Temperature affects leaf litter decomposition in low-order forest streams: field and microcosm approaches

R E S EA RCH AR T I C L E

Temperature affects leaf litter decomposition in low-orderforest streams: field and microcosm approaches

Aingeru Mart�ınez1, Aitor Larrañaga1, Javier Pérez1, Enrique Descals2 & Jes�us Pozo1

1Laboratory of Stream Ecology, Department of Plant Biology and Ecology, University of the Basque Country, Bilbao, Spain; and 2Instituto

Mediterr�aneo de Estudios Avanzados, IMEDEA (CSIC), Esporles (Mallorca), Spain

Correspondence: Aingeru Mart�ınez,

Laboratory of Stream Ecology, Department of

Plant Biology and Ecology, University of the

Basque Country, P.O. Box 644, 48080 Bilbao,

Spain. Tel.: +34 94 601 5939;

fax: +34 94 601 3500;

e-mail: [email protected]

Received 3 June 2013; revised 10 September

2013; accepted 13 September 2013.

Final version published online 17 October

2013.

DOI: 10.1111/1574-6941.12221

Editor: Angela Sessitsch

Keywords

temperature; leaf litter decomposition;

aquatic hyphomycetes; microcosms.

Abstract

Despite predicted global warming, the temperature effects on headwater stream

functioning are poorly understood. We studied these effects on microbial-

mediated leaf decomposition and the performance of associated aquatic hyph-

omycete assemblages. Alder leaves were incubated in three streams differing in

winter water temperature. Simultaneously, in laboratory, leaf discs conditioned

in these streams were incubated at 5, 10 and 15 °C. We determined mass loss,

leaf N and sporulation rate and diversity of aquatic hyphomycete communities.

In the field, decomposition rate correlated positively with temperature. Decom-

position rate and leaf N presented a positive trend with dissolved nutrients,

suggesting that temperature was not the only factor determining the process

velocity. Under controlled conditions, it was confirmed that decomposition

rate and leaf N were positively correlated with temperature, leaves from the

coldest stream responding most clearly. Sporulation rate correlated positively

with temperature after 9 days of incubation, but negatively after 18 and

27 days. Temperature rise affected negatively the sporulating fungi richness and

diversity only in the material from the coldest stream. Our results suggest that

temperature is an important factor determining leaf processing and aquatic

hyphomycete assemblages and that composition and activity of fungal commu-

nities adapted to cold environments could be more affected by temperature

rises. Highlight: Leaf decomposition rate and associated fungal communities

respond to temperature shifts in headwater streams.

Introduction

In forest-shaded streams, where the primary production

is very limited, leaf litter decomposition allows the incor-

poration of carbon and nutrients into secondary produc-

tion (Wallace et al., 1997). In these systems, the

organisms responsible for the processing of detritus are

microbial decomposers, primarily fungi (B€arlocher, 1992;

Gulis & Suberkropp, 2003a, b) and invertebrate detriti-

vores (Grac�a, 2001), which transfer energy to higher tro-

phic levels (Wallace et al., 1997, 1999). Microbial activity

responds to several environmental conditions, such as the

concentration of dissolved nutrients in water (Suberkropp

& Chauvet, 1995; Gulis & Suberkropp, 2003a, b), the

degree of oxygen saturation (Medeiros et al., 2009) and

pH (Dangles et al., 2004). Temperature is clearly another

important parameter stimulating fungal respiration,

mycelia biomass accrual, conidial production and micro-

bial-mediated decomposition (Chauvet & Suberkropp,

1998; Dang et al., 2009; Ferreira & Chauvet, 2011a, b;

Batista et al., 2012; Ferreira et al., 2012; Geraldes et al.,

2012), because warming accelerates chemical reactions

and enhances biological activities (Brown et al., 2004;

Davidson & Janssens, 2006; Davidson et al., 2006).

Decomposition releases CO2 into the atmosphere and,

together with primary production, controls the carbon

fluxes between the biosphere and the atmosphere (Cadish

& Giller, 1997; Cornwell et al., 2008). Therefore, relative

changes in global decomposition and primary production

rates might have implications for the climate (Davidson

& Janssens, 2006). Nevertheless, some processes might

need to surpass certain thresholds of temperature

increases to be observable. Thus, the consequences of the

predicted increase in temperature by the end of this

FEMS Microbiol Ecol 87 (2014) 257–267 ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

MIC

ROBI

OLO

GY

EC

OLO

GY

century (1.1–6.4 °C in global terms; IPCC, 2007) on

microbial communities and on decomposition are

unclear. Moreover, particular species have different ther-

mal ranges within which they can grow, reproduce and

attain their functional roles in the communities (Chauvet

& Suberkropp, 1998; Dallas & Rivers-Moore, 2012). The

predicted temperature increases could surpass the toler-

ance limits of a portion of the species pool and thus

affect differently the activity of assemblages adapted to

different thermal regimes, a subject that has not still

received much attention from the scientists. The few

works focused on this subject in streams have reported a

consistent temperature dependence of microbial respira-

tion among assemblages adapted to different thermal

histories (Sand-Jensen et al., 2007; Perkins et al., 2011).

Nevertheless, we are not aware of studies that tackle this

dependence between temperature and microbial-mediated

leaf decomposition.

The aim of this study was to assess the effects of tem-

perature on microbial decomposition in headwater forest

streams with a different thermal regime. For this, alder

leaf litter was conditioned in three streams that differed

in mean winter temperatures (range: 4.9–9.8 °C), and

incubation was followed either in the streams or in the

laboratory in microcosms at three temperatures (5, 10

and 15 °C). We determined litter decomposition rates

and conidial production of aquatic hyphomycetes

throughout the process. This experimental design not

only allows us to isolate the temperature effects from

other environmental variables, but also enables to test

whether the fungal assemblages adapted to different ther-

mal regimes respond in the same way to shifts in temper-

ature. We hypothesise that (1) temperature will enhance

leaf litter microbial decomposition, (2) the relationship

between microbial decomposition and temperature will

be more difficult to detect in the field, as other environ-

mental factors can also play an important role, and

(3) the leaf decomposition–temperature relationship will

not be dependent on the assemblages adapted to different

thermal regimes.

