrestrial and epiphytic ferns and determining which species are sensitive to the different forest management and the effect of climate change. This, is in addition to the associated mechanisms.

Plant Molecular Biology and Genetics

Behavioral Ecology

Terrestrial

Epiphyte

Ferns

Leaf Stoichiometry

Borneo

Brunei

Leaf stoichiometry – the relative abundance and concentration of leaf elements – is a useful tool for understanding the fitness of a species in a particular environment (Ågren and Weih 2012). It therefore determines the distribution of the plant species across different habitats and the biome as a whole (Sun et al. 2017; Tong et al. 2019). The differences in element concentration and their allometric relationship is important for the terrestrial species with a constant supply of nutrients from the soil and the epiphytes that rely on an irregular supply of nutrients (Zotz and Hietz 2001; Cardelús and Mack 2010).

Plant functioning requires up to 17 essential elements of varying concentrations, with nitrogen (N), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) often required in large quantities. These are referred to as macronutrients (White and Brown 2010). Among these 5 macronutrients, N and P play a major role in plant growth (root morphology) and metabolic functioning (chlorophyll content) (Razaq et al. 2017). Both N and P are tightly linked and, as such, their ratio (N:P) has been suggested to be a major ecological driver. It is used to indicate the nutrient limitations of N and/or P (Koerselman and Meuleman 1996; Güsewell 2004). The crucial function of K, often the second highest element concentration in leaves (Winkler and Zotz 2010), is in the maintenance of the plant’s water status through the regulation of the stomatal aperture (Oddo et al. 2011). Ca has a broad concentration in the leaves (Richardson and Walker 2010) as well as in differing lifeforms (Huang et al. 2019). It is a secondary messenger that transmits environmental change signals to trigger adaptive processes (Ranty et al. 2016). It is also involved in the regulation of transpiration (Song et al. 2008). Mg is an integral component of leaf photosynthesis (Hermans and Verbruggen 2005) and limits plant growth, productivity and metabolism (Baribault et al. 2012).

These elements are important and plant species exhibit differential concentrations in their leaves (Zheng and Shangguan 2007) as a consequence of the differences in their metabolic requirements (Sardans and Peñuelas 2014) and lifeform (Cardelús and Mack 2010). For instance, species from a hotter-drier environment exhibit lower N and P concentrations compared to species from a colder-humid environment, implying a differential nutrient use strategy (Sardans and Peñuelas 2014). Furthermore, epiphytic species often have to rely on a stochastic source of nutrients while terrestrial species have a much more constant supply (Cardelús and Mack 2010). This discrepancy in growth environment has led to the development of distinct leaf stoichiometric profiles between the two lifeforms (Huang et al. 2019). Epiphytes, due to the irregular supply of nutrients, employ what has been proposed as luxury consumption (Winkler and Zotz 2010). The plants accumulate a high concentration of N (Winkler Zotz 2010), and P (Winkler Zotz 2009) in their vacuoles for use beyond the immediate metabolism needs in terms of protein synthesis, enzyme reactions or stomatal regulations.

Despite the overlap in terrestrial and canopy habitat between ferns and angiosperms as well as their distinctive physiological properties (Brodribb and Holbrook 2004; Brodribb et al. 2009), the relative importance of leaf stoichiometry is poorly known, particularly in the tropics. Ecologically, ferns play a range of crucial roles in the regulation of water and in the nutrient balance in the forest ecosystem (Umana and Wanek 2010; Hargis et al. 2019), controlling the regeneration potential of a forest (Coomes et al. 2005) as well as the phytoremediation of soil (Praveen and Pandey 2019). In tropical regions, ferns can be the dominant species in an ecosystem and contribute significantly to nutrient cycling and other ecosystem processes (Amatangelo and Vitousek 2008, 2009).

However, fern phylogenies distinguish the clade called ‘Polypod’ ferns, consisting of the order Polypodiales, which has radiated during the Angiosperm’s early evolution (Schneider et al. 2004; Testo and Sundue 2016). All the other leptosporangiate ferns are grouped into ‘non-polypod’ ferns, diversified over tens of millions of years before the angiosperms (Schneider et al. 2004). The non-polypod ferns include the tree ferns, filmy ferns, water ferns, gleichnenioid, osmundaceous and schizeoid ferns. The non-polypod are archaic compared with the modern polyopod. Therefore, it is relevant to know whether the leaf stoichiometry differs from the phylogenetic different clades of species.

