Effect of leaf litter characteristics on leaf ... - BES journal - Wiley

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Here, we document changes occurring on leaf tissues of three tree species, alder, ... sumption rate of conditioned leaves by the amphipod shredder Gammarus ...
Freshwater Biology (2013) 58, 1672–1681

doi:10.1111/fwb.12158

Effect of leaf litter characteristics on leaf conditioning and on consumption by Gammarus pulex  ER  IC HERVANT AND CHRISTOPHE PISCART N A T A C H A F O U C R E A U , S A R A P U I J A L O N , F R ED UMR5023 Ecologie des Hydrosystemes Naturels et Anthropises, Universite de Lyon, Universite Lyon 1, ENTPE, CNRS, Villeurbanne, France

SUMMARY 1. Terrestrial leaf litter inputs provide an essential energy source for many freshwater organisms. Processing of leaf litter involves several physicochemical and biological factors but always starts with the colonisation of leaves by aquatic hyphomycetes. 2. Here, we document changes occurring on leaf tissues of three tree species, alder, hornbeam and oak, with contrasted leaf properties. Changes in the mechanical properties of leaves and in fungal growth were followed at 10, 25, 35, 45 and 55 days of immersion in natural winter conditions. We hypothesised that fungal growth will be faster and mechanical characteristics will decrease more rapidly in softer, nutrient-rich, than in tougher leaves. In addition, we tested whether the consumption rate of conditioned leaves by the amphipod shredder Gammarus pulex was correlated with the mechanical properties of leaves and their ergosterol content (as a proxy for fungal biomass). 3. Leaf toughness decreased (with different rates for the 3 litter species), whereas the leaf thickness remained stable. The mechanical properties of alder and hornbeam leaves changed similarly, in contrast to oak leaves. Ergosterol contents increased over time more rapidly for alder and hornbeam than for oak leaves for which the fungal growth was delayed. Ergosterol content, leaf toughness and leaf consumption rates by G. pulex were significantly correlated. Leaf consumption rates by G. pulex were higher for soft than for tough leaves. 4. Our results suggest that the consumption of different types of leaves may be delayed according to the time required to acquire conditioning sufficient for shredder consumption. The presence of tough leaves in riparian vegetation may constitute a reservoir of trophic resources available for aquatic organisms at the end of the winter, when soft leaves have been entirely consumed. The diversity of riparian vegetation may hence contribute to sustaining the availability of food resources for adjacent aquatic ecosystems. Keywords: amphipod, biomechanical properties, leaf litter conditioning, organic matter recycling, temperate ecosystems

Introduction Allochthonous organic matter, in the form of senesced leaves of riparian vegetation is a major source of the carbon supporting a detrital food web in headwater streams (Cummins, 1974; Ostrofsky, 1997; Graca et al., 2001; Ribblett, Palmer & Coats, 2005). Hence, litter decomposition is a major component of the carbon cycle in low-order stream ecosystems (Li, Ng & Dudgeon,

2009). Litter decomposition is generally described as a sequence of several interacting processes: the passive leaching of soluble compounds (Ribblett et al., 2005), microbial colonisation or conditioning (Gessner & Chauvet, 1994), fragmentation by invertebrate shredders (Allan, 1996; Graca et al., 2001) and physical abrasion. The first biological actors for leaf litter decomposition are microorganisms, such as fungi and bacteria. In leaf

Correspondence: Natacha Foucreau, UMR5023 Ecologie des Hydrosystemes Naturels et Anthropises, Universite de Lyon, Universite Lyon 1, ENTPE, CNRS; 6 rue Rapha€el Dubois, 69622 Villeurbanne, France. E-mail: [email protected]