Materials and methods

Study sites and stream water characteristics

The field experiment was conducted in three headwater

streams (S1, S2 and S3) with siliceous substrata, flowing

into the Atlantic Ocean (Cordillera Cant�abrica, northern

Spain). The streams showed differences in their daily mean

water temperature (recorded hourly) monitored with

Smart Button temperature data loggers (ACR Systems Inc.,

Surrey, BC, Canada) from October 2010 to April 2011,

which where inversely related to the elevation of each site

(Table 1). The three streams drain forested watersheds. S1

and S2 run mainly through native vegetation (Quercus

robur L. and Fagus sylvatica L. forests), whereas some

upland areas covered with tree plantations of Eucalyptus

globulus Labill. also appear in the watershed of S3

(Table 1); other anthropological land uses are negligible.

From December 2010 to April 2011, water physico-

chemistry was characterised in the three streams on six

occasions. Each time, oxygen saturation, conductivity, pH

(WTW multiparametric sensor) and river flow (Martin

Marten Z30, Current Meter) were measured in situ. Addi-

tionally, water samples were collected from each stream

with polyethylene bottles and transported to the labora-

tory in refrigerated chambers for alkalinity and nutrient

analyses. In laboratory, water samples were immediately

filtered (preweighed 0.7-lm-pore size glass fibre filters,

Whatman GF/F). Subsamples of the filtered water were

used to determine alkalinity by titration to an end pH of

4.5 (APHA, 2005). Nitrate concentration was determined

by capillary ion electrophoresis (Agilent CE); ammonium,

by the manual salicylate method; nitrite, by the sulpha-

nilamide method; and soluble reactive phosphorus (SRP),

by the molybdate method (APHA, 2005).

Incubation of leaves and processing in streams

In October 2010, leaves of alder, Alnus glutinosa (L.)

Gaertner were collected from the forest floor immediately

following natural abscission. Approximately 4 g (�0.25) of

air-dried leaves were placed into mesh bags (dimensions

15 9 20 cm, 0.5-mm mesh size). On 23 February, 22 bags

were fastened to anchored bars in the stream benthos at

each site. After 27 and 54 days of incubation, five bags per

stream were collected and transported to the laboratory.

Leaves were rinsed with stream water (filtered through a

0.2-mm mesh sieve) over a 0.5-mm mesh sieve. The

organic material was oven-dried to obtain the dry mass

(70 °C, 72 h); subsamples from each bag were used for ele-

mental analyses (C, N), and the rest was ashed (500 °C,4 h) to determine the remaining ash-free dry mass

(AFDM). Leaf carbon and nitrogen were determined using

a Perkin-Elmer series II CHNS/O elemental analyser. After

27 days of incubation, 12 extra bags per stream were col-

lected, 20 pairs of leaf discs (one pair per leaf; 20 mm

diameter) per bag were punched out with a cork borer and

used for the microcosm experiment. We used one of these

sets of 20 discs to estimate the initial mass and elemental

composition of microcosm experiment material.

Microcosm assay

Three 36-L tanks were set up as water baths at three

different temperatures (5, 10 and 15 °C). The three tanks

FEMS Microbiol Ecol 87 (2014) 257–267ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

258 A. Mart�ınez et al.

were placed in a refrigerated room at 10 °C constantly. A

recirculating cooler (HL-160CA) was used to cool water

of one of the tanks to 5 °C and a heater circulator (Julabo

EH 17) to heat up water temperature to 15 °C of another

tank. Twelve microcosms (four replicates from each

stream) were placed in each tank. The microcosms con-

sisted of 370-mL glass jars containing 200 mL of filtered

(0.2 lm) water from a different siliceous stream (S4) to

rule out differences in water physicochemical characteris-

tics (pH: 7.8 � 0.17; conductivity: 159 � 3 lS cm�1;

SRP: 18 � 7 lg P L�1; DIN: 204 � 46 lg N L�1; mea-

sured from December 2010 to April 2011 in S4, n = 6).

All glass and plastic material used was previously

autoclaved. In each microcosm, the second set of 20 leaf

discs was introduced and incubated for 27 days. The

microcosms were constantly aerated, agitating the discs,

under a light/dark regime of 12:12 hours. Water was

renewed every 3 days. On three occasions (9, 18 and

27 days of incubation in microcosms), a water volume

of 25 mL was collected from each jar to sample the

hyphomycete conidia suspension, fixed with 2 mL 37%

formalin and stained with a few drops of trypan blue in

lactic acid (0.05%). An aliquot of the suspension was

filtered (Millipore SMWP 5-lm pore size) and stained for

conidial identification to the species level (after Gulis

et al., 2005, and species description catalogues) and quan-

tification under the microscope (5009 magnification).

Sporulation rates were expressed as the number of

conidia per mg dry mass (leaf mass estimated at each

sampling date) and incubation time (no. of conidia mg

dry mass�1 day�1). After 27 days of incubation, coincid-

ing with the retrieval of bags in the field, each set of 20

leaf discs was oven-dried to obtain dry mass (70 °C,72 h). Five dried discs were preserved (�20 °C) for

elemental analyses following the procedure explained

above, and the others were ashed (500 °C, 4 h) to deter-

mine the remaining AFDM.

Statistical analyses

Water temperature comparisons (for the period March

2011–April 2011) were performed with one-way ANOVA

(factor: stream) followed by Tukey’s HSD test (Zar, 2010)

considering mean daily temperature (n = 27) as replicate.