In this study, we aim to elucidate the stoichiometric relationship between terrestrial and epiphytic tropical fern species. Our analyses were based on the hypothesis that there would be optimal concentrations of the leaf elements distinct to each fern lifeform because of their optimal adaptation and ecological traits as part of maximizing fitness in their respective growth environments. As such, we addressed 3 questions, i) Are there differences in the leaf stoichiometric profile between terrestrial and epiphytic fern species? ii) Which element has a limiting effect, based on the allometric relationship, in the species life form? and iii) Do differences in the leaf element concentrations explain the ecological adaptations between the lifeforms and phylogeny?

This study considered 20 fern species belonging to 12 families and 16 genera (Table S1). The species were collected in 3 localities of mixed dipterocarp forest within Brunei Darussalam: Bukit Teraja Forest Reserve (4°18’N, 114°26’E), Kuala Belalong Field Studies Centre (4°32’N, 115°09’E) and Bukit Shahbandar Forest Recreation Park (4°57’N, 114°51’E). The climate in Brunei Darussalam is categorized as tropical equatorial with a mean annual temperature of 27.9°C ranging from 23.8°C – 32.1°C under a humidity of approximately 82% and mean annual rainfall of c. 2722 mm throughout the year (Islam et al. 2019). The first 2 sites were primary forest with minimum or no direct human disturbance, therefore we can consider the species as being in their natural ecological niche. The third site (Bukit Shahbandar), where only the species Platycerium coronarium was sampled, was a disturbed forest with the individuals at the forest margin. However, considering the ecology of the species as an epiphyte exposed to full sun throughout the day, we were confident that there was little alteration and that it did not compromise the species physiology. Moreover, the leaves collected were from mature large individuals. For each species, we collected 5 leaves from 5 individuals to give a total of 25 leaves per species. In 3 species, we had only 4 individuals, Schizaea dichotoma, Asplenium tenerum and Lindsaea borneensis. Only mature leaves were chosen with minimal to no damage and no sign of senescence. The nutritional status of the epiphytic species can rely on the host tree. However, we did not assess the trees where the epiphytes were collected. The fresh leaf samples were placed in sealed plastic bags and later transported, within 4 hours, to the laboratory of the University of Brunei Darussalam. The leaf samples were washed with distilled water prior to oven-drying at 60°C for 24h. The dried leaf samples were milled using a ball mill (Retsch MM400) before proceeding to the chemical analysis. The terrestrial species were collected from the forest floor, whereas the epiphytic species were sampled from the tree trunks belonging to different tree species and rock surfaces in the case of lithophytic Dipteris lobbiana. The lithophytic species was considered to be terrestrial due to the relative similarity in growth habitat to the terrestrial species (Zotz 2016). Following the fern classification, the species were classified as either polypods or non-polypods, totaling 12 and 8 species respectively (Smith et al. 2006; Table S1).

Chemical analysis

Total leaf Nitrogen (N) and Phosphorus (P)

The leaf element analysis for both the total N and total P was conducted following a modified procedure from (Allen et al. 1989) in the user manual for the FIA star 5000. Leaf samples of 0.1g were digested using sulfuric acid and a kjeldahl tablet (CQ-AA18 Thompson and Capper Ltd. Batch 140052) for 2 hours using a block digester at 360°C (BD-46, Lachat instruments, Colorado, USA). The acid-digested solutions were then cooled, filtered with filter paper (Sartorius grade 393), and diluted by adding 50 ml of distilled water in a 50 ml volumetric flask. The leaf N and P concentrations were determined colorometrically using Flow Injection Auto-analyzers (FIA star 5000, Höganäs, Sweden). The concentration values obtained from the FIA were used to calculate the total leaf N and P concentrations.

Total leaf Potassium (K), Calcium (Ca) and Magnesium (Mg)

Leaf samples of 0.2g were digested using a mixture of acid reagents (4.8% H₂SO₄ and H₂O₂) and a LiSO₄ catalyst for 2 hours using a block digester at 360°C (BD-46, Lachat instruments, Colorado, USA; Allen et al., 1989). The mixture contents were then filtered with filter paper (Sartorius grade 393) and diluted by adding 50 ml distilled water in a 50 ml volumetric flask. The total leaf K, Ca and Mg were measured using atomic absorption spectrophotometry (Thermo Scientific ICE 3000) after diluting the acid-digested samples with LaCl₃ for K, Ca and Mg (acid:LaCl₃ in the ratio of 1:1).

