Elena Kazakou
email: elena.kazakou@cefe.cnrs.fr

François Vasseur

Kevin Sartori

Etienne Baron

Nick P Rowe

Denis Vile

Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana

Keywords: Arabidopsis thaliana, gene ontology, environment, functional traits, litter decomposability, natural variation, leaf economics spectrum, glucosinolates

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

Introduction

Tremendous efforts in trait-based ecology have helped us elucidate the drivers of key ecological processes involved in carbon and nutrient cycling, including litter decomposability [START_REF] Chapin | The Mineral Nutrition of Wild Plants[END_REF][START_REF] Coûteaux | Litter Decomposition, Climate and Liter Quality[END_REF]Swift et al., 1998;[START_REF] Cornwell | Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana[END_REF][START_REF] Kazakou | Co-variations in litter decomposition, leaf traits and plant growth in species from a Mediterranean old-field succession[END_REF]. Phenotypic drivers of litter decomposability have been mainly studied by comparative approaches at the interspecific level, which allow generalizations and predictions (Shipley, 2007;[START_REF] Garnier | Plant Functional Diversity. Organism Traits, Community Structure and Ecosystem Properties[END_REF], but remain limited in explanatory power. It is crucial to take into account intraspecific variation to understand the rules that determine community assembly and ecosystem functioning [START_REF] Crutsinger | Comparing intra-and inter-specific effects on litter decomposition in an old-field ecosystem[END_REF][START_REF] Roches | The ecological importance of intraspecific variation[END_REF]. Recent studies showed that there are similar levels of decomposability (Wright et al. 2017). Exploring the effect of genetic variability on the traits underlying these processes can be a way to reconcile ecological and evolutionary drivers (e.g., [START_REF] Donovan | The Evolution of the Worldwide Leaf Economics Spectrum[END_REF]. Here, using a large set of Arabidopsis thaliana natural genotypes, we evaluated how much of the variation in litter decomposability was correlated to genetic variation in (i) morpho-physiological traits, including leaf functional traits and plant life history traits, and (ii) leaf metabolism such as the abundance of secondary metabolites.

Among the different factors expected to impact litter decomposability, i.e. litter mass loss measured in standardized conditions, interspecific variation in leaf structure and physiology has important "after-life effects" on litter quality [START_REF] Cornelissen | Leaf structure and defence control litter decomposition rate across species and life forms in regional floras on two Continents[END_REF][START_REF] Cornelissen | Functional leaf attributes predict litter decomposition rate in herbaceous plants[END_REF][START_REF] Santiago | Extending the leaf economics spectrum to decomposition: evidence from a tropical forest[END_REF][START_REF] Parsons | Plant litter decomposition and nutrient cycling in North Queensland tropical rain-forest communities of differing successional status[END_REF]. In particular, the trade-off between carbon acquisition and nutrient retention at the leaf level (the so-called 'leaf economics spectrum', LES, [START_REF] Wright | The worldwide leaf economics spectrum[END_REF] involves a suite of leaf functional traits that explain variation in leaf decomposability across species [START_REF] Cornwell | Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana[END_REF]. For instance, specific leaf area (SLA; leaf area divided by leaf dry mass) tends to be negatively associated with high foliar concentrations of structural (e.g. lignin) or chemical compounds that promote protection against herbivores or the abiotic environment (Choong et al., 1992 ;Grime et al., 1997). SLA is related to leaf thickness and tissue density, and is positively correlated with litter decomposability across species (e.g., [START_REF] Cornelissen | Leaf structure and defence control litter decomposition rate across species and life forms in regional floras on two Continents[END_REF]Vaieretti et al., 2005;[START_REF] Santiago | Extending the leaf economics spectrum to decomposition: evidence from a tropical forest[END_REF][START_REF] Kurokawa | Leaf herbivory and decomposibility in a Malaysian tropical rain forest[END_REF]. By contrast, leaf dry matter content (LDMC; ratio leaf dry: fresh mass; [START_REF] Kazakou | Co-variations in litter decomposition, leaf traits and plant growth in species from a Mediterranean old-field succession[END_REF][START_REF] Cortez | Plant traits, litter quality and decomposition in a Mediterranean old-field succession[END_REF][START_REF] Kurokawa | Leaf herbivory and decomposibility in a Malaysian tropical rain forest[END_REF] and leaf toughness [START_REF] Cornelissen | Functional leaf attributes predict litter decomposition rate in herbaceous plants[END_REF][START_REF] Cornelissen | Leaf structure and defence control litter decomposition rate across species and life forms in regional floras on two Continents[END_REF][START_REF] Kazakou | Litter quality and decomposability of species from a Mediterranean succession depend on leaf traits but not on nitrogen supply[END_REF] have a negative effect on litter decomposability.

Finally, physical anti-herbivore defence, such as trichome density at the leaf surface, can also affect decomposition and nutrient release [START_REF] Gallardo | Leaf decomposition in two Mediterranean ecosystems of Southwest Spain: influence of substrate quality[END_REF][START_REF] Cornelissen | Functional leaf attributes predict litter decomposition rate in herbaceous plants[END_REF].

Species with long-lived leaves (high leaf lifespan, LLS) are often characterized by higher concentrations of lignin but lower concentrations of N and P (Coley, 1988). Evidence that the litter decomposability depends on the lignin : N ratio as well as on leaf N and C concentrations (Aerts, 1997 ;Cornelissen, 1996;Cornelissen & Thompson,1997) suggests that there is a link between plant physical defence and litter decomposability (Grime et al., 1996 ;[START_REF] Cornelissen | Leaf structure and defence control litter decomposition rate across species and life forms in regional floras on two Continents[END_REF].

As suggested in the review of Chomel et al. (2016), the relationships between morpho-physiological traits and litter decomposability are often idiosyncratic, which can be explained by the key role played by secondary metabolites. Several studies have demonstrated that certain secondary metabolites, especially polyphenolics, can limit growth

Accepted Article

This article is protected by copyright. All rights reserved.

and activity of decomposers, from microorganisms to macroarthropods (Hättenschwiler & Vitousek 2000;Loranger et al. 2002;Chomel et al. 2014). Litter decomposability is generally negatively correlated with the concentration of lignin, a group of complex aromatic polymers present in plant cell walls that is recalcitrant to enzymatic degradation and serves as a structural barrier impeding microbial access to labile carbon compounds [START_REF] Austin | Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana[END_REF]. Most of the studies linking secondary metabolites and decomposability have focused only on lignin, polyphenols and tannins. However, as suggested by Chomel et al. (2016), the secondary metabolites present in the leaf and subsequently in the litter should be considered as a key factor influencing decomposer activity. For instance, variation in the abundance and diversity of defence metabolites could also have after-life effect on tissue degradation by decomposers. Nonetheless, no intraspecific study has investigated and compared the respective role of plant morpho-physiological traits, including leaf traits typically related to decomposability at species level, to a large number of secondary metabolites potentially involved in litter decomposability.

A critical step in trait-based ecology is to evaluate the extent of genetic variation within species. Recent research has emphasized the importance of intraspecific variation on ecosystem functioning, including primary productivity [START_REF] Zhu | Genetic Diversity and Disease Control in Rice[END_REF][START_REF] Crutsinger | Plant Genotypic Diversity Predicts Community Structure and Governs an Ecosystem Process[END_REF], ecosystem stability [START_REF] Hughes | Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana[END_REF][START_REF] Reusch | Ecosystem recovery after climatic extremes enhanced by genotypic diversity[END_REF] and community structure (Violle et al., 2012). Like plant species, different plant genotypes can vary considerably in the quantity and quality of leaf litter produced, creating genotype-specific differences in rates of decomposition and nutrient release [START_REF] Driebe | Cottonwood hybridization affects tannin and nitrogen content of leaf litter and alters decomposition[END_REF]Treseder & Vitousek, 2001;[START_REF] Madritch | Phenotypic diversity and litter chemistry affect nutrient dynamics during litter decomposition in a two species mix[END_REF]2005;Schweitzer et al., 2005;Silfver et al., 2007). Most of the studies on intraspecific variation in decomposability have focused on tree species (for example Silfver et al., 2007 for Quercus and Alnus species and Silfver et al., 2015 for Betula pendula species) or aquatic species [START_REF] Lecerf | Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana[END_REF]. Plants have been compared between few genetically distinct genotypes (Treseder & Vitousek, 2001;[START_REF] Jackson | The within-species leaf economic spectrum does not predict leaf litter decomposability at either the within-species or whole community levels[END_REF], or from hybrids and their parental species [START_REF] Driebe | Cottonwood hybridization affects tannin and nitrogen content of leaf litter and alters decomposition[END_REF][START_REF] Schweitzer | Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana[END_REF]. For instance, Silfver et al. (2007) found that 19 genotypes of Betula pendula had substantial genotypic variation in leaf litter mass loss at the early stages of the decomposition process, and that this variation was associated with genotypic variation in herbivore resistance and leaf concentrations of soluble proteins and total nitrogen (N). [START_REF] Hines | A field facility to simulate climate warming and increased nutrient supply in shallow aquatic ecosystems[END_REF] found that variation in litter chemistry among 12 genotypes of P. australis can be as great as, or greater than, variation reported among species [START_REF] Cornwell | Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana[END_REF] or in

Accepted Article

This article is protected by copyright. All rights reserved.

response to environmental variation (Reich & Oleksyn, 2004;[START_REF] Wright | The worldwide leaf economics spectrum[END_REF][START_REF] Santiago | Extending the leaf economics spectrum to decomposition: evidence from a tropical forest[END_REF]. Finally, [START_REF] Lecerf | Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana[END_REF] showed that intraspecific variation in decomposability of senescent leaves of alder (Alnus glutinosa) was within a range similar to that reported for interspecific variation among co-occurring riparian plant species in Europe.

By constrast, Crustinger et al. (2009) found that Solidago altissima genotypic effects being much weaker than species-level effects as variation among other goldenrod species in decomposition rate was more than twice that of genetic variation within S. altissima.