Other physicochemical characteristics of water for each

stream were analysed using one-way ANOVAs (factor:

stream). The leaf mass losses in the stream were calcu-

lated with a linear model as follows: b = (M0 - Mt)/t,

where b is decomposition linear rate, M0 is the remaining

mass estimated after the first 27 days of incubation, Mt is

the remaining mass at the end of incubation (54 days

since implantation), and t is the incubation time

(27 days). The leaf disc decomposition rate in the micro-

cosms was calculated using the same equation above, in

Table 1. Location, reach characterisation and water physicochemical characteristics of the streams (mean � SE; n = 6). For water temperature,

during winter (from October 2010 to April 2011, n = 199) and during in-stream decomposition time (from March 2011 to April 2011, n = 27),

daily mean values and their range are given

S1 S2 S3

Latitude 43°08′50″N 43°12′36″N 43°18′31″N

Longitude 03°23′36″W 3°15′36″W 03°15′39″W

Altitude (m a.s.l) 920 400 115

Catchment area (km2) 1.87 1.31 2.03

Catchment land use (%)

Native vegetation 94.1 94.0 46.0

Afforested 0 6.0 46.5

Farming 5.9 0 7.5

River banks land use (%)

Native vegetation 93.18 100 70.44

Afforested 0 0 28.67

Farming 6.82 0 0.89

Winter water temperature (ºC) 4.90 (0.21–12.25) 8.21 (3.85–14.50) 9.76 (5.42–15.96)

Experiment water temperature (ºC) 8.03 (4.06–11.35) 10.47 (8.59–13.38) 11.54 (9.17–14.52)

Discharge (L s�1) 84.84 � 43.51 28.32 � 7.96 72.24 � 25.65

pH 6.88 � 0.25 7.50 � 0.18 7.28 � 0.17

Conductivity (lS cm�1) 33.18 � 3.33 89.60 � 7.19 66.32 � 3.97

Oxygen saturation (%) 100.33 � 1.99 101.00 � 1.28 100.67 � 2.25

Alkalinity (meq L�1) 0.13 � 0.02 0.24 � 0.02 0.20 � 0.02

Nitrate (lgN L�1) 120.97 � 42.93 747.21 � 72.72 705.49 � 66.73

Nitrite (lgN L�1) 3.40 � 0.68 3.36 � 0.55 1.26 � 0.18

Ammonium (lgN L�1) 35.33 � 7.10 35.63 � 4.59 44.12 � 8.94

SRP (lgP L�1) 19.18 � 4.31 11.90 � 4.01 17.59 � 4.77


Decomposition and temperature: field and microcosm approaches 259

which the initial AFDM, M0, was obtained for each

microcosm from the group set aside at the beginning of

the experiment, Mt is the final mass, and t is the time of

incubation of the microcosms (27 days). Mass remaining

of discs at 9 and 18 days was estimated assuming that lin-

ear relationship. The rates were also calculated in terms

of degree-days, to standardise them by incubation tem-

perature. Differences in breakdown rates (in terms of days

and degree-days) and leaf nitrogen concentration of the

material incubated in the three streams (both determined

for each leaf bag) were tested with one-way ANOVA, with

stream as a factor. In the microcosms, differences in

decomposition rates (in terms of days and degree-days)

and leaf N concentration of the material incubated in the

different streams were tested with two-way ANOVA (factors:

stream and temperature). Bivariate relationships were

tested by ordinary least square linear regressions. Sporula-

tion rates were compared with three-way ANOVA (factors:

stream, sampling date and temperature). To search for

general differences in fungal assemblages among treat-

ments in the microcosm study, a nonmetric multidimen-

sional scaling (NMDS) taking into account spore

production of all replicates was performed based on the

Bray–Curtis dissimilarity matrix, followed by PERMANOVA

(106 permutations), to test whether there were differences

between source streams and/or between incubation

temperatures in microcosms. When necessary, data were

transformed (log (x+1)), to obtain requirements for para-

metric analyses. All statistical analyses were conducted

using R statistical software (version 2.11.1; R Development

Core Team, 2010).

Results

Stream water characteristics

During the 27 days of in-stream incubation, coinciding

with the microcosm assays [24 March (27 days)–19 April,

2011 (54 days)], only a range of 3.2 °C was observed in

the mean temperature among streams, but temperature

differed significantly (Table 1; ANOVA: F2,78 = 31.93, P <0.001). All streams presented oxygen-saturated waters,

with low mineralisation, circum-neutral pH and low

dissolved phosphorous concentrations (Table 1). How-

ever, the nitrate concentration differed among streams

(ANOVA: F2,14 = 27.02, P < 0.001), S1 presenting lower

values than the other two (Table 1).

Leaf litter processing in the streams

At the end of the in-stream incubation period (54 days),

the mean mass remaining percentage was 79.85% at S1,

60.42% at S2 and 54.70% at S3. The lowest decomposition

rate was measured in the coldest stream (S1), and the high-

est rate, in the warmest one (S3) (Fig. 1; ANOVA:

F2,11 = 12.303, P = 0.002), with a significant positive rela-

tionship between decomposition rate and water tempera-

ture (R = 0.83; P < 0.001; n = 12), and a similar positive

trend with dissolved nitrate (Fig. 1). These differences did

not disappear after standardising the decomposition rates

by accumulated heat, calculated on a degree-day basis

(ANOVA: F2,11 = 9.44, P = 0.004); S1 still showed the lowest

rate (0.019% AFDM degree-days�1), and S3, the highest

rate (0.089% AFDM degree-days�1). Decomposition rates

based on degree-days also showed a positive trend with

dissolved nitrate.

The nutritional quality of leaf litter was the same at the

start of the experiment (day 0) in the three streams. How-

ever, after 27 days, the N concentration on leaves from the

three streams differed (Table 2; ANOVA: F2,12 = 4.872,

P = 0.028). The material from S2 showed the highest nitro-

gen percentage, and S1, the lowest. In the second sampling

(day 54), the material from S3 showed a larger increase in

N than that from the other two streams, and leaf litter from

S1 remained at the lower values (Table 2; ANOVA:

F2,11 = 9.954, P = 0.003). In general, leaf N concentration

showed a positive trend with water temperature and with

the availability of dissolved inorganic nitrogen. Similarly,

decomposition rates were positively correlated with leaf N

concentration at the end of incubation period in stream

(R = 0.65; P = 0.012; n = 12).