Statistical analysis

All element concentrations and ratios were calculated on the basis of mass (mg g^− 1). We assessed the leaf element concentrations between terrestrial and epiphytic fern species as well as between polypod and non-polypod fern species at a 5% significance level. Prior to the analysis, all of the data was checked for homogeneity of variance and normality using the Levene and Shapiro-wilk tests respectively. Assumptions for normality were violated. However, we proceeded to conduct the analyses using the ANOVA test as it is robust in relation to violations of the normality assumption (Blanca et al. 2017). The data that violates the homogeneity of variance assumption was analyzed using the adjusted Welch-ANOVA test. Subsequently, the pairwise correlations between each leaf element in terrestrial and epiphytic species were analyzed by Spearman correlation. Finally, the association between the different leaf element concentrations with the categories of species’ ecological traits and the phylogeny classification were performed using principal component analysis (PCA) using log₁₀-transformed values. The statistical analyses were performed using RStudio Team (2020).

The leaf element concentrations varied across the species (Table S2; Fig. S1). The magnitude of the variations among all of the studied species considered ranged, according to N (10-fold) P (2-fold), K (7-fold), Ca (18-fold), and Mg (13-fold). The variations in the leaf element concentrations between terrestrial and epiphytic species were significantly different for N, P, K, and Ca while no significant difference was detected for Mg. The epiphytic species had a lower leaf N concentration but a higher P, K, and Ca concentration compared to the terrestrial species (Fig. 1). The concentration of leaf Mg between the two lifeforms was similar (2.30 vs 2.40 mg g^− 1 for terrestrial and epiphytic respectively). The first axis of the PCA explained 41.5% of the variation in leaf element concentrations, which corresponds to the K, Ca and Mg elements. The positive side of the first axis loaded the epiphytic species together with the majority of terrestrial species. The second axis of the PCA explained 26.3% of the variation, representing the N and P elements. However, no clear pattern was observed between the two lifeforms (Fig. S2).

The concentrations of the leaf element ratios between the two lifeforms, with the exception of P:K and P:Mg, were all significantly different (Table 1). The terrestrial species constantly had higher values than the epiphytes. The pairwise correlations among the concentrations of N, P, K, Ca and Mg exhibited significant relationships within the terrestrial and epiphytic species (Fig. 2). However, the epiphytic species had four unique correlations which was absent in the terrestrial species and three of them correlated with the availability of phosphorous.

Table 1

Probability value (P) testing between terrestrial and epiphytic fern species regarding the variations in the foliar element ratio of nitrogen: phosphorus (N:P); potassium (N:K); calcium (N:Ca); magnesium (N:Mg) and phosphorus: potassium (P:K); calcium (P:Ca); magnesium (P:Mg). The mean (± SE) foliar element concentrations (mg g^− 1 dry mass) and the F values compared between terrestrial and epiphytic fern species are also presented. The P values in bold suggest significant differences (P ≤ 0.05).
Leaf element ratio	F	P	Terrestrial mean leaf element concentrations	Epiphytic mean leaf element concentrations
N:P	18.41	< 0.001	13.57 ± 1.42	6.48 ± 0.84
N:K	31.68	< 0.001	18.73 ± 1.83	7.28 ± 0.88
N:Ca	36.96	< 0.001	11.00 ± 1.44	2.10 ± 0.24
N:Mg	11.26	0.001	8.99 ± 1.14	4.78 ± 0.52
P:K	0.13	0.72	1.35 ± 0.07	1.30 ± 0.13
P:Ca	26.37	< 0.001	0.97 ± 0.08	0.44 ± 0.06
P:Mg	1.82	0.18	0.75 ± 0.05	0.90 ± 0.09

The concentrations of the leaf elements between the polypod and non-polypod species resulted in a significant difference only for the K concentration, with the polypods displaying a double magnitude concentration (Table S3). The leaf element ratios showed a significant difference in N:P with nearly a double value for polypods, whereas the significant difference in the value of N:K was higher in non-polypods with a difference of 10 units between the fern groups (Table 2). The ratios of P:K, P:Ca and P:Mg were all significantly different with a higher value in the non-polypods. The first axis of the PCA variation in leaf element concentrations corresponds to the K, Ca and Mg elements, whereas the second axis represents the N and P elements. The ordination analysis does not show a clear cluster between the two groups, however the non-polypod species cover the negative quadrant for first and second axes, showing unique traits that polypod species do not have (Fig. S2b).