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Changes in leaf litter over conditioning litter, the fungal biomass is generally one to two orders of magnitude greater than bacterial biomass (Gulis & Suberkropp, 2003b) and, among the fungi, aquatic hyphomycetes are known to play an important role in the decomposition of litter (Kearns & B€ arlocher, 2008; Krauss et al., 2011). Litter decomposition is affected by nutrient concentrations influencing microbial growth (Suberkropp & Chauvet, 1995; Gulis & Suberkropp, 2003a,b; Artigas et al., 2011). Indeed, the leaf elemental stoichiometry (C: N: P) is an important determinant of leaf litter decomposition (Mooshammer et al., 2012) and of the relative roles of bacteria and fungi (e.g. high N/P ratio seems to favour fungi; Guesewell & Gessner, 2009). Physical properties of leaves may also affect microbial decomposers; for example, the possibility of hyphomycete spores attaching to leaves could be influenced by leaf roughness (Kearns & B€ arlocher, 2008). Leaves colonised by microorganisms become more palatable to leaf shredding macroinvertebrates, which are considered the main functional feeding group of low-order streams (Vannote et al., 1980; Graca et al., 2001; Piscart et al., 2009). The joint action of decomposers, shredders and physical fragmentation produces fine particulate organic matter (FPOM), which is used by collector-gatherers and filter-feeders (Heard & Richardson, 1995). Leaf mechanical properties such as toughness (i.e. resistance of a material to crack propagation) and the structure of leaf surfaces are also crucial in the decomposition process (Perez-Harguindeguy et al., 2000; Kearns & B€ arlocher, 2008; Li et al., 2009). For example, C/N ratios and leaf tensile strength, an indicator of physical litter quality, seem to be good indicators of decomposition rates. The leaf toughness contributes largely to the variation in mechanical resistance among tree species (Onoda et al., 2011). The mechanical properties of leaf litter have also been studied through the feeding activity of shredders (Ratnarajah & Barmuta, 2009), and initial leaf toughness influences the feeding activity of shredders (Abelho, 2008). We investigated the effect of the initial thickness and mechanical properties of leaves on leaf litter conditioning and the consequences for consumption by a shredder. We carried out a comparative study to assess temporal changes in the mechanical properties of leaf litter (toughness, specific toughness) from three tree species having leaves of contrasting properties and thickness, the increase in biomass of aquatic hyphomycetes, and consumption rates of conditioned leaf litter by the generalist shredder, Gammarus pulex. We hypothesised that (i) conditioning is slower for thick and tough leaves than for soft ones. Since fungi grow inside the cell wall © 2013 John Wiley & Sons Ltd, Freshwater Biology, 58, 1672–1681

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and alter the leaf structure, we supposed that (ii) the increase in fungal biomass would be negatively correlated with all mechanical properties of leaves with contrasted temporal changes (e.g. speed of fungal growth, alteration of leaf toughness) according to the leaf characteristics. Finally, we hypothesised that (iii) these differences in mechanical properties and on the fungal biomass growth, control rates of leaf consumption by shredders.

Methods Leaves We used three types of leaves differing in toughness: alder (Alnus glutinosa) leaves which are very common along streams and rivers and are considered as soft and readily consumed by aquatic invertebrates (Boyero et al., 2011); and hornbeam (Carpinus betulus) and oak (Quercus robur) leaves which are very common throughout European lowlands. Previous tests have shown that hornbeam leaves have intermediate leaf toughness, whereas oak leaves are hard and require longer exposure to break down (Gulis, Ferreira & Gracßa, 2006). Alder and oak leaves were collected from the same stand of trees near Lyon (45°49′36″N, 05°04′53″E), while hornbeam leaves were collected at a nearby location (45°56′06.6″N, 05°31′45.6″E). Several hundreds of freshly fallen leaves of each species were collected in the autumn, air-dried and stored at 4 °C until needed.