Leaf disc processing and fungal assemblages in

the microcosms

The estimated mean AFDM of the set of discs introduced

in each replicate for incubation in the microcosms was

0.33 g for S1, 0.28 g for S2 and 0.30 g for S3. After incuba-

tion in microcosms, the mass remaining of discs ranged

from 0.22 g at 15 °C of material preconditioned in S2 to

0.30 at 5 °C of material preconditioned in S1. During the

27 days of incubation (24 March–19 April, 2011) and con-

sidering all nine experimental cases (three streams 9 three

temperatures), decomposition rates of leaf discs among

streams did not differ statistically (Fig. 2a and b; ANOVA:

F2,27 = 0.35, P = 0.707). When comparing among temper-

atures, decomposition rates were lowest at 5 °C and highest

at 15 °C (Fig. 2b; ANOVA: F2,27 = 4.10, P = 0.028), appear-

ing a significant positive relationship between decomposi-

tion rate and temperature (R = 0.41; P = 0.014; n = 36).

This relationship was, however, mainly due to the response

of coldest stream (S1), as the regressions in the other two

streams were not significant. The slope of the relationship

was much lower for the incubation in microcosms (0.022)

than for incubation in the field (0.247). The decomposition

rates standardised by accumulated heat (degree-days) were



again different among the three temperatures (ANOVA:

F2,27 = 9.61, P < 0.001), but in contrast to the rates on a

day basis were higher at 5 °C (0.090% AFDM degree-

days�1) than at 10 °C (0.062% AFDM degree-days�1) or

15 °C (0.045% AFDM degree-days�1).

The initial N content of leaf discs (after 27 days of

incubation in streams) in microcosms was different

among the sets from the three streams (Fig. 2c; ANOVA:

F2,11 = 12.03, P = 0.002). Discs from S2 showed a higher

N concentration (4.50%) than those from S3 and S1

(4.10% and 3.87%, respectively). After incubation in the

microcosms, the N concentration of discs from S1 at the

three temperatures and of discs from S3 at 15 °C was

higher than the initial ones, discs from S1 showing the

highest N concentration, and those from S3, the lowest

(Fig. 2c; ANOVA: F2,27 = 7.37, P = 0.003). S1 also regis-

tered the highest leaf N mass remaining at the end of the

microcosm experiment (data not shown). Among temper-

atures, leaf N was lowest at 5 °C (Fig. 2d; ANOVA:

F2,27 = 15,44, P < 0.001), appearing a general significant

positive relationship between N concentration and tem-

perature (R = 0.62; P < 0.001; n = 36). Decomposition

rates did not correlate with the nitrogen concentration of

discs (R = 0.01; P = 0.933; n = 36), in contrast with

those observed in the field.

The sporulation rates were different among streams

(Table 3; ANOVA: F2,81 = 17.47, P < 0.001), with the

assemblage from S1 showing the highest values and that

from S3 the lowest ones. Conidia production also differed

among sampling dates (Fig. 3; ANOVA: F2,81 = 60,47,

P < 0.001), peaking on day 9 of the incubation. The great

interaction time 9 temperature (ANOVA: F4,81 = 26.35,

P < 0.001) showed the changes in the direction of the

relationship between sporulation rate and temperature

with sampling date (Fig. 3). The relationship was positive

for the first sampling (after 9 days of incubation in the

microcosms) and negative for the other two sampling

dates (18 and 27 days). The mean sporulation rate (calcu-

lated for each treatment using the three sampling dates)

was not correlated with decomposition rate.

A total of 34 species of aquatic hyphomycetes were

identified in the microcosms. Independent of the incuba-

tion temperature, the material preincubated in the coldest

stream, S1, showed more species than S2 and S3

(Table 3). Seven species were exclusively found in S1:

only one was exclusive for S2, and three, for S3. The most

evident relationship between species richness and temper-

ature was found in the discs from S1, where richness

appeared to display a negative relationship with increas-

ing incubation temperature (Table 3). Additionally, in

material pre-incubated in this stream, the diversity and

evenness were highest, and there was no clearly dominant

species; that is, five species (Articulospora tetracladia,

Flagellospora curvula, Heliscus lugdunensis, Lemonniera

alabamensis and L. aquatica) were codominant in the

conidial assemblage, each one representing > 10% of the

total sporulation rate. In contrast, only two species dis-

played more than 10% of the total sporulation in material

from S2 and S3, with F. curvula being the species with

the highest number of spores, followed by A. tetracladia.

Only in discs conditioned in the warmest stream, S3, and

incubated at 5 °C this trend was the opposite, with

A. tetracladia being dominant (Table 3). Although the

PERMANOVA showed differences in the fungal assemblages

among source streams (PERMANOVA: PseudoF2,99 = 6.58,

P < 0.001) and incubation temperature in microcosms

(PERMANOVA: PseudoF2,99 = 3.36, P < 0.001), the two-

dimensional nonmetric MDS (Fig. 4) presented clearly

Fig. 1. Relationship between decomposition

rate (% AFDM day�1; mean � SE) with

temperature (left) and dissolved nitrate (right)

in the field experiment.

Table 2. Leaf N concentration (%) during in-stream incubation.

Letters represent statistical differences among streams within each

sampling date (ANOVA and Tukey’s post hoc).

Stream Day 0 Day 27 Day 54

S1 3.08 3.35b 3.44b

S2 3.08 3.68a 3.82a

S3 3.08 3.58ab 4.00a



the great dissimilarities among streams, S1 being different

from the other two.

