Rui Hu

Peng Sun

Hui Yu
email: yuhui@scbg.ac.cn

Yufen Cheng

Rong Wang

Xiaoyong Chen
email: xychen@des.ecnu.edu.cn

Finn Kjellberg

Similitudes and differences between two closely related Ficus species in the synthesis by the ostiole of odors attracting their host-specific pollinators: A transcriptomic based investigation

Keywords: Volatile organic compounds, Agaonidae, Communication, Pollination

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Introduction

Pollinators constitute a strong selective factor acting on floral traits. It may results in the evolution of suites of traits preferentially attractive to particular groups of pollinators [START_REF] Kemp | Dominant pollinators drive nonrandom community assembly and shared flower colour patterns in daisy communities[END_REF]. Such suites of traits constitute pollination syndromes. They have been shown to constitute good predictors of pollinators within regional floras [START_REF] Johnson | Stefan Vogel's analysis of floral syndromes in the South African flora: an appraisal based on 60 years of pollination studies[END_REF], and convergent evolution of flower traits associated with pollinator shifts have been demonstrated (e.g. [START_REF] Rosas-Guerrero | A quantitative review of pollination syndromes: do floral traits predict effective pollinators?[END_REF][START_REF] Smith | Convergent evolution of floral shape tied to pollinator shifts in Iochrominae (Solanaceae)[END_REF]. In addition to flower colour and shape, floral odors constitute an important component of the floral display used by pollinators, as evidenced for individual species [START_REF] Byers | Three floral volatiles contribute to differential pollinator attraction in monkeyflowers (Mimulus)[END_REF] and for plant communities as a whole [START_REF] Kemp | Dominant pollinators drive nonrandom community assembly and shared flower colour patterns in daisy communities[END_REF]. Floral odors may vary in response to pollinator shifts [START_REF] Schiestl | Pollinator-mediated evolution of floral signals[END_REF]. Floral odor and flower colour modification are often due to the modification of regulatory genes and mutation in a single gene can control modification of a given trait [START_REF] Sheehan | Color and scent: how single genes influence pollinator attraction[END_REF][START_REF] Schiestl | Pollinator-mediated evolution of floral signals[END_REF]. Nevertheless, because of the diffuse nature of many pollination systems, the relevance of the pollination syndrome concept has been questioned: determining causality can be challenging [START_REF] Ollerton | A global test of the pollination syndrome hypothesis[END_REF].

Tight species-specific plant-pollinator interactions provide useful model systems to decipher the selective pressures and the adaptive responses underlying the evolution of floral odors and floral odor perception [START_REF] Friberg | Extreme diversification of floral volatiles within and among species of Lithophragma (Saxifragaceae)[END_REF]. Specialized pollination is especially frequent in species rich tropical ecosystems (Johnson and Steiner,2000). Within this context, the mutualism between Ficus and their pollinating fig wasps represents a unique system to investigate the evolution of olfactive signaling by plants and its perception by their pollinators [START_REF] Hossaert-Mckey | Floral scents: their roles in nursery pollination mutualisms[END_REF]. Indeed, this mutualism forms the most extreme example of plant-insect co-diversification documented to date; coevolution between floral odor and its perception is expected [START_REF] Cruaud | An extreme case of plant-insect codiversification: figs and fig-pollinating wasps[END_REF] an ostiole closed by bracts, and oviposit in some flowers, inducing galls in which their offspring develop, and also fertilise other flowers that will produce seeds [START_REF] Weiblen | How to be a fig wasp[END_REF]. Fig wasps only live a few days [START_REF] Weiblen | How to be a fig wasp[END_REF]. It is therefore critical for them to rapidly find receptive figs of their host species. In Ficus, as in nursery pollination systems in general, floral odors play a central role in pollinator attraction [START_REF] Hossaert-Mckey | Floral scents: their roles in nursery pollination mutualisms[END_REF]. [START_REF] Hossaert-Mckey | Chemosensory attraction of fig wasps to substances produced by receptive figs[END_REF][START_REF] Chen | Responses of the pollinating wasp Ceratosolen solmsi marchali to odor variation between two floral stages of Ficus hispida[END_REF][START_REF] Proffit | Signalling receptivity: comparison of the emission of volatile compounds by figs of Ficus hispida before, during and after the phase of receptivity to pollinators[END_REF]. Histological investigations of figs have established that VOC emitting glands are restricted to the ostiolar bracts and, in some Ficus species, the fig wall epidermis [START_REF] Souza | Diversity of fig glands is associated with nursery mutualism in fig trees[END_REF]. In combination with biological observations, these results suggest that the ostiolar bracts could be the main locus of production of the VOCs involved in wasp attraction [START_REF] Souza | Diversity of fig glands is associated with nursery mutualism in fig trees[END_REF].

The VOCs produced by receptive figs are mainly terpenoids, but also phenylpropanoids/benzenoids (or shikimate pathway compounds), and fatty acid derivatives (Grison-Pige et al., 2002a;Grison-Pige et al., 2002b;[START_REF] Chen | Private channel: a single unusual compound assures specific pollinator attraction in Ficus semicordata[END_REF][START_REF] Proffit | Signalling receptivity: comparison of the emission of volatile compounds by figs of Ficus hispida before, during and after the phase of receptivity to pollinators[END_REF][START_REF] Proffit | Specificity of the signal emitted by figs to attract their pollinating wasps: comparison of volatile organic compounds emitted by receptive syconia of Ficus sur and F. sycomorus in Southern Africa[END_REF][START_REF] Soler | Evidence for intersexual chemical mimicry in a dioecious plant[END_REF][START_REF] Hossaert-Mckey | How to be a dioecious fig: chemical mimicry between sexes matters only when both sexes flower synchronously[END_REF]. The biosynthetic pathways at the origin of these compounds are well known in plants, and the enzymes involved have been investigated for a number of plant species [START_REF] Dudareva | Biosynthesis, function and metabolic engineering of plant volatile organic compounds[END_REF]. While some studies have documented the regulation of floral VOC synthesis [START_REF] Dudareva | Biosynthesis, function and metabolic engineering of plant volatile organic compounds[END_REF][START_REF] Muhlemann | Floral volatiles: from biosynthesis to function[END_REF], none of them has analyzed a specialized obligate mutualistic pollination interaction.

In this study, we focus on two closely related Ficus species, F. hirta and F. triloba, that are pollinated by closely related wasp species [START_REF] Yu | Multiple parapatric pollinators have radiated across a continental fig tree displaying clinal genetic variation[END_REF]. The two species co-occur throughout their range [START_REF] Berg | Precursory taxonomic studies on Ficus (moraceae) for the flora of Thailand[END_REF]) so that we may expect selection for signaling differentiation between the two species. We analyze the difference of transcription of candidate genes involved in VOC synthesis in ostiolar bracts between pre-receptive figs and receptive figs of the two Ficus species.

Materials and method

Study species

Ficus hirta Vahl. and Ficus triloba Buch.-Ham. ex Voigt are closely related species, belonging to section Eriosycea, subgenus Ficus. F. triloba was considered as a subspecies of F. hirta [START_REF] Berg | Moraceae (Ficus)[END_REF] but has since been elevated to species rank in a revision of the species group [START_REF] Berg | Precursory taxonomic studies on Ficus (moraceae) for the flora of Thailand[END_REF]. Most individuals of the two species are easily distinguished in the field. F. hirta is shrub that grows to a height of approximately 3 m while F. triloba is a small tree and can be 4-10 m tall. Ficus hirta has small figs, 10-15 mm in diameter, while those of F. triloba are 20-25 mm in diameter. Receptive figs of F. triloba emit a strong sent while the smell of receptive figs of F. hirta is hardly detectable to the human nose.

Sampling and sequencing

Ficus hirta was collected at the South China Botanical Garden, Guangdong Province, China (23°11′N, 113°11′E) while RNA was isolated using TRIzol™ (Tiangen). RNA degradation and contamination was monitored on 1% agarose gels.

RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA). A total amount of 1.5 μg RNA per sample was used as input material for the RNA sample preparations. High-throughput RNA sequencing (RNAseq) was performed on an Illumina Hiseq platform.

Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated.

Assembly and annotation

Clean data was obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from the raw data. All the downstream analyses are based on this clean data. Error rate, Q20, GC-content were calculated.

Transcriptome assembly was accomplished based on all sampled data using Trinity [START_REF] Grabherr | Full-length transcriptome assembly from RNA-Seq data without a reference genome[END_REF] with min_kmer_cov set to 2 and all other parameters set at default. Gene function was annotated using the following databases: Nr (NCBI nonredundant protein sequences), Nt (NCBI non-redundant nucleotide sequences), Pfam (Protein family), KOG (Clusters of Orthologous Groups of proteins), Swiss-Prot (a manually annotated and reviewed protein sequence database), KO (KEGG Ortholog database) and GO (Gene Ontology).

