Jordi Salmona
email: jordi.salmona@gmail.com

Jill K Olofsson

Cynthia Hong-Wa

Jacqueline Razanatsoa

Franck Rakotonasolo

Hélène Ralimanana

Tianjanahary Randriamboavonjy

Uxue Suescun

Maria S Vorontsova

Guillaume Besnard
email: guillaume.besnard@univ-tlse3.fr

Late Miocene origin and recent population collapse of the Malagasy savanna olive tree (Noronhia lowryi)

Keywords:

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

INTRODUCTION

Tropical savannas can be defined as open canopy environments, with variable tree cover and a continuous grassy ground layer (mostly C 4 ) maintained at a climax state by fire regimes and/or herbivores [START_REF] Bond | What limits trees in C 4 grasslands and savannas?[END_REF][START_REF] Lehmann | Deciphering the distribution of the savanna biome[END_REF][START_REF] Archibald | Distribution and determinants of savannas[END_REF]. The contribution of anthropic versus natural factors to the origin and distribution of this habitat remains unclear across the world, and especially so in Madagascar. While the dominant narrative long assumed a major role for human-driven deforestation as a driver of the rise of open canopy environments dominated by grasses (Perrier de la [START_REF] Perrier De | La végétation malgache[END_REF][START_REF] Koechlin | Flore et Végétation de Madagascar[END_REF][START_REF] Burns | Rapid human-induced landscape transformation in Madagascar at the end of the first millennium of the Common Era[END_REF], there is accumulating evidence for an older expansion of Malagasy savannas and the associated fires since the Miocene [START_REF] Bond | The antiquity of Madagascar's grasslands and the rise of C 4 grassy biomes[END_REF][START_REF] Vorontsova | Madagascar's grasses and grasslands: anthropogenic or natural[END_REF][START_REF] Hackel | Grass diversification in Madagascar: in situ radiation of two large C 3 shade clades and support for a Miocene to Pliocene origin of C 4 grassy biomes[END_REF][START_REF] Solofondranohatra | Grass functional traits differentiate forest and savanna in the Madagascar central highlands[END_REF]. Furthermore, Quaternary palynological records support the existence of open habitats long before early human arrival [START_REF] Matsumoto | Late Holocene environments at Lake Mitsinjo, northwestern Madagascar[END_REF]Gasse andVan Campo, 1998, 2001). Fossil records also suggest that a few extinct endemic grazers -giant tortoises, pygmee hippopotamuses, and giant lemurs -may have played a role in the dynamic of Malagasy savannas (e.g. [START_REF] Burney | A chronology for late prehistoric Madagascar[END_REF][START_REF] Virah-Sawmy | Evidence for drought and forest declines during the recent megafaunal extinctions in Madagascar[END_REF][START_REF] Pedrono | Using a surviving lineage of Madagascar's vanished megafauna for ecological restoration[END_REF][START_REF] Goodman | Extinct Madagascar: Picturing the Island's Past[END_REF]Godfrey and Crowley, 2016;Godfrey et al., 2016[START_REF] Samonds | A new late Pleistocene subfossil site (Tsaramody, Sambaina basin, central Madagascar) with implications for the chronology of habitat and megafaunal community change on Madagascar's Central Highlands[END_REF]. Their demise in the last millennia and the rise of pastoralism ca. ~1000 y BP may have deeply impacted ecosystem function [START_REF] Johnson | Ecological consequences of Late Quaternary extinctions of megafauna[END_REF][START_REF] Crowley | A refined chronology of prehistoric Madagascar and the demise of the megafauna[END_REF][START_REF] Veldman | Clarifying the confusion: old-growth savannahs and tropical ecosystem degradation[END_REF][START_REF] Vorontsova | Madagascar's grasses and grasslands: anthropogenic or natural[END_REF][START_REF] Hansford | Early Holocene human presence in Madagascar evidenced by exploitation of avian megafauna[END_REF][START_REF] Godfrey | A new interpretation of Madagascar's megafaunal decline: the "Subsistence Shift Hypothesis[END_REF]. Together with long-term pastoralism, recent dramatic impacts on the Malagasy landscapes due to human driven deforestation are now well documented and undoubtedly played a major role in the more recent expansion of open habitats [START_REF] Harper | Fifty years of deforestation and forest fragmentation in Madagascar[END_REF][START_REF] Dewar | Early human settlers and their impact on Madagascar's landscapes[END_REF][START_REF] Waeber | How effective have thirty years of internationally driven conservation and development efforts been in Madagascar?[END_REF][START_REF] Vieilledent | Combining global tree cover loss data with historical national forest cover maps to look at six decades of deforestation and forest fragmentation in Madagascar[END_REF].

A deeper understanding of savanna origins remains essential to our understanding of habitat mosaics (e.g. savannas, gallery and riparian forests and larger forest blocks) in Madagascar. Such a landscape structure may have favored population isolation and divergence in numerous forest organisms, such as lemurs, chameleons, geckos or plants (e.g. Quéméré et al., 2010;[START_REF] Grbic | Phylogeography and support vector machine classification of colour variation in panther chameleons[END_REF][START_REF] Yoder | Geogenetic patterns in mouse lemurs (genus Microcebus) reveal the ghosts of Madagascar's forests past[END_REF][START_REF] Salmona | Climate change and human colonization triggered habitat loss and fragmentation in Madagascar[END_REF]Aleixo-Pais et al., 2018;[START_REF] Hackel | Grass diversification in Madagascar: in situ radiation of two large C 3 shade clades and support for a Miocene to Pliocene origin of C 4 grassy biomes[END_REF]. In addition, land conservation programs in Madagascar focus on forests in particular [but see references in next section], frequently overlooking open habitats [START_REF] Bond | Ancient grasslands at risk[END_REF] often considered of anthropogenic origin, of low ecological interest and highly impacted by fires and cattle grazing (but see [START_REF] Kull | Isle of Fire: The Political Ecology of Landscape Burning in Madagascar[END_REF].

Perennial savanna plants have evolved dedicated adaptations to survive, reproduce, and compete [START_REF] Simon | Recent assembly of the Cerrado, a neotropical plant diversity hotspot, by in situ evolution of adaptations to fire[END_REF][START_REF] Bond | Ancient grasslands at risk[END_REF][START_REF] Pausas | Unearthing belowground bud banks in fire-prone ecosystems[END_REF][START_REF] Buisson | Resilience and restoration of tropical and subtropical grasslands, savannas, and grassy woodlands[END_REF].

Perennial tussock grasses have evolved to be highly flammable, encouraging fires that kill seedlings of forest plants [START_REF] Simpson | Determinants of flammability in savanna grass species[END_REF][START_REF] Cardoso | Grass species flammability, not biomass, drives changes in fire behaviour at tropical forest-savanna transitions[END_REF]. The frequently observed geophyte strategy allows plants to survive below ground and resprout after fire or grazing, such as some pachypods and numerous grasses in Malagasy savannas [START_REF] Rapanarivo | Taxonomic revision of Pachypodium: Series of revisions of Apocynaceae XLVIII[END_REF][START_REF] Solofondranohatra | Grass functional traits differentiate forest and savanna in the Madagascar central highlands[END_REF]. Tree species have also developed strategies involving protective tissues (e.g. cork) to survive fire. In Madagascar, such a strategy has been reported for tapia (Uapaca bojeri Baill.) and some Sarcolenaceae (e.g. Sarcolaena oblongifolia F. Gérard) [START_REF] Rakotoarisetra | Monographie de Uapaca densifolia dans la Forêt d'Ambohitantely[END_REF][START_REF] Solofondranohatra | Grass functional traits differentiate forest and savanna in the Madagascar central highlands[END_REF].

