Effect of Gap Position on the Heavy Metal Contents of Epiphytic Mosses and Lichens on the Fallen Logs and Standing Trees in an Alpine Forest

Abstract

To understand the role of the forest gaps and epiphytic mosses and lichens in the heavy metal cycles of forest ecosystems, the biomass, concentration, and storage of Cd, Pb, Cu, and Zn in epiphytic mosses and lichens on fallen logs and standing trees from the gap center to the closed canopy of an alpine forest ecosystem on the eastern Tibetan Plateau were investigated. Mosses were the dominant epiphytes on fallen logs and standing trees and contribute 82.1–95.1% of total epiphyte biomass in the alpine forest. A significantly higher biomass of epiphytic mosses and lichens was observed at the gap edge. The heavy metals concentration in mosses and lichens on fallen logs and standing trees varied widely with gap positions. Lower concentrations of Cd, Cu, and Pb were found in the mosses and lichens under the closed canopy, higher concentrations of Cd and Pb were detected in the mosses and lichens at the gap edge, and higher concentrations of Cu were found at the gap center. A significant difference in Zn concentration was observed between the mosses and lichens. No significant differences in Pb or Zn concentrations were observed in the mosses and lichens between the fallen log and standing tree substrates. Furthermore, the epiphytic mosses and lichens at the gap edge accumulated more Cd, Pb, and Cu, whereas the epiphytic lichens on the fallen logs and large shrubs at the gap center accumulated more Zn. In conclusion, gap regeneration accelerates the cycling of heavy metals in alpine forest ecosystems by promoting the growth of epiphytic mosses and lichens on fallen logs and standing trees at gap edges and increasing the concentration of heavy metals in these plants.

1. Introduction

The epiphyte community on coarse woody debris (CWD) and standing trees primarily consists of mosses, lichen, and certain vascular plants [1,2]. As a basic component of most forest ecosystems, epiphytic mosses and lichens on CWD and standing trees play crucial roles not only in promoting biodiversity and holding water [3,4], but also in regulating biogeochemical cycles [5,6,7]. Accordingly, the growth regimes and the elemental concentrations of epiphytic mosses and lichens on CWD and standing trees will to some extent influence the cycling of elements in the forest ecosystem. However, the epiphytic community is sensitive to environmental changes [8,9]. As a consequence, the growth and elemental concentrations of epiphytic plants might be greatly influenced by disturbances to substrates and forests, and these changes may affect the biogeochemical cycles of the forest ecosystem.

The growth of epiphytic mosses and lichens can be affected by the temperature and moisture [10,11], and by atmospheric deposition [12,13]. Within the forest ecosystem, the growth of epiphytes might clearly reflect the differentiation and heterogeneity of hydrothermal conditions [9,14]. Simultaneously, the concentration of elements in epiphytes can elucidate the process of elemental cycles and the distribution of elements in the forest [15,16], particularly as influenced by rainfall [17]. The hydrothermal conditions and the precipitation distribution in forests may be largely attributable to environmental gradients from the gap center of the forest gap to closed canopy [18,19]. As a result, the biomass and concentrations of heavy metals in epiphytic mosses and lichens may vary greatly with the gap position.

Gaps are a major form of disturbance in primary forest ecosystems that dictate the process of forest regeneration [20,21], as well as the biogeochemical cycles as a result of canopy cation exchange and interception [17]. Gaps can alter the microclimate within the forest ecosystem and affect the growth of epiphytic mosses and lichens [11,22,23]. The forest edge has been documented as preferably adapted to the growth of epiphytes because of the “edge effect” [24,25]. In addition, the gap position could affect the concentrations of elements in epiphytic mosses and lichens based on three factors. First, differential leaching and canopy interception inside and outside of the gap can influence the uptake of heavy metals by epiphytic mosses and lichens during the precipitation events [16,26,27]. Second, the nutrients and heavy metals in throughfall and stemflow could be directly absorbed by epiphytic mosses and lichens on standing trees [28,29]. Third, gaps can also influence the uptake of heavy metals by epiphytic mosses and lichens by altering the moisture, temperature, and substrate quality of fallen logs and the bark of standing trees inside and outside the forest gap. To date, however, the effects of gap position on the growth and heavy metal concentrations of epiphytic mosses and lichens on fallen logs and standing trees remain unknown.

