Benoît Perez-Lamarque

Hélène Morlon
email: morlon@biologie.ens.fr

Characterizing symbiont inheritance during host-microbiota evolution: application to the great apes gut microbiota Modeling host-microbiota evolution

Keywords: symbiont transmission, microbiota, molecular evolution, likelihood-based framework, holobiont, great apes

Microbiota play a central role in the functioning of multicellular life, yet understanding their inheritance during host evolutionary history remains an important challenge.

Symbiotic microorganisms are either acquired from the environment during the life of the host (i.e. environmental acquisition), transmitted across populations or species by host-switch (i.e. horizontal transmission), or transmitted across generations with a faithful association with their hosts (i.e. vertical transmission). These different modes of inheritance affect microbes' diversification, which at the two extremes can be independent from that of their associated host or follow host diversification. The few existing quantitative tools for investigating the inheritance of symbiotic organisms rely on cophylogenetic approaches, which require knowledge of both host and symbiont phylogenies, and are therefore often not well adapted to microbial data.

Here, we develop a model-based framework for quantifying the proportion of environmental acquisition, horizontal transmission, and vertical transmission during the evolution of host-associated microbial taxa. We consider a model for the evolution of microbial sequences on a fixed host phylogeny that includes vertical transmission and horizontal host-switches. This model allows estimating the number of host-switches and testing for strict vertical transmission and environmental acquisition. We test our approach using simulations. Finally, we illustrate our framework on gut microbiota high-throughput sequencing data of the family Hominidae and identify several microbial taxonomic units, including fibrolytic bacteria involved in carbohydrate digestion, that tend to be vertically transmitted.

Introduction

Microbiota --host-associated microbial communities --play a major role in the functioning of multicellular organisms [START_REF] Hacquard | Microbiota and host nutrition across plant and animal kingdoms[END_REF]. For example, the gut microbiota plays a significant nutritional role for animals by synthesizing essential nutrients and by helping digestion and detoxification [START_REF] Mcfall-Ngai | Animals in a bacterial world, a new imperative for the life sciences[END_REF]. It is also involved in a broad range of other mutualistic functions important for host protection, development, behavior, and reproduction (Zilber- [START_REF] Zilber-Rosenberg | Role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution[END_REF]. Other less-studied microbiota, such as those found on animal skins or plant roots also play major ecological roles [START_REF] Philippot | Going back to the roots: the microbial ecology of the rhizosphere[END_REF].

Host-microbiota associations have evolved for thousand million years with three major modes of inheritance across phylogenetic host lineages: i) vertical transmission within a host lineage [START_REF] Rosenberg | Microbes Drive Evolution of Animals and Plants: the Hologenome Concept[END_REF], which can happen either by transmission from mother to child (e.g. directly through ovaries during reproduction or at birth), or by social contact while sharing life with related individuals [START_REF] Bright | A complex journey : transmission of microbial symbionts[END_REF] ii) horizontal transmission between unrelated host lineages [START_REF] Henry | Horizontally transmitted symbionts and host colonization of ecological niches[END_REF], which can for example happen through direct interactions, via vectors or via shared habitats [START_REF] Engel | The gut microbiota of insects -diversity in structure and function[END_REF], and iii) environmental acquisition, with microbes coming from the environment independently from other related hosts [START_REF] Bright | A complex journey : transmission of microbial symbionts[END_REF]. The vertical transmission of a given microbial lineage within host lineages can lead to cophylogenetic patterns, with the microbial phylogeny mirroring the host phylogeny (e.g. Helicobacter pylori in humans [START_REF] Linz | An African origin for the intimate association between humans and Helicobacter pylori[END_REF]). Horizontal transmission and environmental acquisition can play key roles in adaptation, for example by allowing host lineages to adapt to new feeding regimes [START_REF] Mckenney | Bamboo specialists from two mammalian orders (Primates, Carnivora) share a high number of lowabundance gut microbes[END_REF][START_REF] Muegge | Diet drives convergence in gut microbiome functions across mammalian phylogeny and within humans[END_REF]. They will tend to erase cophylogenetic patterns linked to vertical transmission. The relative importance of each of the three modes of inheritance depends on the type of host and the type of microbes.

For example, vertical transmission is thought to be far more preponderant in the "core" microbial species, which are shared across hosts regardless of environmental conditions, than in the "flexible" microbial species, facultative and dependent on internal and external conditions [START_REF] Shapira | Gut microbiotas and host evolution: scaling up symbiosis[END_REF].

Quantifying the relative importance of different modes of inheritance during hostmicrobiota coevolution remains a major challenge. Patterns of "phylosymbiosis", i.e. a pattern of concordance between a given host phylogeny and the dendrogram reflecting the similarity of microbial communities across these hosts, is frequently observed [START_REF] Bordenstein | Host biology in light of the microbiome: Ten principles of holobionts and hologenomes[END_REF], for example for great apes gut microbiota [START_REF] Ochman | Evolutionary relationships of wild hominids recapitulated by gut microbial communities[END_REF]. Although these phylosymbiotic patterns suggest that some microbial species within the microbiota are vertically transmitted, such community-wide comparisons of microbiota across hosts do not allow identifying which microbial species are vertically transmitted, nor quantifying the relative importance of the different modes of inheritance across distinct microbial species. More recently, approaches have been developed to apply cophylogenetic concepts to microbial taxa [START_REF] Bailly-Bechet | How long does Wolbachia remain on board?[END_REF][START_REF] Groussin | Unraveling the processes shaping mammalian gut microbiomes over evolutionary time[END_REF]. Cophylogenetic methods were originally developed to study the coevolution between hosts and their symbionts, with the underlying idea that close and long-term associations lead to congruent phylogenies with similar topologies and divergence times [START_REF] De Vienne | Coespeciation vs hos-shift speciation: methodsfor testing, evidence from natural associations and ralation to coevolution[END_REF][START_REF] Page | Trees within trees: Phylogeny and historical associations[END_REF], while processes such as host-switches disrupt this congruence. Cophylogenetic tools either quantify the congruence between symbiont and host trees using distance-based methods (e.g. ParaFit [START_REF] Legendre | A statistical test for host-parasite coevolution[END_REF], PACo [START_REF] Balbuena | PACo: a novel procrustes application to cophylogenetic analysis[END_REF]), or try to find the most parsimonious sets of events (e.g. hostswitches) that allow reconciling both trees (e.g. TreeMap or Jane [START_REF] Conow | Jane: A new tool for the cophylogeny reconstruction problem[END_REF]). In the context of microbiota, Groussin et al. [START_REF] Groussin | Unraveling the processes shaping mammalian gut microbiomes over evolutionary time[END_REF] and [START_REF] Bailly-Bechet | How long does Wolbachia remain on board?[END_REF] have used the ALE program [START_REF] Szöllõsi | Efficient exploration of the space of reconciled gene trees[END_REF][START_REF] Szöllosi | Lateral gene transfer from the dead[END_REF], which was initially designed to solve the gene tree -species tree reconciliation problem. Importantly, all these methods require a reconstruction of the symbionts' trees, which can be problematic for microbiota data typically generated using Next Generation Sequencing (NGS) metabarcoding techniques.

Here, we develop a probabilistic model of host-symbiont evolution, which aims at studying modes of inheritance in the microbiota; our framework does not require building a symbiont phylogeny and allows model comparison. Huelsenbeck et al. [START_REF] Huelsenbeck | A Bayesian framework for the analysis of cospeciation[END_REF] developed a similar approach, focused on host-parasite associations, with a model of cospeciation and host-switches. However, the authors developed an inference framework associated with the joint reconstruction of both host and parasite phylogenetic trees, which is not well adapted to the case when the host phylogenetic tree is robust and the symbionts are represented by a sequence alignment with limited phylogenetic information. We fix the host phylogeny and follow the evolution of individual microbial taxa on the host tree. We compute likelihoods associated with microbial sequence alignments under a model including vertical inheritance and host-switches. We find estimates of the number of host-switches and develop tests for evaluating model support in comparison with scenarios of strict environmental acquisition and strict vertical transmission. We test our approach using simulations and apply it to gut microbiota high-throughput sequencing data of the family Hominidae.

Materials & Methods

HOME: A general framework for studying Host-Microbiota Evolution

From metabarcoding microbiota data to independent alignments Given a host species tree and metabarcoding microbiota data (e.g. rRNA 16S sequences) sampled from each host species, our framework begins by clustering sequences into Operational Taxonomic Units (OTUs) using bioinformatics pipelines. Each OTU is made of distinct microbial populations, each corresponding to a specific host species (Fig. 1A).

We assume as a starting point that there is no within-host genetic variability (we discuss later how we relaxed this assumption), such that each microbial population is represented by a unique sequence. In our analysis of these data, for each OTU and each host, we use the most abundant microbial sequence as the representative sequence. The data we consider thus consists of a series of microbial alignments A, each corresponding to a specific OTU; a given alignment is composed of N-nucleotide long sequences (with potential gaps corresponding to missing data), each corresponding to a specific host. In each alignment, we distinguish the segregating sites (i.e. those that vary in at least one sequence) to those that do not vary across sequences. Some microbial OTUs may not be represented in all host species (i.e. there might be missing sequences in the alignment), which can either be true absences (i.e. the corresponding host species do not host the OTU), or a lack of detection (i.e. the OTU is present but has not been sampled in these host species). Because we cannot distinguish these two possibilities, we simply treat missing sequences as missing data; we do not explicitly model the extinction of symbiotic populations in certain host species, nor the microbial sampling process. We apply our model independently to each alignment.

