Frédéric Legendre

Tony Robillard

Laure Desutter-Grandcolas

Michael F Whiting

Philippe Grandcolas

Phylogenetic analysis of non-stereotyped behavioural sequences with a successive event-pairing method

Keywords:

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Studies on behaviour and phylogeny have a long common history. From the early times, comparative studies have shown that behaviour can be remarkably informative regarding the relationships of taxa, as reviewed by [START_REF] Hinde | The comparative study of species-specific behavior[END_REF]. These comparative studies focused on highly stereotyped and ritualized behaviours, such as courting or nest building [START_REF] Wenzel | Behavioral homology and phylogeny[END_REF]. Being stereotyped, these behaviours were easily compared among species to assess their patterns of evolution [START_REF] Lorenz | Vergleichende Bewegungsstudien an Anatinen[END_REF][START_REF] Hinde | A comparative study of the courtship of certain finches[END_REF][START_REF] Tinbergen | Comparative studies of the behaviour of gulls (Laridae); a progress report[END_REF]. In this context, the homology of stereotyped behaviour has been repeatedly discussed by several seminal papers [START_REF] Baerends | Comparative methods and the concept of homology in the study of behavior[END_REF][START_REF] Atz | The application of the idea of homology to behavior[END_REF][START_REF] Hodos | The concept of homology and the evolution of behavior[END_REF][START_REF] Lauder | Homology, analogy, and the evolution of behavior[END_REF][START_REF] Wenzel | Behavioral homology and phylogeny[END_REF][START_REF] Greene | Homology and behavioural repertoires[END_REF] with reference to the classical criteria of homology proposed by [START_REF] Remane | Die Grundlagen des naturlichen Systems, der vergleichenden Anatomie und der Phylogenetik[END_REF] and especially the criteria of position and special quality. The rationale behind using behaviour for phylogenetic inference was that behavioural acts that are repeatedly observed in various individuals and populations without strong variation or evidence for learning can be presumptively considered as inherited, in the same way as other phenotypic traits, such as morphology or development [START_REF] Wenzel | Behavioral homology and phylogeny[END_REF]. More recently, many studies confirmed the old perception [START_REF] De Queiroz | The usefulness of behavior for phylogeny estimation -levels of homoplasy in behavioral and morphological characters[END_REF][START_REF] Proctor | Behavioral characters and homoplasy: perception versus practice[END_REF][START_REF] Brooks | The nature of diversity. an evolutionary voyage of discovery[END_REF] that behaviour is phylogenetically informative and that it can be studied within such an evolutionary perspective (e.g. [START_REF] Coddington | Orb webs in non-orb weaving ogrefaced spiders (Araneae: Dinopidae): a question of genealogy[END_REF][START_REF] Mclennan | The benefits of communication between comparative ethology and phylogenetic systematics: a case study using gasterosteid fishes[END_REF][START_REF] Wenzel | Application of the biogenetic law to behavioral ontogeny: a test using nest architecture in paper wasps[END_REF][START_REF] Kennedy | Hop, step and gape: do the social displays of the Pelecaniformes reflect phylogeny?[END_REF][START_REF] Johnson | The evolution of postcopulatory displays in dabbling ducks (Anatini): a phylogenetic perspective[END_REF][START_REF] Burmeister | Handbuch der Entomologie. Zweiter Band, zweite Abteilung, Kaukerfe[END_REF][START_REF] Noll | Behavioral phylogeny of corbiculate apidae (hymenoptera; apinae), with special reference to social behavior[END_REF][START_REF] Price | Reconstructing the evolution of complex bird song in the Oropendolas[END_REF][START_REF] Desutter-Grandcolas | Phylogeny and the evolution of calling songs in Gryllus (Insecta, Orthoptera, Gryllidae)[END_REF]Robillard, Höbel & Gerhardt, 2006a). However, treatments of behavioural characters greatly vary among the different studies. In the best case, particular behaviours are used as characters or attributes in phylogenetic analyses, coded 'present' vs. 'absent' [START_REF] Deleporte | Characters, attributes and tests of evolutionary scenarios[END_REF][START_REF] Grandcolas | Phylogenetics and ecology: as many characters as possible should be included in the cladistic analysis[END_REF]. In the worst case, some very broad behavioural classes are mapped onto the phylogenetic trees, leading to potentially biased reconstructions (as reported by [START_REF] Proctor | Behavioral characters and homoplasy: perception versus practice[END_REF][START_REF] Desutter-Grandcolas | Phylogeny and the evolution of calling songs in Gryllus (Insecta, Orthoptera, Gryllidae)[END_REF][START_REF] Grandcolas | The origin of a 'true' worker caste in termites: mapping the real world on the phylogenetic tree[END_REF].

This way to treat behaviour in phylogenetic analyses ignores the fact that behavioural acts do not exist in isolation: they are expressed in a context, usually as an answer to a stimulation by a conspecific or the environment, and they can be combined or repeated. For example, a particular grooming act in a grooming sequence is not equivalent to a similar grooming act in a dyadic (i.e. involving two interacting individuals) agonistic sequence; this grooming act is said to be 'displaced' in the second case [START_REF] Mcfarland | Animal behaviour[END_REF]. Behaviour is therefore better described and behavioural homology can be better established with reference to the criterion of position when the place of different behaviours is considered along the behavioural sequence (Robillard et al., 2006b) and also with reference to the criterion of special quality, as reviewed by [START_REF] Wenzel | Behavioral homology and phylogeny[END_REF]. When behavioural sequences are stereotyped, as in the case of highly ritualized behaviours, the comparisons among species are straightforward as there is only one sequence per species. These sequences may be aligned and analysed as was carried out by ethologists in nonphylogenetic and intraspecific comparisons [START_REF] Abbott | Sequence analysis: new methods for old ideas[END_REF][START_REF] Wilson | ClustalG: Software for analysis of activities and sequential events. Workshop on longitudinal research in social science: a Canadian focus[END_REF][START_REF] Abbott | Sequence analysis and optimal matching methods in sociology. Review and prospect[END_REF][START_REF] Schlich | Analysing intrapersonal variability of travel behaviour using the sequence alignment method[END_REF][START_REF] Van Der Aalst | Workflow mining: a survey of issues and approaches[END_REF][START_REF] Hay | Mining navigation patterns using a sequence alignment method[END_REF]. Alignments of stereotyped behavioural sequences for phylogenetic analysis can be generated in a dynamic way (Robillard et al., 2006b) via direct optimization [START_REF] Wheeler | Optimization alignment: the end of multiple sequence alignments in phylogenetics?[END_REF] or used in a static way in a standard phylogenetic analysis [START_REF] Wilson | ClustalG: Software for analysis of activities and sequential events. Workshop on longitudinal research in social science: a Canadian focus[END_REF]. However, when sequences are not stereotyped within species there is presently no way to use them directly to study species phylogenetic relationships.

The aim of this paper is therefore to propose a new methodology allowing the study of nonsterotyped sequences in a phylogenetic framework and to show its potential by applying it to a typical case of sequences obtained from social interactions. This application is made for some social cockroaches, the behaviour of which has already been much studied [START_REF] Gautier | Processus de différenciation de l'organisation sociale chez quelques espèces de blattes du genre Blaberus: aspects écologiques et éthologiques[END_REF][START_REF] Grandcolas | Les Blattes de la forêt tropical de Guyane Française: structure du peuplement et étude écoéthologique des Zetoborinae[END_REF][START_REF] Van Baaren | Comparison of gregariousness in larvae and adults of four species of zetoborine cockroaches[END_REF][START_REF] Van Baaren | Measurement for solitariness and gregarism: analysing spacing, attraction and interactions in four species of Zetoborinae (Blattaria)[END_REF]van Baaren et al., , 2003a)).

ESTABLISHING A SUCCESSIVE EVENT-PAIRING METHOD TO STUDY BEHAVIOURAL SEQUENCES

The method presented here was inspired by the procedure of event-pairing, which was developed simultaneously by [START_REF] Mabee | Development of the cranium and paired fins in Betta splendens: intraspecific variation and interspecific comparisons[END_REF], [START_REF] Smith | Comparative patterns of craniofacial development in eutherian and metatherian mammals[END_REF] and [START_REF] Velhagen | Analyzing developmental sequences using sequence units[END_REF] to study developmental sequences [START_REF] Bininda-Emonds | From Haeckel to event-pairing: the evolution of developmental sequences[END_REF][START_REF] Jeffery | Analysing developmental sequences within a phylogenetic framework[END_REF][START_REF] Jeffery | A new technique for identifying sequence heterochrony[END_REF]. Using that method, developmental sequences are recoded in all possible pairwise combinations of events, thereby encoding the relative position of each item in the sequence. Each developmental event is coded as possibly occurring before, simultaneously or after any other event [START_REF] Jeffery | A new technique for identifying sequence heterochrony[END_REF]. Event-pairing coding has been recently challenged by [START_REF] Schulmeister | Comparative and phylogenetic analysis of developmental sequences[END_REF] who suggested that treating features of sequences as if they were independent can produce inconsistent reconstructions. This is mainly because developmental sequences are more constrained in terms of temporal linearity than are ethological sequences with respect to the biological process involved. For example, many developmental events cannot occur earlier or later within a sequence because the structures where they should take place are not yet developed or cannot develop twice [START_REF] Schulmeister | Comparative and phylogenetic analysis of developmental sequences[END_REF]. The limitations of event-pairing coding as applied to the study of development are not a problem for non-stereotyped behavioural sequences, where the same event can be expressed several times within the sequence. There is not a one-to-one relationship between a linear sequence and a species, but many different sequences for the same species. Our goal is to code in a phylogenetic context the occurrence and frequency of transitions between two acts among many differently ordered sequences. Only transitions between two successive events are considered here and not the relative position between all pairs of events. This methodology is named the successive event-pairing method to avoid confusion with the event-pairing method.