Discussion

The present study has demonstrated that the combination

of experiments both in the field and in the laboratory pro-

vides complementary information to understand the leaf

litter decomposition in streams. In the field experiment,

despite the good positive fits between leaf processing rate

and water temperature, the differences in dissolved nitrate

among the three streams, higher in warmer ones, do not

allow assume that temperature is the only variable deter-

mining decomposition and suggest a synergetic effect of

both temperature and nitrate. It is often reported that

temperature (Chauvet & Suberkropp, 1998; Dang et al.,

2009; Ferreira & Chauvet, 2011a; Ferreira et al., 2012) and

dissolved nutrients (Suberkropp & Chauvet, 1995; Gulis &

Suberkropp, 2003a, b; Benstead et al., 2009; Duarte et al.,

2009; P�erez et al., 2012) separately enhance processes, such

as microbial-mediated decomposition, fungal production,

and affect fungal performance. However, in natural

systems, the physicochemical processes, the structure and

composition of biotic communities and the activity of the

biota are not determined by a single environmental factor.

In fact, the combined effect of temperature and dissolved

nutrients on stream ecosystems appears to be an important

subject to study because an increase in these two factors in

running waters is predicted in future scenarios associated

with global change (Murdoch et al., 2000). In laboratory

approaches on leaf litter decomposition with alder leaves,

Ferreira & Chauvet (2011b) observed an enhancement of

leaf decomposition due to the synergistic interaction of

temperature and dissolved nutrients. In the present study,

two signs of this interaction arise: (1) the clear difference

in the decomposition rate among the different streams

after the standardisation with degree-days, what means

that other factors are affecting the process, and (2) despite

the narrower range of temperature in field approach

(3.2 °C) than in microcosms (10 °C), variation range of

decomposition rate in field was wider than under labora-

tory conditions. We did not measure sporulation rate nor

fungal biomass in leaves incubated in streams. However,

indirectly, we can assume a fungal growth promotion with

an increase in water temperature and dissolved nitrate

due to the found differences in leaf N concentration, a

relationship widely accepted (Canhoto & Grac�a, 2008;

Webster et al., 2009) because fungal mycelium shows a

higher N concentration than the leaves themselves (Cross

et al., 2005).

(a) (b)

(c) (d)

Fig. 2. Decomposition rate (% AFDM day�1;

mean � SE) (a) and its relationship with

incubation temperature in microcosms (b).

Leaf disc N concentration at the beginning

(black lines) and end of the incubation period

(bars) in microcosms (c) and its relationship

with temperature (d).



Our microcosms approach pursued two objectives: (1)

to isolate the effect of temperature on microbial-mediated

leaf processing from other environmental variables and

(2) to assess whether the activity of microbial assemblages

from streams with different thermal regimes responds in

the same way to temperature. Under laboratory-con-

trolled conditions, our results showed that differences in

decomposition rates and leaf N found in the field study

disappeared (rate) or were opposite (leaf N), whereas

decomposition rate–temperature and leaf N–temperature

relationships were maintained (rate) or even reinforced

(leaf N). The first finding can be explained due to the

pivotal role of dissolved nutrients on decomposition rate

and leaf N mediated by microbial activity (P�erez et al.,

2012), because the availability of dissolved N in micro-

cosms was the same for all treatments. This nitrate con-

centration was 1.79 that of stream S1 and approximately

0.39 that of S2 and S3. Thus, discs from S1 in micro-

cosms doubled the decomposition rate with respect to the

field experiment, whereas S2 and S3 were processed more

Table 3. Contribution (%) of aquatic hyphomycete taxa to the mean (three sampling dates 9 four replicates) total conidial production,

sporulation rate, taxa richness, Shannon diversity and Pielou evenness in the microcosm assay