Furthermore, recent evidence suggests that many non-grass savanna lineages were recruited from nearby ecosystems and evolved in situ adaptations to fire and grazing. This is the case for numerous plant lineages of the Cerrado flora (Brazil), that were likely recruited during the Miocene and Pleistocene [START_REF] Simon | Recent assembly of the Cerrado, a neotropical plant diversity hotspot, by in situ evolution of adaptations to fire[END_REF]. These pieces of evidence again support the local, long persistence of open habitats in different tropical and subtropical regions since at least the Late Miocene [START_REF] Edwards | The origins of C 4 grasslands: integrating evolutionary and ecosystem science[END_REF][START_REF] Strömberg | Evolution of grasses and grassland ecosystems[END_REF].

The Oleaceae genus Noronhia encompasses numerous trees and shrubs endemic to Madagascar (i.e. 87 species are presently recognized in the Malagasy flora), but includes only one savanna species -N. lowryi Hong-Wa -a small shrub that occurs in patches in the savannas of Itremo and surronding massifs of the Central Plateau (Hong-Wa, 2016; Fig. 1).

Noronhia lowryi individuals are often observed in close proximity to one another (Fig. 1) suggesting the occurrence of clonal growth (as already reported for Oleaceae elsewhere in the world; [START_REF] Baali-Cherif | High genetic diversity and clonal growth in relict populations of Olea europaea subsp. laperrinei (Oleaceae) from Hoggar, Algeria[END_REF], a possible geophyte adaptation to fire [START_REF] Bond | Ancient grasslands at risk[END_REF][START_REF] Pausas | Unearthing belowground bud banks in fire-prone ecosystems[END_REF]. Despite being considered endangered (Hong-Wa, 2016), N. lowryi's ecology is still poorly documented. Better understanding of its ecology, phylogeography, and demography will, therefore, help conservation stakeholders develop adequate management strategies.

To evaluate the presence of clonal growth, assess the intra-specific genetic diversity, and the connectivity between distant sites, we investigated nuclear and chloroplast genetic diversity of N. lowryi. We developed 14 nuclear microsatellite loci and used 24 chloroplast microsatellite markers to assess the relative contributions of pollen and seed dispersal. We then inferred the recent demographic history of N. lowryi to evaluate the impact of human colonization on savanna habitat. Finally, from full chloroplast genomes of 11 Noronhia accessions, including the three N. lowryi maternal lineages presented here, we investigated the early origin of this species.

MATERIAL AND METHODS

THE MODEL SPECIES AND HABITAT

Noronhia lowryi Hong-Wa, formerly referred to as "sp1" in Hong-Wa and Besnard (2014), is known to occur at five localities, on marble-quartzite and basement rocks in the Itremo and surronding massifs of the Central Plateau of Madagascar, at an elevation of approximately 1200-1800 m (Figs 1 and 2; Supplementary Data Fig. S1; [START_REF] Hong-Wa | A taxonomic revision of the genus Noronhia (Oleaceae) in Madagascar and the Comoro Islands[END_REF]. Most of its known habitat across the Itremo and Ibity massifs is dominated by C 4 grasses with sparse Tapia (Uapaca bojery) and other woody plants (including N. lowryi), and is typical of one of the Malagasy savanna biomes, punctuated with a few humid forest relics (gallery forests; [START_REF] Alvarado | Comparison of plant communities on the Ibity and Itremo massifs, Madagascar, with contrasting conservation histories and current status[END_REF][START_REF] Alvarado | Achieving sustainable conservation in Madagascar: the case of the newly established Ibity Mountain Protected Area[END_REF][START_REF] Nanjarisoa | Grass survey of the Itremo Massif records endemic central highland grasses[END_REF][START_REF] Goodman | The Terrestrial Protected Areas of Madagascar[END_REF]. Noronhia lowryi is considered endangered due to frequent human-induced fires in these areas and its limited distribution [START_REF] Hong-Wa | A taxonomic revision of the genus Noronhia (Oleaceae) in Madagascar and the Comoro Islands[END_REF]. Noronhia lowryi is generally a small shrub (Hong-Wa, 2016), but when protected from fire on cliffs, it can become a small tree (ca. 3 m; G. Besnard, pers. obs.). Populations generally constitute patches of a few small individuals (Fig. 2). Its flower morphology (especially the long tubular corolla) is suggestive of insect pollination (possibly by moths), while its dry nut-like fruits, 1-2 cm drupes with a very thin fleshy layer and a large seed, are likely to be (or have been) dispersed by animals (e.g. by rodents or lemurs). The Itremo and the Ibity Massifs (Fig. 1) harbour several protected areas, respectively managed by KMCC (Kew Madagascar Conservation Center, 2012) and MBG (Missouri Botanical Garden; [START_REF] Alvarado | Comparison of plant communities on the Ibity and Itremo massifs, Madagascar, with contrasting conservation histories and current status[END_REF][START_REF] Alvarado | Achieving sustainable conservation in Madagascar: the case of the newly established Ibity Mountain Protected Area[END_REF][START_REF] Birkinshaw | Plan de Gestion du Feu pour la Nouvelle Aire Protégée du Massif d'Ibity[END_REF], where the species is presently propagated by seed in restoration programs (Ibity, MBG).

PLANT SAMPLING AND DNA EXTRACTION Seventy-seven samples of N. lowryi were collected at three sites (Fig. 1): Only five individuals were observed and collected in Ibity, as well as in Itremo-West, while a larger population was sampled (n = 67) in the eastern part of the Itremo massif (Supplementary Data Table S1). All observed trees were sampled in the southern part of Itremo-East, while three pairs of individuals were collected every 500 meters in the northern part. Sampling was conducted in 2013 and 2016 by GB, MSV and RJQ. Identifications were further confirmed by CHW. Unfortunately, we were not able to visit the remote sites of Andoharano and Ambohijanaka (Fig. 1). For each sample, we recorded GPS coordinates, collected one leaf dried in silica gel, and a herbarium specimen (deposited at TAN). At Itremo-East, in eight cases, trees were sampled at less than 1.5 m, and suspected of clonal origin (resprout from underground organs; i.e. clonal growth). The same colletor's number was assigned to these samples (no. 6,14,15,17,19,22,30), but neighboring samples were distinguished with an additional letter (e.g. RJQ689-4A; Supplementary Data Table S1). One sample from a herbarium specimen collected in 1996 at Itremo-East was also included in the study.

Total genomic DNA was extracted from ca. 5 mg of dried leaves. Each sample was ground in a 2-ml tube containing three tungsten beads with a TissueLyser (Qiagen Inc., Texas). The BioSprint 15 DNA Plant Kit (Qiagen Inc.) was then used to extract DNA, which was eluted in 200 µl AE buffer.

MARKER DEVELOPMENT

We used shotgun genome sequencing data from two N. lowryi, one N. brevituba, one N. clarinerva, and one N. intermedia [START_REF] Van De Paer | Prospects on the evolutionary mitogenomics of plants: a case study on the olive family (Oleaceae)[END_REF][START_REF] Olofsson | Phylogenomics using low-depth whole genome sequencing: a case study with the olive tribe[END_REF] to assemble 20 nuclear regions with microsatellite motifs (poly GA or GAA) common to the five species. Primers were defined in conserved regions flanking the microsatellite motif to increase their transferability among Noronhia species. After PCR tests and preliminary genotyping analyses, we selected 14 loci (Supplementary Data Table S2). Twelve loci are located in a non-coding part of gene (either intron or 3'/5' untranscribed regions), while one tri-nucleotide microsatellite (Nor-13) corresponds to a coding sequence.