Establishing a matrix of characters coding the occurrence of two successive events in a behavioural sequence is already part of the current statistical analyses of behavioural sequences (Fig. 1). Ethologists build matrices of transition where each cell is filled with the frequency of a transition between two particular acts [START_REF] Martin | Measuring behaviour. An introductory guide[END_REF][START_REF] Gottman | Sequential analysis: a guide for behavioral researchers[END_REF][START_REF] Bakeman | Analyzing interaction: sequential analysis with SDIS And GSEQ[END_REF]. These frequencies are then organized in flow charts generated by hand or according to correspondence analyses [START_REF] Van Der Heijden | Correspondence analysis of longitudinal categorical data[END_REF] to investigate how different acts are organized in different kinds of sequences and to compare them between different species. To adapt this procedure to the successive event-pairing method, we only need to consider that the cells of matrices of behavioural transitions can be used as phylogenetic characters (Fig. 1). This is justified on the basis of the classical criteria of homology applied to comparative ethology [START_REF] Wenzel | Behavioral homology and phylogeny[END_REF]. Homology based on the cells of such matrices fits the criterion of position as it defines a particular succession of two acts, thus specifying the position of one act relative to another. In this context, events A occurring after an event B or after an event C are not considered homologous, strictly speaking: answering by A after B or after C is not the same behaviour and will not necessarily be shown by all species even if the fixed motor pattern involved in displaying A is the same in each case. This is easy to understand if one considers a real example where a species would tend to answer to conspecific aggression by escape, while another species would answer by reciprocal aggres-sion. Ethologists have long known that this kind of difference can be species-specific or common to related species. The occurrence of transitions between two particular acts can be treated as presence-absence characters. The frequencies of transitions can also be used as it is very different to observe that a given transition is very rare or very common. Either a low or a high frequency can be considered characteristic of species and therefore used in phylogenetic analysis as characters. Frequencies can be discretized and coded in different character states using gap coding [START_REF] Archie | Methods for coding variable morphological features for numerical taxonomic analysis[END_REF][START_REF] Stevens | Character states, morphological variation, and phylogenetic analysis: a review[END_REF]. Recently, [START_REF] Goloboff | Continuous characters analysed as such[END_REF] argued that continuous characters need not to be discretized. However, their methodology treats continuous characters as additive characters, which requires an assumption of progressive evolution that we do not want to follow here. The study of quantitative characters has proved to be a 1. Behavioural sequences Sp. 1

1 1 0 2 C 0 2 1 C 0 0 0 D 0 0 1 B 1 0 0 A D B A R Ac 0 0 0 0 D 2 1 0 1 C 1 2 C 0 0 2 B 0 0 0 A D B A R 1 0 0 1 C 2 0 0 C 0 0 1 D 3 0 0 B 1 0 0 A D B A R R 1 1 0 2 C 1 2 0 C 0 0 0 D 1 0 0 B 1 0 0 A D B A

Behavioural transition matrices

1 1 1 1 1 1 1 0 1 1 0 1 1 1 0 0 1 1 1 0 Species 1 Species 2 Species 3 Species 4 2 1 > 0 9 0 > 1 7 1 > 0 4 1 > 0 10 0 > 1 5 0 > 1 3 1 > 0 1 1 > 0 Ac Char.

Figure 1.

A scheme for the establishment of a successive event-pairing method to analyse the evolution of behavioural sequences. In this theoretical example, four species with the same behavioural repertoire (acts A, B, C, D) are studied and three behavioural sequences were observed (1) which are used to build transition matrices (2). For the construction of such matrices, the individuals are alternatively the actor (Ac) and the receiver (R). The presence-absence of transitions within these matrices is used in a second step to characterize each species in a phylogenetic matrix (3) which in turn is analysed to build a phylogenetic tree (4). Char., character; Ind., individual; Sp., species. difficult problem in phylogenetics and the present work is not aimed at solving it. We will focus this work on the most commonly used approaches: discretization and gap coding.

Our method requires that all these behavioural patterns, both the acts and the trends of succession among acts, are largely heritable and that their plasticity and variability are low. This is the most general and necessary assumption that should be substantiated to legitimate phylogenetic studies of behaviour. It can be partly carried out in evaluating the congruence of the phylogenetic tree based on behavioural data with molecular and morphological data. Obviously, assessing the minimal sampling effort is needed to document correctly the behavioural acts, their transitions and frequencies and their independence. These assumptions are no different than for other phenotypic characters (morphology, cytology, etc.), as already argued by [START_REF] Wenzel | Behavioral homology and phylogeny[END_REF].

MATERIAL AND METHODS

AN ILLUSTRATIVE CASE STUDY: GREGARIOUS BEHAVIOUR IN ZETOBORINAE COCKROACHES

Non-stereotyped behavioural sequences are most often observed in the context of social relationships. The observed behavioural sequences are not a series of acts successively emitted by the same individual, a situation which could occur with other behaviours such as territorial displays, grooming activities, etc., but a series of acts emitted by two individuals in alternation. These behavioural relationships are rarely stereotyped and there can be different answers to a particular act from a conspecific, depending on the context, and several different acts can initiate a sequence of interaction between two conspecifics.

Gregarious behaviour in cockroaches is a famous example of presocial behaviour [START_REF] Schal | Behavioural ecology of cockroaches[END_REF][START_REF] Gautier | Relationships between ecology and social behavior in cockroaches[END_REF][START_REF] Nalepa | Postovulation parental investment and parental care in cockroaches[END_REF][START_REF] Grandcolas | The origin of diversity in cockroaches: a phylogenetic perspective of sociality, reproduction, communication and ecology[END_REF][START_REF] Van Baaren | Measurement for solitariness and gregarism: analysing spacing, attraction and interactions in four species of Zetoborinae (Blattaria)[END_REF]van Baaren et al., , 2003b)). It has been recently analysed in a molecular and morphological comparative framework in the subfamilies Zetoborinae and Blaberinae [START_REF] Grandcolas | Les Blattes de la forêt tropical de Guyane Française: structure du peuplement et étude écoéthologique des Zetoborinae[END_REF](Grandcolas, , 1993a(Grandcolas, , b, 1998;;Pellens, Legendre & Grandcolas, 2007a;Pellens et al., 2007b), which provided both a phylogenetic reference and a natural history context for the interpretation of social behaviour observed in the laboratory [START_REF] Grandcolas | Les Blattes de la forêt tropical de Guyane Française: structure du peuplement et étude écoéthologique des Zetoborinae[END_REF][START_REF] Van Baaren | Comparison of gregariousness in larvae and adults of four species of zetoborine cockroaches[END_REF][START_REF] Van Baaren | Measurement for solitariness and gregarism: analysing spacing, attraction and interactions in four species of Zetoborinae (Blattaria)[END_REF]van Baaren et al., , 2003a)). In a first attempt to understand the evolution of social behaviour, relevant categories such as 'gregarious', 'solitary' and 'subsocial', have been mapped onto a phylogenetic tree based on morphology (Grandcolas, 1993a[START_REF] Grandcolas | The evolutionary interplay of social behavior, resource use and anti-predator behavior in Zetoborinae + Blaberinae + Gyninae + Diplopterinae cockroaches: a phylogenetic analysis[END_REF]. Ethological studies have shown that the gregarious behaviours should be analysed in detail and contrasted between species, not only considering broad behavioural categories [START_REF] Grandcolas | Les Blattes de la forêt tropical de Guyane Française: structure du peuplement et étude écoéthologique des Zetoborinae[END_REF][START_REF] Van Baaren | Comparison of gregariousness in larvae and adults of four species of zetoborine cockroaches[END_REF][START_REF] Van Baaren | Measurement for solitariness and gregarism: analysing spacing, attraction and interactions in four species of Zetoborinae (Blattaria)[END_REF]van Baaren et al., , 2003a)). A small group of closely related species has been already well studied and will be used as an example of how the successive event-pairing method can be applied to behavioural sequences to infer a phylogenetic tree congruent with other data and to propose hypotheses of behavioural evolution. We will not discuss the issues of behavioural plasticity and repertoire sampling which have been explored and controlled for in the specific papers cited earlier.

Dyadic interactions in four species -namely Thanatophyllum akinetum [START_REF] Grandcolas | Les Blattes de la forêt tropical de Guyane Française: structure du peuplement et étude écoéthologique des Zetoborinae[END_REF], Schultesia lampyridiformis Roth, 1973, Lanxoblatta emarginata Burmeister, 1838 and Phortioeca nimbata Burmeister, 1838 -have been observed among 11 to 17 groups of six nymphs placed in standard conditions for each species, according to the protocol described in [START_REF] Grandcolas | Les Blattes de la forêt tropical de Guyane Française: structure du peuplement et étude écoéthologique des Zetoborinae[END_REF] and [START_REF] Van Baaren | Measurement for solitariness and gregarism: analysing spacing, attraction and interactions in four species of Zetoborinae (Blattaria)[END_REF]. Each group has been placed in an open-field arena. The observations began 1 h later, lasted 15 min and have been recorded on a Samsung Digital Camcorder VP-D11. The observations have been carried out on nymphs in the middle of their development as this is the most characteristic and intense period of gregarious behaviour (Grandcolas, 1993b;[START_REF] Van Baaren | Comparison of gregariousness in larvae and adults of four species of zetoborine cockroaches[END_REF]. One additional outgroup species Eublaberus distanti Kirby, 1903 from the closely related subfamily Blaberinae has also been observed. Transition matrices have been constructed using the behavioural sequences reported as described in Figure 1. Phylogenetic analyses have been carried out with these behavioural matrices and also by comparison with a morphological and molecular data set. The morphological data are taken from Grandcolas (1993a[START_REF] Grandcolas | The evolutionary interplay of social behavior, resource use and anti-predator behavior in Zetoborinae + Blaberinae + Gyninae + Diplopterinae cockroaches: a phylogenetic analysis[END_REF]. Molecular data for all the species and behavioural data for Eublaberus distanti have been acquired for the present study.