Initial elemental composition of leaves For each tree species, three replicates of three freezedried (Christâ ALPHA 1-4LD) discs (∅16 mm) from three different leaves (N = 3 9 3) were used to measure the elemental ratios of carbon and nitrogen. Total organic carbon and nitrogen were obtained from a FlashEA 1112 elemental analyzer (Thermo Scientific, USA) using aspartic acid (36.09% carbon and 10.52% nitrogen) and nicotinamide (59.01% carbon and 22.94% nitrogen) as calibration standards. Accuracy was checked using an in-house reference material analysed between samples (Leaf standard EMAL; 47.81  0.72% carbon and 2.075  0.024% nitrogen, N = 3). For the extraction and dosage of the total organic phosphorus content, five replicates of 2.0  0.5 mg of dry weight of leaves were made following the method described by Murphy & Riley (1958). All elemental percentages were expressed as percentage of the total dry weight.

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Change in leaf properties during decomposition

(m2). The specific toughness (J m 2 m 1) was calculated as the toughness corrected by leaf thickness (m).

Leaves of the same species and similar sizes were enclosed in fine mesh nylon bags (∅0.5-mm mesh), which excluded most of the invertebrates without interfering with microbial colonisation (Boulton & Boon, 1991). In winter, the bags were immersed in an artificial stream surrounded by deciduous woodland and harbouring a natural diversity of fungi in the University of Lyon (France) campus (Navel et al., 2010). Water temperature was monitored every hour throughout the study period, using a MINILOG8-TDR miniature data logger (WEMCO division, Amirix Systems Inc, Halifax, Canada). Leaves were exposed for at least 35 days. Mechanical properties and ergosterol (see below) were measured on the first day before immersion in the river and then after 10, 25 and 35 days of immersion. Additional measurements were made after 45 and 55 days required for oak leaves to reach a decrease in leaf toughness of at least 50% of the initial value (threshold reached for the other two species after 35 days).

Fungal biomass. The fungal biomass was assessed through ergosterol content (Gessner & Chauvet, 1993). At each sampling date and for each tree species, three replicates of leaf material were freeze-dried (Christâ ALPHA 1-4LD) and weighed to the nearest 0.1 mg. Lipids were extracted twice with methanol (25 : 1 v/w) for 15 min followed by 15 min of sonication (Retsch MM200 Ficher Bioblock; 30 agitations s 1). The extracts were concentrated in pure ethyl acetate to a final concentration of 20 mg mL 1 and ergosterol was quantified by HPLC DAD (1200 series, Agilent Technologies, Santa Clara, USA). Separations were carried out using a Kromasil reverse-phase C18 (RP18) column with an isocratic gradient at 1 mL min 1 of 100% methanol for 30 min. Chromatograms were recorded between 200 and 700 nm, and a specific channel was set at 282 nm for ergosterol quantification.

Leaf mechanical properties. At each sampling date, leaves were removed from the stream and transported in stream water to the laboratory where one piece was cut from each leaf (N = 30) for the biomechanical measurements (see below). The remaining parts of the leaves were replaced in the stream before the end of the day. We measured leaf toughness using ‘punch and die’ tests, which measure the force required to punch a hole through the leaf lamina. Tests were performed on a universal testing machine (Instron 5942, Canton, MA, USA) using a device consisting of a flat-ended cylindrical steel rod (punch, 2.0 mm diameter) mounted onto the moving head of the testing machine, and a stationary base with a sharp edged hole with a 0.1-mm clearance (following Aranwela, Sanson & Read, 1999; Onoda, Schievingand & Anten, 2008). The punch moved downward at a constant speed of 10 mm s 1 and was set to go through the hole without any friction. The leaves were positioned to avoid primary and secondary veins. The force applied to the leaf and the displacement were recorded simultaneously with a frequency of 10 Hz. Leaf thickness (0.01 mm) was measured with a digital thickness gauge avoiding major veins. The force-displacement curve was used to calculate work to punch (called toughness in the rest of the article, J m 2) and specific work to punch (called specific toughness, J m 2 m 1) (Aranwela et al., 1999; Read & Sanson, 2003). Toughness (J m 2) was calculated as the area under the force-displacement curve corrected by the area of the punch

