Mira Sarkis
email: sarkis@telecom-paristech.fr

Cyril Concolato
email: concolato@telecom-paristech.fr

Jean-Claude Dufourd
email: dufourd@telecom-paristech.fr

The Virtual Splitter: Refactoring Web Applications for the Multiscreen Environment

Keywords: C.2.4 [Distributed Systems]: Distributed Applications; D.2.11 [Software Engineering]: [Software Architecture] Application Distribution, Authoring, Multiscreen, Web Application

Creating web applications for the multiscreen environment is still a challenge. One approach is to transform existing single-screen applications but this has not been done yet automatically or generically. This paper proposes a refactoring system. It consists of a generic and extensible mapping phase that automatically analyzes the application content based on a semantic or a visual criterion determined by the author or the user, and prepares it for the splitting process. The system then splits the application and as a result delivers two instrumented applications ready for distribution across devices. During runtime, the system uses a mirroring phase to maintain the functionality of the distributed application and to support a dynamic splitting process. Developed as a Chrome extension, our approach is validated on several web applications, including a YouTube page and a video application from Mozilla.

INTRODUCTION

Recently, intense research activity has been focused on multi-screen scenarios [START_REF] Dufourd | An open platform for multiscreen services[END_REF] [START_REF] Jang | Mobile device-controlled live streaming traffic transfer for multi-screen services[END_REF] inside home environment where cooperation between heterogeneous devices is leveraged. In such a cooperative environment, a "Multi-Screen Application" (MSA) is an application distributed across multiple connected devices, each having a screen, and designed to offer a convivial experience. Examples of MSA are: using a tablet to display additional information synchronized with a TV program, or using the interaction capabilities of smart-phones (e.g., touch screen) jointly with the large screen and processing power of a PC or a TV to display media elements.

Multi-screen applications impose multiple challenges to the application developers. First, they have to design an application that leverages the multi-screen environment and copes with the diversity of devices. Then, they have to determine how the application content will be distributed across devices based on their specific capabilities, to manage the distributed content and to maintain the synchronization and consistency of the distributed content. The use of web technologies helps reducing the complexity of these tasks and increasing the possibility of deploying a ubiquitous application. In this paper, the term application refers to a web application. Many applications were created before the development of the multi-screen concept. Many of them are actually made of different components that could benefit from being distributed. However, few of them were designed in a modular manner that facilitates code distribution.

With such a motivation and focusing on challenges related to authoring MSAs, this paper aims to meet one principal objective: To propose a new approach that reuses existing single-screen applications and refactors them for the multiscreen environment. In contrast to existing works, our approach is based on mapping the application content onto available devices by automatically analyzing the content following the author or the user choices, on splitting the application into two sub-applications and synchronizing them while maintaining the overall application functionality and supporting a dynamic splitting process. The ability for the user to guide this splitting process is novel and opens up many usage scenarios.

This paper is organized as follows. Section 2 compares our approach with related work. Section 3 describes the virtual splitter architecture, including the mapping, annotation, splitting and mirroring phases. An implementation of the solution, an evaluation and a survey of its limitations are given in Section 4. Finally perspectives and conclusions are drawn in Section 5.

STATE OF THE ART

In prior works, a 'WebSplitter' [START_REF] Han | Websplitter: A unifed xml framework for multi-device collaborative web browsing[END_REF] was proposed to split XML-based applications, based on a metadata file. This file is unique for each application and determines which application portions, i.e., which XML elements can be seen on each user device. The splitter requires a middleware proxy that splits the application content into partial views and a clientside component that receives data pushed by the server. The XML splitter architecture is centralized and requires a man-1 Figure 1: Virtual Splitter Architecture ual mapping for each XML element of the application. In his research, Cheng [START_REF] Cheng | Virtual browser for enabling multi-device web applications[END_REF] proposed a virtual browser capable of separating the application logic from its rendering. The logic is kept within a virtual web page. Automatically the virtual browser splits the main DOM tree into multiple DOM trees and maps these trees to corresponding devices as denoted in a hint file that is specific to each application and manually created by the developer. Cross-device operations are executed in a centralized manner depending ultimately on the browser. Bassbouss et al. [START_REF] Bassbouss | Towards a multi-screen application model for the web[END_REF] outlined how to enable traditional applications to become multi-screen-ready. The application is developed as a single-screen application and requires a multi-screen enabled browser. Based on metadata information provided manually by the developer, specific elements are assigned to a remote device while always being shown on the main device.

In contrast to [START_REF] Cheng | Virtual browser for enabling multi-device web applications[END_REF] and [START_REF] Han | Websplitter: A unifed xml framework for multi-device collaborative web browsing[END_REF] our system has a decentralized architecture. Similar to [START_REF] Bassbouss | Towards a multi-screen application model for the web[END_REF] it delivers master-slave applications. The common part for the three previous works is that each application is analyzed and mapped separately and manually by the author (via a hint file or metadata). This means there is no generic analysis method that can be applied to a set of applications. On the contrary, we propose an extensible system that is capable of automatically analyzing the application and mapping its elements, thus simplifying the author's task and involving the end user in mapping her application. The author only has to determine the analysis criterion.

THE VIRTUAL SPLITTER

Overview

A web application consists mainly of HTML, CSS and JavaScript (JS) resources, that are tightly linked. Links exist between elements in the DOM tree (e.g., parent-child, siblings), between the DOM and JS when the JS accesses elements by specific attributes (e.g., id) or by document navigation, between the DOM and the CSS via selectors, etc. In such a context, splitting an application will break some links and cause a failure in the application look and functionality.

In addition, an application presents two dynamic aspects that make the splitting approach more complicated. On one side, splitting a web application requires support for its dynamism since elements are continuously modified, created, moved or removed during runtime. Supporting this dynamism during run-time is essential to ensure the coherence between elements in each of the distributed application. On the other side, automatic partitioning of the application script is a hard task since JS is a flexible and dynamic language characterized by high-order functions, closures, 'eval' function which dynamically evaluates a string expression, etc. In this paper, we take care of the links and dynamicity of the application by focusing on splitting only the HTML document, maintaining links and providing a dynamic splitting phase during run-time while keeping the JS code as a whole running on one device. The following subsections present a detailed description of the virtual splitter architecture as illustrated in Figure 1.

HTML Elements Mapping

The mapping phase is the first phase in our system. Its purpose is to determine which of the application elements map to the devices involved in the multi-screen experience. In any multiscreen scenario, at least two devices are cooperating. The literature refers to the smartTV as the first screen and assigns the expression 'companion screen' [START_REF] Basapur | FANFEEDS: Evaluation of socially generated information feed on second screen as a tv show companion[END_REF] or 'second screen' [START_REF] Tsekleves | Interacting with digital media at home via a second screen[END_REF] to a device providing a means of interaction with the smartTV services. In this work, the 'principal device' is responsible of processing the main application logic, while the 'secondary device' receives processing results only if it is concerned. As depicted in Figure 1, the mapping phase takes as input a query from the application author, when the application is pre-processed offline; or from the user, when the whole process of splitting the application is done at run-time. In our approach, we have envisaged several possible mapping techniques: based on the analysis of HTML elements, their associated semantics and roles, discussed in Section 3.2.1; or based on the visual rendering of elements, discussed in Section 3.2.2. These techniques could be also combined. For instance, mapping only interactive elements placed in a certain region of the screen to the secondary device. This phase is extensible and other analysis techniques could be used here. The query can therefore be either a simple query indicating which mapping technique should be used along with its specific parameters (e.g., element category or position); or a combined query using boolean logic. The output of the mapping phase is two lists of elements, one for each device.