GENOTYPING

Nuclear microsatellites were amplified either in multiplex (pair of loci) or separately (Supplementary Data Table S2) using the method of [START_REF] Schuelke | An economic method for the fluorescent labelling of PCR fragments[END_REF]. For each locus, an 18bp tail of M13 was added to the forward or reverse primer. Each PCR reaction (25 μl) contained 10 ng DNA template, 1× reaction buffer, 2.5 mM MgCl 2 , 0.2 mM dNTPs, 0.15 μmol of one universal fluorescent-labeled M13(-21) primer

(5'TGTAAAACGACGGCCAGT-3'; labeled with one of the three following fluorochromes:

6-FAM, AT550 or AT565; Table S3), 0.2 μmol of the reverse primer(s), 0.5 μmol of the forward primer(s), and 0.5 U of Taq DNA polymerase (Promega). We conducted PCR in a Mastercycler pro PCR System (Eppendorf) for 2 min at 94°C, followed by 25 cycles of 30 s at 94°C, 45 s at 56 or 58°C, and 1 min at 72°C, and then by 10 cycles of 30 s at 94°C, 45 s at 51.5°C, and 45 s at 72°C. The last cycle was followed by a 20-min extension at 72°C. In addition, we investigated the chloroplast genetic (cpDNA) diversity with 24 microsatellites (Supplementary Data Table S3) originally developed for the olive tree by [START_REF] Besnard | Genomic profiling of plastid DNA variation in the Mediterranean olive tree[END_REF]. Three to four loci were simultaneously amplified (except locus 19, that was individually amplified) following the protocol described in [START_REF] Besnard | Genomic profiling of plastid DNA variation in the Mediterranean olive tree[END_REF] and using the universal M13 primer labeled with the YAK fluorochrome. Four nuclear loci and twelve plastid loci PCR products were then multiplexed together with GenScan-600 Liz (Applied Biosystems) and separated on an ABI Prism 3730 DNA Analyzer (Applied Biosystems).

Allele size was determined with Geneious v.9.0.5 [START_REF] Kearse | Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF]. Multistate cpDNA microsatellites were coded by the number of repeated motifs for each allele (e.g. number of T or A), as described by [START_REF] Besnard | Genomic profiling of plastid DNA variation in the Mediterranean olive tree[END_REF]. Genotyping of three individuals (RJQ689-30A, 32 and 46) was repeated to ensure reproducibility. All individuals with ambigous genotypes were also systematically repeated. Nuclear and chlroroplast microsatellite genotypes are available in Supplementary Data Tables S1 andS3.

ASSESSING THE OCCURRENCE OF CLONAL GROWTH

To investigate the occurrence of clones, we first estimated pairwise allelic distance using the R package ape [START_REF] Paradis | APE: Analyses of Phylogenetics and Evolution in R language[END_REF]R Development Core Team, 2014) and considered individuals to be potentially genetically identical when harboring a genetic distance of zero (all shared alleles). We then estimated the probability that identical genotypes could result from independent formation of zygotes (P gen ) following [START_REF] Parks | A study of spatial features of clones in a population of bracken fern, Pteridium aquilinum (Dennstaedtiaceae)[END_REF] using the R package poppr [START_REF] Kamvar | Poppr: an R package for genetic analysis of populations with clonal, partially clonal, and/or sexual reproduction[END_REF]. We further confirmed that identical ramets also share plastid genotypes and excluded clones from subsequent analyses.

GENETIC DIVERSITY

To assess nuclear loci departure from Hardy Weinberg proportions, we estimated Nei F IS statistics [START_REF] Nei | F-statistics and analysis of gene diversity in subdivided populations[END_REF] using the R package demerelate [START_REF] Kraemer | demerelate: calculating interindividual relatedness for kinship analysis based on codominant diploid genetic markers using R[END_REF] and a χ 2 exact test based on 1,000 Monte Carlo permutations of alleles using the R package pegas [START_REF] Paradis | pegas: an R package for population genetics with an integrated-modular approach[END_REF]. To assess the overall, per sampling site and per locus genetic diversity, we estimated allelic richness (A R ; Hurlbert, 1971) using the smallest sampling site size for rarefaction with the R package hierfstat, the number of alleles (A), the observed (H O ) and expected heterozygosities (H E ) with the R package adegenet [START_REF] Jombart | adegenet: aR package for the multivariate analysis of genetic markers[END_REF]. To further assess the effect of drift in the potentially small remaining patches of N. lowryi, we estimated the average individual inbreeding coefficient F with adegenet, from 100 iterations.

GENETIC STRUCTURE

To assess the nuclear genetic structure, we first examined the pairwise site differentiation using a range of population statistics, such as G ST [START_REF] Nei | Estimation of fixation indices and gene diversities[END_REF], G' ST [START_REF] Hedrick | A standardized genetic differentiation measure[END_REF], theta [START_REF] Weir | Estimating F-statistics, for the analysis of population structure[END_REF], D [START_REF] Jost | G ST and its relatives do not measure differentiation[END_REF], and Weir & Cockerham and Nei's [START_REF] Nei | Analysis of gene diversity in subdivided populations[END_REF] with the R package diveRsity [START_REF] Guevara | diverse: an R package to measure diversity in complex systems[END_REF]. Additionally, we investigated the patterns of genetic variance with a Principal Component Analysis (PCA) of allele frequencies. We further assessed the potential presence of substructure using the SnapClust clustering approach [START_REF] Tonkin-Hill | Fast hierarchical Bayesian analysis of population structure[END_REF] with default values, for number of clusters K ranging from one to five and selected the most appropriate K value using the goodness of fit AIC and BIC statistics [START_REF] Akaike | A new look at the statistical model identification[END_REF][START_REF] Schwarz | Estimating the dimension of a model[END_REF]. To assess the potential effect of distance on gene flow, we then investigated patterns of isolation by distance (IBD) in nuclear and chloroplast DNA. To that respect, we conducted a Mantel test [START_REF] Mantel | The detection of disease clustering and a generalized regression approach[END_REF] with 999 permutations, using the R package ade4 [START_REF] Chessel | The ade4 package -I -One-table methods[END_REF], between individual geographic distances and Bruvo's genetic distances [START_REF] Bruvo | A simple method for the calculation of microsatellite genotype distances irrespective of ploidy level[END_REF]) estimated using poppr . We investigated patterns of IBD among all samples including all sampling sites.

F ST
To circumvent possible confounding effects of site substructure on IBD [START_REF] Van Strien | Isolation-by-distance in landscapes: considerations for landscape genetics[END_REF], we also investigated within-site IBD using the largest sampling site Itremo-East. To overcome the possible effects of sampling related individuals and oversampled sites, we repeated IBD, PCA, and clustering analyses with a data subset comprising 37 individuals (Supplementary Data Table. S1). Finally, we investigated cpDNA haplotype relationships using a reduced-median network constructed with Network v.5 [START_REF] Bandelt | Median-joining networks for inferring intraspecific phylogenies[END_REF].

