Lignin Degradation in Foliar Litter ofTwo Shrub Species from the GapCenter to the Closed Canopy in an

Alpine Fir Forest

Wei He, Fuzhong Wu, Wanqin Yang,* Bo Tan, Yeyi Zhao, Qiqian Wu, andMin He

Key Laboratory of Ecological Forestry Engineering, Long-term Research Station of Alpine Forest Ecosystem, Institute of Ecology andForestry, Sichuan Agricultural University, Chengdu 611130, China

ABSTRACT

To understand the effects of forest gaps on lignin

degradation during shrub foliar litter decomposi-

tion, a field litterbag experiment was conducted in

an alpine fir (Abies faxoniana) forest of the eastern

Tibet Plateau. Dwarf bamboo (Fargesia nitida) and

willow (Salix paraplesia) foliar litterbags were placed

on the forest floor from the gap center to the closed

canopy. The litterbags were sampled during snow

formation, snow coverage, snow melting and the

growing season from October 2010 to October

2012. The lignin concentrations and loss in the

litter were measured. Over 2 years, lignin loss was

lower in the bamboo litter (34.64–43.89%) than in

the willow litter (38.91–55.10%). In the bamboo

litter, lignin loss mainly occurred during the first

decomposition year, whereas it occurred during the

second decomposition year in the willow litter.

Both bamboo and willow litter lignin loss decreased

from the gap center to the closed canopy during the

first year and over the entire 2-year decomposition

period. Compared with the closed canopy, the gap

center showed higher lignin loss for both bamboo

and willow litter during the two winters, but lower

lignin loss during the early growing period. Addi-

tionally, the dynamics of microbial biomass carbon

during litter decomposition followed the same

trend as litter lignin loss during the two winters and

growing period. These results indicated that alpine

forest gaps had significant effects on shrub litter

lignin loss and that reduced snow cover during

winter warming would inhibit shrub lignin degra-

dation in this alpine forest.

Key words: alpine forest; freeze-thaw cycle; gap;

lignin degradation; microbial biomass carbon;

shrub foliar litter; snow cover.

INTRODUCTION

Lignin is well known as a recalcitrant component of

the litter substrate and exerts considerable control

over the rate of decomposition (Melillo and others

1982). As one of the primary processes that occurs

during litter decomposition, lignin degradation

plays an important role in terrestrial carbon cycles

and has historically been well studied (Taylor and

Received 17 November 2014; accepted 11 August 2015

Electronic supplementary material: The online version of this article

(doi:10.1007/s10021-015-9921-6) contains supplementary material,

which is available to authorized users.

Author contributions Wanqin Yang and Fuzhong Wu designed the

study. Wei He, Bo Tan, Yeyi Zhao, Qiqian Wu and Min He performed the

research. Wanqin Yang proposed the structure of the paper and Wei He

wrote the paper.

*Corresponding author; e-mail: scyangwq@163.com

EcosystemsDOI: 10.1007/s10021-015-9921-6

� 2015 Springer Science+Business Media New York

others 1989; Cox and others 2001; Austin and

Ballare 2010; Klotzbucher and others 2011). Some

researchers have found that lignin degradation of-

ten occurs in the later stage of decomposition after

the loss of labile components (McClaugherty and

Berg 1987; Rutigliano and others 1996). However,

the litter decomposition process in high latitude

and altitude ecosystems is subject to long periods of

seasonal snow cover (Campbell and others 2005;

Wu and others 2010). Researchers have observed

that some physical and chemical processes during

freeze-thaw events can damage the structure of

lignin or other resistant components (Taylor and

Parkinson 1988; Henry 2007), contributing to lig-

nin degradation even in the early stage of litter

decomposition in cold biomes. Moreover, the depth

of snow cover and its dynamics can influence

freeze-thaw events in the field (Wu and others

2010; Zhu and others 2013), which not only di-

rectly regulate lignin degradation during the cold

season but also influence litter decomposition and

lignin degradation during the warm season as well

by increasing decomposability following winter

decomposition (Baptist and others 2010; Christen-

son and others 2010; Zhu and others 2012).

A debate has also arisen regarding whether forest

gaps can promote or limit litter decomposition.

Zhang and Zak (1995) declared that the litter decay

rate was faster under a closed canopy or in small

gaps than in large gaps in a subtropical forest, but

Denslow and others (1998) found no significant

relationships between gap sizes and litter decom-

position rates in a wet tropical forest. Nevertheless,

a study conducted in cold forests of British Co-

lumbia showed that higher mass loss occurred in

large gaps (Prescott and others 2000). The varia-

tions in climate and litter quality among these

previous studies mean that the available results are

inconclusive with regard to controlling the rates

and dynamics involved in lignin degradation in

high latitude and altitude ecosystems.