Semantic Mapping

As a possible mapping criterion, we describe here a semantic based approach. It is fully automatic, selected by either the author or the user. In the context of multiscreen applications, we analyzed the HTML5 elements defined in the standard to determine their roles and how they could be classified for the purpose of application splitting. We identified four relevant classes: interactive elements (i.e., a, area, button, datalist, form, input, keygen, textarea, nav, optgroup, option, output, select), multimedia elements (i.e., video, audio, source, track), non-interactive, non-multimedia visual elements (i.e., caption, dialog, figcaption, h1 to h6, hgroup, img, kbd, label, legend, object, p, progress) and other elements. As part of the query param-eters, the author indicates one or more class of elements to be moved to the secondary device depending on the characteristics of the available devices. For instance, if a smartTV is present, the system or the user may decide to move only multimedia elements on that device. As another example, if a touch screen is present, the interactive elements may be moved onto that device. This mapping technique produces the lists of elements as follows. First, for each element of the application, the algorithm compares it to the tag names present in the classes indicated in the author query. If the element falls within the indicated classes, it is added to the secondary device list. If the element is a composite element (i.e., div, table, iframe) the algorithm iterates over the its children first. If there is no match and if the element does not belong to the 'other' class, it is added to the primary device list. Then, the algorithm detects any change in the element basic role by checking its attributes, mainly declarative event listeners (e.g., 'onclick') since they are responsible of making elements interactive. A non-interactive element which role is only displaying content i.e., image, becomes interactive if it has an event listener that lets users interact with it. In addition, we exploit the semantic links that are created between HTML elements (e.g., 'for' attribute) by keeping these elements together in the same list.

Screen Region Mapping

We also investigated a region-based approach as a mapping criterion. It is fully automatic but this time based on the application visual rendering. It is selected during runtime by the end-user in her browser. Once the user selects a screen region, the system detects elements within that rectangular region to produce the secondary device list. All other elements are placed in the primary device list.

Annotating Elements

As depicted in Figure 1, the second phase is the annotation. It prepares the application for the splitting phase and takes as input the lists of elements produced by the mapping phase. The annotation algorithm starts by processing each DOM leaf element and sets the value of the 'data-device' attribute to 'device2' for elements in the secondary device list and 'device1' for elements in the primary device list. It should be noted that the previous mapping phase may have populated those lists with only some DOM elements, and not all elements in the tree, for instance only interactive elements or only the elements located in a given region. Thus, each remaining leaf element (resp. parent element) in the DOM tree is annotated with the value of its siblings (resp. its children), if the annotation is the same across siblings (resp. children), or with the value 'dev1&dev2' if they differ, meaning that the element will be present on both devices. As a result, the application is totally annotated: each element contains metadata information, in a 'data-device' attribute, reflecting its target device(s).

Splitting Application Content

After the annotation phase, during run-time, the splitting phase relies on the element metadata information to form two separated applications: a master and a slave application as Figure 1 shows. From the original application, elements annotated with 'device1' or 'dev1&dev2' values are kept visible on the screen of the primary device. Elements annotated with 'device2' value are hidden on the primary device. This forms the master application. These hidden elements serves as a shortcut whenever the application main logic requires reading or modifying elements of the remote application on the secondary device, thus the term 'virtual splitter'. Elements annotated with 'device2' and 'dev1&dev2' values are extracted from the original application and imported to the new slave application running on the secondary device. On the master application, in addition to the retained original application logic, JS code is added in an instrumentation phase and aims at making the master application capable of working synchronously with its slave (see Section 3.5). This code also supports the application dynamism and ensures a dynamic mapping and splitting at run-time. On the slave application, the JS code makes the application capable of collecting user interactions, redirecting them to the master, receiving and integrating changes made to its DOM tree.

Mirroring Application Contents

The virtual splitting phase described in Section 3.4 duplicates some content between the master and slave applications. The role of the mirroring phase is to ensure that the slave application has a DOM tree that is an accurate mirror of the hidden DOM tree in the master application. It is performed as follows: On the 'primary device', any dynamic change affecting elements of the 'secondary device' (e.g., node modification, removal or creation) is mirrored to that device through change messages. Upon receiving a message, the application running on the 'secondary device' updates its DOM tree and integrates this change. On the 'secondary device', any user interaction (e.g., clicks, data inputs) is captured and propagated to the 'principal device' where the interaction handler is processed.

IMPLEMENTATION AND RESULTS

Implementation

We decided to implement the virtual splitter as a Google Chrome extension, first to enable on-the-fly instrumentation of the application without having to change the application itself, and second for the better debugging environment compared to the situation on devices. Master and slave applications are rendered as tabs in the browser and communication between them is done using the postMessage API that is similar to the communication mecanism in COLTRAM [START_REF] Dufourd | An open platform for multiscreen services[END_REF].

As discussed in Section 3.5, to detect relevant changes in the DOM we use the Mutation Summary library1 which is based on the working draft of the Mutation Observer API2 . A Mutation-Summary object is configured to watch changes made to elements with 'device2' annotation. If any change happens to these elements, their descendants or attributes, the extension sends a message to the 'secondary device'. The message contains a list of changes. Each change object consists of the type of change, the concerned node, its position in the DOM tree (i.e., parent and previous sibling node), the concerned attribute(s) and the new value of a text node. However, the Mutation Summary library suffers from some limitations. For instance, it cannot detect changes made to HTML elements using JS functions especially if they are not reflected on the DOM tree. To overcome this limitation, as well as supporting dynamism, we use the Monkey Patching technique [START_REF] Lerner | Supporting dynamic, third-party code customizations in javascript using aspects[END_REF], to extend in JS some native browser functions with custom code, in particular: the 'createElement' function is extended to detect the creation of new elements and to trigger dynamically the mapping, annotation and splitting of these elements; the 'setAttribute' function to update the Mutation Summary configuration, to enable the mirroring of newly created attributes; and the 'addEventListener' function to overcome the limitation of the Mutation Summary library, and to replace an event handler triggered on the slave application to a call to the master application.

Results and Discussion

We tested our system on different applications from simple static pages to dynamic applications, among them: a semantic video application 3 , relying on the Popcorn and JQuery libraries and showing various information (e.g., map, text, images) synchronized with a video. We first used the regionbased mapping. On the video application, we separated the video and Flick'r images from the additional information as Figure 2 shows. This experiment verified the performance of the mirroring phase by maintaining a reliable mirror and the synchronization between both master and slave applications. In addition, no compatibility issues were reported between our instrumented code and the JS libraries. We then used the semantic mapping to split a YouTube page and to separate the interactive class of elements from the other classes (i.e., non-interactive and multimedia). As a result, the video runs on the master with all the comments of users while all buttons, anchors, guide container that proposes additional videos to watch later are moved to the secondary device.