DEMOGRAPHIC HISTORY

The demographic history of N. lowryi was investigated using the R package VarEff [START_REF] Nikolic | Detecting past changes of effective population size[END_REF] that uses an approximate likelihood of the distribution of distance frequencies between alleles in a Monte Carlo Markov Chain framework. This approach offers several advantages: (i) it models several demographic changes, (ii) it implements the three most common microsatellite mutation models, and (iii) it is computationally efficient. This enabled us to test several combinations of parameters such as the number of population size changes, mutation models, the variance of the prior log-distribution of effective sizes, and the maximal distance between alleles. The analyses were performed using each of the three mutation models (single step, two-phase and geometric), allowing three population size changes. Additional parameters are detailed in Supplementary Methods (Supplementary Data). Although this approach makes the assumption that population structure is negligible [START_REF] Chikhi | The confounding effects of population structure, genetic diversity and the sampling scheme on the detection and quantification of population size changes[END_REF]Heller et al., 2012), this was not a major issue for N. lowryi according to our results (see below). Since very little available data can help estimating the generation time, we assumed, from field observations, that N. lowryi individuals are likely to be mature after a couple of years (≥ 3 years), and may last more than a few decades (~ 30-50 years).

However, individuals protected from fire and grazing in cliffs of rock cavities may reproduce over long periods of time. We therefore considered values of 10, 30 and 50 years, to emcompass the possible turnover times between generations. The estimation of the timing of demographic history events is also affected by the assumed mutation rate. We used the value of 5 x 10 -3 close to that used and estimated in olive tree demographic inferences (Besnard et al., 2014). Our results -like most other demographic inferences using genetic data -depend on the assumption that the true mutation rate and generation time do not deviate dramatically from the applied values.

EMPIRICAL EVIDENCE FOR DISPERSAL

To investigate effective gene dispersal events, we estimated potential parentage relationships among individuals. All pairs of individuals with a relatedness S xy [START_REF] Lynch | Estimation of relatedness by DNA fingerprinting[END_REF], estimated in the R package demerelate [START_REF] Kraemer | demerelate: calculating interindividual relatedness for kinship analysis based on codominant diploid genetic markers using R[END_REF], superior or equal to 0.5 (that is expected when an individual is parent of another) were first selected. For each putatively affiliated pairs with one shared allele at each locus, we estimated the probability to observe it by chance, P = ∏ {1-(1-p Axy ) 2 } 2 i , where p A is the frequency of allele A shared by individuals x and y at locus i in the population [START_REF] Hardy | Mendelian proportions in a mixed population[END_REF]. To keep the estimate conservative, when the two alleles at a locus were shared by individuals x and y, we considered the most frequent one.

PLASTOME ASSEMBLY AND PHYLOGENY DATING

We finally estimated the divergence time of N. lowryi maternal lineages revealed in our study paired-end reads were used to reconstruct the whole plastome, following the method described in [START_REF] Zedane | Museomics illuminate the history of an extinct, paleoendemic plant lineage (Hesperelaea, Oleaceae) known from an 1875 collection from Guadalupe Island, Mexico[END_REF]. Alignments were produced using MUSCLE in MEGA v.7 [START_REF] Kumar | MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets[END_REF] followed by manual refinement (particularly in regions with inversions).

One inverted repeat region was removed to avoid considering the same sequence twice. A time-calibrated phylogeny was then obtained from the plastome dataset using Bayesian inference as implemented in BEAST v.2.4.3 [START_REF] Bouckaert | BEAST 2: a software platform for Bayesian evolutionary analysis[END_REF] using a GTR + G + I substitution model, which was identified as the best-fit model with Smart Model Selection v.1.8.1 [START_REF] Lefort | SMS: Smart Model Selection in PhyML[END_REF] in PhyML v.20120412 [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF]. A Yule speciation prior was used, which will adequately describe the plastome tree that is composed mainly of different species. Indeed, 90% of the species are represented by a single individual, while N. lowryi is represented by three very divergent samples that might be considered as independently evolving chloroplast lineages (See Results). We used a relaxed molecular clock with an uncorrelated log-normal distribution of rates [START_REF] Drummond | Relaxed phylogenetics and dating with confidence[END_REF]. Node calibration was realized as reported in [START_REF] Olofsson | Phylogenomics using low-depth whole genome sequencing: a case study with the olive tribe[END_REF].

RESULTS

IDENTIFICATION OF CLONES

We detected identical genotypes on two triplets of ramets (RJQ689-4[A-C] and RJQ689-6[A-C]; Supplementary Data Fig. S2). The probability of encountering such genotypes more than once by chance (according to [START_REF] Parks | A study of spatial features of clones in a population of bracken fern, Pteridium aquilinum (Dennstaedtiaceae)[END_REF] was less than 10 -8 , and was thus due to clonal growth. These two triplets of ramets represent 25% of the eight groups of samples labeled as potentially clonal when collected.

GENETIC DIVERSITY

We found a relatively high overall nuclear genetic diversity in N. lowryi with a mean of 10.2 alleles per locus (N a ) and a mean heterozygosity of 0.59 ( showed relatively low estimates of inbreeding (mean < 0.4) from nuclear microsatellite data, with none of the sampling sites showing particularly high inbreeding levels (Fig. 3). Two microsatellites loci, Nor-8 and Nor-14, show a significant excess of homozygosity in the largest sampled population (Itremo-East; Table 1), suggesting the possible presence of null alleles for these loci.

Similarly to nuclear data, N. lowryi comprises a relatively high chloroplast genetic diversity with 15 polymorphic loci among 24. Five loci (loci #15, 19, 25, 33, 41) showed at least three alleles, including one locus (#19) with within-site polymorphisms. All together, these polymorphisms allowed us to distinguish six chloroplast haplotypes (Supplementary Data Tables S3).

GENETIC STRUCTURE At the nuclear level, none of the site pairwise differentiation indices (G ST , G' ST , theta, D, F ST )

showed significant signal of differentiation among sites (Fig. 4A). Similarly, the reduced representation of the genetic variance in the PCA exhibits no particular clustering pattern of the sampled individuals, neither within nor among sites (Fig. 4B). In addition, the estimation of cluster number (K) best fitting the data showed no consistency at values > 1 (Supplementary Data Fig. S3), and the clustering approach snapclust exhibited no comprehensive signal for K ≥ 2 (Supplementary Data Fig. S4). Finally, we did not find evidence of continuous structure of lower intensity driven by distance (IBD), neither at the regional level (Fig. 4C), nor at the Itremo-East level (Supplementary Data Fig. S5A). Using the "subset" data (Supplementary Data Tables S1), we found very similar PCA, IBD and clustering results (data not shown), suggesting that the results were not influenced by the high number of Itremo-East individuals and the presence of related individuals.

In contrast, we established a clear cpDNA-based differentiation of the three sites, none of which shared a chloroplast haplotype with any other (Fig. 5A). We found a strong (but based only on six haplotypes) signal of cpDNA IBD (Supplementary Data Fig. S5B).

However, we retrieved no signal of IBD when considering Itremo-East data alone (Supplementary Data Fig. S5C). These contradictory signals of cpDNA IBD are likely a consequence of the low number of haplotypes (6 overall, 3 in Itremo-East) and of their peculiar but likely auto-correlated distribution in Itremo-East (Supplementary Data Fig. S6).

DEMOGRAPHIC HISTORY

Using Vareff, all three mutation models exhibit a clear and drastic population size decline within the last 350 generations (Fig. 6; Supplementary Data Fig. S7). Altogether, the three models provide mean estimates of a relatively low current effective population size, with N e < 900 (357-854), and larger ancient population sizes with N e > 4,800 (Supplementary Data Table S5).