Heterogeneity in vegetation cover across the

landscape can also interact with snow to alter snow

cover and the microenvironment and, in turn,

influence lignin degradation. First, the depth of

snow cover often decreases from the gap center to

the closed canopy due to being sheltered by the

alpine forest canopy during winter. We also ob-

served an absence of snow cover patches in some

places under the closed canopy (Wu and others

2014). The much thicker snow cover in the gap

center serves as an insulator that can maintain a

sufficiently warm temperature to support biotic

activities (Campbell and others 2005; Saccone and

others 2013) and contributes to lignin degradation

in litter. Conversely, under thin or no snow cover

at the forest edge and under the closed canopy, the

litter is often exposed to extreme below freezing

temperatures and frequent events of freeze and

thaw. The activity of decomposers is relatively

lower at the forest edge and under the closed ca-

nopy than in the gap center (Freppaz and others

2008; Tan and others 2014), but the freeze-thaw

cycles increase litter decomposability by physical

damage to the lignin structure, which contributes

to lignin degradation. Furthermore, freeze and

thaw events are associated with snow formation

and melting (Schimel and Clein 1996; Groffman

and others 2001), which can also significantly af-

fect lignin degradation (Taylor and Parkinson 1988;

Henry 2007) beneath snow cover. Second, the gap

center often exhibits a higher soil surface temper-

ature during the growing season compared with

the gap edge and closed canopy because it receives

more solar radiation (Zhang and Liang 1995). A

warmer microenvironment (Taylor and others

1989) and adequate sunlight (Austin and Ballare

2010) can theoretically promote the contribution of

decomposers to lignin degradation. Nevertheless,

the litter at the gap edge or under the closed canopy

is more decomposable following physical destruc-

tion of the lignin structure during winter, and this

more decomposable litter may display more rapid

lignin degradation than litter in the gap centers.

Unfortunately, little information is available on

how the process of litter lignin degradation is af-

fected in forest gaps in high latitude and high alti-

tude ecosystems. Therefore, based on these

previous studies, we predicted that litter lignin

degradation would decrease along the snow thick-

ness gradient from the gap center to the closed

canopy during the winter because of the insulation

provided by thicker snow cover and that this rela-

tionship would be reversed during the growing

season because of the more decomposable litter

under the crown canopy after winter.

To test these predictions, a field decomposition

experiment using the freshly senesced foliar litter

from dwarf bamboo (Fargesia nitida) and willow

(Salix paraplesia) was conducted in an alpine fir

forest. The alpine forest, located in the upper

reaches of the Yangtze River and on the eastern

Tibet Plateau, has important roles in conserving

headwaters and soils, supporting biodiversity and

regulating the regional climate (Yang and others

2005). The seasonal snow cover is significant with a

visible snow thickness gradient from the gap center

to the closed canopy in winter, which has direct

and indirect effects on litter decomposition and

other key ecological processes (Wu and others

W. He and others

2010; Zhu and others 2012; Saccone and others

2013). Dwarf bamboo and willow are two domi-

nant understory shrubs in the alpine forests, and

the litterfall and decomposition of these species are

the primary components of forest material cycling

(Yang and Li 1992), though little attention has

been paid to these processes in these species. We

measured the lignin loss and degradation rates

during the decomposition of bamboo and willow

litter during different periods as the snow cover and

temperature dynamics changed over a 2-year per-

iod in three selected gaps from the gap center to the

closed canopy. The objectives were to (1) charac-

terize the effects of a forest gap on shrub litter lig-

nin degradation over different periods during the

decomposition process and to (2) explore the

potential responses of litter lignin degradation and

the related material cycling processes to the re-

duced snow cover that results from warming win-

ters. The results were also expected to provide clear

insight into material cycling processes in high lati-

tude and high altitude forest ecosystems.

MATERIALS AND METHODS

Experimental Design

The study region is located in the Miyaluo Nature

Reserve (102�53¢–102�57¢ E, 31�14¢–31�19¢ N; 2458–4619 m a.s.l.), Li County, Sichuan, southwest China.

The region is a transitional area between the Tibet

Plateau and the Sichuan Basin. The annual precipita-

tion is approximately 850 mm, and the annual mean

temperature ranges from 2�C to 4�C, with maximum

and minimum temperatures of 23�C and -18�C,respectively. The seasonal soil freeze-thaw period be-

gins in early November after the first snowfall, and the

soil remains frozen for 5–6 months. The tree canopy is

dominated by Abies faxoniana and Sabina saltuaria. The

understory shrubs are dominated by Salix paraplesia,

Fargesia nitida, Rhododendron lapponicum, Serberis sar-

gentiana, Sorbus rufopilosa, Rosa sweginzowii and others.

The herbs are dominated by Cacalia spp., Cystopteris

montana, Carex, Cyperus and others. A detailed report

containing soil information can be found in Zhu and

others (2013).

The nylon mesh bag technique (Guo and others

2006) was used to quantify the foliar litter lignin

loss and the lignin degradation rate in three se-

lected gaps, each measuring 25 m (the gap circle

diameter) with similar canopy densities within a

representative fir forest (102�54.72¢ E, 31�15.88¢ N;3582 m a.s.l.) in the nature reserve. Five positions

within each gap, measuring 4 9 4 m, were dis-

tributed from the gap center to the closed canopy

(gap center south, gap center north, canopy edge,

expanded edge and closed canopy) at 3–4 m

intervals to ensure adequate sampling of the

heterogeneous microenvironmental conditions

(Figure 1).