Based on this, we identified a few areas for future improvement. This includes mainly the re-organization of application layout based on devices screen characteristics, the solving of some problems related to the use of relative URLs, the handing of HTML elements such as Canvas that have no inner DOM representation but are controlled by JS code. We will also conduct more systematic testing and validation.

PERSPECTIVES AND CONCLUSIONS

In the multi-screen context, this paper proposed a system to transform existing applications from single-screen to multi-screen applications based on author or user choices. The system consists of an automatic and extensible mapping phase that analyzes the application semantically, visually or a combination of these two, an annotation and a virtual splitting phase that result in master-slave appli-3 see http://popcornjs.org/demo/semantic-video cations. A mirroring phase ensures the correct functionality and synchronization between both parts and a dynamic splitting process. We validated our system on two existing applications: YouTube and semantic-video, and we verified the correct content mapping, synchronization and application functionality. As a future step, we aim at implementing our system in the COLTRAM multi-screen platform and extending this system with a context driven splitting technique that collects information concerning involved devices and creates dynamically adaptive mapping criteria.

Figure 2 :

 2 Figure 2: Splitting The Semantic Video Application Based On The Screen-Region Criterion

see http://code.google.com/p/mutation-summary

see http://www.w3.org/TR/domcore


Ana L Uc I A G O N C

Alv Es

ERI C C HAUVET † , ‡ , FELIX B € AR L O C H E R § Manuel A S Grac

Cristina Canhoto

Ana L Ucia Gonc

Top-down and bottom-up control of litter decomposers in streams

Keywords: consumption, diversity, fungi, shredders, species identity

1. Detritivores preferentially consume certain aquatic hyphomycete species while rejecting others. Fungal identity may therefore be a crucial factor determining stream food-web structure and complexity and extend the impact of microbial diversity to effects up through the food web. 2. In this study, we examined if shredder feeding is affected by the identity of fungi on leaves (bottom-up effects) and if preferences of shredders for particular fungi affect the composition of fungal assemblages (top-down effects). Oak leaf discs were conditioned in microcosms with six individual fungal species previously reported as highly palatable (P), unpalatable/rejected (R) and intermediate (I). Additionally, three microcosms were inoculated with three mixtures of four fungal species, each consisting of a different subset of the six species. 3. Colonised discs were offered to three detritivores with different feeding strategies: Proasellus sp. (Isopoda), Echinogammarus meridionalis (Amphipoda) and Schizopelex festiva (Trichoptera). When offered leaves colonised by single fungal species, consumption rates by E. meridionalis and S. festiva were higher on I, followed by P and, finally, R species. Consumption rates by Proasellus sp. were similar across fungal treatments. Consumption rates by the three invertebrates were also similar across all fungal multispecies treatments, suggesting that invertebrate preferences for, or rejection of, a given fungal species may be masked when it grows in proximity to other fungi. Composition and structure of fungal communities were not significantly affected by the feeding of any of the three invertebrates. 4. Our results suggest that certain combinations of fungal and detritivore species result in unpredictable bottom-up and top-down effects in stream food webs.

Introduction

Riparian vegetation is the main provider of energy, primarily in the form of leaves, to food webs in forested streams [START_REF] Abelho | From litterfall to breakdown in streams: a review[END_REF][START_REF] B€ | The Fungal Community: Its Organization and Role in the Ecosystem[END_REF]. In headwaters, leaves are efficiently retained and converted to living biomass by microbes and leaf-shredding detritivores [START_REF] Abelho | From litterfall to breakdown in streams: a review[END_REF][START_REF] Gessner | Fungal decomposers of plant litter in aquatic ecosystems[END_REF][START_REF] Krauss | Fungi in freshwaters: ecology, physiology and biochemical potential[END_REF]. Aquatic hyphomycetes are a key group of leaf decomposers and an important trophic link between leaf litter and its animal consumers; fungal colonisation increases the palatability of leaves to invertebrates by increasing nitrogen concentration and by partial enzymatic digestion of structural leaf polymers [START_REF] B€ | Digestive enzymes and feeding strategies of three stream invertebrates[END_REF][START_REF] Duarte | Aquatic hyphomycete diversity and identity affect leaf litter decomposition in microcosms[END_REF][START_REF] Canhoto | Interactions between fungi and stream invertebrates: back to the future[END_REF].

In laboratory experiments, invertebrate shredders often preferentially consume fungal mycelia of certain species (B€ arlocher & Kendrick, 1973a). They also discriminate among leaves or leaf patches colonised by different fungal species (B€ arlocher & Kendrick, 1973b;[START_REF] Suberkropp | Comparison of degradative ability, enzymatic activity, and palatability of aquatic hyphomycetes grown on leaf litter[END_REF][START_REF] Suberkropp | Effects of aquatic fungi on feeding preferences and bioenergetics of Pycnopsyche gentilis (Trichoptera: Limnephilidae)[END_REF]. Feeding preferences may be influenced by several interacting factors such as: (i) leaf traits (B€ arlocher & Kendrick, 1973b) and diversity [START_REF] Jabiol | Fungi are involved in the effects of litter mixtures on consumption by shredders[END_REF]; (ii) invertebrate species and/or feeding guilds [START_REF] B€ | Digestive enzymes and feeding strategies of three stream invertebrates[END_REF][START_REF] Arsuffi | Selective feeding by shredders on leaf-colonising stream fungi: comparison of macroinvertebrate taxa[END_REF]; (iii) invertebrate size and developmental stage [START_REF] Suberkropp | Effects of aquatic fungi on feeding preferences and bioenergetics of Pycnopsyche gentilis (Trichoptera: Limnephilidae)[END_REF]; (iv) fungal specific traits, including enzyme production, N content or the presence of attractants or deterrents [START_REF] Arsuffi | Leaf processing capabilities of aquatic hyphomycetes: interspecific differences and influence on shredder feeding preferences[END_REF][START_REF] Rong | Food selection in three leaf-shredding stream invertebrates[END_REF]; (v) environmental factors (e.g. stream pH; [START_REF] Dangles | Effects of stream acidification on fungal biomass in decaying beech leaves and leaf palatability[END_REF]. To date, no single factor has consistently been linked to palatability of mycelia or fungus-leaf complexes.

The effects of consumption by stream-dwelling detritivores on fungal assemblages have generally been overlooked. However, macroinvertebrates have been shown to reduce sporulation rates and richness of aquatic hyphomycete communities on leaves in streams, primarily by shortening the residence time of the leaves (B€ arlocher, 1980). In an insecticide-treated stream, spore numbers were consistently higher than in nearby control streams (i.e. with invertebrates), although the fungal community composition was similar [START_REF] Suberkropp | Aquatic hyphomycetes in insecticide-treated and untreated streams[END_REF]. The opposite, that is stimulation of fungal growth and sporulation in the presence of shredders, was observed on recalcitrant substrata [START_REF] Gonc | The breakdown of Blue Gum (Eucalyptus globulus Labill.) bark in a Portuguese stream[END_REF]. This has been attributed to ammonium excreted by invertebrates [START_REF] Villanueva | Positive effect of shredders on microbial biomass and decomposition in stream microcosms[END_REF]. Other authors reported no changes in fungal biomass, sporulation rates and community composition in the presence of shredders [START_REF] Ferreira | Do invertebrate activity and current velocity affect fungal assemblage structure in leaves?[END_REF][START_REF] Suberkropp | Influence of shredder feeding and nutrients on fungal activity and community structure in headwater streams[END_REF].