EMPIRICAL EVIDENCE FOR SHORT-DISTANCE DISPERSAL

At Itremo-East, we identified 17 pairs of samples sharing at least one allele per nuclear locus (Supplementary Data Table S6; Supplementary Data Fig. S8). The Bonferroni corrected probability that these individual pairs share at least one allele at each locus by chance was relatively low (pBF: 9.6 10 -3 to 1.3 10 -5 ; Supplementary Data Table S6). These observations likely reflecting parent-progeny affiliations were distributed at distances ranging from one meter to ~5.9 km (median = 7.3 m). All the 17 pairs did share a chloroplast haplotype and we thus cannot determine if the parental affiliation is maternal or paternal, except for related individuals RJQ689-6 and RJQ689-7 that uniquely share the rare chlorotype A-3 suggesting a mother-progeny relationship with seed dispersal at ca. 30 m. Most of the related pairs ( 15) are found at distances inferior to 32 m indicative of strong dispersal limitation of propagules. As pollen seems efficiently dispersed (see above), short-distance parent-progeny affiliations may mainly reflect limited seed dispersal.

PHYLOGENETIC RECONSTRUCTION BASED ON PLASTOMES

The phylogeny inferred from plastomes (Supplementary Data Fig. S9) is well resolved, and fully congruent with [START_REF] Olofsson | Phylogenomics using low-depth whole genome sequencing: a case study with the olive tribe[END_REF]. The three N. lowryi maternal lineages form a clade sister to another species collected on the Central Plateau (N. brevituba). The node dating of the Oleeae phylogeny is consistent with [START_REF] Olofsson | Phylogenomics using low-depth whole genome sequencing: a case study with the olive tribe[END_REF]. The crown age of N.

lowryi appears surprisingly old with a split from the N. brevituba lineage ~9.1 Mya [95% confidence: (12.1-6.6)] and a split between Ibity and Itremo sites at ~6.7 Mya (9.3-4.5).

Furthermore, the chloroplast lineages of the two geographically close Itremo sampling sites also show an ancient split [~5.2 Mya (7.7-3.0)]. These old within-species splits do not seem driven by the use of the Yule model, since other within-species datings show, as expected, very recent splits (i.e. Cartrema americana, Chionanthus brassii, and Fraxinus ornus;

Supplementary Data Fig. S9).

DISCUSSION REPRODUCTIVE STRATEGY OF A MALAGASY SAVANNA TREE

Madagascar High Plateau savannas faced increasingly recurrent fires since the advent of agropastoralism ~1,000 y BP [START_REF] Burney | Sporormiella and the Late Holocene extinctions in Madagascar[END_REF][START_REF] Dewar | Early human settlers and their impact on Madagascar's landscapes[END_REF], but also dealt with little predictable climate variations [START_REF] Dewar | Evolution in the hypervariable environment of Madagascar[END_REF], natural fires, and grazing since the expansion and diversification of C 4 grasses ~8-3 Mya [START_REF] Bond | The antiquity of Madagascar's grasslands and the rise of C 4 grassy biomes[END_REF][START_REF] Edwards | The origins of C 4 grasslands: integrating evolutionary and ecosystem science[END_REF][START_REF] Strömberg | Evolution of grasses and grassland ecosystems[END_REF][START_REF] Hackel | Grass diversification in Madagascar: in situ radiation of two large C 3 shade clades and support for a Miocene to Pliocene origin of C 4 grassy biomes[END_REF]. In such environmental conditions, many plants developed geophyte strategies to persist over a long period [START_REF] Bond | Ancient grasslands at risk[END_REF], and clonal growth was expected in the Malagasy savanna olive. From new nuclear microsatellite markers, our results confirm only two out of eight suspected cases of clonal growth (25%) on ramets sampled at close proximity. The area covered by these ramets never exceeded 1 m 2 (Fig. 2C)

and was small when compared to that reported in the Saharan olive, whose surface of the stump can reach 80 m 2 (Baali-Cherif and [START_REF] Baali-Cherif | High genetic diversity and clonal growth in relict populations of Olea europaea subsp. laperrinei (Oleaceae) from Hoggar, Algeria[END_REF]. Whether N. lowryi uses clonal growth to respond to harsh environmental conditions remains an open question, but an efficient sexual reproduction seems to be the prominent reproductive strategy.

MALAGASY SAVANNA OLIVE TREE GENETIC DIVERSITY

Despite the threats faced by N. lowryi, and the low number of known and sampled localities of occurrence (five and three respectively; Fig. 1), it harbours levels of genetic diversity of a sustainable population (H E = 0.63; Table 1; Supplementary Data Table S5), comparable, for instance, to those measured with microstallites in two olive subspecies [START_REF] Baali-Cherif | High genetic diversity and clonal growth in relict populations of Olea europaea subsp. laperrinei (Oleaceae) from Hoggar, Algeria[END_REF]. To our knowldege, the genetic diversity of Malagasy savanna trees has only been assessed in two endangered palm species of Dypsis with amplified fragment-length polymorphisms (AFLPs) [START_REF] Gardiner | Population genetics data help to guide the conservation of palm species with small population sizes and fragmented habitats in Madagascar[END_REF]. A relatively low genetic diversity (H E = 0.233-0.239) was revealed in both species. However, as microsatellites usually reveal higher within-population diversity than AFLPs (e.g. [START_REF] Mariette | Genetic diversity within and among Pinus pinaster populations: comparison between AFLP and microsatellite markers[END_REF][START_REF] Maguire | Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs[END_REF], comparison of genetic diversity between Dypsis and N. lowryi is not possible. The estimated genetic diversity of N. lowryi further suggests that its distribution could be larger than currently known (Fig. 1), with populations to be discovered further away from the main roads and accessible areas. However, the contrast between the relatively high genetic diversity and the low number of known occurrence localities suggests that the estimated genetic diversity may depart from equilibrium and represent a relic of the diversity of an ancient larger population (see also demographic history reconstruction, below). A di-allelic selfincompatibility system maintained in distantly related Oleeae genera (Olea, Phillyrea, and

Fraxinus; [START_REF] Vernet | Evidence for the long-term maintenance of a rare self-incompatibility system in Oleaceae[END_REF][START_REF] Saumitou-Laprade | Elucidation of the genetic architecture of self-incompatibility in olive: Evolutionary consequences and perspectives for orchard management[END_REF] is likely present in Noronhia

[which belongs to the Olea-Phillyrea lineage [START_REF] Olofsson | Phylogenomics using low-depth whole genome sequencing: a case study with the olive tribe[END_REF]], and may have played a role in the maintenance of substantial levels of genetic diversity in N. lowryi, despite its likely recent population size contraction (see below).

CONTRASTING NUCLEAR AND CHLOROPLAST GENETIC MAKEUP MEDIATED BY POLLEN AND SEED MOVEMENTS

The strikingly different patterns of nuclear and chloroplast structure (Figs 4 and 5) across N. lowryi sampling sites can be surprising at first but make sense when interpreted in the light of its biology. Contrasting nuclear and chloroplast genetic differentiation has been reported in numerous plants, notably in the Dalbergia monticola populations from humid forests in eastern Madagascar (Andrianolelina et al., 2019), and such patterns result from contrasted pollen-and seed-mediated gene flow. For N. lowryi, the apparent nuclear panmixia is evidence that pollen movement is not encountering resistance at the scale of this study (~80 km-wide), in contrast to the two Dypsis species from Itremo for which a clear IBD pattern has been observed [START_REF] Gardiner | Population genetics data help to guide the conservation of palm species with small population sizes and fragmented habitats in Madagascar[END_REF]. Our results suggest that N. lowryi's pollen (supposedly dispered mainly by insects) commonly travels long distances. It should be mentioned, however, that the markers used in this study (14 nuclear SSR loci) may lack the power to reveal putative fine differentiations that could be elicited by more intensive genotyping approaches. In contrast, the strong structure revealed by maternally inherited chloroplast markers suggests that the three sampled sites do not exchange seeds. This pattern is in agreement with the deep age of the N. lowryi's chloroplast lineage (6.25 My BP) and suggests that fruits may only be dispersed over short distances (Supplementary Data Table S6), through barochory or by small mammals (rodents). It further suggests that the dry nutlike drupes (with thin fleshy mesocarp and thin endocarp), shared by most Noronhia species (Hong-Wa, 2016), are not primarily spread by birds. Considering its large kernel (seed) size compared to the thin meso-and endo-carp layers, it seems more likely that N. lowryi's seeds are dispersed via non-destructive storage by rodents rather than through (likely destructive) ingestion by animals.