Litter Processing

In September 2010, freshly senesced leaves of

dwarf bamboo (Fargesia nitida) and willow (Salix

paraplesia) were collected from the floor of the

experimental forest. The leaves were air-dried for

more than two weeks at room temperature to en-

sure that the chemical and physical structure of

these litter species was not damaged. When the

litterbags were initially prepared, five samples of

each litter type were oven-dried at 65�C for 48 h to

determine the ratio between the air-dried and

oven-dried mass, which was used to convert the

initial air-dried mass of the litter to its oven-dried

mass. The subsamples were ground (0.3-mm sieve)

and analyzed to determine the initial chemical

composition (C, N, and P contents and lignin and

cellulose concentrations).

Figure 1. Pattern

diagram of litterbags in

the experimental layout

in the selected gaps at the

study sites on the

eastern Tibet Plateau. The

depicted circle (diameter is

25 m) is an example of a

forest gap, and the squares

(4 9 4 m) represent one

possible spatial

arrangement between the

gap and the positions of

the litterbags.

Lignin Degradation in Alpine Forest Gap

The initial litter chemical composition of C, N,

and P was determined as described by Lu (1999).

The C content was determined using the dichro-

mate oxidation-ferrous sulfate titration method

(Wu and others 2010). The levels of N and P were

determined via indophenol-blue colorimetry and

phosphomolybdenum-yellow colorimetry, respec-

tively (Wu and others 2010). All analyses were

conducted in triplicate.

The lignin and cellulose concentrations were

determined according to the acid detergent lignin

method (Vanderbilt and others 2008), with some

modifications. Briefly, fine litter samples (1.0 g)

that were oven-dried and ground were transferred

to digestion tubes and suspended in a solution of

H2SO4 (1.0 mol/l) and cetyltrimethylammonium

bromide (CTAB; 20 g/l; 80 ml). The tubes were

heated at 169�C for 1 h. After cooling, the tubes

were transferred to a sand core funnel (50 ml, G3

specification) and were washed with acetone until

the solution obtained through the suction filtration

was clean. After oven drying at 170�C for 1 h, the

sample and tube were weighed together and were

designated as W1. Subsequently, the sample was

soaked for more than 3 h in an H2SO4 solution

(72%), subjected to suction filtration and washed

with acetone, as described above. The sample was

then oven-dried at 170�C for 1 h. The sample and

tube were then weighed together and were desig-

nated as W2, which was placed in a muffle furnace

(Box Furnace; Lindberg/Blue M, Asheville, NC,

USA) at 550�C for 3 h and weighed after cooling

(designated as W3). The cellulose concentration

was determined from the weight loss difference

between W1 and W2 divided by the sample weight

(1.0 g) and then multiplied by 100, whereas the

lignin concentration was determined as the differ-

ence between the W2 and the W3 measurements.

All analyses were conducted in triplicate.

The samples of air-dried litter (a total of 10 g of

dry weight for each species of litter) were placed in

nylon bags (20 9 20 cm) with a 0.055-mm mesh

on the bottom, a 1.0-mm mesh on the surface and

with the edges sealed. A total of 1500 litterbags (3

gaps 9 5 positions 9 2 species 9 10 sampling

dates 9 5 replicates) were placed on the forest floor

from the gap center to the closed canopy on

October 26, 2010; the bags were placed without

covering them with any soil or litter (the litter on

the top of the soil was also not removed).

To quantify the lignin loss and the lignin degra-

dation rate during each critical period, we divided

the winter and the growing season into the snow

formation period (SF), the snow coverage period

(SC), the snow thawing period (ST), the early

growing period (EG), and the later growing period

(LG). Based on previous observations, the litterbags

were randomly collected after 58 (December 23,

2010), 128 (March 3, 2011), 175 (April 19, 2011),

297 (August 19, 2011), 378 (November 8, 2011),

427 (December 27, 2011), 498 (March 7, 2012),

550 (April 28, 2012), 669 (August 25, 2012), and

734 (October 29, 2012) days of exposure in the

field.

After removing the arthropods and the foreign

roots from the litterbags, the retrieved litter was

separated into two parts. One part was stored in a

refrigerator at 4�C for microbial biomass analysis

(analyses were completed within one week), and

the other part was oven-dried at 65�C for 48 h to

determine the dry mass and the lignin concentra-

tion.