It remains unclear how the composition of fungal assemblages affects feeding by shredders (bottom-up effect) and how fungal assemblages on leaf litter are in turn affected by macroinvertebrate feeding (top-down effect). Because of the importance of the fungus/invertebrate complex as the main driver of litter processing and energy turnover in streams [START_REF] Gessner | Fungal decomposers of plant litter in aquatic ecosystems[END_REF], horizontal (i.e. within trophic levels) and vertical (i.e. between trophic levels) biodiversity effects should be studied concurrently to understand more completely food-web structure and function [START_REF] Hillebrand | Biodiversity in a complex world: consolidation and progress in functional biodiversity research[END_REF][START_REF] Gessner | Diversity meets decomposition[END_REF].

In this study, we investigated whether: (i) the identity of fungal species would affect shredder consumption rates (bottom-up effects) and (ii) the preferences of shredders for particular fungi would affect the composition of the fungal assemblage on leaves (top-down effects). Six fungi were chosen based on earlier studies identifying pairs of palatable (P), unpalatable/rejected (R) and intermediate (I) species. Single and mixed species cultures were used to condition oak leaf discs, which were then offered to three freshwater shredders with different feeding modes: Schizopelex festiva (Sericostomatidae, Trichoptera), a large shredder (high consumer body size:resource ratio), Echinogammarus meridionalis (Amphipoda, Crustacea), a smaller generalist detritivore (low consumer body size:resource ratio), and Proasellus sp. (Isopoda, Crustacea), a litter consumer feeding mainly on leaf surfaces (Grac ßa, Maltby & Calow, 1993a,b;[START_REF] Feio | Food consumption by the larvae of Sericostoma vittatum (Trichoptera), an endemic species from the Iberian Peninsula[END_REF]. Feeding by amphipods has been described as intermediate between that of scrapers and large shredders, given that they rasp and nibble leaves, but with less intensity than other detritivores (Grac ßa et al., 1993a). Some isopods (e.g. Asellus aquaticus) and caddisfly larvae (e.g. Hesperophylax sp. and Limnephilus sp.) with similar feeding strategies have been shown to be selective feeders. They discriminated among patches on the same leaf colonised by different fungal species and consumed them at different rates [START_REF] Suberkropp | Comparison of degradative ability, enzymatic activity, and palatability of aquatic hyphomycetes grown on leaf litter[END_REF][START_REF] Arsuffi | Growth of two stream caddisflies (Trichoptera) on leaves colonized by different fungal species[END_REF]Grac ßa et al., 1993a).

Methods

Microcosm settings

Senescent, freshly fallen leaves of oak (Quercus robur) were collected in autumn near Candal stream (central Portugal) and stored dry. Leaves were cut with a 16mm-diameter cork borer, oven-dried (105 °C, 24 h) and weighed individually (AE0.1 mg). To reduce variability in initial mass, only discs weighing 10.0-13.9 mg were used. Ten sets of 132 randomly chosen discs were reweighed (AE0.1 mg) and placed in individual 500-mL Erlenmeyer flasks with 250 mL of distilled water and autoclaved. Ten additional sets of ten leaf discs were weighed (AE0.1 mg) and placed in test tubes with 25 mL of distilled water. These discs underwent the same treatment to determine a mass loss correction factor due to leaching during sterilisation.

Autoclaved disc sets were transferred to sterilised 1-L glass bottles (microcosms) with 500 mL of nutrient solution (75.5 mg CaCl 2 , 10 mg MgSO 4 .7H 2 O, 0.5 g 3-morpholinopropanesulphonic acid (MOPS), 5.5 mg K 2 HPO 4 and 100 mg KNO 3 L À1 of sterile distilled water; [START_REF] Dang | Magnitude and variability of process rates in fungal diversity-litter decomposition relationships[END_REF]. Microcosms, closed with cotton bungs, were aerated for 24 h at 15 °C through tubing connected to an air pump to allow additional leaching. Contamination was prevented by passing the airflow through sterile cotton bungs. The mineral salt solution in each microcosm was replaced after 24 h and inoculated with a total of 62 500 conidia [START_REF] Treton | Competitive interaction between two aquatic hyphomycete species and increase in leaf litter breakdown[END_REF] equally divided among the fungal species making up the fungal assemblages (see below). Aquatic hyphomycetes were isolated from single conidia in foam or released from submerged leaf litter collected from Candal Stream and from streams in south-western France. The species were ranked as highly palatable (P), unpalatable/rejected (R) or intermediate (I) based on B€ arlocher & Kendrick (1973a,b), [START_REF] Suberkropp | Comparison of degradative ability, enzymatic activity, and palatability of aquatic hyphomycetes grown on leaf litter[END_REF], [START_REF] Arsuffi | Growth of two stream caddisflies (Trichoptera) on leaves colonized by different fungal species[END_REF], [START_REF] Butler | Aquatic hyphomycetes on oak leaves: comparison of growth, degradation and palatability[END_REF], Grac ßa et al. (1993a) and [START_REF] Suberkropp | Effects of aquatic fungi on feeding preferences and bioenergetics of Pycnopsyche gentilis (Trichoptera: Limnephilidae)[END_REF] (Table 1). The following species were used: Flagellospora curvula (P 1 ) and Tetrachaetum elegans (P 2 ), Lemonniera aquatica (R 1 ) and Tetracladium marchalianum (R 2 ), and Clavariopsis aquatica (I 1 ) and Articulospora tetracladia (I 2 ) (Table 2).

To induce conidial release, small agar plugs cut from 14-day-old pure cultures grown on malt extract agar (20 g MEA L À1 distilled water) were placed in Erlenmeyer flasks with 25 mL of nutrient solution (described above) at 15 °C, on an orbital shaker (100 rpm). Three different mixtures of four species were used (one microcosm per treatment): P 1 P 2 I 1 I 2 (preferred and intermediate species), P 1 P 2 R 1 R 2 (preferred and rejected species) and I 1 I 2 R 1 R 2 (intermediate and rejected species). Additionally, six microcosms were inoculated with individual fungal species (P 1 , P 2 , I 1 , I 2 , R 1 and R 2 ), and one microcosm was kept sterile and used to correct for abiotic mass loss. The microcosms were incubated at 15 °C and aerated during 2 weeks (for the first 6 h for periods of 15 min interspersed by periods of 60 min in which the conidia were allowed to settle, followed by continuous aeration). The nutrient solution was replaced after 24 h and then every 2 days. All manipulations were performed aseptically.

After 2 weeks, the 132 leaf discs from each microcosm were harvested. Twenty leaf discs (five sets of four discs) were used for ergosterol extraction, and another 20 discs (five sets of four discs) were used to confirm the presence of fungal species by inducing sporulation, to estimate variability within each microcosm. Seventytwo discs were placed individually on filter paper for 5 s on each side, weighed and immediately used for the feeding experiment. The remaining 20 discs and the 20 leaf discs for ergosterol determination were individually dried on filter paper for 5 s on each side and then weighed, frozen at -20°C, freeze-dried and individually weighed (AE0.1 mg) to determine the relationship of wet to dry mass.

