Dagmar Kudernatsch

Martin Buchmann

Wolfgang Fiedler

Gernot Segelbacher
email: gernot.segelbacher@wildlife.uni-freiburg.de

Extrapair paternity in a German population of the Northern Wheatear (Oenanthe oenanthe)

Keywords:

We here demonstrate the potential of a set of novel microsatellite markers to investigate kin structure and population genetics in the Northern Wheatear (Oenanthe oenanthe). In this study based on 242 individuals from a population in Rhineland-Palatinate (Germany) we found that 26% of the offspring in 46% of the broods were sired by other males than the social fathers. We tested different hypotheses why males engage in extrapair copulations and found that almost all identified genetic fathers originated from directly neighbouring territories. Additionally, we detected two out of 134 offspring in two broods that could not be assigned to their putative mother and thus were probably the result of intraspecific brood parasitism.

Introduction

The Northern Wheatear (Oenanthe oenanthe) is a widespread long distance migrating bird species in Europe and its ecological behaviour has been extensively studied (e.g. Arlt andPärt 2007, 2008;[START_REF] Delingat | Obligatory barrier crossing and adaptive fuel management in migratory birds: the case of the Atlantic crossing in Northern Wheatears (Oenanthe oenanthe)[END_REF].

Wheatears have been considered to be socially monogamous but already [START_REF] Currie | Male and female behaviour and extrapair paternity in the wheatear[END_REF] found that females frequently engage in extrapair copulations (EPC). Such findings have been proved to be common in many other bird species. In more than 75% of socially monogamous passerine species extrapair young (EPY) could be detected with over 11% of offspring being sired by an extrapair male [START_REF] Griffith | Extra pair paternity in birds: a review of interspecific variation and adaptive function[END_REF].

Previous research suggested three main hypotheses for the benefits of such extrapair copulations: (i) the genetic diversity hypothesis [START_REF] Petrie | Extrapair paternity in birds: explaining variation between species and populations[END_REF], (ii) the good genes hypothesis [START_REF] Birkhead | Sperm Competition in Bird[END_REF]) and (iii) the genetic compatibility hypothesis [START_REF] Johnsen | Female bluethroats enhance offspring immunocompetence through extrapair copulations[END_REF]. The genetic diversity hypothesis (i) proposes that females aim to increase the genetic diversity within progeny. This would increase the chance that at least some offspring will survive if environmental conditions change [START_REF] Yasui | The 'genetic benefits' of female multiple mating considered[END_REF][START_REF] Yasui | Female multiple mating as a genetic bet-hedging strategy when mate choice criteria are unreliable[END_REF].

The hypothesis predicts that EPY are distributed randomly among broods and the population's males [START_REF] Griffith | Extra pair paternity in birds: a review of interspecific variation and adaptive function[END_REF]. The good genes hypothesis and the genetic compatibility hypothesis both argue that females seek EPCs with males of higher quality than their social mates. According to the good genes hypothesis (ii) females seek to gain genes of high quality for their offspring, i.e. genes linked to viable or attractive phenotypes (Westneat et al. 1990, Birkhead and[START_REF] Birkhead | Sperm Competition in Bird[END_REF]. EPY thus should not be distributed randomly among broods, but be more frequent in broods where the female is paired with a male of low quality [START_REF] Griffith | Extra pair paternity in birds: a review of interspecific variation and adaptive function[END_REF]. Conversely, males with high quality features are predicted to gain extrapair paternity (EPP) more often than males of low quality [START_REF] Griffith | Extra pair paternity in birds: a review of interspecific variation and adaptive function[END_REF]. The sexy son hypothesis links the good genes hypothesis and the sex allocation theory [START_REF] Trivers | Natural Selection of Parental Ability to Vary the Sex Ratio of Offspring[END_REF]. It predicts a sex-bias among EPY towards males to pass on the attractiveness primarily to the male offspring (Møller andNinni 1998, Dietrich-Bischoff et al. 2006). In the genetic compatibility hypothesis (iii) females will choose males which best complete the female's genome (Kempenaers et al. 1999, Tregenza andWedell 2000). The extrapair father's genome is expected to be very dissimilar to the female's, be very heterozygous, and/or carry rare alleles. The resulting offspring would be more heterozygous [START_REF] Foerster | Females increase offspring heterozygosity and fitness through extrapair matings[END_REF], Stapleton et al. 2007) and be able to express a greater number of gene products, e.g. from the major histoncompatibility complex [START_REF] Johnsen | Female bluethroats enhance offspring immunocompetence through extrapair copulations[END_REF][START_REF] Fossøy | Multiple genetic benefits of female promiscuity in a socially monogamous passerine[END_REF]). The hypothesis predicts higher EPP rates in genetically more related pairs, and that the EPF is less related to the female than her social partner [START_REF] Griffith | Extra pair paternity in birds: a review of interspecific variation and adaptive function[END_REF].

Several studies seem to support the genetic benefits hypotheses (e.g. [START_REF] Johnsen | Female bluethroats enhance offspring immunocompetence through extrapair copulations[END_REF][START_REF] Foerster | Females increase offspring heterozygosity and fitness through extrapair matings[END_REF] although [START_REF] Akςay | Extrapair paternity in birds: review of the genetic benefits[END_REF] found that half of the studied species fail to support the good genes and the genetic compatibility hypothesis and moreover, that the type of genetic benefit varies across species, between close related species and even within the same species.

In our study on wheatears we explore the different hypotheses by firstly testing a set of novel microsatellite markers for kinship analysis in this species and by identifying potential EPY in broods. When arriving back from their breeding grounds males generally establish territories a few days before females arrive and both parents care for the young equally [START_REF] Menzel | Die neue Brehm Bücherei -Der Steinschmätzer (Oenanthe oenanthe)[END_REF]. Breeding pairs defend their territories aggressively and both, females and males, may expel their equivalent sex [START_REF] Menzel | Die neue Brehm Bücherei -Der Steinschmätzer (Oenanthe oenanthe)[END_REF][START_REF] Currie | Male and female behaviour and extrapair paternity in the wheatear[END_REF]. During the period of egg laying males are following females closely [START_REF] Currie | Male and female behaviour and extrapair paternity in the wheatear[END_REF]. Despite the social monogamy of the species it still remains unclear why extra pair copulations occur frequently in this species.