Microbial Biomass

The microbial biomass carbon (MBC) was mea-

sured via the chloroform fumigation incubation

method (Vance and others 1987), with some

modifications. Briefly, samples of fresh retrieved

litter (0.5 g) were transferred to triangle bottles

(50 ml), which were placed into a vacuum drying

oven (DZF-6090; Shanghai cable spectrum instru-

ment Co., Ltd.) and incubated for 24 h with chlo-

roform (filtered with distilled water in a separatory

funnel three times) under a vacuum. The chloro-

form was removed, and the samples were subjected

to the vacuum treatment four times to ensure the

full release of the residual chloroform from the

samples. The triangle bottles were subsequently

sealed with plastic wrap and oscillated for 30 min

after the addition of 20 ml of a K2SO4 solution

(0.5 mol/l). The samples were filtered through fil-

ter paper, and the measured liquid was collected. A

5-ml aliquot of each liquid sample was transferred

to a digestion tube, and 10 ml of a potassium

dichromate-sulfuric acid mixture (0.018 mol/l

K2Cr2O7 and 12 mol/l H2SO4) was added per tube.

The tubes were heated at 169�C for 15 min and

were then transferred and titrated using a FeSO4

solution (0.05 mol/l). The treatment of the corre-

sponding un-fumigated samples was initiated at the

addition of the K2SO4 solution (0.5 mol/l), as de-

scribed above. The microbial biomass carbon con-

tent of the litter was calculated as the difference

between the chloroform-fumigated and the un-

fumigated samples (calculated as dry weight), with

the difference corrected with a conversion factor of

0.38 (Vance and others 1987). All analyses were

conducted in triplicate.

W. He and others

Microenvironment Measurements

The snow thickness was measured with a ruler on

each sampling day. The ambient temperatures in

the litterbags and of the atmosphere were mea-

sured every 2 h using iButton DS1923-F5 Re-

corders (Maxim/Dallas Semiconductor, Sunnyvale,

CA, USA), which were placed in one litterbag at

each sample location (gap center south, gap center

north, canopy edge, expanded edge and closed ca-

nopy) and up in one shrub tree.

A freeze-thaw cycle occurred whenever the

temperature dropped below 0�C for at least 3 h,

which was then followed by an increase above 0�Cfor at least 3 h, or vice versa (Konestabo and others

2007). To characterize the temperature dynamics of

each critical period, we calculated the average

temperature (AT) and the frequency of the freeze-

thaw cycle (FFTC) from the daily mean tempera-

tures and the number of freeze-thaw cycles per

period, respectively.

The remaining lignin content (ML), the lignin loss

(L) and the lignin degradation rate per 30 days (V)

of the litter were calculated as follow:

MLt ¼ Mt � Ct;

Lt %ð Þ ¼ MLðt�1Þ�MLt

� �=ML0 � 100;

Vt %ð Þ ¼ Ltð%Þ=Dt � 30;

where Mt is the remaining mass of the litter when

sampled; Ct is the concentration of the lignin when

sampled; MLt and ML(t– 1) are the remaining lignin

contents between the current and previous sam-

pling dates, respectively; ML0 is the initial lignin

content, and D4t is the number of days between the

current and previous sampling dates.

Statistical Analyses

The differences between the initial substrates of the

two species were evaluated using an independent

sample t test with an alpha level of 0.05. To test the

effects of gap-position (gap center south, gap center

north, canopy edge, expanded edge and closed ca-

nopy) on litter lignin loss, a one-way ANOVA was

conducted, with gap position as the main effect and

lignin loss in each period as the dependent variable.

Significant differences in litter lignin loss among

the positions during each decomposing period and

in the litter lignin degradation rate at each position

among the different decomposing periods were

determined using one-way ANOVA and least sig-

nificant difference tests (LSD). The relationships

between abiotic or biotic factors (AT and FFTC or

MBC, respectively) and the lignin loss among gap

positions in each period were determined using

Pearson correlation coefficients. All analyses were

performed in the SPSS statistical software program

(version 17.0).

RESULTS

Microenvironment Across LandscapePositions

Snow cover formed after the litterbags were put in

place and persisted throughout the winter of the

first year (Figure 2). The snow cover depth visibly

decreased from the gap center to the closed canopy

throughout the two winters (from the gap center,

on average, of 20.53 cm, to 12.55 cm, to 5.62 cm,

to 1.52 cm and to 0 cm under the closed canopy).

Figure 2. The effects of

the gaps on the thickness

of snow cover in the

alpine fir forest of the

eastern Qinghai-Tibet

Plateau (mean ± SE,

n = 5).

Lignin Degradation in Alpine Forest Gap

The varying depths of snow cover resulted in dif-

ferent temperature dynamics across the landscape

positions (Figure 3). The average temperature in-

creased from –1.61�C to –0.75�C and from –1.39�Cto –0.03�C following the decrease in the thickness

of the snow cover from the gap center to the closed

canopy in the two winters (from 0 to 175 d and 378

to 550 d of exposure), respectively. By contrast, the

average temperature decreased from 8.77�C to

6.68�C and from 12.38�C to 9.65�C from the gap

center to the closed canopy in the two growing

seasons (175 to 378 d and 550 to 734 d of expo-

sure), respectively. Thicker snow cover resulted in

fewer freeze-thaw cycles compared with thinner or

the absence of snow cover at the forest edge and

under the closed canopy during SC (snow coverage

period) of the first year and during the ST (snow

thawing period) of both years (see Supplementary

Material Table S1; on average, with thicker snow

cover, there were 18 fewer freeze-thaw cycles for

the first SC and 31 fewer for the two ST periods).