Differential gene expression analysis

Gene expression levels were estimated for each sample using the program RNA-Seq by Expectation-Maximization (RSEM, [START_REF] Li | RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome[END_REF]. Differential gene expression analysis between pre-receptive and receptive figs was performed using the DESeq R package (1.10.1). To control for false discovery rate, the resulting P values were adjusted using the Benjamini and Hochberg approach. Genes with an adjusted P-value <0.05 and fold chang >2 found by DESeq were considered to be differentially expressed. Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) was implemented using the GOseq R packages based on the Wallenius non-central hyper-geometric distribution [START_REF] Young | Gene ontology analysis for RNA-seq: accounting for selection bias[END_REF]. Enrichment of differential expression genes in KEGG pathways was analyzed using the KOBAS software [START_REF] Mao | Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary[END_REF].

In F. hirta, the low detected numbers of differentially expressed genes between pre-receptive and receptive figs was caused by the low Pearson correlation of gene expression between FhirtaB4, which is an outlier in almost all the pairwise comparisons, and the two other samples collected at receptive stage, FhirtaB2 and FhirtaB3 (see results).

Therefore, an additional analysis was done, including only the two samples FhirtaB2 and FhirtaB3. To compare variation in the level of expression of individual genes in pre-receptive and receptive fig ostiolar bracts, reads per kilo base per million mapped reads (RPKM) of the two phases were compared.

Real-time PCR

To confirm upregulation of genes at fig receptivity, 8 terpene synthases of F. hirta and 13 terpene synthases of F. triloba were amplified using real-time PCR. For the two species, samples were dissected and immediately put into Sample Protector for RNA/DNA (Takara, Japan). They were transferred to -20 °C as rapidly as possible. RNA was extracted using the PRC1 buffer of the HiPure Plant RNA Mini Kit (Magen, China) according to the manufacturer instructions.

Then 500 ng RNA per sample were used as input material for first-strand cDNA synthesis by the Reverse Transcriptase M-MLV (RNaseH-) (Takara, Japan). Real-time PCR was carried out in the presence of the double-strand DNA-specific dye SYBR Green I (SYBR Green Premix Ex Taq, Takara, Japan) and monitored in real-time with the Roche LightCycler480 system (Roche, Switzerland). The primers were designed according to the sequences to get 70-300 bp products and the specificity of the primers was monitored by the melting curve of the real-time PCR program.

Ortholog and Ka/Ks analysis

The coding sequence of each putative unigene was extracted based on the BLASTX results. The ESTSCAN software was used to determine the coding directions of sequences for which no alignment was obtained. The resulting coding sequences extracted from putative unigenes were translated into amino acid sequences using the standard codon table.

Self-to-self BLASTP was conducted for all amino acid sequences with a cut-off E-value of 1e -5 . Orthologous groups were constructed from the BLASTP results with OrthoMCL v2.0.3 [START_REF] Li | OrthoMCL: identification of ortholog groups for eukaryotic genomes[END_REF] using default settings.

To assess roles of Darwinian positive selection on the evolution of the genes, we used the CodeML program from the PAML package v4.8a [START_REF] Yang | PAML 4: phylogenetic analysis by maximum likelihood[END_REF] to perform positive selection tests. Ka is the number of nonsynonymous substitutions per non-synonymous site and Ks is the number of synonymous substitutions per synonymous site. The ratio Ka/Ks (ω) of orthologous gene pairs is an indicator of selective pressure acting on a protein-coding gene and were estimated. Signature of positive selection is indicated by ω > 1 [START_REF] Yang | Codon-substitution models for detecting molecular adaptation at individual sites along specific lineages[END_REF][START_REF] Yang | Bayes empirical bayes inference of amino acid sites under positive selection[END_REF][START_REF] Zhang | Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level[END_REF].

Results

Sequencing and de novo assembly

For F. hirta pre-receptive and receptive figs, we obtained 43, 311, 613 and 44, 910, 372 clean reads amounting to 6.50G and 6.74G, respectively. For F. triloba pre-receptive and receptive figs, we obtained 42, 566, 854 and 44, 320, 990 clean read amounting to 6.38G and 6.65G, respectively (Table S1). The Q20 of all the samples were above 93% and the GC content of all the samples were comprised within the interval 45-47% (Table S1). For F. hirta, 111,065 unigenes were obtained with an average size of 1048 bp, and for F. triloba, 83,407 unigenes were obtained with an average size of 1032 bp. The length distribution of the unigenes is shown in Fig. S1. The Pearson correlation coefficients show high correlation in gene expression of a plant between pre-receptive and receptive figs. This is especially true for F. triloba for which the three highest correlation values correspond to the three within plant-between stage comparisons (Fig. S2).

Sample FhirtaB4 is an outlier presenting the lowest correlation coefficients with other samples. Further, sample FhirtaB4 presents the highest number of reads and the lowest GC content of all the samples confirming its outlier status (Table S1).

Annotation and classification of the assembled unigenes

Annotation results are summarized in Table S2 and Fig. S3. 61,456 (55.33% of the total) unigenes of F. hirta and 45,894 (55.02% of the total) unigenes of F. triloba were annotated in at least one database. The top hit species for F. hirta and F. triloba were the same, namely Prunus mume, Prunus persica, Vitis vinifera, Pyrus x, and Theobroma cacao (Fig. S4). These five taxa and Ficus are members of the rosid clade of the Angiosperms. The GO category (GO), the unigene function categories (KOG) and the KEGG database distributions of the unigenes of F. hirta and F. triloba were highly similar (Fig. 1, Fig. 2A, Fig. 2B). The identified pathways include the metabolism of terpenoids, lipids, and amino acids, which are volatile organic compounds (VOCs) and/or precursors of VOCs.

Volatile organic compound synthase genes

For F. hirta, we found 205 unigenes coding for 51 enzymes involved in terpenoid biosynthesis (Table S3). Among these, 99 unigenes coded for 29 terpenoid backbone synthases, 44 unigenes coded for 9 sesquiterpenoid and triterpenoid synthases, 44 unigenes coded for 10 diterpenoid synthases and 18 unigenes coded for 3 monoterpenoid synthases. For F. triloba, we found 175 unigenes coding for 47 enzymes involved in terpenoid biosynthesis (Table S3). Among these, 77 unigenes coded for 28 terpenoid backbone synthases, 47 unigenes coded for 9 sesquiterpenoid and triterpenoid synthases, 37 unigenes coded for 8 diterpenoid synthases and 14 unigenes coded for 2 monoterpenoid synthases (Table S3).

All the terpene backbone enzymes expressed in F. hirta were also expressed in F. triloba, except for isopentenyldiphosphate delta-isomerase (IDI), which catalyzes the reversible conversion of IPP and DMAPP. The two species expressed the same sesquiterpene and triterpene synthases, but F. hirta expressed one more monoterpene synthase ((-)alpha-terpineol synthase) and two more diterpene synthases (ent-cassa-12, 15-diene 11-hydroxylase and ent-copalyl diphosphate synthase) than F. triloba.

Phenylpropanoid/benzenoid pathway genes and fatty acid derivatives synthase genes were also annotated (Table S4).

For F. hirta, we found 251 unigenes encoding 17 synthases involved in phenylpropanoid/benzenoid synthesis, and 91 unigenes encoding 14 synthase involved in alpha-linolenic acid metabolism pathways. For F. triloba, we found 238 unigenes encoding 16 synthases involved in phenylpropanoid/benzenoid synthesis, and 75 unigenes encoding 14 synthase involved in alpha-linolenic acid metabolism pathways. Overall, F. hirta and F. triloba expressed the same phenylpropanoid/benzenoid pathway synthases, except for one additional synthase (caffeoylshikimate esterase) expressed by F. hirta. The two species expressed the same fatty acid derivatives synthase.

Differentially expressed unigene analysis

For F. hirta, among the 60,299 unigenes detected in both pre-receptive and receptive figs, 187 (0.31%) unigenes were found to be differently expressed. Out of these, 79 unigenes were up-regulated in the receptive phase while 108 unigenes were down-regulated. For F. triloba, among the total 53,267 unigenes detected in both pre-receptive and receptive figs, 1413 (2.65%) unigenes were found to be differently expressed. Out of these, 773 unigenes were up-regulated in receptive phase and 640 unigenes were down-regulated. In F. hirta, we did an additional analysis excluding the outlier, sample FhirtaB4. All the following differential gene expression analyses in F. hirta are based on this reduced dataset.