Modeling the evolution of an OTU on a host phylogeny

We consider the evolution of a given microbial OTU on a host phylogeny T (Fig. 1); T is assumed to be a known, ultrametric, rooted and binary n-tips tree. The model is defined as follows:

(i) Vertical transmission: From an ancestral microbial population at the root of the host phylogeny represented by a N-nucleotide long sequence with Nv "variable" sites (i.e. those that can experience substitutions), substitutions occur along host branches.

Following classical models of molecular evolution [START_REF] Strimmer | Genetic distances and nucleotide substitution models[END_REF], we assume that each variable nucleotide evolves independently from the others according to a substitution model with a rate μ that is supposed to be the same for all variable nucleotides and constant along the evolutionary branches (strict-clock model). The substitution model is represented by a continuous-time reversible Markov process, characterized by an invariant measure π (i.e. the vector of base frequencies at equilibrium) and an instantaneous transition rate matrix Q between different states [START_REF] Strimmer | Genetic distances and nucleotide substitution models[END_REF].

At a host speciation event, the two daughter host lineages inherit the microbial sequence from the ancestral host, after which microbial populations on distinct host lineages evolve independently.

(ii) Host-switches: A discrete number (ξ) of host-switches happens during the evolution of the OTU on the host tree. The switches occur from a "donor" branch, with a probability proportional to its branch length, and at a time uniformly distributed on the branch, to a "receiving" branch, with equiprobability among the co-existing branches (we do not consider the phylogenetic proximity from the donor branch). When a hostswitch happens, for convenience we assume that the microbial sequence from the donor host replaces that of the receiving host and the microbial sequence from the donor host remains unchanged.

Each series of host-switches on T defines a tree of microbial populations TB that summarizes which populations descended from which ones and when their divergences occurred (Fig. 1). In the absence of host-switches (ξ = 0), TB and T are identical. When host-switches occur, they break the congruence between TB and T (e.g. Fig. 1C). Hence, the model can be decomposed in two steps: first, host-switches generate TB from T; second, a sequence (representing a microbial population) evolves on TB with a constant substitution rate.

Likelihood computation and inference

We develop a likelihood-based framework in order to fit the above model to data comprising a given (fixed) tree T of hosts and an alignment 𝐴 " of microbial sequences characterizing populations of a given microbial OTU for these hosts (here the alignment 𝐴 " is reduced to the segregating sites). This will allow estimating the number of switches 𝜉 % on the host tree. The probability of the alignment assuming that the substitution rate is μ and that there are ξ switches is given by: L(𝐴 " |µ, ξ) = . L(𝐴 " |µ, TB) 𝑑TB TB

(1)

where L(𝐴 " |µ, TB) is the probability of the alignment assuming that the substitution rate is μ and the microbial tree is TB, and the integral is taken over the space of trees obtained with ξ switches on T. In practice, we compute this integral using Monte Carlo simulations: we simulate a large number (S) of microbial trees obtained with ξ switches on T (see next section), compute for each TB the probability of the alignment assuming that the substitution rate is μ, and sum these probabilities:

L(𝐴 " |µ, ξ)~ 1 𝑆 5 L(𝐴 " |µ, TB) TB (2)
This approximate expression converges to the exact integral form when S is large.

We compute the probability L(𝐴 " |µ, TB) of the sequence alignment 𝐴 " on a given microbial tree TB using the Felsenstein pruning algorithm [START_REF] Felsenstein | Evolutionary trees from DNA sequences: A maximum likelihood approach[END_REF]. We take into account the possibility of gaps in the microbial alignment, considering them as "missing values" by pruning off the tips of the tree with a gap [START_REF] Truszkowski | Maximum likelihood phylogenetic inference is consistent on multiple sequence alignments, with or without gaps[END_REF]. First, we choose the model of DNA substitution between the K80, F81, and HKY matrices from the alignment reduced to segregating site (𝐴 " ) using the function modelTest (R package phangorn) and based on a BIC selection criterion: this function estimates Q and π directly from AS, where Q, the reversible transition rate matrix, depends on the invariant measure π. We also obtain estimates of the transition/transversion rate ratio κ (K80 and HKY) and of the base frequencies at equilibrium π (F81 and HKY) from these models. Second, we compute the probability of the alignment at each nucleotide position ν using the pruning algorithm. For a given segregating site among 𝐴 " , let P(t) be the vector of probabilities of states A, C, G and T at time t. P(t) is given by P(t) = M(t) * P(0) where P(0) = (1 < , 1 = , 1 > , 1 ? ) with 1A equals 1 if A is the initial nucleotide is A and 0 otherwise, and M(t) = e ABC . Let P D (s) be the probability of the alignment corresponding to the clade descending from node s in the phylogeny for nucleotide ν. We have:

P D (leaf) = (1 < , 1 = , 1 > , 1 ? ) and P D (s) = IM(t J )P D (s J )K. IM(t M )P D (s M )K (3)
Where s1 and s2 are the two nodes descending from s and t1 and t2 are their respective times of divergence. We iterate this pruning calculation from the leaves to the root of the tree, and obtain the probability of the alignment at the nucleotide position ν:

L D = π P D (root) (4)
Because we consider only segregating sites, we condition this probability on the occurrence of at least one substitution. The probability of a substitution happening on a tree TB of total branch length B is given by (1 -e TBU ). Finally, the probability of the alignment AS is obtained by multiplying the probabilities corresponding to each nucleotide. Hence the probability of the variable alignment AS is given by:

L(𝐴 " |µ, T V ) = (1 -e TBU ) TW X Y L Z W X [\J ( 5
)
where NS is the number of segregating nucleotides.

In practice, we used S = 10 4 and plotted the resulting value of L(𝐴 " |µ, ξ) with an increasing number of trees TB to ensure that S was large enough to obtain a reliable approximation of the likelihood. For each ξ, we find μ that maximizes L(𝐴 " |µ, ξ). Finally, we repeat these analyses for a range of realistic ξ values (typically ξ = [0, 1, 2, ... , 2n] )

and deduce the couple of parameters 𝜉 % and µ ^ that maximizes the probability of the alignment. Low 𝜉 % values are indicative of OTUs that are transmitted mostly vertically, while high 𝜉 % values are indicative of those that perform frequent host-switches.

Simulations of host-switches: from T to TB

Each switch is characterized by its "donor" branch, by its position on the branch, and by its "receiving" branch. A switch replaces the existing microbial sequence in the receiving host, and creates a new branching event in the microbial tree TB. Four types of switches can occur and each of them results in different rules to obtain TB from T (Fig. 2):

(i) the switch occurs just after the root on the host tree, before any other speciation event: TB is obtained from T by re-dating the root of the tree to the time of the hostswitch. This switch does not change the topology of the tree (i.e. it only affects the branch lengths).

(ii) the switch occurs from an internal branch to a branch directly related to the root, i.e.

one of the sequences originating at root no longer has descendants in the current sequences: TB is obtained from T by re-rooting the tree to the most recent common ancestor to all the current microbial sequences. This switch changes both the topology of the tree and the branch lengths.

(iii) the switch occurs between 2 sister lineages: TB is obtained from T by re-dating the divergence between the two sister lineages to the time of the host-switch. This switch only affects the branch lengths of the tree.

(iv) the switch occurs between 2 distantly related lineages and the receiving branch is not related to the root: TB is obtained from T by an internal reorganization of the tree.

This switch changes both the topology of the tree and the branch lengths.

Technically, in order to reduce computation time, we simulated a "bank of trees" with ξ switches on the host tree and use these same trees in our different analyses.

Model selection

In addition to the general model fitting procedure described above, we designed two model selection procedures: the first aims at testing whether the presence of horizontal switches is statistically supported (versus a simpler model with only strict vertical transmission); the second aims at testing support for a model with a limited number of host-switches versus environmental acquisition (OTUs that are environmentally acquired will provide high µ ^ and ξ % estimates and could thus be interpreted as frequent horizontal transmissions with high substitution rates instead of environmental transmission).

In order to test support for a scenario with horizontal host-switches versus strict vertical transmission, we compute 𝐿 `= L(𝐴 " |µ ^, T), the likelihood corresponding to the best scenario of evolution of the microbial sequences directly on the host tree (i.e. no switch)

and compare it to the likelihood 𝐿 J = LI𝐴 " aµ ^, ξ % K corresponding to the best scenario with horizontal transmission, using a likelihood ratio test. In order to test support for a scenario with horizontal host-switches versus environmental acquisition, we test its support when compared to a scenario where microbial populations are acquired at random by host species (thereafter referred to as a scenario of "independent evolution"): we randomize R times the host-microbe association and run our model on each of these randomized data. Next, we analyze the rank of ξ % and µ ^ estimated from the original alignment in the distribution of ξR and μR estimated from the randomized alignments. Ideally, we would perform a large number of randomizations (e.g. R>100)

and directly compute p-values from the ranks of ξ % and µ ^. However, for computational reasons we used only R=10 randomized alignments and chose to reject the hypothesis of independent evolution if ξ % < ξR and µ ^ < μR for all R. Conversely, if the estimated number of switches ξ or the substitution rate μ are ranked within the distribution of ξR and μR, we consider that a scenario of independent evolution cannot be rejected.