Methods

Study site and field methods

242 blood samples were obtained from a northern wheatear population in Rhineland-Palatinate, Germany (approximately 49°30'N, 8°10'E), during the breeding seasons 2005 and 2006. About 150 breeding pairs nest in a vineyard area of about 15km². The breeding pairs are inhomogeneous distributed in the area with a density of up to 38 breeding pairs per km 2 (see [START_REF] Buchmann | Die Brutbiologie des Steinschmätzers (Oenanthe oenanthe) auf intensiv genutzen Flächen in Rheinland-Pfalz[END_REF]. Breeding birds were trapped with small nest traps when they left their nests, other birds using spring net traps. Breeding birds could be defined with great accuracy as social parents as almost all birds in the population were colour-ringed and parents were recorded at their nests several times during the period of nest building period, incubation and feeding the young (see [START_REF] Buchmann | Die Brutbiologie des Steinschmätzers (Oenanthe oenanthe) auf intensiv genutzen Flächen in Rheinland-Pfalz[END_REF], Buchmann et al. 2009). Measurements for body mass were taken using a spring scale with a precision of 0.1g. Wing length was measured to the nearest 0.05 mm using a wing ruler and tarsus length was measured to the nearest 0.5mm using a calliper. Age of the birds was estimated by plumage features and birds were classified as second year or older ones. Juveniles were sampled in the nests when they were between 6 and 12 days old (mean 7.4 ± 1.9). Blood from all birds was taken from the brachial vein and transferred in 1 ml Queen's lysis buffer [START_REF] Seutin | Preservation of avian blood tissue samples for DNA analysis[END_REF]. Some samples were transferred in 1 ml 70% Ethanol. All samples were stored at 4°C until DNA extraction.

DNA methods

DNA was extracted from blood according to a standard salt extraction method [START_REF] Miller | A simple salting out procedure for extracting DNA from human nucleated cells[END_REF] with modifications: (i) The extraction buffer contained 10 mM Tris-HCl pH 8, 10 mM EDTA pH 8, 200 mM NaCl, (ii) a digestion of 40 μl of blood sample was carried out for one hour at 55°C in a total volume of 400 μl which contained 330 μl extraction buffer, 2% SDS and 10 μg/μl Proteinkinase K, (iii) all centrifugation steps were performed at 13,000 rpm for five minutes, (iv) for precipitation one volume cooled Isopropanol was added, followed by a centrifugation step and transfer of the supernatant into a new vessel. The pellet was washed twice with 70% Ethanol and eluted in 100μl Buffer AE (Qiagen). The offspring's sexes were identified using the primers 2550F and 2718R [START_REF] Fridolfsson | A simple and universal method for molecular sexing of non-ratite birds[END_REF] in a total volume of 10 μl containing 1 x mi-Hot Taq-buffer (Metabion), 10 pmol of each primer, 0.2 mM of each nucleotide, 1.5 U mi-Hot-Taq (Metabion) and 1 μl of a 1:20 dilution from the stock DNA. The PCR profile comprised an initial denaturation step of 95°C for 5 minutes, followed by a touch-down scheme with the annealing temperature being lowered 0.5°C per cycle, starting at 54°C until 48°C was reached. Additional 20 cycles at that temperature were followed by a final step of 5 minutes at 72°C. PCR products were resolved in 1.2% agarose gels and stained with SYBR Gold (Invitrogen). Individual genotyping was performed as described in [START_REF] Kudernatsch | Isolation of ten tetranucleotide microsatellite loci in the Northern Wheatear (Oenanthe oenanthe)[END_REF] for the loci oe2, oe4, oe5, oe6, oe7, oe8, oe9, oe10, oe11, oe12.

Parentage analyses

In For loci oe2, oe9, and oe11 the presence of possible null alleles can´t be ruled out (see [START_REF] Kudernatsch | Isolation of ten tetranucleotide microsatellite loci in the Northern Wheatear (Oenanthe oenanthe)[END_REF]) which might result in false exclusion of paternity. To assign offspring genotypes to the parents' genotypes, individuals with only one band at these loci were therefore not regarded as homozygotes, but as heterozygotes with one unknown loci (indicated by a zero). Parentage was determined using the likelihood based approach in CERVUS 3.0 [START_REF] Kalinowski | Revising how the computer program CERVUS accomodates genotypic error increases success in paternity assignment[END_REF], available at http://www.fieldgenetics.com). First, we assessed maternity and if confirmed, mothers were included into the offspring file as known parent. Exclusion probability was very high (> 0.999). An individual was assigned as the genetic father, if the probability values estimated by CERVUS suggested it with a confidence level of at least 80% or if the social father had the highest LOD score of all potential male birds tested. The chosen confidence level describes that at least 80% of the parentage assignments are reliably correct.

Heterozygosity and genetic similarity

Standardized heterozygosity was calculated for each individual (see [START_REF] Coltman | Parasite-mediated selection against inbred Soay sheep in a freeliving island population[END_REF]. Genetic similarity between individuals was calculated using the method of Queller and Goodnight (1998) implemented in the IDENTIX 1.1 software [START_REF] Belkhir | IDENTIX, a software to test for relatedness in a population using permutation methods[END_REF], http://www.genetix.univ-montp2.fr). The loci oe2, oe9 and oe11 were excluded for the calculation of genetic similarity and heterozygosity because of the possible presence of null alleles.

Statistical analyses

Statistical analyses were performed using the R 2.5.1 software (R Development Core Team 2007, http://www.r-project.org). Data from 2005, 2006 and both seasonal broods were pooled for all analyses. The distribution of EPP among broods was analysed as described in [START_REF] Bouwman | Older male reed buntings are more successful at gaining extrapair fertilizations[END_REF], based on [START_REF] Sokal | Extrapair paternity in hooded warblers[END_REF] and [START_REF] Perreault | Patterns and correlates of extrapair paternity in American redstarts (Setophaga ruticilla)[END_REF]. We compared the heterozygosity of within-pair young (WPY) with that of their maternal half-siblings using a generalised linear mixed model (GLMM, Laplace method). Family identity was included as a random factor and the status of offspring (WPY or EPY) as a fixed factor.

Heterozygosity, the response variable, was considered as proportion data and handled as described in [START_REF] Crawley | The R Book[END_REF]. High levels of overdispersion made a modification of the model with quasi-binomial rather than binomial errors necessary. We compared males in parameters of body size and body condition. It proved that body size is better represented by a combination of parameters rather than solely by univariate variables [START_REF] Freeman | Univariate Metrics Are Not Adequate to Measure Avian Body Size[END_REF]. Therefore we conducted a principal components analysis including measurements of tarsus and wing length. Values of the first ordinary axis were determined as 'body size' for all individuals. Since the data of tarsus and wing length was not scaled identically, a correction factor integrated in R was applied. The body condition of males was calculated as residuals from ordinary least squares of body mass versus body size (Figure 1; see [START_REF] Schulte-Hostedde | Restitution of mass-size residuals: Validating body condition indices[END_REF]. Rare alleles were determined by first calculating frequencies for each allele per locus using the Genepop 4.0 software (Raymond and Rousset 1995, http://genepop.curtain.edu.au). Allele frequencies were root transformed to converge a binomial distribution. . We identified the first quartiles of the frequencies at each locus and considered alleles as 'rare' if their frequency was less than the value of the first quartile. First quartiles of allele frequencies per locus were identified using the summary option in R.

Results

Parentage analyses

In total, 26% of offspring were not the genetic offspring of their social fathers. Split in both years EPP-rate was 25% ( 24 We identified two clutches in which one of the nestlings genetically did not match the female of the breeding pair. Both cases were observed in second broods 2005. In one of the cases both genetic parents could be assigned and their territory was located about 300 metres away from the nest site with two other territories being located in between.