Excluding sample FhirtaB4, many more unigenes were differentially expressed (3007; 5.0%), out of which 1487 were upregulated and 1520 were down-regulated in receptive figs. To detect metabolic pathways that are particularly active at fig receptivity, the KEGG enrichment of the up-regulated genes was analyzed (Fig. 3 and Table S5). In F. hirta, the up-regulated pathways included ribosome, phenylpropanoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, terpenoid backbone biosynthesis, carotenoid biosynthesis, monoterpenoid biosynthesis, isoflavonoid biosynthesis, brassinosteroid biosynthesis, and so on (Fig. 3A). In F. triloba, the up-regulated pathways included plantpathogen interaction, fatty acid elongation, galactose metabolism, terpenoid backbone biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, monoterpenoid biosynthesis, alpha-linolenic acid metabolism, ether lipid metabolism, and phenylpropanoid biosynthesis (Fig. 3B). Up-regulated genes of F. trilobawere enriched in fatty acid elongation, alpha-linolenic acid metabolism, ether lipid metabolism, which were different from that of F. hirta.

Differentially expressed volatile organic compound synthase genes

For F. hirta, several genes involved in VOC synthesis were upregulated at fig receptivity (Table 1 andTable S6).

Twenty-four differentially expressed unigenes belonged to the phenylpropanoid pathways. The following enzyme coding genes were up-regulated. Five genes that encoded 4-coumaroyl-CoA ligase (4CL) which could catalyze the formation of cinnamoyl-CoA, coumaroyl-CoA and caffeyoyl-CoA. These were precursors of phenylpropanoid compounds, such as cinnamaldehyde, p-coumaraldehyde, caffeylaldehyde. Among the differentially expressed genes, there were also two caffeic acid O-methyltransferase (COMT), that catalyzing the methaltransfer in precursors of the dehydes, and five dehydrogenase, that catalyzing the transformation of dehydes into alcohols. These dehydes and alcohols could constitute the VOCs produced by the phenylpropanoid pathways. Seven unigenes encoded peroxidases [START_REF] Mei | Cotton GhPOX1 encoding plant class III peroxidase may be responsible for the high level of reactive oxygen species production that is related to cotton fiber elongation[END_REF], which catalyzing the degrading of volatile phenylpropanoid compounds. In terpene synthesis, eight backbone genes, five sesquiterpene (four TPS1 and one alpha-farnesene synthase), two triterpene and three monoterpene genes (TPS14) were differentially expressed (Table 1). These results suggest that phenylpropanoid and terpene synthesis may constitute an important component of receptive fig odor in F. hirta.

For F. triloba, differentially expressed genes belonged to several types of VOC biosynthesis pathways especially the terpene pathway. In the terpenoid synthesis pathway, 13 unigenes were up-regulated at fig receptivity (Fig. 4 and Table 1). These synthases included 5 backbone synthases, and 8 TPS family unigenes, including 4 sesquiterpene, 1 triterpene and 2 monoterpene genes (TPS14). Different from F. hirta, 6 fatty acid derivatives synthesis pathway genes were upregulated, including lipoxygenase (LOX2S), hydroperoxide dehydratase (AOS), allene oxide cyclase (AOC), and acetyl-CoA acyltransferase (ACAA1) (Table S6). These were all jasmonate synthesis pathway enzymes. Jasmonate could generate methyl-jasmonate by jasmonate O-methyltransferase.

Hence, F. hirta and F. triloba presented similitudes and differences in the up-regulation at receptivity of genes involved in metabolic pathways that can be associated with the production of VOCs. In addition, three unigenes encoding ATPbinding cassette transporters (ABC transporters) also show a differential expression pattern between prereceptive and receptive figs of F. triloba (Table S5), while in F. hirta no genes in this pathway show significantly different expression.

These three unigenes include two ATP-binding cassettes, subfamily B (MDR/ TAP), and one ATP-binding cassette, subfamily A (ABC1).

Confirmation of differential gene expression by real-time PCR

To confirm transcriptome data, ostiolar bracts of pre-receptive and receptive figs were subjected to real-time PCR.

Eight supposedly upregulated terpene synthases unigenes of F. hirta and 13 terpene synthases unigenes of F. triloba were selected (Table S7). For F. hirta, 6 unigenes showed consistency between the real-time PCR and the transcriptomic data (75% consistency, Fig. 5 and Table S7) while this number was 8 for F. triloba (61.5% consistency, Fig. 6 and Table S7). The expression of 4 F. hirta synthases that were shown to be up-regulated in the ostiolar bracts of receptive figs was compared between tissues by amplifying the genes in extracts from the epidermis of fig walls from receptive fig. The expression of these genes was only upregulated in the ostiolar scales (Fig. S5). 3.7. Sequence divergence of orthologs between the two species Among 3322 identified pairs of orthologous genes (Table S8), 583 orthologous pairs of genes had Ka/Ks values larger than 1, and 1174 had Ka/Ks values below 0.1 and the remaining 1565 genes had intermediate Ka/Ks values (Table S9).

Hence, 583 orthologous pairs presented signatures of positive selection. These positively selected genes were clustered to photosynthesis, RNA transport, ubiquinone and other terpenoid-quinone biosynthesis, folate biosynthesis, nucleotide excision repair, flavonoid biosynthesis, plant hormone signal transduction, and so on (Table S10). A single gene directly involved in VOC synthesis, HMGR (OG06988 in Table S10), a terpene backbone synthase in the MVA pathway, presented a signature of positive selection.

Discussion

The results support the hypothesis that the ostiole is an important locus of receptive fig odor production. Indeed our transcriptomic results, supported by real-time PCR, show that a series of genes involved in VOC synthesis are up-regulated in the ostioles of receptive figs. [START_REF] Yu | De novo transcriptome sequencing in Ficus hirta Vahl. (Moraceae) to investigate gene regulation involved in the biosynthesis of pollinator attracting volatiles[END_REF] described the transcriptome of female flowers of F. hirta, as female flowers were previously thought to be the locus of VOC synthesis. They analyzed terpenoid pathway genes and discovered several differentially expressed genes. However, only genes down-regulated at receptivity were evidenced, suggesting no role of female flowers in the VOCs responsible for wasp attraction [START_REF] Yu | De novo transcriptome sequencing in Ficus hirta Vahl. (Moraceae) to investigate gene regulation involved in the biosynthesis of pollinator attracting volatiles[END_REF]. This agrees with histological data on a series of Ficus species [START_REF] Souza | Diversity of fig glands is associated with nursery mutualism in fig trees[END_REF]. Further, the low expression in the fig wall epidermis of F. hirta of terpene synthase genes that were strongly expressed in the ostiole of receptive figs suggests a central role of the ostiole in the production of receptive fig odor.

The two Ficus species largely expressed genes encoding the same synthase. In the VOC synthesis pathways, comparatively to F. triloba, F. hirta expressed 4 additional enzymes involved in terpenoid biosynthesis, one additional synthase in the phenyl/benzenoid pathway and the two species expressed the same fatty acid derivative synthases.

Further, only one terpene backbone synthase presented signatures of positive selection. Limited signals of selection on gene sequences were expected as these are classical plant biosynthetic pathways [START_REF] Dudareva | Biosynthesis, function and metabolic engineering of plant volatile organic compounds[END_REF]. Therefore, interspecific differentiation in receptive fig odor is most probably mainly based on gene regulation. Indeed, the two species presented some striking differences in which genes were up-regulated at receptivity: the detected number of up-regulated unigenes for F. hirta, and F. triloba were, respectively, 18 versus 13 in the terpenoid pathway, 24 versus 0 in the phenyl/benzenoid pathways and 0 versus 6 in the fatty acid pathway. Hence, variation in gene expression seems to be the main mechanism allowing rapid divergence of receptive flower odors, as in other systems [START_REF] Sheehan | Color and scent: how single genes influence pollinator attraction[END_REF][START_REF] Schiestl | Pollinator-mediated evolution of floral signals[END_REF]. Nevertheless, the large numbers of up-regulated terpene synthesis genes suggest that they are important components of receptive fig odors of both the two species. Methyl-jasmonate or its derivate might be important components in VOCs of receptive figs of F. triloba.

Expression profiles, especially in F. triloba, presented strong within plant correlation between pre-receptive and receptive figs (Fig. S2). This result suggests that different plants were in different physiological conditions. This difference in gene expression among plants should be compared with the among plants homogeneous up-regulation of VOC synthesis and other metabolic pathways in receptive figs. In F. hirta and in F. triloba, a large numbers of metabolic pathways were up-regulated at receptivity, indicating an important change in metabolism between prereceptive and receptive figs. Unigenes encoding primary metabolism, photosynthesis, brassinosteroid biosynthesis and plant hormone signal transduction were also differentially expressed. Photosynthesis provides the energy to the primary metabolism. Primary metabolism, such as starch and sucrose metabolism, glycerophospholipid metabolism, and fatty acid elongation, provide the material and energy basis required for secondary metabolism [START_REF] Aharoni | Metabolic engineering of the plant primary-secondary metabolism interface[END_REF].

