S Nys

S Vijgen

K Magerman

R Cartuyvels

Comparison of Copan eSwab with the Copan Venturi Transystem for the quantitative survival of Escherichia coli, Streptococcus agalactiae and Candida albicans

Comparison of Copan eSwab with the Copan Venturi

Transystem for the quantitative survival of , and

Introduction

Swab transport systems should preserve the viability and stability of micro-organisms in clinical specimens through-out the transport and storage process. Most swab collection devices currently on the market are prepared with rayon, Dacron or cotton fibres wound onto the tip of the swab shaft. The flocked swab (Copan Diagnostics, Italy) is a nylon-tipped swab prepared by a spray-on flocked fibre technique, developed for the transport of bacteria, yeast and viruses [START_REF] Van Horn | Comparison of 3 swab transport systems for direct release and recovery of aerobic and anaerobic bacteria[END_REF][START_REF] Van Horn | Comparison of the Copan ESwab system with two Amies agar swab transport systems for maintenance of microorganism viability[END_REF]. This technique provides stronger capillary action and strong hydraulic uptake of liquids, which should result in better specimen collection. This design should also provide more efficient uptake and release of specimen material and, therefore, less entrapment of specimens. The new transport device, eSwab, is the combination of the new nylon-flocked swab with the modified liquid Amies transport medium. Up until now, only two studies describe the comparison of this new flocked transport system with the current systems available for the recovery and maintenance of aerobic and anaerobic bacteria [START_REF] Van Horn | Comparison of 3 swab transport systems for direct release and recovery of aerobic and anaerobic bacteria[END_REF][START_REF] Van Horn | Comparison of the Copan ESwab system with two Amies agar swab transport systems for maintenance of microorganism viability[END_REF]. Both studies describe the performance of eSwab for the strains described in the CLSI document M40-A [START_REF]Quality control of microbiological transport systems; Approved Standard[END_REF], i.e. fastidious organisms and strains difficult to culture. These data are also indicated on the package insert provided by Copan. On easy-growing aerobic bacteria or yeasts however, no information is available yet. Therefore, in this study, eSwab was compared with an Amies agar swab system for the maintenance of viability and the quantitative survival of Escherichia coli, Streptococcus agalactiae and Candida albicans in vitro.

Materials and methods

Strains

The three strains tested were E. coli ATCC 25922, S. agalactiae ATCC 13813 and C. albicans ATCC 90028.

Quantitative elution

The quantitative elution method, as described in the CLSI document M40-A [START_REF]Quality control of microbiological transport systems; Approved Standard[END_REF], was used to evaluate the performance of eSwab (480CE, Copan Diagnostics, Italy) and the Copan Venturi Transystem (CVT, 108C.USE, Copan Diagnostics, Italy). For each microorganism, inoculated swabs were preserved at room temperature (RT) and 4°C for 0 (15 min after inoculation), 6, 24 and 48 h. Also, three mixtures of E. coli and S. agalactiae (900/100 µL, 500/500 µL and 100/900 µL) were tested under the same conditions.

In each experiment, strains were freshly sub-cultured twice onto Columbia agar plates supplemented with 5% sheep blood (COL5%, 221165, BD Diagnostics, US) and incubated in 5% CO 2 at 35°C. In the single-strain experiments, an initial organism suspension of 0.5 McFarland standard (approximately 10 8 cfu/mL) in sterile saline (0.85% NaCl w/v) was prepared and 10-fold-diluted to obtain a suspension of 10 7 cfu/mL (also used as the positive control suspension). For each time point (n=4) and each temperature (n=2), one swab was inoculated with 100 µL of the 10 7 -cfu/mL suspension for the three organisms tested and allowed to absorb the fluid completely (∼30 s). Subsequently, all swabs (24 CVT and 24 eSwab) were closed in their respective tube, i.e. eSwab or CVT, and stored at the proper conditions. This experiment was performed in triplicate. The swabs for time point zero (T= 0) were processed 15 min after inoculation.

The CVT swabs were mixed well into 1 mL of sterile saline in order to obtain ∼10 6 cfu/mL, as already present in the eSwab liquid medium. Subsequently, the CVT suspension and eSwab medium were serially 10-fold-diluted. A 100-µL aliquot of each dilution was inoculated to duplicate COL5% and spread over the entire surface using a sterile L-spatula (174CS01, Copan Diagnostics, Italy). Plates were incubated for 18-24 h in 5% CO 2 at 35°C. Colony counts were obtained by reading plates with 30 to 300 countable colonies. A series of 10-fold dilutions of the positive control suspension was used as growth controls.

In the mixed experiments of both E. coli and S. agalactiae, an initial organism suspension of 0.5 McFarland (approximately 10 8 cfu/mL) in sterile saline was prepared and 10-fold-diluted to obtain a suspension of 10 7 cfu/mL. Of both 10 7 -cfu/mL suspensions, a mixture was made with ratios of 900/100 µL, 500/500 µL and 100/ 900 µL E. coli/S. agalactiae, respectively. These mixed suspensions were also used as positive growth controls. Swabs were further inoculated and processed with the mixtures as described above for the single-organism suspensions. This experiment was performed in triplicate. After preservation for 0, 6, 24 or 48 h at RT or 4°C, a 100-µL aliquot of the diluted sample was inoculated onto duplicate MacConkey (221270, BD Diagnostics, US) and CNA agar (221353, BD Diagnostics, US) for the enumeration of E. coli and S. agalactiae, respectively. MacConkey plates were incubated for 18-24 h at 35°C in ambient air, whereas CNA plates where incubated in 5% CO 2 at 35°C. Colony counts were obtained by reading plates with 30 to 300 countable colonies. A series of 10-fold dilutions of the positive control suspension was used as growth controls.

