William C Beckerson

Ricardo C Rodríguez De La Vega

Fanny E Hartmann

Marine Duhamel

Tatiana Giraud

Michael H Perlin
email: michael.perlin@louisville.edu

Cause and Effectors: whole genome comparisons reveal shared but rapidly evolving effector sets among host-specific plant-castrating fungi

Keywords: Functional Proteomics, Effectors, Small Secreted Proteins, Host Specificity, Fungal Pathogens Importance summary

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Host specialization is a phenomenon well documented in many fungal pathogen/plant host systems [START_REF] Sánchez-Vallet | The genome biology of effector gene evolution in filamentous plant pathogens[END_REF], which most often occurs through host shifts [START_REF] Vienne | Cospeciation vs host-shift speciation: methods for testing, evidence from natural associations and relation to coevolution[END_REF]. The ability to infect a new host is determined by the protein-protein interactions that occur at the pathogen/host interphase. For pathogens to be successful, they must not only be able to colonize the host, but must also work around a gauntlet of host defense responses, as well as manipulate the host to their advantage.

Pathogens accomplish these ends through the deployment of many secreted effectors [START_REF] Lanver | Ustilago maydis effectors and their impact on virulence[END_REF][START_REF] Anderson | Plants versus pathogens: an evolutionary arms race[END_REF][START_REF] Whisson | A translocation signal for delivery of oomycete effector proteins into host plant cells[END_REF].

It has been understood for several decades that plant pathogens utilize secreted effectors to infect their hosts [START_REF] Sánchez-Vallet | The genome biology of effector gene evolution in filamentous plant pathogens[END_REF][START_REF] Albersheim | Proteins from plant cell walls inhibit polygalacturonases secreted by plant pathogens[END_REF], including the maize pathogen member of the "smut fungi", Ustilago maydis [START_REF] Lanver | Ustilago maydis effectors and their impact on virulence[END_REF]. To defend against these pathogens, plants continuously evolve to recognize pathogen-associated molecular patterns and trigger a variety of immune responses [START_REF] Jones | The plant immune system[END_REF].

Reciprocally, there is an ongoing selective pressure for plant pathogens to adapt to their host by developing new effectors, or otherwise alter the composition of their secretomes, to evade detection and find new ways to manipulate the host to their advantage. Secretomes can thus evolve rapidly, not only during host shift events but also due to intra-specific coevolution [START_REF] Meile | A fungal avirulence factor encoded in a highly plastic genomic region triggers partial resistance to Septoria tritici blotch[END_REF]. It is, however, still unclear whether changes in secretomes leading to host specialization and local adaptation primarily involve effector gene gains/losses or changes in their sequences. Repeatinduced point mutations (RIP) is a fungal defense mechanism against transposable elements that has been suggested to play a role in effector diversification in fungi harboring effectors in regions rich in repetitive elements [START_REF] Fudal | Repeat-induced point mutation (RIP) as an alternative mechanism of evolution toward virulence in Leptosphaeria maculans[END_REF][START_REF] Van De Wouw | Evolution of linked avirulence effectors in Leptosphaeria maculans is affected by genomic environment and exposure to resistance genes in host plants[END_REF]. RIP indeed acts via mutations of repeated sequences at specific target sites and can "leak" on neighbor genes [START_REF] Fudal | Repeat-induced point mutation (RIP) as an alternative mechanism of evolution toward virulence in Leptosphaeria maculans[END_REF][START_REF] Van De Wouw | Evolution of linked avirulence effectors in Leptosphaeria maculans is affected by genomic environment and exposure to resistance genes in host plants[END_REF].

Host specialization following host shift is particularly common in the fungal pathogen species complex Microbotryum violaceum [START_REF] Refregier | Cophylogeny of the anther smut fungi and their caryophyllaceous hosts: prevalence of host shifts and importance of delimiting parasite species for inferring cospeciation[END_REF]. Microbotryum species are basidiomycete smut fungi that complete their life cycle in the anthers of their respective host plants, replacing the pollen with their own fungal spores [START_REF] Schäfer | The illustrated life cycle of Microbotryum on the host plant Silene latifolia[END_REF]. Originally described as a single species, these "anther smuts" are now understood to represent a complex of species [START_REF] Perlin | Molecular approaches to differentiate subpopulations or formae speciales of the fungal phytopathogen Microbotryum violaceum[END_REF][START_REF] Gac | Evolution of reproductive isolation within a parasitic fungal species complex[END_REF], most being highly specific to particular species of the Caryophyllaceae family, also known as "pinks" [START_REF] Hood | Distribution of the anther-smut pathogen Microbotryum on species of the Caryophyllaceae[END_REF]. Intra-specific coevolution has also been suggested to occur based on local adaptation patterns, where host plants were more resistant to their local sympatric anther-smut pathogen than to those from geographically distant populations of the same species [START_REF] Kaltz | Local mal-adaptation in the anthersmut fungus Microbotryum violaceum to its host plant Silene latifolia:evidence from a crossinoculation experiment[END_REF][START_REF] Feurtey | Strong phylogeographic co-structure between the anther-smut fungus and its white campion host[END_REF].

To infect their hosts, Microbotryum fungi, like many other plant pathogens, employ an array of effector proteins to block plant immune response and otherwise manipulate the host during infection [START_REF] Perlin | Sex and parasites: genomic and transcriptomic analysis of Microbotryum lychnidis-dioicae, the biotrophic and plant-castrating anther smut fungus[END_REF][START_REF] Kuppireddy | Identification and initial characterization of effectors of an anther smut fungus and the potential host target proteins[END_REF]. While the specificity of the various Microbotryum species to their corresponding host plants has been extensively described [START_REF] Gac | Evolution of reproductive isolation within a parasitic fungal species complex[END_REF][START_REF] Hood | Distribution of the anther-smut pathogen Microbotryum on species of the Caryophyllaceae[END_REF][START_REF] Vienne | Hybrid sterility and inviability in the parasitic fungal species complex Microbotryum[END_REF], the molecular basis for host specialization and coevolution within the complex has just recently begun to be explored [START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeorgraphic structure[END_REF][START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF][START_REF] Aguileta | Finding candidate genes under positive selection in non-model species: examples of genes involved in host specialization in pathogens[END_REF]. Understanding the changes that have occurred in the secretomes of these host-specific species will broaden our understanding of the mechanisms behind coevolution, host-shifts and emergent diseases. Furthermore, Microbotryum species offer a unique model system to study host shifts and specialization, with multiple host-specific and closely related pathogens (24), which is not often the case in agriculturally propagated crops.

To test whether host-specific or locally-adapted closely-related pathogens mainly differed in their secretomes by gene gains/losses or by rapid evolution of shared effectors, we compared the secretomes of three Microbotryum species, two sister species, M. lychnidis-dioicae and M.

silenes-dioicae, and a more distantly related relative, M. violaceum var paradoxa. We sought to identify sets of core secreted proteins (i.e., orthologous genes encoding secreted proteins shared by all species), that likely play a major role in the pathogenicity of the species complex as a whole. We also sought to identify species-specific effectors and effectors evolving under positive selection and highly expressed in planta, thus perhaps involved in host specificity. To further our understanding of coevolution and local adaptation, we compared the secretomes of two M. lychnidis-dioicae strains collected from geographically distant populations belonging to distinct genetic clusters that have shown contrasted infection patterns consistent with plant local adaptation [START_REF] Feurtey | Strong phylogeographic co-structure between the anther-smut fungus and its white campion host[END_REF]. We also investigated whether the most frequent changes among host-specific species or locally-adapted clusters involved mostly the gain/loss of secreted proteins or the diversification of shared proteins. As RIP-like footprints have been detected in Microbotryum fungi [START_REF] Hood | Repeat-induced point mutation and the population structure of transposable elements in Microbotryum violaceum[END_REF], we also tested whether sequence divergence in genes under positive selection and/or in genes encoding secreted proteins could have been facilitated by RIP.

Results

Overview of Microbotryum predicted secretomes

Analysis of the three Microbotryum secretomes revealed inventories of SPs of similar sizes in all three species. Initial prediction identified around 600 genes with signal peptides in each species (Figure 1). Utilizing sequence-based criteria of cellular localization and secretory signals, we kept 302, 371, and 418 SPs in M. violaceum var paradoxa, M. silenes-dioicae and M. lychnidisdioicae, respectively, for further analysis.

FIGURE 1

Over 85% of the predicted SPs were clustered into 453 orthologous groups, 225 comprising exclusively predicted SPs (645 SPs), henceforth called "SP-only", and 239 in which at least one member was not predicted as SP (298 SPs), henceforth called "SP-mixed" (Figure 2).

Over two thirds of the predicted SPs belonged to orthologous groups with genes in all three species (753 predicted SPs in 163 SP-only and 177 SP-mixed groups). Further, 190 predicted SPs belonged to orthologous groups shared by only two species. Only 148 SPs (i.e., 14% of the total) had no ortholog in two of the species and were therefore classified as species-specific SPs (62 in M. violaceum var paradoxa, 44 in M. lychnidis-dioicae and 42 in M. silenes-dioicae).

Predicted SPs were significantly depleted in species-specific genes in all three species (Chisquare with Yates correction p ≤ 0.0002). We classified as "core-secretome" 47% of the predicted SPs (513 genes belonging to 163 SP-only orthologous groups with members in all three species). In 118 SP-mixed orthologous groups with single-copy members in all three species, secretion signals were predicted in the orthologs of a single species, orthologs being non-SPs in the two other species; such orthologous groups will be referred to as "monoSP" hereafter (Figure 2 and Supplemental File SF1).

FIGURE 2

The majority of SPs for each species were smaller than the median length of all predicted proteins in the three species (57%, 68% and 65% of SPs were smaller than 361 amino acids for M. lychnidis-dioicae, M. silenes-dioicae, and M. violaceum var paradoxa, respectively; Figure 3a and Supplemental File SF1 3b). This trend was further observed when analyzing the subset of core SPs (Figure 3 and supplemental file SF1).