Hence, this increase in gene expression may provide the metabolic background for a burst in VOC synthesis.

Phytohormones play important roles in development and metabolism of plants. Brassinosteroid biosynthesis genes were up-regulated in both F. hirta and F. triloba (Table S5). Jasmonate synthase genes, including LOX2S, AOS, AOC, and ACAA1, were up-regulated in F. triloba. Further, transcription factor genes involved in plant hormone signal transduction such as TGA, DELLA, SAUR, ERF1, SnRK2, MYC2, were also up-regulated in F. triloba (Table S11). [START_REF] Boachon | Natural fumigation as a mechanism for volatile transport between flower organs[END_REF], and as receptive fig odors are known to accumulate in the fig cavity [START_REF] Souza | Diversity of fig glands is associated with nursery mutualism in fig trees[END_REF].

Three unigenes encoding ATP-binding cassette transporters (ABC transporters) also show a differential expression pattern between prereceptive and receptive figs of F. triloba. ABC transporters are a transport system superfamily involved in translocation of substrates across membranes [START_REF] Rice | Diversity in ABC transporters: type I, II and III importers[END_REF]. Because VOCs are lipophilic liquids with low molecular weight and high vapour pressure at ambient temperatures [START_REF] Pichersky | Biosynthesis of plant volatiles: nature's diversity and ingenuity[END_REF], it has long been assumed that volatiles passively diffuse out of the cells through cell membranes [START_REF] Eberl | Releasing plant volatiles, as simple as ABC[END_REF].

However, recent calculations have shown that for many plant volatiles, the internal concentrations could reach high values (up to 100 mM), which could lead to the buildup of toxic amounts of volatiles within the membranes [START_REF] Widhalm | Rethinking how volatiles are released from plant cells[END_REF]. Further, in Petunia hybrida, PhABCG1 was demonstrated to facilitated active transport of volatiles across the plasma membrane [START_REF] Adebesin | Emission of volatile organic compounds from petunia flowers is facilitated by an ABC transporter[END_REF]. Hence, the up-regulated ABC transporters may be involved in VOC release by receptive figs. We did not detect variation in ABC transporters expression differentiation in F. hirta. This may be due to differences in ostiole structure between the two species, leading to different constraints on odor emission.

Alternatively, other transporters may be involved in F. hirta.

Conclusions

This study provides the first analysis of transcriptome variation in ostiolar bracts between pre-receptive and receptive

Fig

  Fig wasps breed within figs, the closed urnshaped receptacles characteristic of Ficus. The interior of the fig is lined with flowers. Pollinating wasps are attracted by receptive figs (ready to be pollinated), enter the receptive fig through

  Fig wasps are attracted by volatile organic compounds (VOCs) released by receptive figs, while they are not attracted by the VOCs released by figs that are not yet receptive or past receptivity

F

  . triloba was collected nearby, at Dinghu Mountain, Guangdong Province, China (23°09′~23°11′N, 112°30′~112°33′E). To avoid pollinator visitation, figs were bagged before fig receptivity. Ostiolar bracts were dissected from pre-receptive and receptive figs without pollinated, and were put into Sample Protector for RNA/DNA (Takara). For F. triloba, each sample consisted of bracts from two figs from one tree. Three replicates from three trees were collected. For F. hirta, because of the smaller figs and smaller ostiolar bracts, every sample consisted of bracts from 10 to 12 figs. Two samples were collected from one tree and a third sample was collected from another tree.

  fig odor emission, then signaling between ostiole and receptive female flowers is required, and the up-regulated phytohormone coding genes may be involved. Signaling between ostiole and flowers could be mediated by VOCs accumulating in the fig cavity as such a process is documented in other flowers[START_REF] Boachon | Natural fumigation as a mechanism for volatile transport between flower organs[END_REF], and as receptive

  figs in two Ficus species. The results agree with the hypothesis that the ostiole is the locus of production of the receptive fig odors that are responsible for pollinating wasp attraction. The results suggest that differences in receptive fig odors between related species are primarily determined by variation in gene expression regulation. Regulation based receptive fig odor differentiation between species suggests a potential for rapid evolution of this interspecific barrier.

Fig. 2 .Fig. 3 .

 23 Fig. 2. The distributions of unigene function categories predicted by KOG (A) and KEGG (B) for F. hirta and F.triloba.

Fig. 4 .

 4 Fig. 4. An overview of terpenoid VOC biosynthesis pathway unigenes and differentially expressed genes in F. triloba. Enzyme are shown in box, number between bracket is the number of unigenes encoding this enzyme. Bold box includes the upregulated unigene encoded enzymes. DXP, 1deoxy-D-xylulose 5-phosphate; DXS, 1-deoxy-Dxylulose-5-phosphate synthase; DXR, 1-deoxy-Dxylulose-5-phosphate reductoisomerase; MCT, 2C-methyl-Derythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-C-methylD-erythritol kinase; MECPS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase: HDS, (E)-4hydroxy-3-methylbut-2-enyl-diphosphate synthase. IDS, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; AACT, acetyl-CoA C-acetyltransferase; HMGS, hydroxymethylglutaryl-CoA synthase; HMGR, hydroxymethylglutaryl-CoA reductase (NADPH); MVK, mevalonate kinase; PMK, phosphomevalonate kinase; MPDC, diphosphomevalonate decarboxylase; FPPS, farnesyl diphosphate synthase; GGPS, geranyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase. FLDH, farnesol dehydrogenase; SQLE, squalene monooxygenase.

Fig. 6 .

 6 Fig. 6. Real-time PCR of some of terpenoid synthesis pathway genes of F. triloba. FeGAPDH was used as reference gene. A corresponds to pre-receptive figs, and B to receptive figs. Fe represents F. triloba; TPS14 and TPS15, homologue of terpene synthase 14 gene of Arabidopsis; GO, gibberellin 2-oxidase; TPS1, TPS2, and TPS3, homologue of terpene synthase 1 gene of Arabidopsis.

Table 1

 1 Unigenes

			Gene_id	Name	Abbrevation in this paper	Homologous genes of Arabidopsis
	F. hirta	Backbone synthase	Cluster-12957.42642	1-deoxy-D-xylulose 5-phosphate synthase	DXS	DXS
			Cluster-12957.32504	hydroxymethylglutaryl-CoA synthase	HMGS	HMGS
			Cluster-12957.17615	4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase	HDS	HDS
			Cluster-12957.36388	geranylgeranyl pyrophosphate synthase	GGPPS	GGPPS
			Cluster-12957.31617	hydroxymethylglutaryl-CoA reductase	HMGR	HMGR
		Sesquiterpene synthase Cluster-12957.13509	farnesol dehydrogenase	FLDH	FLDH
			Cluster-12957.3618	(-)-germacrene D synthase	TPS1	TPS1
			Cluster-12957.2277	(-)-germacrene D synthase	TPS3	TPS1
			Cluster-12957.3619	valencene/7-epi-alpha-selinene synthase	TPS2	TPS1
		Triterpene synthesase	Cluster-12957.59461	squalene monooxygenase	SQLE	SQLE
		Monoterpene synthase	Cluster-14540.0	(3S)-linalool synthase	TPS14	TPS14
			Cluster-12957.2989	(3S)-linalool synthase	TPS15	TPS14
		Diterpene synthase	Cluster-12957.25452	gibberellin 2-oxidase	GO
	F. triloba Backbone synthase	Cluster-21669.40714	prenylcysteine alpha-carboxyl methylesterase	PCME	PCME
			Cluster-21669.41527	geranylgeranyl reductase	chlP	chlP
			Cluster-21669.67549	mevalonate kinase	MVK	MVK

encoding terpenoid pathway synthase up-regulated in receptive compared with pre-receptive phase of F.hirta and F. triloba

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31670395; 31630008), Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) (GML2019ZD0408), Guangzhou Science and Technology Plan Project ( 201707010351), the National Natural Science Foundation of China (31971568) and Province Natural Science Foundation of Guangdong (c20140500001306). FK is supported by LIA MOST.

Data archiving statement

Raw sequence data has been submitted to NCBI's Short Read Archive (SRA). The accession number is PRJNA492455 for F. triloba and PRJNA491590 F. hirta.

Author contributions

Declaration of competing interest

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.


Simone P Teixeira

Marina F B Costa

João Paulo Basso-Alves

Finn Kjellberg

Rodrigo A S Pereira

The synstigma turns the fig into a large flower

Keywords:

The synstigma is a structure formed by clusters of two to several stigmas, whether in the same or between different flowers. Although rare in angiosperms, synstigmas are found in c. 500 out of the c. 750 Ficus spp. (Moraceae). This floral structure is associated with fig-fig wasp pollinating mutualism.