Detecting transmitted OTUs

Based on the analyses above and our definition of modes of inheritance, we sort the OTUs into two different categories: the vertically and/or horizontally transmitted OTUs called transmitted OTUs (those that reject the hypothesis of independent evolution), and the environmentally acquired OTUs called independent OTUs (those that do not reject the hypothesis of independent evolution). In practice, there is no universal similarity threshold that will provide the "right" biological unit delineation across all microbial groups [START_REF] Sanders | Stability and phylogenetic correlation in gut microbiota: Lessons from ants and apes[END_REF] (Fig. S1). "Over-splitting" a biological unit using a similarity threshold that is too high for that biological unit will reduce statistical signal (each sub-unit will be represented in fewer hosts) and will miss host-switches between sub-units (given that sub-units will be analyzed independently). "Over-merging" OTUs using a similarity threshold that is too low will tend to blur a signal of transmission, and will over-estimate mutation rates, because alignments will mix sequences from distinct biological units. By using several clustering thresholds, we can hope to find one that properly delimitates biological units. Given that vertical transmission tends to be erased by improper delimitation, if it is detected for at least one threshold, then it suggests that it is the "right" threshold and that vertical transmission does indeed occur.

Implementation

All the scripts of our model are written in R (R Core Team 2018), using the packages ape, phangorn and phytools for the manipulations of phylogenetic trees [START_REF] Paradis | APE: Analyses of phylogenetics and evolution in R language[END_REF][START_REF] Revell | phytools: An R package for phylogenetic comparative biology (and other things)[END_REF][START_REF] Schliep | phangorn: Phylogenetic analysis in R[END_REF] and are freely available on GitHub (https://github.com/hmorlon/PANDA) and in the R package RPANDA [START_REF] Morlon | RPANDA: an R package for macroevolutionary analyses on phylogenetic trees[END_REF]. We also used the packages parallel, expm, ggplot2, reshape2 and R2HTML for the technical aspects of the scripts. All outputs of our model (e.g. parameter estimation and model selection) are concatenated in a user-friendly HTML file with different formats (e.g. tables, values, pdf plot and diagrams). We provide a tutorial in https://github.com/BPerezLamarque/HOME/blob/master/README.md.

Testing our approach with simulations

We performed a series of simulations to test the ability of our approach to recover simulated parameter values and evolutionary scenarios. We calibrated our choices of tree size, alignment size and parameter values so as to obtain simulated data comparable to those of the great ape-microbiota data (Fig. S6 and Table S2). We considered 3 independent host trees of size n=20 (T1, T2, and T3) simulated under a Yule model (no extinction) using the function pbtree from phytools. We scaled these trees to a total branch length of 1. On each of these host trees, we considered a scenario of strict vertical transmission (ξ=0), scenarios with host-switches ξ=[1, 2, 3, 5, 7, 10], and a scenario of environmental acquisition; each of these scenarios were obtained by simulating the corresponding microbial trees TB. For the scenario of strict vertical transmission, TB=T. For scenarios of host-switches, 15 TB per ξ value were derived from T. For the scenario of environmental acquisition, 20 TB were simulated under a Yule model independently from T, using the same procedure as above. Finally, we simulated on each TB the evolution of microbial sequences of a total length N=300 with a proportion of variable nucleotides x=0.1, using our own codes. We simulated the K80 stochastic nucleotide substitution process with a ratio of transition/transversion rate κ=0.66 and three different values of substitution rate (μ=0.5, 1 or 1.5). We simulated 20

alignments A per substitution rate on T for the scenario of strict vertical transmission (180 alignments total), and 1 alignment per TB per substitution rate for the scenarios of host-switch (135 alignments per ξ value) and environmental acquisition (180 alignments). Thereafter we call "ξ-switches alignment" an alignment simulated with ξ switches on T and "independent alignment" an alignment simulated independently from T.

We applied our inference approach to each simulated couple of T and A and compared the estimated parameters (𝜉 % , µ ^, and κ ^) to the simulated values. We used mixed linear models with the host tree (T1, T2, and T3) as a random effect (R package nlme). We tested homoscedasticity and normality of the model residuals and considered a p-value of 0.05 as significant. We also evaluated the type I and type II errors associated with our tests of strict vertical transmission and environmental acquisition.

Empirical application: great apes microbiota

We illustrate our approach using data from Ochman et al. [START_REF] Ochman | Evolutionary relationships of wild hominids recapitulated by gut microbial communities[END_REF]; this paper is one of the first paper testing hypotheses about co-diversification in the wellstudied clade of great apes (using phylosymbiotic patterns), and the associated data has been used in other papers aimed at studying codiversification [START_REF] Sanders | Stability and phylogenetic correlation in gut microbiota: Lessons from ants and apes[END_REF] Ochman et al. [START_REF] Ochman | Evolutionary relationships of wild hominids recapitulated by gut microbial communities[END_REF] extracted DNA from the fecal samples, PCRamplified the DNA for the 16S rRNA V6 gene region using universal primers, and finally sequenced the PCR product using 454 (Life Sciences/Roche). They obtained 1,292,542 reads after sequence quality trimming and barcodes removal. Gut microbiota are now sequenced with more resolution than was possible at the time of the Ochman paper, but not necessarily for entire clades. These data provide a good illustration of our approach.

We obtained the reads from Dryad (http://datadryad.org/resource/ doi:10.5061/dryad.023s6). We used python scripts from the Brazilian Microbiome Project (BMP, available on http://www.brmicrobiome.org/) [START_REF] Pylro | Brazilian Microbiome Project: Revealing the unexplored microbial diversity -challenges and prospects[END_REF] which combines scripts from QIIME 1.8.0 [START_REF] Caporaso | QIIME allows analysis of high-throughput community sequencing data[END_REF] and USEARCH 7 [START_REF] Edgar | UPARSE: Highly accurate OTU sequences from microbial amplicon reads[END_REF] as well as our own bash codes. We merged raw reads from all the hosts and processed them step by step:

(i) Dereplication: we discarded all the singletons and sorted the sequences by abundance using USEARCH commands derep_fulllength and sortbysize (ii) Chimera filtering and OTU clustering: we removed chimers and clustered sequences into OTUs using the -cluster_otus command of the UPARSE pipeline [START_REF] Edgar | UPARSE: Highly accurate OTU sequences from microbial amplicon reads[END_REF]. We chose a 1.0, 3.0 or 5.0 OTU radius (the maximum difference between an OTU member sequence and the representative sequence of that OTU), which corresponds to a minimum identity of 99%, 97% and 95%. We performed an additional chimera filtering step using uchime_ref with the RDP database as a reference (http://drive5.com/uchime/rdp_gold.fa).

(iii) Taxonomic assignation: we assigned taxonomy using a representative sequence for each OTU generated (with -cluster_otus), using assign_taxonomy.py from QIIME and the latest version of the Greengenes database (http://greengenes.secondgenome.com), or using BLAST when Greengenes did not assign taxonomy with enough resolution.

(iv) Mapping reads to OTUs and OTU table construction: we used the usearch_global command to map all the reads from the different samples to these taxonomy-assigned

OTUs. Then we used make_otu_table.py and BMP scripts to build the OTU table (a list of all the OTUs with their abundance by host individual).

(v) Core-OTUs selection: we selected the "core" OTUs as the ones that occurred in at least 75% of the host individuals, using the compute_core_microbiome.py script from QIIME.

(vi) Making intra-OTU alignments: discarding few OTUs that had unvaried alignments, we obtained 130 OTUs at 95%, 110 OTUs at 97%, and 66 OTUs at 99% similarity thresholds (Table S1). For each OTU, we built the bacterial alignment by selecting for each host individual the most abundant sequence among all the reads mapped to that OTU. We considered that the microbial genetic variability within each host individual (hereafter referred to as "intra-individual variability") is mainly due to PCR and sequencing artefacts, so we neglected it (Fig. S7).

Finally, we applied our approach to each core OTU independently, and to the nexus tree of the 26 host individuals, constructed with mitochondrial markers provided in the supplementary data of the article, scaled to a total branch length of 1. We used this individual-level tree instead of the species-or sub-species level tree in order to increase tree size (there are only 7 subspecies in our great apes tree); this approach also provides a way to account for microbial genetic variability within host subspecies (hereafter referred to as "intraspecific variability"). We arbitrarily resolved intra subspecies polytomies by assigning quasi-null branch lengths (10 -4 ) to the corresponding branches. We classified the OTUs into "transmitted" and "independent"

OTUs"; among the transmitted OTUs, we distinguished those where the transmission is strictly vertical, and for the others we recorded the estimated number of switches. In order to get an idea of the proportion of the microbiota that is transmitted we also recorded the number of reads corresponding to the transmitted OTUs.

Accounting for intra-host genetic variability

Our treatment of the great ape data illustrates an approach to account for intra-host microbial genetic variability: instead of running HOME on a species-level host tree (with a single representative microbial sequence per host species), it can be run on an individual-level host tree, with arbitrarily small intra-specific branch-lengths. Because this usage of HOME is slightly different from the case envisioned in our description of the approach, we tested its behavior. We simulated the evolution of microbial alignments on the great apes sub-species tree with a range of intraspecific variability similar to the range observed in the great apes alignments. For each OTU alignment, we defined intraspecific variability (V) as the mean nucleotidic diversity within host subspecies (computed using Nei's estimator [START_REF] Ferretti | Neutrality tests for sequences with missing data[END_REF]))

divided by the total nucleotidic diversity computed on the entire alignment. We simulated a total of 180 alignments according to 3 scenarios: strict vertical transmission (ξ=0), transmission with 5 host-switches (ξ=5), and environmental acquisition. For every scenario, we simulated intraspecific variability by extending the stochastic process generating nucleotidic substitution on every sequence for a time range that allowed to obtain levels of intraspecific variability that corresponded to the empirical level of intraspecific variability. We ran HOME on each of these simulated alignments and evaluated its performance, in terms of parameter estimation and model selection, when there was no intraspecific variability (V=0), low and intermediate intraspecific variability (0<V<0.5), and high intraspecific variability (V>0.5).

case of the test of environmental acquisition, the power ranges from 100% for ξ=1 to 60% for ξ=10, and it would decrease further with more switches (Fig. 4B). In both cases, the power increases when the substitution rate μ is larger (Fig. S4).