The log 10 of the average cfu recovered was determined for the growth controls (100%) and for each swab type, temperature and time combination, and based upon a cfu comparison with the growth controls expressed in % recovery. According to the CLSI guideline M40-A [START_REF]Quality control of microbiological transport systems; Approved Standard[END_REF], for samples stored at RT, no more than a 3 log 10 decline in cfu is accepted. At RT, for overgrowth, no limit for acceptance is defined. For samples stored at 4°C, no more than a 1 log 10 increase in cfu and 3 log 10 decline in cfu is acceptable.

Results

The recovery of the three micro-organisms separately was higher in eSwab (97-100%) as compared to CVT (86-96%) at RT for time point 0 h and remained at the same level after 6 h of preservation (Table 1). Preservation beyond 6 h resulted for both E. coli and C. albicans in proliferation of the micro-organism in both transport systems. S. agalactiae recovery, on the other hand, remained stable throughout the experiment at RT. The preservation of eSwab and CVT at 4°C prevented the proliferation of E. coli and C. albicans (Table 2). The recovery after preservation with eSwab was higher (>94%) as compared to CVT (77-94%) throughout the whole experiment (4°C) for the three micro-organisms tested. Both transport systems, however, did stay within the 3 log 10 decrease limit defined by the CLSI.

For the mixed culture of E. coli and S. agalactiae, similar results were found as compared to the recovery of the microorganisms separately (Tables 3 and4). At RT, eSwab showed a higher recovery for both micro-organisms after 0 and 6 h preservation as compared to CVT. Preservation beyond 6 h resulted for E. coli in proliferation for both transport systems. For S. agalactiae, a decline in the percentage cfu recovered was found for both transport systems, with eSwab still showing the highest recovery rate. The different concentrations of micro-organisms mixed together did not seem to influence the processes occurring at RT in both transport systems. At 4°C, the recovery of E. coli in eSwab ranged between 89-103%, depending on the micro-organism concentration in the experiment. S. agalactiae recovery in eSwab fluctuated between 84-99%, independently of its concentration. CVT recovery rates were lower throughout the whole experiment for both micro-organisms tested.

Discussion

Specimen collection and transportation is a cornerstone of good diagnostic performance in the Clinical Microbiology laboratory. Viability must be maintained, as well as the relative proportions of all micro-organisms present in the clinical specimen [START_REF] Van Horn | Evaluation and comparison of two Stuart's liquid swab transport systems tested by the CLSI M40 method[END_REF]. Therefore, in this study, we evaluated the performance of the new flocked swab with liquefied Amies transport medium for the quantitative survival of E. coli, S. agalactiae and C. albicans in vitro. Furthermore, we analysed the in vitro survival of E. coli and S. agalactiae in mixed cultures with different proportions of each bacteria. To our knowledge, this is the first study evaluating the performance of eSwab for the detection and survival of easy-growing aerobic bacterial strains and mixed cultures in vitro.

In vitro, eSwab met the CLSI acceptance criteria at both storage temperatures for the three isolates tested. Although no significant differences were detected between both transport devices, the recovery rate was higher in eSwab as compared to the cotton swab for both the single-micro-organism experiments and the mixed cultures. At refrigerated temperature, the recovery rates were very stable over time for eSwab (recovery of 90% and higher). At RT on the other hand, both for E. coli and C. albicans, overgrowth could be detected from 6-24 h after inoculation of both transport systems. Van Horn et al. also observed heavy overgrowth of Pseudomonas aeruginosa after RT storage for eSwab and other transport devices tested [START_REF] Van Horn | Comparison of the Copan ESwab system with two Amies agar swab transport systems for maintenance of microorganism viability[END_REF]. Although the CLSI does not have criteria on the percentage overgrowth allowed [START_REF]Quality control of microbiological transport systems; Approved Standard[END_REF], this phenomenon could lead to the misinterpretation of mixed infections and misleading culture results in laboratory practice. As for other swabs, a prompt transport to the laboratory of eSwabs is, therefore, recommended.

One limitation of the in vitro study might be the use of the quantitative elution method as proposed by the CLSI, as this method does not reflect typical laboratory swab practices. However, the advantage of this method over the roll plate method (which resembles more closely routine laboratory practices) is that it provides quantitative data that can more easily detect performance characteristic differences between the swab transport systems [START_REF] Van Horn | Comparison of 3 swab transport systems for direct release and recovery of aerobic and anaerobic bacteria[END_REF][START_REF] Van Horn | Comparison of the Copan ESwab system with two Amies agar swab transport systems for maintenance of microorganism viability[END_REF].

One can conclude that eSwab did not only meet the criteria set by the CLSI for a transport device for microbiological culture, but as its recovery rate in vitro was higher than currently available systems, it might also enhance the sensitivity of bacterial culture in the future.

Table 2

 2

	Recovery of single
	organisms from CVT
	and eSwab at 4°C

Table 3

 3 Recovery of mixed organisms from CVT and eSwab at RT

	Mixture	Organism	Swab system	log 10 (cfu/ml, %) recovered
	(E. coli/S. agalactiae)	log 10 (cfu/mL) growth control		0h	6h	24h	48h
	900/100	E. coli, 8.2	CVT	7.6 (93)	7.0 (85)	9.1 (110)	10.8 (131)
			eSwab	7.9 (97)	8.6 (105)	11.3 (137)	11.0 (133)
		S. agalactiae, 7.1	CVT	5.9 (83)	5.7 (79)	5.2 (73)	5.0 (69)
			eSwab	7.3 (102)	7.3 (102)	7.3 (102)	5.4 (75)
	500/500	E. coli, 8.5	CVT	6.4 (75)	6.5 (77)	9.0 (106)	10.2 (120)
			eSwab	7.4 (87)	7.6 (90)	10.4 (123)	12.5 (147)
		S. agalactiae, 8.2	CVT	6.4 (78)	5.8 (71)	5.8 (71)	4.7 (58)
			eSwab	7.3 (89)	7.2 (88)	6.8 (83)	5.6 (68)
	100/900	E. coli, 8.3	CVT	6.7 (81)	7.2 (87)	10.1 (123)	11.1 (134)
			eSwab	7.4 (89)	8.2 (99)	10.6 (128)	10.9 (132)
		S. agalactiae, 9.2	CVT	6.6 (72)	6.3 (69)	6.3 (69)	5.8 (64)
			eSwab	8.3 (90)	8.2 (90)	7.1 (78)	6.1 (67)

Table 4

 4 Recovery of mixed organisms from CVT and eSwab at 4°C

	Mixture	Organism	Swab system	log 10 (cfu/ml, %) recovered
	(E. coli/S. agalactiae)	log 10 (cfu/mL) growth control		0h	6h	24h	48h

Conflict of interest statement All authors: no conflicts.