Fallen logs and standing trees are two important substrates for epiphytic mosses and lichens in most forest ecosystems [2,3]. Fallen logs represent a major source of CWD [1], and fallen logs distributed within the forest ecosystem provide a more favorable and continuous habitat for the growth of epiphytic mosses and lichens, as well as the growth of other substrates [30]. Theoretically, fallen logs exposed to different gap positions will experience different hydrothermal and light regimes, and thereafter foster different species of epiphytic mosses and lichens. Consequently, the growth and heavy metal concentration of epiphytic mosses and lichens on fallen logs may vary with forest microhabitat. Similarly, the moisture, temperature, and light regimes of the bark of the standing trees also vary with gap positions and influence the growth of the epiphytic mosses and lichens [31]. Additionally, the epiphytic mosses and lichen on standing trees can be affected by stemflow water [17]. However, little information is available on the differential effects of gap positions on the growth and heavy metal absorption of epiphytic mosses and lichens between fallen logs and standing trees.

The alpine forest located in the upper reaches the of Yangtze River fulfills a crucial role in retaining water, promoting biodiversity, conserving soil, and sequestering atmospheric carbon dioxide [4,32]. In particular, a thinner mineral soil layer, thicker organic layer, and higher CWD stocks have been found in the primary Minjiang fir (Abies faxoniana) forest ecosystems [33], and fallen logs and epiphytic bryophytes and lichens function as important sinks for water and nutrients [4,34]. Furthermore, the regeneration of the Minjiang fir forest is driven by gaps created by normal and abnormal tree death from events such as frequent mountain hazards, snow disaster, and natural death [35]. Consequently, gaps may have underlying roles in adjusting to the growth of epiphytic bryophytes and lichens through changes in the moisture, temperature, and light regimes. However, the effects of gaps and substrates (fallen logs and standing trees) on the biomass and heavy metal uptake of epiphytic mosses and lichens remain unknown. We hypothesized that (1) the biomass of epiphytes would be highest on fallen logs at the gap edge because the fallen logs and gap edges provided more favorable growth conditions; and (2) the epiphytic mosses and lichens in the center of the gap would present higher concentrations and accumulations of heavy metals because of the distribution of precipitation in the alpine forest, whereas the effects of fallen logs and standing trees on heavy metal concentrations of epiphytic mosses and lichens would vary greatly with gap position.

To test these hypotheses, we investigated the biomass of epiphytic mosses and lichens on fallen logs and standing trees (including large shrubs) in the gap center, at the gap edge, and under a closed canopy in the primary Minjiang fir forest ecosystem in the upper reaches of the Yangtze River. Simultaneously, the concentrations of Cd, Pb, Cu, and Zn in epiphytic mosses and lichens were measured, and the storage of these elements was calculated. The objective of this study is to understand the role of epiphytic mosses and lichens with regard to gap regeneration and heavy metal cycles in the alpine forest ecosystem.

2. Materials and Methods

2.1. Site Description

The study was conducted at the Long-term Research Station of the Alpine Forest Ecosystems of Sichuan Agricultural University, which is located in the Miyaluo Nature Reserve (102°53′–102°57′ E, 31°14′–31°19′ N; 2458–4619 m above sea level) in Lixian County, Sichuan Province, southwestern China. This region is a transitional area between the Sichuan Basin and the Tibetan Plateau. The annual precipitation is approximately 850 mm, and the annual mean temperature is 3 °C, with a maximum and minimum temperature of 23 °C (July) and −18 °C (January), respectively. In the alpine zone, the primary Minjiang fir (Abies faxoniana) forest is well conserved. The shrubs layers are dominated by Salix paraplesia and Rhododendron lapponicum, and the herbaceous layers are dominated by Festuca ovin, Carex spp., Cystopteris montana, Berberis sargentiana, and Parasenecio forrestii. The fern species Adiantum capillus-junonis and Athyrium austro-orientale are mainly observed on the ground and Elaphoglossum yunnanense is mainly observed on the trunks. The lichen genera are dominated by Peltigera Pers, Parmelia Ach., Everniastrum, and Lobaria Schreb. The mosses are dominated by Hylocamium splendens, Actinothuidium hookeri, and Thuidium cymbifolium.