Fungal biomass

Five sets of four leaf discs in each treatment were freeze-dried and used to determine concentration in ergosterol as a measure of fungal biomass (as described in Gessner &[START_REF] Gessner | Ergosterol-to-biomass conversion factors for aquatic hyphomycetes[END_REF][START_REF] Grac | Methods to Study Litter Decomposition. A Practical Guide[END_REF]Gessner, 2005). Ergosterol was converted to fungal biomass assuming 5.5 lg ergosterol per mg fungal dry mass (DM) [START_REF] Gessner | Ergosterol-to-biomass conversion factors for aquatic hyphomycetes[END_REF]. Fungal biomass was expressed in mg per g leaf DM.

Sporulation of aquatic hyphomycetes

Five sets of four leaf discs of each treatment were used to measure sporulation of aquatic hyphomycetes after day 14 of conditioning. They were incubated in 100-mL Erlenmeyer flasks with 25 mL of nutrient solution on a shaker (100 rpm; 15 °C; 48 h). Conidial suspensions were preserved with 2 mL of 37% formalin. Subsamples were filtered (Millipore SMWP, 5 lm pore size, Billerica, MA, U.S.A). Trapped spores were stained with 0.05% cotton blue in lactic acid (60%), identified and counted at 200 9 [START_REF] Grac | Methods to Study Litter Decomposition. A Practical Guide[END_REF]. Sporulation rates were converted to spore volumes using the species-specific conversion factors from [START_REF] B€ | Effects of leaf size and decay rate on colonization by aquatic hyphomycetes[END_REF] and to conidial mass by the formula: M = À0.058 9 V 2 + 641 9 V, where M is the conidial mass (ng), and V is the corresponding conidial volume in lm 3 [START_REF] Baldy | Microbial dynamics associated with leaves decomposing in the main stem and floodplain pond of a large river[END_REF].

Invertebrate feeding experiment

Leaf discs inoculated with each one of the nine combinations of fungal species were individually offered to 12 specimens in separate containers of one of the following invertebrate species collected in streams from Central Portugal: Proasellus sp. (Fail stream; 40°36'24.89"N, 07°58'30.83"W; 325 m asl), Echinogammarus meridionalis (Redinha; 39°58'43.43"N, 08°34'23.94"W; 98 m asl) and Schizopelex festiva (M uceres; 40°32'01'' N, 08°09'15'' W; 210 m asl). Thirty litres of stream water from each stream were filtered (Glass fibre filters; Millipore APFF, 0.7 lm pore size) and used in the experiments. Individuals were acclimatised in aerated filtered stream water for 15 days and fed ad libitum with oak litter conditioned in the stream for 2 weeks.

Invertebrates were individually allocated to plastic containers (70 mm diameter 9 85 mm high) with 200 mL of stream water. Aeration was continuously provided with aquarium pumps. The bottom of the containers was covered with a fine layer of ashed (550 °C; 6 h) stream sediment from the original stream. Each microcosm was supplied with two leaf discs, one leaf disc was exposed to invertebrate feeding while the other was enclosed in a small fine mesh bag, attached to the edge of each container and used as control. The experiment was run at 15 °C with a 12 : 12 dark:light photoperiod.

After two (S. festiva) or four days (Proasellus sp. and E. meridionalis), when a mean of 50% of the exposed disc's surface area remained, the experiment was stopped and the remaining litter mass was recovered. All discs (control and exposed) were frozen at À20 °C, freeze-dried and weighed (AE0.1 mg). Initial DM of leaf discs was determined from the linear regression between wet and dry disc mass (see above). Consumption was calculated as the difference between control and exposed discs in each microcosm. Consumers were removed from each container, dried at 105 °C for 48 h and weighed to the nearest 0.1 mg. Individual relative consumption rates (RCR) for each leaf treatment were expressed as consumption per mg invertebrate DM per day.

Molecular analysis

To compare the composition of the fungal assemblage on control discs before exposure to invertebrates and on discs after exposure, the presence and relative contributions to total biomass of individual fungal species were assessed with denaturing gradient gel electrophoresis (DGGE). Band intensities on the gel were estimated by measuring pixel density with ImageJ (http://rsbweb.nih.gov/ij/ by National Institutes of Health).

DNA from four leaf discs from each treatment (control and invertebrate-exposed discs) was extracted with the UltraClean Soil DNA kit (MoBio Laboratories, Solana Beach, CA, U.S.A) according to the manufacturer's instructions, and DNA was pooled to obtain one value per treatment. The ITS2 region of fungal ribosomal DNA was amplified with the primer pair ITS3GC and ITS4 [START_REF] Nikolcheva | Seasonal and substrate preferences of fungi colonizing leaves in streams: traditional versus molecular evidence[END_REF]. For polymerase chain reaction (PCR), 0.5 lL of each primer, 12.5 lL of Taq DNA polymerase, 9.5 lL of nuclease-free water and The 380-to 400-bp amplification products of each treatment were analysed with the DCode TM Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA, U.S.A). Samples of 20 lL PCR products were loaded on 8% (w/v) polyacrylamide gels in 19 Tris-Acetate-EDTA (TAE) with a denaturing gradient from 30 to 70% (100% denaturant corresponds to 40% formamide and 7 M urea). A DNA mixture from pure cultures of the aquatic hyphomycete species (F. curvula, T. elegans, L. aquatica, T. marchalianum, C. aquatica and A. tetracladia) was used to calibrate the DGGE gels. The gels were run in 19 TAE at 55 V for 16 h at 56 °C and stained with 19 gelRed.

Data analysis

Initial DM of discs was calculated from linear regressions between specific wet/dry mass for each microcosm (R 2 ≥ 0.75). Mycelial biomass from ergosterol and DNA banding intensity, total conidial mass, as well as relative consumption rate for the three invertebrate species were compared among initial fungal assemblages by one-way ANOVA. Tukey's tests were applied for post hoc multiple comparisons whenever relevant (STATISTICA 7). Data were transformed (log (x + 1)) when necessary to satisfy assumptions of normality and homoscedasticity [START_REF] Zar | Biostatistical Analysis[END_REF].

DGGE banding intensities of fungal assemblages on oak leaf discs before and after invertebrate exposure (control versus exposed leaf disc) were analysed by nonmetric multidimensional scaling (NMDS) ordination for each invertebrate species, based on the Bray-Curtis similarity index of log(x + 1)-transformed banding intensities data (PRIMER v6; [START_REF] Clarke | Primer v5: User Manual/ Tutorial[END_REF]. Analyses of similarity (one-way ANOSIM) and hierarchical clusters were performed among mixed treatments.