Financial support All authors: no financial support.


Thibaud Gruber

Laurent Nagle
email: laurent.nagle@u-paris10.fr

F T Bairlein

Gruber

Territorial reactions of male Yellowhammers (Emberiza citrinella) toward a specific song structure

Keywords: Emberiza citrinella, Song structure, Territory defence

Male songbirds assess their rivals by listening to their songs and, in some species, are especially sensitive to certain parts of the song. Yellowhammers (Emberiza citrinella) sing songs with different structures consisting of an initial phrase of stereotyped and repeated notes in a trill, and a terminal phrase, present or not, which consists of low or high frequency whistles that we called elements. We analyzed 1,055 songs from 25 different males and found that four different structures could be distinguished in a French Yellowhammer population. We found that the frequency ranges of the elements were more constant across individuals than the ranges of the notes, and among them, the low frequency element was nearly twice as conserved as the high frequency element. Then, in the wild, we tested the territorial responses of males to the four different structures and found that the full song structure, which is composed of the initial phrase followed by a high frequency element and a low frequency element, induced stronger territory defence responses from the males than any of the other structures. As only the structure containing the initial phrase and the low frequency element also induced a strong response, we propose that the main element responsible for the enhanced reaction of the males is the low frequency element, which is consistent with the fact that it is more conserved than the other constituents.

Introduction

Male passerine songs are believed to have two functions in sexual selection: territory defence and mate attraction [START_REF] Catchpole | Bird song: biological themes and variations[END_REF]. Their songs are more or less complex in their structure and composition, and repertoire sizes also vary according to the species [START_REF] Marler | Three models of song learning: evidence from behavior[END_REF][START_REF] Todt | Acquisition and performance of song repertoires: ways of coping with diversity and versatility[END_REF]. It has been suggested that, depending on the species, either the choice of song type [START_REF] Luschi | Song-type function during territorial encounters in male Cetti's warblers Cettia cetti[END_REF] or the song pattern [START_REF] Beecher | Song-type matching between neighbouring song sparrows[END_REF][START_REF] Rutkowska-Guz | Song structure and variation in yellowhammers Emberiza citrinella from western Poland[END_REF]) could have a specific function. It has also been shown that some elements of a song are directly responsible for a specific response from birds, notably in males [START_REF] Nagle | The influence of song mode on responses of male American redstarts[END_REF][START_REF] Rehsteiner | Singing and mating success in water pipits: one specific song element makes all the difference[END_REF]. Passerines may vary their song structure according to the intended audience: depending on the species, long, complex, and variable songs appear to be used for mate attraction, while short, repeated, and stereotyped songs seem mostly aimed at other males [START_REF] Catchpole | Sexual selection and the evolution of complex songs among European warblers of the genus Acrocephalus[END_REF][START_REF] Catchpole | Why do birds sing?[END_REF][START_REF] Krebs | The significance of song repertoires: the Beau Geste hypothesis[END_REF][START_REF] Slater | Chaffinch song repertoires-observations, experiments and a discussion of their significance[END_REF].

Yellowhammers (Emberiza citrinella) possess both structures in their songs: an initial phrase, composed of repeated syllables of one or two notes, and a terminal phrase, composed of one or two whistles (which we will here refer to as elements) which are not always present [START_REF] Hansen | Song variations in the yellowhammer, Emberiza citrinella[END_REF]. The different song structures produced by the males are also produced at different rates [START_REF] Hiett | Song repertoires and seasonal song in the yellowhammer, Emberiza citrinella[END_REF]. Finally, this species is characterized by a small repertoire of 1-4 song types [START_REF] Hansen | Neighbour-stranger song discrimination in territorial yellowhammer Emberiza citrinella males, and a comparison with responses to own and alien song dialects[END_REF][START_REF] Hiett | Song repertoires and seasonal song in the yellowhammer, Emberiza citrinella[END_REF][START_REF] Osiejuk | Repertoire and temporal organization of song bouts in the yellowhammer (Emberiza citrinella) from Western Poland[END_REF][START_REF] Rutkowska-Guz | Song structure and variation in yellowhammers Emberiza citrinella from western Poland[END_REF]). Given the different singing features found in this species, and following previous work by Rutkowska-Guz and [START_REF] Rutkowska-Guz | Song structure and variation in yellowhammers Emberiza citrinella from western Poland[END_REF], we presumed as a preliminary hypothesis that any meaningful signal would most likely be found in the structural variation of the song itself rather than in the song type choice.