When HOME is applied to an individual-level host tree in order to account for intraspecific microbial genetic variability, Type I error rates associated to the test of environmental acquisition remain very low regardless of the magnitude of the variability (Fig. S5). The confidence in the estimation of the parameters (ξ and μ) remains good for low values of intraspecific variability (V<0.5), but decreases with increasing variability (V>0.5). The type I error rate associated to the test of strict vertical transmission increases with increasing variability, and the power of the two tests decreases with increasing variability.

Modes of inheritance in the great apes microbiota

Applying HOME to great apes gut microbiota data, we found that among the core OTUs with at least one segregating site, approximately 9 in 10 OTUs are environmentally acquired while 1 in 10 is transmitted (Fig. 5A); more specifically, the ratios of transmitted OTUs (and strictly vertically transmitted OTUs) were the following:

12(8)/130 at 95%, 12(10)/110 at 97%, and 4(4)/66 at 99%. In terms of relative abundance, 108,206 unique sequences in 1,292,542 (8.4%) belonged to transmitted OTUs (and 1,184,336 sequences, 91.6%, to strictly vertically transmitted ones, Table S3). Almost half of these sequences (49,508) were from an Acinetobacter bacterium (Moraxellaceae family); another important pool of these sequences was from the family Prevotellaceae (28,843 reads). In total, 12 bacterial families (in 27) contained OTUs that were transmitted, including Veillonellaceae, Lachnospiraceae, Ruminococcaceae and Paraprevotellaceae (Fig. 5B, Table S4). Some of these families (e.g. Desulfurococcaceae,

Pelobacteraceae, Rhodocyclaceae and Eubacteriaceae) were entirely made of a transmitted OTU, while others also had many OTUs and/or sequences that were not transmitted (e.g. Ruminococcaceae, Lachnospiraceae and Coriobacteriaceae).

The sequence length and proportion of segregating sites of OTUs inferred as transmitted were similar to those of other OTUs (Fig. S6 and Table S2), suggesting that HOME is not biased towards detecting vertical transmission in OTUs with specific characteristics.

However, the intraspecific variability of OTUs inferred as transmitted tend to be smaller than that of other OTUs (Table S5 and Fig. S7), which is consistent with our simulation results showing that the power to detect vertical transmission decreases with increasing intraspecific variability.

Discussion

We developed a likelihood-based approach for studying the inheritance of microbiota during the evolution of their hosts from metabarcoding data. We showed using simulations that even relatively short reads can help identify modes of inheritance, without the need to build a microbial phylogenetic tree. Applying our model to great apes microbiota data, we identified a set of transmitted gut bacteria that account on average for 8.4% of the total gut microbiota.

Our combination of model fitting and hypothesis testing helps identify modes of inheritance. We see the estimate of the number of switches as a good indicator of modes of inheritance (from faithful vertical transmission for low ξ values to horizontal transmission and environmental acquisition for high ξ values) rather than as an accurate estimation of past host-switches. We have indeed shown that ξ tends to be underestimated when quite many switches are simulated on a fixed host tree. In nature this underestimation may be even more pronounced, as our model ignores hostswitches that happened in lineages not represented in the phylogeny, as a result of either extinction or undersampling [START_REF] Szöllosi | Lateral gene transfer from the dead[END_REF]. In line with these results, we find that the hypothesis of vertical transmission is often not rejected when there are in fact host-switches. On the other hand, we can also estimate a positive ξ from data simulated under strict vertical transmission; however in this case, a model with hostswitches will in general not be selected when compared to a model of strict vertical transmission. Hence, if the hypothesis of strict vertical transmission is rejected, one can conclude with confidence that host-switches occurred (or that the microbial unit was environmentally acquired). Similarly, the hypothesis of independent evolution is often not rejected when the transmission is actually vertical with rather frequent hostswitches, and rarely rejected in scenarios of environmental acquisition, such that when it is rejected, one can conclude with confidence that the microbial unit is transmitted.

Said differently, our approach is conservative in its identification of transmitted OTUs;

and when an OTU is identified as being transmitted, our approach is conservative in its identification of switches.

When it occurs, the support for vertical transmission of a given microbial unit arises from a phylogenetic signal in microbial sequences (i.e. a congruence between the phylogenetic similarity of host species and the molecular similarity of the microbes they host). However, such congruence can also arise from processes not accounted for in our model, such as geographic or environmental effects; for example, if there is a phylogenetic/molecular signal in the geographic or habitat distribution of hosts/ microbes, or if the host environment creates microbial selective filters, this could result in a phylogenetic signal in microbial sequences that could be misleadingly interpreted as vertical transmission. We have not evaluated the robustness of our approach to such effects. Future developments could involve reconstructing ancestral areas/habitats or host environments on the host phylogeny in order to distinguish a phylogenetic signal truly driven by vertical transmission versus other effects.

In the construction of the model, we have made the important assumption that there is no microbial genetic variability within host species, such that each microbial OTU is represented by at most one sequence in each host. This is quite unlikely in natural microbial populations where multiple microbial strains can colonize a host species [START_REF] Louca | High taxonomic variability despite stable functional structure across microbial communities[END_REF], and this also prevents incorporating in our model horizontal hostswitches without replacement (i.e. the persistence of both ancestral and newly-acquired symbionts in a lineage). In our empirical application, we tackled this limitation by representing each host species by several individuals, using approximately zero-length branches to split conspecifics in the host phylogeny. Although our simulations show that the statistical power of our tests decreases strongly when intraspecific variability is high, they also show that the hypothesis of environmental acquisition is rarely rejected when the acquisition is indeed environmental. Hence, HOME is unlikely to misleadingly identify transmitted OTUs, especially in the presence of intraspecific variability.

Another (more satisfying) approach would be to directly account for intraspecific variability in microbial sequences in the likelihood computation; this could for example be done by representing the data by --at each tip of the host phylogeny and for each nucleotide --a vector of probabilities of states A, C, G and T representing the intra-host relative abundance of the four bases at the given nucleotidic position. In this case, we would directly use the variation given at the level of amplicon sequence variants (ASVs) [START_REF] Callahan | DADA2: High-resolution sample inference from Illumina amplicon data[END_REF].

There are several other developments that would significantly improve the approach.

For example, accounting for extinction and missing species in the host phylogeny would provide a better representation of past host-switches. Also, rather than considering each OTU as an independently evolving unit, it would be interesting to account for interactions between these units, that can for example lead to competitive exclusion [START_REF] Koeppel | Species matter: the role of competition in the assembly of congeneric bacteria[END_REF] or interdependency (e.g. adaptive gene loss [START_REF] Morris | The Black Queen Hypothesis: Evolution of dependencies through adaptive gene loss[END_REF]), and are crucial aspects of microbial community assembly. Finally, incorporating dynamics of extinctions and recolonizations of a symbiont across host clades would extend the time scale of application of the approach to hundreds of millions of years [START_REF] Shapira | Gut microbiotas and host evolution: scaling up symbiosis[END_REF]. Indeed, while ignoring such dynamics is reasonable for studying microbial evolution at small evolutionary scales such as within great apes [START_REF] Ochman | Evolutionary relationships of wild hominids recapitulated by gut microbial communities[END_REF], it would not be reasonable at larger evolutionary timescales such as across invertebrate or vertebrate species [START_REF] Brooks | Phylosymbiosis: relationships and functional effects of microbial communities across host evolutionary history[END_REF].

In the great apes gut microbiota, we identified OTUs representing 8.4% of the total number of reads that are transmitted across generations during millions of years of evolution. Given the low phylogenetic signal in the geographic distribution of the hosts (see [START_REF] Ochman | Evolutionary relationships of wild hominids recapitulated by gut microbial communities[END_REF]), these OTUs are likely truly transmitted vertically. Thus, our results suggest that the phylosymbiosis pattern observed by Ochman et al. [START_REF] Ochman | Evolutionary relationships of wild hominids recapitulated by gut microbial communities[END_REF] is partially driven by vertically transmitted bacteria, as suggested by [START_REF] Sanders | Stability and phylogenetic correlation in gut microbiota: Lessons from ants and apes[END_REF]. Still, the major part of the microbiota is constituted of bacteria that are environmentally acquired and therefore evolving independently from the great apes phylogeny [START_REF] Moeller | Sympatric chimpanzees and gorillas harbor convergent gut microbial communities[END_REF]. We found transmitted OTUs in 12 microbial families, including Lachnospiracea, Coriobacteriaceae, Paraprevotellaceae, Rhodocyclaceae, and Alcaligenaceae. This illustrates the utility of our approach, which offers the advantage of investigating the whole microbiota without an a priori on which families might be transmitted; this is a good complement to approaches that focus on few candidate families, such as in Moeller et al.'s study [START_REF] Moeller | Cospeciation of gut microbiota with hominids[END_REF]. In the later study, the authors amplified 3 primer-specific families (Bacteroidaceae, Bifidobacteriaceae, and Lachnospiracea) and showed that phylogenies representing the Bifidobacteriaceae and Bacteroidaceae were congruent with the apes phylogeny, suggesting that codiversification occurred in these two families. Unfortunately, neither Bifidobacteriaceae nor Bacteroidaceae were represented in the core OTUs in Ochman et al.'s data, even with a 95% similarity threshold: those bacteria were either not sampled, badly processed during DNA extraction and PCR, wrongly taxonomically annotated, or too divergent to be merged into core OTUs defined at 95%. Conversely, while Moeller et al. did not find any signal of co-phylogeny in the Lachnospiraceae family, we found 3 transmitted OTUs belonging to this family. However, they investigated the phylogenetic relationships between all the strains of Lachnospiraceae and whether they match the phylogenetic tree of great apes. This illustrates the utility of our approach, which investigates transmission modes of separate OTUs within bacterial families, rather than considering in a single evolutionary framework all the sequences from the same family.