Test of the genetic benefits hypotheses

Distribution of EPY among broods:

EPY were not allocated randomly among broods: the distribution significantly deviated from a binomial distribution (Table 1, χ 2 =32.19, d.f. = 7, p<0.001). Twice as many broods as expected did not contain any EPY. Only a few broods contained one or two EPY. There were more broods than expected which contained numerous EPY (Table1).

Distribution of EPF among broods:

Four females chose only one male in addition to their pair male as the father for their offspring. Several males were chosen as EPF's in another four broods. One male bird was especially successful in obtaining EPC's:

Besides being a father in his own brood he was also the father of young in two other broods. In his own brood only one out of three young was sired by him.

when breeding with an older male (n = 3 old males). Old males in the population were significantly larger than young males (Wilcoxon signed rank test, W=61, p < 0.001, n = 15 young, 27 old males).

Sex allocation theory:

The entire data set comprised 134 nestlings of the northern wheatear. Overall sex ratio was insignificantly biased towards females (46% males; Wilcoxon signed rank test, W=167, p = 0.18). In broods containing EPY and WPY, there was no bias towards one sex in EPY compared to their maternal half sibs (Wilcoxon's matched pairs test, V=28, p = 0.55, n = 11 dyads, Figure 3a). However, broods which contained no EPY displayed significantly more females than broods with a combination of EPY and WPY (Wilcoxon signed rank test, W = 85, p=0.04, n = 11 for broods only containing WPY, n = 10 for broods containing at least some EPY, Figure 3b).

Is there a seasonal difference in EPP rates?

In all families except one we found equal or insignifcantly lower EPP rates in the second brood of the year (Wilcoxon's matched pairs test, V = 22.5, p = 0.18, n = 10 dyads).

Discussion

Frequency of extra -pair offspring

The EPP rates of the northern wheatear in this study (29% of offspring in 46% of broods) are above the mean value for socially monogamous birds (11% of offspring in 29% of broods, [START_REF] Griffith | Extra pair paternity in birds: a review of interspecific variation and adaptive function[END_REF]. Interestingly our results exceed those of an earlier study in the northern wheatear (11%, n= 73 offspring, [START_REF] Currie | Male and female behaviour and extrapair paternity in the wheatear[END_REF]. [START_REF] Currie | Male and female behaviour and extrapair paternity in the wheatear[END_REF] were studying birds on Bardsey Island, which is located three kilometres of the mainland´s coast. EPP rates are often unexpectedly low within island populations probably due to lower sexual selection processes (Griffith 2000) or to genetic depletion. However, a higher density of birds (27 territory males/100ha on Bardsey Island, up to 38 males/100ha in our study area) may have also been led to higher EPY rates in our study area.

Genetic diversity hypothesis

The genetic diversity hypothesis predicts that females aim to mate with multiple males and that EPP is distributed randomly among broods. In support of the hypothesis, we found that some females chose more than one male as the father for EPY. On the other hand, a similar number of females chose only single males as fathers for EPY. This tends to support the good genes and the genetic compatibility hypotheses, but opposes the genetic diversity hypothesis. Another contradictory result is that EPY were not distributed among broods randomly. The most crucial factor to reject the genetic diversity hypothesis is that EPY were not more heterozygous than WPY. However, Most of the studies on EPP in birds found only weak evidence for this hypothesis [START_REF] Kempenaers | Extrapair paternity and egg hatchability in tree swallows: evidence for the genetic compatibility hypothesis?[END_REF] or evidence to reject the hypothesis (e.g. [START_REF] Bouwman | How female reed buntings benefit from extrapair mating behaviour: testing hypotheses through patterns of paternity in sequential broods[END_REF][START_REF] Ockendon | Extrapair paternity in an insular population of house sparrows after the experimental introduction of individuals from the mainland[END_REF].

Good genes hypothesis

The good genes hypothesis predicts that EP Males are of higher quality, which might be reflected by larger body mass, age or attractive plumage features. In our study, we did not find better body conditions in extrapair males, i.e. they did not have larger body mass. This contradicts the results of [START_REF] Currie | Male and female behaviour and extrapair paternity in the wheatear[END_REF], which stated that males with a better body condition are preferred based on 8 broods with genetic and social fathers known.

Contradictory relations within the same species have been described before (e.g. Kempenaers et al. 1997[START_REF] Charmantier | Do extrapair paternities provide genetic benefits for female blue tits Parus caeruleus?[END_REF], and this might indicate that study sites differ and not all females may choose the same criteria (compare Akςay and Roughgarden 2007).

We did not find differences in EPP rates in nests of young and old territory males. Old males were larger than young males; but body size and age are not independent factors.

There was no sex bias towards one sex in the young northern wheatears. The sex ratio in broods which contained EPY and WPY was equal, too. However, broods without EPY tended to contain more females.

However, this bias in our study needs to be evaluated with a larger sample size as the direction of sex allocation differs between species and even between populations of the same species (reviewed in [START_REF] Komdeur | Adaptive sex allocation in birds: the complexities of linking theory and practice[END_REF]. Nestling mortality could not be detected in our study area (number of eggs matched always the number of hatched young).

Genetic compatibility hypothesis

Females, which follow the genetic compatibility hypothesis are supposed to choose extrapair males which are genetically more dissimilar, very heterozygous and/or carry rare alleles. But those criteria did not influence the female's mate choice in our study. Thus, we reject the hypothesis for the northern wheatear in the studied population. However, it has recently suggested that females selecting genetically compatible fathers for their offspring offer a possible general explanation for the function of extrapair paternity [START_REF] Griffith | Female infidelity and genetic compatibility in birds: the role of the genetically loaded raffle in understanding the function of extrapair paternity[END_REF].

Sexy neighbours

In this study, EP males usually originated from directly neighbouring territories. Several studies documented similar findings (e.g. [START_REF] Perreault | Patterns and correlates of extrapair paternity in American redstarts (Setophaga ruticilla)[END_REF][START_REF] Kempenaers | Extrapair paternity and egg hatchability in tree swallows: evidence for the genetic compatibility hypothesis?[END_REF][START_REF] Johnsen | Female bluethroats enhance offspring immunocompetence through extrapair copulations[END_REF][START_REF] Segelbacher | Extrapair young despite strong pair bonds in the European Nuthatch (Sitta europaea)[END_REF][START_REF] Bouwman | Older male reed buntings are more successful at gaining extrapair fertilizations[END_REF][START_REF] Garvin | Immune response of nestling warblers varies with extrapair paternity and temperature[END_REF], suggesting that females may gain non-genetic benefits from extrapair males, e.g. additional hunting grounds, participation in raising the young or anti-predator defence [START_REF] Møller | Sperm competition and sexual selection: a meta-analysis of paternity studies in birds[END_REF]. Males residing at nearby territories might on the other hand just have more chances to approach the female for an EPC than males from more distant territories. If neighbouring males have more chances to court a female than other males of more distant territories do, then pair males do basically only compete against neighbouring males in terms of attractiveness instead of competing against the most attractive males in the whole population. Therefore it can be assumed that extrapair males do not necessarily need to be more attractive than the neighbouring female's social mate's to successfully obtain extrapair offspring.