FIGURE 3

Signal peptide clusters and yeast secretion trap results

The clustering of the signal peptides of predicted SPs resulted in 280 groups with two or more sequences at 75% sequence identity (823 sequences out of the 1091 predicted SPs). The signal peptides tested here together with the four previously tested [START_REF] Kuppireddy | Identification and initial characterization of effectors of an anther smut fungus and the potential host target proteins[END_REF] are representative of the signal peptides of 28 predicted SPs in the three Microbotryum species under study (Figure 4). To test whether the predicted secretion signals can indeed direct secretion, we used an invertasedeficient mutant of Saccharomyces cerevisiae. Such mutants can grow on glucose but not on sucrose unless transformed with a plasmid containing the invertase gene with a functional secretion signal, which allows the invertase to cleave extracellular sucrose into glucose and fructose in the medium. Cells of the invertase-deficient mutant SEY6120 of S. cerevisiae were transformed with pYST-0 vectors containing each tested signal peptide region upstream and inframe with the invertase gene. As evidenced by the ability of their respective secretion signals to allow SEY6120 to grow on medium containing sucrose as the sole carbon source, all 9 predicted secreted proteins that have been tested so far using yeast secretion trap have been confirmed to be secreted (Figure 4 and reference19). Interestingly, protein 12964 from M. violaceum var paradoxa, was originally filtered out of our list of predicted SPs, due to the prediction that it is GPI-anchored to the membrane. Nevertheless, in this assay using only the secretion signal of the protein, invertase was secreted, suggesting that our conservative approach to estimating secretion may initially filter out membrane proteins with potential functional components outside the fungal cell.

FIGURE 4

Interspecies comparison of Microbotryum predicted secretomes

As expected due to their phylogenetic placement, the orthologous proteins of M. silenes-dioicae and M. lychnidis-dioicae were more similar (median identity 98.7%) than either of the two sister groups compared to M. violaceum var paradoxa (median 86.9% for M. lychnidis-dioicae / M. violaceum var paradoxa and 87.1% for M. silenes-dioicae / M. violaceum var paradoxa).

Orthologous SPs, including those belonging to the core secretome, were significantly less similar to one another than control non-SPs from single-copy orthologous groups of similar lengths (Wilcoxon rank sum test with continuity correction p < 7e-7 for all three pairwise betweenspecies comparisons, Figure 5). Out of the 150 single-copy orthologous groups with a SP predicted in each of the three species, i. [START_REF] Perlin | Sex and parasites: genomic and transcriptomic analysis of Microbotryum lychnidis-dioicae, the biotrophic and plant-castrating anther smut fungus[END_REF][START_REF] Juge | Plant protein inhibitors of cell wall degrading enzymes[END_REF]. Glycosyl hydrolases (GHs) (PF00295.17 and PF00704.28) were found in the core and monoSP orthologous groups, despite an overall paucity of GHs represented among M. lychnidis-dioicae genes [START_REF] Perlin | Sex and parasites: genomic and transcriptomic analysis of Microbotryum lychnidis-dioicae, the biotrophic and plant-castrating anther smut fungus[END_REF]. Enzymes of these particular families are interesting due to their ability to hydrolyze pectin, a process important in both pathogenic and saprophytic fungi life stages [START_REF] Sprockett | Evolutionary analysis of glycosyl hydrolase family 28 (GH28) suggests lineage-specific expansions in necrotrophic fungal pathogens[END_REF].

Intraspecific comparisons of Microbotryum predicted secretomes

We further investigated footprints of positive selection using McDonald-Kreitman (MK) tests that compare the amount of variation within a species (polymorphism) to the divergence between species (substitutions) at two types of sites, synonymous and non-synonymous. A ratio of nonsynonymous to synonymous polymorphism within species lower than the ratio of nonsynonymous to synonymous differences between species indicates positive selection [START_REF] Mcdonald | Adaptive protein evolution at the Adh locus in Drosophila[END_REF].

We performed three pairwise species comparisons between M. violaceum var paradoxa, M. lychnidis-dioicae and M. silenes-dioicae, using 148 core, 115 monoSP and 314 control orthologous groups. We used population genomics data from 20, 18, and 4 isolates from M. lychnidis-dioicae, M. silenes-dioicae, and M. violaceum var paradoxa, respectively (22, 29, 30; supplemental table ST1). Figure 5A shows the locations where the isolates were sampled. The Substitutions, on the other hand, were more frequent between M. lychnidis-dioicae Lamole and M. lychnidis-dioicae 1318 strains in predicted SPs than in control genes (Wilcox rank sum test with continuity correction p = 2.537e-05, Figure 5C and supplemental file SF4).

FIGURE 5

Genomic context of predicted SPs

In contrast to some other plant pathogenic fungi with effectors frequently located in repeat-rich regions, we did not find genes encoding predicted SPs to be significantly closer to transposable elements than other genes (Figure 6) and found no evidence for genome compartmentalization into AT-rich or GC-rich regions in any of the three genomes analyzed, extending previous observations [START_REF] Perlin | Sex and parasites: genomic and transcriptomic analysis of Microbotryum lychnidis-dioicae, the biotrophic and plant-castrating anther smut fungus[END_REF]. We nevertheless estimated the frequency of sites potentially affected by the RIP-like mechanism reported in Microbotryum fungi, targeting TTG and CAA trinucleotides.

We calculated a RIP index that takes values above one when there is an excess of TTG and CAA trinucleotides over the corresponding target sites not affected by RIP (TCG and CGA), controlling for local sequence composition (see Methods). The coding regions of predicted SPs did not show any significant excess of RIP-affected trinucleotides, regardless of whether the orthologous groups showed signs of positive selection (Figure 6). Our RIP-index measure was negatively correlated with distance to transposable elements (TEs), indicating RIP leakage to TE-neighboring regions. The RIP index was not correlated with the ratio between nonsynonymous and synonymous substitutions (Figure 6), indicating that the RIP-like mechanism does not play a significant role in the diversification of genes under positive selection in Microbotryum fungi.

FIGURE 6

Expression of predicted SPs across infection stages

We focused our analysis on M. lychnidis-dioicae Lamole genes expressed in at least one of the five infection stages or three mating conditions for which we retrieved expression data [START_REF] Perlin | Sex and parasites: genomic and transcriptomic analysis of Microbotryum lychnidis-dioicae, the biotrophic and plant-castrating anther smut fungus[END_REF][START_REF] Toh | Transcriptional analysis of mating and pre-infection stages of the anther smut, Microbotryum lychnidis-dioicae[END_REF]32). Among the 2,840 genes fulfilling this condition, we found 135 and 58 predicted SPs from the single-copy core and monoSP orthologous groups, respectively, and compared their expression profiles to 232 genes from the non-SP control group (same length distribution but not predicted as potential effectors). Hierarchical clustering of expression profiles across infection stages grouped the genes into low (31 genes, median log2FC range -7.35 -4.15), medium (117 genes, median log2FC range 0.0 -1.8), high (29 genes, median log2FC range 9.19 -12.40), and no change (248 genes, median log2FC 0) average gene expression across infection stages. We found no major changes in expression of core, monoSP or non-SP genes across three mating conditions. Predicted SPs from the core orthologous groups were enriched among genes with high or low average expression across infection stages, respectively 19 and 18 out of 135 core SPs compared with 7 and 6 out of 232 control genes (Fisher's two tailed exact test p = 1.8E-3 and 1.1E-3, respectively; Figure 7). In line with the pattern observed across all predicted SPs, we could infer the function of only 14 core and 7 monoSP genes with either high or low average expression. Glycosyl hydrolases, often involved in pathogenesis [START_REF] Sprockett | Evolutionary analysis of glycosyl hydrolase family 28 (GH28) suggests lineage-specific expansions in necrotrophic fungal pathogens[END_REF], were among the most common hits (supplemental files SF1 and SF5).

DISCUSSION

Microbotryum secretomes appeared as largely shared among species, i.e., with few gene gains/losses. Instead, we found SPs to be rapidly evolving as these were more differentiated among species and more often under positive selection compared to non-SP genes, indicating that many SPs likely evolved under diversifying selection among species parasitizing different hosts. Such rapid evolution was also indicated by the low percentage of SPs matching Pfam domains (31-47%), a percentage that decreased to less than 20% for the small secreted proteins.

Such a finding regarding the lack of identifiable Pfam domains of a substantial proportion of SPs is consistent with previous reports in other smut pathogens and is a hallmark of secreted effectors involved in host-specificity [START_REF] Jones | Bioinformatic prediction of plant-pathogenicity effector proteins of fungi[END_REF]. Diversifying selection in Microbotryum SPs is likely due to coevolution within species, local adaptation or specialization to different hosts, involving rapid changes in the sequences of secreted proteins to avoid detection in the plant and, more generally, to counteract evolving host defenses. Such a hypothesis is reinforced by the finding that SPs under positive selection were more often highly expressed in planta than non-SP genes.

Although we found few species-specific SPs or with copy-number variation, these accessory SPs may also be involved in coevolution, local adaptation, and/or host specialization [START_REF] Plissonneau | Pangenome analyses of the wheat pathogen Zymoseptoria tritici reveal the structural basis of a highly plastic eukaryotic genome[END_REF][START_REF] Schuster | Comparative analyses of secreted proteins in plant pathogenic smut fungi and related basidiomycetes[END_REF].

The results from the intraspecific comparison between the two M. lychnidis-dioicae strains shed further light on coevolution and local adaptation. We indeed found SPs to be more differentiated than non-SPs between two strains from genetically differentiated populations.