Among the families in which we found transmitted OTUs, some are well known for having mutualistic properties. For example, the Lachnospiraceae, Paraprevotellaceae and Rhodocyclales families are involved in breaking down complex carbohydrates in the gut; they have even evolved to a fibrolytic specialization in gut communities [START_REF] Biddle | Untangling the genetic basis of fibrolytic specialization by lachnospiraceae and ruminococcaceae in diverse gut communities[END_REF]. These vertically transmitted fibrolytic bacteria, which have been co-evolving for millions of years with the great apes, may be one of factors that allowed frequent and rapid dietary shifts during the evolutionary history of hominids [START_REF] Hardy | The importance of dietary carbohydrate in Human evolution[END_REF][START_REF] Head | Sympatric chimpanzees (Pan troglodytes troglodytes) and gorillas (Gorilla gorilla gorilla) in Loango National Park, Gabon: dietary composition, seasonality, and intersite comparisons[END_REF][START_REF] Muegge | Diet drives convergence in gut microbiome functions across mammalian phylogeny and within humans[END_REF]. However, why these particular bacteria are faithfully vertically transmitted while other digesting gut bacteria seem largely environmentally acquired remains unclear.

Microbiota data is being collected across multiple hosts at an unprecedented scale. Our approach allows identifying, among numerous microbial units most of which are environmentally acquired, those that are vertically transmitted and potentially coevolving with their hosts. The current implementation of our model is entirely adapted to applications to other datasets using different sequencing techniques, clustering methods and de-noising algorithms. Being able to identify vertically transmitted microbial units is an important step towards a better understanding of the role of microbial communities on the long-term evolution of their hosts.

Figures 853 854 Figure 1 :CBFigure 3 :

 85413 Figures

Acknowledgments

The authors thank Ana Alfonso Silva, Leandro Aristide, Julien Clavel, Carmelo Fruciano, Eric Lewitus, Sophia Lambert, Odile Maliet, Marc Manceau, Olivier Missa, and Guilhem Sommeria-Klein for helpful comments on the article. They also thank Florian Hartig, Marc-André Selosse and Florent Martos for helpful discussions. This work was supported by the Centre national de la recherche scientifique (CNRS), the grant PANDA from the European Research Council (ERC-CoG) attributed to H.M., the Ecole Doctorale FIRE -Programme Bettencourt, and a doctoral fellowship from the École Normale Supérieure de Paris attributed to B.P.L.

Data Accessibility Statement

The implementation of HOME is available on github (https://github.com/hmorlon/PANDA) and in the R package RPANDA [START_REF] Morlon | RPANDA: an R package for macroevolutionary analyses on phylogenetic trees[END_REF]. We provide a tutorial and scripts to prepare the data in https://github.com/BPerezLamarque/HOME/blob/master/README.md.

The sequences used in our empirical applications are available in https://doi.org/10.5061/dryad.023s6/3.

Data citation

Sanders JG, Powell S, Kronaue DJC, Vasconcelos HL, Fredrickson ME, Pierce NE (2014) Data from: Stability and phylogenetic correlation in gut microbiota: lessons from ants and apes. Dryad Digital Repository. https://doi.org/10.5061/dryad.023s6

Results

Performance of HOME

Testing the performance of HOME using intensive simulations, we find a reasonable ability to recover simulated parameter values (Fig. 3). Estimates of the number of switches 𝜉 % are highly correlated with simulated values ξ, although the approach tends to overestimate the true number of switches when there are very few (less than 2) and to underestimate this number when there are many (Fig. 3A). The linear regression confirms these results 𝜉 % = 2.15 (Fdl=606=1015, p-value <0.0001) + ξ * 0.58 (Fdl=606=141, pvalue <0.0001). The ability to recover the true number of switches does not depend on the simulated substitution rate (Fdl=606=0.2601, p-value=0.61; Fig. S2). The substitution rate is rather well estimated (Fig. 3B), although it tends to be slightly overestimated when the simulated number of switches exceeds 3 (slope 0.04; Fdl=606=45.9, p-value<0.0001; Fig. 3B). The simulated transition/transversion rate ratio κ is well estimated (median ± s.d. =0.68 ± 0.17), although it is slightly underestimated when the substitution rate is high (slope of -0.015; Fdl=606=12, p-value=0.0007). For alignments simulated independently from the host tree, the approach estimates a high number of switches (median ± s.d. = 16 ± 6.2, Fig. 3A), and highly overestimates the substitution rate (Fig. 3B). The type of host tree (T1, T2 or T3) has little impact on the estimation of ξ (it explains less than 3% of the total variance, Fig. S2), μ (around 10%, Fig. S3) and κ (less than 0.01%).

Our model selection procedure has very low type I error rates, and type II error rates that depend on the situation (Fig. 4): the hypothesis of strict vertical transmission was nearly never rejected when transmission was indeed strictly vertical (1/180, type I error= 0.0056%) and always rejected under environmental acquisition (Fig. 4A); conversely, the hypothesis of independent evolution was almost always rejected when transmission was strictly vertical (1/180) and almost never rejected under environmental acquisition (3/180, type I error= 0.017%, Fig. 4B). While the type I error rates of the two tests are low, their power to detect a scenario of strict vertical transmission with host-switches is variable. In the case of the test of strict vertical transmission, the power ranges from 95% for ξ=10 to 45% when ξ=1 (Fig. 4A). In the

Author Contributions


P Polovodov

Sophie Eliet

O Haenssler

Gilles Dambrine

Kamel Haddadi

Didier Theron

A near-field scanning microwave microscope in a scanning electron microscope: design and challenges

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


HAL is

Introduction

Twice at the turning point of the millennium, I wrote a chapter on the situation of foundations in France in books directed by Helmut Anheier [START_REF] Anheier | Private Funds and Public Purpose, Philanthropic foundations in International Perspectives[END_REF]Schlüter, Then and Walkenhorst, 2001). These comparative books showed that among European countries, France had, with Ireland, the less developed foundation sector contrasting with its post-war revival in the bulk of European countries. Has this position fundamentally changed twenty years later? And have the historical, juridical and sociological reasons then evoked to explain the scarcity of foundations in France been altered nowadays?

The foundation has a two millennium tradition in Europe: philanthropy in Antiquity was advocated by writers such as Plato, Epicurus, Seneca and Cicero. The high school of Aristotle and the library of Alexandria were examples of outstanding foundations. In the middle Ages foundations were created at the initiative of the Church and through the donations of the nobility and rich guilds or brotherhoods. They ran hospices, hospitals, orphanages, monasteries, colleges and universities. In the 17 th and 18 th centuries monarchs across Europe were suspicious of the extension of mortmain property secluded from productive economy and escaping inheritance taxes. In France, Louis XIV and Louis XV restricted the rights of existing foundations and prohibited new ones This millenary tradition was brutally eradicated in France by the 1789 Revolution while in other European countries foundations were gradually modernized. The reasons for this eradication are multiple: abolition of the privileges while foundations are the work of the privileged; state monopoly on activities of general interest proclaimed by the Le Chapelier Act that fight guilds as well as other intermediate entities and finally impecuniosities of the revolutionary government to finance wars against European countries. Therefore the Jacobin state, in conflict with the Church favorable to the monarchy, expelled the congregations and either sold the properties of the Church and its foundations to the rising bourgeoisie, nationalized and secularized some of them--hospitals especially--or replaced others by a state model, such as universities.

This eradication was durable. An often infused by the government shy resurgence of some foundations can be observed at the end of 19 th century, following the 1887 Institut Pasteur, such as Fondation Curie, Fondation Rothschild . During the interwar period, Fondation Deutsch de la Meurthe and many foreign foundations were at the origin of the Cité Universitaire, an international hall of residence for foreign students in Paris. After WW2, André Malraux, the first Minister of culture encouraged the creation of foundations to promote culture. In 1969, Malraux was also at the origin of Fondation de France, the main multipurpose grant-making foundation in France. However there was a total absence of the so-called "boom of foundations" as elsewhere in Europe or United States in the years [START_REF] Pomey | Traité des fondation d'utilité publique[END_REF]-1990[START_REF] Archambault | Country report: France[END_REF][START_REF] Archambault | Pourquoi les fondations sont-elles si rares en France[END_REF] Of course this historical background is permanent and at the origin of the durable scarcity of foundations in France. However, the legislation has become more favorable to donations and foundation creation in the first decade of this millennium, as seen hereafter in a first part. Has France's lagging behind its European neighbors been caught up in the 21st century? This issue will then be discussed with the most recent empirical data. Finally, the chapter shows that foundations are in competition with a large associative network; the French remain suspicious of foundations and wealthy individuals influence, as this specific feature can be illustrated by the controversies surrounding donations to rebuild Notre-Dame after the 2019 fire.