2.2. Experimental Design

Centered on forest gap (each of the gap circle diameters is longer than 25 m), three 100 × 100 m plots were established at least 500 m apart in the primary Minjiang fir forest ecosystem according to our previous investigations. The average tree age is about 130 years. In each plot, three 20 × 20 m subplots were selected at the gap center, gap edge, and under closed canopy, respectively. Forest gaps formed in the last two decades and were naturally occurring [35]. In each subplot, we classified fallen logs that were greater than 1 m in length or height and 10 cm in diameter and upright trees with diameters of 15 to 45 cm and with more than 50% epiphytic cover on the trunk were selected [1,36]. The survey data and sample information on the subplots are listed in

Table 1. Study site positions and epiphyte composition and diversity on FL (fallen log) and ST (standing tree, including large shrubs) in the gap center, at the gap edge, and under the closed canopy.

Gap Position		No. of Substrates	No. of Epiphytic Species		Shannon-Wiener Index	Simpson Index	Species Evenness	Dominant Moss Species	Dominant Lichen Genera
			Mosses	Lichens
Gap Center	FL	18	9	5	2.32	0.82	0.84	Actinothuidium hookeri, Dicranum scoparium, Entodon cladorrhizans, Hylocomium splendens, Hygrohypnum smithii, Leucodon sciuroides, Neckera pennata, Barbella enervis	Peltigera (Pers.), Parmelia (Ach.), Everniastrum, Lobaria (Schreb)
	ST	21	8	4	2.07	0.82	0.78
Gap Edge	FL	22	10	4	2.14	0.81	0.76
	ST	29	11	4	2.52	0.89	0.87
Closed Canopy	FL	18	10	5	1.99	0.74	0.69
	ST	41	11	4	2.40	0.86	0.81

. The lichens were only identified to the genus.

2.3. Sampling and Chemical Analysis

In August 2015, mosses on the trunks of fallen logs and standing trees were collected from 20 cm × 20 cm quadrats at different cardinal directions on the trunks [37]. The superficial area of the trunk was estimated as a circular cone that was cut vertically to the bottom. We then visually assessed the percentage of moss cover on each trunk. The epiphytic lichens were collected in their entirety. Since most of the moss species grow together, we cannot separate them from moss blocks, especially roots. As a result of this, we only classified mosses and lichens into two broad categories.

To measure the element concentrations, the oven-dried samples were ground in a mill with a 0.3 mm mesh screen, and the powdered epiphytic samples were digested with a concentrated acid mixture of HNO₃-HClO₄ (5:1, v/v) and heated to 160 °C for 5 h. After digestion, the Cd, Pb, Cu, and Zn concentrations were determined by atomic absorption spectrometry (AAS, AA-7000, Shimadzu Corporation, Kyoto, Japan). The concentration and total amount of heavy metals were calculated for epiphytic mosses and lichens.

2.4. Calculations

All biomass results were based on oven-dried (24 h at 65 °C) weights. The biomass of each epiphytic categories was calculated for fallen logs and standing trees. The biomass of the samples was calculated as follows:

$$\begin{gathered} {\mathrm{E}}_{\mathrm{biomass}}=Q_{biomass}\times T_{area}\times C_1 \\ {\mathrm{M}}_{\mathrm{biomass}}={\displaystyle {\sum }_{i=1}^n{E}_{biomass}}/{\displaystyle {\sum }_{i=1}^{n}S} \end{gathered}$$

(1)

where E_biomass is the total biomass of epiphytes on each trunk (fallen log or standing tree) (kg), Q_biomass is the mean value of biomass of epiphytes in a 20 cm × 20 cm quadrat on a trunk (kg), T_area is the superficial area of the trunk (m⁻²), C₁ is the degree of cover of epiphytes on the trunk (%), M_biomass is the biomass of epiphytes at each gap position at the site (kg m⁻²), and S is the total surface area of the trunk (m⁻²).