Intraspecific brood parasitism

We detected two individual offspring which were not genetically not matching the female of the breeding pair.

This could be explained by potential intraspecific brood parasitism. Additional support for such a behaviour comes from field observations, where females were observed to aggressively expell other females from their territories. This might be a reaction in order to keep them from laying an egg into their clutch. We have observed the breeding male and female during the breeding period several times at their nest (nest building, incubating, feeding) and thus assume that any potential errors in catching non breeding birds and misclassifying the mother of the clutch are very low in the our described cases.

In our study we demonstrate the potential of a novel set of microsatellite markers to investigate the breeding biology of wheatears. Although our sampling size is relatively small to test the different hypotheses in detail, we are convinced that further studies will have the potential to fully explore these questions data using our marker set.

Figure 1

Relationship between body mass and body size for males (r 2 = 0.15, P = 0.009, n = 44). The body size index was obtained from a principal component analysis based on tarsus and wing length.

  2005 we sampled 15 complete families with a total of 99 offspring: 77 young hatched in first clutches and 22 young in second clutches from five of the families (overall mean 4.95 ± 1.1 SD, Appendix 1). In 2006 five additional families with first and second broods were sampled. They comprised a total of 35 offspring of which 20 young hatched in first clutches and 15 in second clutches (overall mean 3.89 ± 1.7 SD offspring per brood, Appendix 1). For parentage analysis we included additional individuals captured in the breeding area (21 females and 25 males in 2005, 46 males and 8 females in 2006). Based on field observations in our study area we estimated that we sampled about 30% of all resident males in 2005 and about 50% in 2006. When analysing extra pair paternity for males in 2005 we only analysed broods in which the breeding female could be genetically assigned as the mother of the clutch (n=97). Median hatching date for first broods was not different in our sampled broods as compared to the whole study population (1995: median 18.5. for population (n=37), median for sampled broods 17.5. (n=15)).

  /97) in 2005 and 29% (10/35) in 2006. At least one EPY occurred in 46% of the broods, comprising 50% (10/20) of broods in 2005 and 38% (3/8) of broods in 2006. Genetic fathers could be assigned for 42% of EPY in 2005 (12 offspring in 5 broods) and for 30% of EPY in 2006 (3offspring in 3 broods).

Fremdvaterschaften

  von Steinschmätzern (Oenanthe oenanthe) in Rheinland Pfalz führten wir Vaterschaftsanalysen an 242 Individuen mit neuen genetischen Mikrosatellitenmarker durch. In 46% aller untersuchten Nester fanden wir Jungvögel, die nicht vom Männchen des Brutpaars gezeugt wurden. Insgesamt konnten 26% aller Jungvögel auf andere Männchen zurückgeführt werden. Außerdem konnten wir in zwei Nestern Jungvögel beobachten, die nicht vom brütenden Weibchen stammten und damit wahrscheinlich ein Ergebnis von Nestparasitismus sind. Wir testeten verschieden Hypothesen warum Weibchen Fremdvaterschaften suchen und fanden fast alle nachgewiesenen Väter in direkt benachbarten Territorien.

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3 Boxplots showing the brood sex ratio for within-pair young (WPY) and extrapair young (EPY) in (a)

Table 1

 1 Distribution of extrapair young (EPY) among broods of different sizes. Shown are the observed values and the expected values for a binomial distribution (in brackets). There was a divergence from binomial distribution: χ =32.19, d.f. = 7, p<0.001.

	Number of EPY per brood

Spatial analysis of potential fathers

The distance between the territory of extrapair male and the nesting site of the female was less than 500 metres (mean 250 m ± 150 m SD, n=7) in all cases except for one. The EP father usually originated from directly neighbouring territories (n=6). In one case the territory of the EP father located 2,550 metres away from the female's nesting site.

Genetic similarity, heterozygosity and rare alleles:

We found a significant negative correlation between the genetic similarity of parents and the heterozygosity of their young, i.e. genetically dissimilar parents produced more heterozygous young than genetically similar parents (Pearson's product-moment correlation; t=-2.37, p=0.03, n=27 mate combinations). Yet, EP fathers were not more dissimilar to females than their social mates (Wilcoxon matched pairs test, V = 18, p=0.73, n = 8 dyads, Figure 2a), nor were they more heterozygous than WPF's (Wilxocon matched pairs test, V=15, p=0.73, n=8 dyads). Congruously, the WPY did not differ in heterozygosity from their maternal half-sibs (GLMM, χ 2 = 0.98, d.f. = 1, p = 0.32, n = 10 dyads) and their paternal half-sibs (GLMM, χ 2 = 0.47, d.f.=1, p=0.49, n=4 dyads). The heterozygosity of WPY and EPY is compared in Figure 2b. EP fathers did not display more rare alleles than within the breeding pair (Wilcoxon matched pairs test, V = 1.5, p = 0.57).

Are fatter, larger and older males sexier?

EP fathers tended to be larger than males of the breeding pair (Wilcoxon's matched pairs test, V=5, p=0.08, n = 8 dyads, Figure 2c). Male social mates in families without EPP were not larger than in families with EPP occuring (Wilcoxon signed rank test, W = 56, p = 0.65, n = 9 without EPY, 11 with EPY). The body condition of EPF's did not differ from that of WPF's (Wilcoxon's matched pairs test, V=10, p=0.31, n = 8 dyads, Figure 2d). The age of males does not explain the occurrence of EPP: Males of the second calendar year were betrayed as often as older ones (χ 2 = 1.36, d.f. = 7, p = 0.99, n=8). Nevertheless, females with young mates as breeding partners more often choose older males for EPC (n = 4 out of 5) and never choose younger ones Appendix 1. List of families included in this study. Presented are the year of sampling, the number of offspring and extrapair young (EPY) in first and second broods. The broods in which juveniles were detected which did not match the putative female are indicated with an asterisk.


Marilisa Barranca

Simona Fontana

Simona Taverna

Giovanni Duro

Isabelle Zanella- Cleon

Michel Becchi

Giacomo De Leo

Riccardo Alessandro

Prof Alessandro Riccardo

Proteomic analysis of Parietaria judaica pollen and allergen profiling by an immunoproteomic approach

Keywords: allergens, Parietaria judaica, immunoproteome, pollen

Parietaria judaica pollen is one of the most common causes of airway allergic disease in the Mediterranean area. Proteome analysis of mature Parietaria judaica pollen by two-dimensional gel electrophoresis (2-DE) and mass spectrometry allowed to establish the first reference proteome map of this weed. Proteins involved in a variety of cellular functions as well as the occurrence of allergens were detected. By using 2-DE and immunoblotting with sera from Parietaria judaica allergic patients we obtained a more detailed characterization of Parietaria judaica allergen profile so to improve our comprehension of the pathogenesis of pollen-induced allergic reaction.