RECENT DEMOGRAPHIC HISTORY OF N. LOWRYI Our inferences of N. lowryi's demography through time suggest that its effective population size dramatically declined approximately 350 generations ago. To our knowledge this is the first demographic collapse of a Malagasy plant documented from genetic data. The exact timing of such population decrease is, however, difficult to infer with precision since it depends on accurate knowledge of generation time. Additional studies on the average time necessary to replace reproducing individuals of a population will help resolve the reason(s) of N. lowryi decline. At this stage, two alternative explanations can be given: (1) Assuming a generation time of 10 years, this major decline would have occurred within the last four millennia, and could originate from a change in fire regime. Indeed, since the human colonization of Madagascar during the Early Holocene if not earlier [START_REF] Hansford | Early Holocene human presence in Madagascar evidenced by exploitation of avian megafauna[END_REF]but see: Anderson, 2019;Douglas et al., 2019), the frequency of fire ignition may have continuously risen as a consequence of population density [START_REF] Guyette | Dynamics of an anthropogenic fire regime[END_REF]. Major fire peak traces, likely due to the advent of agro-pastoralism, have been dated to the last millennium [START_REF] Burney | Sporormiella and the Late Holocene extinctions in Madagascar[END_REF][START_REF] Dewar | Early human settlers and their impact on Madagascar's landscapes[END_REF][START_REF] Voarintsoa | Three distinct Holocene intervals of stalagmite deposition and nondeposition revealed in NW Madagascar, and their paleoclimate implications[END_REF]. In an interconnected open habitat, a long-lasting continuous rise in ignition frequency may have had earlier heavy impacts [START_REF] Archibald | Evolution of human-driven fire regimes in Africa[END_REF]). It's also possible that a drought event occurring at or since the Mid-Holocene transition [START_REF] Burney | Late Holocene environmental changes in arid southwestern Madagascar[END_REF][START_REF] Kiage | Late Quaternary paleoenvironmental changes in East Africa: a review of multiproxy evidence from palynology, lake sediments, and associated records[END_REF][START_REF] Virah-Sawmy | Evidence for drought and forest declines during the recent megafaunal extinctions in Madagascar[END_REF] may have exacerbated fire ignition frequency and spread intensity, and acted synergistically with human-driven impact as suggested for other species in various regions of Madagascar [START_REF] Goodman | Extinct Madagascar: Picturing the Island's Past[END_REF][START_REF] Salmona | Climate change and human colonization triggered habitat loss and fragmentation in Madagascar[END_REF][START_REF] Godfrey | A new interpretation of Madagascar's megafaunal decline: the "Subsistence Shift Hypothesis[END_REF]; (2) Alternatively, considering generation times of 30 or 50 years, this decline would coincide with the Last Glacial Maximum (LGM; that ended ~12 kya in the area). On the central highlands, at the onset of the Holocene, the major vegetation shift from Ericaceous bush to grass-dominated habitat (Gasse andVan Campo, 1998, 2001) likely impacted N. lowryi populations.

Furthermore, vegetative reproductive strategies may have dominated during unfavourable periods (altering generation time), allowing long-term maintenance of surviving individuals, as already reported, for instance, in the Saharan olive [START_REF] Baali-Cherif | High genetic diversity and clonal growth in relict populations of Olea europaea subsp. laperrinei (Oleaceae) from Hoggar, Algeria[END_REF]. A bottleneck signal could thus reflect a strong decline of the population during the LGM, with isolated, relic patches of individuals subsequently reconnecting when conditions became favourable at the onset of the Holocene.

HIGH PLATEAU SAVANNA ORIGINS AND THE ESTABLISHMENT OF N. LOWRYI

Our phylogenetic work, built on previous larger-scale efforts [START_REF] Olofsson | Phylogenomics using low-depth whole genome sequencing: a case study with the olive tribe[END_REF], suports the monophyly of N. lowryi, complementing nuclear data (this work) and field evidence (Hong-Wa, 2016) to confirm N. lowryi a distinct unique lineage, despite its geographic discontinuity (Fig. 1). Furthermore, the antiquity of the N. lowryi clade (~6.7 Mya) does surprisingly co-occur with the documented global and local Miocene grassland expansion [START_REF] Bond | The antiquity of Madagascar's grasslands and the rise of C 4 grassy biomes[END_REF]Stromberg, 2011;[START_REF] Hackel | Grass diversification in Madagascar: in situ radiation of two large C 3 shade clades and support for a Miocene to Pliocene origin of C 4 grassy biomes[END_REF]. Although our phylogenetic analyses based on full plastomes are not yet comprehensive, they identified a taxon occurring in humid forests (N. brevituba) as sister to N. lowryi. Altogether these results suggest adaptations to grazed and fire-prone savanna habitat evolved in situ, from a forest ancestor during the rise of the savanna biome, as also described for the Cerrado (Brazil; [START_REF] Simon | Recent assembly of the Cerrado, a neotropical plant diversity hotspot, by in situ evolution of adaptations to fire[END_REF]. Finally, the deep chloroplast divergence estimated among the three sites (~6.7-5.2

Mya) suggests that seed-mediated dispersal between these has been very limited over the past million years. The environmental factors that prevented seed dispersers from ensuring connectivity at this regional scale remain to be identified. An ancient fragmented distribution (on marble-quartzite soils) combined with a low seed dispersal by small mammals (e.g. rodents) could explain such a strong chloroplast DNA diferentiation.

ON THE CONSERVATION OF A WOODY SPECIES IN THE HIGH PLATEAU SAVANNAS

There is little doubt, on the basis of these results, that N. lowryi should remain "Endangered" as suggested by [START_REF] Hong-Wa | A taxonomic revision of the genus Noronhia (Oleaceae) in Madagascar and the Comoro Islands[END_REF]. Although the species has now been recorded in five locations (Fig. 1), the new localities in Itremo-West and Ibity do not drastically affect the distributions estimated by [START_REF] Hong-Wa | A taxonomic revision of the genus Noronhia (Oleaceae) in Madagascar and the Comoro Islands[END_REF]. It now includes two protected areas (i.e. Itremo and Ibity), a positive contribution to the conservation of N. lowryi. However, our estimates of effective population size (N e ~ 350-850) suggest that the IUCN criteria C2a are also applicable, leading to the following updated set of criteria [EN B1ab(i,ii,iii,iv,v) + 2ab(i,ii,iii,iv,v) + C2a(i,ii)]. The very low estimated seed dispersal distance additionally suggests that N. lowryi is unlikely to recolonize areas from which it was recently extirpated.

Furthermore, the lack of knowledge on its potential seed disperser(s) still limits our ability to improve seed dispersal via indirect conservation actions (i.e. endemic rodents conservation).