Therefore, the significance of within-species variation is still poorly known (Silfver et al., 2009;2015), although it is essential to perform intraspecific analysis where phylogenetic correlation do not obscure causation. For instance, using a large number of genotypes covering a wide geographical range would enable quantitative genetic analysessuch as genome-wide association studies (GWAS)and population genetics analyses, a prerequisite for inferring evolution and adaptation. To date, the genes and molecular pathways that influence litter decomposability still remain unknown.

The model species Arabidopsis thaliana is promising to examine the phenotypic and genetic drivers of litter decomposability. Indeed, A. thaliana has been widely studied in molecular biology, cell biology and quantitative genetics because of its small stature and short life cycle (e.g., Clark et al., 2006;[START_REF] Platt | The Scale of Population Structure in Arabidopsis thaliana[END_REF][START_REF] Horton | Genome-wide patterns of genetic variation in worldwide Arabidopsis thaliana accessions from the RegMap panel[END_REF][START_REF] Alonso-Blanco | 1,135 genomes reveal the global pattern of polymorphism in Arabidopsis thaliana[END_REF]. The genomic sequencing of natural populations in this species allows performing GWAS to examine the genetic architecture of litter decomposability and identify candidate genes. Combined with its wide distribution across contrasted climates and its important phenotypic variability, A. thaliana is also becoming a species of interest for traitbased ecology. For instance, recent studies have shown the same patterns of trait covariations in A. thaliana and across species, such as relationships between SLA, LNC, leaf lifespan and photosynthetic rate (Vasseur et al., 2012;Vasseur et al., 2018b). In addition, high-throughput metabolomics has been successfully performed across hundreds of A. thaliana genotypes [START_REF] Wu | Mapping the Arabidopsis metabolic landscape by untargeted metabolomics at different environmental conditions[END_REF]. This generated massive and unprecedented data to investigate the role that leaf metabolites could have on specific ecological processes. For instance, glucosinolates are important secondary metabolites involved in plant defence among species of the Brassicaceae family. In A. thaliana, they are notably involved in plant defence against herbivores, bacteria, and fungi [START_REF] Kliebenstein | Comparative analysis of quantitative trait loci controlling glucosinolates, myrosinase and insect resistance in Arabidopsis thaliana[END_REF][START_REF] Reichelt | Benzoic acid glucosinolate esters and other glucosinolates from Arabidopsis thaliana[END_REF]. However, their potential role in litter decomposability remains to be elucidated.

In the present study, we hypothesized that litter decomposability is related to the heritable variation of underlying traits, and thus genes. We tested this hypothesis using 211

Version postprint

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. (2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Accepted Article

This article is protected by copyright. All rights reserved.

genotypes of A. thaliana and addressed four questions. First, does genetic variation for litter decomposability occurs within a global set of genotypes of A. thaliana? Second, at the within-species level, is litter decomposability better explained by morpho-physiological traits or by secondary metabolites? Third, is intraspecific variation in litter decomposability correlated with climate at the genotypes' collection sites? Finally, is litter decomposability heritable and what are the genes involved in this after-life process?

Materials and Methods

Plant material and experimental design

We randomly sampled 211 genotypes from the RegMap collection (Fig. 1, Table S1) [START_REF] Horton | Genome-wide patterns of genetic variation in worldwide Arabidopsis thaliana accessions from the RegMap panel[END_REF]. Seeds used in the present study were provided by Fabrice Roux (Laboratoire des interactions plantes microorganisms LIPM, Auzeville). Plants were grown in three replicates in a semi-controlled greenhouse environment. Seeds were sown on organic soil and stratified at 4 °C for four days. At the emergence of the first two true leaves, plants were transplanted in 300 mL individual pots filled with a 1:1 (v:v) mixture of loamy soil and organic compost (Neuhaus N2). Pots were randomly distributed among three blocks (one replicate per block) that were rotated every day. All pots were watered twice a week. Additional light provided ca. 65 µmol m -2 s -1 photosynthetic photon flux density (PPFD) at plant height. Photoperiod and temperature were kept constant at 12 h day length, and 18/16 °C day/night, respectively.

Litter collection and decomposition assay

Plant litter was collected at the moment of maximum leaf senescence for each genotype. As

A. thaliana retain dead leaves on the rosette, we cut off leaves that were dead in order to prevent decomposition by fungi when litters were in contact with the soil. Litters were carefully cleaned, then air-dried and stored in the laboratory. The decomposability of each litter sample was assessed using a standard laboratory bioassay in microcosms as described by Wardle et al. (2002) (for a detailed description see Supplementary Methods S1).

The percentage of the initial litter mass remaining after incubation (LMR, %) was calculated as: LMR= (Litter mass initial -Litter mass final )/Litter mass initial * 100, where Litter mass initial is the dry litter leaf mass at the beginning of incubation and Litter mass final the dry litter leaf mass after the 60 days of incubation in the microcosms.

Version postprint

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. ( 2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Accepted Article

This article is protected by copyright. All rights reserved.

Measurement of plant morpho-physiological traits

Plant morpho-physiological traits, including leaf functional traits and plant life history traits,

were measured using standardized procedures in three replicates per genotype [START_REF] Pérez-Harguindeguy | New handbook for standardize measurement of plant functional traits worldwide[END_REF]. For each plant, leaf lifespan (LLS) was estimated as the average number of days from leaf emergence to full senescence using daily pictures of one plant replicate per genotype where three consecutive leaves were tracked from emergence to full senescence. One adult, fully-exposed and non-senescent leaf was harvested from each individual (n = 3 per genotype) at bolting stage. Harvested leaves were water-saturated in deionised water at 4 °C for 24 h, scanned for leaf area, measurement (LA, mm 2 ) and weighed. Specific leaf area (SLA, m 2 kg -1 ) and leaf dry matter content (LDMC, mg g -1 ) were calculated as the ratio between leaf area and leaf dry mass, and between leaf dry mass and saturated fresh mass, respectively. Average trichome density (cm -2 ) was estimated from trichome counts on adaxial and abaxial leaf sides of two 28 mm 2 zones of the harvested leaf.

We determined the leaf nitrogen content (LNC, %) on one leaf replicate per genotype with an et al., 2012, 2018a, 2018b).

Leaf metabolites dataset

Quantitative variation in untargeted metabolitesi.e. all measurable metaboliteshas been measured recently across 309 genotypes by [START_REF] Wu | Mapping the Arabidopsis metabolic landscape by untargeted metabolomics at different environmental conditions[END_REF]. Wu and colleagues reported variation for 4,182 metabolites in non-stressing conditions. 91 genotypes were common to [START_REF] Wu | Mapping the Arabidopsis metabolic landscape by untargeted metabolomics at different environmental conditions[END_REF] and the present study. Due to missing data, only 2,132 metabolites were retained for subsequent analyses here. Four genotypes among the 91 had missing data for one or several metabolites, which were inputted with the 'missMDA' package in R. We first investigated whether there were correlations between decomposition rate and metabolites

Version postprint

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. ( 2019 4, see later). In doing so, our approach was voluntarily not oriented toward a specific class of metabolites, but rather to test if there is an effect of metabolites on decomposability over the broadest range of metabolites that we could use. To compare the role of different classes of metabolites, we used the set of 103 identified metabolites from [START_REF] Wu | Mapping the Arabidopsis metabolic landscape by untargeted metabolomics at different environmental conditions[END_REF], and investigated the correlations between each metabolite and LMR.

Spearman's coefficients of correlation were reported, after grouping by metabolite class (amino acid -amine, coenzyme, flavonoid, glucoside -sugar, glucosinolate, nucleosidenucleotide and their derivative, organic acid, and phenylpropanoid). For convenience, amines and amino acids were treated together, as well as sugars and glucoside, and nucleotide, nucleoside and their derivatives. Only the classes containing at least three identified metabolites were kept for the analysis.

Statistical analyses

For each trait, genotypic means (trait mean by genotype) were estimated from linear models (package 'lsmeans'). Principal Component Analyses (PCA) were performed separately on morpho-physiological traits (morpho-physiological PCA), metabolites (metabolomic PCA)

and climate variables (climatic PCA) using the 'ade4' package. The morpho-physiological PCA was performed across the 91 genotypes with both morpho-physiological traits and metabolites, using leaf area, leaf resistance to shearing (LRS), SLA, LDMC, leaf lifespan (LLS), bolting time (FT), nitrogen concentration (LNC) and trichome density. Metabolomic PCA was also performed on the same 91 genotypes, using the 2,132 untargeted metabolites measured by [START_REF] Wu | Mapping the Arabidopsis metabolic landscape by untargeted metabolomics at different environmental conditions[END_REF]. Climatic PCA was also perfomed on the 91 accessions, using the climate variables at the collection points of the genotypes extracted from WorldClim database (http://www.worldclim.org/bioclim). For each PCA, we retained all principal components (PCs) that explain more than 5% of total genetic variability (Fig. S2) for subsequent analyses.

To quantify the respective role of functional traits and metabolites on litter decomposability, we performed a multivariate random forest model, using the 'randomForest' package in R, which allows model classification based on Breiman's random forest algorithm [START_REF] Breiman | Random Forests[END_REF]. All PCs were scaled (standard deviation = 1) and centered (mean = 0) prior to analysis. In addition, and following previous approaches (e.g., Shantz & Burkepile, 2014; [START_REF] Barnagaud | Species' thermal preferences affect forest bird communities along landscape and local scale habitat gradients[END_REF][START_REF] Leonard | Determinants of the occurrence of unburnt forest patches: Potential biotic refuges within a large, intense wildfire in south-eastern Australia[END_REF]Welti & Joern, 2014), we

Accepted Article

This article is protected by copyright. All rights reserved.

fitted with linear regressions all possible combinations of explanatory variables (i.e. morphophysiological PCs and metabolomic PCs) nested within the full model, using the dredge function in the 'MuMIn' package (Barton, 2012). Then, we plotted the averaged coefficients fitted for each explanatory variable across all possible models. This approach is powerful, as it helps to minimize loss of information contained in the data [START_REF] Schielzeth | Simple Means to Improve the Interpretability of Regression Coefficients[END_REF]. Moreover, since predictor variables (i.e. PCs) were rescaled before analysis, the distribution of these fixed effects can be interpreted as effect sizes. That is, a coefficient with value +1 indicates that a 1 SD increase in the predictor variables (i.e. PCs) directly causes a 1 SD increase in the response variable (i.e. LMR), after accounting for any other predictor variables [START_REF] Blonder | Structural and defensive roles of angiosperm leaf venation network reticulation across an Andes-Amazon elevation gradient[END_REF]. We followed the same procedure for climate PCs. All analyses were performed using R 3.15 (R Core Team, version 3.5.3, 2019).