Leaf consumption by a shredder Gammarus pulex is a dominant generalist shredder in many European fresh waters (Pinkster, 1972). This species plays an essential role in leaf litter degradation in streams given its feeding ecology and high abundance (Dangles & Malmqvist, 2004; Piscart et al., 2009, 2011a, b). To avoid any site effects, three populations of G. pulex were sampled from three different habitats: a spring near Dijon (47°24′13″N, 04°52′57″E); a 2-m-wide stream near Lyon (45°49′47″N, 05°04′53″E) and a 10-m-wide stream near Saint-Maurice-de-Gourdans (45°49′07″N, 05°14′47″E). For each site, using a hand-net, around 500 adult individuals of each sex were collected by separating precopula pairs in the field. Individuals were maintained in separate 10-L tanks with aerated site water for 7 days at 12 °C and 12 : 12 h light/dark cycle to standardise their physiological parameters. During this period, individuals were fed every 2 days with crustacean food (JBL NovoCrabsâ). After this standardisation period, animals of each population were placed individually in 7-cm-diameter plastic cups with 60 mL of filtered water from their own site (N = 20 per sex, per population and per tree species; total = 20*2 (sex)*3 populations*3 tree species = 360). Three weighed discs (∅16 mm) from conditioned leaves of the same tree species were added to each cup to reduce variability due to between-leaf differences in thickness or hardness. After 10 days, the remaining leaf material and the amphipod were freeze-dried (Christâ ALPHA 1-4LD), and the © 2013 John Wiley & Sons Ltd, Freshwater Biology, 58, 1672–1681

Changes in leaf litter over conditioning difference between the initial leaf mass and the final leaf mass was used to compute the leaf consumption rate. To take into account the different levels of toughness among the three leaf types, measurements of the leaf consumption rate were made for leaves conditioned by immersion during 10 days for alder, 25 days for hornbeam and 35 days for oak leaves. Mass losses due to factors other than consumption (e.g. microbial consumption, leaching) were estimated in discs (N = 12 per tree species) incubated in the same conditions, but with no shredders. These control discs were also used to convert the initial fresh mass of discs into initial dry mass and compared to the final dry mass of discs to compute the leaf consumption rate. Leaf consumption rates were calculated as initial dry leaf mass-final dry leaf mass minus the mean microbial consumption/amphipod dry mass/ 10 days. The leaf consumption rate was hence expressed in mg d 1 mg 1.

Statistical analyses Differences between tree species with regard to thickness, toughness, specific toughness, ergosterol content, elemental composition and the C/N ratios were tested before conditioning (at T0) using one-way ANOVAs, with tree species as the fixed factor and the Tukey’s HSD test for pairwise comparisons between tree species. Then, the comparisons between thickness and mechanical properties (toughness and specific toughness) on different dates for each tree species were made using a mixed-effects model with tree species as the fixed factors, dates as the predictor factors and leaf number as a random effect. To take into account the repeated measures, this analysis was made on the same leaves on successive dates. Post hoc tests (Tukey contrast tests) were used for pairwise comparisons between dates. In addi-

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tion, for the three tree species, we determined the time (T50) corresponding to the number of days needed for a decrease by 50% of the initial value of the toughness or specific toughness according to a logistic regression. Spearman’s correlations were used to study the change in the mean value of ergosterol content of each species over time. The mean percentage increase in ergosterol content (in comparison with T0) was determined, only after 10 and 25 days for alder leaves: the ergosterol content remained stable after 25 days. The mean percentage decreases in thickness, toughness and specific toughness compared to the T0 value were determined. For the three tree species, Spearman tests were also used to assess the correlation between the percentage increase in ergosterol content and the percentage decreases in thickness, toughness and specific toughness. Finally, Spearman’s correlations were used to test for one date, the relation between the leaf litter consumption rates of G. pulex, the ergosterol content and the mechanical properties of the tree species. The differences in leaf consumption rates between the tree species were tested using a two-way ANOVA with the tree species and populations of G. pulex as the fixed factors. Tukey’s HSD tests were used for multiple comparisons. All tests were performed using STATISTICA 7.1 software (StatsoftTM, Tulsa, USA), except for mixed-effect models, which were performed with RTM 2.15.1 statistical software (R Development Core Team, 2011).