The synstigma structure and pollen tube pathways were studied in six Ficus spp. from Ficus section Americanae to test the hypothesis that the synstigma allows pollen grains deposited on a stigma to emit pollen tubes that can grow laterally and fertilize surrounding flowers. Syconia containing recently pollinated stigmas were collected and dissected, and the stigmas were processed for analyses with light and scanning and transmission electron microscopy. The arrangement of the synstigmas across species can be spaced or congested, with the number of stigmas per synstigma ranging from two to 20. Contact between the stigmas in a synstigma occurs by the intertwining of the stigmatic branches and papillae; their union is firm or loose. The pollen tube grows through live cells of the transmitting tissue until reaching the ovule micropyle. Curved pollen tubes growing from one stigma to another were observed in five out of the six species studied. The curvilinear morphology of pollen tubes probably results from competition by pollen between the stigmas composing a synstigma via chemotropic signals. The synstigma appears to be a key adaptation that ensures seed production by flowers not exploited by the fig wasps in actively pollinated Ficus spp.

Introduction

A synstigma is a rarely reported structure of angiosperms formed by the clustering of two or more stigmas originating from a single flower or from different flowers. Records of synstigmas have been reported in: early-diverging angiosperms (Amborellaceae, Austrobaileyaceae, Illiciaceae, Nymphaeaceae, Schisandraceae) and magnoliids (Annonaceae, Calycanthaceae, Monimiaceae, Siparunaceae), in which the gynoecium is apocarpic, but contacted by an extragynoecial compitum [START_REF] Endress | Evolutionary diversification of the flowers in angiosperms[END_REF][START_REF] Endress | Ancestral traits and specializations in the flowers of the basal grade of living angiosperms[END_REF]; Rosa setigera Michx. (Rosaceae), in which the apocarpic gynoecium is contacted by an intrastylar compitum [START_REF] Kemp | Morphological differences and changes of the in short-lived flowers of Rosa setigera Michaux and their relationship to dioecy[END_REF]; for two species of Procris Comm. ex Juss. (Urticaceae) in which the congested arrangement of the pistillate inflorescence could promote the union of surrounding stigmas for the shared reception of pollen grains [START_REF] Weberling | Morphology of flowers and inflorescences[END_REF]; and, apparently, for Typhaceae, in which there is a union of groups of four flowers [START_REF] Carvalho | Synflorescence morphology of species of Typha L. (Typhaceae): anatomical and ontogenetic bases for taxonomic applications[END_REF] and pollen dispersed in tetrads [START_REF] Nicholls | The function of pollen tetrads in Typha (Typhacae)[END_REF].

The clade with the largest number of records so far is Ficus L. (Moraceae), with 500 species predicted to form synstigmas [START_REF] Verkerke | Structure and function of the fig[END_REF]Jousselin, Rasplus & Kjellberg, 2003b;[START_REF] Jousselin | The functional implications of active and passive pollination in dioecious figs[END_REF]BassoAlves et al., 2014;[START_REF] Teixeira | Morphological diversity and function of the stigma in Ficus species (Moraceae)[END_REF]. A synstigma can be a continuous structure inside a syconium (the urn-shaped inflorescence of Ficus, the fig), composed of all stigmas of all flowers [START_REF] Galil | On the pollination ecology of Ficus sycomorus in East Africa[END_REF] or the result of contact between two or more stigmas of distinct flowers, acting as a common surface for pollen germination [START_REF] Jousselin | The functional implications of active and passive pollination in dioecious figs[END_REF][START_REF] Jousselin | Flower specialization in a passively pollinated monoecious fig: a question of style and stigma?[END_REF], 2003a,).

Ficus is taxonomically and ecologically diverse [START_REF] Bruun-Lund | Rush hour at the museumdiversification patterns provide new clues for the success of figs (Ficus L., Moraceae)[END_REF][START_REF] Clement | Evolution and classification of figs (Ficus, Moraceae) and their close relatives (Castilleae) united by involucral bracts[END_REF], encompassing c. 750 of the 1100 species described for the family [START_REF] Berg | Moraceae diversity in a global perspective[END_REF]. This high level of diversity seems to result from the obligatory mutualistic interaction established with small Agaonidae wasps [START_REF] Kjellberg | Biology, ecology and evolution of fig pollinating wasps (Chalcidoidea: Agaonidae)[END_REF]. However, the fig tree and the fig wasp have divergent reproductive interests. The reward for the pollinating wasp is the oviposition site (i.e. flower ovaries) to rear its brood. Fertilized flowers are assumed to be better quality food for the wasp larvae than unfertilized ones, creating a selective pressure from the wasp side for the evolution of active pollination, i.e. the pollinating fig wasp actively collects pollen in its natal fig, stores it in specialized thoracic structures called 'pollen pockets' and later actively deposits it on stigmas of the flowers into which they oviposit [START_REF] Jousselin | The functional implications of active and passive pollination in dioecious figs[END_REF].

The active pollination in Ficus could have a high cost for plants on seed set if wasps only pollinate the flower into which they oviposit. This effect may generate a selective pressure from the plant side for the development of the synstigma, which could then be interpreted as an adaptation to guarantee seed production in some of the fig flowers. As such, active pollination should be seen coevolving with other features, such as the spatial arrangement of the flowers within the syconium. In passively pollinated species (those in which pollinating wasps have no behaviour of collecting/depositing pollen), the synstigma is not present and the flowers that receive the wasp eggs and the seed-producing flowers are morphologically different, especially regarding the length of the stigmas. The longer-styled flowers have long filiform stigmatic branches projecting into the fig cavity that brush the body of the wasp and collect pollen in the process, whereas the shorter-styled flowers have short filiform stigmatic branches stigmas that do not receive pollen (Jousselin et al., 2003a[START_REF] Jousselin | Flower specialization in a passively pollinated monoecious fig: a question of style and stigma?[END_REF][START_REF] Teixeira | Morphological diversity and function of the stigma in Ficus species (Moraceae)[END_REF].

There is a strong correlation between the presence of synstigma and active pollination in both monoecious and gynodioecious species of Ficus (Jousselin et al., 2003b). In monoecious Ficus spp., the flowers are arranged in several layers so that some ovaries are closer to the inner wall of the syconium and other ovaries are closer to the central cavity of the syconium. The length of the styles varies accordingly so that the stigmas may unite at the same level, forming a synstigma. Both active and passive pollinating wasps oviposit shorter-styled flowers, probably because these flowers are more suitable for the offspring development [START_REF] Anstett | Unbeatable strategy, constraint and coevolution, or how to resolve evolutionary conflicts: the case of the fig/wasp mutualism[END_REF] and less exposed to parasites [START_REF] Al-Beidh | Parasites and mutualism function: measuring enemy-free space in a fig-pollinator symbiosis[END_REF]. Longer-styled flowers generally do not receive wasp eggs and may produce a seed if their embryo sac is fertilized [START_REF] Jousselin | The functional implications of active and passive pollination in dioecious figs[END_REF].

The hypothetical function of the synstigma in Ficus spp. is to allow pollen grains deposited on the stigma of a flower to emit pollen tubes that will grow laterally through nearby stigmas and fertilize non-pollinated neighbouring flowers, thus promoting seed production [START_REF] Jousselin | The functional implications of active and passive pollination in dioecious figs[END_REF].

Despite the paradigm of the functional significance of the synstigma for mutualism, there are no detailed studies of the morphological structure or its functional significance in relation to pollen germination.

In this study, we aimed to test the hypothesis that the synstigma allows pollen grains deposited on a stigma to emit pollen tubes that can grow laterally and fertilize surrounding flowers, favouring the production of seeds and thus balancing the costs and benefits of the fig-fig wasp mutualism. To that end, the detailed structure of the synstigma and the pollen tube pathways were studied in six monoecious, actively pollinated species of Ficus section Americanae Miq.

Material and methods

Six species of Ficus subgenus Urostigma (Gasp.) Miq. section Americanae were selected for this study (Table 1), and at least two individuals from each species were sampled. The species were chosen to cover a range of syconium sizes to allow the exploration of one possible source of variation associated with syconium size that can impose physical constraints on stigma arrangement and affect pollen grains/pistillate flowers ratio.

At least six syconia were collected from each species at the stage when the pistillate flowers presented receptive stigmas. To follow the entire course of the pollen tube through the pistil, figs were collected at three successive stages: (1) syconium containing live pollinating wasps; (2) syconium containing dead wasps and clear and turgid stigmas and (3) syconium containing dead wasps and decaying brownish stigmas.