Quantitative genetics analysis

Classical ('monogenic') GWAS (genome-wide association studies) were performed on 214,050 biallelic SNPs from the publicly available RegMap genetic panel [START_REF] Horton | Genome-wide patterns of genetic variation in worldwide Arabidopsis thaliana accessions from the RegMap panel[END_REF]. GWAS exploits natural genotypic variation and enables the analysis of significant associations between hundreds of thousands of single-nucleotide polymorphisms (SNPs) and specific phenotypes [START_REF] Horton | Genome-wide patterns of genetic variation in worldwide Arabidopsis thaliana accessions from the RegMap panel[END_REF]. GWAS was carried out using EMMAX [START_REF] Kang | Variance component model to account for sample structure in genome-wide association studies[END_REF] implemented in the online platform easyGWAS (https://easygwas.ethz.ch/; [START_REF] Grimm | easy GWAS': Cloud based platform for comparing the results of genome-wide association studies[END_REF], with minimum allele frequency at each SNP above 5%, and which including the two first axes of a PCA performed on SNPs as covariates to take into account population structure in A. thaliana. Significance threshold that considers multiple testing correction was determined with the Bonferroni method (α = 0.05 or 0.1).

Polygenic GWAS were performed with Bayesian Sparse Linear Mixed model (BSLMM) implemented in EMMA [START_REF] Zhou | Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana[END_REF], which accommodates both poly-and oligogenic architectures. Chip heritability (h 2 ) was calculated from BSLMM models as the sum of phenotypic variance explained by all SNPs, assuming genetic additivity. Given the importance of metabolome PC3, we performed BSLMM and GO analysis to examine the biological functions associated with this axis. By contrast with monogenic GWAS, BSLMM models two effect hyperparameters, a basal effect, alpha, that captures the fact that many SNPs contribute to the phenotype, and an extra effect, beta, which captures the stronger effect of only a subset of SNPs. We summed alpha and beta to estimate total SNP effect and investigate the correlation between SNP effects on LMR and metabolome PC3, respectively.

We then used the 1% top-SNPs (i.e. 0.5% at the lower tail and 0.5% at the upper tail) of the

Version postprint

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. ( 2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Accepted Article

This article is protected by copyright. All rights reserved.

distribution of SNP effect to identify the genes that contributed most to the phenotypic effect.

Gene names were extracted from SNPs position using gene annotation along the A. thaliana genome from the TAIR10 GFF3 file. This resulted in 1,910 top-genes in total, for LMR, and 3,398 top-genes for metabolome third PC axis. We then performed Gene Ontology (GO) analysis using the AgriGO [START_REF] Du | agriGO: a GO analysis toolkit for the agricultural community[END_REF] and REVIGO online tools to visualize significant enrichment in GO categories (Supek et al., 2011).

Results

Genetic variability in litter decomposability and morpho-physiological traits

We found a marked variation in litter decomposability among the 211 genotypes of A.

thaliana (Fig. 2): litter mass remaining (LMR) ranged from 21 to 57 %. Significant variability was also found in morpho-physiological traits (Fig S1 ): trichome density was the most variable trait (25-fold variation; from 0 to 24 trichomes per cm 2 , Fig. S1H). LES traits were also very variable, notably leaf dry matter content (LDMC) (5.4-fold variation between 65 and 351 mg g -1 , Fig. S1C) and leaf nitrogen content (LNC) (20.8-fold variation between 0.4% and 8%, Fig. S1F), while specific leaf area (SLA) and leaf life span (LLS) varied 5 and 3.5 times (from 12.3 to 91.3 m 2 kg -1 and from 15 to 53.5 d, respectively) (Fig. S1D andS1G).

In addition, flowering time and leaf area varied between 25 and 113 days, and between 104 and 811 mm 2 , respectively (Fig. S1A andS1B).

Comparing the effect of plant morpho-physiological traits and metabolites on litter decomposability

The two first PCs of the morpho-physiological PCA explained together 80% of the multivariate genetic variability (62% and 18% for PC1 and PC2, respectively; Fig. 3A), while PC3 and PC4 explained 10% and 5%, respectively, of total genetic variability. PC1 represented the leaf economics spectrum: positive PC1 values were associated with acquisitive strategies characterized by high specific leaf area (SLA) and high leaf nitrogen content (LNC), but short lifespan, low leaf dry matter content (LDMC) and low structural resistance. Inversely, negative PC1 values were associated with conservative strategies characterized by opposite leaf trait values. PC2 was mainly explained by variation in leaf area (Fig. 3A). The four first PCs of the metabolomic PCA together explained 36% of metabolomic variability (PC1: 13%, PC2: 11%, PC3: 7%, PC4: 5%; Fig. 3B).

We then used random forest models and multiple model evaluation to compare the respective role of morpho-physiological trait variation and metabolomic variation on litter

Version postprint

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. ( 2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Accepted Article

This article is protected by copyright. All rights reserved.

mass remaining (LMR). Models were performed on the 91 genotypes subset, using the first five PCs of both morpho-physiological traits and metabolomic PCA. Random forest model indicated that PC3 of the metabolomic PCA was the main contributor of litter mass remaining (LMR), followed by the second PCA axis of morpho-physiological traits (Fig. 3C).

By comparison, intraspecific variations related to the leaf economics spectrum and structural resistance (morpho-physiological PC1) had only small effects on litter mass remaining (LMR) variability among genotypes, even less than morpho-physiological PC4 and metabolomic PC2 (Fig. 3C). Consistently, multiple model evaluation suggested that metabolomic PC3 had the strongest effect (+40%) on litter mass remaining (LMR), while morpho-physiological PC1 had no significant effect (Fig. 3D). Multiple model evaluation also suggested that morpho-physiological PC2 had a significant negative effect on litter mass remaining (LMR), suggesting that bigger leaves had higher decomposability (i.e. lower LMR).

Using a set of 103 metabolites identified from [START_REF] Wu | Mapping the Arabidopsis metabolic landscape by untargeted metabolomics at different environmental conditions[END_REF], we explored the role of different classes of metabolites on leaf decomposability (Fig. 3; Table S2). Results suggest that glucosinolates, a group of secondary metabolites involved in plant defence in Brassicaceae like A. thaliana, also had globally a negative effect on decomposability, as illustrated by the positive correlations between individual glucosinolates and LMR ( 14glucosinolates out of 32 were significantly and positively correlated with LMR, P < 0.05; Table S2). In addition, amino acids composition also had a notable effect on decomposability, with some amino acids being negatively correlated with LMR (e.g., Lglutamic acid, indole-3-lactic acid, homoserine; Table S2) and others being positively correlated (e.g., L-tryptophan, methionine, valine; Table S2). The same result was found for flavonoids, nucleotides/nucleosides and organic acids (Fig. 4). Finally, phenylpropanoids were on average negatively correlated with LMR, although none of the correlation with the underlying metabolites was significant (P > 0.05; Table S2).

Effect of climate of origin on litter decomposabiilty

The PCA on the 19 climate variables that describe the environment of origin of each genotype (Fig. 5A) showed that PC1 was mainly associated to temperature variables (with those related to winter temperature as top contributors), while with PC2 mainly accounted for precipitation variables (with those related to summer precipitation as top contributors).

Together, PC1 and PC2 accounted for 62% of variability (PC1 = 35%, PC2 = 27%). Rainfallrelated variables at collection sites (PC2) had a small but significant effect on litter mass

Version postprint

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. ( 2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Accepted Article

This article is protected by copyright. All rights reserved.

remaining (LMR (Fig. 5B), suggesting that adaptation to water availability leads to variation in leaf properties that can alter decomposability. However, the lack of correlation between climatic PC2 and metabolomic PC3 (r 2 = 0.01, P = 0.30) suggests that the changes in leaf properties associated with precipitation are independent from the metabolites identified here as associated with litter decomposability (metabolomic PC3). Finally, morpho-physiological PC1 was negatively correlated to climatic PC1, relating species with high acquisitive strategies to warmer temperatures than species with conservative strategies (Table S3).

Genetic bases of litter decomposability

Chip heritability of litter mass remaining, a measure of narrow-sense heritability (h 2 ), was estimated to 0.25 with BSLMM. This indicated a substantial genetic control of litter decomposability, presumably because the underlying determinants of litter decomposability (e.g., leaf metabolome) have a strong genetic basis. No SNP was significantly associated with LMR along the genome after multiple testing correction (Fig. 6A), which can be explained by a large number of genes contributing to the phenotype, each with small effecti.e. a 'polygenic' architecturerather than few genes with strong effect on the phenotype, as classically tested under the monogenic GWAS. We found that the 1,910 top-genes involved in LMR were indeed significantly enriched for specific cellular components such as proteins localized in plasma membrane and chloroplast envelope (Fig. 6B). In addition, we also found a significant enrichment in the biological processes related to post-embryonic development, post-translational protein modification and transport (Fig. S3C). Finally, the molecular functions of genes involved in litter decomposability were significantly enriched for functions related to ATP binding and ATPase activity, as well as transmembrane transporter activity (Fig. S3E).

Given the importance of metabolomic PC3, we performed BSLMM and GO analysis to examine the biological functions associated with this axis. Interestingly, GO analysis of the 1% top-genes revealed a significant enrichment for proteins localized in plasma membrane and chloroplast envelope (Fig. 6C), similarly to what has been found for LMR (Fig. 6B).