The sampling effort for behaviour has been critically evaluated with respect to the previous behavioural studies that were conducted and published on the same insect species (e.g. [START_REF] Van Baaren | Measurement for solitariness and gregarism: analysing spacing, attraction and interactions in four species of Zetoborinae (Blattaria)[END_REF]. Accumulation curves for the occurrence of transitions according to the number of observations have been computed to show that the sampling effort is large enough to observe the transitions, either uncommon or frequent, occurring in each species.

Character independence has also been evaluated by checking whether frequencies of transitions involving a similar behavioural act are not misleadingly correlated. This can be easily tested with a c 2 goodnessof-fit test [START_REF] Chatfield | Analysing sequences of behavioural events[END_REF][START_REF] Zar | Biostatistical analysis[END_REF], which verifies whether the frequency of a transition between two acts can be determined by the total frequency of each act involved. Basically, this test compares expected frequencies with observed frequencies. Following [START_REF] Zar | Biostatistical analysis[END_REF], some data have been pooled together in order to have an average expected frequency of at least six, which avoids bias in c 2 computation.

PRIMERS, PCR AND SEQUENCING

Leg muscle tissue was excised from roach specimens preserved in 100% ethanol. DNA was extracted using the Qiagen DNeasy protocol for animal tissue. Mitochondrial ribosomal DNA large subunit (16S, ~385 bp), nuclear ribosomal DNA small subunit (18S, ~1875 bp) and nuclear ribosomal DNA large subunit (28S) domains A (~360 bp) and C (~330 bp) were amplified. 18S was amplified and sequenced in four overlapping fragments corresponding to GA, AD1D2, BCE and EF domains. PCR reactions were lead on a DNA Engine DYAD TM , Peltier Thermal Cycler with the following conditions: an initial heating step of 94 °C for 2 min followed by 40 cycles of 94 °C for 60 s, 55 °C for 60 s and 72 °C for 75 s. Then a final elongation at 72 °C was carried out over 7 min. The different primers used which have already been published are listed in Table 1. Electrophoresis gel was used to visualize PCR products and to check that there was no contamination as a result of a negative control. PCR products were purified via the Montage PCR 96 Cleanup Kit (Millipore) and sequenced using ABI Big Dye 3.1 with the following sequence profile: 27 cycles of 96 °C for 10 s, 50 °C for 5 s and 60 °C for 4 min. Sequencing reactions products were purified with Sephadex TM columns and fractionated on an ABI 3730 XL DNA sequencer. Each sequence was edited using Sequencher 4.0 [START_REF] Genecodes | Sequencher[END_REF] Even if repertoires are basically identical among species and therefore cannot convey a great deal of phylogenetic information, their analysis has been carried out for a matter of comparison (analysis A). Using the successive event-pairing method, a second phylogenetic analysis has been performed on the presence-absence of transitions taken as characters (analysis B). In a third analysis (analysis C), characters based on the discretized frequencies of the transitions were added to the B data set. The marginal frequencies of each initiating act have been calculated. For instance, in species 1, one event A and two events C have been observed in answer to an event B (Fig. 1). Then, the frequencies for the transitions B/A, B/B, B/C and B/D are 0.33, 0.00, 0.67 and 0.00, respectively. All the frequencies for the same initiating act have been pooled throughout all the species to form a distribution. Each distribution of frequencies was discretized and coded using gap coding [START_REF] Archie | Methods for coding variable morphological features for numerical taxonomic analysis[END_REF][START_REF] Stevens | Character states, morphological variation, and phylogenetic analysis: a review[END_REF].

The morphological data set has been taken from the matrix of 78 characters used by Grandcolas (1993a), considering only the five species of the present study. As it represents few parsimony-informative characters (9), it has been analysed together with the molecular data (analysis D). The molecular data set represents portions of genes 16S, 18S and 28S. Molecular sequences, which present a very low variability in length, were aligned using Muscle 3.6 [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF]. Finally, behavioural data (including frequencies) have been combined together with the morphological + molecular data set (analysis E). All characters were equally weighted and coded as non-additive. All parsimony analyses have been performed using PAUP4.0b10 [START_REF] Swofford | PAUP* -phylogenetic analysis using parsimony (*and others methods)[END_REF] with an exhaustive search to ensure finding the most parsimonious tree. Consistency index (CI; [START_REF] Kluge | Quantitative phyletics and the evolution of anurans[END_REF], retention index (RI; [START_REF] Farris | The retention index and the rescaled consistency index[END_REF] and number of parsimony-informative characters were recorded. Bremer support values were computed with the help of TreeRot.v2b [START_REF] Sorenson | TreeRot, version 2[END_REF]. Bootstrap values were computed for 1000 replicates using PAUP4.0b10. Character optimizations on the phylogenetic trees were performed with fast procedure (i.e. accelerated transformation ACCTRAN) using Winclada version 1.00.08 [START_REF] Nixon | Winclada version 1.00.08[END_REF].

Because the monophyly of the ingroup has been established previously (Grandcolas, 1993a[START_REF] Grandcolas | The evolutionary interplay of social behavior, resource use and anti-predator behavior in Zetoborinae + Blaberinae + Gyninae + Diplopterinae cockroaches: a phylogenetic analysis[END_REF], the ingroup was designated as monophyletic in tree visualization.

Analyses C and E include characters based on the frequencies of transitions and consequently include inapplicable characters. If a particular behavioural transition is not observed in one or several species, the character based on its frequencies is inapplicable for that or those species. Those characters were coded with a dash ('-') in the matrices but were interpreted as missing data during the tree search. This 'reductive coding' better reflects the information content of the data [START_REF] Strong | Character coding and inapplicable data[END_REF].

The behavioural data were tested for significant structure using the permutation tail probability test (PTP; [START_REF] Faith | Could a cladogram this short have arisen by chance alone? On permutation tests for cladistic structure[END_REF] and the g1 statistics [START_REF] Hillis | Discriminating between phylogenetic signal and random noise in DNA sequences[END_REF][START_REF] Hillis | Signal, noise, and reliability in molecular phylogenetic analyses[END_REF] in PAUP4.0b10 [START_REF] Swofford | PAUP* -phylogenetic analysis using parsimony (*and others methods)[END_REF]. However, even if there is a structure in a phylogenetic tree based on behavioural data, this structure can be a reflection of common evolutionary ecological pressures rather than of phylogenetic relationships [START_REF] Kennedy | Hop, step and gape: do the social displays of the Pelecaniformes reflect phylogeny?[END_REF]. If the behavioural tree is congruent with a tree based on other data sets (morphological + molecular here), it is most likely to be as a result of a common phylogenetic signal in the different data sets. Behavioural and morphological + molecular trees were tested for congruence using the triplets tree comparison metric (Symmetric Difference of triplets, SDt hereafter) as implemented in the software Component v2.0 [START_REF] Page | COMPONENT User manual (release 2.0)[END_REF]; the lower the value, the more congruent the trees. This value can be compared with a null distribution calculated after generating all topologies with five leaves unrooted (options 'generate all' and 'tree-to-tree distances/triplets/SD').

RESULTS

Twenty-four different acts were identified from the behavioural sequences of the different species (Tables 2 and3). Eublaberus distanti and Schultesia lampyridiformis have the largest behavioural repertoire (19 acts), whereas Lanxoblatta emarginata and Phortioeca nimbata have the smallest and the same one (15 acts). The outgroup E. distanti displays some autapomorphic acts and notably the sudden jump (SJ) and the act PS (when an individual puts its pronotum under the other individual and stands up suddenly), both of which are known in Blaberus, the sister genus of Eublaberus [START_REF] Gautier | Processus de différenciation de l'organisation sociale chez quelques espèces de blattes du genre Blaberus: aspects écologiques et éthologiques[END_REF].