The aim of this study was to assess the existence of song structure specificity in male Yellowhammers in a territory defence context. More precisely, we wanted to know if any of the structures that can naturally be found in Yellowhammer songs was more likely to induce a territorial answer from another male Yellowhammer. It has been shown that male birds extract information from vocal interaction to assess rivals [START_REF] Mennill | Do male black-capped chickadees eavesdrop on song contests? A multi-speaker playback experiment[END_REF][START_REF] Naguib | Nightingales respond more strongly to vocal leaders of simulated dyadic interactions[END_REF][START_REF] Peake | Male great tits eavesdrop on simulated male-to-male vocal interactions[END_REF]), so we expected the males to react differently to the different structures, for instance, if one particular structure presented a particular threat to them. The song structures in Yellowhammer differ the most in their terminal phrase, especially by the presence or not of a low frequency element [START_REF] Hansen | Song variations in the yellowhammer, Emberiza citrinella[END_REF]. Its presence allows us to discriminate easily between the song structures, making the song longer and more complicated, we hypothesized that it would constitute a key feature to take into account for the males.

Firstly, we analyzed the songs of a French Yellowhammer population to characterize their singing pattern, which differs among geographic areas [START_REF] Hiett | Song repertoires and seasonal song in the yellowhammer, Emberiza citrinella[END_REF]. Then, we conducted a playback experiment on the same population, during which we recorded several territorial reactions-i.e., latency and distance of approach to the loudspeaker and time spent in the vicinity of the loudspeaker [START_REF] Brooks | Individual recognition by song in whitethroated sparrows. I: discrimination of songs of neighbors and strangers[END_REF]-to the four different song structures that had been characterized with the previous analysis. Finally, we compared these territorial responses to the four structures in order to determine if a particular structure or a certain element it contained induced an enhanced territorial response.

Methods

Subjects and study area

The study was carried out in the municipality of Lassigny, Oise, Picardie, France (coordinates of the center of the study area, Lagny: 49°37 0 N, 2°55 0 E). Playback experiments were conducted on 19 male Yellowhammers, a species commonly found in this region along forest edges and in tree lines surrounded by farmland. Males were found in territories identified by one of the authors (L.N.). Each male was tested with the songs of a distant male whose territory was far enough from its own territory so that this bird would not be possibly heard, thereby avoiding any neighborhood effect. Moreover, the latter male was chosen so that his song would not overlap with the tested male's repertoire to provide a new stimulus. For all the subjects, songs were recorded before and after the playback, in order to make sure that the song used to test him was not part of his repertoire, as well as to check that the same male was tested on two consecutive days of testing, as these birds were not banded. Playbacks were conducted between 10 April and 5 May 2007, during the breeding period [START_REF] Peakall | Nest records of the yellowhammer[END_REF]. No significant difference in the Yellowhammers' responses, either vocally or behaviorally, was found between April and May, independently of whether the experiment was conducted in April or May.

Song structure and terminology

Yellowhammer songs have two distinct parts [START_REF] Hansen | Song variations in the yellowhammer, Emberiza citrinella[END_REF] which have been named initial phrase and terminal phrase [START_REF] Hiett | Song repertoires and seasonal song in the yellowhammer, Emberiza citrinella[END_REF]. The initial phrase is the repetition of one or two notes (which are called syllables). The variety of this initial segment is referred to by the song type [START_REF] Hansen | Song variations in the yellowhammer, Emberiza citrinella[END_REF]. The terminal phrase can contain either a high frequency element or a low frequency element or both. Following Hiett and Catchpole's terminology (1982) we named:

• No terminal phrase (NTP, Fig. 1a): the structure where only the initial phrase is present. • No high frequency element (NHE, Fig. 1b): the structure where the initial phrase is followed by a terminal phrase constituted only of the low frequency element. • No low frequency element (NLE, Fig. 1c): we created and named this new category for a structure that was found in the French studied population but was not present in [START_REF] Hiett | Song repertoires and seasonal song in the yellowhammer, Emberiza citrinella[END_REF]. We chose this name because it contains the initial phrase followed by a terminal phrase comprised only of the high frequency element. • Full song structure (FSS, Fig. 1d): the structure where the initial phrase is followed by a terminal phrase constituted of both the high frequency and the low frequency elements.

Additionally, we found that the high frequency element could be found either in a short version or a longer version (Fig. 2) whereas the low frequency element is always long. Finally the nature of the high frequency element was not found to be linked with the song type: we found that this high frequency element could be found in either a long form or a short form in a same song type, and even produced by the same bird.

Song analysis and playback stimuli

Stimuli were created from songs of 25 males of the same population recorded between 22 March and 8 June 2006 and during two periods of the day: between 0700 and 1200 and between 1625 and 1945 hours, respectively (UTC ? 1). Birds were recorded with a Telinga Pro 6 Phantom parabola and microphone and a Nagra Ares-BB ? digital recorder. Songs were firstly analyzed using Avisoft SASLab Pro 2 (for download of newest versions, http://www.avisoft.com/downloa_.htm). A total of 1,055 songs were analyzed, with a mean number ± standard deviation (SD) of 40.3 ± 29.7 songs per male. Length, type, frequency ranges, and structure were measured for each song. Song abruptions were not taken into account during the analysis. Minimum and maximum song and interval duration were calculated according to [START_REF] Naguib | Effects of different levels of song overlapping on singing behaviour in male territorial nightingales (Luscinia megarhynchos)[END_REF] method. To generate stimuli, we selected 19 different full song structures (FSS) from 19 different males that we edited in order to obtain the four structures described previously (see Fig. 1) using Avisoft SASLab Pro 2. These 19 song structures were selected to test the highest possible number of song types with the highest possible number of singers. Each selected song thus differed from the others by its singer to avoid pseudoreplication [START_REF] Kroodsma | Design of song playback experiments[END_REF]. Moreover, 17 of them differed by their song type. The two remaining selected songs were of two previously used types, but sung by a different singer and with a different high frequency element than found in the first one: i.e., with a long high frequency element or a short high frequency element, depending on the singer (Fig. 2). As short and long high frequency elements were found at the same rate during the analysis of the 25 males' song types, we selected 9 ''short high frequency element'' types and 10 ''long high frequency element'' types. Previous analyses of Yellowhammer songs based on the recordings for the 25 males (for a total length of 3 h 50 min) indicated that the mean inter-song interval within a bout was of 7.93 ± 1.52 s and the mean song duration was of 2.05 ± 0.31 s. Therefore, we built sequences of one song from one male and with a given structure (FSS, NHE, NLE, or NTP) followed by the length of silence required to make up a total of 10 s. This sequence was repeated 30 times in order to build a 5-min stimulus. In summary, the stimuli consisted of 17 song types plus 2 types already used but with a different high frequency element, all sung by different males. Each one was presented to a single male who was only exposed to this type, but within the four different structures (FSS, NHE, NLE, and NTP) edited from the initial FSS. All the stimuli were normalized via Audacity to the same amplitude.