Moreover, the gap center had a relatively higher

average temperature than the expanded edge and

the closed canopy throughout the 2 years (see

Supplementary Material Table S1; on average, the

temperature in the gap center was 1.83�C higher

than in the expanded edge and the closed canopy

throughout the 2 years).

Initial Leaf Litter Chemistry

The N, P, and lignin contents and the N/P and

lignin/cellulose ratios were significantly lower in

bamboo litter than in willow litter, whereas the

cellulose content and the C/N, C/P and lignin/N

ratios were significantly higher in bamboo litter,

although no significant differences in foliar litter C

were observed between the two species (Table 1).

Influence of Landscape Position on LitterLignin Decomposition and MBC

The remaining mass, expressed as a percentage of

the initial mass, decreased as decomposition pro-

ceeded (Figure 4A, B). The percent remaining mass

increased from the gap center to the closed canopy

during each specific period, although a significantly

lower remaining mass was observed in willow litter

than in bamboo litter at each corresponding posi-

tion. A higher lignin concentration during the

decomposition of both bamboo and willow litter

was found in the gap center than at the expanded

edge or under the closed canopy during the later

decomposition stage in this study period (Fig-

ure 4C, D). The remaining lignin, expressed as a

percentage of the initial lignin, began to decrease

after exposure, which continued until the end of

the study in both species at all landscape positions

(Figure 4E, F). The remaining lignin was higher at

the expanded edge and under the closed canopy

than in the gap center in each specific period, with

the exception of a higher remaining lignin content

in willow litter in SF (snow formation period) and

SC in the first year in the gap center.

Gaps significantly affected MBC as decomposi-

tion proceeded in both litter species, although the

MBC content was higher in the willow litter than

in the bamboo litter for most decomposition peri-

ods (Figure 5). Higher MBC during both bamboo

and willow litter decomposition was detected in the

Figure 3. Dynamics of

the average temperature

in ambient foliar litter

and in the atmosphere in

the alpine fir forest of the

eastern Qinghai-Tibet

Plateau from October 26,

2010, to October 29, 2012

(a total of 734 days of

exposure in the field).

W. He and others

gap center than at the expanded edge or under the

closed canopy during the SF, SC and ST in both

years and during the LG (later growing period) in

the second year of decomposition. In contrast, MBC

was lower in the gap center than at the expanded

edge or under the closed canopy during the EG

(early growing period) of both years.

Influence of the Season on Litter LigninDecomposition and MBC

Over the 2-year decomposition period, lignin loss

ranged from 34.64 % to 43.89 % for bamboo litter

and from 38.91 % to 55.10 % for willow litter from

the closed canopy to the gap center, respectively

(Figure 6C, F). Lignin loss mainly occurred during

the first decomposition year in the bamboo litter

but during the second decomposition year in the

willow litter (Figure 6B, E). Lignin loss showed an

obvious decreasing trend from the gap center south

to the closed canopy throughout the entire 2-year

period, during the first year for both litters and

during the second year for bamboo litter; however,

in the second year, willow litter had the highest

and the lowest lignin loss values in the canopy edge

and the closed canopy, respectively (Figure 6B, C,

E, F). Compared with the closed canopy, the gap

center experienced higher lignin loss in both litter

species during the two decomposition winters

(Figure 6A, D). The bamboo litter lignin loss during

the first growing season and the willow litter lignin

loss during the second growing season generally

increased from the gap center to the closed canopy,

although higher lignin loss was observed in the gap

center during the first growing season in the willow

litter and the second growing season in the bamboo

litter (Figure 6A, D). Lignin degradation rate fol-

lowed similar patterns to lignin loss (%) (see Sup-

plementary Material Figure S2, S3).

Among the ten periods (see Supplementary

Material Figure S1), the first LG resulted in the

highest lignin loss in bamboo litter regardless of

forest position. However, the highest lignin loss in

willow litter in the gap center was observed during

the second SC and at the expanded edge and under

the closed canopy during the second EG. There

were only a few differences in the bamboo litter

lignin loss from the gap center to the closed canopy

during the first SF and the second decomposition

year (see Supplementary Material Figure S1a).

Compared with other forest positions, the gap

center showed higher lignin loss in bamboo litter

during the first SC and ST but lower lignin loss

during the first EG. Compared with the bamboo

litter, the willow litter’s lignin loss was lower in the

Table

1.