The concentration (C₂) of Cd, Pb, Cu, and Zn in epiphytes (mg kg⁻¹) was calculated using the following formula:

$${\mathrm{C}}_2=(\rho \times V)/m$$

(2)

where ρ is the measured content (mg L⁻¹), V is the sample volume (L), and m is the weighed sample mass during the sample processing (g).

The total store of heavy metals (T) in epiphytes at each gap position (mg m⁻²) was calculated using the following formula:

T = M_biomass × C₂

(3)

2.5. Statistical Analyses

One-way ANOVA (analysis of variance) and LSD (least significant difference) tests were used to evaluate the differences in Cd, Pb, Cu, and Zn concentrations of epiphytic mosses and lichens at different gap positions, as well as the storage of Cd, Pb, Cu, and Zn in epiphytes. Significance was observed at the 0.05 level. The effects of gap position, epiphytic categories, and substrate on the concentrations and total accumulation of heavy metals were analyzed using a multivariate analysis of variance (MANOVA). All of the statistical analyses were performed using the SPSS software package (standard release version 22.0 for Windows, SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Moss and Lichen Biomass

The total biomass of the epiphytes was 310.08 ± 159.45 kg ha⁻¹ on the fallen logs, and 93.09 ± 40.36 kg ha⁻¹ on the standing trees. Mosses dominated the epiphytic plant community and contributed 82.1% to 95.1% of the total epiphytic biomass. The gap edge had a higher biomass of epiphytic mosses and lichens than the gap center and the closed canopy, although the epiphytic lichen biomass varied greatly with growth substrate and gap position (

Table 2. Epiphytic biomass on fallen logs and standing trees in the gap center, at the gap edge, and under the closed canopy, and the ratio of each epiphytic biomass to the total epiphytic biomass in the alpine primary Minjiang fir forest ecosystem.

	Gap Center		Gap Edge		Closed Canopy
	Biomass (g m−2)	Ratio (%)	Biomass (g m−2)	Ratio (%)	Biomass (g m−2)	Ratio (%)
Fallen logs
Mosses	599.3	82.1	1235.7	88.7	1217.9	94.1
Lichens	85.7	11.8	110.9	7.9	40.0	3.1
Ferns	1.9	0.2	3.6	0.2	1.9	0.1
Herbs	42.7	5.9	43.3	0.3	35.9	2.7
Total	729.6	100.0	1393.5	100.0	1295.7	100.0
Standing trees
Mosses	689.9	95.1	800.4	83.6	350.9	89.7
Lichens	36.2	4.9	156.7	16.3	29.9	7.6
Ferns	-	-	-	-	2.6	0.6
Herbs	-	-	-	-	7.4	1.8
Total	726.1	100.0	957.1	100.0	390.8	100.1

).

3.2. Heavy Metal Concentrations in Mosses and Lichens

As shown in Figure 1, the concentration of heavy metals in mosses and lichens varied widely with gap position. Higher concentrations (p < 0.05) of Cd, Pb, and Zn were found in mosses at the gap edge, and the highest (p < 0.01) Cu concentrations in epiphytic mosses were observed on large shrubs at the gap center. Higher Cd and Pb concentrations were detected in the epiphytic lichens on fallen logs in the gap center and on standing trees at the gap edge, respectively. Epiphytic lichens on both fallen logs and large shrubs had the highest Cu concentrations in the gap center, and the lowest Zn concentrations in epiphytic lichens were observed at the gap edge. Moreover, neither the substrate nor the epiphytic categories had a significant effect on either Pb or Zn concentrations in epiphytic mosses and lichens, although a significant interaction was observed between the substrate and epiphytic categories (p < 0.01) on the Pb concentrations in epiphytes (

Table 3. Multivariate analysis of variance (MANOVA) for the effect of gap position, substrate, and categories on the heavy metal concentrations in epiphytic mosses and lichens on fallen logs and standing trees/large shrubs in the alpine Minjiang fir forest ecosystem.