In that respect, our analyses suggest that the current seed propagation of N. lowryi in a restoration program (Ibity, MBG) is not likely to cause a particularly negative out-breeding effect. Indeed, the distant sites do not show particular structure and are already likely exchanging genes at relatively high rates through pollen dispersal. Ongoing management of fire and grazing activities at Itremo and Ibity (Kew Madagascar Conservation Centre, 2012; [START_REF] Birkinshaw | Plan de Gestion du Feu pour la Nouvelle Aire Protégée du Massif d'Ibity[END_REF]C. Birkinshaw, pers. comm.) are also likely to be among the most efficient measures to protect current N. lowryi's populations. These actions will likely benefit the entire diverse endemic biota present at both sites [START_REF] Alvarado | Comparison of plant communities on the Ibity and Itremo massifs, Madagascar, with contrasting conservation histories and current status[END_REF][START_REF] Alvarado | Management impacts on fire occurrence: A comparison of fire regimes of African and South American tropical savannas in different protected areas[END_REF], which hopefully still includes N. lowryi's seed dipersers.

CONCLUSIONS

Ancient in situ adaptation and recent demographic collapse of an endangered woody plant highlight the antiquity and uniqueness of Malagasy savannas. Building upon accumulating evidence of a climate-driven expansion of Malagasy savannas since the Late Miocene [START_REF] Bond | The antiquity of Madagascar's grasslands and the rise of C 4 grassy biomes[END_REF][START_REF] Vorontsova | Madagascar's grasses and grasslands: anthropogenic or natural[END_REF][START_REF] Hackel | Grass diversification in Madagascar: in situ radiation of two large C 3 shade clades and support for a Miocene to Pliocene origin of C 4 grassy biomes[END_REF] Phylogenetic relationships between the six detected haplotypes were analyzed with a reducedmedian network [START_REF] Bandelt | Median-joining networks for inferring intraspecific phylogenies[END_REF] based on the dataset given in Supplementary Data Table S4. This analysis allowed distinguishing three lineages (namely A, B, C). For the distribution of chloroplast haplotypes at a finer scale (for Itremo-East) see Supplementary Data Fig. S6.

(

  see below). The Oleeae phylogeny based on full plastomes recently reconstructed by[START_REF] Olofsson | Phylogenomics using low-depth whole genome sequencing: a case study with the olive tribe[END_REF] was re-used and complemented with five new Noronhia accessions:Noronhia lowryi (RJQ686-1; Itremo-West, Madagascar), Noronhia brevituba (MSV1929-7; Ankafobe, Madagascar), Noronhia candicans (GB385-2017; Binara, Madagascar), Noronhia spinifolia (GB124-2017; Binara, Madagascar), and Noronhia mannii (White 886; Gabon). This sampling thus allowed us to consider the three maternal lineages of N. lowryi revealed in our study (see below), as well as another Noronhia species (N. brevituba) that occurs in the northern Central Plateau (gallery forests), plus five Malagasy species from low elevation forests. In addition, we analysed one representative species of the two Continental African lineages of Noronhia (i.e. N. peglerae and N. mannii; Hong-Wa and[START_REF] Hong-Wa | Intricate patterns of relationships in the olive family as inferred from multi-locus plastid and nuclear DNA sequence analyses: a close-up on Chionanthus and Noronhia (Oleaceae)[END_REF]. DNA was extracted from the five additional accessions, quality checked, and sequenced at the Genopole platform of Toulouse as detailed in[START_REF] Olofsson | Phylogenomics using low-depth whole genome sequencing: a case study with the olive tribe[END_REF]. For each accession,

  , our results show for the first time that the ancient establishment of open habitats also included woody plants and highlight the need for a clear and evidence-based change in narrative concerning the origin of savannas in Madagascar. In addition, our results stress the urgency of conservation of this unique and antique forsaken habitat, likely collapsing at an unprecedented and underestimated pace. median network reconstruction with the NETWORK software.

Figure 1 .

 1 Figure 1. Distribution of Noronhia lowryi and location of sampled sites. The sampled region is located in Madagascar Central High Plateau (green dot). Samples used in this study are represented with orange dots. Herbarium records of N. lowryi are represented by red triangles [from Hong-Wa (2016), the Tropicos database and the Museum national Histoire Naturelle (MNHN) of Paris]. Protected areas are represented in green and national roads in red. Notethat the protected area encompassing the site of Andoharano in the center-south of the map is considered since 2008 as a potential site to develop a protected area. We have no particular information suggesting that it is currently managed.

Figure 2 .

 2 Figure 2. Sampling of Noronhia lowryi in the eastern part of the Itremo massif. (A) General view of the landscape; (B) Fruiting individual (RJQ689-10); (C) Three neighbor ramets (RJQ689-4A, B and C) with the same genotype supporting a case of clonal growth; and (D) Three neighbor ramets (RJQ689-30A, B and C) with distinct genotypes. (Photo credit: Jan Hackel).

Figure 3 .

 3 Figure 3. Inbreeding and genetic diversity in Noronhia lowryi. (A) Levels of averaged individual inbreeding. Most individuals have a relatively low inbreeding coefficient (< 0.4) from nuclear microsatellites data, and none of the three sampling sites show particularly high inbreeding levels. (B) Loci diversity expressed by the observed diversity plotted against the expected diversity. The graph shows that loci deviates very little from Hardy-Weinberg expectations for sites with large sample sizes. (C) Boxplot of sampling sites diversity represented by the observed (H O ) and expected diversity (H E ) across loci. The figure shows that the three sampling sites show overall similar diversity levels. IE: Itremo-East; Ib: Ibity; IW: Itremo-West; all: overall estimates.

Figure 4 .

 4 Figure 4. Noronhia lowryi's spatial nuclear genetic structure. (A) Measures of genetic divergence among sites with their confidence interval (dashed arrows). Abscise labels represent pairs of compared sites, IE: Itremo-East; Ib: Ibity; IW: Itremo-West. None of the divergence statistics differ from the null expectation under Hardy-Weinberg equilibrium (zero, horizontal black line). (B) Principal Component Analysis (PCA) of the genetic variance showing that the sampling origin does not drive nuclear genetic variance. (C) Graphic representation of the relationship between genetic and geographic distances (isolation by distance) suggesting that nuclear genetic differentiation of N. lowryi is not influenced by distance.

Figure 5 .

 5 Figure 5. Spatial distribution of chloroplast genetic diversity in Noronhia lowryi. Pie charts indicate the frequency of each haplotype in the three populations. Their size is proportional to the number of genotyped individuals. No haplotypes are shared among sampling sites.

Figure 6 .

 6 Figure 6. Noronhia lowryi's demographic history inferred with VarEff (Nikolic and Chevalet, 2014). Mode (black line), and kernel density (color scale) of the posterior distribution of the effective population size (N e ) over time (in generations), inferred under three distinct mutation models: (A) Single Step Mutation Model (SMM), (B) Geometric Mutation Model (GMM), and (C) Two-Phase Mutation Model (TPM). The results only support a demographic decline between 300 and 400 generations ago. See also Supplementary Data Fig. S7 for a more extensive description of demographic history results.

  Table 1). Furthermore, this diversity is similar among sites with allelic richness A r , expected H E and observed heterozygosities H O comprised within narrow ranges (A r = [3.9-4.2], H E = [0.53-0.59], H O = [0.52-0.63]; Table 1), despite strong discrepancies in sampling size (63 vs 5; Table 1). Furthermore, all individuals

Table S5 .

 S5 VarEff posterior effective population size (N e ) estimates. Table S6. Parent affiliation and short distance dispersal in Noronhia lowryi. Figure S1. Sampling of Noronhia lowryi in the western Itremo and Ibity massifs.Figure S2. Identification of clones using pairwise allelic genetic distances. Noronhia lowryi genetic clustering analysis results. Figure S5. Representation of the relation between genetic and geographic distances within the Itremo-East site. Figure S6. Spatial distribution of chlorotypes in Noronhia lowryi at the Itremo-East site. Figure S7. Noronhia lowryi demographic history inferred with VarEff. Figure S8. Geographic representation of Noronhia lowryi pairs of samples sharing at least one allele per locus. Figure S9. Dated maximum-likelihood phylogenetic tree inferred from the alignment of whole plastomes using BEAST. Supplementary Methods 1. Description of the VarEff analysis.