Results Initial values of mechanical properties and chemical characteristics of leaves Before conditioning, the mechanical properties of leaves varied between species, regardless of the parameter considered (Table 1). The thicknesses of alder leaves were

Table 1 Initial means ( SD) of mechanical properties, ergosterol contents, percentages of carbon, nitrogen and phosphorus and C/N ratios, of leaf litter at T0 (before colonisation by aquatic fungi) Alnus glutinosa Thickness (mm) Toughness (J m 2) Specific toughness (J m 2 m 1) Ergosterol content (mg g 1) %C %N %P C/N Results of the P < 0.05.

ANOVA

0.19 189 1.05 106 1.30 48.2 2.7 0.05 18.3

       

Carpinus betulus a

0.03 45a 2.5 105a 0.50a 2.4a 0.3a 0.02a 1.7a

0.11 167 1.57 106 0.35 47.8 2.1 0.04 23.2

       

b

0.01 40a 3.8 105b 0.08b 1.3a 0.3a 0.02a 2.9a,b

Quercus robur 0.20 356 1.79 106 0.09 44.3 1.0 0.03 46.07

       

0.02a 101b 4.2 105c 0.12b 1.9a 0.3b 0.01a 16.2b

test are presented with letters indicating significant differences between tree species for each parameter considered, for

© 2013 John Wiley & Sons Ltd, Freshwater Biology, 58, 1672–1681

N. Foucreau et al. alder and hornbeam leaves (P = 0.06) and between hornbeam and oak leaves (P = 0.81), but the C/N ratio of oak leaves was significantly higher than that of alder leaves (P < 0.05).

not significantly different from oak leaves (P = 0.13), but were lower than hornbeam leaves (P < 0.001). However, the toughness values for alder and hornbeam leaves did not differ significantly (P = 0.41), but were significantly lower than for oak leaves (P < 0.001). The specific toughness of the three species differed significantly (F2,88 = 35.8, P < 0.001), being lowest in alder (P < 0.001) and highest in oak (P < 0.05). The initial content of ergosterol in leaves differed for the three tree species (Table 1, F2,6 = 13.45, P = 0.006). The ergosterol content in alder leaves was higher than for hornbeam and oak leaves (P < 0.05, P < 0.01, respectively), with the latter two not significantly different (P = 0.57). The elemental composition of leaves (i.e. C, N and P) did not vary among species (P > 0.05), except for the %N, which was significantly lower for oak leaves (Table 1, P < 0.05). The C/N ratio was similar between

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Leaf thickness did not change during decomposition for alder leaves (Fig. 1a, F3,87 = 1.30, P = 0.28). The thickness of hornbeam varied slightly (Fig. 1b, F3,86 = 5.33, P < 0.05), but did not differ between T0 and after 35 days and the thickness of oak leaves (Fig. 1c) decreased slightly over time (F5,145 = 9.36, P < 0.001). Both toughness and specific toughness decreased over time for all tree species (Fig. 1, P < 0.001). However, toughness and specific toughness values decreased significantly after 25 days of conditioning for alder and hornbeam leaves

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Fig. 1 From left to right, mean ( SD) of thickness (mm), toughness (J m 2), specific toughness (J m 3) of leaf litter over the course of the experiment for the leaves of three tree species (a) Alnus glutinosa, (b) Carpinus betulus and (c) Quercus robur. Letters indicate significant differences. Curves on toughness and specific toughness graphics corresponds to logistic regression models. © 2013 John Wiley & Sons Ltd, Freshwater Biology, 58, 1672–1681

Changes in leaf litter over conditioning (a) 3.5

Ergosterol content (in mg.g–1)

(Fig. 1a,b), but only after 35 days for oak leaves (Fig. 1c). The time taken to a 50% decrease in toughness and specific toughness (Table 2) was similar for alder and hornbeam leaves (35 and 36 days) and longer for oak leaves (49–51 days).