Source Variance	df	F-Value				p-Value
		Cd	Pb	Cu	Zn	Cd	Pb	Cu	Zn
Gap position (G)	2	61.922	294.544	355.286	10.114	<0.001 **	<0.001 **	<0.001 **	0.001 **
Substrate (S)	1	12.618	0.001	95.685	0.023	0.002 **	0.971	<0.001 **	0.882
categories (C)	1	12.058	0.01	43.216	3.483	0.002 **	0.922	<0.001 **	0.074
G × S	2	3.955	3.476	105.848	7.997	0.003 *	0.047 *	<0.001 **	0.002 **
G × C	2	24.765	26.201	24.837	10.579	<0.001 **	<0.001 **	<0.001 **	0.001 **
S × C	1	0.002	16.611	6.896	0.96	0.968	<0.001 **	0.015	0.337
G × S × C	2	3.322	46.457	36.185	2.668	0.053	<0.001 **	<0.001 **	0.09

Significant effects: * p < 0.05, ** p < 0.01; n = 36.

). A correlation was observed between the gap position and substrate and gap position and epiphytic categories for all heavy metal concentrations measured in the epiphytes. However, the Cd and Zn concentrations of epiphytes were not significantly affected by the interactions among gap position, substrate, and epiphytic categories (Table 3).

3.3. Heavy Metal Storage in Mosses and Lichens

Heavy metal storage in mosses and lichens varied significantly with gap position, growth substrate, and element. The epiphytic mosses stored more heavy metals than the epiphytic lichens (Figure 2). The epiphytic mosses and lichens at the gap edge stored more Cd and Pb, although the epiphytic lichens on fallen logs in the gap center stored more (p < 0.01) Cd. The Cu storage in epiphytic mosses and lichens varied significantly with gap position, and the lowest Cu storage in both the epiphytic mosses and lichens was found under the closed canopy. In contrast, higher Zn storage was found in the epiphytic mosses on fallen logs at the gap edge and under the closed canopy. Furthermore, the MANOVA indicated that the gap position, substrate, categories, and their combined interactions had significant effects on Cd, Pb, Cu, and Zn storage in the epiphytic mosses and lichens on fallen logs and standing trees; however, the effects of the substrate and the interaction between the substrate and categories were not significantly related to Cd storage in epiphytes (

Table 4. Multivariate analysis of variance (MANOVA) for the effects of gap position, substrate, and categories on heavy metal storage in epiphytic mosses and lichens on fallen logs and standing trees/large shrubs in the alpine Minjiang fir forest ecosystem.

Source Variance	df	F-Value				p-Value
		Cd	Pb	Cu	Zn	Cd	Pb	Cu	Zn
Gap position (G)	2	119.720	66.648	84.401	11.540	<0.001 **	<0.001 **	<0.001 **	<0.001 **
Substrate (S)	1	0.004	7.221	25.446	61.483	0.952	0.013 *	<0.001 **	<0.001 **
categories (C)	1	404.133	239.213	341.470	314.549	<0.001 **	<0.001 **	<0.001 **	<0.001 **
G × S	2	12.769	5.553	117.384	46.752	<0.001 **	0.01 **	<0.001 **	<0.001 **
G × C	2	101.484	22.317	53.907	28.819	<0.001 **	<0.001 **	<0.001 **	<0.001 **
S × C	1	0.298	10.212	27.297	68.095	0.590	0.004 **	<0.001 **	<0.001 **
G × S × C	2	22.564	14.228	156.704	38.892	<0.001 **	<0.001 **	<0.001 **	<0.001 **

Significant effects: * p < 0.05, ** p < 0.01; n = 36.

).

4. Discussion

Our results supported the hypothesis that mosses dominate the epiphytic communities on fallen logs and standing trees in the Minjiang fir alpine forest and that fallen logs and standing trees at the gap edge had a higher biomass of epiphytic mosses and lichens. The relatively wet, cool forest environment in the coniferous-dominated stands provides favorable growing conditions for many mosses [22]. The well-documented “edge effect” may also create more favorable light and moisture conditions for epiphytes at the gap edge. In contrast, epiphytes on fallen logs and large shrubs in the gap center are exposed to and may suffer from harsh light and wind conditions, which could lead to desiccation and plant damage [11]. Similarly, a lower biomass of epiphytic mosses and lichens on fallen logs and standing trees under the closed canopy could result from the weaker light.