Table 2

Probability value (P) testing between polypod and non-polypod fern species regarding the variations in the foliar element ratio of nitrogen: phosphorus (N:P); potassium (N:K); calcium (N:Ca); magnesium (N:Mg) and phosphorus: potassium (P:K); calcium (P:Ca); magnesium (P:Mg). The mean (± SE) foliar element concentrations (mg g^− 1 dry mass) and the F values compared between polypod and non-polypod fern species are also presented. The P values in bold suggest significant differences (P ≤ 0.05).
Leaf element ratio	F	P	Polypod mean leaf element concentrations	Non-polypod mean leaf element concentrations
N:P	8.49	0.005	16.64 ± 2.34	9.08 ± 1.13
N:K	6.00	0.02	14.24 ± 1.63	23.24 ± 3.30
N:Ca	1.18	0.28	7.82 ± 1.07	10.21 ± 1.92
N:Mg	3.13	0.08	9.25 ± 1.49	16.28 ± 3.68
P:K	22.90	< 0.001	1.10 ± 0.07	2.66 ± 0.32
P:Ca	10.77	0.002	0.76 ± 0.07	1.47 ± 0.21
P:Mg	10.29	0.003	0.69 ± 0.05	1.49 ± 0.25

The leaf element concentration in tropical terrestrial and epiphytic fern species had a high level of variability in relation to both the lifeform and species (Table S2; Fig. S1). This high variability among species, in relation to lifeform and species evolution, has also been confirmed for tropical ferns (Watkins, Philip and Cardelús, 2007) orchids and bromeliads (Cardelús and Mack, 2010) as well as subtropical species of bryophytes, lichens and spermatophytes (Huang et al., 2019). The discrepancies between the terrestrial and epiphyte species are due to the differences in habitat, where terrestrial species have access to a larger resource pool in the soil floor whereas epiphytic species have to rely on more stochastic sources of nutrients (Zotz and Hietz 2001; Cardelús and Mack 2010; Chen et al. 2019). This also sometimes depends on the host tree (Cardelús and Mack 2010). Among these elements, N and Ca have the greatest difference with the mean difference between terrestrials and epiphytes being 7.8mg g^− 1 for N and 3.3mg g^− 1 for Ca. The high variability of the element concentration among the different species and lifeforms shows that certain elements are a limiting factor for some species. The use of an element ratio is important to show the relationship between the important elements and to indicate the contribution of the limiting factor. The N:P ratio in the two lifeforms (13.57–6.48 mg g^− 1) shows that the N-P nutrition status is significantly different between terrestrial and epiphytes tropical ferns. According to the previous studies, the N:P ratio is rather inconsistent with averages ranging from 12.1 ± 10.5 (Zotz and Hietz 2001) to 16.1 ± 5.8 (Zotz 2004). The values of 14 and 16 are considered to be the threshold used to indicate the limitation of the allometric relationship between N and P (Koerselman and Meuleman 1996). In epiphytic species, an N:P ratio > 12 indicates a P-limitation (Wanek & Zotz 2011). In this study, terrestrial ferns with a value of 13.57 mg g^− 1 can have a limitation of N and P or a co-limitation of both elements; epiphytic fern with a much lower value at 6.48 mg g^− 1 are most certainly under the limitations of both elements in their biological development. The allometric relationship between terrestrial and epiphytic species shows that for terrestrial ferns, there is a mean average of N at 19.2 mg g^− 1 and P at 1.6 mg g^− 1 and a mean average of N at 11.4 mg g^− 1 and P at 1.9 mg g^− 1 for epiphytes. Therefore, the disproportion between the two elements is higher for terrestrial and lower for epiphytic ferns respectively. This is also confirmed by the much stronger statistically significant P values for N (< 0.001) compared to P (0.02) between the terrestrial and epiphytic species.