The syconia were cut in half and fixed in FAA 50 [START_REF] Johansen | Plant microtechnique[END_REF], FNT [START_REF] Lillie | Histopathologic technic and practical histochemistry[END_REF] or Karnovsky solution [START_REF] Karnovsky | A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy[END_REF]. The total number of flowers per syconium was estimated by counting flowers in one syconium of each species. The structure of the synstigma was described by observing the samples using stereoscopic, scanning electron microscopy (SEM, surface examination) and light microscopy (LM, anatomical examination). For the surface examination, small portions of syconia were dehydrated in an ethanolic series, critical point dried in a Bal-Tec CPD 030 apparatus, mounted on a metallic support on carbon tape and/or adhesive glue and sputtered with gold in a Bal-Tec SCD 050 apparatus. Observations were performed using Jeol JSM 5200 and Jeol JSM6610LV SEM at 25 kV.

For the anatomical examination, small portions of syconia were dehydrated in an ethanol series, fixed in Leica histological resin [START_REF] Gerrits | The application of glycol methacrylate in histotechnology; some fundamental principles[END_REF] and longitudinally sectioned (5 μm) using a Leica RM 2245 rotary microtome. The sections were stained with 0.05% toluidine blue in phosphate buffer (pH 6.3) [START_REF] O'brien | Polychromatic staining of plant cell walls by toluidine blue O[END_REF], and the slides were mounted at the time of observation with distilled water and a glass coverslip. Observations and photographs were taken with a Leica DFC 320 digital camera coupled with a Leica DM 4500 B light microscope.

The emission and growth of pollen tubes in the stigma of a flower and between flowers were observed in fresh or fixed materials under LM. Pistils were softened and cleared in 20% sodium hydroxide at 60 °C for 20-40 min, washed in distilled water, crushed in 0.1% aniline blue in 0.1 M of K3PO4 buffer [START_REF] Martin | Staining and observing pollen tubes in the style by means of fluorescence[END_REF], mounted between slide and coverslip and observed under a Leica DM 5000 B epifluorescence light microscope with filter A (360-470 nm).

The ultrastructure of the growing pollen tube was studied using transmission electron microscopy (TEM). Samples of stigmas were fixed in Karnovsky's solution in 0.075 M phosphate buffer (pH 7.2-7.4) for 24 h, washed in 0.075 M phosphate buffer (pH 7.2-7.4) [START_REF] Karnovsky | A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy[END_REF], post-fixed in 1% osmium tetroxide in 0.075 M phosphate buffer (pH 7.2-7.4) for 1 h, gradually dehydrated in acetone solution and embedded in Araldite 6005. Subsequently, semi-thin sections (0.5 μm) were obtained using a Leica Reichert ultramicrotome and stained with 0.05% toluidine blue [START_REF] O'brien | Polychromatic staining of plant cell walls by toluidine blue O[END_REF].

Ultrathin sections (60 nm) were obtained using a Leica Reichert ultramicrotome, stained with 2% uranyl acetate solution for 15 min [START_REF] Watson | Staining of tissue sections for electron microscopy with heavy metals[END_REF] and lead citrate for 15 min [START_REF] Reynolds | The use of lead citrate at high pH as an electron-opaque stain in electron microscopy[END_REF], observed and documented using a Philips EM 208 transmission electron microscope.

The degree of cohesion between the stigmas followed a manual criterion of separation of the stigmas: loose, indicating that the stigmas were just touching each other with no effort required to separate them; and firm, for those that required some effort to separate them. The stigmatic papillae were characterized as short when they were the same height as the epidermal cells of the style, and long when they were at least twice the height of these cells.

Results

The syconium and its flowers

The studied Ficus spp. have syconia of different shapes and sizes (Table 2). All contain developing staminate flowers (Fig. 1A), short-styled pistillate flowers and longstyled pistillate flowers (Fig. 1B,C). The shorter-styled pistillate flowers are interspersed with longer-styled pistillate flowers (Fig. 1C), with all stigmas converging at the same level in the inner side of the syconium (Fig. 1C,D). The pistillate flowers have a unilocular ovary with one ovule, a style positioned laterally to the ovary and a stigmatic surface formed by one or two filiform branches partially covered by papillae (Fig. 1E,F).

Stigma and synstigma

In all species, the disposition of stigmas varied from a solitary stigma to groups of contiguous stigmas (synstigmas) histologically connected or not to each other, with variation in (1) the number of stigmas forming each synstigma, (2) the proximity between the synstigmas and (3) the degree of cohesion between the stigmas of a synstigma (Table 2, Figs 2 and3).

The number of stigmas per cluster ranged from two to 20 in F. citrifolia Mill., F. eximia Schott, F. obtusifolia Kunth and F. roraimensis C.C.Berg and from two to ten in F. carautana J.Neves & Emygdio and F. pertusa L.f. (Table 2). The number of flowers composing a synstigma does not depend on the number of flowers existing in the syconium, as exemplified in F. obtusifolia (Table 2). A qualitative analysis indicates that stigmas are in closer proximity in F. eximia, resulting in a more congested distribution in the syconium compared to the other species (cf. Fig. 2E with Figs 2A, C, 3A, C, E; Table 2).

The contact between the stigmas occurs through the intertwining of branches and papillae in all species (Table 2; Figs 2B,D,F,3B,D,F). This intertwining may be firm or loose, regardless of the size of the papillae (see Table 2). In F. carautana, stigmatic papillae are short (Fig. 2B), and the degree of cohesion between stigmas is loose. In the other species in which contact is also loose, stigmatic papillae are long (Figs 2D, F, 3B, D, F; Table 2).

Pollen and pollen tube

The species exhibited little variation in pollen and pollen tube morphology and in the behaviour of the growing pollen tubes in the pistil. In each species, these characteristics did not vary between longerand shorter-styled flowers and even in previously oviposited flowers.

The pollen grain deposited on the stigmas is small, c. 8-10 µm. It germinates rapidly on the stigmatic papillae in almost all species at the first subphase (presence of living wasp) (Fig. 4A-C, E). Except for F. pertusa, the germination is slightly slower and was only observed at the second subphase (presence of dead wasp). The pollen tube grows through the exudate produced as a result of the degeneration of stigmatic cells, reaching the style at the third subphase (presence of dead wasp and decaying brownish stigmas) (Fig. 4D,E). It continues to grow through the intercellular spaces of the transmitting tissue (Fig. 4D,E) that occupies the central portion of the style until it reaches the ovary (Fig. 4F).

In five of the six species (F. carautana, F. eximia, F. obtusifolia, F. pertusa and F. roraimensis) pollen tubes were observed growing from one stigma towards the adjacent stigma (Fig. 5). The pollen tubes in these cases did not grow straight, instead exhibiting small curvatures (Fig. 5D,E). Pollen tubes were observed in the style of oviposited and non-oviposited flowers.

The pollen tube grows through intercellular spaces of the style without degradation of the transmitting tissue cells (Fig. 6). The cells of the transmitting tissue at the time of passage of the pollen tube are intact, with secretory activity, thin walls (Fig. 6), large vacuole (Fig. 6), central lobed nucleus (Fig. 6C) and peripheral cytoplasm, containing amyloplasts (Fig. 6A,C), rough endoplasmic reticulum (Fig. 6B,D), mitochondria (Fig. 6A,D) and numerous vesicles (Fig. 6A,C). The pollen tube has a dense cytoplasm (Fig. 6), is thick-walled, structured in an external stratum (probably pecto-cellulosic) and an internal stratum (probably callosic) (Fig. 6D). No cytoplasmic connections were observed between the pollen tube and adjacent cells of the transmitting tissue, but the walls of both appear to be closely linked (Fig. 6).