A more favorable legislation: 2003 and 2009 Acts

At the end of the last century, two recent laws gave a legal personality to foundations that were often confused with associations before: cooperatives or mutual societies by a simpler and independent of the state approval process. The founder has to devote a five-year renewable sum of at least €152,000, that has to be consumed during the five years period. Corporate foundations cannot receive donations and legacies.  Because of the difficulty to create a RUP foundations, many individuals and some companies opted for a non autonomous legal status. These "sheltered foundations" with no minimum endowment are mainly hosted by Fondation de France or Institut de France, a public institution that houses the five academies. They have their own boards, deciding of their grants or activities, while the host institution usually manages their assets.

During the millennium decade the foundation landscape has changed dramatically since 2003 Patronage Act. This law, simplified the creation of a RUP foundation, enhanced tax incentives to donations and later new kinds of specialized foundations were proposed to the potential founders.

The 2003 Patronage Act includes administrative simplifications to the establishment of a RUP foundation and removal of the minimum permanent endowment that was formerly an important obstacle. In case of foundations devoted to temporary causes an expendable endowment can be authorized by the Council of State.

But the main change is the spectacular reinforcement of tax incentives for donors to RUP and business foundations:  For individual donations, the tax credit on income tax rose from 40 per cent up to 66 per cent, with a cap rising from 6 per cent up to 20 per cent of taxable income. The donation over the cap can be deducted from income tax during the following five years. This last point is of course intended to enhance the creation of foundations. In addition since 2009, donations have been 75 per cent deducible from the wealth tax (Impôt sur la Fortune), with a cap of €50,000.  Tax deductibility of companies giving rose from 33 per cent up to 60 per cent while the limit on authorized donations goes from 2.5 to 5 per thousand of turnover. The French tax deductibility is now he highest in Europe.

At the end of the millennium decade, four new legal statuses of foundations were proposed to the potential founders in the field of research and higher education: scientific cooperation foundation, partnership foundation, university foundation and hospital foundation. These new foundations have different economic models according to their by-laws, sometimes with option for the founder between flow foundations, expendable or permanent endowment foundations.

In 2008 finally endowment funds, inspired by the Anglo-Saxon pattern, were proposed to potential founders. In opposition to the four new kind of foundations above, they are multipurpose or generalist entities. They are very easy to create, without an a priori control of the government and no minimum endowment was initially required.

These new incentives to create foundations show that the state's historical suspicion of foundations vanished. Though the above mentioned change in the foundation statutes and tax encouragements were proposed by right-wing governments, the following left-wing government did not suppress them. The legal vacuum until the end of the 1980s has been replaced by abundance, even overflow, of different legal statutes. Has this change provoked a foundation boom in France, later but similar to what is observed in other European countries? .

3. Foundations to-day and recent evolutio 3.1 the landscape of foundations to-day

The total number of foundations, including endowment funds, is 4858 in 2017, as shown in Table 1. However, in comparison with 87,000 foundations in USA, 25, 000 in Germany, 12,000 in UK, 14,000 in Denmark, 18,000 in Sweden, the French philanthropic sector remains underdeveloped and has not yet caught up its historical lagging behind. Even among the Southern countries, the number of foundations is less than in Italy (6,200) or Spain (8,900) In 2017, the French foundations assets totalize €26.5 billion, up 31 per cent since 2013. FRUPs hold 75% of these assets. In comparison, the level of foundations total assets is €770 billion in USA and in North-Western Europe: €100 billion for German foundations, € 70 billion for British, 44 billion for Danish and 50 billion for Swedish foundations. In southern Europe, Italian foundations inherited the assets of public banks up to €85 billion and Spanish foundations hold €22 billion assets, with the same order of magnitude than their French counterparts. Foundations have therefore a lesser economic role in France than in other European countries (Jevakhoff & Cavailloles, 2017) French foundations annual expenditure is €7.4 billion. Expenses of FRUPs, the most ancient part of the philanthropic sector, represent 88% of this total expenditure. They are operating foundations, hiring the bulk of the sector's employees while other foundationscorporate, sheltered or new legal forms-and endowment funds as well, are mainly distributive or grantmaking. All kind of foundations hire 86,000 employees. To complete the economic model of French philanthropic sector, sources of its income are the following in 2014:  Investment income: 8%  Fees and sales, per diem reimbursements and other third party payments: 42% (coming roughly half from the private sector, half from the public sector)  Public grants and subsidies: 10%  Private giving: 30%  Miscellaneous and n e c: 8% The part of investment income is limited because the assets of French foundations are not very high comparing to their foreign counterparts and part of them is rapidly consumed in grant-making foundations. Of course operating foundations have a higher part of commercial revenue, coming mainly from contracts with the public sector, because they run health, social services or education establishments. Grant-making foundations rely mainly on private and public donations or subsidies.

Analysis of the recent growth of the foudations

The total number of foundations, including endowment funds, shows apparently a spectacular growth after decades of quasi stagnation. This number was apparently multiplied by seven within 27 years that means an average annual growth during the period of 7.3 per cent.

These figures are in fact misleading. Table 3 breaks down the evolution between two subperiods, the second one including all the favorable new legislation above described.

The first remark is that endowment funds explain the main part of the acceleration of the 2001-2017 sub period growth. Since 2009 endowment funds mushroomed: easy to create, with no a priori public control and no minimum endowment, half of these funds have no endowment at all! Therefore since 2014 a minimum endowment of €15,000 is required to prevent such empty shells. The average annual growth for the 2001-2017 falls from 9.7 to 4.8 per cent when endowment funds are excluded. For the whole 1990-2917 periods, it is down from 7.3 to 4.5 per cent and the acceleration between the two sub periods is limited. Regarding the heavyweights of the foundation sector, the public utility foundations, their growth is lesser than all other kinds of foundations and do not accelerate after the 2003 Patronage Act that missed its main target. Potential founders did not choose to create a FRUP despite the higher tax credit, the simpler process and the possibility of giving one year of income through the deferral to five subsequent years of the gift above the threshold.

In opposition, corporate foundations have a two-digit growth rate during the whole period. Corporate foundations do not exist before 1990 and the first sub period beginning from scratch shows its highest level. Companies that create foundations were mainly public corporations, cooperatives or mutual societies. During the second sub-period, private standard corporations benefited more than individuals of the most favorable tax credit in Europe to create corporate foundations. Sheltered foundations continue to grow at the same rate during the two periods and the host foundations multiplied without a shift for more independence towards PUF due to the lowering of the minimum endowment. Finally, new forms of foundations did not explode like endowment funds and remain in a modest number.

To conclude, corporations were no doubt more eager than individuals to benefit of the higher tax benefits provided by the 2003 Patronage Act to create foundations. Despite its recent growth, it is unlikely that French philanthropic sector will catch-up its neighbors' level in the next decade.

4-Hypothetical causes of the persistent disaffection of the French with regard to the foundations

Among the causes of the scarcity of foundations in France cited in Archambault 2004, two of them vanished: on the one hand, state's historical suspicion has been replaced by acknowledgement that "the state has no monopoly on public good", as said Jacques Chirac, prime minister in 1986; on the other hand, the legal vacuum has been replaced by many legal statutes of foundations and funds, unequally easy to create.

However association remains the simplest legal entity to be created and the most independent of the state in its governance and control. While more than 70,000 associations are created every year, it is less than one hundred for endowments funds or foundations. About 1,500,000 associations are spread throughout the French territory when the foundations are less than 5,000, with a high concentration on the Parisian area. French people like and trust associations and dislike or ignore foundations as many polls show it.

In fact foundation appears as an institution less adapted than association to the French culture for several reasons. Firstly, operating foundations are part of the Catholic Church charitable tradition dating back to the Middle-Ages and some of them have kept this religious reference in their title and this is why they are considered by a large part of the French as remains of the past or simply overlooked. Associations are partly relevant to this religious tradition, but their historical background is more complex: they are also heirs of the medieval guilds, 19 th Century workers' movements and 20 th Century Olympic movement, social protection, minority groups' advocacy and environment defense movements. Secondly, foundations have no democratic governance, based on the principle "one person, one vote in General Assembly, such as cooperatives, mutual societies and associations. That is why it is questionable that foundations belong to the Social and solidarity economy (SSE) as the 2014 Act on SSE decided. According to the CIRIEC perimeter of SSE, only foundations created by its organizations are part of SSE (Chaves and[START_REF] Chaves | The European Social Economy. Concept and dimensions of the Third sector, CIRIEC[END_REF]1012). Thirdly, the publicprivate partnership began in the 1960 to 1980 years with the main associations working in the welfare activities: health and social services, education, culture, sports and recreation. Of course operating foundations working in these fields benefited also of public funding, but they were never leaders in the partnerships. In addition, there are no community foundations in France as in other European countries because the central and local governments cannot create foundations. Therefore, large associations play in France the role that foundations do in other European and American countries. However in recent years, some fundraising associations became foundations and this change is encouraged by the 2014 SSE Act. 4 shows the evolution of the number of establishments with staff in foundations and associations and the number of their employees over the 2005-2015 decade. Obviously, associations have the lion's share in the French nonprofit sector regarding the number of establishments with staff and employees. But foundations are larger -55 employees in average-than associations -10 employees in average--. Foundations are growing quickly over the two sub-periods while associations are growing smoothly during the 2005-2010 subperiod while their establishments decrease and their employees are steady during the 2010-2015 sub-period. Two explanations of this contrasted evolution: on one hand, there is a reduction of public funding since 2010 that impact more associations than foundations who are less dependent on public money. On the other hand, some associations merged to cope with these restricted income, other created endowments funds or foundations to capture donations and a few large fundraising associations changed their legal statute to become foundations, such as Petits Frères des Pauvres or Fondation pour la Recherche sur le Cancer.