Playback protocol

Playback experiments were conducted between 0710 and 1354 hours, between 10 April and 5 May 2007, on 19 males found in the field. Whenever a male was found singing in his territory, we installed a Elipson C10 AL loudspeaker on a hypothetic line drawn between two posts he had been observed singing from, 10 m from one of the two spots. We delimitated a 10-m diameter around this spot that we called the zone. This installation did not result in much stress for the bird that either stayed where it was or moved nearby and started singing again. Hearing the bird was a confirmation of its presence nearby so that we could make sure it was still in its territory and would hear the playback. We considered that it did also provide a way to control the proximity to the zone (and therefore, their possibility to reach the zone as soon as possible) for all the birds at the start of the experiment, a parameter which may otherwise have interfered with the latency analysis. As soon as the installation was completed, we began the broadcast of one group of stimuli (4 structures from the same song type) from a database built in an Archos Gmini XS100 MP3 Player in WAV file format. The MP3 player and the speaker had been previously adjusted to play the songs with a power of 80 dB at 1 m distance. A 5-min stimulus from a single song type was played to the subjects, followed by a 15-min break, before receiving another 5-min stimulus from the same type but with a different structure. The same protocol was used on the same male the following day, during the same period of the day, with the two remaining structures to be tested to conduct the playback in the same environmental conditions. Structure order during the broadcast was randomized for each male.

For each session (i.e., a sequence of 30 repetitions of the same stimulus), we measured three different variables:

• Latency: delay between the beginning of the playback and the arrival of the male inside the zone; • Duration: time the male spent inside the zone;

• Distance to the loudspeaker: the closest distance to the loudspeaker where the bird alighted.

Statistical analysis

An analysis of variance (ANOVA) was conducted to assess if the order of play and the structures had an effect on the birds' answer, i.e., if they approached the loudspeaker or not. A Friedman test was used to compare the distribution of structures according to song types. We analyzed the distribution of values for each tested variable (latency, duration, distance) with a multivariate general linear model (MGLM), followed by pairwise multiple comparison procedures (Student-Newman-Keuls method) to compare each structure with the others. All the birds were present in their territory and heard by the experimenters at the start of the playback and reacted to at least one structure. We used a Levene test to assess the equality of variances between song frequency ranges. Therefore, we could assess the consistency of the frequency of components of the song among individuals. A Wilcoxon test was used to compare males' responses to the nature (short or long) of the high frequency element. Finally an ANOVA with repeated measures was used to assess the impact of high frequency element (short or long) propagation on males' responses. 1). Both analysis by male and by song type showed that FSS was the most common structure that could be found in the population: 23 males out of 25 (92%) presented this structure at least for one type, and 19 song types out of 23 (82%) were recorded with this structure (Table 1). Even though only 1 male (4%) was recorded with the four different structures, 18 out of 25 presented at least three different structures (including FSS). Furthermore, 2 out of 23 types were recorded with the four structures, but 13 out of 23 (56%) were recorded at least with three different structures (including FSS).

Song type selection has no influence on the composition of the terminal phrase of the song Inside each of the 23 song types, we counted the number of exemplars of each structure (NTP, NHE, NLE, and FSS) that could be found for the song type in the recorded files.

A Friedman test showed that the song type had no significant influence on the composition of the terminal phrase (X 2 21 = 25.226, P = 0.238). Song type selection is therefore not responsible for the terminal phrase choice and males can finish their songs (no terminal phrase, one or two final elements) independently of the song type.

Basic songs parameters and conservation of the final element in population

The analysis of the 1,055 songs allowed us to draw out the basic song parameters of the studied population, and to build the playback stimuli as close as possible to the mean ranges (Table 2). Mean song duration (2.05 ± 0.31 s) and interval duration (7.93 ± 1.52 s) combined to make a unit roughly 10 s in duration that was repeated 30 times to build one stimulus sequence as described in ''Methods''. The mean number of repetitions of the note in the initial phrase (10.35 ± 1.76) was also taken into account to build the stimuli.

For each song, we measured the high and low extreme frequencies of both notes (the repeated, stereotyped constituents of the initial phrase) and elements (the two possible constituents of the terminal phrase) (see Fig. 2). Mean ranges were determined for the four different constituents: low frequency note (LN), high frequency note (HN), low frequency element (LE), and high frequency element (HE) (Table 3). In order to assess whether the variation around the mean range was significantly different between notes and elements, we used a Levene test to assess the homoscedasticity of the ranges. We found that the standard deviations around the mean were not significantly different between HN and LN (P = 0.88), but were significantly different between LN and HE (P = 0.007), and between LE and HE (P = 0.008). In conclusion, the standard deviation around the mean range of the low frequency element is significantly smaller (1.75 as small) than the standard deviation around the mean range of the high frequency element, which is itself significantly smaller (1.7 as small) than the standard deviations of either high or low frequency notes (Fig. 3).