These findings further support the idea that coevolutionary pressures may be causing divergence in effectors between differentiated populations of pathogens. In fact, the populations from South and Eastern Europe were genetically differentiated in both M. lychnidis-dioicae and its host plant Silene latifolia, and the plant showed local adaptation to the fungus [START_REF] Feurtey | Strong phylogeographic co-structure between the anther-smut fungus and its white campion host[END_REF], indicating the occurrence of coevolution. Gene presence-absence polymorphisms in M. lychnidis-dioicae, corresponding to the pathogen and host phylogeographic structure [START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeorgraphic structure[END_REF], and numerous selective sweeps across the genome [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF], further supported the existence of coevolution. In contrast with several crop pathogens (e.g., 36, 37), neither presence-absence polymorphisms nor selective sweep regions were enriched in predicted SPs, even though nearly 10% of SPs were found located within recent selective sweeps in M. lychnidis-dioicae, which suggests recent adaptive events involving some SPs.

The identification of a set of shared and conserved SPs, i.e., the 126 core-secretome orthologous groups without positive selection, was also interesting, providing a starting point to search for effectors that play a central role in the common pathogenicity traits of these fungi, e.g., the effectors that allow the fungi to migrate to the plant anthers, to induce stunted ovary and pseudoanther development in female flowers, and to eliminate and replace host pollen with fungal spores. The observed differential expression of core secreted proteins further narrows the search for these central effectors and points to sets of genes within the secretome that may play other central roles in the fungal life cycle, including the secretion of extracellular enzymes for carbon source metabolism. Indeed, phosphatases, peptidases, lipases and glycosidases accounted for half of the Pfam annotations of core-secretome orthologous groups with no signs of positive selection (20 out of 38). While such enzymes are clearly associated with fungal pathogens [START_REF] Brown | Secreted biomolecules in fungal plant pathogenesis[END_REF][START_REF] Monod | Secreted proteases from pathogenic fungi[END_REF][START_REF] Keyhani | Lipid biology in fungal stress and virulence: Entomopathogenic fungi[END_REF], they are often found in animal [START_REF] Brown | Secreted biomolecules in fungal plant pathogenesis[END_REF][START_REF] Monod | Secreted proteases from pathogenic fungi[END_REF], and necrotrophic plant pathogens [START_REF] Sprockett | Evolutionary analysis of glycosyl hydrolase family 28 (GH28) suggests lineage-specific expansions in necrotrophic fungal pathogens[END_REF][START_REF] Reis | Molecular and functional characterization of a secreted lipase from Botrytis cinerea[END_REF][START_REF] Gacura | Comparison of pectindegrading fungal communities in temperate forests using glycosyl hydrolase family 28 pectinase primers targeting Ascomycete fungi[END_REF], rather than in biotrophic fungi. On the other hand, the up-regulation of many carbohydrate active enzyme genes related to cell wall degradation was also seen in both wheat stem and poplar rust, P. graminis and M. larici-populina, respectively [START_REF] Duplessis | Obligate biotrophy features unraveled by the genomic analysis of rust fungi[END_REF]. In the case of M. lychnidis-dioicae, GH28 polygalacturonase domain-containing proteins were up-regulated during infection and were among the proteins with signs of positive selection enriched in the core secretome and monoSP orthologous groups. Since polygalacturonase is required for the pathway implicated in pollen dehiscence [START_REF] Wang | A global analysis of the polygalacturonase gene family in soybean (Glycine max)[END_REF], this is consistent with a fundamental role for such enzymes in the pathogenic lifestyle of anther-smut fungi.

Future research with Microbotryum will utilize these findings to better understand the function of the most promising SP candidates, by identifying their targets within each host. Such research geared towards identifying the targets of secreted effectors from M. lychnidis-dioicae in its corresponding host plant, Silene latifolia, has already made progress [START_REF] Kuppireddy | Identification and initial characterization of effectors of an anther smut fungus and the potential host target proteins[END_REF]. For instance, we identified here MvSl-1064-A1-R4_MC02g04003 as part of the core secretome undergoing diversifying selection across species. We also found its transcript among the most highly expressed across infection stages. Its predicted protein product (residues 21-156) has been shown

to interact with two host proteins in yeast two-hybrid assays [START_REF] Kuppireddy | Identification and initial characterization of effectors of an anther smut fungus and the potential host target proteins[END_REF]. Extension of such work to analyze candidate effectors herein identified through in silico studies should add new insights into their relevance in host preference and the evolution of the Microbotryum species complex.

By narrowing down the genomes and identifying prime candidates that are likely to play a major role in the pathogen's life cycle, this work helps to bridge the gap between the quickly expanding availability of Microbotryum genomes (24, 30, 45) and the emerging cellular and molecular biology work being done to understand the role of effectors in this system [START_REF] Kuppireddy | Identification and initial characterization of effectors of an anther smut fungus and the potential host target proteins[END_REF].

More generally, this study showed that the molecular changes that lead to different host ranges between closely related plant pathogens, or different locally-adapted genetic clusters, involved little gene gains/losses in their secretome but instead rapid evolution of shared secreted proteins. This represents a significant advance in our understanding of pathogen evolution and may contribute to understanding host shifts and emergent diseases.

Materials and Methods

Comparative genomics

To analyze the relationship between various predicted effectors, we performed genomic analyses on the following available genomes, obtained using Pacific Bioscience (PacBio) single molecule real time sequencing: GCA_900015465.1 for M. lychnidis-dioicae Lamole a 1 (Italy) (45), GCA_900015495.1 for M. violaceum var paradoxa from Silene paradoxa 1252 a 1 [START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF], and QPIF00000000 for M. silenes-dioicae 1303 a 2 [START_REF] Branco | Evolutionary strata on young mating-type chromosomes despite the lack of sexual antagonism[END_REF]. These genomes were selected for comparison due to their relationship to one another; M. lychnidis-dioicae strains and M. silenesdioicae are sister species, able to infect one another's host in the greenhouse, although to a lesser degree than their natural host [START_REF] Gibson | Performance of a hybrid fungal pathogen on pure-species and hybrid host plants[END_REF] and very little in natural populations (47), while M. violaceum var paradoxa serves as an outgroup, unable to infect either of the sister species' hosts or vice versa [START_REF] Vienne | Hybrid sterility and inviability in the parasitic fungal species complex Microbotryum[END_REF].

In total, we used eight sequence-based prediction tools to identify potential effectors by searching each genome for genes with hallmarks for secretion and without conflicting cellular localization predictions. The initial list of putative secreted proteins (SPs) were generated by running the entire genomes through SignalP 4.0 [START_REF] Petersen | SignalP 4.0: discriminating signal peptides from transmembrane regions[END_REF]. In order to increase the stringency of this analysis, the SPs must then have passed the following criteria to rule out potential localization or retention in various membranes within or on the cell, similar to the previously published protocol for M. lychnidis-dioicae [START_REF] Perlin | Sex and parasites: genomic and transcriptomic analysis of Microbotryum lychnidis-dioicae, the biotrophic and plant-castrating anther smut fungus[END_REF]. Potential transmembrane domains were predicted with TMHMM [START_REF] Krogh | Predicting transmembrane protein topology with hidden Markov model: application to complete genomes[END_REF] and Phobius [START_REF] Käll | Advantages of combined transmembrane topology and signal peptide predition -the Phobius web server[END_REF]. Only gene models with none or a single transmembrane domain prediction overlapping the signal peptide prediction were considered further [START_REF] Perlin | Sex and parasites: genomic and transcriptomic analysis of Microbotryum lychnidis-dioicae, the biotrophic and plant-castrating anther smut fungus[END_REF][START_REF] Petersen | SignalP 4.0: discriminating signal peptides from transmembrane regions[END_REF]. Prosite was used to screen for predicted endoplasmic reticulum retention signals, while PredGPI (51) was used to screen for potential glycosylphosphatidylinositol anchors, and NucPred (52) was used to screen for nuclear localization signals in the predicted protein (Figure 1).

Gene models predicted to be secreted and without conflicting localization predictions (i.e., negative for transmembrane domains, endoplasmic reticulum retention, GPI-anchoring, and nuclear localization) were further screened using additional criteria to identify strong predictive footprints of secretion in the signal peptide region. To qualify as a SP, the candidates must also have passed stringent cutoff values for secretion, listed in Figure 1 We searched the resulting putative SPs among the orthologous groups reconstructed previously [START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF]. Briefly, the orthologous groups were obtained using mcl (54) to cluster highscoring blastp matches between all gene models predicted in 15 haploid genomes from eight Microbotryum species, previously parsed with orthAgogue [START_REF] Ekseth | orthAgogue: an agile tool for the rapid prediction of orthology relations[END_REF]. We classified a predicted SP as a species-specific SP if there was no ortholog in two of the species being considered. For predicted SP belonging to orthologous groups, we distinguished between species-specific, twoor three-way orthologous groups (i.e., predicted as SP in a single, in two or in three species, respectively) and between orthologous groups composed exclusively by predicted SP (SP-only)

and those containing at least one gene model not predicted as SP (SP-mixed). We defined the "core secretome" as the full set of predicted SPs belonging to SP-only three-way orthologous groups (i.e., present and predicted as SPs in all three species). Conversely, we defined as "accessory secretome" the predicted SPs that were either species-specific or belonged to SPmixed or two-way SP-only orthologous groups (i.e., were not present in all species or not predicted as SP in all species; Figure 2). Together, the core and accessory secretomes make up the "pan-secretome", i.e., the full set of predicted SP in all species considered.

Pfam domain annotation

We searched Pfam release 32 [START_REF] El-Gebali | The Pfam protein families database in 2019[END_REF] against the translated gene models of all predicted SP and their homologs with hmmsearch from the hmmer 3.1b1 suite (http://hmmer.org). Hits with an Evalue smaller than 1e-3 were considered significant. The results were then categorized by size as well as presence/absence of a predicted Pfam domain (supplemental file SF1).