Introduction

Parietaria judaica belongs to the Urticaceae family, is commonly found in urban and rural areas and its pollen is one of the main causes of allergy in the Mediterranean area [START_REF] Masullo | Respiratory allergy to parietaria pollen in 348 subjects[END_REF].

Parietaria judaica pollen allergens have been identified and characterized by immunochemical and molecular-biological techniques [START_REF] Amoresano | Assignment of disulphide bridges in Par j 2.0101, a major allergen of Parietaria judaica pollen[END_REF][START_REF] Stumvoll | Identification of cross-reactive and genuine Parietaria judaica pollen allergens[END_REF]. Par j 1 and Par j 2 represent two major allergens belonging to a family of glycoproteins known as nonspecific lipid transfer proteins [START_REF] Salcedo | Plant nonspecific lipid transfer proteins as food and pollen allergens[END_REF]. Recently, the presence of minor allergens like profilin (Par j 3) and 2-EF-hand (Par j 4) proteins have been also described [START_REF] Asturias | PCR-based cloning and immunological characterization of Parietaria judaica pollen profilin[END_REF][START_REF] Bonura | Isolation, expression and immunological characterization of a calcium-binding protein from Parietaria pollen[END_REF]. However, other proteins, specific isoforms or posttranslational modifications (PTM) of known allergens can contribute to hypersensitivity reactions in allergic individuals. The accurate diagnosis of hypersensitive patients as well as the in-deep knowledge of the mixtures for a effective immunotherapy depend on the molecular characterization of all allergenic components from a biological source.

Proteomic profiling of pollen extracts coupled with classical immunological approaches have been applied to examine the presence of novel allergenic components in maize, grass, and other species [START_REF] González-Buitrago | Proteomic approaches for identifying new allergens and diagnosing allergic diseases[END_REF][START_REF] Petersen | Proteome analysis of maize pollen for allergy-relevant components[END_REF]). An understanding of P. judaica pollen proteome would provide insight in the molecular nature of allergens as well as of other proteins involved in the functional specialization of pollen. Using 2-DE followed by LC-MS/MS analyses, we generated the first reference map of the P. judaica mature pollen proteome. In total, we identified 70 protein spots including isoforms, and by using an immunoproteomic approach with serum from P. judaica allergic patients, we have obtained a more detailed characterization of Parietaria judaica allergen profile so to improve our comprehension of the molecular determinants of P. judaica-induced allergic reaction. In particular, we have evidenced that the IgE from patients recognise Par j 1 and the Par j 2 P2 allergens while they don't recognise the polypeptide Par j 2 P8. These results could provide important indications to develop new and more efficient vaccines for allergic patients.

Materials and methods

Pollen collection and protein extraction

Pollen from Parietaria judaica was purchased from Allergon (Ängelholm, Sweden) and extracted as previously described [START_REF] Alessandro | Production of an egg yolk antibody against Parietaria judaica 2 allergen[END_REF]. Briefly, the pollen grains were defatted with diethyl ether, extracted in PBS at 4°C, dialysed against water and finally lyophilised and stored at -80°C until use.

2-DE, image acquisition and analysis

Aliquots of the lyophilised proteins, 60 µg for analytical gels and 1mg for preparative gels, were solubilized in a buffer containing 8 M Urea, 4% w/v CHAPS, 40 mM TRIS and 65 mM DTE and a trace amount of bromophenol blue.

The first dimension IEF was carried out using immobilized pH gradient (IPG) strips, pH 3-10 non linear and pH 4-9 linear, 18 cm long. The first dimension, equilibration steps and the second dimension were performed as previously described [START_REF] Fontana | Comparative proteome profiling and functional analysis of chronic myelogenous leukemia cell lines[END_REF].

Gels were stained with ammoniacal silver nitrate for analytical gels [START_REF] Hochstrasser | Methods for increasing the resolution of two-dimensional protein electrophoresis[END_REF] or comassie staining for preparative gels. The gels were scanned by densitometer ImageScanner II (GE Healthcare Life Science, NJ, USA ) and the ImageMaster 2D Platinum 6.0 software (GE Healthcare Life Science, NJ, USA) was used to perform the qualitative and quantitative analysis of the 2-DE maps. Human serum was used as internal standard for isoelectric point (pI) and Molecular Weight (MW) calibration of the 2-DE gel with pH 3-10 non linear gradient [START_REF] Bjellqvist | The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences[END_REF].

In-gel protein digestion

Coomassie blue or silver-stained protein spots were excised from the preparative gels and cut into 1-mm pieces. In-gel digestion was performed as described by Shevchenko et al [START_REF] Shevchenko | Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels[END_REF]

Patient sera

For the identification of IgE-reactive proteins, sera from patients (n = 10) sensitized to Parietaria judaica whose radioallergosorbent test (RAST) exhibited scores over 3, were used. Control sera were from healthy, non-allergic subjects and from subjects sensitized to other allergic sources (dust mite, Olea and Cupressus). All sera were stored at -80 °C until use.

Immunoblotting

For immunoblot analysis, 2-DE gel was transferred to nitrocellulose membrane (Hybond-ECL, Amersham Bioscience, Little Chalfont, UK) and the membrane was incubated in blocking solution (5% w/v non-fat dry milk, 20 mM Tris, 140 mM NaCl, 0.1% Tween-20) (TBST) at room temperature. After several washings in TBST buffer, the filters were incubated overnight at room temperature with sera from allergic patients diluted 1:10 followed by horseradish peroxidase (HRP)-conjugated rabbit anti human IgE (1:2000 dilution). After washings in TBST buffer, immunocomplexes were detected by the enhanced chemiluminescence detection system (Super Signal, Pierce, Rockford, IL, USA).

Results and Discussion

Proteome map of Parietaria judaica pollen

Parietaria judaica represents the main cause of allergy in the Mediterranean area where up to 80% of pollinosis patients are sensitized against the proteins composing the P. judaica pollen. With the aim to obtain a comprehensive and detailed analysis of Parietaria judaica pollen content, we extracted total protein from 1.5 gr of mature pollen. Aliquots of the protein samples were subjected to 2-DE analysis and representative silver-stained 2D gel is shown in Fig. 1. Good reproducibility and resolution were achieved throughout the experimental procedures. On average 975 protein spots were clearly resolved using IPG strips pH 3-10 non-linear.