Unfavorable inheritance legislation is also evoked as a persistent obstacle to the creation of foundations by will. If the testator has children, between half and three quarter of her/his estate is reserved for the children, according to their number. Most founders of FRUP or sheltered foundations are alive and they are younger nowadays than two decades ago. . Despite the more favourable attitude of the public powers, resistance to foundations remains in administrative culture and public opinion as well. The idea that state and local authorities have a monopoly on public interest activities remains alive and is detrimental to both corporate and RUP foundations. In administrative culture, however, the Jacobin belief in the superiority of state in provision of medical, social, cultural or educational services is still alive. The traditional view that for-profit enterprises should not engage in activities of general interest continues to prevail as well in administration and public opinion. Endowments to corporate foundations are then considered by a large part of the French population as taxexempt alternatives to advertising expenses or "social/green washing". More generally, corporate patronage is seen as an undue levy on resources that could have alternately been spent on wage increases or on taxes financing public services. Even tax credits for households giving can be viewed as gifts to the richer part of the population 1 .

 These ambiguous behaviours can be illustrated by the recent controversies on individual and corporate donations to rebuild Notre-Dame cathedral after its partial destruction by fire on April 15 th 2019. Notre-Dame is the most popular religious building even for the most secular part of the population 2 because it is linked to the French history and literature. During the three hours of the burning of the roof framing, built in medieval timber, the bulk of the French were rooted to their TV sets till the firemen said that the main part of Notre-Dame was saved. This time of national union between believers and non-believers was short. The next day, giving pledges flowed towards Fondation de France and Heritage Foundation; the richest French people, Arnault (LVMH), Pinault (Kering) and Bettancourt (L'Oréal) gave €100,000 or 200,000 in a competition to be the most generous and the following controversies, in newspapers, the Internet and pubs as well, included at least these two points:

1

In spite of the fact that tax exemptions to support giving are rather fair in France. In 1996, tax credits replaced reductions of taxable income, unfair with a progressive income tax that gives a larger tax-exemption to the higher incomes.

2

France is with Denmark the country where religious observances are the less. Official surveys on religious affiliation are sedom. In the most recent one 45% of the French said that they have no religion, 43% said they are Catholic, 8% Muslim, 2% Protestant, 2% belonged to other religions (Jewish, Orthodox, Buddhist…) (INSEE-INED, 2008)  -It is easy to find €1 billion in a few days for an historical building famous all over the world and not for the needy population (or the "yellow jackets") or other less famous parts of our heritage  -The richest are generous with public money as tax credits are 66% or 75% of their gifts. To reply to this objection, the above cited donors, or their corporate foundations, said that they will waive their tax credits (maybe they were over the cap because of previous donations)

This dispute is specific to a society with a passion for equality and a high level of distrust (Algan, ) especially of the wealthiest part of the population. However civil society is a school of trust and citizenship. the recent growth of foundations may be an opportunity for them to be more clearly part of civil society.

5-Conclusion

Though it is unlikely that French foundations will catch up their more advanced neighbour countries in the next decade, there are clear signs of a change in the world of foundations. New foundations are more grant-making and less operating; they are more flow foundations spending a higher part of their assets. New founders are often businessmen having sold their start-up, they are younger and eager to follow by themselves the management and impact of their foundation.

In a country with the highest level of taxes and social contributions, state cannot do everything. Foundations could have a greater role in the detection and the answer to new needs in the field of health, research, education, culture and social services. They could more than they do now help associations to meet their lack of resources and be more efficient. They have to learn to cooperate with public powers at all levels and to acknowledge the legitimacy of elected entities. More community foundations would help to a better knowledge of the administrative culture by foundations and of the hybrid culture of foundationsworking for general interest with business management efficiency--by civil servants. Having to please neither to their shareholders as corporations or voters as central or local governments, and even to their members as associations, foundations can experiment new and unpopular fields, alternatives to jail for example. These innovations if successful could be scaled-up by the state. The mix of the world of foundations with other worlds would benefit to the whole society.



Fondation reconnue d'utilité publique (RUP hereafter

   A 1987 Act defines a foundation as "the legal act through which one or several individuals or legal entities decide the irrevocable allocation of estate, rights or resources for a nonprofit making activity of general interest" The establishment of a

	) is long, complicated and
	centralized. It requires a high minimum endowment of €762,200 that is not
	expendable. Government representatives must sit in its board up to one third. RUP
	foundations benefit a tax-exempt status and can receive tax deducible donations and
	legacies exempt from inheritance tax;
	 According to a 1990 Act, corporate foundations are created by corporations,

Table 1 Number of foundations (1990-2017)

 1

		Years	1990 1995 2001 2010 2014 2017
	Public Utility Foundations	393 445 471 578	634 638
	(FRUPs)
	Corporate Foundations	-	25	67 250	344 400
	Sheltered foundations	326 404 571 811 1161 1242
	New foundations	-	-	-	47	89	84
	Endowment funds	-	-	-	162 1842 2494
	Total Foundations and	719 874 1109 1848 4071 4858
	Endowment funds
	Total	Foundations,	719 874 1109 1686 2229 2364
	without Endowment Funds

Source Archambault 2004

, for the three first years; de Nervaux (2015 and 2019) for recent years

Table 2 Employees in foundations, by activities, 2015/12/31

 2 Table 2 breaks down the total number of employees by activities: social services come first, then hospitals and other human health services which was first historically. Education and research, with mainly Pasteur Institute, have a lesser economic weight. Information and communication gather the bulk of grant-making foundations employees

	Activities	Number	%
	Social services	39,086	45.6%
	Health	27,636	32.2%
	Education & research	9,336	10.9%
	Information &	6,826	8.0%
	Communication
	Other	2,887	3.3%
	Total	85,771	100%

Source INSEE-CLAP Tableaux harmonises de l'économie sociale

Table 3 Evolution of foundations : average annual growth during sub periods

 3

		1990-2001	2001-2017	1990-2017
	Public Utility Foundations	1.7%	1.9%	1.8%
	Corporate Foundations	17.9%	11.8%	13.4%
	Sheltered foundations	5.2%	5.0%	5.1%
	New foundations	-	8.6%
	Endowment funds		57.7%
	Total Foundations and	4%	9.7%	7.3%
	Endowment funds
	Total Foundations, without	4%	4,8%	4,5%
	Endowment Funds
	Personal calculation after table1

Table 4 Number of employers establishments and employees in foundations and

 4

	associations, 2005-2015
	Years	2005	2010	2015
	Foundations, establishment s	900	1,267	1,568
	growth		40.1%	23.8%
	Foundations employement	53,844	67,933	85,771
	growth		26.2%	27.7%
	Associations, establishments	172,463	188,810	186,713
	growth		9.5%	-1.1%
	Associations employment	1,625,163	1,840,864	1,852,083
	growth		13.3%	0.6%
	Source INSEE CLAP

Table

The recent evolution of foundations in France

Edith Archambault (Paris1 Pantheon-Sorbonne University)


Sofia Papastamkou

Antonio Castro

Sichani Rojas

Anna-Maria

Designing Multilingual Digital Pedagogy Initiatives: The Programming Historian for English, Spanish, and French speaking DH Communities

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

Designing Multilingual Digital Pedagogy Initiatives:

The Programming Historian for English, Spanish, and French speaking DH Communities Sofia Papastamkou (sofia.papastamkou@univ-lille.fr) 1 , Antonio Rojas Castro (antonio.rojas-castro@bbaw.de) 2 , Anna-Maria Sichani (a.sichani@sussex.ac.uk)

Increasing Access

The Programming Historian aims to publish accessible tutorials targeted for a global audience. For that reason, authors are encouraged to address technological, linguistic and cultural barriers:

• methods & tools should be accessible to international audiences and reproducible in languages other than the original (e.g. multilingual documentation, different character sets for text analysis tools...);

• primary sources and alternative datasets from outside their geographical expertise can be suggested for readers to explore;

• specific cultural references, idiomatic expressions, or tones might not register for all audiences;

• historical persons, organizations, or events specific to a particular culture shall be clarified and explained in detail;

• examples of code and metadata shall use internationally recognized standards for date and time formats.

Three editions

Open access is not only about copyright issues and internet connection! Language also shapes access to information on the internet. By translating and publishing original tutorials in Spanish and French, The Programming Historian aims to make open educational resources accessible for a diverse community of scholars:

• English edition contains 80 original tutorials;

• Spanish edition contains 44 tutorials (42 translated tutorials and 2 original tutorials); • French edition contains 2 translated tutorials. All our tutorials including translations are peer-reviewed by experts! Bibliography

3 1 IRHiS, CNRS/University of Lille, 2 BBAW, 3 University of Sussex About the project

	The Programming Historian is a community-led pedagogy
	initiative launched in 2008 for Humanities scholars that
	aims to publish open-access peer-reviewed tutorials in
	English, Spanish and French on a wide range of digital
	tools, techniques, and workflows.
	It is now a proudly multilingual open access journal
	involving a large team of editors, authors, translators and
	reviewers, that requires software development, conducts
	community surveys and solicits community input.