Latency and distance variables depend on the song structure during playback experiment Birds reacted similarly to the different stimuli during playback, independently of their moment of broadcasting (first or second during the first or the second day of playback): 15 males out of 19 reacted to the first stimuli played and 14 out of 19 reacted to the second stimuli during the first day of testing, while 13 males out of 19 reacted to the first stimuli and 14 out of 19 reacted to the second stimuli the second day. It also appears that the selected protocol based on two tests per day during 2 days did not affect the birds' responses to the playback: they reacted actively to the stimuli in all cases, independently of when they were broadcast either on the first day or the second, first or second in order (ANOVA, F 3,72 = 0.16, P = 0.923) and independently of the structure that was broadcast (ANOVA, F 3,72 = 1.459, P = 0.235).

No interaction was found between the two parameters. Overall, 7 birds did not react to NTP (4 in first series, 3 in second series), 6 birds did not react to NLE (3 in first series, 3 in second series), 5 birds did not react to NHE (3 in first series, 2 in second series), and only 2 birds did not react to FSS (both in second series). 9 birds reacted to all the structures proposed.

Latency

The distribution of the latencies showed that they differed significantly (MGLM, F 3,72 = 7.034, P \ 0.001) according to the song type (NTP, NHE, NLE, FSS). Pairwise multiple comparisons showed that most of the latency reactions were different from each other when compared pairwise. NLE and NTP were not significantly different: NLE versus NTP: q = 1.001, P [ 0.05, but NHE and NLE were significantly different: NHE versus NLE: q = 4.294, P \ 0.05, and NHE and FSS were significantly different: FSS versus NHE: q = 4.717, P \ 0.05 (Fig. 4a).

Duration

The distribution of the durations inside the zone showed no significant differences between structures (MGLM, F 3,72 = 2.404, P = 0.074; Fig. 4b).

Distance

The distribution of the distances showed that they differed significantly (MGLM, F 3,72 = 4.765, P = 0.004) according to the song type (NTP, NHE, NLE, FSS). The pairwise multiple comparisons showed a significant difference when FSS was compared with all the other structures: FSS versus NTP: q = 4.384, P \ 0.05, FSS versus NLE: q = 4.077, P \ 0.05, FSS versus NHE: q = 4.48, P \ 0.05. Other structures compared pairwise showed no significant differences for this parameter: NTP versus NHE: q = 2.833, NTP versus NLE: q = 2.622, NHE versus NLE: q = 1.610 (Fig. 4c).

The nature of the high frequency element has no effect on the male Yellowhammer's response

When comparing the responses of males to a short (s, n = 9) or a long (l, n = 10) high frequency element in the no low frequency element (NLE) structure, we did not find any significant difference for any of the tested variables: latency (Mann-Whitney U test: U = 37.5, N 1 = 9, N 2 = 10, P = 0.549), duration (Mann-Whitney U test: U = 36, N 1 = 9, N 2 = 10, P = 0.497) or distance (Mann-Whitney U test: U = 36.5, N 1 = 9, N 2 = 10, P = 0.497). Moreover, when comparing latency responses between NLE structures and their corresponding NTP structure (NLEs with corresponding NTPs and NLEl with corresponding NTPl), no interaction was found between the nature of the high frequency element and the effect of the structure (ANOVA with repeated measures, no effect was found for the structure: F 1,17 = 1.14, P = 0.301, nor for the high frequency element length: F 1,17 = 0.996, P = 0.332, and no interaction between structure and high frequency element length was found: F 1,17 = 0.037, P = 0.85). In summary, the difference in latency response between NLEl and NTPl was not significantly different from the difference between NLEs and NTPs.

Discussion

In this study, we have shown that in Yellowhammers, the full song structure (FSS) elicits more territorial reactions from males than the three other structures that are found in the studied population. Even though the birds reacted similarly to any species-specific stimuli broadcast suggesting that a male was intruding their territory, they reacted more strongly in the case of the FSS structure. The fact that birds reacted equally to all types of structures is explained by the fact they considered any intrusion a threat and therefore went to look for the intruder; however, the degree of intensity they showed in their answer depended on the structure that was broadcast. There were significant structure effects for both the latency of responses and the distance to the loudspeaker: males whose territory was being intruded upon reacted faster and came closer to the emission source when the FSS was broadcast than they did for the other structures. There were no significant differences in the duration of the stay of males inside the 10-m zone around the loudspeaker. This variable may not have been appropriate for this protocol. A better indicator of the male's motivation may be the duration of stay during and after the playback, possibly the time birds would wait after the opponent has left to ensure he does not return. However, only a few males out of the 19 tested stayed in the zone until the end of the test, regardless of the structure that was broadcast. A possible explanation is that when the birds arrived in the zone and did not find another male Yellowhammer, they became disinterested in the playback, whatever its structure, and left the zone.

Both latency and distance to the loudspeaker were found to be respectively shorter and smaller when FSS was broadcast, and the latency found when NHE was broadcast was also significantly smaller than when NLE or NTP were broadcast. Although not significantly different from the values found for NLE and NTP, the mean distance value found for NHE is smaller than the mean values found for NLE and NTP, so that we can form the hypothesis that the main element which is responsible for the male reaction in the FSS is the low frequency element. This hypothesis is supported by the fact that FSS is not found in some Yellowhammer populations where NHE can be found [START_REF] Hansen | Song variations in the yellowhammer, Emberiza citrinella[END_REF][START_REF] Kaiser | Der Gesang der Goldammer und die Verbreitung ihrer Dialekte[END_REF].

Two questions can be raised from our results: is the low frequency element directly responsible for the Yellowhammer reaction, or is it the duration of the FSS which is responsible for the enhanced reaction observed by males, and in this case, would the nature of the low frequency element be purely epiphenomenal with this exaggerated reaction?