InitialQuality

oftheFoliarLitterof

Fa

rges

ian

itid

aand

Sa

lix

pa

rap

lesi

a(m

ean±

SD,

n=5)

Species

C(g

kg-1)

N(g

kg-1)

P(g

kg-1)

C/N

C/P

N/P

Lignin

(%)

Cellulose

(%)

Lignin/C

ellulose

Lignin/N

Fa

rges

ia

nit

ida

317.71a±

16.60

9.02b±

0.12

0.94b±

0.07

35.23a±

1.38

339.80a±

9.11

9.66b±

0.64

14.79b±

0.62

12.97a±

0.48

1.14b± 0.00

16.40a±

0.47

Sa

lix

pa

rap

lesi

a

371.89a±

31.55

14.33a±

0.26

1.28a±

0.06

25.93b±

1.74

290.72b±

10.31

11.23a±

0.36

21.79a±

1.02

10.60b±

1.04

2.06a± 1.11

15.20b±

0.44

Dif

fere

nt

low

erca

sele

tter

sin

dic

ate

asi

gnifi

can

tdif

fere

nce

bet

wee

nsp

ecie

sw

ith

inth

esa

me

vari

able

(in

dep

enden

tsa

mple

st

test

,P<

0.0

5).

Lignin Degradation in Alpine Forest Gap

gap center than in the closed canopy during the SF

and EG of the first decomposition year and during

the SF, ST and EG of the second decomposition

year, but was higher in the gap center during other

decomposition periods (see Supplementary Mate-

rial Figure S1).

Additionally, forest gaps exerted significant ef-

fects on lignin loss in the willow litter in all

decomposition periods and on lignin loss in the

bamboo litter in the majority of the decomposition

periods (except for the first SF and SC and the

second ST and EG) (see Supplementary Material

Figure 4. The effects of the gaps on the remaining mass (% of initial) (A, B), lignin concentration (C, D) and remaining

lignin content (% of initial) (E, F) in the foliar litter of Fargesia nitida and Salix paraplesia in the alpine fir forest of the

eastern Qinghai-Tibet Plateau from October 26, 2010, to October 29, 2012 (a total of 734 days of exposure in the field).

W. He and others

Figure S1, Table 2). Lignin loss in the foliar litter

from bamboo and willow was positively correlated

with the average temperature in the two SCs and

the first LG, whereas these parameters were nega-

tively correlated in willow in the second EG (Ta-

ble 3). Lignin loss was only positively correlated

with the frequency of freeze-thaw cycles in the

second SC (in both species) and the second LG (in

willow litter), whereas these parameters were

negatively correlated in the first EG and the second

SF (in both species) and in the first SF and ST (in

willow litter) (Table 3). In addition, litter lignin loss

was significantly related to MBC in the first SC and

EG for bamboo and in the second SC and EG for

willow (Table 3).

DISCUSSION

Lignin Decomposition across LandscapePositions

The prediction that litter lignin loss in alpine forests

would decrease from the gap center to the closed

canopy during winter was well supported by the

results of the present study, which corroborates

previous findings that snow cover can promote

mass loss in litter (Wu and others 2010; Zhu and

others 2012). There are other possible underlying

mechanisms that could explain the observed pat-

tern of litter lignin loss during winter. Theoreti-

cally, specialized biota (mainly fungi) are able to

synthesize extracellular enzymes, which is a bio-

Table 2. Results of the One-way ANOVA for the Effects of Gap Positions (Gap Center South, Gap CenterNorth, Canopy Edge, Expanded Edge and Closed Canopy) (df = 4) on Lignin Loss (%) in Foliar Litter

Species 1st year 2nd year

Snow

formation

period

Snow

coverage

period

Snow

thawing

period

Early

growing

period

Later

growing

period

Snow

formation

period

Snow

coverage

period

Snow

thawing

period

Early

growing

period

Later

growing

period

Fargesia nitida 0.107 2.414 27.106** 9.178** 10.765** 15.560** 5.607* 1.889 3.207 4.935*

Salix paraplesia 5.711* 16.860** 22.440** 42.954** 29.573** 19.101** 344.329** 24.133** 35.009** 33.978**

Note: values presented are F values; significant effects: * P < 0.05; ** P < 0.01; n = 15.

Table 3. Correlation Coefficients (r) between the Average Temperature (�C), Frequency of Freeze-thawCycles (times) or Microbial Biomass Carbon (mg kg-1 of dry mass) and Foliar Litter Lignin Loss (%) duringEach Decomposition Period for the First and Second Years

Decomposition

period

Fargesia nitida Salix paraplesia

Average

temperature

Frequency of

freeze-thaw

cycle

Microbial

biomass

carbon

Average

temperature

Frequency

of freeze-

thaw cycle

Microbial

biomass

carbon

1st year

Snow formation period 0.030 0.098 0.017 –0.436 –0.597* –0.577*

Snow coverage period 0.541* –0.025 0.606* 0.668* –0.224 0.472

Snow thawing period 0.229 –0.470 0.290 –0.500 –0.826** 0.177

Early growing period –0.466 –0.733** 0.574* –0.398 –0.669** 0.436

Later growing period 0.526* –0.301 –0.444 0.840* –0.434 –0.711**

2nd year

Snow formation period 0.013 –0.730** –0.031 –0.468 –0.540* –0.151

Snow coverage period 0.566** 0.763** 0.001 0.936** 0.826* 0.723**

Snow thawing period 0.165 –0.476 0.245 –0.283 0.211 –0.345

Early growing period –0.133 N.A. –0.037 –0.836** N.A. 0.468

Later growing period –0.145 –0.221 0.073 0.210 0.926** 0.540*

Significant effects: * P < 0.05; ** P < 0.01; n = 15.