Protein identification

To obtain a first reference proteomic map of P.j pollen, 100 protein spots were excised from Coomassie blue stained gels and among these, 70 protein spots (corresponding to 21 different proteins) were identified by LC-MS/MS technology (Table 1; Supplementary Material). Identified proteins ranged in calculated molecular mass from 92 to 7.5 kDa and in calculated pI from 4.30 to 7.10. All proteins were designated with arbitrary spot numbers as shown in Fig. 1. While the identification of proteins by proteomics methodology has found extensive application with organisms which have a complete sequence of their genomes already available, cross species identification has been used to identify proteins from several plant species [START_REF] Faulkner | Proteomic identification of putative plasmodesmatal proteins from Chara corallina[END_REF][START_REF] Fernando | Characterization of pollen tube development in Pinus strobus (Eastern white pine) through proteomic analysis of differentially expressed proteins[END_REF][START_REF] Wang | Isolation and proteomic analysis [corrected] of cell wall-deficient Haematococcus pluvialis mutants[END_REF]. In fact, in our work, due to the poor protein and DNA sequence database coverage in Parietaria judaica (only 7 proteins are present in Uni-Prot database for this plant), MS analyses allowed the recognition of 25 protein spots from P.j and 45 from other species (Table 1; Supplementary Material). Multiple spots corresponding to the same protein were also identified, as described in other proteomic studies, and reasonably corresponding to posttranslational modifications, protein degradation, different isoforms derived from different genes of a multigene family or products of alternatively spliced mRNAs. Fig. 2 shows the distribution of P.j pollen proteins according to their functional classification. Briefly, the proteins were grouped in four distinct functional classes: proteins involved in carbon metabolism and energy generation, cytoskeletal and calcium-binding proteins, stress-related proteins, pollen allergens and proteins involved in the methionine metabolism. As concerns specifically P.j allergens, we identified 11 spots (37-47) correspond to Par j 2 P2 protein, 13 spots (49-61) correspond to Par j 2 P8 protein, while 1 spot (36) corresponds to Par j 1 P1 protein. A search of Par j 1 and 2 protein sequence, using specific softwares supported by ExPaSy Proteomics Server evidenced both O-glycosylation sites and phosphorylation sites (http://ogpet.utep.edu/OGPET and http://www.cbs.dtu.dk/services/NetPhos ).

In particular, Par j 1 P1 protein has 1 glycosylation sites and 5 phosphorylation sites, Par j 2 P8 protein has 5 phosphorylation sites and Par j 2 P2 protein has 3 phosphorylation sites. These posttranslational modifications do not change significantly the molecular weight of a protein but they may be responsible of the shifts in isoelectric points. At the same time, post-translational modifications might have an influence on the Ig binding so that the knowledge of the structure of allergens is important to improve diagnosis and therapy of allergy [START_REF] Ferreira | Modified recombinant allergens for safer immunotherapy[END_REF].

Identification of IgE reactive spots by 2-DE and pollen allergens

Although molecular biology and immunological methodologies have allowed the identification of Par j 1 and Par j 2 as the major pollen components responsible of allergic reaction and profilin as a minor allergen (Asturias et al.), the study of P.j pollen allergens have never been completed by using an immunoproteomic approach. Since Par j 1 and Par j 2 are basic proteins, to improve the separation of their several isoforms, the pollen protein samples were run on IPG strips 6-9 linear, whih allows a better separation of polypeptides with a pI higher than 7. Figure 3A shows a representative Coomassie-stained 2 D gels and corresponding immunoblot obtained by using pooled sera of allergic patients (Fig. 3B). In order to better characterize the biochemical identities of immunoreactive spots (Fig. 3A, # 1-7) they were excised from Coomassie stained 2-DE gel and subjected to LC-ESI MS/MS. Table II in Supplementary Material shows the results of protein spot identifications. Protein spots n° 1 an 2 are isoforms of Par j 1 allergen, protein spots n° 3-5 are isoforms of Par j 2 P8 while protein spots n° 6 and 7 are isoforms of Par j 2 P2 allergen. Fig. 3B, shows a representative western blot using the pooled sera from patients. In particular, the spot

Figure Legends

Figure 1 Representative proteomic map of Parietaria judaica pollen, proteins (60 µg) were loaded on IPG strips (18 cm, 3.5-10 nonlinear pH). The second dimension was performed on a vertical linear-gradient slab gel (9-16%). Gel was silver stained.

  with minor modifications. De-staining was obtained by successive washes with 20 mM NH 4 HCO 3 buffer and H 2 O/CH 3 CN (50/50) mixture for Coomassie blue. For proteolytic digestion, the gel was treated by 5-15 µl of a trypsin solution (20ng/ul in 50 mM NH 4 HCO 3 ) for 5 h at 37°C. The resulting tryptic peptides were extracted from the gel by centrifugation and the supernatant fraction was recovered. A second extraction step was performed using 10-15 µl H 2 O/CH 3 CN/HCOOH (60/36/4; v/v/v) mixture for 30 min and, finally, all extracts were pooled. For LC-ESI/MS/MS experiments, extracts were dried in a vacuum concentrator and re-suspended in 0.1 % TFA solution. NanoLC/nanospray/tandem mass spectrometry (LC-ESI/MS/MS) Experiments were performed on a Q-STAR XL instrument (Applied Biosystems, Courtaboeuf, France) equipped with a nanospray source using a distal coated silica-tip emitter (FS 150-20-10-D-20, New Objective, USA) set at 2300V. Information Dependent Acquisition (IDA) mode allowed +2 to +4 charged peptide ions within a m/z 400-2000 survey scan mass range to be analyzed for subsequent fragmentation. MS/MS spectra were acquired in the m/z 65-2000 range. The collision energy was automatically set by the software (Analyst 1.0) and was related to the charge of the precursor ion. The MS and MS/MS data were recalibrated using internal reference ions from a trypsin autolysis peptide at m/z 842.510 [M+H] + and m/z 421.759 [M+2H] 2+ . Deconvolution of the TOF MS spectra were done with Bayesian Peptide Reconstruct tool of Analyst. Multicharged ions in 700 to 1800 m/z range with 0.2 Da mass tolerance were used to calculate glycopeptide masses from 2000 to 4000 Da. Tryptic peptides were separated using an Ultimate-nanoLC (Dionex, Voisins Le Bretonneux, France) with a C 18 PepMap micro-precolumn (5µm; 100Ǻ; 300 µm x 5 mm ; Dionex) and a C 18 PepMap nano-column (3µm; 100Ǻ; 75 µm x 150 mm ; Dionex). After a 3 min. wash with 0.05% TFA in H 2 O/CH 3 CN (98/2) of the injected sample solution on the micro-precolumn, the chromatographic separation was developed using a linear 60 min gradient from 0 to 50 % B, where solvent A was 0.1 % HCOOH in H 2 O/CH 3 CN (95/5) and solvent B was 0.08 % HCOOH in H 2 O/CH 3 CN (20/80) at approximately 200 nl/min flow rate. Protein identifications were performed using the Paragon TM Algorithm thorough search mode in ProteinPilot TM software (Applied Biosystems, Courtaboeuf, France).

  indicated with arrows corresponds to Par j 1 and spot included in the square corresponds to Par j 2 P2. Control experiments, carried out with pooled sera from 10 nonsensitized subject, indicated the specificity of antibody detection (Figure3 C). Interestingly, no reaction was ever observed against Par j 2 P8 suggesting that Par j 2 P2, together with Par j 1, are the main allergenic components present in Parietaria judaica pollen.