StreamS1 S2 S3

Temperature (ºC) 5 10 15 5 10 15 5 10 15

Alatospora acuminata ‘subulate’* 0.7 0.6 0.1 0.2 0.4 0.2 0.1 < 0.1

A. acuminata ‘pulchelloid’* 0.8 0.4 1.2 0.4 0.5 0.2 0.5 0.8 0.1

A. acuminata (neotype)* 2.1 1.0 0.5 1.3 4.2 1.0 2.5 1.0 0.6

A. pulchella Marvanov�a 0.2 < 0.1 < 0.1 0.1

Anguillospora crassa Ingold < 0.1

A. filiformis Greathead 0.6 1.2 0.6 1.2 2.4 1.0 9.4 9.7 0.9

A. longissima (Sacc. & Syd.) Ingold < 0.1 < 0.1 0.1 0.1 0.0 < 0.1

A. tetracladia Ingold 24.1 20.0 30.1 14.3 25.3 10.2 51.6 16.2 15.9

Clavariopsis aquatica De Wildeman 0.9 3.2 0.3 0.1 0.3 < 0.1 0.0 0.1

Culicidospora aquatica R.H. Petersen 1.4 1.1 0.6 0.1 0.1

Dendrospora erecta Ingold < 0.1

Flagellospora curvula Ingold 30.9 22.6 17.1 79.7 60.6 79.9 25.6 58.2 62.3

Fontanospora eccentrica (R.H. Petersen) Diko 0.2 0.1 < 0.1 0.1 < 0.1

Goniopila/Margaritispora† < 0.1 0.2 < 0.1

Gyoerffyella oxalidis Vanev < 0.1

Heliscella stellata (Ingold & Cox) Marvanov�a 0.3

Heliscus lugdunensis Sacc. & Th�erry 11.2 18.3 22.7 1.0 3.4 2.4 2.0 1.7 1.1

Lemonniera alabamensis Sinclair & Morgan-Jones 14.0 5.1 14.0 0.2 < 0.1

L. aquatica De Wildeman 10.5 25.3 11.5 0.2 < 0.1 0.2 < 0.1

L. terrestris Tubaki < 0.1 < 0.1

Lunulospora curvula Ingold < 0.1 0.4 5.5 9.9

Mycofalcella calcarata Marvanov�a, Om-Kalth. & Webster 0.9 0.4 0.1 < 0.1 < 0.1

Stenocladiella neglecta Marvanov�a & Descals 0.1 0.1 < 0.1

Tetrachaetum elegans Ingold < 0.1 0.8 2.4 3.9 0.3 3.4 4.8

Tetracladium furcatum Descals 0.1 0.1

T. maxilliforme (Rostr.) Ingold < 0.1

T. setigerum (Grove) Ingold 0.2 < 0.1 0.1

Tricladium angulatum Ingold < 0.1

T. attenuatum Iqbal 0.1 0.1 0.2 0.1

T. biappendiculatum (Arnold) Vanev < 0.1 < 0.1

T. chaetocladium Ingold < 0.1 0.2 0.4 0.6 7.4 2.7 4.2

T. splendens Ingold 0.1 < 0.1 0.1 < 0.1 < 0.1

Varicosporium elodeae Kegel 0.4 0.2 0.9 0.1 < 0.1

Variocladium giganteum (Iqbal) Descals & Marvanov�a 0.1 < 0.1 0.1 < 0.1

Mean sporulation rate (no. conidia mg�1 day�1) 39.3 85.4 99.4 49.8 122.0 58.6 18.6 25.2 65.9

Richness 26 23 21 14 13 15 18 17 17

Shannon diversity 2.74 2.67 2.55 1.09 1.69 1.17 1.98 2.04 1.79

Pielou evenness 0.58 0.59 0.58 0.29 0.46 0.30 0.47 0.50 0.44

Data for the five predominant taxa (> 10%) for each treatment are highlighted in bold.

*Segregated forms.†Aggregated taxa entries (see explanation in Pozo et al., 2011).



slowly. In parallel, leaf N of discs from S1 increases,

whereas that of S2 and S3 decreases along the incubation

in microcosms. Additionally, although net N immobilisa-

tion was not observed, the highest leaf N mass remaining

at the end of the microcosm experiment was shown by

S1. The second finding, the increase in decomposition

rate and leaf N (indirect measurement of microbial

growth on decomposing leaves) with temperature within

the considered range (5–15 °C) was expected and is in

agreement with other reported results (Ferreira &

Chauvet, 2011a; Batista et al., 2012; Ferreira et al., 2012;

Geraldes et al., 2012). However, the most interesting

result is that the clearest response of acceleration of leaf

decomposition with temperature was shown by the

material preconditioned in the coldest stream, contrasting

with our initial hypothesis. Thus, our results are opposite

to those reported by other authors (Sand-Jensen et al.,

2007; Perkins et al., 2011), who found that respiration of

microbial assemblages from different thermal regimes

responded to temperature in a similar way.

Sporulation rates in our study (after 5–8 weeks of

incubation, 1–3 weeks under microcosm conditions)

showed typical values for this stage of incubation, which

are lower than those usually reported for sporulation

peaks (after 2–4 weeks of incubation; Gessner, 1997;

Schlickeisen et al., 2003; P�erez et al., 2012). The sporula-

tion rates of hyphomycetes appear to be affected by tem-

perature, as clear relationships were observed between

both variables in the three samplings. There is not agree-

ment with this relationship in the literature, because

while some authors report an enhancement of sporulation

rates with an increase in temperature (Chauvet &

Suberkropp, 1998; Ferreira & Chauvet, 2011a), some oth-

ers do not observe this link (Fernandes et al., 2009, 2012;

Geraldes et al., 2012). Furthermore, in the present study,

an increase in sporulation rates with temperature rising

only appeared after 9 days of incubation in microcosms

(36 days in total), the effect being the opposite on the

consecutive sampling dates (18 and 27 days in micro-

cosms). Low temperatures appeared to maintain a con-

stant spore production throughout the decomposition

process. This might have functional consequences in the

field, as the success in the colonisation of new substrata

might be a function of spore production and the time

interval in which a relatively high production of spores is

maintained. A positive relationship between conidia pro-

duction and decomposition rate has been reported in

some cases (e.g. Maamri et al., 2001; P�erez et al., 2012)

Fig. 3. Relationship between sporulation rate (no. conidia mg�1 day�1) associated with leaf discs and temperature in microcosms for the three

sampling dates. Note log10 scale used in y-axis.

Fig. 4. Nonmetric multidimensional scaling (NMDS) ordination of

fungal assemblages taking into account the spore production of all

replicates.



but not in others (e.g. Pozo et al., 2011; Mendoza-Lera

et al., 2012). In this study, where fungal sporulation was

examined for leaves incubated in microcosms, the mean

sporulation rate did not show a significant relationship

with the corresponding decomposition rate.

Temperature influences metabolic activity (Brown et al.,

2004), but also plays a key role in determining community

structure (Anderson-Glenna et al., 2008; Lear et al., 2008),

species distribution (Castella et al., 2001) and interspecific

relationships (Jiang & Morin, 2007). Hyphomycete assem-

blages in microcosms were more different among source

streams than among incubation temperatures in terms of

richness, evenness and diversity. B€arlocher et al. (2008)

and Fernandes et al. (2012) observed that warming nega-

tively affects fungal richness. In the present study, the

richest assemblage was found in leaf discs from the coldest

site. Additionally, incubation in microcosms at higher

temperatures (10 and 15 °C) only reduced the richness

and the diversity of the coldest stream. This suggests that

species composition of fungal assemblages from cold

headwater streams is more sensitive to the predicted global

rise in water temperature, possibly as a consequence of

surpassing the thermal threshold of certain species to sur-

vive. However, caution is required with any interpretation

of this result, as temperature changes influence rare spe-

cies and in both directions: some of these species disap-

peared, but others appeared at higher temperature

incubations (see Table 3). The difference in the response

of species to warming can critically influence and has con-

sequences not only for species distributions, but also for

critical ecosystem processes such as litter decomposition

(Dang et al., 2009). Nevertheless, contrary to observations

by Duarte et al. (2006) and P�erez et al. (2012), our taxa

richness was not accompanied by differences in leaf litter

decomposition rates, which can be explained by the high

ecological redundancy of hyphomycete species (Dang

et al., 2005; Pascoal et al., 2005; Ferreira & Chauvet,

2012). However, a change in the structure of fungal

assemblages could affect overall detritus breakdown

because detritivore activity is enhanced by the presence or

dominance of certain fungal species (Suberkropp et al.,

1983; Arsuffi & Suberkropp, 1984).