Signal peptide clustering and experimental validation

We clustered the predicted signal peptide sequences with CD-HIT (57) allowing for up to five amino acid differences (non default options: -c 0.75 -l 5). We tested if predicted signal peptides could direct the secretion of the Suc2 invertase employing a yeast-based secretion trap method [START_REF] Kuppireddy | Identification and initial characterization of effectors of an anther smut fungus and the potential host target proteins[END_REF][START_REF] Lee | A yeast secretion trap assay for identification of secreted proteins from eukaryotic phytopathogens and their plant hosts[END_REF]. Six signal-peptide encoding sequences, as determined by SignalP Invertase deficient (suc2 -) Sacchromyces cerevisiae strain (SEY 6210 -MATαleu2-3, 112 ura3-52 his-Δ200 trp1-Δ901 lys2-801 suc2 -Δ9 GAL) cells were transformed with the constructs using the Frozen-EZ Yeast transformation II kit TM from Zymo Research. Cells were then suspended in water and spread onto synthetic drop (SD) out, SD/-Leu (Clontech) selection plates with either sucrose as the sole carbon source or glucose as a control. Resulting colonies from the sucrose plates were grown overnight in 3 ml of SD/-Leu broth with sucrose and 10 μL of undiluted, 10-fold dilutions, and 100-fold dilutions were spotted onto SD/-Leu with glucose or sucrose as the carbon source and incubated for 2 days at 30 °C. Clones harboring functional signal peptides with the reconstituted invertase activity were able to grow on sucrose as the sole carbon source. Untransformed mutant yeast strain SEY 6210 and transformed SEY 6210 cells with empty pYST-0 vector were used as negative controls. Plasmid DNA was extracted from the positive clones and used to retransform E. coli. The constructs were again checked for the presence of signal peptide sequence by DNA sequencing (Eurofins, Louisville, KY).

Tests for positive selection

We focused our selection analysis on single-copy three-way orthologous groups with one or three predicted SP. We found 163 three-way SP-only orthologous groups, among which 150 were single-copy orthologous groups (i.e., single-copy three-way SP-only orthologous groups or single-copy core secretome). Furthermore, 118 single-copy orthologous groups retained a single predicted SP after annotation (i.e., single-copy three-way SP-mixed orthologous groups from the accessory-secretome, hereafter abbreviated as monoSP). As a first method to test for positive selection, we compared evolutionary codon models M8 and M8a (59) on 150 core and 118 monoSP single-copy orthologous groups using SELECTON [START_REF] Doron-Faigenboim | Selecton: a server for detecting evolutionary forces at a single amino-acid site60[END_REF]. To check whether positive selection was more or less frequent in SPs compared to other (non-SP) genes, we performed the same test in 314 randomly picked single-copy three-way orthologous groups without predicted SP and with the same length distribution as predicted SPs. The evolutionary model M8, in which a proportion of sites are drawn from a category with dN/dS ratio greater than one, i.e., allowing for sites undergoing positive selection, was tested against M8a, in which no site is allowed to have a dN/dS ratio larger than one, i.e., does not allow for positive selection, using a likelihood ratio test with one degree of freedom to determine the statistical probability that the genes evolve under positive selection [START_REF] Stern | Advanced models for detecting positive and purifying selection using a Bayesian inference approach[END_REF]. We adjusted chi-squared p-values using Bonferroni's correction for multiple testing in R considering 582 tests.

We also performed McDonald-Kreitman (MK) tests to infer the existence of positive selection [START_REF] Mcdonald | Adaptive protein evolution at the Adh locus in Drosophila[END_REF]. MK tests contrast levels of polymorphism and divergence to test for a departure from neutrality in terms of non-synonymous substitutions (i.e., rapid amino-acid changes) while controlling for gene-specific mutation rates. MK tests estimate α, the fraction of amino acid substitutions that were driven by positive selection. To analyze within-species polymorphism, we used genome sequences previously obtained with Illumina paired-end sequencing technology for populations of the three focal species M. lychnidis-dioicae, M. silenes-dioicae and M. violaceum var paradoxa [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF][START_REF] Whittle | Recent and massive expansion of the mating-type-specific region in the smut fungus Microbotryum[END_REF][START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF]. We downloaded raw data publicly available from the NCBI Short Read Archive (SRA) under the BioProject IDs PRJNA295022, PRJNA269361 and PRJEB16741. Four major genetic clusters were identified in Europe in M. lychnidis-dioicae [START_REF] Badouin | Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates[END_REF], and we only considered strains belonging to the largest cluster in North Western Europe so that population subdivision does not bias selection inferences. A list of the isolates used in the analysis is presented in supplemental table ST1. We processed the raw genome data of 18 M. silenes-dioicae, 20 M. lychnidis-dioicae, and four M. violaceum var paradoxa isolates to build pseudo-alignments sequences of gene coding sequences within each species using as reference genomes the assemblies reported in GCA_900015465.1 for M. lychnidis-dioicae, GCA_900120095.1 for M. silenes-dioicae and GCA_900015485.1 for M. violaceum var paradoxa. First, reads were trimmed for quality (length >50; quality base >10) using the Cutadapt v1.12 software [START_REF] Martin | Cutadapt removes adapter sequences from highthroughput sequencing reads[END_REF]. We mapped Illumina reads against the reference genomes of each species using bowtie2 v2.1.0 (63) and filtered for PCR duplicates using picard-tools (http://broadinstitute.github.io/picard). We realigned reads, called for SNPs and filtered them for quality, high genotyping rate (>90%) and minor allele frequency (>10%) using GATK version 3.7 [START_REF] Mckenna | The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data[END_REF] and vcftools version 0.1.13 (65) as described previously [START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeorgraphic structure[END_REF][START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF]. We built pseudoalignments sequences of gene coding sequences from the VCF file produced by GATK using a customized script. For each strain, reference nucleotides were replaced by their variants in the reference sequence. We used MUSCLE [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF] and translatorX (67) to perform codon-based alignments of gene coding sequences among and between species. We used the MKT() and get.MKT() functions in the POPGENOME Rpackage [START_REF] Pfeifer | POPGENOME: an efficient Swiss army knife for population genomic analyses in R[END_REF] to perform MK tests.

With these tools, we performed three comparisons. We tested for positive selection comparing polymorphism and divergence of 148 core secretome and 115 monoSP orthologous groups for (1) M. violaceum var paradoxa against M. lychnidis-dioicae and M. silenes-dioicae strains;

(2) M. silenes-dioicae against M. violaceum var paradoxa strains; and (3) M. lychnidisdioicae against M. violaceum var paradoxa strains. We excluded from the analyses genes having multiple (paralogous) copies. No neutrality index or α value could be computed for 27 orthologous groups in the pairwise species comparison (1), 67 orthologous groups in the pairwise species comparison (2) and 67 orthologous groups in the pairwise species comparison (3), due to lack of synonymous or non-synonymous polymorphism. We performed the same three pairwise comparisons with 314 genes from the control group described above. No neutrality index or α value could be computed for 30, 99 and 84 in the control pairwise comparisons (1), ( 2) and (3), respectively. We assessed significance of positive selection for genes having a neutrality index inferior to 1 and a positive α value using a Fisher test (p-value < 0.05).

Footprints of RIP (repeat-induced point mutations)

We investigated the extent of RIP-like footprints in Microbotryum genomes with a per-gene RIPindex defined as the ratio of t over n (RIP-index=t/n), with t being the sum of TTG and CAA trinucleotides (forward and reverse potentially RIP-affected targets; 24) divided by the sum TCG and CGA (forward and reverse non RIP-affected targets), and n being the sum of all other nontarget trinucleotides [ACG]TG and CA[CGT] divided by the sum of [ACG]CG and CG[CGT], to control for contextual sequence composition. A RIP-index greater than one thus represents an excess of potentially RIPed sites controlling for the base composition. We compared the distribution of per-gene RIP-index values between genes predicted to encode SPs and those not predicted to encode SPs (non-SPs), and considering whether or not the genes belonged to orthologous groups undergoing positive selection.

Genomic landscape analyses

We used OcculterCut v1.1 [START_REF] Testa | OcculterCut: A Comprehensive Survey of AT-Rich Regions in Fungal Genomes[END_REF] to determine if Microbotryum genomes harbored AT-rich regions. Contigs suspected to contain mitochondrial sequences were removed from the assemblies prior to the analysis using the mito_filter.sh script, available as part of the OcculterCut distribution (https://sourceforge.net/projects/occultercut). Transposable elements locations for M. lychnidis-dioicae and M. silenes-dioicae were retrieved from a previous study [START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeorgraphic structure[END_REF] and predicted in M. violaceum var paradoxa using the same TE centroid sequence database [START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeorgraphic structure[END_REF]. Distance to TE was parsed with bedtools [START_REF] Quinlan | BEDTools: a flexible suite of utilities for comparing genomic features[END_REF].

Intraspecific secretome comparison between M. lychnidis-dioicae isolates from differentiated populations

For analyzing the genome-wide intraspecific variation in secretomes, a second genome (assembly GCA_003121365.1) of M. lychnidis-dioicae isolated in Olomouc, Czech Republic, and abbreviated as M. lychnidis-dioicae 1318, was analyzed [START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeorgraphic structure[END_REF]. We used blastp and orthAgogue to obtain high-scoring pairs between gene models of M. lychnidis-dioicae 1318 and the entire gene model set analyzed previously [START_REF] Branco | Multiple convergent supergene evolution events in mating-type chromosomes[END_REF] and re-ran the mcl algorithm. We then parsed the extended orthologous groups to identify the M. lychnidis-dioicae 1318 gene models homologous to the M. lychnidis-dioicae Lamole SPs identified in this work. We compared the frequency of synonymous and non-synonymous single nucleotide substitutions in codon-based pairwise alignments of M. lychnidis-dioicae Lamole and M. lychnidis-dioicae 1318 genes corresponding to the core secretome or to the non-SP control single-copy orthologous groups.