Transition matrices (Appendix S1) have been constructed using the behavioural sequences. Accumulation curves showed that the total number of observations is large enough to observe all frequent or uncommon transitions, the number of which has reached a plateau (e.g. for Eublaberus distanti, Fig. 2). According to the matrices, S. lampyridiformis was the most active cockroach with more than 1300 behavioural transitions (Appendix S1) representing a mean of 65 interactions/h. Conversely, P. nimbata and E. distanti were the less active (c. 30 interactions/h). We found no correlation between the amount of activity and the number of kinds of transitions among the five species, which shows again that there is no bias related to a sampling effect depending on different species activities (r = 0.70, P > 0.15). According to the behavioural matrices (Appendix S1), some transitions were never observed (empty cells of the matrices), while others were rarely or commonly observed (cells filled with a small or a large integer, respectively). As a mean, the behavioural sequences include from 6 to 7 acts depending on the species, ranging from very short (two acts) to quite long (25 acts). Transition matrices show that many transitions are not observed in some species and that others are rare or frequent. Statistical independence among different transitions for a same species was assessed according to c 2 goodness-of-fit tests which were highly significant (Eublaberus: 3. Behavioural repertoires of the five cockroaches used in this study (also established according to [START_REF] Gautier | Processus de différenciation de l'organisation sociale chez quelques espèces de blattes du genre Blaberus: aspects écologiques et éthologiques[END_REF][START_REF] Grandcolas | Les Blattes de la forêt tropical de Guyane Française: structure du peuplement et étude écoéthologique des Zetoborinae[END_REF][START_REF] Van Baaren | Measurement for solitariness and gregarism: analysing spacing, attraction and interactions in four species of Zetoborinae (Blattaria)[END_REF]van Baaren et al., , 2003a, b, b)

MT AC MA CB PS PP KL SP SA SD SJ BI Eublaberus distanti X X X X X X X X Lanxoblatta emarginata X X X X X X X Phortioeca nimbata X X X X X X X Thanatophyllum akinetum X X X X X X X X Schultesia lampyridiformis X X X X X X X X X GD GA TP FP RO WD ES WP GA GL WA NO Eublaberus distanti X X X X X X X X X X X Lanxoblatta emarginata X X X X X X X X Phortioeca nimbata X X X X X X X X Thanatophyllum akinetum X X X X X X X X X Schultesia lampyridiformis X X X X X X X X X X
72, c 2 0.05 = 92.8, P << 0.05; Schultesia: c 2 = 908.6, ddl = 144, c 2 0.05 = 173.0, P << 0.05; Thanatophyllum: c 2 = 250.7, ddl = 56, c 2 0.05 = 74.5, P << 0.05). This trend can be easily observed in the transition matrices where the values within the same rows or lines are very different.

The phylogenetic analysis based on behavioural repertoires of the different species (Table 3) resulted in four most parsimonious trees (six informative characters, L = 15 steps, CI = 0.80, RI = 0.50), the strict consensus of which is totally unresolved (Fig. 3A). Even if the repertoires are rich, their phylogenetic analysis is not decisive enough for establishing a resolved tree.

Phylogenetic matrices were built up from the transitions matrices as explained in Figure 1. The data set (B) based on the presence-absence of transitions taken as characters comprised 576 characters, 74 of which were parsimony informative (i.e. 12.8 %). Its analysis resulted in a single most parsimonious tree (L = 217 steps; CI = 0.86, RI = 0.58; Fig. 3B). The addition of characters based on frequencies of the transitions (analysis C) gave also a single most parsimonious tree with the same topology and similar indices (L = 300 steps; CI = 0.87, RI = 0.55; Fig. 3C). The matrix comprised 1152 characters, with 89 parsimony-informative characters (7.7 %). In both trees, no zero-length branches were reconstructed which means that behavioural data bring information at different levels. The analysis of morphology + molecular data set yielded one most parsimonious tree with a slightly different topology: the positions of S. lampyridiformis and T. akinetum are inverted with regard to the analyses B and C (L = 351 steps, CI = 0.88, RI = 0.50; Fig. 3D). The matrix comprised 3034 characters among which 88 were parsimony informative (2.9 %). Finally, the combined analysis resulted in one most parsimonious tree similar to those based on behavioural characters (L = 659, CI = 0.86, RI = 0.48; Fig. 3E). Among the 4186 characters, 177 were parsimony informative (4.3 %). This latter topology showed maximal bootstrap values and very high Bremer values (18 and 24). Statistics of all those analyses have been summarized in Table 4. They demonstrated that behavioural data were not more homoplastic than molecules and morphology and that behaviour brought proportionally more informative characters (up to 12.8 % of informative characters vs. 2.9 %). The g1 statistics and the PTP test indicated that behavioural data were highly structured (data set B: g1 = -1.406, P < 0.01; PTP test for 1000 replicates, P = 0.001) and the comparison between trees of the analyses B and D resulted in a SDt value of 0.2. Compared with the null distribution, this result suggested that those two trees (behavioural and morphological + molecular) were rather congruent. Only 12 pairwise comparisons between topologies out of 105 have a SDt value lower than 0.2.

The comparison of the trees and their statistics produced by the analyses B and C revealed to what extent presence-absence of behavioural transitions and their frequencies were informative. First, the B data set included 74 informative characters, whereas the C data set included 89 informative characters. Therefore, frequencies brought few supplementary informative characters (89 -74 = 15) when compared with presence-absence of behavioural transitions. Second, Bremer values increased (increment of one for both clades) which means that the two data sets are not contradictory. However, frequencies gave less support to the clade [T. akinetum, (L. emarginata -P. nimbata)] than did the presence-absence of behavioural transitions. The comparison of branch lengths confirmed this point. Indeed, the increase of branch length between the two topologies was, in proportion, the smallest for this clade. On the contrary, frequencies brought relatively more information than presence-absence of behavioural transitions for the clade (L. emarginata -P. nimbata) with a Bremer support value of one for 15 informative characters vs. a Bremer support value of one for 74 informative characters, respectively (see Table 5).

Using the topology retrieved in the combined analysis (Fig. 3), we have looked at the behavioural transitions supporting different nodes of the tree.

First, every branch was supported by a reasonable amount of changes. By comparison with repertoires which were basically identical for all species, this means that transitions offered a large amount of information with common states and differences among every species and group of species.

Second, acts supporting Zetoborinae with respect to the Blaberinae outgroup corresponded mainly to absences of agonistic or avoiding acts: slip ones pronotum under the other and stand up suddenly (PS), push the other with ones pronotum (PP), sudden jump (SJ) and sudden withdrawal of the antennae (WA), move away but stop in proximity (WP), rotation (RO) or freezing posture (FP), respectively. In the other way, Zetoborinae displayed other 'negative' acts in answer to 'positive' sollicitation (transitions which are not expressed by Eublaberus). Those 'negative' acts were: stilt posture with antennation (SA), goes down (GD), goes down and hides its antennae (GA) and withdrawal (WD).

Third, species with contrasted social behaviour (e.g. the solitary T. akinetum vs. other gregarious species) did not show a special amount of difference but particular changes. The behavioural repertoire of T. akinetum revealed only one autapomorphic behaviour: the absence of stilt posture (SP). Therefore, despite its solitary behaviour, Thanatophyllum did not display an especially idiosyncrasic repertoire with regard to gregarious species. However, according to the successive event-pairing analysis, we were able to determine that T. akinetum was the only species displaying the following behavioural transitions, all of which limiting inter-individual interactions: move towards/bite (MT/BI), antennal contact/ leg kick (AC/KL), stilt posture with antennation/ move away but stop in proximity (SA/WP), bite/ escape (BI/ES), rotation/leg kick (RO/KL), move away but stop in proximity/goes down and hides its antennae (WP/GA), nothing/goes down and hides its antennae (NO/GA).

We also looked at its gregarious sister group, composed of two closely related species which were supposed to have very similar social gregarious behaviour, L. emarginata and P. nimbata. These species were not only very close morphologically, they also lived in similar habitats (under loose bark of trees, Grandcolas, 1993b) and exhibit the same behavioural repertoire. Thirteen transitions supported this clade and notably five transitions involving acts of antennation (antennal contact and mutual antennation, AC and MA, respectively). Moreover, on the five characters based on frequencies and supporting this clade, four involved antennation as an answer to an act: move towards (MT) twice, stilt posture with antennation (SA) and nothing (NO).

DISCUSSION

BEHAVIOURAL SEQUENCES IN PHYLOGENETIC ANALYSES

Since Whitman (1898), [START_REF] Heinroth | Beobachtungen bei der zucht des zigenmelkers (Caprimulgus europaeus L.)[END_REF], [START_REF] Lorenz | Vergleichende Bewegungsstudien an Anatinen[END_REF] and others pioneered the comparative study of behaviour, many phylogenetic analyses of behaviour have been carried out. The concept of behavioural homology has been discussed and finally found to be similar to the homology of other phenotypic characters (see [START_REF] Baerends | Comparative methods and the concept of homology in the study of behavior[END_REF][START_REF] Atz | The application of the idea of homology to behavior[END_REF][START_REF] Hodos | The concept of homology and the evolution of behavior[END_REF][START_REF] Lauder | Homology, analogy, and the evolution of behavior[END_REF][START_REF] Wenzel | Behavioral homology and phylogeny[END_REF]. As morphological and molecular characters, behavioural ones proved to be phylogenetically informative when they were accurately defined and adequately sampled. As emphasized by [START_REF] Wenzel | Behavioral homology and phylogeny[END_REF], all these analyses took benefit and became more rigorous with the rise of phylogenetic methods, such as cladistics.

However, in spite of these very significant advances, the great majority of phylogenetic analyses of behaviour still considered behavioural acts in isolation (see [START_REF] Wenzel | Behavioral homology and phylogeny[END_REF] for a review). Behavioural acts are regarded most often as equivalent if they are similar, even if they occur in different sequences or different places within the sequences. This is a much lower standard than for other kinds of characters (molecules, morphology) where the position of the feature is always carefully established (i.e. DNA sequence alignment, position criterion for morphological structures).

We recently emphasized that the most up-to-date phylogenetic algorithms designed to compare DNA sequences -such as direct optimization [START_REF] Wheeler | Optimization alignment: the end of multiple sequence alignments in phylogenetics?[END_REF][START_REF] Wheeler | Dynamic homology and phylogenetic systematics: a unified approach using POY[END_REF] -can be successfully applied to the stereotyped behavioural sequences (Robillard et al., 2006b). In the present paper, we make one more step and propose to compare non-stereotyped and, therefore strictly speaking, not species-specific sequences. Thus, instead of focusing directly on the relationships among different behavioural sequences to infer relationships among species, we aimed at inferring relationships among characteristic parts of these sequences by applying a method of successive event-pairing which codes successions of acts.