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Leaf consumption rate Leaf consumption rate by G. pulex was significantly negatively correlated with the specific toughness but not with the thickness, nor with the toughness, and positively with the content of ergosterol (Table 3). Leaf consumption rates differed significantly between the tree species (F2,299 = 41.8, P < 0.001). Consumption rates on alder leaves were significantly higher (0.0739  0.0058 mg d 1 mg 1) than on hornbeam (0.0334  0.0041 mg d 1 mg 1, P < 0.001) and oak leaves (0.0177  0.0026 mg d 1 mg 1, P < 0.001), while consumption on hornbeam leaves was significantly higher than on oak leaves (P < 0.05; Fig. 3).

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Fungal biomass in leaves tended to increase during decomposition (Fig. 2). Ergosterol content in alder leaves reached 2.41 mg g 1 after 25 days and decreased slightly to 2.02 mg g 1 after 35 days (Fig. 2a). In hornbeam leaves, ergosterol increased over time (r = 0.91, P < 0.001) to a maximum value of 2.20 mg g 1 after 35 days (Fig. 2b), which was similar to the values of alder by the same time, but higher than that observed for oak leaves (0.16 mg g 1; Fig. 2c). Even after 55 days, the maximum ergosterol content in oak leaves (1.32 mg g 1) was still lower, but the increase over time was significant (r = 0.91, P < 0.01). The percentage increases in ergosterol content in leaves were not significantly correlated with the percentage thickness (r = 0.13, P = 0.71), but were highly correlated with the percentage decreases in toughness and specific toughness (r = 0.81, P < 0.01 and r = 0.78, P < 0.01; respectively).

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3 2.5 2 1.5 1 0.5 0 0

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Time (days) Fig. 2 Mean ( SD) content of ergosterol (mg g 1) over the course of the experiment for the leaves of three tree species (a) Alnus glutinosa, (b) Carpinus betulus and (c) Quercus robur.

Table 2 Results of regression logistic models applied to toughness and specific toughness values and T50 values (time in days when initial toughness was reduced by 50%) for leaves of three tree species. In the model, the variable x represents the time (in days)

Toughness Specific toughness

Model T50 Model T50

Alnus glutinosa

Carpinus betulus

Quercus robur

0.15x + 5.28*** 36.2 day 0.12x + 4.39*** 35.3 day

0.12x + 4.22*** 36.7 day 0.10x + 3.32*** 34.8 day

0.22x+10.90*** 49.3 day 0.32x + 16.02*** 50.5 day

***The symbol corresponded to P < 0.001 for the models. © 2013 John Wiley & Sons Ltd, Freshwater Biology, 58, 1672–1681

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Discussion Initial values of mechanical properties and chemical characteristics of leaves The leaves from the three tested species differed in their initial chemical and mechanical parameters. Oak leaves were tougher than alder and hornbeam leaves. Otherwise, oak leaves had lower %N than alder and hornbeam. Breakdown rates are considered a function of leaf N content and are often predicted based on C/N ratios (Webster & Benfield, 1986; Gessner, Chauvet & Dobson, 1999). Indeed, high C/N ratio in leaves may slow the breakdown rate (see Enriquez, Duarte & Sand-Jansen, 1993 cited in Ardon & Pringle, 2008). In our study, the initial C/N ratio was higher for oak than for alder leaves, with an intermediate ratio for hornbeam leaves suggesting different expected breakdown rates according to tree species. Otherwise, the higher initial mechanical resistance of oak leaves compared to the others could be explained by the higher proportion of tissues conferring a structural resistance to leaves, such as lignified tissues (Moustafa, Stout & Bradley, 1968; Evert, 2006; Table 3 Correlations (Spearman) between mean thickness and mechanical properties of leaves from three tree species (pooled) and mean leaf consumption rates (in mg g 1 d 1) three populations (pooled) of G. pulex N

Mean leaf consumption rate (mg leaf.day–1.mg amphipod–1)

Consumption rate and thickness Consumption rate and toughness Consumption rate and specific toughness Consumption rate and ergosterol content

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0.474

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Carpinus betulus

Quercus robur

Fig. 3 Mean ( SE) leaf consumption rate (mg leaf day 1 mg amphipod 1) of leaves by gammarids for the leaves of three tree species, Alnus glutinosa, Carpinus betulus and Quercus robur.