Metabolomic PC3 was also significantly enriched for 1,3-beta-D-glucan, a key polymer of cell wall composition. Moreover, we estimated with BSLMM the SNP effects on each individual variable (i.e. metabolomic PC3 and LMR), and inspected their correlation (Fig. 6D). As expected many SNPs had a null effect on each variable, generating a cross along the zero axes when plotting the relationship between SNP effects on each variable in Fig. 6D.

However, we found that SNP effects on metabolomic PC3 were significantly and positively

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. ( 2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Accepted Article

This article is protected by copyright. All rights reserved.

correlated with SNP effects on LMR (r = 0.05, P < 0.001; Fig. 6D), which suggests that differences in litter decomposability across A. thaliana genotypes are at least in part explained by variation in metabolomic PC3, such as the chemical properties of plasma membranes and cell wall.

Discussion

In this study, we examined the drivers of litter decomposability within species by comparing the role of plant morpho-physiological traits and leaf metabolites among A. thaliana genotypes. Our results showed that litter decomposability vary by 2.7-fold and is partly heritable (h 2 = 0.25). This heritable variation of litter decomposability can be attributed to adaptation to different precipitations across the distribution range of A. thaliana, and it is mainly due to genetic variations in leaf metabolites rather than the genetic variations in plant morpho-physiological traits typically found at the species level.

The intraspecific range of variation in litter decomposability observed in this study is comparable to the variability found in several interspecific analyses. For example, litter mass remaining (LMR) from several herbaceous species measured in similar conditions (leaf litter incubated in microcosms for 90 days with standardized soil and controlled temperature and soil humidity) ranged from 30% for Crepis foetida to 92% for Carex humilis (3-fold) (data from Kazakou et al., 2007 and[START_REF] Bumb | Traits determining the digestibility-decomposability relationships in species from Mediterranean rangelands[END_REF]. Previous studies at the interspecific level have demonstrated that litter decomposability is partly controlled by tissue nutrient concentration and by the density of structural material in the leaf [START_REF] Cornelissen | Leaf structure and defence control litter decomposition rate across species and life forms in regional floras on two Continents[END_REF]Wardle et al., 2002[START_REF] Cornwell | Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana[END_REF]. For instance, nutrient concentration in green leaves, such as nitrogen, carbon or phosphorus content, are likely to be linked with litter nitrogen content or with the litter lignin:nitrogen ratio [START_REF] Wright | The worldwide leaf economics spectrum[END_REF]. Traits such as leaf dry matter content (LDMC) might also reflect structural support and defence against herbivores, which makes them good predictors of litter quality and thus litter decomposability across species [START_REF] Cornelissen | Functional leaf attributes predict litter decomposition rate in herbaceous plants[END_REF][START_REF] Kazakou | Co-variations in litter decomposition, leaf traits and plant growth in species from a Mediterranean old-field succession[END_REF]. Indeed, positive correlations were observed, always at the interspecific level, between leaf dry matter content (LDMC) and lignin content [START_REF] Wright | The worldwide leaf economics spectrum[END_REF][START_REF] Kazakou | Co-variations in litter decomposition, leaf traits and plant growth in species from a Mediterranean old-field succession[END_REF]Quested et al., 2007;Fortunel et al., 2009). Together, these traits are part of the leaf economics spectrum that describes resource-use strategies of living leaves [START_REF] Wright | The worldwide leaf economics spectrum[END_REF]. We thus tested the hypothesis that, within species, litter decomposability can be predicted by a set of leaf traits in a manner similar to what has been observed across species, such as traits related to the leaf economics spectrum. In particular, we expected a positive relationship between resource-

Accepted Article

This article is protected by copyright. All rights reserved.

acquisitive strategies and litter decomposability. Our results showed that the leaf economics trade-off was indeed reflected by leaf trait covariations across the set of genotypes studied, in a similar way as at the interspecific level. This variation was captured by morphophysiological PC1: rapid acquisition of resources is generally correlated with high specific leaf area (SLA) and leaf nitrogen content (LNC), while high leaf dry matter content (LDMC), high leaf resistance to shearing (LRS) and long leaf life span (LLS) reflect a resource conservation strategy. Interestingly, both flowering time and trichome density were negatively correlated with these traits, suggesting that late-flowering plants have conservative strategies with abundant trichomes at the leaf surface. This relationship is consistent with the analysis of ecological strategies among A. thaliana genotypes (Vasseur et al., 2018).

However, despite the large range of variation in functional leaf traits, our initial hypothesis was not supported by our analyses. Neither particular leaf traits nor morpho-physiological PC1 explained the intraspecific variation in litter decomposability in A. thaliana.

Secondary metabolites could also impact litter decomposability and therefore nutrient turnover. For example, numerous studies have pointed out the role of secondary metabolites (lignin, cellulose, hemicellulose) as regulating factors of litter decomposability [START_REF] Berg | Litter decomposition and organic matter turnover in northern forest soils[END_REF], but the role of secondary metabolites still remain largely underexplored. Our results showed that a large number of metabolites, together grouped along metabolomic PC3, was the main contributor of the variation of litter mass remaining (LMR) in A. thaliana. Furthermore, genetic analysis showed that the top-genes associated with litter mass remaining (LMR) variation between genotypes are mainly localized in cell and organelle envelopes. Interestingly, the same enrichment was observed for the genes associated with metabolomic PC3, and SNP effects measured for litter mass remaining (LMR) and metabolomic PC3 were significantly correlated. Thus, litter decomposition in A. thaliana seems to be mainly driven by the abundance and diversity of metabolites, notably those related to the chemical composition of cell and organelle envelopes. In addition, glucosinolates were also negatively with the rate of leaf decomposition across genotypes pointing the role of defence metabolites in after-life effects.

However, we have to notice that our results were based on the secondary metabolites identified by [START_REF] Wu | Mapping the Arabidopsis metabolic landscape by untargeted metabolomics at different environmental conditions[END_REF] and other unidentified metabolites may play an important role to the decomposition process. The remarkable diversity of secondary compounds that have been quantified from the model plant A. thaliana, present tremendous challenges for the study and interpretation of secondary metabolism.

We found that intraspecific variation in litter decomposability is shaped by numerous genes along the genome, each with relatively weak effect (i.e. a polygenic architecture). Monogenic GWAS did not reveal any significant association, presumably because no gene had an effect strong enough

Accepted Article

This article is protected by copyright. All rights reserved.

to reach significance threshold after multiple testing correction. This polygenic architecture of leaf decomposability is consistent with a prominent role of secondary metabolites, which are numerous and diverse, and encoded by many genes. We also found a positive correlation between climatic PC2 and litter mass remaining (LMR). Hence, drier environments might have favored genotypes with higher LMR (i.e. lower decomposability). This positive relationship between precipitation and litter decomposability corroborates interspecific findings where leaves with recalcitrant molecules are selected in mesic conditions (Quested, 2008;[START_REF] Cornwell | Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana[END_REF]. A complicating factor is that the temperature and water sensitivity of litter decomposability is also dependent on the quality of the carbon compounds in litter: litter enriched in C-based secondary compounds (i.e. low quality) is more sensitive to temperature and precipitation change than litter with high amounts of-quality C compounds [START_REF] Fierer | Litter quality and the temperature sensitivity of decomposition[END_REF]. However, our analysis suggests that variation in litter mass remaining associated with adaptation to water availability is independent of the class of metabolites involved in decomposition, as climatic PC2 and metabolomic PC3 were not correlated. A possible explanation is that adaptation to water availability relies on diverse metabolites that were not associated with a specific metabolomic PC, and not with the axis related to cell and organelle envelopes (PC3) in particular. This relation is consistent with recent findings that showed the polygenic architecture of drought adaptation in A. thaliana [START_REF] Exposito-Alonso | The rate and potential relevance of new mutations in a colonizing plant lineage[END_REF]. Together, our results suggested that selection for resistance to specific stresses during plant life can have after-life effects on important ecological processes such as microbial activity and nutrient cycling.

Further studies will be required to identify precisely the metabolites having an after-life effect.

Conclusion

We find a large genetic variability in litter decomposability as well as in morpho-physiological traits, but they were not correlated with each other. By contrast, metabolites located in cell membranes and envelopes played a significant role in litter decomposability. In addition, metabolites related to plant defence, such as glucosinolates, were also negatively correlated with decomposability. Thus, our results illustrated the prominent effect of secondary metabolites on after-life processes at the intraspecific level. Further explorations of the associations between decomposability and secondary metabolites (and especially glucosinolates) are needed, as well as the genes and physiological mechanisms that explain the variation in litter decomposability along environmental gradients. In this perspective, manipulating specific genes related to glucosinolates biosynthesis, for instance using mutants and/or introgression lines, offers a promising avenue to confirm the role of these metabolites in litter decomposability. Moreover, using reciprocal transplants of different genotypes

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. ( 2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Accepted Article

This article is protected by copyright. All rights reserved.

along specific environmental gradients (related to temperature, precipitation and/or nutrient availability), to measure changes in metabolomic profiles and decomposition rate, would allow teasing apart the effects of different environmental conditions on litter decomposability and its metabolic drivers.

  elemental analyzer(model EA 1108; Carlo Erba Instruments, Milan, Italy). According toOnoda et al. (2011) an approximation of the structural resistance to shearing (LRS, J m -1 ) was measured using a protocol similar to that of[START_REF] Ang | Novel way of measuring the fracture toughness of leaves and other thin films using a single inclined razor blade[END_REF] using a single blade cutting device mounted on a portable Instron inspec 2200, instrumented device that measured the force (N) and distance of the downward cutting blade, (Instron Engineering Corp., Canton, MA, USA). Leaves were cut in the middle position of the leaf length, from the leaf margin up to but not including the midrib. The horizontal distance cut was calculated via the vertical displacement and the angle of the blade set to 30° from the horizontal. Flowering time (FT) was measured at bolting stage (first emergence of the floral bud) and was included because it represents a major trait associated with plant life history and resource-use strategies (Vasseur

  all metabolites measured inWu et al. (Fig. 3C, D, see later). Then, we went further by looking more specifically to a subset of 103 metabolites for which functions were identified in[START_REF] Wu | Mapping the Arabidopsis metabolic landscape by untargeted metabolomics at different environmental conditions[END_REF] (Fig.

). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Accepted Article

This article is protected by copyright. All rights reserved. over

  Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. (2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Acknowledgements

We thank Fabrice Roux for providing the seeds of some of the genotypes used in this study. This work was supported by INRA and CNRS. This publication has been written with the support of the Agreenskills fellowship programme which has received funding from the EU's Seventh Framework Programme under the agreement N° FP7-609398 (Agreenskills contract 3215 'AraBreed' to FV).

Accepted Article

This article is protected by copyright. All rights reserved. Schweitzer JA, Joseph K. Bailey JK, Hart SC, Whitham TG. 2005. Non-additive effects of mixing cottonwood genotypes on litter decomposition and nutrient dynamics. Ecology 86 (10): 2834-40. Shantz AA, Burkepile DE 2014. Context-dependent effects of nutrient loading on the coralalgal mutualism. Ecology 95: 1995-2005. Shipley B. 2007. Comparative Plant Ecology as a Tool for Integrating Across Scales. Annals of Botany 99 (5): 965-966. Silfver T, Mikola J, Rousi M, Roininen H, Oksanen E. 2007. Leaf litter decomposition differs among genotypes in a local Betula pendula population. Oecologia 52 (4): 707-14. Silfver T, Roininen H, Oksanen E, Rousi M. 2009. Genetic and environmental determinants of silver birch growth and herbivore resistance. Forest Ecology and Management 257 (10): 2145-49. Silfver T, Paaso U, Rasehorn M, Rousi M, Mikola J. 2015. Genotype × herbivore effect on leaf litter decomposition in Betula pendula saplings: ecological and evolutionary consequences and the role of secondary metabolites. Plos one 10(1): 1-15. Supek F, Bošnjak M, Škunca N, Šmuc T. 2011. REVIGO summarizes and visualizes long lists of gene ontology terms. PloS one 6.7: e21800. Swift MJ, Andrén O, Brussard M, BRiones MM, Couteaux E, Ekschmitt A, Kjoller A, Loiseai P, Smith P. 1998. Global Change, Soil Biodiversity, and Nitrogen Cycling in Terrestrial Ecosystems: Three Case Studies. Global Change Biology 4 (7): 729. Treseder KK, Vitousek PM. 2001. Effects of soil nutrient availability on investment in acquisition of N and P in Hawaiian rain forests. Ecology 82 (4): 946-54. Vaieretti MV, Pérez Harguindeguy N, Gurvich DE, Cingolani M, Cabido M. 2005. Decomposition dynamics and physico-chemical leaf quality of abundant species in a montane woodland in Central Argentina. Plant and Soil 278 (1-2): 223-34. Vasseur F, Violle C, Enquist BJ, Granier C, Vile D. 2012. A common genetic basis to the origin of the leaf economics spectrum and metabolic scaling allometry. Ecology Letters 15: 1149-1157 Vasseur F, Exposito-Alonso M, Ayala-Garay OJ, Wang G, Enquist BJ, Vile D, Violle C, Weige D. 2018a. Adaptive diversification of growth allometry in the plant Arabidopsis thaliana. Proceedings of the National Academy of Sciences 115(13): 3416-3421.

Version postprint Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. (2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983 Accepted Article This article is protected by copyright. All rights reserved. Vasseur F, Sartori K, Baron E, Fort F, Kazakou E, Segrestin J, Garnier E, Vile D, Violle C. 2018b. Climate as a driver of adaptive variations in ecological strategies in Arabidopsis thaliana. Annals of Botany 122 (6): 935-945. Violle C, Enquist, BJ, McGill BJ, Jiang L, Albert, CH, Hulshof C, Jung V, Messier J. 2012. The return of the variance: intraspecific variability in community ecology. Trends in Ecology and Evolution. 27(4): 244-52. Wardle DA, Bonner KI, Barker GM. 2002. Linkages between plant litter decomposition,

Accepted Article

This article is protected by copyright. All rights reserved.

Version postprint

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. ( 2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Accepted Article

This article is protected by copyright. All rights reserved.

Version postprint

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. ( 2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Accepted Article

This article is protected by copyright. All rights reserved.

Version postprint

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. ( 2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Accepted Article

This article is protected by copyright. All rights reserved.

Version postprint

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. ( 2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Accepted Article

This article is protected by copyright. All rights reserved.

Version postprint

Comment citer ce document : Kazakou, E., Vasseur, F., Sartori, K., Baron, Rowe, N., Vile, D. ( 2019). Secondary metabolites have more influence than morphophysiological traits on litter decomposability across genotypes of Arabidopsis thaliana . New Phytologist, 224 (4), 1532-1543. , DOI : 10.1111/nph.15983

Authors contributions

EK, FV, KS, EB and DV planned and designed the research. EK, FV, KS, EB, NR performed experiments, conducted fieldwork, analysed data. EK and FV wrote the manuscript. EK and FV contributed equally to this work.

Accepted Article

This article is protected by copyright. All rights reserved. S1. List of accessions with measured phenotypic traits. S2. Correlation of LMR with leaf traits, leaf-traits PCs and metabolomic PCs. S3. Sixty top contributors of metabolomic PC3.

Supplemental

Supplemental Table

Supplemental Table

Supplemental Table

Accepted Article

This article is protected by copyright. All rights reserved.

metabolomic and morpho-physiological PCs on LMR (n = 91). All PCs were scaled prior to analysis.

Accepted Article

This article is protected by copyright. All rights reserved.


B A Rakha

M. S Ansari

S Akhter

E Blesbois

Allah Bushra
email: bushrauaar@gmail.com

Rakha

Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study

Keywords: Semen characteristics, Age, Season, Quantitative semen quality, Qualitative semen quality, Indian red jungle fowl

The reproductive potential of the adult males is expected to vary with age/season and largely differ not only in closely related avian species but even in subspecies, breeds and/or strains of the same species. Thus, it is pre-requisite to have knowledge of seminal parameters to achieve maximum production potential of at-risk species for ex situ in vitro conservation programs. A 4-year study was designed to evaluate the effect of age and season (spring, summer, autumn and winter) on semen characteristics of Indian red jungle fowl (Gallus gallus murghi) in retrospective manner. Semen ejaculates (n=1148) were regularly collected from eight adult Indian red jungle fowls from 6 to 54 months of age. Quantitative and qualitative semen parameters viz; volume (µL), concentration (1x10 9 mL -1 ), total sperm number per ejaculate (1x10 9 mL -1 ), motility (%), viability (%), plasma membrane integrity (%), acrosome integrity (%) and semen quality factor were recorded. A chronological increasing trend with age of most sperm quantitative and qualitative traits (semen volume, sperm concentration, total sperm number per ejaculate, plasma membrane integrity, viability, acrosomal integrity, semen quality factor) was observed. The highest values were observed at four years of age (P < 0.05) with the exception of sperm motility that was not affected by the age. Spring was the best season for sperm parameters viz; volume, motility, plasma membrane integrity, viability and acrosomal integrity (P < 0.05), however a remarkable sperm production was noticed all over the year. It is concluded that Indian red jungle fowl exhibit an evolution of sperm production that greatly differs in many points from other fowl sub-species. It is suggested that semen ejaculates of highest quality achieved for semen banking at the age of four year in the spring season.

Introduction

The red jungle fowl, a member of family Phasianidae and genus Gallus is considered one of the ancestors of the domestic fowl [START_REF] Delacour | The pheasants of the world[END_REF][START_REF] Gautier | Gallus gallus" (On-line), Animal Diversity Web[END_REF][START_REF] Eriksson | Andersson LIdentification of the yellow skin gene reveals a hybrid origin of the domestic chicken[END_REF]. The red jungle fowl is enlisted as least concern species in IUCN Red List of Threatened Species [START_REF]IUCN Red list of threatened species 2015-4[END_REF] but its population is continuously declining in its native habitat [START_REF] Gautier | Gallus gallus" (On-line), Animal Diversity Web[END_REF][START_REF] Gallus Gallus | The IUCN Red List of Threatened Species[END_REF]. Red jungle fowl is considered as endangered in Singapore list of Threatened Plant and Animal Species, facing high risk of extinction in the wild [START_REF] Wang | An annotated checklist of birds of Singapore[END_REF]. From five fowl subspecies, Indian red jungle fowl (Gallus gallus murghi) is native to Northern India and Azad Jammu and Kashmir, Pakistan [START_REF] Subhani | Population status and distribution pattern of red jungle fowl (Gallus gallus murghi) in Deva Vatala National Park, Azad Jammu & Kashmir, Pakistan: A pioneer study[END_REF][START_REF] Rakha | Semen characteristics of the Indian Red Jungle Fowl (Gallus gallus murghi)[END_REF][START_REF] Rakha | Impact of ejaculate frequencies on the quality of Red Jungle Fowl (Gallus gallus murghi) semen[END_REF][START_REF] Rakha | Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen[END_REF]. The Indian red jungle fowl population is found in restricted areas in fragments, and is more likely to collapse of genetic diversity and population decline due to sudden unexpected threats [START_REF] Gautier | Gallus gallus" (On-line), Animal Diversity Web[END_REF][START_REF] Peterson | Genetic endangerment of wild Red Jungle fowl Gallus gallus?[END_REF][START_REF] Mukesh | Identification of galliformes through forensically informative nucleotide sequencing (FINS) and its implication in wildlife forensics[END_REF][START_REF] Kumar | Genetic diversity and population structure analysis between Indian red jungle fowl and domestic chicken using microsatellite markers[END_REF]. The factors contributing in population reduction are habitat destruction, egg collection, predation and genetic hybridization with domestic fowl [START_REF] Subhani | Population status and distribution pattern of red jungle fowl (Gallus gallus murghi) in Deva Vatala National Park, Azad Jammu & Kashmir, Pakistan: A pioneer study[END_REF][START_REF] Kumar | Genetic diversity and population structure analysis between Indian red jungle fowl and domestic chicken using microsatellite markers[END_REF].