The method is conceptually very straightforward by coding the occurrence of transitions between the acts in the sequences. It applies a better and more accurate concept of behavioural homology, each act being considered according not only to its special quality but also to its position within sequences.

Three main assumptions are implied and must be discussed with regard to behavioural characters: heritability, sampling and independence. All kind of characters used in phylogenetic analyses, either morphological, molecular or behavioural, should be checked from this point of view. In practice, this is usually made only for non-traditional characters such as behavioural or physiological ones, the variability of which is intuitively more questioned by scientists [START_REF] Wenzel | Behavioral homology and phylogeny[END_REF]. Heritability is the first and most basic concern as non-heritable traits would be nonsensical if used in a phylogenetic context of descent with modification (see Grandcolas & D'Haese, 2003 for a review). With the exception of breeding and genetic studies, the only a priori way to assess the heritability of characters is to control for epigenetic effects by observing every species in the same conditions and by varying and repeating the conditions of observation. A posteriori, phylogenetic congruence between behaviour and other markers, including those molecular ones reputed to be neutral, is also a mean to assess heritability. In the present case, both criteria have been employed: repertoires and kinds of interactions have been found stable in repeated studies [START_REF] Gautier | Processus de différenciation de l'organisation sociale chez quelques espèces de blattes du genre Blaberus: aspects écologiques et éthologiques[END_REF][START_REF] Grandcolas | Les Blattes de la forêt tropical de Guyane Française: structure du peuplement et étude écoéthologique des Zetoborinae[END_REF][START_REF] Van Baaren | Measurement for solitariness and gregarism: analysing spacing, attraction and interactions in four species of Zetoborinae (Blattaria)[END_REF]van Baaren et al., , 2003a) ) and the present study) and the different data sets are reasonably congruent.

As for the sampling effort, behaviour is not more difficult to sample than morphology or molecules and it only requires to have living specimens placed in controlled and relevant conditions and to follow classical protocols (e.g. [START_REF] Martin | Measuring behaviour. An introductory guide[END_REF][START_REF] Wenzel | Behavioral homology and phylogeny[END_REF]. In our case, accumulation curves were computed showing that the sampling of behavioural transitions has reached a plateau in every species, which allowed a sound interspecific comparison. Given that samples are large enough, the successive eventpairing method also has the advantage to bring more characters -potentially up to a square power morethan the acts considered in isolation.

Character independence has been mentioned for a long time as a potential problem in phylogenetics, but also as one without solution. The most obvious cases of dependence between characters must be checked for and discarded, but some dependence will necessarily exist between characters observed in a same organism which cannot be extirpated [START_REF] Wiley | Phylogenetics. the theory and practice of phylogenetic systematics[END_REF][START_REF] Simmons | Artifacts of coding amino acids and other composite characters for phylogenetic analysis[END_REF]. For instance, molecular phylogenetic studies usually do not consider that base pairs in the stem regions of ribosomal RNA are not truly independent. This problem does not occur with our case for the successive eventpairing method as shown by the c 2 goodness-of-fit tests.

Finally, this method successfully proved to be efficient and informative according to the present example of sequences of dyadic interactions in cockroach groups taken from a well-studied case in the literature [START_REF] Van Baaren | Comparison of gregariousness in larvae and adults of four species of zetoborine cockroaches[END_REF][START_REF] Van Baaren | Measurement for solitariness and gregarism: analysing spacing, attraction and interactions in four species of Zetoborinae (Blattaria)[END_REF]van Baaren et al., , 2003a)). In contrast with the undecisive analysis of the repertoires which were very similar, the analyses of the event-pairing data provided a high number of independent characters, which resulted in a phylogenetic tree fully resolved and highly consistent, not much different and not less consistent than the one retrieved with morphology and molecules. For example, 60 characters (or 89 with the data set including frequencies) potentially support the monophyly of the Zetoborinae, a subfamily which is well supported based on other data [START_REF] Roth | The male genitalia of Blattaria. III. Blaberidae: Zetoborinae[END_REF]Grandcolas, 1993a[START_REF] Grandcolas | The evolutionary interplay of social behavior, resource use and anti-predator behavior in Zetoborinae + Blaberinae + Gyninae + Diplopterinae cockroaches: a phylogenetic analysis[END_REF]Pellens et al., 2007a, b). Our study also refuted the old and recurrent belief that behaviour is more homoplastic than other phenotypic traits, as already argued by [START_REF] Mclennan | The benefits of communication between comparative ethology and phylogenetic systematics: a case study using gasterosteid fishes[END_REF], [START_REF] Wenzel | Behavioral homology and phylogeny[END_REF][START_REF] De Queiroz | The usefulness of behavior for phylogeny estimation -levels of homoplasy in behavioral and morphological characters[END_REF] and [START_REF] Proctor | Behavioral characters and homoplasy: perception versus practice[END_REF]. Consistency and retention indices and numbers of informative characters were similar between analyses C (behaviour) and D (morphology and molecules). In brief, our analysis suggested that even non-stereotyped sequences, which are usually considered more variable and less species-specific, also contain phylogenetic information. It is worth noting that phylogenetic analysis of habitats in the subfamily Zetoborinae revealed much less consistency than the present behavioural analysis [START_REF] Grandcolas | The evolutionary interplay of social behavior, resource use and anti-predator behavior in Zetoborinae + Blaberinae + Gyninae + Diplopterinae cockroaches: a phylogenetic analysis[END_REF]Pellens et al., 2007a), in that there was more homoplasy in habitat changes than in social behaviour for this clade.

GREGARIOUS BEHAVIOUR IN ZETOBORINAE COCKROACHES

Behavioural transitions analysed via our method included many informative characters, few of which were homoplastic. These characters also supported relationships among species in a way which did not appear biased, as also shown by the comparison with morphological and molecular analyses. For example, species did not cluster in broad behavioural categories or potentially non-natural classes, such as gregarious vs. solitary species. The solitary species Thanatophyllum akinetum was not the sister group of gregarious species, but it was nested within ingroup taxa, suggesting that some synapomorphic behavioural transitions were shared by two gregarious species and the solitary one, but not with the other gregarious species, including the outgroup. Additionally, the analysis identified some autapomorphic characters which made sense in the context of solitary life habits. With the same perspective, the acts supporting Zetoborinae -both the solitary and gregarious -with respect to the gregarious Blaberinae outgroup corresponded mainly to absences of agonistic or avoiding acts in response to varied acts such as antennations or moving away (PS, PP, SJ and WA, WP, RO or FP respectively). Zetoborinae displayed other negative acts in answer to positive sollicitation (transitions which are not expressed by Eublaberus). Those negative acts were SA (agonistic), GD, GA, RO, WD (avoiding). Thus, Eublaberus and the Zetoborinae studied here displayed different agonistic and avoiding acts in several particular situations. This suggests that Zetoborinae were more disposed than Eublaberus to display promoting acts (mostly antennation) in front of negative acts and that they gained the behaviour of displaying 'avoiding' acts (GD, GA, RO, WD) to stop interactions rather than agonistic behaviours (or that Eublaberus lost it and acquired aggressive behaviour). Zetoborinae did not appear to be prone to more or less social behaviour than Eublaberus; they displayed a different kind of social behaviour. Finally, Lanxoblatta and Phortioeca, two genera known to be closely related [START_REF] Roth | The male genitalia of Blattaria. III. Blaberidae: Zetoborinae[END_REF]Grandcolas, 1993a[START_REF] Grandcolas | The evolutionary interplay of social behavior, resource use and anti-predator behavior in Zetoborinae + Blaberinae + Gyninae + Diplopterinae cockroaches: a phylogenetic analysis[END_REF], clustered together on the basis of similar behavioural transitions involving antennation.

Using non-stereotyped behaviours in phylogenetic analyses opens new avenues for studies in behaviour evolution. In contrast to restricted stereotyped behaviours such as courtships or nest-building, non-stereotyped behaviours represent most of the behavioural activity of many species, such as feeding, foraging, playing, interacting, etc. [START_REF] Mcfarland | Animal behaviour[END_REF]. Since long ago, these non-stereotyped behaviours were generally considered as less species-specific in their characteristics and composed of acts often widespread in related species, hence not chiefly adequate for comparative studies (e.g. [START_REF] Hinde | The comparative study of species-specific behavior[END_REF]. Conversely, they were commonly analysed in psychological or sociological studies where the analysis of non-stereotyped sequences is a frequent and important matter [START_REF] Abbott | Sequence analysis: new methods for old ideas[END_REF][START_REF] Abbott | Sequence analysis and optimal matching methods in sociology. Review and prospect[END_REF][START_REF] Schlich | Analysing intrapersonal variability of travel behaviour using the sequence alignment method[END_REF][START_REF] Elzinga | Sequence similarity. a non-aligning technique[END_REF][START_REF] Van Der Aalst | Workflow mining: a survey of issues and approaches[END_REF][START_REF] Hay | Mining navigation patterns using a sequence alignment method[END_REF].

The successive event-pairing methodology and the present case study show that non-stereotyped behaviours are informative from a phylogenetic and evolutionary point of view. This brings many characters potentially useful to study phylogenetic relationships, the adequacy of which can be established with a simple but careful preliminary statistical treatment. Once the phylogenetic tree is reconstructed, the occurrence of each kind of transition among two acts can be mapped on the tree to understand where and how it evolved, allowing for the more detailed elucidation of behavioural evolution.