Discussion

The synstigma turns the fig into a large flower with an extragynoecial compitum

The fig inflorescence containing many small uniovulate flowers could be functionally compared to a large flower with an apocarpous gynoecium. However, this configuration does not present the inherent disadvantage of apocarpy in terms of pollination efficiency. Indeed, the synstigma acts as [START_REF] Endress | Syncarpy and alternative modes of escaping disadvantages of apocarpy in primitive angiosperms[END_REF][START_REF] Endress | Convergent elaboration of apocarpous gynoecia in higher advanced dicotyledons (Sapindales, Malvales, Gentianales)[END_REF]. a common surface for pollen germination, growth The compitum is a region of the pistil where the and competition of pollen tubes, analogous to the pollen tubes converge [START_REF] Endress | Evolutionary diversification of the flowers in angiosperms[END_REF], allowing the function of a compitum in syncarpous gynoecium distribution of pollen tubes between carpels [START_REF] Carr | The functional significance of syncarpy[END_REF] and acting as a site of pollen tube selection [START_REF] Endress | Syncarpy and alternative modes of escaping disadvantages of apocarpy in primitive angiosperms[END_REF][START_REF] Erbar | Pollen tube transmitting tissue: place of competition of male gametophytes[END_REF][START_REF] Lora | The diversity of the pollen tube pathway in plants: toward an increasing control by the sporophyte[END_REF]. Thus, the synstigma of Ficus is functionally analogous to a special type of compitum, the extragynoecial found in some groups, formed in the external surfaces of the gynoecia [START_REF] Endress | Noncarpellary pollination and 'hyperstigma' in an angiosperm (Tambourissa religiosa, Monimiaceae)[END_REF][START_REF] Endress | Syncarpy and alternative modes of escaping disadvantages of apocarpy in primitive angiosperms[END_REF][START_REF] Endress | Diversity and evolutionary trends in the floral structure of Tambourissa (Monimiaceae)[END_REF]Endress et al., 1983;[START_REF] Endress | Gynoecium structure and evolution in basal angiosperms[END_REF][START_REF] Vieira | Oxypetalum banksii subsp. banksii: a taxon of Asclepiadaceae with an extragynoecial compitum[END_REF][START_REF] Lyew | Pollen tube growth in association with a drytype stigmatic transmitting tissue and extragynoecial compitum in the basal angiosperm Kadsura longipedunculata (Schisandraceae)[END_REF][START_REF] Du | Intercarpellary growth of pollen tubes in the extragynoecial compitum and its contribution to fruit set in an apocarpous species, Schisandra sphenanthera (Schisandraceae)[END_REF][START_REF] Lau | Stigmatic exudate in the Annonaceae: pollinator reward, pollen germination medium or extragynoecial compitum?[END_REF].

A common type of extragynoecial compitum involves abundant mucilage secreted by the stigmatic papillae, as commonly found in some groups of earlydiverging angiosperms [START_REF] Endress | Noncarpellary pollination and 'hyperstigma' in an angiosperm (Tambourissa religiosa, Monimiaceae)[END_REF][START_REF] Endress | Evolutionary diversification of the flowers in angiosperms[END_REF][START_REF] Endress | Diversity and evolutionary trends in the floral structure of Tambourissa (Monimiaceae)[END_REF][START_REF] Endress | Gynoecium structure and evolution in basal angiosperms[END_REF][START_REF] Wang | Extragynoecial pollen-tube growth in apocarpous angiosperms is phylogenetically widespread and probably adaptive[END_REF][START_REF] Endress | Ancestral traits and specializations in the flowers of the basal grade of living angiosperms[END_REF][START_REF] Lau | Stigmatic exudate in the Annonaceae: pollinator reward, pollen germination medium or extragynoecial compitum?[END_REF]. The mucilage serves as a germination medium and aids in the selection and distribution in Monimiaceae [START_REF] Endress | Noncarpellary pollination and 'hyperstigma' in an angiosperm (Tambourissa religiosa, Monimiaceae)[END_REF][START_REF] Endress | Syncarpy and alternative modes of escaping disadvantages of apocarpy in primitive angiosperms[END_REF]. However, as of pollen tubes between the carpels. An extreme the synstigma of Ficus does not produce abundant case of this condition is the 'hyperstigma' described secretion [START_REF] Heslop-Harrison | The receptive surface of the angiosperm stigma[END_REF][START_REF] Ramírez-Benavides | Pollination analogies between Orchidaceae, Ficus (Moraceae) and Asclepiadaceae[END_REF] present study), synstigma cohesion is mainly the result of the physical intertwining of the stigmatic branches and papillae [START_REF] Teixeira | Morphological diversity and function of the stigma in Ficus species (Moraceae)[END_REF]. As such, the synstigma is functionally analogous to single flowers with carpels post-genitally and distally united, as found in Malvaceae, Apocynaceae and Sapindales (Endress et al., 1983;[START_REF] Vieira | Oxypetalum banksii subsp. banksii: a taxon of Asclepiadaceae with an extragynoecial compitum[END_REF].

Competition among flowers within a syconium causes changes in pollen tube morphology

We detected no differences between floral morphs in characteristics related to pollen grain germination and pollen tube growth, suggesting that the plant has no mechanism to favour preferential fertilization of long-styled pistillate flowers. Similarly, we did not find any variation in the behaviour and morphology of the pollen tubes between Ficus spp. having different numbers of flowers per syconium and different degrees of cohesion of the synstigma. A looser synstigma or a synstigma formed of few stigmas could be predicted to favour the formation of branched pollen tubes in an attempt to broaden the resources for tube growth [START_REF] Maheshwari | An introduction to the embryology of angiosperms[END_REF]. Branched pollen tubes were found in F. citrifolia, a species with loose synstigmas, but as no pollen tubes were observed growing between flowers, it was not possible to check the presence of this condition. In F. carautana, F. obtusifolia and F. roraimensis, pollen germination was rapid (at the first subphase, when living wasps are present), as observed in the species presenting a firm synstigma and similar to that observed in the passively pollinated F. carica, which does not form synstigmas and in which pollen tubes were observed on the style 24 h after pollination [START_REF] Beck | Breeding system in Ficus carica, the common fig. II. pollination events[END_REF].

The rapid pollen germination in the studied Ficus spp. probably results from the release of tricellular pollen grains from the anthers (documented in F. elastica Roxb. ex Hornem.; [START_REF] Zhang | Examination of the cytoplasmic DNA in male reproductive cells to determine the potential for cytoplasmic inheritance in 295 angiosperm apecies[END_REF], resulting in short-lived pollen [START_REF] Dafni | Pollen viability and longevity: practical, ecological and evolutionary implications[END_REF]. Our results suggest that the pollen tube in Ficus is heterotrophic during its growth, depending on external resources provided by the exudates released by the stigmatic cells and the transmitting tissue. This idea is supported by the observation that pollen tubes in Ficus grow through intercellular spaces and not by penetration/degradation of transmitting tissue cells. Indeed, heterotrophic pollen tube growth through intercellular spaces has been reported for many other taxa [START_REF] Maheshwari | An introduction to the embryology of angiosperms[END_REF][START_REF] Mulcahy | The two phases of growth of Petunia hybrida (Hort. Vilm-Andz.) pollen tubes through compatible styles[END_REF][START_REF] Taylor | Pollen germination and tube growth[END_REF][START_REF] Graaf | Pollen and pistil in the progamic phase[END_REF][START_REF] Lersten | Flowering plant embryology[END_REF]Malhó, 2006).

The curvilinear morphology of the pollen tubes growing laterally in the synstigma has also been observed in F. fulva (fig. 2b in [START_REF] Jousselin | The functional implications of active and passive pollination in dioecious figs[END_REF]. We suggest that stigmas composing the synstigma compete for pollen tubes, by producing chemotrophic signals. These signals would indicate which stigmas have not yet been contacted by pollen and, thus, would be more attractive to germination. The stigmatic papillae, after the deposition and germination of the pollen grain, undergo physical and chemical changes that alter their receptivity and the consequent ability to attract and sustain the growth of pollen tubes [START_REF] Adhikari | Fertilization in flowering plants: an odyssey of sperm cell delivery[END_REF], interfering with their direction. Other two alternative explanations seem less plausible. (1) The connection between the stigmas could expose the pollen tube to the independent, simultaneous chemical signals emitted by the egg cells [START_REF] Herrero | Changes in the ovary related to pollen tube guidance[END_REF][START_REF] Okuda | Pollen tube guidance by attractant molecules: LUREs[END_REF] from the female gametophyte of the flower in which the pollen was deposited and by the egg cells present in surrounding flowers, united by the synstigma. These conflicting signals might change the direction of the pollen tube growth, resulting in numerous short bends along the pollen tube that meanders over the synstigma. However, although important in the guidance of the growing pollen tube, the signals from the female gametophyte cells are restricted to the ovarian and ovule tissues. The beginning of the pollen tube trajectory, through the stigma and style, is mediated by signals from the sporophyte [START_REF] Zheng | The long journey of pollen tube in the pistil[END_REF]. ( 2) Incompatibility mechanisms among flowers located near the pollinated flower might generate the atypical pollen tube morphology (see [START_REF] Lee | Patterns of fruit maturation: a gametophytic competition hypothesis[END_REF]. This is also unlikely, because the production of viable seeds in neighbouring flowers and the occurrence of natural and manipulative hybridization [START_REF] Condit | An interspecific hybrid of Ficus[END_REF][START_REF] Parrish | Genetic evidence for natural hybridization between species of dioecious Ficus on island populations[END_REF] indicate that incompatibility mechanisms are absent in Ficus.