Figure 2

 2 Figure 2 Pie chart representing the percentage distribution of identified pollen proteins within functional classes

Acknowledgments M.B. was a fellow of Regional Agency for Environmental Prevention, ARPA Sicilia. This work was supported by ARPA Sicilia; Università di Palermo (International Cooperation) to R.A.; ex 60 % MURST to R.A. and to G.D.L.

The data presented in our study show for the first time the identification of Parietaria judaica pollen allergens through bidimensional western blot thus representing satisfactory tools for immunological characterization and classification of the proteins from a raw plant extract.


Pietro Carlozzi
email: p.carlozzi@ise.cnr.it

Cristina Pintucci

Raffaella Piccardi

Arianna Buccioni

Sara Minieri

Maurizio Lambardi

Green energy from grown at low to high irradiance values

Keywords: green energy, bio-H 2, biodiesel, hydrogen production rate, R. palustris

Introduction

Purple non-sulfur photosynthetic bacteria can decompose organic acids by using light energy and nitrogenase in a photofermentation process [START_REF] Basak | The prospect of purple non-sulfur (PNS) photosynthetic bacteria for hydrogen production: the present state of the art[END_REF]. Green energies (bioH 2 and biodiesel) needs of cultural systems to be produced. Bioreactor design, hydrodynamic aspects and light distribution are key points to improve H 2 yield.

Improvements could also be achieved through the genetic modification [START_REF] Nath | Improvement of fermentative hydrogen production: various approaches[END_REF]. [START_REF] Chisti | Biodiesel from microalgae[END_REF] reported: "Biodiesel derived from oil crops is a potential renewable and carbon neutral alternative to petroleum fuels. Unfortunately, biodiesel from oil crops, waste cooking oil and animal fat cannot realistically satisfy even a small fraction of the exiting demand for transport fuels". A review of biodiesel production from oleaginous microorganisms was recently carried out by [START_REF] Ratledge | Microbial and algal oils: Do they have a future for biodiesel or as commodity oils?[END_REF]. Purple, non-sulfur photosynthetic bacteria, such as Rhodopseudomonas palustris, is able to use irradiance more efficiently than do crop plants, and oil production from bacteria could be improved under adequate or stressed culture conditions.

The purpose of this study is to investigate a co-production of bio-H 2 and rich biomass content of lipids for biodiesel production by means of Rhodopseudomonas palustris (strain 42OL) grown at low irradiance (LI) (36, 56 and 76 W m -2 ), at both medium (MI) (151 and 320 W m - 2 ) and high irradiance (HI) (500 and 805 W m -2 ). The relationship between light intensity and both hydrogen production rate and the total energy conversion efficiency has also been investigated.

Material and methods

Description of the cultural system

A cylindrical glass photobioreactor [internal diam = 9.6 cm; working volume (V) = 1.07 l] was held at 30 ± 0.2 °C. All irradiance was measured at the surface of the photobioreactor using a flat glass surface (10 x 16 cm) an averaged from nine different location points. The heat exchanger-Plexiglas basin was not filled of water during the irradiance measurements.

The average radial depth of water between the Plexiglas (inside wall) and the photobioreactor (outside wall) was 7.3 cm. The gas produced by bacteria was trapped in a graduated glass column [START_REF] Carlozzi | Fed-batch operation for bio-H 2 production by Rhodopseudomonas palustris (strain 42 OL)[END_REF].

Organism and culture conditions

Rhodopseudomonas palustris (strain 42OL) was taken from the culture collection of the former Centro di Studio dei Microrganismi Autotrofi of Florence. This centre is now part of the Institute of Ecosystem Study, CNR, Florence -Italy. The bacterium was grown at 30 °C on a medium previously described in the literature [START_REF] Carlozzi | Biomass production and studies on Rhodopseudomonas palustris grown in an outdoor, temperature controlled, underwater tubular photobioreactor[END_REF], but with glutamic acid at 0.865 g l -1 and malic acid at 3.26 g l -1 . The initial pH of the medium was 6.8.

All experiments were carried out under continuous light, at irradiances of 36, 56, 75, 151, 320, 500, and 803 W m -2 . The photobioreactor irradiated from one side was operated in a fedbatch mode [START_REF] Carlozzi | Fed-batch operation for bio-H 2 production by Rhodopseudomonas palustris (strain 42 OL)[END_REF]. This feeding strategy was used for long-term investigations (max = 408 h); otherwise, the yields produced (biomass and H 2 ) would have ceased for lack of macronutrients.

Analytical methods

Dry biomass was determined by using the empirical equation of [START_REF] Carlozzi | Biomass production and studies on Rhodopseudomonas palustris grown in an outdoor, temperature controlled, underwater tubular photobioreactor[END_REF].

Growth was determined from the bacteriochlorophyll (Bchl) concentration [START_REF] Carlozzi | Growth characteristics of Rhodopseudomonas palustris cultured outdoors, in an underwater tubular photobioreactor and investigation on photosynthetic efficiency[END_REF]. Cultures were irradiated with a 250 W OSRAM Power-star HQI-TS lamp, and the irradiance was measured using a Quantum/Radiometer/Photometer (model LI-185B, LI-COR, Lincoln, Nebraska, USA). In order to determine organic acid concentrations in the bacteria cultures, a HPLC was utilised. After disposable syringe filter units (MFS-13 mm, 0.45 µm pore size) were used to remove the cells, the supernatant was tested for malic acid.

The HPLC was equipped with a C18 analytical column (250 mm x 4.6 mm) and run at 25 °C.

The mobile phase was 0.1% H 3 PO 4 , at 1 ml min -1 .

Gas and lipid evaluation

The gas mixture produced (CO 2 and H 2 ) was first made to flow into a basin containing a saline solution absorber of NaOH, which stripped CO 2 and then the hydrogen was trapped in a calibrated column, where it was collected and the volume measured to determine hydrogen production. No CO 2 was found inside the calibrated column. This was checked by sampling 0.1 ml from the calibrate column and injecting it into a Perkin-Elmer Autosystem gas chromatograph equipped with a thermal conductivity detector (TCD) and a Silica Gel 60/80 Grade 12 column (Alltech, Derfield). The carrier gas was He; pure H 2 was used as the standard. Total lipid extraction and the fatty acid profile were determined in accordance with [START_REF] Pushparaj | Fatty acid composition of antarctic cyanobacteria[END_REF].