ACKNOWLEDGEMENTS

Figure 2 .

 2 Figure 2. Accumulation curve of the number of different transitions according to the number of observation sessions for the species Eublaberus distanti.

  c 2 = 380.3, ddl = 56, c 2 0.05 = 74.5, P << 0.05; Lanxoblatta: c 2 = 297.3, ddl = 81, c 2 0.05 = 103.0, P << 0.05; Phortioeca: c 2 = 222.1, ddl =

Figure 3 .

 3 Figure 3. Analysis A, strict consensus of the four most parsimonious trees obtained (L = 15, CI = 0.80, RI = 0.50) with the data set based only on the behavioural repertoires. Analyses B-E, most parsimonious trees obtained with the four different data sets (B, presence-absence of behavioural transitions, L = 217, CI = 0.86, RI = 0.58; C, presence-absence and frequencies of behavioural transitions, L = 300, CI = 0.87, RI = 0.55; D, morphological and molecular data set, L = 351, CI = 0.88, RI = 0.50; E, molecular, morphological and behavioural data -including frequencies, L = 659, CI = 0.86, RI = 0.48). Numbers above and below branches are branch lengths (under fast optimization) and bootstrap/Bremer values, respectively.

  This study has been developed in the course of a PhD granted by Ministère de la Recherche to Frédéric Legendre. Molecular work was partly made in the laboratory of Michael Whiting in Brigham Young University during a stay in Provo (Utah) supported by the programme pluriformation 'Etat et structure phylogénétique de la biodiversité actuelle et fossile' directed by Philippe Janvier and by NSF DEB-0120718 (MFW). Part of the behavioural observations has been carried out in Station biologique de Paimpont (UMR 6552 CNRS, Université de Rennes I) with facilities provided by Pierre Deleporte. The methodology has been presented in a talk at the Hennig XXV meeting in Oaxaca, Mexico owing to a grant of Ecole Doctorale 392 'Diversité du Vivant'. All the people having made possible this work in these different ways are warmly thanked. We are also indebted to Eric Guilbert, Cyrille D'Haese and two anonymous referees for their comments on previous versions of this manuscript.

Table 1 .

 1 PCR primers names and sequences with the seven targeted portions of DNA

	Genes	Primers	Sequences (5′-3′)	Sources
	16S	16SAr	CGCCTGTTTATCAAAAACAT	Xiong & Kocher (1991)
		16SF	TTACGCTGTTATCCCTAA	Kambhampati (1995)
	18S GA	1F	TACCTGGTTGATCCTGCCAGTAG	Giribet et al. (1996)
		b5.0	TAACCGCAACAACTTTAAT	Whiting et al. (1997)
	18S AD1D2	2F	AGGGTTCGATTCCGGAGAGGGAGC	Hillis & Dixon (1991)
		b2.9	TATCTGATCGCCTTCGAACCTCT	Jarvis, Haas & Whiting (2004)
	18S BCE	a1.0	GGTGAAATTCTTGGACCGTC	Whiting et al. (1997)
		7R	GCATCACAGACCTGTTATTGC	Whiting (2002)
	18S EF	a3.5	TTGTGCATGGCCGYTCTTAGT	Whiting (2002)
		9R	GATCCTTCCGCAGGTTCACCTAC	Giribet et al. (1996)
	28S A	Rd1.2a	CCCSSGTAATTTAAGCATATTA	Whiting (2002)
		Rd3b	CCYTGAACGGTTTCACGTACT	Jarvis et al. (2004)
	28S C	28SA	GACCCGTCTTGAAGCACG	Whiting et al. (1997)
		28SB	TCGGAAGGAACCAGCTAC	Whiting et al. (1997)

Table 2 .

 2 List of the different behaviours displayed by the cockroaches (also established according to[START_REF] Gautier | Processus de différenciation de l'organisation sociale chez quelques espèces de blattes du genre Blaberus: aspects écologiques et éthologiques[END_REF][START_REF] Grandcolas | Les Blattes de la forêt tropical de Guyane Française: structure du peuplement et étude écoéthologique des Zetoborinae[END_REF][START_REF] Van Baaren | Measurement for solitariness and gregarism: analysing spacing, attraction and interactions in four species of Zetoborinae (Blattaria)[END_REF] van Baaren et al., , 2003a, b) , b)

	Behaviours promoting	MT	Moving towards the other individual
	interactions	AC	Antennal contact with the body of the other individual
		MA	Mutual antennation
		CB	Climbing onto the body of the other individual with one to six legs
	Behaviours favouring	PS	An individual puts its pronotum under the other and stands up suddenly
	departure or a break	KL	An individual kicks the other with one leg
	in the interactions	PP	An individual pushes the other with its pronotum
		SP	Stilt posture. An individual rises on its legs
		SA	Stilt posture combined with antennal movement
		SD	Rapidly after a stilt posture, an individual goes down
		BI	An individual bites the other
		SJ	An individual jumps suddenly towards the other
		GD	An individual goes down
		GA	An individual goes down and hides its antennae
		RO	Rotation: turning away from the other individual (without significant
			displacement of the centre of gravity of the body)
		WD	Withdrawal. An individual moves away from the other
		WP	An individual moves away from the other but stops in proximity
		ES	Escape. An individual moves quickly away from the other
		WA	Sudden withdrawal of the antenna(e)
		TP	Tilt posture. An individual gives way of legs on its stimulated side
		FP	Freezing posture. An individual does not move at all
	Behaviours without	GA	Grooming behaviour of the antenna
	particular significance	GL	Grooming behaviour of the leg
		NO	Nothing. The individual does nothing notable
	Table

Table 5

 5

included the partitioned Bremer support values and the number of informative characters for each partition. It revealed that behaviour (including frequencies) brought more than 80% of the signal [(32 + 2)*100/42 = 80.95]. Even when the support of each partition was normalized by the number of informative characters, behavioural data appeared the most informative together with morphological data.

Table 4 .

 4 Phylogenetic analyses with their data sets, results and statistics

							Number of	% of
			Number				informative	informative
	Analyses	data sets	of MPT	L (steps)	CI	RI	characters	characters
	A	Repertoire	4	15	0.80	0.50	6	25.0
	B	Behaviour	1	217	0.86	0.58	74	12.8
	C	Behaviour including frequencies	1	300	0.87	0.55	89	7.7
	D	Molecular and morphology	1	351	0.88	0.50	88	2.9
	E	Behaviour, molecular and morphology	1	659	0.86	0.48	177	4.3
	CI, consistency index; L, length of the MPT; MPT, most parsimonious trees; RI, retention index.

Table 5 .

 5 Partitioned Bremer values

					Number of
		Node	Node		informative
	Partitions	(L, P)	[T, (L, P)]	S Bremer	characters	%	%
	Behaviour	1	31	32	74	1.03	0.91
	Behaviour (frequencies only)	1	1	2	15	0.32
	Morphology	6	1	7	9	1.85	0.22
	16S	1	-5	-4	4 6	-0.21
	18S	4	-2	2	15	0.32
	28S	5	-2	3	18	0.40
	TOT	18	24	42	177

'S Bremer' is the total Bremer support for each partition and '%' is the percentage of Bremer values that each partition supports normalized by the number of parsimony informative characters. Then, for each partition i, %i = [(S Bremer i/S Bremer TOT*100)/Ni]; with Ni the number of informative characters for the partition i. For the last column, partitions are: behaviour including frequencies and morphology + molecular

SUPPLEMENTARY MATERIAL

The following material is available for this article online: Appendix S1. Transitions matrices of the five cockroaches.


Jean-Marc Rouaboul

Christian Mougin

Patrick Ravanel

Michel Tissut

Georges Mrlina

Jean-Pierre Calmon

EFFECTS OF N,N'-BIS-(4-TRIFLUOROMETHYLPHENYL)-UREA ON ISOLATED PLANT MITOCHONDRIA AND THYLAKOID MEMBRANES

Keywords: Solanum tuberosum, Solanaceae, Spinacia oleracea, Chenopodiaceae, phenylurea, plant mitochondria, thylakoids, uncoupling activity

The N,N'-bis-(4-triftuoromethylphenyl)-urea is demonstrated to be a powerful uncoupler of ATP formation in plant mitochondria and thylakoid membranes. In mitochondria, the full uncoupling effect was obtained at a concentration of 3 µM uncoupling agent in the medium, which corresponded to an inner mitochondrial concentration of 7.2 nmol uncoupling agent mg -1 protein. The full uncoupling concentration was displaced from 3 to 0.6 µM when bovine serum albumin was omitted from the reaction medium. Up to 10 µM, the studied compound acted as a strictly selective uncoupler. At concentrations higher than 10 µM, a selective inhibition of electron transfer occurred at the level of the external NADH dehydrogenase of the inner mitochondrial membrane; the I50 value was 25 µM. In thylakoid membranes, the full uncoupling effect was obtained for a concentration of 0.6 µM uncoupling agent in the medium, which corresponded to a concentration of 25 nmol uncoupling agent mg -1 chlorophyll. The addition of bovine serum albumin (0.1%) to the reaction medium reversed this effect. For concentrations greater than 2 µM, inhibition of PSII electron transfer was observed. Full inhibition was obtained at 15 µM uncoupling agent and corresponded to 650 nmol uncoupling agent mg -1 chlorophyll. In marked contrast with classical uncouplers such as CCCP (m-chlorophenylhydrazone) or substituted phenols, the N,N'-bis-(4-triftuoromethylphenyl)-urea cannot be classified among the protonophoric class of uncouplers, due to its inability to exchange protons at pHs between 2 and 9. Its uncoupling action must therefore be attributed either to conformational changes of a transmembrane protein present both in mitochondria and in thylakoid membranes, or to a modification of the arrangement of the phospholipid membrane bilayers.