Both duration and propagation of the different songs are affected by their composition: a longer song like an FSS song could thus elicit more reactions than a shorter song like an NTP song. If duration was the only characteristic assessed by males (so that a longer song would provoke an enhanced reaction), we would have obtained significantly different results for the NLE songs, depending on whether they had a long or a short high frequency element. No significant difference was found between these two ''sub-structures''. Moreover, the high frequency element can be found either in a long form or a short form, which constitute two groups in our data: NLEs (for the song type with a short high frequency element) and NLEl (for the song type with a long high frequency element). The comparison of the relative difference between the paired NTP and NLE structures also supports the claim that duration is not a sufficient factor to explain the enhanced reaction from the birds. If duration was the most important factor taken into account by birds, their reactions to NLE structures relatively to the paired NTP structure should be stronger if the NLE contains a long high element rather than a short high element, as the long high element is longer than the short high element. We did not observe any significant differences between the two situations, therefore making it unlikely that duration is the only factor driving the birds' reactions. Concerning propagation, FSS is composed of trills and whistles, and it has been suggested in the literature that whistles have better long-range transmission [START_REF] Slabbekoorn | Birdsong and sound transmission: the benefits of reverberations[END_REF][START_REF] Wiley | Adaptations for acoustic communication in birds: sound propagation and signal detection[END_REF] than broadband trills [START_REF] Naguib | Reverberation of rapid and slow trills: implication for single adaptations to long-range communication[END_REF], and signal degradation is also thought to affect song recognition [START_REF] Naguib | Assessment of auditory distance in a territorial song bird: accurate feat or rule of thumb?[END_REF]. Therefore, FSS songs should be heard more easily because of less signal degradation and better propagation. However, NLE structures also contain the high frequency element, which is a whistle, and its propagation properties should then also increase the males' reaction, compared with the reaction to the NTP structure. As for duration, if propagation alone was responsible for bird attraction, NLEl should be more attractive in relation to its NTPl version than NLEs to its respective NTPs version, because of a difference of length and propagation; [START_REF] Slabbekoorn | Birdsong and sound transmission: the benefits of reverberations[END_REF], indeed, suggested that the longer the whistle, the bigger the behavioral response. The differences between NLEl and NTPl on the one hand, and between NLEs and NTPs on the other hand, are not significantly different, indicating that the propagation and length properties of the whistles are not the only factor responsible for the enhanced male reaction. The low frequency element seems therefore to be the main factor responsible for this enhanced reaction because of both its presence and its nature. Another argument in favor of this hypothesis is the significant difference found in approaching reactions: no propagation property can be proposed as a relevant explanation to the smaller distance observed in the case of FSS broadcasting once the bird is in the zone. A higher motivated reaction to a specific structure or element seems to be the most natural hypothesis, as it has already been shown in other species (e.g., Water Pipits Anthus spinoletta; [START_REF] Rehsteiner | Singing and mating success in water pipits: one specific song element makes all the difference[END_REF].

Here, we have shown that male Yellowhammers react more strongly to the most complete structure than to the single stereotyped and repeated structure. It had been suggested in the literature [START_REF] Catchpole | Sexual selection and the evolution of complex songs among European warblers of the genus Acrocephalus[END_REF][START_REF] Catchpole | Why do birds sing?[END_REF]) that the complete structure was designated to attract females rather than to function in intrasexual selection. We think our result is compatible with this idea. It is indeed possible that the same song structure can be directed to two different receivers and can be interpreted in two different manners.

Females would assess the richness of the song as a good indicator of male value, and assess a potential genitor, which has already been suggested in another playback experiment on Great Tits (Parus major) [START_REF] Otter | Do female great tits (Parus major) assess males by eavesdropping? A field study using interactive song playback[END_REF], when, at the same time, males could assess the value of a potential rival. Although our study was aimed mainly at male behavior, we observed in some cases that the female came along with the tested male she was paired with. We did not find any ornithological data suggesting that female Yellowhammers contribute to the defence of the territory with the male, but we know that female Yellowhammers assess their mate by flying above the already defined territories and choosing the one to which they are the most attracted [START_REF] Spitz | Bruant jaune[END_REF]. Therefore, we cannot say if the females were attracted to the playback itself or if they came to help the male defend the territory.

We think that both the low frequency element and the high frequency element play an active part in sexual selection. Although we found that males react more strongly to the FSS structure, maybe because they interpret it as a more aggressive signal, we think that the presence of the terminal phrase is itself an answer to the intersexual selection problem. The presence of this terminal phrase indeed multiplies by four the number of song types that a male can produce. This is of central importance in a species whose repertoire is small [START_REF] Hansen | Neighbour-stranger song discrimination in territorial yellowhammer Emberiza citrinella males, and a comparison with responses to own and alien song dialects[END_REF][START_REF] Hiett | Song repertoires and seasonal song in the yellowhammer, Emberiza citrinella[END_REF], especially since it has been shown that females prefer bigger repertoires in this species [START_REF] Baker | Sexual response of female yellowhammers to differences in regional song dialects and repertoires sizes[END_REF]. Therefore, the presence of the terminal phrase in Yellowhammers could have been developed as a means to enrich a repertoire in a species where juveniles mainly learn their repertoire from their parents and those in the neighborhood of their natal area, and not from the neighborhood of the breeding area [START_REF] Hiett | Song repertoires and seasonal song in the yellowhammer, Emberiza citrinella[END_REF]; therefore, we cannot expect an imitation process to enrich this repertoire. The presence of the terminal phrase could have thus been developed as a means to ''cheat'' on the size of the repertoire, which would not be surprising in passerines, where resident males of a given area are thought to sing different songs to discourage newcomers by giving the impression of a crowded area (see the Beau Geste effect; [START_REF] Krebs | Song repertoires and territory defence in the great tit[END_REF][START_REF] Krebs | The significance of song repertoires: the Beau Geste hypothesis[END_REF].

Finally, we found that the low frequency element was more conserved in the population than any other song constituents. The standard deviation around its mean range was indeed nearly half the standard deviation around the mean range of the high frequency element and three times less than the standard deviation around the mean ranges of the notes. We find it unlikely that a physiological constraint could be the main reason for this smaller variation, given that some of the low frequency notes were found in the same range of variation and with even lower extreme frequencies (unpublished data). Therefore, we think that the low frequency element is an important part of the male song in the Yellowhammer and that this element is more constrained to be produced within a certain range.