In contrast, epiphytic ferns exhibit a slightly significant (P = 0.02) higher P concentration relative to terrestrial ferns (Fig. 1). This pattern contradicts the studies where the P is scarce in other epiphytic species such as bromeliaceae (Zotz and Richter 2006; Winkler and Zotz 2009), orchidaceae (Zotz 2004), cyanolichens (Benner et al. 2007), ferns (Huang and Lin 2016) and lichens (Benner and Vitousek 2007). Epiphytic plants have evolved mechanisms to cope with this predicament such as the provision of a luxury consumption of P and storing more P in the form of phytin than metabolically required in the case of P scarcity (Winkler and Zotz 2009). Moreover, P is readily resorbed into green leaves from senescing leaves in epiphytic species (Zotz 2004). We can predict that the higher leaf P concentration in epiphytic ferns, relative to their terrestrial counterpart, is related to their ability to store and resorb P in their leaves. The allometric relationship between N and P can be perceived as the plants resorbing more N (or P) when they are N (or P) starved in terms of a state of nutrient scarcity and imbalance (Han et al. 2013) (Table 1). In other words, plants actively resorb more of the limited nutrient. This justifies the P differences between terrestrial and epiphyte ferns. However, the correlation between P and K, Ca, and Mg shows a negative trend which has important metabolic implications for the species (Fig. 2).

The epiphytes had the highest values for K and Ca (Fig. 1) while not significant for Mg. This study shows that the Ca concentration was significantly different between the fern lifeforms, whereby epiphytic ferns maintain a greater Ca concentration than terrestrial ferns. Important to notice that two epiphytic species have an exceptional high concentration of Ca (Fig. S1) whereas the rest are within the interval of the terrestrial species. However, the difference in Ca concentration in epiphytic ferns is double the amount found in terrestrial species (5.70 mg g^− 1 vs 2.36 mg g^− 1). The precedent study has reported that the Ca concentration in ferns is consistently lower when compared to woody angiosperms (Amatangelo and Vitousek 2008). Similarly, the Ca concentration was higher in terrestrial angiosperm species from sites close to the study area (soil total Ca = 0.15 mg g^− 1). This has been related to the thicker and more sclerophylleous leaves found in forests with drier climatic conditions, compared with the thinner leaves in forests with more humid climatic conditions (Metali et al. 2015). The epiphytes considered in this study, except for Asplenium tenerum, have thick leaf tissues, specifically Antrophyum callifolium and Platycerium coronarium. The investment in the higher Ca acts as form of structural reinforcement in the cell walls and membrane of thick sclerophylleous leaves (White and Broadley 2003), with elaborate traits used to acquire nutrients in a dry and nutrient poor environment (Watkins and Cardelús 2012). The greater Ca concentration in epiphytic ferns likely relates to their structural requirement in terms of their cell walls and membrane regarding the sclerophyll adaptation in dry and nutrient poor environments. Secondly, the exchangeable Ca in the soil of mixed dipterocarp forests is very low (0.01 mg g^− 1; Metali et al. 2015) which suggests the possibility of epiphytic ferns obtaining Ca from their hosts. Ca acts as an intracellular messenger that transmits signals to detect changes in the environment, triggering the necessary adaptive responses (Ranty et al. 2016). Epiphytic ferns are exposed to frequent changes in the environmental factors, therefore the higher leaf Ca in epiphytic ferns could promote adaptations in a stressful habitat. This was confirmed by the lower ratios of N:Ca and P:Ca used to promote adaptive growth (Table 1).

One of the crucial roles of potassium in terrestrial plants is the maintenance of leaf water content through the regulation of the stomatal aperture (Oddo et al. 2011). Plants retain a higher K concentration and low N:K ratio in their leaves to alleviate the inhibition of growth under water stress (Sardans et al. 2016). In addition, a higher leaf thickness and better water storage capacity has been linked to a higher K concentration (Lin and Yeh 2008). In this study, epiphytic ferns maintain a greater K concentration in their leaves as well as exhibiting a significantly lower N:K ratio compared with terrestrial ferns (Table 1, Fig. 1). Furthermore, the higher leaf K concentration in epiphytic ferns might relate to their ability to efficiently resorb K more than terrestrial ferns (Suriyagoda et al. 2018). Epiphytic bromeliaceae efficiently absorb K and maintain it in their green leaves through the means of foliar trichomes. They retain in their leaves as a form of luxury consumption storage (Winkler and Zotz 2010). In other epiphytes such as lichens and bryophytes, they retain lower K concentrations and a high N:K ratio because they can survive under more extreme conditions (Huang et al. 2019). It is likely that epiphytic species experience frequent and long periods of drought in an epiphytic habitat which promotes the activation of mechanisms to effectively take up K and conserve it in the green leaves through efficient resorption in order to sustain growth under water-stress conditions.