Introduction

Many substituted phenylureas are well known as PSII inhibitors, acting efficiently on light-dependent electron transfer in chloroplasts [START_REF] Fedtke | Biochemistry and Physiology of Herbicide Action[END_REF]. In some technical preparations of diuron [N- (3,4-dichlorophenyl)-N'-dimethyl-urea] an impurity is present which acts as a potent uncoupler [START_REF] Blein | [END_REF]. Its structure was established as being that of N,N'-bis- (3,4-dichlorophenyl)-urea. The physiological interest of this molecule led us to synthesize many derivatives of the same chemical family, including N,N'-bis-(4trifluoromethylphenyl)-urea which was found to be a very toxic molecule towards isolated plant mitochondria and thylakoid membranes.

Results

Synthesis and physical constants of N,N'-bis-(4-trijluoromethylphenyl)-urea

Aniline is known to form symmetrical phenylureas in combination with phenylisocyanate [START_REF] Blein | [END_REF][3][4]. Therefore, N,N'-bis-(4-triftuoromethylphenyl)-urea (USpCF3) was synthesized by using an aniline derivative with the appropriate substitution pattern and a phenylisocyanate with the same substitution pattern on the phenyl ring (see Experimental). The USpCF3 was crystallized several times and its structure carefully characterized: UV EtOH max (log ): 264 (4.7); 1 H NMR (200 MHz, DMSO-d6, TMS); 7.63 (8H, dd, H-2 and H-3), 9.2 (2H, s, N-H); 13 C NMR (50MHz, DMSO-d6); 152.2 (s, C=O), 143.2 (s, C-1), 122.5 (q, CF3), 127.2 (s, C-4), 118.2 (s, C-2), 126.0 (d, C-3).

The partition coefficient (P) of USpCF3 was determined by a conventional octanol-water measurement (-log P=3.8), and its solubility in distilled water was found to be close to 5 µM. However, the presence of bovine serum albumin (BSA) in the mitochondrial suspension medium greatly increased the solubility of USpCF3, showing that urea is able to bind to this protein. The solubility increase is also observed when BSA is replaced by mitochondria or chloroplasts.

Uncoupling activity of USpCF3

Mitochondrial oxygen consumption for electron transfer from succinate to O2, in the presence of ADP, in a medium containing 0.1% BSA (state III = 262 nmol O2 min -1 mg -1 mitochondrial protein) is shown in Fig. 1A. When all the ADP had been utilized, the oxygen consumption (state IV) fell to 37 nmol O2 min -1 mg -1 protein and the addition of USpCF3 produced a sharp increase in the rate of electron transfer. Both CCCP (5 µM) and ADP (200 µM), if added at this stage, had no effect. These findings demonstrate that the compound under study, at 3 µM, is able to fully uncouple electron transfer. The same uncoupling effect was observed when succinate was replaced by -ketoglutarate, exogenous NADH or pyruvate. The spectrophotometric measurement of mitochondrial swelling [5], confirms the existence of this uncoupling activity (Fig. 2). A rapid passive swelling in NH4CI or NH4NO3 isoosmotic medium was obtained upon addition of USpCF3, indicating the entry of H + into the mitochondrial matrix space. The lowest concentration which causes complete uncoupling is 3µM.

Half (D50) and full (D100) uncoupling effects were obtained for concentrations in the medium of 5.9 nmol USpCF3 mg -1 protein and 13.6 nmol USpCF3 mg -1 protein, respectively. When the mitochondrial protein concentration in the medium changed from 0.2 to 1.2 mg protein ml -1 , the concentrations in the medium which induced D50, (as expressed by the total amount of USpCF3 introduced in the reaction vessel), remained at a level of 5.8 ± 0.4 nmol mg -1 protein.

Cautiously broken class A chloroplasts gave rise to true class C chloroplasts which were unable to perform the Mehler reaction. After addition of methylviologen, they carried out the light-dependent transfer of electrons from water to methylviologen, the re-oxidation of which led to the formation of hydrogen peroxide. This reaction led to an oxygen consumption which was greatly increased by the addition of 0.6 µM USpCF3 (Fig. 3A). This increase remained light-dependent, and a further addition of NH4CI (5 mM), used as reference uncoupler, did not change the rate of oxygen consumption. USpCF3, therefore, totally uncouples the photophosphorylation system. Under our conditions (23 µg chlorophyll), this full uncoupling effect corresponds to a concentration in USpCF3 of 26 nmol mg -1 chlorophyll in the medium. D50 was obtained with 13 nmol USpCF3 mg -1 chlorophyll in the medium. The same result was obtained when PSI operates alone (ascorbate+DPIP as electron and proton donor; methylviologen, as electron acceptor; in the presence of a PSII inhibitor). This uncoupling effect probably explains the full inhibition of O2 evolution in class A chloroplasts which was obtained at 0.6 µM USpCF3 (Fig. 3C), the inhibitory effect of USpCF3 on the photodependent electron transfer being unable to explain such an inhibition of O2 evolution (Fig. 3B, see below).

In order to see if the uncoupling effect of USpCF3 on phosphorylation processes can be explained by the existence of H + transmembrane transport (as is the case of the classical protonophoric uncouplers such as CCCP [6], pentachlorophenol [7] or platanetin [8]), the ability of USpCF3 to change from a neutral to an ionized form was studied. In order to do this, UV spectral shifts dependent on pH changes (between 9 to 2) were looked for. No significant spectral variation of the product was observed when changing the pH of the medium (Fig. 4A). This indicates that USpCF3 remains in a stable neutral form, whatever the pH. The same result was obtained by the classical method of pKa measurement (Fig. 4B). Under the same experimental conditions, the reference protonophore uncoupler CCCP presented a pKa value of 5.3 with both methods (Fig. 4).

The uncoupling effect of USpCF3 in mitochondria was greatly increased when BSA was omitted from reaction medium. Under this condition, D100 decreased from 3 to 0.6 µM (Fig. 1B). A further addition of 0.1% BSA at this stage re-established oxidative phosphorylation. This indicates the reversibility of the uncoupling activity induced by USpCF3 (Fig. lC). Similar results were also obtained with class A chloroplasts and thylakoïd membranes (results not shown).

The high UV specific absorbance of USpCF3 [EtOH max (log ):): 264 (4.7)] allowed us to measure its partition rate between the medium and the mitochondria when the mitochondrial concentration increased (Fig. 5A). In a reaction medium devoid of BSA, USpCF3 at 5 µM attained its solubility limit. In the presence of BSA and mitochondria, the solubility of USpCF3 can easily reach 20 µM and an equilibrium was established between the medium and the mitochondria, dependent on the concentration of the mitochondrial suspension. For an amount of mitochondrial protein between 1 and 1.4 mg ml -l , 0.4 µM USpCF3 remains in the medium containing BSA 0.1%.

When the concentration of USpCF3 in the medium increased from 1 to 20 µM, the percentage of product bound to a quantity of mitochondria corresponding to 0.22 mg protein ml -l remained almost constant and close to 53% of the added product (Fig. 5B) suggesting that, in this range of concentration, no saturation takes place inside the mitochondria. Under these conditions, it is possible to calculate the amount of product bound to mitochondria, in order to obtain either a 50 or a 100% uncoupling effect. These values are close to 3.1 (= 5.9 x 0.53) nmol USpCF3 mg -1 protein for D50 and 7.2 (= 13.6 x 0.53) nmol USpCF3 mg -1 protein for D100.

Inhibitory activity of USpCF3

Mitochondrial electron transfer from the respiratory substrate to O2 was studied in an uncoupled state (presence of 5 µM CCCP), using either malate, succinate or exogenous NADH as substrates. If an inhibitor acts on the common part of the electron pathway, (between the quinone pool and O2), it lowers the rate of oxygen consumption in the same manner, whatever the substrate oxidized. In contrast, when an inhibitor acts preferentially on one of the flavoprotein dehydrogenases (before the quinone pool), the decrease in oxygen consumption differs from one substrate to another. The I50 values obtained were 100 and 130 µM for malate oxidized at pH 6.5 and 7.5 respectively, 50 µM for succinate and 25 µM for exogenous NADH (with 0.28 mg protein m -l in the medium). The oxidation of this latter substrate was fully inhibited at a concentration of 200 µM USpCF3, which corresponds to 715 nmol USpCF3 mg -1 protein. All these results show that USpCF3 is a moderate inhibitor of electron transfer in mitochondria. The greatest effect was shown when exogenous NADH is the substrate, suggesting a semi-selective effect on the external NADH dehydrogenase of the inner membrane. Under these conditions, I50 was obtained for a concentration in the medium of 89.3 nmol USpCF3 mg -1 protein. This value is 15fold higher than the value obtained for D50.

In thylakoid membranes, USpCF3, at concentrations greater than 5 µM, inhibits electron transfer through PSII. The I50 value was obtained at 6 µM (Fig. 3B) which corresponds to a concentration of 260 nmol USpCF3 mg -1 chlorophyll in the medium. This value is 25fold higher than D50. 100% inhibition is obtained at 650 nmol USpCF3 mg -1 chlorophyll (=65 nmol USpCF3 mg -1 thylakoidal protein).