•

  Crymble, A., 'Identifying and Removing Gender Barriers in Open Learning Communities: The Programming Historian' in: Blended Learning In Practice (Autumn, 2016), 49-60. • Gibbs, F., 'Editorial Sustainability and Open Peer Review at Programming Historian', DH Commons, Vol. 1 (2015). • Smith, M. S., 'Opening Education', Science, 323(2), 89-93 (2009). Open access | Open peer review | Open ethos • Full language initiatives • Contact zone: writing for a global audience • Inclusivity: gender-inclusive writing • Ad hoc translations • Neutral political policy

	Internationalisation strategy
	•
	Follow us / join us / Síguenos /
	Únete / Suivez-nous / Rejoignez-
	nous
	programminghistorian.org
	@proghist

• Sichani, A.-M., Baker, J., Afanador Llach, M. J., Walsh, B., 'Diversity and Inclusion in Digital Scholarship and Pedagogy: The Case of The Programming Historian', Insights, (2019).


Petr Polovodov

Olaf Haenssler

Didier Théron

Christophe Boyaval

Sophie Eliet

Gilles Dambrine

Kamel Haddadi
email: kamel.haddadi@univ-lille.fr

Building a near-field scanning millimeter-wave microscope integrated in a scanning electron microscope

Keywords: Near-field scanning microwave microscopy (NSMM), scanning electron microscope (SEM), evanescent waves, millimeter-wave

A near-field scanning millimeter-wave frequency microscope built inside a scanning electron microscope is described. The proposed instrumentation can provide simultaneous atomic force and millimeter-wave images together with the scanning electron microscopy image of the probe / sample interaction. In contrast with conventional scanning microwave microscopy tools, the instrumentation proposed opens the way to achieve traceable measurements by tackling issues encountered in traditional near-field scanning microwave microscopy tools. First, water meniscus in the vicinity of the probe tip is eliminated to reduce parasitic capacitance. Secondly, targeted frequency up to 110 GHz results in confined electric fields in the vicinity of the probe tip to enhance the lateral resolution. Finally, visualization of the tip / sample interaction offers unique possibility to support the measured millimeter-wave signals by fine FEM modeling.

I. INTRODUCTION

The introduction of near-field scanning microwave microscopy (NSMM) tools have pioneered many applications, notably including mapping and quantitative measurement of the electrical properties of materials and devices at micro and nano scale [START_REF] Anlage | Principles of nearfield microwave microscopy[END_REF]- [START_REF] Zhang | Microwave atomic force microscopy imaging for nanometer-scale electrical property characterization[END_REF]. Basically, the scanning microwave microscope (SMM) consists of an atomic or scanning tunneling microscope combined with a vector network analyzer (VNA) [START_REF]Scanning Microwave Microscopy (SMM) Mode[END_REF]- [START_REF]Scanning Microwave Impedance Microscopy (sMIM)[END_REF]. The tip scans across the sample, emitting a microwave signal scattered by the material, altering its amplitude or/and phase properties.

Most of the reported NSMM tools operate in the microwave frequency range, i.e. 300MHz-30GHz. Consequently, there is a natural mismatch between the probe tip size (tens of nm) compared to the wavelength of operation in the order of the cm. When the tip of the probe is held close to the surface of a sample, at a distance separation much smaller than the wavelength of operation, the evanescent electric fields are highly confined in space. In contrast with traditional methods based on propagating waves, microscopy techniques result in high lateral and depth resolutions governed by the tip size [10]- [START_REF] Haddadi | Sensitivity and accuracy analysis in scanning microwave microscopy[END_REF] There is an urgent need to extend the frequency capabilities of microwave microscopy tools to achieve sub-50nm spatial resolution combined with sub-aF electrical resolution. In this effort, a new NSMM is developed with broadband capabilities up to 110 GHz. Consequently, the new instrumentation offers the possibilities to adapt the lateral and depth resolutions by proper choice of the frequency of interest in the range 1-110 GHz. In addition, the instrumentation has been designed to operate inside a scanning electron microscope for environment control and water meniscus elimination [START_REF] Weeks | Direct imaging of meniscus formation in atomic force microscopy using environmental scanning electron microscopy[END_REF]- [START_REF] Calleja | Size determination of fieldinduced water menisci in noncontact atomic force microscopy[END_REF]. Finally, SEM image of the tip / sample interaction offers the opportunity to achieve fine FEM modeling [START_REF] Haddadi | Near-field scanning millimeter-wave microscope combined with a scanning electron microscope[END_REF]- [START_REF] Haddadi | Combined scanning microwave and electron microscopy: A novel toolbox for hybrid nanoscale material analysis[END_REF]. In Section II, the design, fabrication and validation of the probing structure is presented with a preliminary targeted frequency of 40 GHz. In Section III, the system integration that requires specific design considerations is discussed.

II. MICROSTRIP PROBING STRUCTURE

The NSMM-cantilever consists of a modified 12Pt400B microwave probe from Rocky Mountain Nanotechnology® (RMN) to achieve 40 GHz frequency operation. This probe consists initially in an ultra-sharp solid platinum probe tapered down to 50 nm and attached to a ceramic substrate. The probe is modified to support a TEM propagation mode through a microstrip structure. To that end, a conductive gold line of 200 µm is placed under the platinum, and continues to the back of the chip as the transmission line (Fig. 1). The back side is then completely covered with gold over an adhesive layer. This NSMM-Cantilever is embedded into a waveguide structure (both coplanar and microstrip structures have been realized for further comparisons) which can be exchanged in case of destroyed tips by using a solder-less PCB mount connector of Rosenberger Corp. with a clamping and screwing mechanism (Fig. 2). The NSMM-probe has been assembled and millimeter-wave characterization has been performed using a vector network analyzer to measure the return loss (Fig. 3). From the experimental results, Fig. 3 shows magnitude of the reflection coefficient better than -10 dB up to 40 GHz in accordance with simulated data (not shown here). It has to be noticed that that three resonances occur in the measured response. A time-domain analysis has been used to identify the origins of the resonances at 7.7GHz: transition connectorsubstrate, 23.1 GHz: Cantilever beam and 37.8 GHz: transition substrate-cantilever. Although the impact of the resonances is minor, optimization of the transitions is under investigation to limit resonance effects.

III. SYSTEM INTEGRATION

The block diagram of the system is described in Figure 4. The hemisphere above the sample is mainly occupied by the electron column and leaves only a very small gap accessible for the SMM to achieve a working distance of 20 mm. The system presented here has been designed to be as compact as possible. The SMM stage is mounted on the chamber vertically to the electron beam of the SEM. The SMM consists of two main components, a sample scanning stage and a probing unit (Fig. 4). The sample scanning stage is composed of an AFM scanner positioned onto a coarse positioning stage. The positioning stage consists of three piezo driven linear actuators manufactured by Smaract GmbH with scanning ranges of 26 mm in X and Y directions and 11 mm in Z direction. The actuators enable closed-loop operation with less than 10 nm resolution and 25 nm repeatability in any direction. The AFM scanner ANSxyz100/Std from AttocubeTM operates in open-loop on scanning ranges of 505024 µm 3 with sub-nm positioning resolution and 0.1% scan repeatability (Fig. 5). The measuring equipment, i.e. SMM integrated into SEM, has been developed and is operational. Fig. 6 gives a picture of the equipment.

IV. CONCLUSION

This study was focused on design, fabrication and measurement of new NSMM-cantilevers connected to microstrip based PCBs. These NSMM-cantilevers have been integrated into a SEM. The new instrument has been fully automated using nano-positionning to provide simultaneous atomic force and millimeter-wave images of the sample under investigation.

Fig. 1 .

 1 Fig. 1. Scanning electron image of the NSMM-cantilever manufactured by RMN®.

Fig. 2 .

 2 Fig. 2. NSMM-cantilever integration in a dedicated PCB board.

Fig. 3 .

 3 Fig. 3. Return loss mag(S11) of the modified NSMM-probe.

Fig. 4 .

 4 Fig. 4. Sketch of the proposed millimeter-wave NSMM.

Fig. 5 .

 5 Fig. 5. Photography of the millimeter-wave NSMM integrated inside a Tescan Mira XMU SEM.

Fig. 6 .

 6 Fig. 6. Millimeter-wave NSMM integrated into a SEM. The preliminary experimental demonstration considers the capacitance reference kit developed my MC2 Technologies® and uses a modified probe from Rocky Mountain Nanotechnologies® [Fig7(a)]. The topography, amplitude and phase images are simultaneously scanned over 30 x 30 µm2 at the scan rate of 5.8 µm/s (0.19 line/s in a single direction) with 512 x 512 pixels resolution. Fig. 7(b) demonstrates 2D AFM image of the reference kit of the device under test.

ACKNOWLEDGMENT

This work has received funding from the European Union Horizon H2020 Programme (H2020-NMBP07-2017) under grant agreement n°761036 MMAMA. This work used the facilities within the EQPX ExCELSiOR ( www.excelsiorncc.eu ) funded by the National Research Agency (ANR).