At the phylogenetic level, element concentration showed no great difference with the exception of K (polypod 1.59 mg g^− 1 and non-polypod 0.88 mg g^− 1; P < 0.001; Table S3). The two clades showed a significant allometric difference regarding the N:P and N:K relationship (Table 2). The older non-polypod ferns, despite having a similar concentration of P, tend to have a much more limited allometric relationship compared with N. At the same time, non-polypods have a consistently higher ratio for K, Ca and Mg, indicating the different metabolic utilization of P compared with the more evolved polypods. The high ratio values of the elements in non-polypod ferns suggests that they have a better capacity to accumulate the elements in high proportions. They could also have a lower metabolic capability which results in a higher ratio of accumulation compared with polypod ferns. From the ordination analysis despite a clear difference between the two groups we can observe that the negative quadrant is represented only by non-polypod therefore they show to have some physiological capability to cope with low level concentration of elements in their leaves.

In conclusion, we investigated the leaf element concentration of tropical ferns and revealed that there are different concentrations between the terrestrial and epiphyte fern species. The Ca concentration in epiphytic species and the potential relation with sclerophylleous leaf ecology and the transpiration aspect could be used to develop more implications in the understanding of epiphytic species in a tropical habitat. The epiphytic species with a high variability in element concentration and potential luxury consumption have adapted to an environment with a much more variable and severe condition compared to the terrestrial species. Archaic ferns use the elements in different proportions compared with more evolved ferns. This shows that they can have important implications for their ecology as they often occur in restricted habitats with more specific micro-climatic conditions. These results can be used as basis to assess the potential risk to the performance of epiphytic species in forested habitat under different management styles and due to the changing climate. Moreover, despite this range of adaptation to rough conditions, climate change scenario reports (Gómez González et al. 2017) that there will be areas with an increase in drought conditions. This has clearly relevant implications for conservation for species that have a small population size, that contribute in terms of richness and that provide a habitat and symbiosis for other species.

Funding

This research was supported by the Government of Brunei Darussalam through the Ministry of Education (Brunei Government Scholarship to S.P.S.).

Acknowledgements

We would like to acknowledge the Department of Forestry, Brunei Darussalam, for the provision of species sampling in the forest reserves. We are grateful to the research assistants Nurhazwani Sirun, Nur Fauziah Haji Yahya for their assistance in species sampling as well as Nur Aqilah Haji Zainal Ariffin in species sampling and editing of Fig. 2. We also like to thank the lab technicians from the Environmental and Life Sciences Department for their logistic support. Moreover, we would like to acknowledge Dr. Faizah Metali for the help during the leaf element digestion and analysis process.