Our results show that temperature enhances leaf litter

decomposition mediated by microbial activity. Moreover,

it appears that temperature has a more direct effect on

activity and specific composition of communities adapted

to cold environments, suggesting differential responses

of fungal communities to climatic change. Caution is

needed, however, when studying temperature–decomposi-

tion relationships in streams within a slight range of

thermal regimes, because of the synergistic effect of

temperature and dissolved nutrients on the leaf litter

processing.

Acknowledgements

This study was funded by the Spanish Ministry of Science

and Innovation (projects CGL2010-22129-C04-01 and

CGL2011-23984) and by the Basque Government

(Research Grant IT-302-10). The authors thank the tech-

nicians of SGIker’s SCAB Service and of the University of

The Basque Country, UPV/EHU, for the nitrate measure-

ments. A. Mart�ınez was granted by the Basque Govern-

ment. The authors want to thank anonymous reviewers

for their help in improving the manuscript.

References

Anderson-Glenna MJ, Bakkestuen V & Clipson NJW (2008)

Spatial and temporal variability in epilithic biofilm bacterial

communities along an upland river gradient. FEMS

Microbiol Ecol 64: 407–418.APHA (2005) Standard Methods for the Examination of Water

and Wastewater, 21st edn. American Public Health

Association, American Water Works Association, and Water

Environment Federation, Washington, DC.

Arsuffi TL & Suberkropp K (1984) Leaf processing capabilities

of aquatic hyphomycetes: interspecific differences and

influence on shredder feeding preferences. Oikos 42:

144–154.B€arlocher F (1992) The Ecology of Aquatic Hyphomycetes.

Springer, Berlin.

B€arlocher F, Seena S, Wilson KP & Williams DD (2008)

Raised water temperature lowers diversity of hyporheic

aquatic hyphomycetes. Freshw Biol 53: 368–379.Batista D, Pascoal C & C�assio E (2012) Impacts of warming

on aquatic decomposers along a gradient of cadmium stress.

Environ Pollut 169: 35–41.Benstead JP, Rosemond AD, Cross WF, Wallace JB, Eggert SL,

Suberkropp K, Gulis V, Greenwood JL & Tant CJ (2009)

Nutrient enrichment alters storage and fluxes of detritus in

a headwater stream ecosystem. Ecology 90: 2556–2566.Brown JH, Gillooly JF, Allen AP, Savage VM & West GB

(2004) Toward a metabolic theory of ecology. Ecology 85:

1771–1789.Cadish G & Giller KE (1997) Driven by Nature: Plant Litter

Quality and Decomposition, p. 432. CAB Int, Wallingford.

Canhoto C & Grac�a MAS (2008) Interactions between fungi

and stream invertebrates: back to the future. Novel

Techniques and Ideas in Mycology (Sridhar S, B€arlocher F &

Hyde KD, eds), Fungal Divers Res Ser 20: 205–325. Hong

Kong University Press, Hong Kong.

Castella E, Adalsteinsson H, Britain JE et al. (2001)

Macrobenthic invertebrate richness and composition along a

latitudinal gradient of European glacier-fed streams. Freshw

Biol 46: 1811–1831.Chauvet E & Suberkropp K (1998) Temperature and

sporulation of aquatic hyphomycetes. Appl Environ

Microbiol 64: 1522–1526.



Cornwell WK, Cornelissen JHC, Amatangelo K et al. (2008)

Plant species traits are the predominant control on litter

decomposition rates within biomes worldwide. Ecol Lett 11:

1065–1071.Cross WF, Benstead JP, Frost PC & Thomas SA (2005)

Ecological stoichiometry in freshwater benthic systems:

recent progress and perspectives. Freshw Biol 50: 1895–1912.Dallas HF & Rivers-Moore NA (2012) Critical Thermal Maxima

of aquatic macroinvertebrates: towards identifying

bioindicators of thermal alteration. Hydrobiologia 679: 61–76.Dang CK, Chauvet E & Gessner MO (2005) Magnitude and

variability of process rates in fungal diversity–litterdecomposition relationships. Ecol Lett 8: 1129–1137.

Dang CK, Schindler M, Chauvet E & Gessner MO (2009)

Temperature oscillation coupled with fungal community

shifts can modulate warming effects on litter decomposition.

Ecology 90: 122–132.Dangles O, Gessner MO, Guerold F & Chauvet E (2004)

Impacts of stream acidification on litter breakdown:

implications for assessing ecosystem functioning. J Appl Ecol

41: 365–378.Davidson EA & Janssens IA (2006) Temperature sensitivity of

soil carbon decomposition and feedbacks to climate change.

Nature 440: 165–173.Davidson EA, Janssens IA & Luo YQ (2006) On the variability

of respiration in terrestrial ecosystems: moving beyond Q10.

Glob Change Biol 12: 154–164.Duarte S, Pascoal C, C�assio F & B€arlocher F (2006) Aquatic

hyphomycete diversity and identity affect leaf litter

decomposition in microcosms. Oecologia 147: 658–667.Duarte S, Pascoal C, Garab�etian F, C�assio F & Charcosset JY

(2009) Microbial decomposer communities are mainly

structured by trophic status in circumneutral and alkaline

streams. Appl Environ Microbiol 75: 6211–6222.Fernandes I, Uzun B, Pascoal C & C�assio F (2009) Responses

of aquatic fungal communities on leaf litter to temperature–change events. Int Rev Hydrobiol 94: 410–419.