Light conversion efficiency

The total energy conversion efficiency (η′) was determined using the following equation: 33.61 ρH 2 VH 2 + (P B (-∆P B )) η′ (%) = - --------------------------------------------------x 100 (1)

I A tH 2 + (MA c (-∆MA)) + (GA c (-∆GA))
where ρH 2 is the concentration of H 2 (g l -1 ); VH 2 is the volume of H 2 produced (l); P B is the total biomass produced (g) (ash = 4.5% of dry biomass; (-∆P B ) is the heat of combustion of ash-free biomass (dw) (kcal g -1 ); I is the irradiance (W m -2 ); A is the irradiated area of the photobioreactor (m -2 ), which was calculated as being ½ of the cylindrical reactor surface (2πr i h), with r i and h indicating, respectively, the internal radius and the height of the cylindrical reactor; tH 2 is the evolution time (h); MA c is the malic acid consumed (g); (-∆MA) is the heat of combustion of MA (kcal g -1 ); GA c is the glutamic acid consumed (g), and (-∆GA) is the heat of combustion of GA (kcal g -1 ).

Results and discussion

Growth of R. palustris and H 2 production are shown in Fig. 1. Both bio-H 2 and dry biomass productivities and the total energy conversion efficiency versus the irradiance are shown in Fig. 2. The total H 2 produced in the photobioreactor (working volume: 1.07 l) increased in accordance with the irradiance up to 4.2 l(H 2 ) l culture -1 at 320 W m -2 . By increasing the irradiance further, the H 2 yield decreased by about 7.5%. The average H 2 production rate (HPR av ) was 12.3 ml l -1 h -1 320 W m -2 . [START_REF] Chen | Improved phototrophic H 2 production with Rhodopseudomonas palustris WP3-5 using acetate and butyrate as dual carbon substrates[END_REF] predicted an overall H 2 photoevolution rate of 12.6 ml l -1 h -1 by using acetate and butyrate as dual carbon substrates.

The yield of H 2 achieved in this study was about 33 % higher than that reported by Carlozzi and Lambardi, 2009, and the H 2 photoevolution rate was within the range of those cited in the literature for use of the same microorganism species [START_REF] Chen | Hydrogen production by indigenous photosynthetic bacterium Rhodopseudomonas palustris WP3-5 using optical fiber-illuminating photobioreactors[END_REF][START_REF] Eroğlu | Hydrogen production by Rhodobacter sphaeroides O.U.001 in a flat plate solar bioreactor[END_REF].

The highest cumulative H 2 was achieved at 320 W m -2 , while the highest dry biomass was attained at 500 W m -2 . This was because the biomass production competed with the H 2 photoevolution.

The total energy conversion efficiency was at its highest (6.9 %) at 36 W m -2 , and went progressively down as the irradiance increased. In 2009, Obeid et al. wrote: "The conversion efficiency of light energy to hydrogen is the key factor in the development of a biological process devoted to hydrogen production". The η′ of 6.9% is among the highest values cited in the literature [START_REF] Kondo | Efficient hydrogen production using a multilayered photobioreactor and a photosynthetic bacterium mutant with reduced pigment[END_REF][START_REF] Basak | Photofermentative hydrogen production using purple non-sulfur bacteria Rhodobacter sphaeroides O.U.001 in an annular photobioreactor: a case study[END_REF].

Biomasses of R. palustris harvested at the end of each fed-batch cycle, during bio-H 2 photoevolution, were analysed in order to determine the total lipid content. The results are shown in Fig. 3. The lipid content 36-39 % of the dry biomass when the irradiance was within the 56-151 W m -2 range. A further increase in the irradiance caused a relevant and progressive drop in the lipid content in the R. palustris biomasses: the minimum content ( 22%) was reached when the irradiance was at its highest (803 W m -2 ). With an irradiance of less Fig. 2. Bio-H 2 and dry-biomass productivities and the total energy conversion efficiency achieved at seven different irradiances (36, 56, 75, 151, 320, 500 and 803 W m -2 ).

Bars ± standard deviation.

Fig. 1

 1 Fig.1

Fig. 2

 2 Fig.2

Fig. 1 .

 1 Fig.3

Fig. 3 .

 3 Fig. 3. Lipid content in dry-biomass of R. palustris grown from 36 to 803 W m -2 during

Fig. 4 .

 4 Fig. 4. (A) Solar radiation and (B) predicted results of the monthly oil production from R.

Acknowledgements

We wish to thank Edoardo Pinzani for his management of the cultural system and Angelo Sacchi and Sandro Dodero for their technical assistance.

than 56 W m -2 , the lipid content of the biomass dry weight also decreased. The major fatty acid of R. palustris grown inside a tubular underwater photobioreactor (UwTP) [START_REF] Carlozzi | Biomass production and studies on Rhodopseudomonas palustris grown in an outdoor, temperature controlled, underwater tubular photobioreactor[END_REF] were 16:0 (17 %); 16:1 (7 %); 18:1 (73 %); 18:0 (3 %). Our data are similar to the four R. palustris strains cited by [START_REF] Imhoff | Lipids, quinones and fatty acids of anoxygenic phototrophic bacteria[END_REF]. Co-production of both bio-H 2 and biodiesel, as biofuels, is proposed in this study. For biodiesel production, triglycerides are transeterified with methanol. The reaction occurs stepwise: triglycerides are first converted to diglycerides, then to monoglycerides, and lastly to glycerol + methyl esters (biodiesel) [START_REF] Chisti | Biodiesel from microalgae[END_REF]. The author listed fourteen microalgal species with their respective oil contents. The oil content we found in R. palustris biomasses can be included in the group of the five microalgal species, with the highest oil content, reported by [START_REF] Chisti | Biodiesel from microalgae[END_REF].

Bacteria accumulate fewer lipids than do microalgae, and the average oil content is about 20 -40% of the dry biomass [START_REF] Meng | Biodiesel production from oleaginous microorganisms[END_REF]. Microorganisms that can accumulate lipids at more than 20 % of their biomass are defined as oleaginous species [START_REF] Ratledge | The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms[END_REF]. Since the lipid content of the R. palustris, that we investigated was within the range of 22-39 % of the dry-biomass, we could define our bacteria strain as began an oleaginous species. [START_REF] Carlozzi | Biomass production and studies on Rhodopseudomonas palustris grown in an outdoor, temperature controlled, underwater tubular photobioreactor[END_REF] reported, outdoors, a production of R. palustris dry biomass of 64 g m -2 day -1 . On the basis of Carlozzi's investigations, a projection of the monthly oil production over the years can be made from R. palustris grown outdoors in the climatic conditions of Florence, Italy (latitude 43° 46′ N, longitude 11° 15′ E) (Fig. 4).

Projections on bio-H 2 production, outdoors, were not carried out, because we did not have enough experimental results, even though a first, very short investigation has recently been carried out [START_REF] Carlozzi | Indoor and outdoor photobiological hydrogen production by Rhodopseudomonas palustris, strain 42OL[END_REF]. To reduce the greenhouse gas effect, oil production (biodiesel) from plants, crops, microalgae, yeast and, recently, bacteria has already been suggested [START_REF] Ratledge | The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms[END_REF][START_REF] Chisti | Biodiesel from microalgae[END_REF]Rittmann, 2008). Nevertheless, we would remark that this was the first time, which a non-sulfur photosynthetic bacteria (R. palustris)

has been proposed as a potential oil source for biodiesel production.