Ågren GI, Weih M (2012) Plant stoichiometry at different scales: Element concentration patterns reflect environment more than genotype. New Phytol 194:944–952.
Allen S, Grimshaw H, Parkinson J, Quarmby C (1989) Chemical analysis of ecological materials. Oxford, UK: Blackwell Scientific Publications.
Amatangelo KL, Vitousek PM (2008) Stoichiometry of ferns in Hawaii: Implications for nutrient cycling. Oecologia 157:619–627.
Amatangelo KL, Vitousek PM (2009) Contrasting predictors of fern versus angiosperm decomposition in a common garden. Biotropica 41:154–161.
Baribault TW, Kobe RK, Finley AO (2012) Tropical tree growth is correlated with soil phosphorus, potassium, and calcium, though not for legumes. Ecol Monogr 82:189–203.
Benner JW, Conroy S, Lunch CK, Toyoda N, Vitousek PM (2007) Phosphorus fertilization increases the abundance and nitrogenase activity of the cyanolichen Pseudocyphellaria crocata in Hawaiian montane forests. Biotropica 39:400–405.
Benner JW, Vitousek PM (2007) Development of a diverse epiphyte community in response to phosphorus fertilization. Ecol Lett 10:628–636.
Blanca MJ, Alarcón R, Arnau J, Bono R, Bendayan R (2017) Non-normal data : Is ANOVA still a valid option ? Psicothema 29:552–557.
Brodribb TJ, Holbrook NM (2004) Stomatal protection against hydraulic failure: A comparison of coexisting ferns and angiosperms. New Phytol 162:663–670.
Brodribb TJ, McAdam SAM, Jordan GJ, Feild TS (2009) Evolution of stomatal responsiveness to CO2 and optimization of water-use efficiency among land plants. New Phytol 183:839–847.
Cardelús CL, Mack MC (2010) The nutrient status of epiphytes and their host trees along an elevational gradient in Costa Rica. Plant Ecol 207:25–37.
Chen LC, Wang LJ, Martin CE, Lin TC (2019) Mediation of stemflow water and nutrient availabilities by epiphytes growing above other epiphytes in a subtropical forest. Ecohydrology 12.
Coomes DA, Allen RB, Bentley WA, et al. (2005) The hare, the tortoise and the crocodile: The ecology of angiosperm dominance, conifer persistence and fern filtering. J Ecol 93:918–935.
Gómez González DC, Quiel CR, Zotz G, Bader MY (2017) Species richness and biomass of epiphytic vegetation in a tropical montane forest in western Panama. Trop Conserv Sci 10:9–11.
Güsewell S (2004) N:P ratios in terrestrial plants: Variation and functional significance. New Phytol 164:243–266.
Han W, Tang L, Chen Y, Fang J (2013) Relationship between the relative limitation and resorption efficiency of nitrogen vs phosphorus in woody plants. PLoS One 8:1–11.
Hargis H, Gotsch SG, Porada P, Moore GW, Ferguson B, Stan JT Van (2019) Arboreal epiphytes in the soil-atmosphere interface: how often are the biggest “buckets” in the canopy empty? Geosci 9.
Hermans C, Verbruggen N (2005) Physiological characterization of Mg deficiency in Arabidopsis thaliana. J Exp Bot 56:2153–2161.
Huang GZ, Lin TC (2016) Response of two epiphyte species to nitrogen and phosphorus fertilization at a humid subtropical rainforest in northeastern Taiwan. Taiwan J For Sci 31:293–304.
Huang JB, Liu WY, Li S, et al. (2019) Ecological stoichiometry of the epiphyte community in a subtropical forest canopy. Ecol Evol 9:14394–14406.
Islam SN, Mohamad SMBH, Azad AK (2019) Acacia spp.: Invasive trees along the Brunei Coast, Borneo. Coastal Research Library. 455–476.
Koerselman W, Meuleman AFM (1996) The Vegetation N:P Ratio: a New Tool to Detect the Nature of Nutrient Limitation. J Appl Ecol 33:1441.
Lin CY, Yeh DM (2008) Potassium nutrition affects leaf growth, anatomy, and macroelements of Guzmania. HortScience 43:146–148.
Metali F, Salim KA, Tennakoon K, Burslem DFRP (2015) Controls on foliar nutrient and aluminium concentrations in a tropical tree flora : phylogeny, soil chemistry and interactions among elements. New Phytol 280–292.
Oddo E, Inzerillo S, Bella F La, Grisafi F, Salleo S, Nardini A (2011) Short-term effects of potassium fertilization on the hydraulic conductance of Laurus nobilis L. Tree Physiol 31:131–138.
Praveen A, Pandey VC (2019) Pteridophytes in phytoremediation. Environ Geochem Health, doi:10.1007/s10653-019-00425-0.