Lignin Degradation in Alpine Forest Gap

logical process that accounts for most lignin

degradation (Meentemeyer 1978; Swift and others

1979). One well-known contributing mechanism is

that as the snowpack accumulates, frost can affect

the abundance and activity of microbes, particu-

larly increasing fungal activity and the fungi-to-

bacteria ratio (Haei and others 2011). Additionally,

continuous snow cover can provide a stable mi-

croenvironment that protects decomposer com-

munities and insulates soil organisms from

extremely cold temperatures (Sharratt and others

1999; Bokhorst and others 2013). Furthermore, as

the snow thaws, the increased moisture can be

beneficial for microbial activity (Hicks Pries and

others 2013). The abundance and activities of

decomposers can decrease with the decreasing

snow cover depth from the gap center to the closed

canopy (Tan and others 2014) (Figure 5), causing a

decrease in litter lignin loss associated with the

decreasing snow cover depth during winter.

However, the prediction that litter lignin loss

would increase from the gap center to the closed

canopy during the growing season was only par-

tially supported. Lignin loss did not follow the ex-

pected trend during the first growing season in the

willow litter or during the second growing season

in the bamboo litter, exhibiting higher values in the

gap center (Figure 6A, D). Interestingly, in the gap

center, both of the litter species exhibited higher

lignin loss during the early growing season and

lower lignin loss during the later growing season

compared with values in the expanded edge and

closed canopy (see Supplementary Material Fig-

ure S1). These changes may be attributed to the

following combined factors. The gap center has

greater sunlight exposure and precipitation than

the adjacent closed canopy during the growing

season, but evaporation occurs more quickly due to

the high solar radiation during the early growing

season (Ritter 2005). Comparatively, in the

microenvironments of the expanded edge and

closed canopy, evaporation is reduced, and the

temperature is more suitable (Zhang and Liang

1995). Both of these factors ultimately affected

decomposer activities and communities, which in

turn influenced the loss of litter lignin (Zhang and

Liang 1995) (Figure 5). Additional decomposable

litter, induced by strong freezing and thawing in

winter, can also contribute to the litter lignin loss

during the growing season in the expanded edge

and closed canopy due to the lack of snow coverage

during winter. However, as the decomposition

proceeded during the later growing season, the

ambient environment became more mild and

stable (see Supplementary Material Table S1, Fig-

ure 3), and the warmer microenvironment and

sufficient moisture favored the recovery of

decomposer abundance and contributed to lignin

degradation (Figure 5); as a result, sufficient pre-

cipitation and adequate sunlight in gap centers

could accelerate the contribution of decomposers to

lignin degradation more. In addition, litter in the

gap center received more solar radiation, thus

causing further photodegradation of the litter lig-

nin than in the expanded edge and closed canopy

(Austin and Ballare 2010).

Seasonal Effects on Litter LigninDecomposition

We also observed that lignin loss in the bamboo

litter mainly occurred during the first decomposi-

tion year but that the loss in the willow litter oc-

Figure 5. The effects of the gaps on the microbial bio-

mass carbon (MBC) content of Fargesia nitida and Salix

paraplesia foliar litter during different periods of decom-

position in the alpine fir forest of the eastern Qinghai-

Tibet Plateau from October 26, 2010, to October 29, 2012

(a total of 734 days of exposure in the field).

W. He and others

curred during the second decomposition year

(Figure 6B, E). The observed variation could be

closely related to the significantly higher initial

lignin content found in willow litter compared with

that in bamboo litter (Table 1). Although lignin loss

in bamboo litter (expressed as a percentage of the

initial lignin content) was higher than in willow

litter in the first year, the absolute loss of lignin

(gram) was higher in willow litter (Figure 4). Fur-

thermore, a higher lignin content is often associ-

ated with lower contents of labile components, as it

has been hypothesized that lignin surrounds labile

litter components in plant cell walls (Berg and

McClaugherty 2003). These labile components can

be a good potential substrate for microbes and

other decomposers (Cleveland and others 2014),

which in turn, control lignin degradation. As a re-

sult, variations in litter lignin contents and other

quality characteristics cause variations in lignin

loss. Accordingly, compared with the rapid lignin

degradation of the bamboo litter during the first LG

and second SF, the willow litter lignin degradation

rate was higher during the second SF and SC than

during other specific periods in the current study

(see Supplementary Material Figure S3). The find-

ings agree with the previous conclusion that litter

with a lower initial lignin content can display ear-

lier rapid lignin loss (Talbot and Treseder 2012).