Discussion

Our results show that N,N'-bis-(4-trifluoromethyl-phenyl)-urea (USpCF3) is one of the most powerful uncouplers of ATP formation known at the present time, acting both on mitochondria and on thylakoid membranes. The result of this action is a proton transfer through the biological membranes, leading to a collapse of the proton gradient. This gradient is required for the biosynthesis of chloroplastic and mitochondrial ATP. The efficiency of USpCF3 is comparable to the protonophore reference uncoupler CCCP [6] and better than pentachlorophenol [7]. The effectiveness of USpCF3 can be compared to that of platanetin, a natural lipophilic flavonoid extracted from plane-tree buds [8]. To obtain a full uncoupling effect, the amount of product associated with mitochondria must reach 7.2 nmol mg -1 protein. The great lipophilicity of the molecule under study (-log P = 3.8) suggests that the major part of USpCF3 linked to the mitochondria must be associated with the lipophilic components of the membrane. We can roughly estimate that the internal concentration of the product inside the mitochondrial membrane should be higher than 5 mM for full uncoupling to take place. In fact, if we consider that the lipidic part of the mitochondria represents approximately 30% of the dry weight [START_REF] Douce | Mitochondria in Higher Plants. Structure, Function and Biogenesis[END_REF], and that all the bound USpCF3 is associated with this lipophilic fraction, the USpCF3 lipidic concentration can be estimated to be close to 6 nmol mg -1 lipid. This represents 5.4 mM if we consider the lipid density as being close to 0.9. All the other well-known uncouplers, which are all very lipophilic compounds, probably also accumulate in great concentrations inside the biological membranes. As a consequence, it seems highly unlikely that the uncoupling effect can be obtained through high affinity binding to membrane components [START_REF] Bakker | [END_REF]11]. In comparison, it should be emphasized that the specific binding of PSII inhibitors (such as phenmedipham) to the thylakoidal D1 protein is close to 2 nmol mg -1 chlorophyll, which corresponds to 0.2 nmol mg -1 protein [12].

One particularly interesting point which emerged from this study is the role played by BSA on the uncoupling effects of the studied compound. It seems likely that it binds to BSA, maybe through hydrogen bonds between the-NH-and-SH or the S-S sites of the protein [13,14].

During the course of our experiments, the presence of small molecules coming from the altered mitochondria (even after a Percoll gradient purification) decrease the coupling rate (state III-state IV ratio) by an increase in the H + membrane porosity. In the reaction medium, BSA traps these small molecules. The role played by BSA in our experiments has therefore to be attributed, in part, to this binding of small molecules, and, in addition, to the binding of the studied product, as previously shown with other lipophilic compounds [15,16]. Thus, BSA greatly decreases the concentration of USpCF3 inside the mem-brane of the organelles. It is suggested that this latter effect is associated with the presence of a -NH-group of the urea, as it is associated with the presence of a hydroxyl group in the case of phenols [6].

The most noteworthy characteristic of USpCF3 is that this powerful uncoupler remains non-ionizable at each of the pH values studied. That means that this compound is unable to transport H + through biological membranes, and that it cannot act in the same way as CCCP [17], phenolic compounds [6] or platanetin [8]. Its mode of uncoupling action may be to bring about either a con-formational change in the transmembrane proteins, leading to H+ permeability [18,19] or a change in the phospholipidic bilayer organization and/or activity [START_REF] Blein | [END_REF]. This latter point is presently under study using artificial bilayer vesicles.

Experimental

Synthesis N,N'-Bis-(4-trifluoromethylphenyl)-urea(USpCF3) was obtained by reaction of 4trifluoromethylphenylisocyanate (0.024 M; 4.48 g) with 4-trifluoromethyl-aniline(0.024 M; 3.86 g), in dry Et2O, at room temp., for 6 hr, in the presence of triethylamine. White crystals of USpCF3 were filtered under vacuum. After recrystallization from EtOAc, 3.13 g USpCF3 were obtained.

Control of purity

TLC (CHCl3-EtOAc, 7:3) and reversed phase HPTLC (MeOH-H2O, 17:3) respectively on commercial silica gel plates F254 and RP18F254 (Merck) were used to check the purity of the USpCF3.

pKa measurements

Due to the low water solubility of USpCF3, an ethanolic soln (EtOH-H2O, 3:1) was used for pKa determination experiments. The pKas were firstly determined by spectrophotometric methods. The UV spectrophotometric shifts of bands II and I were analysed to determine the pH value for which the equilibrium between the ionized and the neutral form was 50%. The pKas were also determined using a titrimetric method. Solns (50 ml) containing l mM of the product were automatically titrated with 0.01 M HCI. The pKa corresponds to the half-neutralization value of the studied product. In this ethanolic soln, CCCP was not completely ionized, so 0.5 ml NaOH (0.1 M) was added to the soln.

Log P measurement.

Experimental values were obtained as previously described [21].

Fig. 1 .

 1 Fig. 1. Polarographic traces showing the full uncoupling activity of USpCF3 in potato tuber mitochondria. (A) In the presence of BSA (0.1%) in the reaction medium; (B) in the absence of BSA; (C) reversibility of the uncoupling effect. M: Mitochondria (220 µg protein m1 -1 medium), S: succinate 6 mM +ATP 0.3 mM; ADP: 200 µM; KCN: 30 µM; CCCP: 5 µM. Numbers on traces refer to nmol O2 consumed min-1 mg -1 protein.

Fig. 2 .Fig. 3 .

 23 Fig. 2. Swelling of isolated mitochondria in the presence of USpCF3 in NH4Cl or NH4NO3 isoosmotic medium. The decrease in absorbance (A) of the mitochondrial suspension (0.24 mg protein ml -1 medium; BSA 0.1 %) induced by USpCF3 was measured at 540 nm, CCCP was used as a reference uncoupler. U: uncoupler.

Fig. 4 .

 4 Fig. 4. pK determination for USpCF3 and CCCP. (A) By spectral shift measurements. Spectral changes were measured at 264 nm for USpCF3 (EtOH max for the molecular form) and at 380 nm for CCCP (EtOH max for the ionized form); (B) by titrimetry. Curves were obtained by addition of HCl (0.01 M) in CCCP and USpCF3 soln (1 mM, in EtOH-H2O, 3:1). Reference trace without product: dotted lines.

Fig. 5 .

 5 Fig. 5. (A) Partition of USpCF3 between mitochondria and medium. (A) When mitochondrial concentration increases in 1 ml medium containing 5 nmol USpCF3 and (B) when USpCF3 concentration increases in 1 ml medium containing 220 µg mitochondrial protein. All experiments were performed in the presence of BSA 0.1%.

Acknowledgement

The authors thank Dr Mourad Kaouadji for his spectroscopic analysis of USpCF3.

Preparation of chloroplasts

Spinach leaves (Spinacia oleracea L. var. Monstrueux de Viroflay) were homogenized in ice-cold extraction medium (0.3 M mannitol, 25 mM sodium pyrophosphate, EDTA 2 mM, BSA 1g 1 -1 , pH 7.9). The homogenate was filtered and intact class A chloroplasts were prepared by centrifugation [22] and•further purified on a Percoll density gradient.

Class C chloroplasts (thylakoids) were prepared, in dist. H2O, by gentle osmotic shock of intact chloroplasts, just prior to the experiments.

Preparation of mitochondria.

Mitochondria from potato tubers (Solanum tuberosum L.) was prepared by methods previously described [23].

O2 exchange measurements

O2 exchange was followed polarographically at 25°, using a Clark-type electrode system.

For plant mitochondria, the reaction medium contained 0.3 M mannitol, 5 mM MgCl2, 10 mM KCl, 10 mM Pi buffer, and in some cases, 0.1% BSA. All incubations were carried out at pH 7.2. For measuring photosynthetic O2 exchange, the reaction medium contained 0.3 mannitol, 50 mM MOPS (morpholinopropane sulphonic acid), 2 mM EDTA, pH 7.6.

The release of O2 by class A chloroplasts was measured after the addition of 0.5 mM NaHCO3 and 0.1 mM Na2HPO4. In thylakoid membranes (23 µg chlorophyll ml -1 medium), electron transfer was measured in the presence of the electron acceptor methylviologen (paraquat, 0.1 mM). NH4Cl (5 mM) was used as a reference uncoupler. KCN (0.1 mM) was added to inhibit any possible catalase activity.

Uncoupling test for thylakoids.

Measurements of the uncoupling effect were made on light-driven electron flow through PSII +PSI. The uncoupling effect gave rise to an increase in the O2 consumption rate. A 100% uncoupling effect was obtained when the addition of NH4Cl (5 mM) did not induce further increases O2 consumption.

Uncoupling test for mitochondria.

A suspension of intact mitochondria was energized. After a state III-state IV transition, the uncoupling effect of the product added at this stage corresponds to an increase in the oxidation rate. A 100% uncoupling effect was obtained when the rate of O2 was not further stimulated by the addition of CCCP (5 µM) or ADP (200 µM). Mitochondrial swelling. Mitochondrial swelling was measured, as previously described [5], by the apparent absorbance decrease at 540 nm. The reaction medium contained 100 mM NH4NO3 or NH4Cl, 15 mM Tris-HCI, 0.1% BSA, pH 7.2. A rapid passive swelling was induced by uncouplers.

Protein and chlorophyll measurements.

Protein contents were determined according to ref. [24] and chlorophyll concentrations were measured as described in ref. [25].