The decline in Indian red jungle fowl needs special attention; several reports suggest adopting appropriate ex situ conservation strategies [START_REF] Gautier | Gallus gallus" (On-line), Animal Diversity Web[END_REF][START_REF] Peterson | Genetic endangerment of wild Red Jungle fowl Gallus gallus?[END_REF][START_REF] Mukesh | Identification of galliformes through forensically informative nucleotide sequencing (FINS) and its implication in wildlife forensics[END_REF]. For ex situ in vitro conservation, semen banking is considered one of the most applicable options for bird species that are facing continuous decline in their natural habitat and the threats cannot be easily mitigated and/or ameliorated [START_REF] Blanco | Species variation in osmotic, cryoprotectant, and cooling rate tolerance in poultry, eagle, and peregrine falcon spermatozoa[END_REF][START_REF] Łukaszewicz | Attempts on freezing the Greylag (Anser anser L.) gander semen[END_REF][START_REF] Blesbois | Semen cryopreservation for ex situ management of genetic diversity in chicken: creation of the French avian cryobank[END_REF][START_REF] Blesbois | Predictors of success of semen cryopreservation in chickens[END_REF][START_REF] Blanco | Comparative cryopreservation of avian spermatozoa: benefits of non-permeating osmoprotectants and ATP on turkey and crane sperm cryosurvival[END_REF][START_REF] Blanco | Comparative cryopreservation of avian spermatozoa: effects of freezing and thawing rates on turkey and sandhill crane sperm cryosurvival[END_REF][START_REF] Rakha | Evaluation of Tris-citric acid, skim milk and sodium citrate extenders for liquid storage of Punjab Urial (Ovis vignei punjabiensis) spermatozoa[END_REF]. Cryo-banking of valuable male germplasm could be an important assisted reproductive biotechnology to maintain genetic diversity and in vitro ex situ conservation program [START_REF] Rakha | Semen characteristics of the Indian Red Jungle Fowl (Gallus gallus murghi)[END_REF][START_REF] Rakha | Impact of ejaculate frequencies on the quality of Red Jungle Fowl (Gallus gallus murghi) semen[END_REF]. It is well established that avian semen can be stored in frozen state for infinite period of time without significant decline in fertilizing potential [START_REF] Blesbois | Predictors of success of semen cryopreservation in chickens[END_REF][START_REF] Holt | Basic aspects of frozen storage of semen[END_REF][START_REF] Blesbois | Freezing avian semen[END_REF]. The characteristics of the semen ejaculate viz; volume, concentration, motility, viability, plasma membrane and acrosome integrity, semen quality factor determine the reproductive potential of the cocks either used in natural or artificial breeding program [START_REF] Rakha | Semen characteristics of the Indian Red Jungle Fowl (Gallus gallus murghi)[END_REF][START_REF] Rakha | Impact of ejaculate frequencies on the quality of Red Jungle Fowl (Gallus gallus murghi) semen[END_REF][START_REF] Saacke | Semen quality in relation to semen preservation[END_REF][START_REF] Siudzińska | Effect of semen extenders and storage time on sperm morphology of four chicken breeds[END_REF][START_REF] Lierz | A novel method for semen collection and artificial insemination in large parrots (Psittaciformes)[END_REF]. Nevertheless, semen quality varies with age and season of collection in birds that may affect fertilizing capacities accordingly [START_REF] Sexton | Effects of dietary energy on semen production, fertility, plasma testosterone, and carcass composition of broilerbreeder males in cages[END_REF][START_REF] Anderson | The semen of animals and its use for artificial insemination[END_REF][START_REF] Oguntunji | The influence of seasonal variation on performance of a commercial laying strain in a derived savanna environment in Nigeria[END_REF]. The success of semen banking depends Comment citer ce document : Rakha, B., Ansari, M., Akhter, S., Blesbois on successful cryopreservation and acceptable frozen thawed semen quality and fertility outcomes that depend on the initial quality of the ejaculate to be used in processing for cryopreservation [START_REF] Blesbois | Predictors of success of semen cryopreservation in chickens[END_REF][START_REF] Rakha | Evaluation of Tris-citric acid, skim milk and sodium citrate extenders for liquid storage of Punjab Urial (Ovis vignei punjabiensis) spermatozoa[END_REF][START_REF] Ansari | Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa[END_REF]. The reproductive performance of the male fowl varies with age and seasons; largely differ not only in closely related bird species but also even in subspecies, breeds and/or strains of the same species [START_REF] Elagib | The effects of age and season on semen characteristics of white leghorn cocks under Sudan conditions[END_REF]. Scarce available literature depicted immense variations in semen characteristics of five subspecies of red jungle fowl [START_REF] Rakha | Semen characteristics of the Indian Red Jungle Fowl (Gallus gallus murghi)[END_REF][START_REF] Malik | Evaluation of the ejaculate quality of the red jungle fowl, domestic chicken, and bantam chicken in Malaysia[END_REF]. The knowledge about retrospective seminal parameters is therefore the perquisite to achieve maximum production potential of at-risk species for ex situ in vitro conservation.

Therefore, present, 4-year retrospective study was designed to elucidate the effect of age and season (autumn, spring, summer and winter) on semen characteristics (motility, semen volume, sperm concentration, total sperm number per ejaculate, viability, semen quality index, plasma membrane and acrosome integrity) of Indian red jungle fowl.

Materials and methods

Experimental birds

The chicks of Indian red jungle fowl were reared at Semen collection of eight cocks was performed on alternate days, depending on individual cock quality and libido.

Semen collection and quantitative evaluation

The birds were subjected to semen collection training through abdominal massage as described by Burrows and Quinn [START_REF] Rakha | Semen characteristics of the Indian Red Jungle Fowl (Gallus gallus murghi)[END_REF][START_REF] Burrows | A method of obtaining spermatozoa from the domestic fowl[END_REF][START_REF] Rakha | Comparison of extenders for liquid storage of Indian red jungle fowl (Gallus gallus murghi) spermatozoa[END_REF]. The successful ejaculate was collected after 4 weeks of training. Semen was collected from individual birds in a graduated plastic tube. A total of 1148 ejaculates were studied during the 4-year study period (2012)(2013)(2014)(2015). Semen volume was measured in microlitres using micropipette.

Sperm progressive motility

Sperm motility of each ejaculate was determined as described by Zemjanis [START_REF] Zemjanis | Diagnostic and therapeutic technique in animal reproduction[END_REF] by mixing 10 µL of semen samples in 500 µL of phosphate buffer saline (pH 7.2, 300 mOsm/kg). The percentage of motile sperm was subjectively evaluated on a scale ranging from 0 to 100% by putting a drop of semen sample on a pre-warmed glass slide (37 o C) under phase contrast microscope (x400, Olympus BX20, Japan).

Sperm concentration

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6
Neubauer haemocytometer (Marienfeld, Germany) under phase contrast microscope (x400, Olympus BX20, Japan) throughout the 4-year study period [START_REF] Rakha | Semen characteristics of the Indian Red Jungle Fowl (Gallus gallus murghi)[END_REF].

Total sperm number per ejaculate

Total sperm number per ejaculate was obtained by multiplying the total volume with the concentration of ejaculates obtained during the study period [START_REF] Rakha | Semen characteristics of the Indian Red Jungle Fowl (Gallus gallus murghi)[END_REF][START_REF] De Reviers | Predicting the adult dailysperm output after the first ejaculates in cockerels raised under different photoschedules[END_REF].

Plasma membrane integrity

Plasma membrane integrity of spermatozoa was assessed by using hypo-osmotic swelling test (HOS) as described by Santiago-Moreno et al. [START_REF] Santiago-Moreno | Use of the hypo-osmotic swelling test and aniline blue stainingtoimprove the evaluation of seasonal sperm variation in native Spanish freerange poultry[END_REF]. The HOS solution was prepared by adding 1 g of sodium citrate to 100 mL of distilled water. Semen (25 µL) was mixed with 500 µL of HOS solution (100 mOsm/kg) and incubated at 37 o C for 30 minutes. A drop of incubated solution was placed on a pre warmed (37 o C) slide and fixed in buffered 2%

glutaraldehyde. The spermatozoa showing swollen heads, swollen and coiled tails were classified as normal spermatozoa having intact plasma membrane. A total of 200 spermatozoa were counted at four separate fields under a phase-contrast microscope (1000x with oil immersion).

Sperm viability

Viability (% Live / total sperm) of Indian Red Jungle Fowl spermatozoa was examined by adding eosin-nigrosin to the lake glutamate solution [START_REF] Bakst | Determination of sperm concentration II. Establishing a standard curve. Techniques for semen evaluation, semen storage, and fertility determinationEd[END_REF]. Twelve drops of stain were mixed with 1 drop of semen. A smear was made on a glass slide, fixed and air dried. A total of 200 spermatozoa were studied per slide under a phase-contrast microscope (1000x with oil immersion). The mixture provides a clear background in the smear to enhance the contrast of white and pink. Unstained and straight sperm were considered as "live and morphologically normal" while the pinkish stained as "dead " sperm.

Acrosomal integrity

Comment citer ce document : Rakha, B., Ansari, M., Akhter, S., Blesbois Acrosomal integrity of Indian Red Jungle Fowl spermatozoa was assessed through Giemsa stain [START_REF] Jianzhong | Methods and effects of Hongshan cock spermatozoa cryopreservation[END_REF]. The stain was prepared by adding Giemsa (3g) and phosphate buffer saline at pH 7.0 (2 mL) into 35 mL water. Smear was prepared by taking a drop of semen sample on a clean glass slide, dried and fixed in neutral formal-saline (5% formaldehyde) for 30 min.

Fixed slides were kept in Giemsa stain for 1.5 hrs. Sperm with normal acrosome appeared to be evenly stained; abnormal spermatozoa were unevenly stained while spermatozoa having ruptured acrosome remained unstained. A total of 200 spermatozoa were observed at least in four separate fields under a phase-contrast microscope (x400; Olympus BX20, Japan) at magnification of1000x with oil immersion.