Per-site substitution numbers were calculated as the sum of substitutions divided by the length of the nucleotide alignment.

Analysis of gene expression level across infection stages and mating conditions

We retrieved gene expression data across M. lychnidis-dioicae Lamole infection stages on Silene latifolia and phytol-induced mating conditions from previous studies [START_REF] Perlin | Sex and parasites: genomic and transcriptomic analysis of Microbotryum lychnidis-dioicae, the biotrophic and plant-castrating anther smut fungus[END_REF][START_REF] Toh | Transcriptional analysis of mating and pre-infection stages of the anther smut, Microbotryum lychnidis-dioicae[END_REF]32) as average log2 fold change (log2FC) against the mated (non-infection) condition (n=2-4 for each of the eight conditions analyzed). We obtained the one-to-one gene model correspondences between long-and short-read sequencing-based assemblies of the same M. lychnidis-dioicae Lamole strain as best reciprocal hits with blastp. We focused our analysis on predicted SPs from the core and monoSP orthologs, using gene models from the control set described above for comparisons.

Only genes with a Benjamini-Hochberg's adjusted p-value lower than 1e-5 in at least one condition were considered. Clustering and plotting was performed in R with the heatmap.2 function of the gplots package using 10 bins for colouring the log2FC values and clustering by mean values per row. Pie charts were generated with the pie function of R base.

Plotting, statistical tests and figures

Unless otherwise stated all plots and statistical tests were performed in R version 3. the light green background, all genes are predicted as secreted. In the core secretome, all three species have at least one predicted SP; in the species-specific orthologous groups, predicted SPs were represented in a single species (i.e., paralogous genes); in the accessory two-way (a2way) groups, one species did not have any ortholog in our reconstruction. In the SP-mixed orthologous groups at right, with the yellow background, not all orthologs were predicted as secreted; for example, in the monoSP group, a single species had predicted secreted proteins in the monocopy orthologous group. The box color key corresponds to the ratio of predicted SPs over the total number of genes in a given orthologous group per species, with a gradient from blue when all orthologs in all three species are predicted as secreted to dark gray when no ortholog is predicted as secreted. cells are able to grow on sucrose as the sole carbon source. Different dilutions of cells were made (undiluted, diluted 10x or 100x) to better distinguish differences, if any. B) Amino acid sequences and species range of signal peptides tested here and in a previous study [START_REF] Kuppireddy | Identification and initial characterization of effectors of an anther smut fungus and the potential host target proteins[END_REF]. Cells under the "SP/gene count" columns follow the same color scheme as in Figure 2. Microbotryum species abbreviations are as in Figure 2. The signal peptide with the code 12964 in panel A corresponds to a protein from M. violaceum var paradoxa predicted to be GPI-anchored to the membrane.

Supplementary materials

  Cause and Effectors: whole genome comparisons reveal shared but rapidly evolving effector sets among host-specific plant-castrating fungi William C Beckerson, Ricardo Rodriguez de La Vega, Fanny E Hartmann, Marine Duhamel, Tatiana Giraud, Michael Perlin To cite this version: William C Beckerson, Ricardo Rodriguez de La Vega, Fanny E Hartmann, Marine Duhamel, Tatiana Giraud, et al.. Cause and Effectors: whole genome comparisons reveal shared but rapidly evolving effector sets among host-specific plant-castrating fungi. mBio, 2019, 10, pp.e02391-19. 10.1128/mbio.02391-19. hal-02303825 Introduction

  MK tests indicated signatures of within-species positive selection in eight core secretome orthologous groups and fifteen monoSP orthologous groups (supplemental file SF3). Out of the 23 orthologous groups with signatures of positive selection detected using MK tests, six were also detected to evolve under positive selection in the SELECTON analysis (supplemental file SF1). Five orthologous groups were found undergoing intraspecific positive selection in all three comparisons. Intraspecific selection tests on control non-SP orthologous groups revealed that 11 underwent positive selection. While core SPs showed no excess of fixed non-synonymous polymorphisms, monoSPs were enriched in genes evolving under within-species positive selection (15 out of 115 monoSPs versus 11 out of 314 non-SP genes, Fisher's exact test p = 0.0008147).When we compared two well-assembled M. lychnidis-dioicae genomes, those of the Lamole and 1318 strains, originating from two differentiated populations maladapted to their sympatric hosts[START_REF] Feurtey | Strong phylogeographic co-structure between the anther-smut fungus and its white campion host[END_REF], we only found 29 Lamole M. lychnidis-dioicae SPs without a corresponding 1318 M. lychnidis-dioicae gene (12 predicted SPs in 10 orthologous groups and 17 species/strain-specific SPs). In addition, we found 11 orthologous groups for which gene model counts were different between the 1318 and Lamole M. lychnidis-dioicae strains. The ratio of SP-containing orthologous groups with gene count polymorphisms between M. lychnidis-dioicae strains was significantly smaller than the genome-wide ratio (21/357 SPs vs 2642/12277 all genes, Chi-square with Yates correction p < 1e-11). We found few predicted SPs within genome regions showing presence/absence polymorphism within species as analyzed previously[START_REF] Hartmann | Gene presence-absence polymorphism in castrating anther-smut fungi: recent gene gains and phylogeorgraphic structure[END_REF] in both M. lychnidis-dioicae Lamole (five) and M. silenes-dioicae (two).

FIGURE 7

 7 FIGURE 7

  , for at least three of the following four tests: a predicted secretion signal by TargetP (53), a D-score of greater than 0.43 for the neural network [NN], a secretion probability of greater than 0.8 for the hidden Markov model [HMM] from SignalP3.0, and predicted secretion by Phobius.

  4.1 software, were amplified by PCR. Standard PCR cycle was used with initial denaturation set at 94 °C for 4 min and 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s and final extension time of 5 min at 72 °C. The purified fragments were then subcloned into a TOPO vector using an Invitrogen TOPO TA Cloning ® kit, and subjected to restriction digestion with Eco RI and Not I enzymes. The digested fragments were then purified and cloned into the pYST-0 vector, upstream and in-frame with an invertase coding sequence, SUC2. The presence of each signal peptide encoded in-frame with the SUC2 coding region was confirmed by DNA sequencing (Eurofins, Louisville, KY).

  6.1 (71 R Core Team, 2019). Final layout of the figures was produced with Inkscape version 0.92.3. ACKNOWLEDGMENTS. This work was supported by European Research Council [GenomeFun 309403 grant] to [TG]; by National Institute of Health (NIH) [sub-award #OGMB131493C1] to [MHP] from [P20GM103436] to [Nigel Cooper, PI]; and by the 2016-2017 STEM Chateaubriand Fellowship to [WCB]. The contents of this work are solely the responsibility of the authors and do not represent the official views of the NIH. Author Contributions: WCB, TG, and MHP conceived the project. WCB, RCRdlV, TG, and MHP wrote the paper with the input of all authors. WCB performed initial bioinformatic analysis and tested candidate signal peptides. RCRdlV analyzed the data and prepared the final draft. FEH analyzed population genomics data. MD analyzed RIP footprints. WCB, FEH, and RCRdlV generated the figures and tables.

Fig. 1 .

 1 Fig. 1. Procedural framework for predicting secreted proteins in three Microbotryum

Fig. 2 .

 2 Fig. 2. Comparison between the secretomes from three Microbotryum species. A) Key to the

Fig. 3 .

 3 Fig. 3. Overview of predicted SP (secreted protein) and non-SP homologs. A) Length

Fig. 4 .

 4 Fig. 4. Experimental validation of predicted signal peptides. A) Yeast secretion trap analysis

Fig. 5 .

 5 Fig. 5. Inter-and intra-specific comparisons of Microbotryum secretomes. A) Sampling

Fig. 6 .

 6 Fig. 6. Investigation of the impact of RIP (repeat-induced point mutations) on gene

Fig. 7 .

 7 Fig. 7. Relative expression of Microbotryum lychnidis-dioicae genes across infection stages

  Pale gray boxes represent missing genes in a given orthologous group. B) Stacked bar plots of gene counts in the different categories described in the panel A, with the same terminology, light colors correspond to non-SP homologs of predicted SPs. C) Areaproportional Venn diagram of predicted SP and non-SP homologs, also including speciesspecific genes. Each area is annotated with six-cell blocks with the number and proportion of predicted SPs in SP-only and SP-mixed orthologous groups, respectively, colored following the same gradient as in panel A. Numbers at the bottom of the blocks correspond to the number of SP-only (left) or SP-mixed orthologous groups (right). Rows in the blocks correspond to M.

lychnidis-dioicae, M. silenes-dioicae, M. violaceum var paradoxa, from top to bottom. Venn diagram was obtained with BioVenn

[START_REF] Hulsen | BioVenn -a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams[END_REF]

. Abbreviations for all panels: a2way, accessory SP two-way orthologous groups; Core, orthologous groups in which all members are predicted as SP and with at least one gene in each species; mixSP, orthologous groups with both SP and non-SP genes not including monoSP; monoSP, orthologous groups with one gene in each species but with a single predicted SP; MvSl, M. lychnidis-dioicae; MvSd, M. silenes-dioicae; MvSp M. violaceum var paradoxa; SP-mixed, orthologous groups with at least one gene not predicted as encoding a SP; SP-only, orthologous groups in which all genes are predicted as encoding SPs.