Results

Microbial conditioning

Mycelial biomass (based on ergosterol) associated with the leaf discs differed significantly among fungal treatments (one-way ANOVA, F 8,36 = 75.40, P < 0.001; Fig. 1), with T. elegans (P 2 ) exhibiting the lowest (Tukey's test, P = 0.088) and L. aquatica (R 2 ) the highest values (Tukey's test, P = 0.008). In individual cultures, mycelial biomass tended to be lower in microcosms containing leaf discs inoculated with P than with I and R species, which also affected the fungal biomass in mixed cultures; when P species were present in the inoculum, fungal biomass was significantly lower (Tukey's test, P < 0.036). Fungal biomass in mixed cultures was lower than the biomass in three of the six pure cultures (Tukey's test, P < 0.001).

In DGGE gels, the bands obtained from control discs of some fungal species (F. curvula and T. elegans, P 1 /P 2 , and A. tetracladia and L. aquatica, I 1 /R 2 ) were very close to each other, impairing the ability to differentiate among all species within an assemblage. The abovementioned tendency for lower biomass in monocultures with P, followed by I and R species, was also apparent here. However, total fungal biomass of all mixtures tended to be equal or higher than in monocultures (oneway ANOVA, F 8,18 = 10.26, P < 0.001; Fig. 1).

Total conidial mass produced on day 14 was affected by the composition of initial inocula (one-way ANOVA, F 8,34 = 315.79, P < 0.001; Fig. 2), with F. curvula (P 1 ) and C. aquatica (I 2 ) exceeding all others. By contrast, T. elegans (P 2 ) produced fewer spores in single cultures than in mixed assemblages. The total conidial mass of each species in mixed cultures was generally lower than in

P 2 I 1 I 2 R 1 R 2 P 2 P 1 P 1 I 1 I 2 P 2 R 1 R 2 I1I 2 R 1 R 2
Fig. 1 Mycelial biomass from ergosterol ( ; n = 5, AESE) and DNA banding intensity in denaturing gradient gel electrophoresis gels ( ; n = 3, AESE) associated with oak leaf discs in microcosms, incubated with mono-or multicultures of six aquatic hyphomycete species for two weeks. P 1 -Flagellospora curvula, P 2 -Tetrachaetum elegans, I 1 -Clavariopsis aquatica, I 2 -Articulospora tetracladia, R 1 -Lemonniera aquatica and R 2 -Tetracladium marchalianum, where P, R and I indicate preferred, rejected and intermediate preference by consumers from literature. Significant differences (P < 0.05) among treatments are indicated by different letters (lower-case letters for mycelial biomass, upper-case letters for total fungal biomass). monocultures. Clavariopsis aquatica (I 2 ) tended to be dominant, while mixed assemblages with R species tended to be co-dominated by more than one species (data not shown).

Invertebrate feeding

Proasellus consumption rates were not significantly affected by aquatic hyphomycete assemblages (one-way ANOVA, F 8,33 = 0.61, P = 0.77; Fig. 3a). Echinogammarus meridionalis consumed more of leaf discs colonised by monocultures of I and P rather than R species (one-way ANOVA, F 8,31 = 16.68, P < 0.001; Fig. 3b). No significant differences were detected among consumption rates of discs inoculated by combinations of four species (Tukey's test, P > 0.082). Consumption of these discs ranged from low to intermediate compared with discs inoculated with monocultures.

Finally, consumption by S. festiva was higher from discs with a monoculture of C. aquatica (I 2 ) over those with T. elegans (P 2 ) (Tukey's test, P = 0.014; Fig. 3c). This shredder had similar consumption rates on leaves colonised by the three fungal assemblages.

Effects of invertebrate feeding activities on fungal assemblages

Based on DNA band intensities, fungal assemblages in leaf discs were not significantly modified by the feeding activities of any animal species tested (one-way ANO-SIM, R 2 > 0.088, P > 0.80; Fig. 4a-c). There was more than 80% similarity between fungal assemblages on control discs and those exposed to invertebrates.

Discussion

When discs colonised by single fungal species were offered in individual microcosms, the consumption rate of two of the invertebrates (E. meridionalis and S. festiva) differed with fungal species, suggesting selective feeding. Earlier studies suggested that both F. curvula and T. elegans should be of high, and C. aquatica, of intermediate palatability (B€ arlocher & Kendrick, 1973a,b;[START_REF] Suberkropp | Comparison of degradative ability, enzymatic activity, and palatability of aquatic hyphomycetes grown on leaf litter[END_REF][START_REF] Arsuffi | Growth of two stream caddisflies (Trichoptera) on leaves colonized by different fungal species[END_REF][START_REF] Butler | Aquatic hyphomycetes on oak leaves: comparison of growth, degradation and palatability[END_REF][START_REF] Suberkropp | Effects of aquatic fungi on feeding preferences and bioenergetics of Pycnopsyche gentilis (Trichoptera: Limnephilidae)[END_REF]. However, these three fungi were not favoured by Proasellus or E. meridionalis, suggesting that the invertebrate preferences for fungal species are species-specific. By contrast, all invertebrates consumed statistically identical amounts of leaf material colonised by the three combinations of four fungi. In all three invertebrates, consumption tended to be higher on one or more of the single-culture discs than on any of the mixed-culture discs (although the difference was not significant with Proasellus sp.). Thus, invertebrate preference or rejection of a given fungal species may be masked when it grows in mixtures with other fungi. This may have implications for fungal growthdepending on the identity of neighbouring colonies, some intermingling with non-related mycelia may be beneficial, rather than an interference. Non-preferential feeding was also reported in Pycnopsyche gentilis (Trichoptera): consumption of leaf patches colonised by different fungal species did not differ significantly [START_REF] Suberkropp | Effects of aquatic fungi on feeding preferences and bioenergetics of Pycnopsyche gentilis (Trichoptera: Limnephilidae)[END_REF]. Other studies, however, showed that isopods (e.g. Asellus aquaticus), amphipods (e.g. Gammarus pseudolimnaeus and Gammarus pulex) and caddisfly larvae (e.g. Hesperophylax sp. and Limnephilus sp.) detected and discriminated among fungal species offered as mycelium or growing on various leaf species (B€ arlocher & Kendrick, 1973a;[START_REF] Suberkropp | Comparison of degradative ability, enzymatic activity, and palatability of aquatic hyphomycetes grown on leaf litter[END_REF][START_REF] Arsuffi | Growth of two stream caddisflies (Trichoptera) on leaves colonized by different fungal species[END_REF]Grac ßa et al., 1993a). This discrepancy may be related to the fact that, in previous studies, different fungal species occurred on well-separated leaf patches and their hyphae did not intermingle (B€ arlocher & Kendrick, 1973a,b;[START_REF] Suberkropp | Comparison of degradative ability, enzymatic activity, and palatability of aquatic hyphomycetes grown on leaf litter[END_REF][START_REF] Arsuffi | Growth of two stream caddisflies (Trichoptera) on leaves colonized by different fungal species[END_REF]but see Chung & Suberkropp, 2009). In our study, we simultaneously inoculated four fungal species onto leaf discs, presumably allowing intimate mixing. This may present a more realistic scenario, since up to 12 species can be found on a single leaf in the field [START_REF] Shearer | Comparison of three techniques for the study of aquatic hyphomycete communities[END_REF]. On the other hand, the design of our experiments did not give the consumer a true choice among food items within the same microcosm, which may accentuate differential feeding (Table 1).