The synstigma of Ficus section Americanae does not form a continuous surface as observed in Ficus section Sycocarpus Miq. (fig. 2a in [START_REF] Jousselin | The functional implications of active and passive pollination in dioecious figs[END_REF]. Instead, figs of the studied species had several synstigmas formed by groups of two to 20 flowers. The selective pressures for variation in the number of flowers that compose each synstigma are obscure. We tried to explore one potential reason for variation by investigating species in which syconia covered a large range in size, reflected by their number of flowers, but we did not find any obvious connection. Natural history data related with pollination behaviour could provide insight into this question, as pollen availability seems to be a limiting factor in Ficus [START_REF] Kjellberg | Some pollinators are more equal than others: factors influencing pollen loads and seed set capacity of two actively and passively pollinating fig wasps[END_REF][START_REF] Wang | Has pollination mode shaped the evolution of Ficus pollen?[END_REF]. Also, the wasp pollination behaviour largely affects the pollen distribution inside the syconium, because in the active pollination pollen grains are deposited in batches on some stigmas.

Outlook

The presence of pollen tubes growing from one stigma to another in F. carautana, F. eximia, F. obtusifolia, F. pertusa and F. roraimensis supports the hypothesis that synstigmas act as distributors of pollen tubes to flowers situated near those that have been pollinated, thus modifying the pattern of fertilization and promoting wasp offspring without seed predation or, at least, reducing seed predation.

In this way, we expand the preliminary information about distribution of pollen tubes by synstigmas in F. fulva Reinw., F. condensa King [START_REF] Jousselin | The functional implications of active and passive pollination in dioecious figs[END_REF], F. microcarpa L.f., F. salicifolia Vahl and F. sur Forssk. (Jousselin et al., 2003a). Pollen tubes were also observed in the style of oviposited flowers, confirming that pollinating wasp larvae found in the syconium of monoecious Ficus spp. with active pollination may develop at the expense of the plant embryo and endosperm and may therefore be affected by embryo sac fertilization (Jansen-González, [START_REF] Jansen-González | Mutualism from the inside: coordinated development of plant and insect in an active pollinating fig wasp[END_REF].

Precise observations of wasp behaviour are now needed to better understand the roles of the stigmatic structure for pollen deposition behaviour, actual locus and pattern of pollen deposition.

Figure 1 .

 1 Figure 1. Morphological aspects of the flowers of Ficus citrifolia (A, C, D), F. obtusifolia (B) and F. pertusa (E). A, Longitudinal section of a staminate flower highlighting the young developing anthers. B, Surface view of a short-styled pistillate flower and a long-styled pistillate flower. C, Interspersed arrangement of short-styled and long-styled pistillate flowers within a syconium showing the ovaries at different levels and stigmas at the same level. D, Overview of half a syconium cut longitudinally showing the pistillate flowers with stigmas at the same level. E, Longitudinal section of a pistillate flower showing the anacrostylous ovary with one locule and one ovule and papillate stigma. F, Detail of stigmatic papillae. Symbols: a = anther, lsf = long-styled flower, o = ovary, ov = ovule, p = papilla, s = stigma, ssf = short-styled flower, st = style. Scale bars: A, E = 20 µm; B, C = 500 µm; D = 2 mm; F = 100 µm.

Figure 2 .

 2 Figure 2. Arrangement within a syconium and anatomy of the synstigma of Ficus carautana (A, B), F. citrifolia (C, D) and F. eximia (E, F). There is a variation in the proximity between synstigmas featuring a more spaced (A, C) or more congested (E) distribution. Note a connecting region between the papillae of two stigmas (B, D, F). Symbols: c = connecting region, s1, s2 = stigma 1 and 2. Scale bars: A, C, E = 500 µm; B, D, F = 50 µm.

Figure 3 .

 3 Figure 3. Arrangement within a syconium and anatomy of the synstigma of Ficus obtusifolia (A, B), F. pertusa (C, D) and F. roraimensis (E, F). There is a variation in the proximity between synstigmas featuring a more spaced distribution (A, C, E). Note a connecting region between the papillae of two stigmas (B, D, F). Symbols: c = connecting region, s1, s2 = stigma 1 and 2. Scale bars: A, C, E = 500 µm; B, D, F = 50 µm.

Figure 4 .

 4 Figure 4. Epifluorescent images of the pollen tube growth through the stigma, style and ovary of Ficus citrifolia (A, D, F), F. eximia (B), F. roraimensis (C) and F. carautana (E). A-C. Pollen grains on the stigma and pollen tubes growing through stigmatic papillae. D, E, Pollen tubes on the style traversing the transmitting tissue. F, Pollen tube reaching the ovary. Symbols: gp = germinating pollen grain, o = ovary, pt = pollen tube, s = stigma, st = style. Scale bars: A, B = 100 µm; C = 50 µm; D, F = 200 µm; E = 20 µm.

Figure 5 .

 5 Figure 5. Stigma views (LM) of Ficus carautana (A), F. eximia (B), F. obtusifolia (C), F. pertusa (D, E) and F. roraimensis (F). Pollen tube growing from one stigma towards the adjacent one. Note the pollen grains germinating on the stigma (E, F). Symbols: s1, s2, s3 = stigma 1, 2 and 3, st = style, gp = germinating pollen grain, pt = pollen tube. Scale bars: A = 50 µm; B, C, E, F = 100 µm; D = 20 µm.

Figure 6 .

 6 Figure 6. Ultrastructure (TEM) of the pollen tube growing through the style of flowers of Ficus citrifolia (A, D), F. obtusifolia (B) and F. pertusa (C). A, B, Pollen tube surrounded by intact cells of the transmitting tissue. Note the electron-dense content of the pollen tube. C, Detail of two cells of the transmitting tissue showing thin walls, large vacuoles, amyloplasts, central lobed nucleus. Note the tip of a pollen tube located between the walls of the two transmitting tissue cells. D, Detail of the pollen tube showing the thick wall, the innermost stratum consisting of translucent substance (callose). Note a transmitting tissue cell with a large vacuole, mitochondria and an extensive rough endoplasmic reticulum. Symbols: a = amyloplast, cw = callosic wall, mi = mitochondria, nu = nucleus, pt = pollen tube, pw = pollen tube wall, rer = rough endoplasmic reticulum, tt = transmitting tissue, v = vacuole, ve = vesicle. Scale bars: A = 5 µm; B, C = 3 µm; D = 1 µm.

Table 1 .

 1 Information about sampled species of Ficus section Americanae

	Species	Collection site	Collection date	Voucher

Table 2 .

 2 Information on stigma / synstigma morphology in the studied species of Ficus sect. Americanae. Symbols: fec = short styled flower; fel = long styled flower.

			Number of
	Species	Approximate number of flowers in a syconium	branches per stigma fec fel	Arrangement	Synstigma characteristics number Flower Type of intertwining	Size of stigmatic papillae	Degree of cohesion between stigmata
	F. carautana	258	1-2	1-2	spaced	2-10	branches/papillae	short	loose
	F. citrifolia	494	1-2	1-2	spaced	2-17	branches/papillae	long	loose
	F. eximia	532	1-2	1-2	congested	2-20	branches/papillae	long	firm
	F. obtusifolia	1738	2	1-2	spaced	2-15	branches/papillae	long	loose
	F. pertusa	210	1-2	1-2	congested	2-11	branches/papillae	long	firm
	F. roraimensis	440	1-2	1-2	spaced	2-20	branches/papillae	long	loose

Acknowledgements

We thank Edimárcio da Silva Campos (FCFRP/USP), Maria Dolores S. Ferreira (Tuca), José A.

Maulin and

Maria Teresa P. Maglia (FMRP/USP) for technical assistance and Camila D. Souza and Luciano Palmieri (University of São Paulo, Ribeirão Preto, Brazil) for the Ficus samples, Dewey Litwiller (Language Centre at the University of Saskatchewan) for the English revision and Dr Louis Ronse De Craene and an anonymous reviewer for the critical reading of the manuscript. This work was supported by FAPESP (grant numbers 2012/02374-2 and 2014/07453-3), FAPERJ (grant numbers E-26/202.411/2017 and E-26/202.412/2017), CNPq (grant numbers 305793/2018-7, 303493/2015-1 and 302806/2019-9) and CAPES (code 001).

F. carautana L.J.Neves & Emygdio Corumbá, MS, Brazil 03/2012 R.A.S.Pereira et al. 178 (SPFR) F. citrifolia Mill. Ribeirão Preto, SP, Brazil 09/2011 S.P.Teixeira et al. 79 (SPFR) F. eximia Schott Ribeirão Preto, SP, Brazil 04/2012 R.A.S.Pereira et al. 138 (SPFR) F. obtusifolia Kunth Galia, SP, Brazil 10/2011 R.A.S.Pereira et al. 158 (SPFR) F. pertusa L.f. Ribeirão Preto, SP, Brazil 07/2011 R.A.S.Pereira et al. 127 (SPFR) F. roraimensis C.C.Berg Manaus, AM, Brazil 07/2012 Alcohol collection (FCFRP/USP)