Goncßalves, Gracßa & Callisto, 2007). Ostrofsky (1997) showed that total phenolics, %N and % lignin explained 50% of the variation of the breakdown rates for many tree species. Moreover, we found an initial difference in the ergosterol content of freshly fallen leaves, suggesting that the colonisation of leaves by fungi may strongly differ among tree species before and perhaps after the arrival of leaves in aquatic environments. The step of conidial attachment on the surfaces of leaves (the reproductive forms for these fungi) could be a part of the explanation since it is known to depend on leaf roughness (Kearns & B€ arlocher, 2008). If the initial roughness of leaves is reduced in tough leaves, the colonisation rate may be affected. In particular, the cuticular wax of leaves (Onoda, Richards & Westoby, 2012), and some composite cell wall cellulosic-microfibrils set in a hemicellulose or lignin matrix (Lucas et al., 2000; Read & Sanson, 2003), constituting leaf tissues could influence the toughness and the roughness of leaf surfaces, and consequently the initial colonisation by conidia.

Structural changes during leaf conditioning Contrary to our expectation, the mean thickness remained quite stable over time for hornbeam and alder leaves and decreased slightly only for oak leaves, suggesting that the biomass of fungi did not have a strong effect on this parameter. In addition, the secondary veins (only major veins were avoided for the measures) may constitute resistant areas that are therefore less colonised by hyphomycetes due to their richness in structural elements, particularly lignin. Therefore, even if the area between the veins was affected over time by aquatic fungi, any decrease in thickness may only appear late in the conditioning process. Leaf thickness seems insufficient for detecting early changes in the mechanical properties of leaf litter. Contrary to the thickness, both the toughness and the specific toughness, relating to the energy needed to punch leaf material, decreased strongly over time. Such changes attributed to colonisation by aquatic hyphomycetes have already been described (e.g. Goncßalves et al., 2007; Li et al., 2009; Ligeiro et al., 2010). The toughness and the specific toughness values of alder and hornbeam leaves were affected after 20 days, whereas these values remained stable until 35 days for oak leaves. Compared to alder and hornbeam, oak leaves required almost twice as much time to exhibit a 50% reduction in their toughness. The value of toughness represents the structural resistance of leaves, which depends on the thickness and © 2013 John Wiley & Sons Ltd, Freshwater Biology, 58, 1672–1681

Changes in leaf litter over conditioning on the material resistance (here, the specific toughness). Since the pattern of toughness is similar to the pattern of specific toughness over time, and since the leaf thickness did not particularly change through time, the change in the resistance of leaves (toughness) seemed to be more due to the change in material resistance (specific toughness), which is probably due to the degradation of structural elements. This observation is congruent with our hypothesis that differences among tree species would be linked to different chemical compositions of leaf tissues. Moreover, it is well known that cellulose and lignin degradation are slow (Fioretto et al., 2005), so the composition in leaf tissues conferring a material resistance seemed to be a good predictor of the leaf breakdown rate. The ergosterol content increased over time with different patterns according to tree species. The ergosterol content increased in alder leaves immediately after the exposure to fungal conidia and remained stable after 35 days of conditioning. For hornbeam leaves, the ergosterol content was still increasing at this time, but had only just started to increase for oak leaves. Therefore, the increase in ergosterol content appears to be faster in alder and hornbeam leaves than in oak leaves. For example, eucalyptus leaves have physical (mainly leaf cuticle) and chemical (polyphenols, oils) barriers which retard both colonisation and growth of some aquatic hyphomycetes species (Canhoto & Gracßa, 1999). Our results highlighted a strong correlation between the production of ergosterol and the decrease in toughness and specific toughness for three leaf species. This relation could partially explain the impact of the leaf colonisation by aquatic hyphomycetes on degradation rates, which are directly linked to both the consumption of leaf tissues by hyphomycetes and the increase in hydraulic fragmentation.