Lignin is well-known to have low degradability

(Melillo and others 1982). McClaugherty and Berg

(1987) found that the absolute lignin content de-

creased in the early period of decomposition only in

litter with a high initial lignin concentration

(>30%). For the litter species with a low initial

lignin concentration, the relative lignin content

increased before an absolute decrease (Fioretto and

others 2005), which was caused by an increase in

lignin-like compounds (microbial by-products)

produced by soil microorganisms during decom-

position, and these lignin-like compounds might

mask the slight lignin degradation that occurred

during decomposition (Brandt and others 2010;

Song and others 2011). Inconsistent with this

finding, both litter species in this study showed

immediate absolute lignin loss when exposed for

only 58 d (see Supplementary Material Figure S1),

although these litter types had relatively low initial

lignin concentrations (14.79% in bamboo and

21.79% in willow). The results in this case seem to

suggest that seasonal snow cover with frequent

freeze-thaw events had a significant impact on the

litter lignin structure prior to its degradation by

biological processes, leading to accelerated lignin

degradation in the high altitude forest ecosystem.

Additionally, although the bamboo litter showed

few differences from the gap center to the closed

canopy during the SF (see Supplementary Material

Figure S1), the statistical analyses showed that the

gap-mediated freeze-thaw events during this peri-

od had a significant influence on the lignin loss of

Figure 6. The effects of gaps on lignin loss in Fargesia nitida and Salix paraplesia foliar litter during different periods of

decomposition in the alpine fir forest of the eastern Qinghai-Tibet Plateau. W1, first winter period; G1, first growing

period; W2, second winter period; G2, second growing period. Bars indicate the standard error. Different lowercase letters

indicate significant differences (P < 0.05) among the litterbag positions during the same decomposition period.

Lignin Degradation in Alpine Forest Gap

willow litter (Table 2). This finding implied that

freeze-thaw events and other factors that occurred

during the early period of litter decomposition

might have a large and significant effect on the

litter of species with larger leaves and relatively

higher lignin content than on those with smaller

leaves and lower lignin content.

Furthermore, the litter lignin degradation rate

did not always increase with an increasing fre-

quency of freeze-thaw cycles over the 2-year

decomposition period (see Supplementary Material

Figure S3, Table S1), which indicated that the

change in litter quality could play a more critical

role in litter lignin degradation during the later

stages of decomposition (Preston and others 2009a;

Preston and others 2009b; Wu and others 2010;

Zhu and others 2012). Nevertheless, the average

temperature was positively correlated with the lit-

ter lignin degradation rate for most of the sample

periods, except for the early growing season, be-

cause high solar radiation was a extreme factor that

created a drier microenvironment by increasing

evaporation and lowering moisture, which was not

beneficial to the growth and activity of microbes

(Sariyildiz 2008).

CONCLUSIONS

The lignin loss and degradation rates in the foliar

litter of the two examined shrub species decreased

from the gap center to the closed canopy over the

first year and overall for the entire two-year

decomposition period. Compared with the gap edge

and closed canopy, the gap center exhibited higher

litter lignin loss during winter and the later grow-

ing season but exhibited lower litter lignin loss

during the early growing season as both bamboo

and willow litter decomposition proceeded. The

bamboo litter, with a lower initial lignin content,

exhibited earlier and more rapid lignin loss than

willow litter. The results of this study provided

clear insight into the litter lignin degradation pro-

cess from the gap center to the closed canopy,

suggesting that seasonal snow cover has significant

impacts on litter lignin degradation and that re-

duced snow cover resulting from winter warming

could limit litter lignin loss in this alpine forest

ecosystem.

ACKNOWLEDGEMENTS

We are grateful to the anonymous reviewer and to

Dr. Jennie DeMarco for their constructive com-

ments, as well as to Xiangyin Ni and Bin Wang for

their help with the field sampling and laboratory

analyses. The National Natural Science Foundation

of China (31170423 and 31270498), the National

Key Technologies R&D Program (No. 2011BAC

09B05) and the Program of Sichuan Youth Sci-tech

Foundation (Nos. 2012JQ0008 and 2012JQ0059)

supported this work.

Compliance with Ethics Statement Wecertify that this article represents original workthat has never been published and is not underconsideration for publication elsewhere. No datawere fabricated or manipulated (including ima-ges) to support our conclusions. No data, text, ortheories by others were presented as if they wereour own. The submission is explicitly from allcoauthors whose names appear on the paper andwho contributed sufficiently to the scientific workand therefore share the collective responsibilityand accountability for the results. We also declarethat the coauthors have no conflicts of interest.Informed consent was obtained from all individ-ual participants included in this study. Addi-tionally, a permit was granted from the WesternSichuan Forestry Bureau to conduct scientificexperiments in the Miyaluo Nature Reservebeginning in March 2006. The leaf litter collectedfor this study was only sampled on a very limitedscale and therefore had negligible effects onbroader ecosystem function. Moreover, this re-search was conducted in compliance with thelaws of the People’s Republic of China. The re-search did not involve measurements on humansor animals, and no endangered or protected plantspecies was involved.

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