Sperm quality factor

Sperm quality factor (SQF) of each semen ejaculate was calculated to check the number of spermatozoa potentially capable of fertilization in ejaculate. Sperm quality factor was calculated by the following formula [START_REF] Siudzińska | Effect of semen extenders and storage time on sperm morphology of four chicken breeds[END_REF].

SQF = Sperm concentration ቀ ୠ୧୪୪୧୭୬ ୫ ቁ x

Discussion

In conservation programs, the success of AI and semen cryopreservation depends on the initial semen quality of ejaculate [START_REF] Blesbois | Predictors of success of semen cryopreservation in chickens[END_REF]. Semen evaluation, based on in vitro measurement of sperm motility, plasma membrane integrity, viability, acrosomal integrity and semen quality factor (SQF) provides an indirect clue about the fertility potential of the males [START_REF] Lierz | A novel method for semen collection and artificial insemination in large parrots (Psittaciformes)[END_REF][START_REF] Rakha | Semen characteristics of the Indian Red Jungle Fowl (Gallus gallus murghi)[END_REF][START_REF] Saacke | Semen quality in relation to semen preservation[END_REF][START_REF] Siudzińska | Effect of semen extenders and storage time on sperm morphology of four chicken breeds[END_REF]. The production of high quality semen ejaculates depends on the reproductive cycle of the species; furthermore, species or subspecies specific variations depend on the birds, their age, their environment including climate changes such as seasons [START_REF] Elagib | The effects of age and season on semen characteristics of white leghorn cocks under Sudan conditions[END_REF][START_REF] Santiago-Moreno | Use of the hypo-osmotic swelling test and aniline blue stainingtoimprove the evaluation of seasonal sperm variation in native Spanish freerange poultry[END_REF][START_REF] Santiago-Moreno | Characterization of red-legged partridge (Alectoris rufa) sperm: seasonal changes and influence of genetic purity[END_REF][START_REF] Bah | Semen characteristics of local breeder cocks in the Sahel region of Nigeria[END_REF][START_REF] Dumpala | The effect of semen storage temperature and diluent type on the sperm quality index of broiler breeder semen[END_REF][START_REF] Obidi | Seasonal variations in seminal characteristics of Shikabrown breeder cocks[END_REF][START_REF] Tabatabaei | Correlation between age of rooster and semen quality in Iranian indigenous broiler breeder Chickens[END_REF][START_REF] Shanmugam | Effect of age on semen quality in naked neck and dwarf chicken under tropical climatic conditions[END_REF][START_REF] Sonseeda | Effects of Environmental Factors, Ages and Breeds on Semen Characteristics in Thai Indigenous Chickens: A One-year Study[END_REF][START_REF] Churchil | Semen quality parameters, their interrelationship and post-washing sperm attributes of Rhode Island Red roosters[END_REF].

M A N U S C R I P T A C C E P T E D

In contrast to domestic fowl, we showed for the first time in the present study that the highest production and quality of semen of Indian red jungle fowl is not reached until the fourth year of age and spring stays the best season.

Our results greatly differ from the observations made in most domestic fowl as Long et al. [START_REF] Long | Rooster semen cryopreservation: effect of pedigree line and male age on postthaw sperm function[END_REF] reported a decrease in sperm concentration from 1 to 1.5 years of age, a wellknown fact in the breeding practice. Nevertheless, this situation would be modulated in local breeds of tropical area as Chotesangasa [START_REF] Chotesangasa | Effects of mating ratio, cock number in the flock and breeder age on fertility in Thai native chicken flock[END_REF] and Sonseeda et al. [START_REF] Sonseeda | Effects of Environmental Factors, Ages and Breeds on Semen Characteristics in Thai Indigenous Chickens: A One-year Study[END_REF] found an increase in Thai indigenous chicken sperm concentration between 1 and 2 years of age. However, our results with Indian red jungle fowl appeared to be "extreme" as 4 years of age is the best in terms of sperm concentration and volume. Semen volume has already been reported to differ with the age in Leghorns [START_REF] Elagib | The effects of age and season on semen characteristics of white leghorn cocks under Sudan conditions[END_REF]. Contrary to our findings, broiler males showed maximum semen volume in the first year of age and then decreased with age, a difference with here, one of the presumed ancestors, the red jungle fowl. As a way of consequence, the total number of sperm per ejaculate increased with the age, and was observed highest when the Indian red jungle fowls were 4 years old. This leads to suggest an increase in the efficiency of sperm production with the age in this wild fowl. Many hypotheses may arise from this observation including suggestion of increasing adaptation of the species to its environment, nutrition, and management conditions. However, due to low mean volume of semen, total sperm number per ejaculate (billion) were still lower in Indian red jungle fowl compared to Malaysian red jungle fowl [START_REF] Malik | Evaluation of the ejaculate quality of the red jungle fowl, domestic chicken, and bantam chicken in Malaysia[END_REF] and domestic fowls [START_REF] Long | Rooster semen cryopreservation: effect of pedigree line and male age on postthaw sperm function[END_REF][START_REF] Hafez | Reproduction in Farm Animals[END_REF]. Would there be a sort of "compensation" of this low daily sperm production through and increasing of the longevity of sperm production?

In addition to the increase of sperm production with the age, it was evident that the functionality of the sperm produced by Indian red jungle fowl increased with the age to reach a maximum at 4 years as most of the quality parameter measured here (Hypo-osmotic test, livability, acrosome integrity) were at their optimum at 43-54 months of age. More precisely, Indian red jungle fowl spermatozoa showed greater resistance to the hypo-osmotic solution at 37°C at 4 years of age. These changes would indicate that sperm plasma membrane increases its "plasticity" with the age and thus its functionality. Indeed, the response of spermatozoa towards hypo-osmotic solution in terms of swollen head and coiled tail is due to the movement of physical and biochemical compounds which play essential roles in maintaining the viability and fertilizing ability of sperm [START_REF] Zavos | Effects of various degrees of supercooling and nucleation temperatures on fertility of frozen turkey spermatozoa[END_REF][START_REF] Daader | Response of spermatozoa of different breeds of rabbits to hypo osmotic swelling test[END_REF][START_REF] Ramu | The hypo-osmotic swelling test for evaluation of sperm membrane integrity[END_REF]. Shanunagum et al. [START_REF] Shanmugam | Effect of age on semen quality in naked neck and dwarf chicken under tropical climatic conditions[END_REF] reported that response of spermatozoa to the hypo-osmotic environment at one year of age was maximum compared to 6 months of age; and described that age and genetic factors contribute to the quality of semen in naked neck and dwarf chicken. But none of these studies examined 3 or 4 years old fowl, thus limiting the potential comparison with our study.

Sperm viability and the integrity of the sub-cellular fraction of sperm such as the acrosome are of absolute significance for the success of fertilization. In the present study, viability and acrosome integrity were highest at the age of 43-54 months, a fact that is opposite to the results described in previous reports. Indeed, decreases in viability and sperm morphological integrity have previously been reported to coincide with the end of a reproductive period between 1 and 1.5 years of age in free-range poultry [START_REF] Shanmugam | Effect of age on semen quality in naked neck and dwarf chicken under tropical climatic conditions[END_REF], turkeys [START_REF] Douard | Reproductive period affects lipid composition and quality of fresh and stored spermatozoa in turkeys[END_REF] and roosters [START_REF] Kelso | Lipid and antioxidant changes in semen of broiler fowl from 25 to 60 weeks of age[END_REF][START_REF] Cerolini | Relationship between spermatozoan lipid composition and fertility during aging of chickens[END_REF]. Such decrease has been related to changes in lipid composition and decrease in fertility [START_REF] Cerolini | Relationship between spermatozoan lipid composition and fertility during aging of chickens[END_REF]. However, for these parameters also, the comparison between the literature results and our results is limited since older ages were not explored in earlier studies. One hypothesis to explain results could be due to late optimal sexual maturity in Indian red jungle fowls. The SQF is dependent on the quantitative parameters of semen and on one qualitative parameter, the number of morphologically normal and live spermatozoa. In the present study, the SQF was significantly higher at 43-54 months of age compared to other ages. As discussed earlier, the Indian red jungle fowl is a resident of Temperate and Tropical forest and developed a high tolerance to environmental variations that could be "cost- intensive" for the animal metabolism and consequently the sexual maturation. We suggest that the Indian red jungle fowl would get fully mature at later ages compared to other fowls [START_REF] Malik | Evaluation of the ejaculate quality of the red jungle fowl, domestic chicken, and bantam chicken in Malaysia[END_REF].

Sperm motility was the only quality factor that was not affected by the age in the present study, partially confirming the results obtained by Sonseeda et al. [START_REF] Sonseeda | Effects of Environmental Factors, Ages and Breeds on Semen Characteristics in Thai Indigenous Chickens: A One-year Study[END_REF] on Thai indigenous chicken of over 30 months. Sperm motility is a predictive parameter of viability and quality of semen. However, in the present study, this parameter did not follow the evolution of the viability measurements. This could indicate a specific reproductive maturation process where the different functions of the sperm do not reach the maturity at the same time; the signaling pathways and flagellar structures related to motility being fully mature much earlier than plasmatic membranes and acrosome attributes.

The sperm production, volume and concentration of sperm were higher in spring and low in the other seasons with a remarkably low semen volume in summer. The sperm production is highly species-specific and even varies among different strains or breeds [START_REF] Churchil | Semen quality parameters, their interrelationship and post-washing sperm attributes of Rhode Island Red roosters[END_REF],

and may vary considerably throughout the year and seasons among avian species [START_REF] Elagib | The effects of age and season on semen characteristics of white leghorn cocks under Sudan conditions[END_REF][START_REF] Bah | Semen characteristics of local breeder cocks in the Sahel region of Nigeria[END_REF].

Environmental factors such as photoperiod and temperature affect semen production. In mild climates with large range of photoperiods/temperatures, domestic fowl sperm production is totally stopped for some months during autumn/winter and reaches maximum during spring.