Samory Houzangbe
email: samory.houzangbe@ensam.eu

Olivier Christmann
email: olivier.christmann@ensam.eu

Geoffrey Gorisse
email: geoffrey.gorisse@ensam.eu

Simon Richir
email: simon.richir@ensam.eu

Effects of Voluntary Heart Rate Control on User Engagement in Virtual Reality

Keywords: Virtual Reality, Biofeedback, Engagement, User Study Human-centered computing-Interaction paradigms-Virtual reality; Human-centered computing-Interaction design process and methods-User centered

The usage of biofeedback in Virtual Reality (VR) is becoming more and more important in providing fully immersive experiences. With the rapid evolution of physiological monitoring technologies it is important to study how different modalities of biofeedback can alter user experience. While previous studies use biofeedback as an additional interaction mechanic. We created a protocol to assess heart rate control competency and used the results of said protocol to immerse our participants in a VR experience where the biofeedback mechanics are mandatory to complete a game. We observed consistent results between our competency scale and the participants' mastery of the biofeedback game mechanic in the VR experience. We also found that the biofeedback mechanic has a significant impact on engagement.

INTRODUCTION

The use of physiology in Virtual Reality (VR) has been studied in order to bring more out of the experience. The general consensus seems to point toward the fact that the usage of physiological biofeedback can bring more user engagement in VR experiences [START_REF] Sra | Breathvr: Leveraging breathing as a directly controlled interface for virtual reality games[END_REF]. However, biofeedback generally takes the role of an additional mechanic that the user can neglect [START_REF] Houzangbe | Fear as a biofeedback game mechanic in virtual reality: Effects on engagement and perceived usability[END_REF]. That is why we want to study how a voluntary biofeedback game mechanic can influence user experience in Immersive Virtual Environments (IVE). Thus, it is important to provide a personalized experience, based on competency, and study how mandatory biofeedback affects user experience.

The mechanics of voluntary heart rate control have been thoroughly studied, notably by Sroufe [START_REF] Sroufe | Effects of depth and rate of breathing on heart rate and heart rate variability[END_REF]. The results of his studies show that it is possible to influence one's own heart rate with breathing and that a training strategy facilitate the acquisition of mastery over physiological control. Moreover Larkin et al. [START_REF] Larkin | The effect of feedback-assisted reduction in heart rate reactivity on videogame performance[END_REF] demonstrate that the inclusion of biofeedback, coupled with training, increase even more the learning of heart rate control.

In order to study the effect of a voluntary heart rate biofeedback on user engagement in VR, we conduct a two-part study. The first part consists in determining the ability of the participants to control their own heart rate and classifying them in different skill groups. The second part of our experience exposes the participants to an IVE where they have to control their heart rate to complete the game. We analyze the participants reported feeling of engagement in regard to their competency.

VOLUNTARY HEART RATE CONTROL VR GAME

To quantify one's own heart rate control competency we developed a simple desktop application that proposes a series of heart rate control exercises consisting in accelerating or lowering the heart rate during the longest possible period of time, each exercise last 1:30 minutes. The application is split in three parts. The first is a blank screen informing the participants that the calibration of their heart rate baseline is being performed (based on the work of Houzangbe et al. [START_REF] Houzangbe | Fear as a biofeedback game mechanic in virtual reality: Effects on engagement and perceived usability[END_REF]). The second part is a training phase, the screen shows different breathing patterns that the participants have to follow (a fast and shallow breathing pattern and a slow a deep breathing pattern), in order to observe the effects of these patterns on their heart rate. Finally, the experiment begins. It consists in a series of 8 exercises where the participants have to either increase their heart rate or lower it (inspired by the work of Manuck et al. [START_REF] Manuck | Role of feedback in voluntary control of heart rate[END_REF]). During an exercise the participants have to maintain, for the maximum of time, their heart rate in a designated zone, based on their baseline value. Fifty-seven participants completed this experiment.

To measure how well the participants fare in the different conditions we calculated the mean time they spent in the designated heart rate zone for each round of the trials. Based on those time values we calculate time intervals that allow us to classify the participants in categories ranging from 1 (weak) to 4 (strong) for each trial type and compute a mean value of those scores to have a definite result on the 57 participants' ability: (1) 21 didn't succeed in controlling their heart rate, (2) 2 are considered weak at controlling their heart rate, (3) 13 are considered regular at controlling their heart rate, (4) 13 are considered good at controlling their heart rate and (5) 8 are considered exceptional at controlling their heart rate.

Following a similar pattern than previously described we develop an immersive virtual reality game that will take advantage of the participants voluntary heart rate control as a game mechanic. The game consists in a series of 8 heart rate control exercises, one for each level in the game. The participants unlock the end of each level by reaching certain heart rate values during a defined time. From the results of the first study presented in this paper we elected the heart rate thresholds to be: (1) reaching the baseline value and lower, when reducing one's own heart rate and (2) reaching the baseline value plus 20 BPM and higher, when increasing one's own heart rate.

The game is partly inspired by Portal1 . Placed in a futuristic experimental laboratory, the game consists of a succession of rooms that each possesses its unique mechanics and interaction (Figure 2). The game starts in a furnished room with a floating drone in a corner. For our mandatory biofeedback game mechanic to be adapted to each participant, we start every experiment with a calibration phase, the same one presented in the first study. The calibration takes place in the starting room, in the VR environment, during that time the participants can explore the room but are invited not to be too active. Once the calibration is over, the drone explains the context and gives the instructions to the participants.

To give feedback to the participants on their heart rate, each room has a specific interaction that is linked to it. The different interactions are described in the next section. To induce a high connection from the participant with the virtual environment we wanted to tie the effect of heart rate to a "logical" consequence in the virtual environment [START_REF] Groenegress | The physiological mirror-a system for unconscious control ofavirtual environment through physiological activity[END_REF] (e.g. the higher the heart rate the bigger the fire, the lower the heart rate the lower the volume of gas...).

From the 57 participants of the previous experiment we select 30 persons to participate in the second part of the experiment. They are classified as two competency groups for the rest of the study. The "Low Control Group" (LCG) comprised of 8 participants that didn't succeed the heart rate control and 8 that are considered regular at controlling their heart rate (N=16). The other group, the "High Control Group" (HCG) is composed of 8 participants that are good at controlling their heart rate and 6 that are exceptional at controlling their heart rate (N=14). Participants experienced both difficulties of the game, in the same order. Firstly, the normal difficulty (ND) then the enhanced difficulty (ED). To study the effects of this new mechanic on user engagement we built a questionnaire evaluating different factors of engagement.

RESULTS AND DISCUSSION

The results of our mixed between-within analysis of variance showed that there was significant interaction between the difficulty and the level of the participants for the question "How fun was the gaming experience?" (Wilks' Lambda=.759, F(1, 28)=8.894, p=.006, partial eta squared=.241). We first analyzed the results depending on the competency group for this question. When looking in detail we found no significant differences between the difficulty levels for the LCG. On the other hand, the HCG reported significantly different scores depending on the difficulty (p=.014, Z=-2.449), the score being higher for the second round (M(ED)=3.86, SD=.864) than for the first round (M(ND)=3.43, SD=.756).

We observed some significant influence of the round over the scores for questions "How successful did you feel when you overcame the obstacles in the game?" (Wilks' Lambda=.862, F(1, 28)=4.491, p=.043, partial eta squared=.138; M(ND-all)=3.63, SD=1.19; M(ED-all)=4.10, SD=.92) and "How much did you find that your skills were gradually improving through the course of overcoming the challenges in the game?" (Wilks' Lambda=.830, F(1, 28)=5.723, p=.024, partial eta squared=.170; M(ND-all)=3.30, SD=1.02; M(ED-all)=3.60, SD=1.13). We note that the participants felt more rewarded during the second round. Looking into more details, using a Wilcoxon signed ranks test, these results are especially observable for the participants in the HCG, which reported significantly higher personal gratification for the questions "How much did you feel that the game constantly motivated you to proceed further to the next level?" (p=.025, Z=-2.236; M(ND-HCG)=3.36, SD=.842; M(ED-HCG)=3.71, SD=.726) and "How much did you find that your skills were gradually improving through the course of overcoming the challenges in the game?" (p=.034, Z=-2.121; M(ND-HCG)=3.29, SD=.91; M(ED-HCG)=3.71, SD=1.14). We can report that the participants in the HCG felt more rewarded during the ED condition as it "felt more challenging, the difficulty was more satisfying".

These results allow us to highlight that the level of engagement is indeed linked to the levels of competency and difficulty. Indeed, the participants that were rated as highly capable of influencing their heart rate reported overall better results in the different factors of engagement. Interestingly we can clearly observe that the participants in the HCG are more engaged when the difficulty of the game is higher compared to the participants in the LCG who prefer the normal difficulty.

This work presented a two-part experiment designed to study the effect of a mandatory heart rate control mechanic on user engagement in a VR experience. We were able to directly translate the results of the first part of our study into a VR experience. The usage of direct voluntary heart rate biofeedback had a significant impact on the level of engagement. The participants having better control over their heart rate reporting higher results, despite voluntary heart rate control being the only interaction mechanic.

Figure 1 :

 1 Figure 1: Illustrations of the different rooms of the VR game.

ValveCorporation -2007


Maha Abboud

Philippe Hoppenot
email: p.hoppenot@iut.univ-evry.fr

Emmanuel Rollinde
email: emmanuel.rollinde@u-cergy.fr

Enhancing mathematics and science learning through the use of a Human Orrery

Keywords: Instructional innovation, STEM education, Enactivism, Astronomy

We present in this paper an innovative approach to introduce STEM topics in the classroom through the context of the Solar System. Our pedagogical tool consists in a Human Orrery modeling at a human scale the Solar System dynamics. While learners walk along planetary orbits, they enact different mathematical and scientific notions. Previous experimentations were centered on science concepts and have shown an enhanced motivation and well-being and a better understanding. We discuss here the connection with the cognitive science theory of enactivism and with the activity theory, both already used in the field of mathematics education. New pedagogical sequences are proposed that deeply combine mathematics and science contents. Those will be set up in a classroom context before the CERME conference.