Fernandes I, Pascoal C, Guimar~aes Pinto R, Sousa I & C�assio

F (2012) Higher temperature reduces the effects of litter

quality on decomposition by aquatic fungi. Freshw Biol 57:

2306–2317.Ferreira V & Chauvet E (2011a) Future increase in

temperature more than decrease in litter quality can affect

microbial litter decomposition in streams. Oecologia 67:

279–291.Ferreira V & Chauvet E (2011b) Synergistic effects of water

temperature and dissolved nutrients on litter decomposition

and associated fungi. Glob Change Biol 17: 551–565.Ferreira V & Chauvet E (2012) Changes in identity of the

dominant species in aquatic hyphomycete assemblages do

not affect litter decomposition rates. Aquat Microb Ecol 66:

1–11.Ferreira V, Gonc�alves AL & Canhoto C (2012) Aquatic

hyphomycete strains from metal–contaminated and

reference streams might respond differently to future

increase in temperature. Mycologia 104: 613–622.

Geraldes P, Pascoal C & C�assio F (2012) Effects of increased

temperature and aquatic fungal diversity on litter

decomposition. Fungal Ecol 5: 734–740.Gessner MO (1997) Fungal biomass, production and

sporulation associated with particulate organic matter in

streams. Limnetica 13: 33–45.Grac�a MAS (2001) The role of invertebrates on leaf litter

decomposition in streams – a review. Int Rev Hydrobiol 86:

383–393.Gulis V & Suberkropp K (2003a) Effect of inorganic nutrients

on relative contributions of fungi and bacteria to carbon

flow from submerged decomposing leaf litter. Microb Ecol

45: 11–19.Gulis V & Suberkropp K (2003b) Leaf litter decomposition

and microbial activity in nutrient enriched and unaltered

reaches of a headwater stream. Freshw Biol 48: 123–134.Gulis V, Marvanov�a L & Descals E (2005) An illustrated key to

the common temperate species of aquatic hyphomycetes.

Methods to Study Litter Decomposition: A Practical Guide, 1st

edn (Grac�a MAS, B€arlocher F & Gessner MO, eds),

pp. 153–168. Springer, Dordrecht.IPCC (Intergovernmental Panel on Climate Change) (2007)

Climate Change 2007: The Physical Science Basis.

Contribution of the Working Group I to the Fourth

Assessment Report of the Intergovernmental Panel on Climate

Change. Cambridge University Press, Cambridge, UK.

Jiang L & Morin PJ (2007) Temperature fluctuation facilitates

coexistence of competing species in experimental microbial

communities. J Anim Ecol 76: 660–668.Lear G, Anderson MJ, Smith JP, Boxen K & Lewis GD (2008)

Spatial and temporal heterogeneity of the bacterial

communities in stream epilithic biofilms. FEMS Microbiol

Ecol 65: 463–473.Maamri A, B€arlocher F, Pattee E & Chergui H (2001) Fungal

and bacterial colonization of Salix pedicellata leaves decaying

in permanent and intermittent streams in Eastern Morocco.

Int Rev Hydrobiol 86: 337–349.Medeiros AO, Pascoal C & Grac�a MAS (2009) Diversity and

activity of aquatic fungi under low oxygen conditions.

Freshw Biol 54: 142–149.Mendoza-Lera C, Larra~naga A, P�erez J, Descals E, Mart�ınez A,

Moya O, Arostegui I & Pozo J (2012) Headwater reservoirs

weaken terrestrial–aquatic linkage by slowing leaf–litterprocessing in downstream regulated reaches. River Res Appl

28: 13–22.Murdoch PS, Baron JS & Miller TL (2000) Potential effects of

climate change on surface water quality in North America.

J Am Water Resour Assoc 36: 347–366.Pascoal C, C�assio F & Marvanov�a L (2005) Anthropogenic

stress may affect aquatic hyphomycete diversity more than

leaf decomposition in a low–order stream. Arch Hydrobiol

162: 481–497.P�erez J, Descals E & Pozo J (2012) Aquatic hyphomycete

communities associated with decomposing alder leaf litter in

reference headwater streams of the Basque Country

(northern Spain). Microb Ecol 64: 279–290.



Perkins DM, Yvon-Durocher G, Demars BOL, Reiss J, Pichler

DE, Friberg N, Trimmer M & Woodward G (2011)

Consistent temperature dependence of respiration across

ecosystems contrasting in thermal history. Glob Change Biol

18: 1300–1311.Pozo J, Casas J, Men�endez M et al. (2011) Leaf–litter

decomposition in headwater streams: a comparison of the

process among four climatic regions. J North Am Benthol

Soc 30: 935–950.R Development Core Team (2010) R: A Language and

Environment for Statistical Computing. R Foundation for

Statistical Computing, Vienna.

Sand-Jensen K, Pedersen NL & Søndergaard M (2007) Bacterial

metabolism in small temperate streams under contemporary

and future climates. Freshw Biol 52: 2340–2353.Schlickeisen E, Tietjen TE, Arsuffi TL & Groeger AW (2003)

Detritus processing and microbial dynamics of an aquatic

macrophyte and terrestrial leaf in a thermally constant,

spring–fed stream. Microb Ecol 45: 411–419.

Suberkropp K & Chauvet E (1995) Regulation of leaf

breakdown by fungi in streams: influences of water

chemistry. Ecology 76: 1433–1445.Suberkropp K, Arsuffi TL & Anderson JP (1983) Comparison

of degradative ability, enzymatic activity, and palatability of

aquatic hyphomycetes grown on leaf litter. Appl Environ

Microbiol 46: 237–244.Wallace JB, Eggert SL, Meyer JL & Webster JR (1997) Multiple

trophic levels of a forest stream linked to terrestrial litter

inputs. Science 277: 102–104.Wallace JB, Eggert SL, Meyer JL & Webster JR (1999) Effects

of resource limitation on a detrital–based ecosystem. Ecol

Monogr 69: 409–442.Webster JR, Newbold JD, Thomas SA, Valett HM &

Mulholland PJ (2009) Nutrient uptake and mineralization

during leaf decay in streams – a model simulation. Int Rev

Hydrobiol 94: 372–390.Zar JH (2010) Biostatistical Analysis, 5th edn. Prentice Hall,

Upper Saddle River.



Documents

Temperature affects leaf litter decomposition in low-order forest streams: field and microcosm approaches