In subtropical desert and in Saharan climates with a smaller range of annual photoperiods, sperm production may be severely affected by the extremely hot temperatures and lack of precipitation of the summer [START_REF] Elagib | The effects of age and season on semen characteristics of white leghorn cocks under Sudan conditions[END_REF][START_REF] Peltoniemi | Seasonal and management effects on fertility of the sow: a descriptive study[END_REF]. These environmental cues have also been expressed in a breed specific manner. Some breeds show consistent semen production throughout the year as in Thai chicken held in tropical humid climates [START_REF] Sonseeda | Effects of Environmental Factors, Ages and Breeds on Semen Characteristics in Thai Indigenous Chickens: A One-year Study[END_REF][START_REF] Aengwanich | Effects of high environmental temperature on blood indices of Thai indigenous chickens, thai indigenous chickens crossbred and broilers[END_REF][START_REF] Penfold | Seasonal patterns of LH, testosterone and semen quality in the Northern pintail duck (Anas acuta)[END_REF].

Our study shows that the seasonal sperm production of Indian red jungle fowl is not so far from the Thai indigenous chicken "model", but also shows characteristics close to the "Saharan model" with summer variations potentially related to the high temperatures of the Pakistan summer. Indeed, deleterious effects of high temperatures on germ cells have already been described [START_REF] Duangduen | Effect of hsp70 Genotypes on Thermotolerance in Thai Native Chicken and Broilers[END_REF]. The results of the present study are in agreement with previous studies on Northern pintail ducks and Nigerian local domestic chicken in which high summer temperatures caused temporary reduction in sperm production [START_REF] Bah | Semen characteristics of local breeder cocks in the Sahel region of Nigeria[END_REF][START_REF] Tabatabaei | Correlation between age of rooster and semen quality in Iranian indigenous broiler breeder Chickens[END_REF][START_REF] Duangduen | Effect of hsp70 Genotypes on Thermotolerance in Thai Native Chicken and Broilers[END_REF]. The low volume of semen in hot summer could tentatively be related to the absence of cutaneous heat evaporation that cannot mitigate the stressful events of heat disruption in summer season [START_REF]The signifiance of meteorology in animal production[END_REF].

In our study, spring was identified as the best season for the SQF that mixes quantitative and viability sperm traits and for many other sperm quality traits (motility, viability, acrosome integrity). In mild climates, the lengthening of photoperiod in spring season acts as an environmental cue, stimulates the secretion of FSH, LH and testosterone, which play important role in spermatogenesis and semen production [START_REF] Thurston | Spermiogenesis in commercial poultry species: anatomy and control[END_REF]. Photoperiod increase has previously been reported to increase the number of morphologically normal and live sperm as well as motility and SQF in domestic fowls such as free range poultry, Leghorns and others [START_REF] Elagib | The effects of age and season on semen characteristics of white leghorn cocks under Sudan conditions[END_REF][START_REF] Sexton | New Poultry Semen Extender 2. Effect of the Diluent Components on the Fertilizing Capacity of Chicken Semen Stored at 5°[END_REF][START_REF] Giesen | Beltsville poultry semen extender. 9. Effect of storage temperature on turkey semen held eighteen hours[END_REF]. At the opposite, poor livability and viability of sperm in winter or autumn have directly been associated with the shortening of the photoperiod in freerange poultry [START_REF] Santiago-Moreno | Influence of Season on the Freezability of Free-Range Poultry Semen[END_REF] and turkey [START_REF] Douard | Reproductive period affects lipid composition and quality of fresh and stored spermatozoa in turkeys[END_REF]. Poor motilities were also reported in correlation with loss of energy and mitochondrial integrity in shortened photoperiod compared to spring collected semen [START_REF] Long | Avian semen cryopreservation: what are the biological challenges?[END_REF]. Seasonal differences in mitochondrial permeability [START_REF] Savina | Bioenergetic parameters of lamprey and frog liver mitochondria during metabolic depression and activity[END_REF] and its membrane phospholipids composition [START_REF] Martin | Effect of day length on oxidative capacities of mitochondria from red muscle of rainbow trout (Oncorhynchus mykiss). Comparative Biochemistry and Physiology Part A[END_REF] could also contribute to explain the greater sensitivity of sperm in time of short photoperiod [START_REF] Santiago-Moreno | Influence of Season on the Freezability of Free-Range Poultry Semen[END_REF].

In the "Saharan climate," the semen quality of Nigerian shika brown in terms of live and normal sperm was reported maximum in winter season, which is in contrast to the present study [START_REF] Rekwot | ,x Sperm plasma membrane integrity (%) Autumn 16[END_REF]. The reason of these differences could possibly be attributed to heterogeneity of

  document : Rakha, B., Ansari, M., Akhter, S., Blesbois, E. (2017). Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study. Theriogenology, 99, 105-110. DOI : 10.1016/j.theriogenology.2017.05.016 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Comment citer ce document : Rakha, B., Ansari, M., Akhter, S., Blesbois, E. (2017). Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study. Theriogenology, 99, 105-110. DOI : 10.1016/j.theriogenology.2017.05.016 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Comment citer ce document : Rakha, B., Ansari, M., Akhter, S., Blesbois, E. (2017). Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study. Theriogenology, 99, 105-110. DOI : 10.1016/j.theriogenology.2017.05.016 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Comment citer ce document : Rakha, B., Ansari, M., Akhter, S., Blesbois, E. (2017). Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study. Theriogenology, 99, 105-110. DOI : 10.1016/j.theriogenology.2017.05.016 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Comment citer ce document : Rakha, B., Ansari, M., Akhter, S., Blesbois, E. (2017). Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study. Theriogenology, 99, 105-110. DOI : 10.1016/j.theriogenology.2017.05.016 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Comment citer ce document : Rakha, B., Ansari, M., Akhter, S., Blesbois, E. (2017). Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study. Theriogenology, 99, 105-110. DOI : 10.1016/j.theriogenology.2017.05.016 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

  The data on ejaculate volume, sperm concentration and total sperm number per ejaculate of red jungle fowl from 6 to 54 months of age are given in Table1. Semen volume, sperm concentration and total sperm number per ejaculate was recorded significantly (P<0.05) higher at 43-54 months of age followed by 31-42, 19-30 and 6-18 months of age.The highest (P<0.05) semen ejaculate volume, sperm concentration and total sperm number were recorded in spring season followed by winter, autumn and summer from 6 to 54 months of age.The data on qualitative semen quality parameters viz; motility, plasma membrane integrity, viability, acorosme integrity and semen quality factor were given in Table2. Sperm motility did not differ (P>0.05) with increase in age from 6 to 54 months of age.

		8
		ACCEPTED MANUSCRIPT
	3.	Results
		M A N U S C R I P T
		A C C E P T E D

ejaculate volume ሺmLሻ x normal livespermatozoa 100

2.3 Statistical analysis

The data were analyzed with statistical software Megastat ® version 7.25 Mc-Graw-Hill New Media, New York, for excel. The descriptive statistical parameters (mean, standard error of mean) were calculated for different age groups and seasons. To compare differences between seasons and age groups, data were analyzed using analysis of variance in completely randomized design with unequal replications. Post hoc comparisons between the means were done though least significant difference test to determine differences among individual groups at different ages and seasons. Differences were considered statistically different when P<0.05, and presented as Mean ± SEM. Comment citer ce document : Rakha, B., Ansari, M., Akhter, S., Blesbois, E. (2017). Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study. Theriogenology, 99, 105-110. DOI : 10.1016/j.theriogenology.2017.05.016 Nevertheless, an increase was observed in plasma membrane integrity, viability, acrosome integrity and semen quality factor with the increasing age. The highest (P<0.05) values for aforementioned parameters were recorded during 43-54 months of age compared to 31-42, 19-30 and 6-18 months of age. A highly positive (P<0.05) effect of spring season was recorded on most of the quality parameters with the exception of motility that was equivalently efficient in autumn, spring and summer and only altered in winter.

  Comment citer ce document : Rakha, B., Ansari, M., Akhter, S., Blesbois, E. (2017). Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study. Theriogenology, 99, 105-110. DOI : 10.1016/j.theriogenology.2017.05.016

	12
	ACCEPTED MANUSCRIPT
	M A N U S C R I P T
	A C C E P T E D

  among breeds, strains and species-specificity[START_REF] Long | Rooster semen cryopreservation: effect of pedigree line and male age on postthaw sperm function[END_REF], but also to the specific Saharan climate. The terrible dry and hot summer would have a significant role in the adaptation of the reproductive season, the best season of reproduction being consequently moved to winter. Regarding this parameter, the "Pakistan" environment of the Indian red jungle fowl presents a more classical but "mixed" seasonal effect since sperm production is present all over the year.It is concluded that Indian red jungle fowls exhibit better semen quality in spring season and chronological increasing trend with maximal quality at the age of 43-54 months in terms of volume, sperm concentration, total sperm number per ejaculate, motility, viability, semen quality factor, plasma membrane and acrosome integrity. It is suggested that semen ejaculates of maximal quality can be achieved at the age of four years in the spring season.

	13
	ACCEPTED MANUSCRIPT
	the semen quality
	M A N U S C R I P T
	A C C E P T E D

Comment citer ce document : Rakha, B., Ansari, M., Akhter, S.,

Blesbois

, E. (2017). Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study. Theriogenology, 99, 105-110. DOI : 10.1016/j.theriogenology.2017.05.016 These elements are very important in order to build an AI program for the conservation of genetic diversity of the Indian red jungle fowl. Comment citer ce document : Rakha, B., Ansari, M., Akhter, S., Blesbois, E. (2017). Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study. Theriogenology, 99, 105-110. DOI : 10.1016/j.theriogenology.2017.05.016

Version postprint

Comment citer ce document : Rakha, B., Ansari, M., Akhter, S., Blesbois • Semen quantitative and qualitative traits increased with the increasing age.

• Spring was the best season for the production of high quality ejaculate at 4-years of age.

• The reproductive potential of IJRF is highly variable in many points from its descendants.