P 2 I 1 I 2 R 1 R 2 P P 1 2 I 1 I 2 P 1 P 2 R 1 R 2 I 1 I 2 R 1 R 2
I 2 I 1 P 2 P 1 R 1 R 2 P 1 P 2 I 2 I 1 P 1 P 2 R 2 R 1 I 1 I 2 R 2 R 1
Colonisation patterns by individual fungal species in mixed inocula strongly depend on the identity of co-colonisers, with direct repercussions on growth patterns and reproductive output [START_REF] B€ | Intraspecific hyphal interactions among aquatic hyphomycetes[END_REF][START_REF] Yuen | Interspecific interactions among tropical and subtropical freshwater fungi[END_REF][START_REF] Ferreira | Contamination by uranium mine drainages may affect fungal growth and interactions between fungal species and strains[END_REF]. In the current study, mycelial biomass (based as ergosterol) and species-specific conidial production in mixtures were generally lower than in the respective monocultures, suggesting some degree of mutual inhibition [START_REF] Pascoal | Realized fungal diversity increases functional stability of leaf-litter decomposition under zinc stress[END_REF]. The intensities of DGGE bands of amplified fungal DNA confirmed the presence of the appropriate species, even when spore production was minimal (T. elegans). However, fungal biomass estimated by DGGE band densities diverged from biomass estimated by ergosterol (Fig. 1). This is not unexpected since ergosterol concentrations of fungal mycelia can vary by a factor of up to 14, depending on species and culture conditions [START_REF] Charcosset | Effect of culture conditions on ergosterol as an indicator of biomass in the aquatic hyphomycetes[END_REF]

P 2 R 1 R 2 control; -P 1 P 2 R 1 R 2 exposed; -I 1 I 2 R 1 R 2 control; -I 1 I 2 R 1 R 2 exposed.
content strongly depends on fungal age [START_REF] Grimmett | Does the growth rate hypothesis apply to aquatic hyphomycetes?[END_REF]. This illustrates the need for new and more discriminatory quantitative approaches, such as quantitative real-time or in situ PCR, to overcome the current difficulty of assessing the relative abundances of fungal species and their spatial distribution within leaves or leaf patches [START_REF] Krauss | Fungi in freshwaters: ecology, physiology and biochemical potential[END_REF].

It is generally accepted that the relationships between invertebrates and fungal assemblages are affected not only by the identity of fungal species but also by the substratum quality, with fungal conditioning becoming more important on recalcitrant leaves [START_REF] Jabiol | Fungi are involved in the effects of litter mixtures on consumption by shredders[END_REF]). We therefore expected clear feeding preferences in our experiments with oak leaves. While this was the case with discs inoculated with single fungal species, there was no selective feeding on discs with several species, suggesting that all fungal assemblages guaranteed a level of conditioning sufficient to stimulate shredder consumption (either to acquire the resources, i.e. leaf and fungi, or simply to acquire enzymatic activity), independently of the species involved. Several previous studies have also failed to relate consistently palatability of a fungus-leaf combination to selected parameters, such as lipids, proteins, fungal biomass or activities of various degradative enzymes (e.g. [START_REF] Arsuffi | Selective feeding by stream caddisfly (Trichoptera) detritivores on leaves with fungal-colonized patches[END_REF]. [START_REF] Rong | Food selection in three leaf-shredding stream invertebrates[END_REF] suggested that a combination of nutritional improvements due to fungal colonisation (macronutrients such as proteins, lipids, carbohydrates, as well as leaching or possibly fungal inactivation of leaf toxins) is important in food choice. Once these changes have reached a threshold, more specific repellents and attractants, or possibly micronutrients, may fine-tune the feeding preferences of invertebrates. A further complication is the fact that even minor changes in incubation temperature or nutrient concentration in the medium can drastically affect palatability and nutritional value of fungal mycelia [START_REF] Nikolei | Vergleichende Untersuchungen zur Fortpflanzung heterogener Gallm€ ucken under experimentellen Bedingungen[END_REF].

The presence of species that were preferred when offered as monocultures did not consistently influence the feeding activity of the three shredders on leaves with mixed cultures. This suggests that the invertebrates (despite their distinct feeding modes) were unable to discriminate among species grown in mixtures and/or that fungal assemblages have common emergent properties that compensate for the inherent variability of individual fungal species (e.g. composition, stoichiometry). It has been suggested that shredders have different physiological and behavioural adaptations that compensate for stoichiometrical imbalances between food and consumers (essentially C : N ratios; [START_REF] Tuchman | Nutritional quality of leaf detritus altered by elevated atmospheric CO 2 : effects on development of mosquito larvae[END_REF][START_REF] Canhoto | Interactions between fungi and stream invertebrates: back to the future[END_REF]. However, the actual impact of invertebrate feeding on fungal biomass and assemblages was assumed to depend on the characteristics and number of the species involved (e.g. composition, physiological condition and efficiency of the digestive process; [START_REF] Cross | Ecological stoichiometry in freshwater benthic systems: recent progress and perspectives[END_REF].

Different modes of feeding can shift the composition of the fungal assemblage by selectively removing some mycelia and ignoring others, and S. festiva did induce some shifts in the quantity of DNA of the I 1 I 2 R 1 R 2 treatment. However, we did not observe any significant changes in the fungal assemblages of control and invertebrate-exposed discs. In our experiments, we had a maximum of four fungal species, whose biomasses were fairly evenly distributed, and both control and treatment discs were examined after identical incubation periods. This prevented us from observing all potential effects of top-down control by invertebrates that might occur in the field, such as the occurrence of late arrivals on discs inaccessible to invertebrates. In addition, invertebrates only had access to one food source at a time, disallowing actual choices between fungus/leaf combinations, which might have influenced overall species distribution in the entire system. Our design also ignored the potential impact of faster decomposition (reduced residence time) often observed in the presence of invertebrates [START_REF] Suberkropp | Influence of shredder feeding and nutrients on fungal activity and community structure in headwater streams[END_REF]. Based on the 'more individuals hypothesis' [START_REF] Evans | Dissecting the species-energy relationship[END_REF], we would expect greater overall fungal diversity on leaves whose breakdown is delayed, as might happen in the absence of shredding invertebrates.

The general absence of top-down effects of invertebrates on fungi was unexpected, in view of previously reported strong preferences among patches (or leaf discs) colonised by different fungal species (e.g. B€ arlocher & Kendrick, 1973a,b;[START_REF] Suberkropp | Comparison of degradative ability, enzymatic activity, and palatability of aquatic hyphomycetes grown on leaf litter[END_REF][START_REF] Arsuffi | Growth of two stream caddisflies (Trichoptera) on leaves colonized by different fungal species[END_REF]Grac ßa et al., 1993a;[START_REF] Jabiol | Fungi are involved in the effects of litter mixtures on consumption by shredders[END_REF]. This suggests that certain combinations of fungi and detritivores result in unpredictable bottomup and top-down effects in stream food webs.