Ranty B, Aldon D, Cotelle V, Galaud JP, Thuleau P, Mazars C (2016) Calcium sensors as key hubs in plant responses to biotic and abiotic stresses. Front Plant Sci 7:1–7.
Razaq M, Zhang P, Shen HL, Salahuddin (2017) Influence of nitrogen and phosphorous on the growth and root morphology of Acer mono. PLoS One 12:1–13.
Richardson SJ, Walker LR (2010) Nutrient ecology of ferns. Fern Ecology. 111–139.
Sardans J, Alonso R, Carnicer J, Fernández-Martínez M, Vivanco MG, Peñuelas J (2016) Factors influencing the foliar elemental composition and stoichiometry in forest trees in Spain. Perspect Plant Ecol Evol Syst 18:52–69.
Sardans J, Peñuelas J (2014) Climate and taxonomy underlie different elemental concentrations and stoichiometries of forest species: the optimum. Plant Ecol 215:441–455.
Schneider H, Schuetipelz E, Pryer KM, Cranfill R, Magallón S, Lupia R (2004) Ferns diversified in the shadow of angiosperms. Nature 428:553–557.
Smith AR, Pryer KM, Schuettpelz E, Korall P, Schneider H, Wolf PG (2006) A classification for extant ferns. Taxon 55:705–731.
Song WY, Zhang Z Bin, Shao HB, et al. (2008) Relationship between calcium decoding elements and plant abiotic-stress resistance. Int J Biol Sci 4:116–125.
Sun L, Zhang B, Wang B, et al. (2017) Leaf elemental stoichiometry of Tamarix Lour. species in relation to geographic, climatic, soil, and genetic components in China. Ecol Eng 106:448–457.
Suriyagoda LDB, Rajapaksha R, Pushpakumara G, Lambers H (2018) Nutrient resorption from senescing leaves of epiphytes, hemiparasites and their hosts in tropical forests of Sri Lanka. J Plant Ecol 11:815–826.
Testo W, Sundue M (2016) A 4000-species dataset provides new insight into the evolution of ferns. Mol Phylogenet Evol 105:200–211.
Tong R, Zhou B, Jiang L, Ge X, Cao Y, Yang Z (2019) Leaf nitrogen and phosphorus stoichiometry of chinese fir plantations across China: A meta-analysis. Forests 10:1–16.
Umana NH-N, Wanek W (2010) Large canopy exchange fluxes of inorganic and organic nitrogen and preferential retention of nitrogen by epiphytes in a tropical lowland rainforest. Ecosystems 13:367–381.
Watkins JE, Cardelús CL (2012) Ferns in an angiosperm world: Cretaceous radiation into the epiphytic niche and diversification on the forest floor. Int J Plant Sci 173:695–710.
Watkins JE, Philip J, Cardelús CL (2007) The influence of life form on carbon and nitrogen relationships in tropical rainforest ferns. Oecologia 153:225–232.
White PJ, Broadley MR (2003) Calcium in plants. Ann Bot 92:487–511.
White PJ, Brown PH (2010) Plant nutrition for sustainable development and global health. Ann Bot 105:1073–1080.
Winkler U, Zotz G (2009) Highly efficient uptake of phosphorus in epiphytic bromeliads. Ann Bot 103:477–484.
Winkler U, Zotz G (2010) ‘And then there were three’: Highly efficient uptake of potassium by foliar trichomes of epiphytic bromeliads. Ann Bot 106:421–427.
Wright IJ, Westoby M, Reich PB (2002) Convergence towards higher leaf mass per area in dry and nutrient-poor habitats has different consequences for leaf life span. J Ecol 90:534–543.
Zheng S, Shangguan Z (2007) Spatial patterns of leaf nutrient traits of the plants in the Loess Plateau of China. Trees - Struct Funct 21:357–370.
Zotz G (2004) The resorption of phosphorus is greater than that of nitrogen in senescing leaves of vascular epiphytes from lowland Panama. J Trop Ecol 20:693–696.
Zotz G (2016) Plants on Plants – The Biology of Vascular Epiphytes.
Zotz G, Hietz P (2001) The physiological ecology of vascular epiphytes: Current knowledge, open questions. J Exp Bot 52:2067–2078.
Zotz G, Richter A (2006) Changes in carbohydrate and nutrient contents throughout a reproductive cycle indicate that phosphorus is a limiting nutrient in the epiphytic bromeliad, Werauhia sanguinolenta. Ann Bot 97:745–754.

Tropical Terrestrial And Epiphytic Ferns Have Different Leaf Stoichiometry With Ecological Implications.

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Chemical analysis

Total leaf Nitrogen (N) and Phosphorus (P)

Total leaf Potassium (K), Calcium (Ca) and Magnesium (Mg)

Statistical analysis

Results

Discussion

Declarations

Funding

Acknowledgements

References

Supplementary Files

Status:

Version 1