Zusammenfassung

Territoriale Reaktionen ma ¨nnlicher Goldammern (Emberiza citrinella) auf eine spezifische Gesangsstruktur Ma ¨nnliche Singvo ¨gel scha ¨tzen ihre Rivalen ein, indem sie deren Gesang zuho ¨ren, und bei einigen Arten reagieren sie besonders empfindlich auf bestimmte Teile des Gesangs. Goldammern (Emberiza citrinella) produzieren Gesa ¨nge mit unterschiedlichen Strukturen, die aus einer anfa ¨nglichen Phrase stereotyper und wiederholter Noten in einem Triller und einer abschließenden Phrase (mal vorhanden, mal nicht) bestehen, die aus niedrig-oder hochfrequenten Pfiffen zusammengesetzt ist, die wir Elemente genannt haben. Wir haben 1,055 Gesa ¨nge von 25 unterschiedlichen Ma ¨nnchen aus einer franzo ¨sischen Goldammern-Population untersucht und herausgefunden, dass vier verschiedene Strukturen unterschieden werden konnten. Wir haben beobachtet, dass sich die Frequenzbereiche der Elemente verschiedener Individuen sta ¨rker a ¨hnelten als die Notenbereiche und das niederfrequente Element nahezu doppelt so konserviert war wie das hochfrequente Element. Wir haben dann im Freiland die territorialen Reaktionen von Ma ¨nnchen auf die vier verschiedenen Strukturen getestet und herausgefunden, dass die Gesamt-Gesangsstruktur (FSS), die aus der Anfangsphrase gefolgt von einem hoch-und einem niederfrequenten Element besteht, sta ¨rkere Territoriumsverteidigungsreaktionen der Ma ¨nnchen hervorrief als irgendeine der anderen Strukturen. Da die Struktur, welche nur die Anfangsphrase und das niederfrequente Element umfasst, ebenfalls eine starke Antwort auslo ¨ste, schlagen wir vor, dass das Hauptelement, das fu ¨r die sta ¨rkere Reaktion der Ma ¨nnchen verantwortlich ist, das niederfrequente Element ist, was mit der Tatsache in Einklang steht, dass es sta ¨rker konserviert ist als die anderen Komponenten.

Fig. 1

 1 Fig. 1 Sonograms of Yellowhammer (Emberiza citrinella) song showing the four different structures that were found in the studied population. a No terminal phrase (NTP) where only the initial phrase is present, b no high frequency element (NHE) where the initial phrase is followed by a low frequency element only, c no low frequency element (NLE) where the initial phrase is followed by a high frequency element only, d full song structure (FSS) where the initial phrase is followed both by high and low frequency elements. NTP, NHE and NLE were built from FSS structure, using Avisoft SASLab Pro 2 and Audacity programs

Fig. 3 Fig. 4

 34 Fig. 3 Box plots presenting the distributions of frequencies of the four main constituents of the Yellowhammer song in a French population: high frequency note (HN), low frequency note (LN), high frequency element (HE), and low frequency element (LE)

Table 1

 1 Song count and proportion of the different song structures found in a population of 25 French male Yellowhammers (Emberiza citrinella) presenting 23 different song types

	Song structure	Song count	%	No. of males a	% b	No. of song types a	% b
	Full song structure	679		64.4	23	92	19	82
	No low element	107		10.1	18	72	15	65
	No high element	23		2.2	6	24	5	22
	No terminal phrase	246		23.3	22	88	18	78
	Total	1,055		100
	a Number of males (and song types) observed with the song structure
	b Corresponding percentage relative to the total number of males (25) and song types (23)
	Table 2 Basic song parameters of the studied French	Variable		Number of songs	Mean ± SD	Minimum	Maximum
	Yellowhammer population, based on 25 males		Song duration (s) Interval duration (s)	1,048 1,022	2.05 ± 0.31 7.93 ± 1.52	1.73 6.40	2.36 9.45
			Repetition (n)		1,048	10.35 ± 1.76	5	17
			High high note (kHz)	1,043	7.23 ± 0.53	5.68	8.52
			Low high note (kHz)	1,043	4.67 ± 0.48	2.92	6.45
			High low note (kHz)	1,048	5.73 ± 0.68	3.44	8.69
			Low low note (kHz)	1,048	3.73 ± 0.55	2.06	7.32
			High high element (kHz)	1,033	8.76 ± 0.26	6.35	9.64
			Low high element (kHz)	1,033	7.03 ± 0.31	5.42	9.04
			High low element (kHz)	1,028	5.87 ± 0.25	4.99	6.71
			Low low element (kHz)	1,028	4.33 ± 0.17	3.61	5.08

Table 3

 3 Mean frequency ranges and associated standard deviations found for notes and elements in a French Yellowhammer population

	Variable		Minimum	Maximum	Mean	Standard deviation	Coefficient of variation
	High note (kHz)		0.71		4.29	2.60	0.81	0.31
	Low note (kHz)		1.04		3.78	2.00	0.81	0.40
	High element (kHz)		0.82		2.48	1.62	0.47	0.27
	Low element (kHz)		0.25		1.96	1.49	0.27	0.18
		5
	Frequency (kHz)	4 3 2 1
		0
		HN	LN	HE	LE
		Song constituent

J Ornithol (2010) 151:645-654

Acknowledgments We thank Franck Ramus and Timothe ´e Poisot for their advice on statistical analysis. We thank Franck Ramus for useful advice and comments on early versions of the manuscript. We thank Madelaine Krehm and Marie Monbureau for their help with the translation and Andrew Goodall for proof-reading of the manuscript.