The results also indicated that the biomass of epiphytic mosses and lichens on the fallen logs was significantly higher than on the standing trees. This finding could be explained by the ability of epiphytes to more effectively extract water and nutrients from the fallen logs [1]. Furthermore, large shrubs in the gap center had a higher biomass of epiphytic mosses than those under the closed canopy, which reflected the higher coverage and greater thickness of mosses on tree trunks in the gap center than in the closed canopy [4].

Gap position had a strong effect on the concentration and storage of Cd, Pb, Cu, and Zn in epiphytic mosses and lichens on fallen logs and standing trees in the alpine forest, although these effects varied with the heavy metal type, which supports our second hypothesis. First, the highest and lowest concentrations of Cd and Pb in epiphytic mosses and lichens were observed at the gap edge and under the closed canopy, respectively, which was attributed to the canopy intercepting heavy metals from atmospheric precipitation [7,19,38]. Rainfall is a major source of heavy metals [39,40]; thus, epiphytic mosses and lichens under the closed canopy absorb less Cd and Pb than those at the gap edge. In addition, the washing effect of precipitation may result in lower Cd and Pb concentrations in epiphytic mosses at the gap center [41]. This could be proven by the lower concentration of Cd and Pb in mosses on fallen logs than large shrub because fallen logs are subjected to a stronger washing effect when happening on heavy rainfall.

In addition, the thicker epiphytic moss layer with a looser structure could accelerate the transfer of Cu from the upper layer to lower layer when the leaching process occurs in the gap edge and closed canopy [42]. Nevertheless, the evapotranspiration with solar radiation in the gap center can promote the uptake of Cu in epiphytic moss from bottom to top [43]. Thereby, the highest concentrations of Cu in epiphytic mosses on the large shrubs and fallen logs were found respectively at the gap center and gap edge, and the lowest Cu concentrations in epiphytic mosses on standing trees and fallen logs were both found under the closed canopy. Furthermore, the highest and lowest Zn concentrations in the epiphytic mosses on fallen logs and standing trees were observed at the gap edge and gap center, respectively, but the highest and lowest Zn concentrations in epiphytic lichen were observed at the gap center and gap edge, respectively. The difference of Zn concentrations between mosses and lichens could depend on the uptake mechanism of Zn in epiphytic mosses and lichens, and the higher temperature and light-stimulated in the gap center would promote the uptake of Zn in some lichens [44]. Certainly, Cu and Zn are two microelements necessary for plant growth and reproduction, and the differences in physiological requirements for particular elements between epiphytic mosses and lichens could explain the differential response of heavy metal concentrations in epiphytic mosses and lichens to gap position [45,46].

In our study, epiphytic mosses stored more Cd, Pb, Cu, and Zn than epiphytic lichens, primarily because of the significant difference in biomass between epiphytic mosses and lichens, with the epiphytic mosses presenting a higher biomass. The higher leaf surfaces and higher biomass of mosses can promote heavy metal accumulation [47]. Moreover, the epiphytic mosses and lichens at the gap edge stored more Cd and Pb, which is attributable to both the higher biomass of the epiphytes and the higher Cd and Pb concentrations in the epiphytic mosses and lichens at the gap edge. Similarly, the epiphytic mosses in the gap center stored more Cu than the mosses at the gap edge, which was primarily because of the higher Cu concentrations in the epiphytic mosses at the gap center. However, the lowest Zn storage in the epiphytes resulted from a lower Zn concentration in the mosses and the lower biomass of epiphytic mosses and lichens. Significant differences were not observed in Zn storage in the epiphytes between the gap edge and the closed canopy because the Zn concentrations in the epiphytes, which were affected by the gap position, varied widely with the type of epiphyte, although the growth substrates at the gap edge had a higher biomass of epiphytic mosses and lichens.

5. Conclusions

Mosses dominated the epiphytic community, and fallen logs at the gap edge had the highest biomass of epiphytic moss in the alpine forest. Overall, the gap position had a strong effect on the concentration and storage of heavy metals in the epiphytic mosses and lichens at the study site, although the effect varied depending on the type of heavy metal. Furthermore, interactions between the gap position and growth substrate determined the concentration and storage of heavy metals in the epiphytic mosses and lichens. This study showed that the heavy metals cycle and accumulation were unique in alpine forest where forest gaps have been playing a more crucial role in the distribution of elements than other factors.