Fig. 2

 2 Fig. 2 Total conidial mass from oak leaf discs in microcosm, inoculated with individual and mixtures of six aquatic hyphomycete species for two weeks (n = 5, AESE). P 1 -Flagellospora curvula, P 2 -Tetrachaetum elegans, I 1 -Clavariopsis aquatica, I 2 -Articulospora tetracladia, R 1 -Lemonniera aquatica and R 2 -Tetracladium marchalianum. Significant differences (P < 0.05) of conidial mass among different treatments are indicated by different letters.

Fig. 3

 3 Fig. 3 Relative consumption rates (RCR) of (a) Proasellus sp. (b) E. meridionalis and (c) S. festiva fed oak leaf discs conditioned with individual and mixtures of the six aquatic hyphomycete species (n = 12, AESE). P 1 -Flagellospora curvula, P 2 -Tetrachaetum elegans, I 1 -Clavariopsis aquatica, I 2 -Articulospora tetracladia, R 1 -Lemonniera aquatica and R 2 -Tetracladium marchalianum. Significant differences (P < 0.05) of mycelial biomass among different treatments are indicated by different letters.

Fig. 4

 4 Fig. 4 Ordination (non-metric multidimensional scaling) of mixed fungal assemblages in oak leaf discs before and after invertebrate exposure (control versus exposed leaf disc) to (a) Proasellus sp. (b) Echinogammarus meridionalis and (c) Schizopelex festiva, based on Bray-Curtis similarity matrix of fungal DNA banding.-P 1 P 2 I 1 I 2 control; -P 1 P 2 I 1 I 2 exposed; -P 1 P 2 R 1 R 2 control; -P 1 P 2 R 1 R 2 exposed; -I 1 I 2 R 1 R 2 control; -I 1 I 2 R 1 R 2 exposed.

Table 1

 1 Summary of food consumption experiments. The various food sources were offered separately (in separate containers) or together (in one container). Preferences are listed below as rankings

	Reference	Invertebrate consumer	Measured variable(s) as function of fungal species	Food offered
	Arsuffi & Suberkropp, 1986	Hesperophylax magnus	Relative consumption rates (% d À1 ):	Separately
			1. Flagellospora curvula
			2. Alatospora acuminata
			3. Tetracladium marchalianum
			4. Lemonniera aquatica
		Psychoglypha sp.	Relative consumption rates (% d À1 ):	Separately
			1. Heliscus lugdunensis
			2. Flagellospora curvula
			3. Alatospora acuminata
			4. Articulospora inflata*
			5. Tetracladium marchalianum
			6. Lemonniera aquatica
	B€ arlocher & Kendrick, 1973a	Gammarus pseudolimnaeus	Daily consumption of fungal mycelium (mg d À1 )	Together
			1. Clavariopsis aquatica
			2. Fusarium sp.
			3. Tricladium angulatum
			4. Anguillospora long ıssima
			5. Cladosporium sp.
			6. Alternaria sp.
			7. Tetracladium marchalianum
			8. Flagellospora curvula
			9. Aspergillus niger
	Butler & Suberkropp, 1986	Psychoglypha sp.	% of fungal-colonised leaves consumed:	Together
			1. Flagellospora curvula
			2. Alatospora acuminata
			3. Clavariopsis aquatica
			4. Articulospora inflata
			5. Filosporella annelidica
			6. Tetracladium marchalianum
			7. Lemonniera aquatica
			8. Heliscus lugdunensis
	Chung & Suberkropp, 2009	Pycnopsyche gentilis (third instar larvae)	Degree of shredding (% loss of patch	Together
			area by feeding of fungal patches)
			1. Articulospora tetracladia
			2. Tetrachaetum elegans
			3. Alatospora acuminata
			4. Anguillospora filiformis
	Grac ßa et al., 1993a	Gammarus pulex	% of fungal-colonised leaves consumed:	Together
			1. Anguillospora longissima
			2. Heliscus lugdunensis
			3. Tricladium angulatum
			4. Articulospora tetracladia
			5. Fusarium sp.
			6. Lemonniera aquatica
			7. Tetracladium marchalianum
			8. Cylindrocarpon sp.
			9. Tetracladium setigerum
		Asellus aquaticus	% of fungal-colonised leaves consumed:	Together
			1. Tetracladium marchalianum
			2. Anguillospora longissima
			3. Heliscus lugdunensis
			4. Tetracladium setigerum
			5. Tricladium angulatum
			6. Fusarium sp.
			7. Cylindrocarpon sp.
			8. Articulospora tetracladia
			9. Lemonniera aquatica

Table 1 (

 1 Continued) Geniculospora inflata (Ingold) Sv. Nilsson ex Marvanov a & Sv. Nilsson.

	Reference	Invertebrate consumer	Measured variable(s) as function of fungal species	Food offered
	Rong et al., 1995	Gammarus tigrinis	Daily consumption of fungal-colonised	Together
			leaves(mg leaf/mg animal d À1 )
			1. Anguillospora filiformis
			2. Clavariopsis aquatica
			3. Tetracladium marchalianum
		Tipula caloptera	1. Anguillospora filiformis	Together
			2. Clavariopsis aquatica
			3. Tetracladium marchalianum
		Pycnopsyche guttifer	1. Anguillospora filiformis	Together
			2. Tetracladium marchalianum
			3. Clavariopsis aquatica
	Suberkropp et al., 1983	Hesperophylax sp.	% of fungal-colonised leaves consumed:	Together
			1. Alatospora acuminata
			2. Flagellospora curvula
			3. Heliscus lugdunensis
			4. Tetracladium marchalianum
			5. Anguillospora pseudolongissima
			6. Clavariopsis aquatica
			7. Tetrachaetum elegans
			8. Articulospora inflata*
			9. Lemonniera aquatica

*

Table 2

 2 Palatability list of six fungal taxa according to the literature (see text for detailed citations)

	Fungal species	Palatability	Designations
	Flagellospora curvula	Highly palatable	P 1
	Tetrachaetum elegans	Highly palatable	P 2
	Clavariopsis aquatica	Intermediate palatable	I 1
	Articulospora tetracladia	Intermediate palatable	I 2
	Lemonniera aquatica	Unpalatable/rejected	R 1
	Tetracladium marchalianum	Unpalatable/rejected	R 2

© 2014 JohnWiley & Sons Ltd, Freshwater Biology, 59, 2172-2182

Acknowledgments

We thank Ana Virg ınia L ırio for the assistance in the field and laboratory experience, and Ivan Grimmett with help in molecular techniques in Mount Allison University (supported by an NSERC Canada grant to FB). Financial support by the Portuguese Science and Technology Foundation (reference SFRH/BD/47089/2008) to ALG is gratefully acknowledged. This study was supported by the European Regional Development Fund (ERDF) through the COMPETE -Operational Factors of Competitiveness Program (POFC-COMPETE) and national funds through FCT -Foundation for Science and Technology, under the project 'Predicting the effect of global warming on stream ecosystems' (FCT Ref: PTDC/CLI/67180/2006; COMPETE Ref: FCOMP-01-0124-FEDER-007112) and by the Pessoa exchange programme between Portugal and France (PHC 22808 TB) from the French Ministry of Foreign and European Affairs.

Declaration of authorship

All authors conceived and designed the experiments. ALG performed the experiments and wrote the first draft of the manuscript, and all authors collaborated on writing the final version.