Leaf consumption rate by shredders Leaf litter consumption rates by shredders are known to be linked to both leaf toughness, with soft leaves being fed on preferentially (Li & Dudgeon, 2008; Ratnarajah & Barmuta, 2009), and to colonisation by aquatic hyphomycetes (Arsuffi & Suberkropp, 1989; Graca, Maltby & Calow, 1993; Graca et al., 2001). Our results are congruent with all of these observations, with a leaf consumption rate that was negatively correlated with the leaf toughness and positively correlated with the content of ergosterol produced by hyphomycetes. Several factors are involved in this relationship. Firstly, soft leaves may © 2013 John Wiley & Sons Ltd, Freshwater Biology, 58, 1672–1681

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be more easily consumed by shredders. Secondly, the ergosterol production may stimulate the leaf consumption rate of shredders, which are able to discriminate between leaf material colonised by fungi and uncolonised leaf material (Graca et al., 1993). Thirdly, we found a positive interaction between the mechanical properties of leaves and the production of ergosterol. In addition, we found a strong effect of the toughness of leaves on the conditioning process over time. This result suggested that the exposure time was more discriminant for the consumption of leaves by the shredder G. pulex than the type of leaves, which is consistent with the observation of Ligeiro et al. (2010) on leaf colonisation by stream shredders. We may assume that in tough leaves ergosterol content increases and toughness decreases during the conditioning process, resulting in leaves reaching the same condition as soft leaves. Another explanation could be a decrease over time in the concentration in leaves of secondary compounds (e.g. polyphenols), which negatively affect the palatability of leaves (Assmann et al., 2011). In conclusion, our results suggest that soft leaves are quickly available for microorganisms and shredders, whereas hard leaves may constitute a reservoir of organic matter that is usable later, in the spring or summer. A field study by Abelho (2008) dealing with the degradation of three contrasting litter species in term of toughness (soft leaves, medium leaves and tough leaves) supports this idea. Indeed, the colonisation of macroinvertebrates (shredders species) tended to occur earlier and in higher numbers on soft leaves, and only later shredder densities were higher on tougher leaves. Thus the diversity of riparian vegetation could be very important for stream ecosystem functioning by sustaining a source of organic matter over time. Moreover, breakdown rates of mixtures of different litter species entail multiple changes in litter breakdown rates (Lecerf et al., 2011). In addition, mixing litter species of contrasting degradability may improve microhabitat structure and persistence through time, which could in turn modify consumer-resource interactions and consequently impact the litter breakdown rate (Kominoski et al., 2009). Our study suggests that a change in the diversity of riparian vegetation, in term of leaf toughness, induced by land use or climate change could shorten leaf litter availability across seasons. For example, in a climate change context, the replacement of soft leaves from trees living in temperate environments by harder leaves from trees adapted to more arid conditions could delay the availability of the feeding resource several months after abscission and should be taken into account in riparian management.

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Acknowledgments We thank Marion Javal and Marie-Rose Viricel for the acquisition of data and Felix Vallier (UMR CNRS 5023, Universite Lyon 1) for his help in constructing the equipment for biomechanical measurements. Thanks to Floriant Bellvert (CESN, UMR CNRS 5557, Universite Lyon 1) for helping us during ergosterol assays, Florian Mermillod-Blondain and Laurent Simon (UMR CNRS 5023) for, respectively, data on phosphorus and carbon and nitrogen contents. Comments provided by two anonymous referees on an early version of the manuscript were most appreciated. Finally, we are grateful to the national research agency for funding (program Wetchange ANR-09-CEP-006-01 of the National Research Agency - Agence Nationale de la Recherche-ANR).

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(Manuscript accepted 2 April 2013)