Introduction

STEM literacy is a crucial issue for European educational policies today as emphasized by successive trans-European studies since the Rocard report (2006). Numerous difficulties have been revealed by science and mathematics education researchers in knowledge acquisition of scientific notions of movements, velocity, proportionality and functions. Many of them arise from the gap between children's sensorial or everyday life experiences and the abstract scientific explanations. Furthermore, the concept of speed with its related concepts of distance and time/duration and its links with proportionality and graphs has not been studied extensively from a cross cultural point of view.

The study presented in this paper explores the use of astronomy to teach mathematics and physics. We consider that astronomy should not be taught and considered in education science as an independent subject but as a specific application of general laws of physics and of application of mathematics concepts. The European ROSE project (Sjoberg & Schreiner, 2010) has shown that the subject that interests both girls and boys was found to be life outside earth. We confirm in our experiments that the study of the Solar System motivates students to do sciences, while a large number of mathematical subjects may be easily introduced through the observation and understanding of planets' motion. The tool that we use to this purpose is a Human Orrery modeling at a human scale the Solar System dynamics in ways that can enhance the learner's perception of mathematics and physics concepts in a real world apparatus.

In the following sections, we will provide a description of this tool and its affordance and the potentials within a STEM approach. We will also present our theoretical frame and the outcomes of first preliminary experiments. This background presentation enables us then to introduce a large scale project in progress and to give some examples of settings that are being currently designed in order to be implemented, observed and evaluated in several European countries.

The Human Orrery

An Orrery is a mechanical instrument showing circular orbits of the planets. The first Orrery to be created at a human scale was done in Japan (Dynic Astropark) and then at the Armagh Observatory [START_REF] Asher | The Human Orrery: a new educational tool for astronomy[END_REF]. The design of a Human Orrery is made such that users may walk along orbits of different bodies around the Sun that is located at the center of the design. The Human Orrery that we use is printed in a large map of 12m by 12m (see Figure 1). It allows one to follow the orbits of the inner planets (Mercury, Venus, Earth and Mars) and Jupiter; the inner planets are located inside the asteroid belt that is materialized with a grey color together with the orbit of the largest asteroid known, Cérès. The highly eccentric orbits of two comets are also used: Encke (the smallest elliptical orbit) and Chury. This choice of objects illustrates different type of movements while keeping the size of the Orrery reasonable. Earth is at one meter from the Sun, while Jupiter's orbit has a diameter of 10,5m. The orbits of all bodies are materialized by dots at constant intervals of times, with accurate elliptical shapes. Note that, orbits are near-circular for the five planets and Ceres. The interval of time may be different for each orbit, but is always a multiple of 16 terrestrial days: For Earth, there are 23 dots separated by 16 days, which would make a period of 368 days. For Jupiter, there are 54 dots separated by 80 days, which makes a period of 4320 days instead of the real period of 4332,59 days. A sound (either a clock or hand claps) is heard regularly. The interval of time between two sounds corresponds to 16 terrestrial days. Every user makes one step during this time interval. Then, the person that enacts Earth walks from one point to the next (distance) in one step (duration), while "Jupiter" has to do five steps (five times 16 days) to reach the next point. All rules are described in details in [START_REF] Rollinde | Learning Science Through Enacted Astronomy[END_REF]. By acting according to those rules, the movements on the Human Orrery illustrate the correct relative velocities of all Solar System bodies.

Both constructing and enacting on Human Orrery involve topics from mathematics and science and enhance a STEM approach of scientific notions usually perceived as abstract concepts by students. We shall discuss in more details the use of the Human Orrery and its future perspective for STEM education after a discussion of the theoretical framework underlying this innovative pedagogy.

Theoretical framework

The use of a Human Orrery in education is based on the assumption that bodily perceptions help the learning of abstract concepts. In other words, "abstract symbols used in formal education-words and syntax in reading, numbers and operators in math-need to be grounded in bodily experience" [START_REF] Glenberg | Embodiment as a unifying perspective for psychology[END_REF]. In the context of the Solar System, the objects under scrutiny can neither be touched nor seen directly in the classroom, duration is very long, and very little is expected to happen in the classroom situation. By physically enacting the objects and their movements, the situation changes completely: dynamic properties are revealed and experienced; the invisible and impalpable acquire a certain degree of "palpability" allowing learners to deeply understand them as walking along the Human Orrery.

The cognitive science theory of enaction [START_REF] Varela | The Embodied Mind: Cognitive Science and Human Experience[END_REF]) may provide a theoretical foundation to those assumptions. [START_REF] Torrance | In search of the enactive: Introduction to special issue on enactive experience[END_REF] describes enactivism as the nature of the mind, defined by its relation to the world: "(…) the organism's world is "enacted", brought forth by sensorimotor activity: world and organism are co-determined, they co-emerge. [For the mind], awareness of its self and of its world is a central feature of its lived embodiment in the world." Transposed into Mathematics Education (we refer the reader to the review by [START_REF] Reid | The coherence of enactivism and mathematics education research: A case study[END_REF], enactivism theory has been used successfully, for example to explain the emergence of formal mathematical thought from premathematical experiences in the course of a lesson sequence on three-dimensional geometry for 6year-old children [START_REF] Roth | Geometry as objective science in elementary school classrooms: Mathematics in the flesh[END_REF]. Similarly, in the context of a numerical sequence, Radford (2014) shows that, "through an intense interplay between various sensorial modalities and different signs, the students' perception and the concomitant mathematical thinking have gained a theoretical dimension that they did not have before". More generally, learning can be facilitated to the extent that lessons are created that map to and activate sensorimotor systems (see for example [START_REF] Johnson-Glenberg | Effects of Embodied Learning and Digital Platform on the Retention of Physics Content: Centripetal Force[END_REF] in the context of digital platforms). This line of argument may sound contrary to early researches in science education that rather tend to show that our observations and interactions with the world often produce naïve conceptions in conflict with formal physical laws (e.g. [START_REF] Hestenes | Force concept inventory[END_REF]. Difficulties in knowledge acquisition in science seem to arise from the gap between children's sensorial experiences and the abstract scientific explanations. We make here the assumption that it is necessary to promote a "mindful attention to perception" [START_REF] Varela | The Embodied Mind: Cognitive Science and Human Experience[END_REF]). In the context of the Human Orrery, our goal is to build a complex learning world filled by metaphors: a simple map becomes the empty interplanetary space; series of dots become the elliptical structure of an orbit; peers become planets with different velocities; duration is heard while distance is felt… The Human Orrery (a human-scaled technological object) acquires a meaning through the learner's cognition and his/her mindful perception of this world and thus activates new cognition.

To theoretically ground our observations, analyses of teaching and learning within the Human Orrery context we plan to make use of Activity Theory as used today in Mathematics education [START_REF] Abboud | Use of activity theory to make sense of mathematics teaching: a dialogue between perspectives[END_REF][START_REF] Vandebrouck | Mathematics classrooms students' Activities and Teachers' Practices[END_REF]. [START_REF] Roth | Situated cognition[END_REF] acknowledge that original works on Activity Theory by "Soviet social psychologists explicitly ground their ideas in theories in which thinking, acting and environment are part of the same analytic unit." Indeed, the Activity Theory, in particular the notion of subject-object interactions mediated by an instrument enables to study the activities of subjects (teachers and learners) using tools (the Orrery itself but also the set of technological tools involved) and their impact on the processes of learning physical and mathematical concepts at stake when experiencing the Human Orrery. Yet, current use of Activity Theory in education does not mention the role of bodily perception. "Mental processes (speaking, thinking, reading or writing) need to be understood in terms of complex ecologies: neuromuscular and physiological processes AND cultural historical origin and nature" [START_REF] Roth | Situated cognition[END_REF]. Our intention is thus to work towards an "Embodied Activity Theory" which would be a framework adapted to our research and that would develop over time in a dynamic interaction between analyses of data and design and use of theoretical tools.

Outcomes from first experiments

The main use of Human Orrery in the literature has been the introduction of astronomy in open schooling contexts (e.g. [START_REF] Francis | Using role-playing games to teach astronomy: An evaluation[END_REF][START_REF] Asher | The Human Orrery: a new educational tool for astronomy[END_REF][START_REF] Lopresto | Assessment of a Solar System Walk[END_REF]. Since 2014, our team focuses on the use of a "human Orrery" to learn science rather than astronomy only in different pedagogical context, from primary school to University. Through our initiative, seven Human Orreries were built in France (one for a science center, one in a public place in Paris, five in primary or secondary schools), one was drawn in a Lebanon school and one map was purchased by a science center in Vietnam. Human Orreries are used thereafter by different teachers (physics, mathematics, technology and sports) in those seven places plus about five other schools who use our trap only. Questionnaires and interviews were used to quantify the amount of learning and the students' motivation. The sequences are discussed and improved after each application. They have also been used in teacher education for two years, and may be obtained upon request to the authors. More details on these first experiments and derived conclusions as described here may be found in [START_REF] Rollinde | Learning Science Through Enacted Astronomy[END_REF].

The implemented sequences have focused on the one side on the motivation and interests of the learners and on the other side on specific scientific notions. Among the key concepts introduced through the Human Orrery are kinematic ones. The embodiment of duration and length allows to alleviate the confusion related to the use of those concepts in the common language. The manipulation of those two concepts to compute the velocity requires to deal with three variables, which is known to be source of cognitive overload. As an example, almost every groups express a confusion between large velocity and small orbital period, or between small period and small perimeter; they all reduce the reasoning to a simple comparison between two variables. The enaction is expected to lighten the abstract mathematical task by providing diverse sensory-motor experiences as proposed in the sequence described in the next section. Additional difficulties encountered regularly include: the Moon that cannot be seen given the scale used (she would be behind the disc of the Earth); the confusion between 24h and one year for one orbital period of Earth (which is strengthened by the 23 dots used for the orbit of Earth); the very low speed of Jupiter is also a source of astonishment. Comments made by undergraduate students actually showed their surprise that so much may be learnt with this tool. For instance, one student declares: "There was joy, emotions. Here, it is wonderful what is going on'. She continues then: 'Universe is not made of mathematics numbers and equation of physics, this is something else, and it has to be understood in another way." Another student suggested that "illustrating a concept with a movement makes it more real. We feel that we live it.".

Project and examples of settings

The authors of this paper aim at incorporating more STEM contents into future works. We propose in the following an example of such settings, that we are currently designing, tackling the study of speed's meaning and proportionality factor. More examples could be given particularly concerning the construction of the Human Orrery and associated mathematical notions at play, but this goes beyond the scope of these proceedings.

A detailed enacted sequence on speed

In the science class, speed is understood as the length of a displacement for a given time unit (e.g. year or second). This sequence will provide a way to enact three concepts (time, distance and speed) using bodily perceptions and tools. In the mathematic class, speed is understood as the proportionality factor in the relationship between distance and duration. [START_REF] Ayan | Middle school students' reasoning in nonlinear proportional problems in geometry[END_REF] have shown that proportionality relationship is often approached by superficial characteristics of the problems, namely the monotonous relation expected between two variables, and lack clear, quantitative, arguments. [START_REF] Modestou | Cognitive and metacognitive aspects of proportional reasoning[END_REF] express the metacognitive aspect that underlines the awareness of (non)proportionality and may explain this lack of coherence in reasoning about proportionality. There is thus an obvious need for a renewed approach to proportionality and speed.

The sequence we designed uses the Human Orrery, a clock that sounds regularly (each sound is called a clap) with a given tempo, blue ropes and red ropes and markers (to materialize different positions along the ropes). There are four stages: (i) Enaction of space and time while walking freely on the Orrery under spatial and time constraints. (ii) Following a right planetary walk to enact different displacements for different durations. (iii) Enaction of the speed of the planets and the proportionality relationship. (iv) Determination of the speed of the planets (optional). The first two stages can easily be implemented in primary school; the second two are more adapted to lower secondary school.

Enact space and time

The initial proposed goal is to invent a coherent way to walk on the Human Orrery. The action is then to walk, while focusing on two perceptions: looking and hearing. The students' task explained by the teacher may be: "you have to find a way to walk coherently while accounting for dots on the map and claps. At the end, you will have to explain your choices." We expect that different rules may be proposed. For instance, each step should last the duration between two claps or each step should go from one dot to the next one, whether the students focus on what they hear or what they see. According to the rules proposed, we seek for answers to questions such as: Are they going to make a connection with speed too? Are they going to use local rules (their steps only) or global rules (the entire orbits or their relation with each other)? Are they going to describe what they are doing or global observations (who is going faster or when to end)? All those possible and expected actions (or wording here) will be useful to understand what is enacted and how.

According to the effective actions, different proximity-in-action [START_REF] Abboud | Use of activity theory to make sense of mathematics teaching: a dialogue between perspectives[END_REF]) may be foreseen to ensure coherence among the group. The students' activity could either remains on a minima level e.g. by proposing to focus on one set of dots that follow one orbit or at on a maxima level, e.g. by going faster or slower and to propose different ways to use the rhythm or the dots

Enact the planets' movement

The first action will probably end up with different solutions for a coherent walk. In the process, most learners should have noticed that the drawing is related to the Solar System. The teacher confirms and describes the different orbits. The learners want to know and understand their own role in this planetary walk. The next goal is then to measure their movement while walking as planets.

The associated task requires organisation and mathematical skills: "You will work by groups of 3. Each group chooses either Earth or Jupiter. You start to walk when you hear one sound and continue to walk until you hear "n" additional sounds. You must then find a way to show to the entire class the length of your displacement in order to compare or combine the different measures". The number "n" is explicitly given but is different for each group. For Earth, some group will work with 3 claps, some with 7 claps. For Jupiter, some will work with 5 and some with 9. Those numbers are chosen so that none is a simple combination of others by the use of addition, in order to favour the use of multiplication later on. To achieve this task, each group has a rope (blue for Earth and red for Jupiter) and markers that can be attached to the rope.

Due to the complexity of the task (follow the rules, do not measure the line but the circular length, keep record of the measured length for comparison), specific proximity-in-action are required to go through it. First, every group has to respect the planetary rules. Then, the length of a circular displacement will be measured with the rope and some markers. For groups that have completed the first task, the teacher may propose to measure the distance while starting from another point in the same orbit or to do the same measurement for another orbit with a different rope. It is important to ask them to walk and measure simultaneously, in order to enact the displacement together with the duration.

Enact the proportionality relationship between length of displacement and duration

To keep the learners focused on the subject of planets, the next goal is directly related to planets while the action will be made on ropes only. By this shift, the reasoning is made slightly more abstract. The required task is not a physical action but a reasoning one in order to answer the question: "how do the speeds of Jupiter and Earth compare?" This may sound like a simple task if it was a simple account of what they have played. Indeed, it is to compare the speed of Jupiter and Earth during the planetary walk. To avoid this shortcut, the teacher asks for a material, or visual, proof using the ropes. This reformulated task becomes complex and fully associate science and mathematics skills and concepts.

A first prediction may be searched for: How should the length of the rope of the faster planet compare to the slower one (a minima)? Such a comparison requires using a common duration, or the same number of sounds. This is our first sub-task. Let's call N the common number of claps associated to this common duration. N may be equal to 10 (this number may be obtained with the available lengths through the sum of 7+3 for Earth and the product 2 times 5 for Jupiter).

The next sub-task is then to derive the length that would have had the rope of the two planets for this common duration. The teacher proposes then to measure the length of the displacement after one clap only. To realise this task, one has to divide the rope in N parts and then use the ruler… or use the ruler for the full length of the rope and divide by N!

Enact a non-proportionality relationship

Exposing learners to a non-proportional situation is a way to face the well-known bias of proportionality and to encourage students to develop a critical mind. The Human Orrery provides us with an interesting situation: the case of a comet. Unlike the planets' orbits which are near-circular, the orbit of the comet Encke is a highly eccentric ellipse. During the movement on its orbit, the speed of the comet varies strongly: learners have to make either very large steps near the Sun or cannot even put one feet between two dots far away from the Sun. This usually creates either laugh or stress but never leave the learners insensitive in that case, duration and travelled distance are thus related monotonously, but are not proportionally linked. Using the same methodology as for Earth and Jupiter, learners will discover that the length of the rope measured for different initial position and different duration is not proportional to the duration.

The examples of pedagogical sequence that we presented above are the first of a set of settings that will be designed and implemented in several countries and in different cultural contexts as part of a European project we are currently setting up. We note that activities on the Human Orrery may also be used as an introduction to more formal mathematical activities; for example on the derivation of Kepler laws using power laws and interpolation or on the understanding of Newton laws using differential equations.

Conclusion and Perspectives

The perspective that we adopt in our project is that of STEM being taught through an interdisciplinary approach that incorporates an embodied dimension and draws on real-world modeling. The use of the Human Orrery enables students to enact or "experience" scientific concepts and the dynamics of their properties by evolving in an adapted environment. Until recently, we only focused on the dissemination of Human Orreries and on a proof-of-concepts in terms of motivation and interest. Different sequences have been co-constructed by teachers and researchers and have led to preliminary positive results on learning too. The current dynamic of the project is to motivate more classes from different levels and teachers from subjects other than mathematics and physics such as art, technology, music, languages (native or foreign), physical education and sport. In 2018-2019, four additional classes have decided to join the project in France: two primary schools in the context of interdisciplinary projects and one secondary school through a project on Mars involving science and technology. At the European level, we are expecting to submit a H2020 project involving research in mathematics and science didactics together with science centers (museum, observatories and associations). Our next objective is then to design sequences that fit into the STEAM approach and that will be used as experimental situations for the theoretical framework of enactivism, complemented with the framework of Activity Theory in the context of science education. Our focus will be as much on the didactical learning as on the motivation and awareness of the learners during the activity.

Figure 1 -

 1 Figure 1 -Left: The map of the Human Orrery (inner planets and one comet only, see text for details). Right: Pupils walking along the different orbits up to Jupiter.


Xénia Philippenko

Gonéri Le Cozannet

Lydie Goeldner

Approaches to integrate people's representations of climate change within adaptation pathways

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

Adaptation : acceptability and timescales

Auteurs

  Xénia Philippenko, BRGM / Gonéri Le Cozannet, BRGM / Lydie Goeldner-Gianella, Université de Paris 1 / Ywenn De La Torre, BRGM Relevant references : • Haasnoot M., et al., Dynamic adaptive policy pathways: A method for crafting robust decisions for a deeply uncertain world, 2013. • Hinkel J., et al., The ability of societies to adapt to twenty-first-century sea-level rise, 2018 • Eisenack K., et al., Explaining and overcoming barriers to climate change adaptation, 2014 • Friesinger S., Bernatchez P., Perceptions of Gulf of St. Lawrence coastal communities confronting environmental change: Hazards and adaptation, Québec. Canada, shown that people's representations of climate change can either facilitate climate policies or act as a barrier to successful adaptation. Here, we explore how to integrate people's perception and representations of their territories, of climate change and adaptation within the adaptation pathways framework, and we present a preliminary application in Saint-Pierre and Miquelon, a French oversea territory affected by sea-level rise, erosion and flooding. résidents perceive an urgency for adaptation. The results show a demand for immediate action and are willing to allocate important ressources.COAST OF THE TOWN OF STlargely preferred, as these are perceived most efficient to preserve residences and infrastructures. Partial relocation is considered as a potential solution only for few assets. people would accept the disappearance of the island. Some of them insist on the need to accept this phenomenon now, but the other are divided on the timing. largely preferred, but 3 diverging adaptation pathways are designed by the population. Some of them may coexist, according to people's perception.