	Figure S3. Most likely number of panmictic clusters in Noronhia lowryi. Figure S4.

Table 1 .

 1 Noronhia lowryi genetic diversity and microsatellite summary statistics by sampling site and on the whole sample. Number of genotypes

ACKNOWLEDGMENTS

We thank the Direction Générale du Ministère de l'Environnement et des Forêts de Madagascar, Madagascar's Ad Hoc Committee for Fauna and Flora and Organizational Committee for Environmental Research (CAFF/CORE) for permission and support to perform this study. We thank the local communities of Ibity and Itremo for their warm reception and support. We warmly thank the many local guides and cooks for sharing their incomparable expertise and help in the field, misaotra anareo jiaby. This work was partly funded through an ERA-NET BiodivERsA project: INFRAGECO (Inference, Fragmentation, Genomics, and Conservation, ANR-16-EBI3-0014). We also thank the LABEX TULIP (ANR-10-LABX-0041) and CEBA (ANR-10-LABX-25-01), and the LIA BEEG-B (Laboratoire International Associé -Bioinformatics, Ecology, Evolution, Genomics and Behaviour, CNRS). Travel funding was provided by a DREIC project (VSR28AFRIQ; UPS). We are grateful to the Get-Plage sequencing (in particular Sandra Fourré) and Genotoul bioinformatics (BioinfoGenotoul) platforms Toulouse Midi-Pyrenees for sequencing services and providing computing resources. We thank Lounes Chikhi and Pascal-Antoine Christin for comments on an early version of the draft, Jan Hackel for help in the field, and Roselyne Etienne and Claire Latapie for lab assistance. Finally, we are greatful to reviewers for their insightfull comments that helped substantially improve the quality of the manuscript.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article at the publisher's website: https://academic.oup.com/biolinnean/article/129/1/227/5626515


Jean-Marc Sabaté

Michel Veyrac

François Mion

Laurent Siproudhis

Philippe Ducrotté

Franck Zerbib

Jean-Charles Grimaud

Michel Dapoigny

François Dyard

Benoit Coffin

Sabaté Jean-Marc

Veyrac Michel

Mion Fran

Siproudhis Laurent

Ducrotté Philippe

Zerbib Franck

Grimaud Jean-Charles

Dapoigny Michel

Dyard Fran

Coffin Benoit

H Pato-Gastroent Rologie

M Dico-Chirurgicale

Marc Sabat
email: marc.sabate@lmr.aphp.fr>

Relationship between rectal sensitivity, symptoms intensity and

Keywords: Adolescent, Adult, Aged, Female, Humans, Irritable Bowel Syndrome, complications, physiopathology, Male, Middle Aged, Muscle Contraction, physiology, Pain Measurement, Pain Threshold, physiology, Prognosis, Prospective Studies, Quality of Life, psychology, Questionnaires, Rectum, physiopathology Irritable Bowel syndrome, rectal sensitivity, quality of life

est destinée au dépôt

INTRODUCTION

Irritable bowel syndrome (IBS) is a common functional bowel disorder characterized by abdominal pain or discomfort associated with altered bowel habits and/or abdominal bloating and/or defecation disorders. The pathophysiology of IBS is probably multifactorial, 1 associating visceral hypersensitivity, micro-inflammation and psychosocial factors. It has been suggested that rectal hypersensitivity, i.e. 2

the lower threshold for pain or discomfort in response to distension, could be a biological marker of IBS. If the evidence of rectal 3 hypersensitivity in IBS patients has been extensively reported, the relationship between this hypersensitivity and the intensity of abdominal symptoms has been poorly investigated. Recent data have suggested that a significant relationship between rectal hypersensitivity and the severity of IBS symptoms could exist while others failed to demonstrate such a correlation. . . 4-8 9 10 IBS, as a chronic condition, has a significant negative impact on quality of life (QoL) which is at least equivalent to that demonstrated in other chronic gastrointestinal disorders such as gastro-oesophageal reflux disease or in non-digestive diseases, such as depression or diabetes mellitus. QoL alterations have been correlated with IBS symptom intensity. However, the relationships between pain 11 12 threshold determined during rectal distension and an alteration in QoL in IBS patients has been poorly evaluated.

The primary objectives of this multicentre prospective study were to search for a relationship between the rectal threshold to distension and IBS symptom intensity defined by the irritable bowel severity scoring system. The secondary objectives were to look for a 13 correlation between rectal threshold to distension and alterations in QoL measured by the Functional Digestive Disorder Quality of Life Questionnaire (FDDQL) and/or the different items of the questionnaires of IBS intensity and of the FDDQL. 14

PATIENTS AND METHODS

Patients

Following approval from the Saint Germain en Laye Ethics Committee, the study was conducted from June 2003 to December 2004 in IBS patients, aged 18 to 65 years, who provided written informed consent in eight French academic hospitals. Patients could be included if they fulfilled Rome-II IBS criteria. Specifically, they had to experience abdominal pain/discomfort with two of the following 1 characteristics for at least 12 weeks (not necessarily consecutive) during the previous 12 months: 1) relief with defecation; 2) onset associated with a change in stool frequency; 3) onset associated with a change in stool form. Each patient underwent a complete clinical evaluation to exclude organic disease and only patients with a normal total colonoscopy within the previous 5 years could be included. Patients were excluded if they had undergone abdominal surgery other than appendectomy or hernia repair. Analgesics, antispasmodics, laxatives and antidiarrhoeal agents were stopped at least 7 days before the experimental protocol.

Questionnaires

IBS severity scoring system

Each patient was asked to complete the irritable bowel syndrome severity scoring system initially developed and validated by Francis et al. of which the French version has been previously validated. This self-administered questionnaire is composed of: 1) two items 13 12 concerning the presence of abdominal pain and bloating (response yes or no); 2) four visual analogue scales measuring intensity of pain, bloating, relief following defecation, and impact of symptoms on general quality of life; 3) an item on the number of days of suffering during the preceding 10 days. This questionnaire produces a quantitative score ranging from 0 to 500 used to determine the severity of symptoms with a score < 75 defined patients in remission; between 75 and 175 mild IBS; between 176 and 300 moderate IBS; and > 301 severe IBS.

Quality of life questionnaire

QoL was measured by using the previously validated French version of the FDDQL questionnaire. It is a 43 item self-administered 14

questionnaire that provides a profile with eight dimensions (stress scale, daily activities, coping with disease, anxiety, alimentation, sleep, discomfort, control) as well as a global score which can vary from 0 (poor QoL) to 100 (good QoL).

Rectal distension and pain threshold

Rectal distensions were performed with a 5 cm latex balloon (maximal volume 300 mL, Marquat, 94470 Boissy Saint Leger, France). None of the patients had previous rectal sensory testing. A small water enema of less than 50 mL was performed before rectal testing in patients with presence of stool in the rectum. The balloon was lubricated and inserted into the rectum. The distal attachment site of the balloon was 4 cm from the anal verge. The proximal opening of the tube was linked to a 100 mL syringe used to inflate the balloon with air. Each rectal distension was performed by slowly injecting air and lasted from 30 to 45 seconds from zero millilitres to the distending volume and deflated. A 2-min rest period was observed between each distension. Distensions were performed by 10 mL-stepwise increments from 0 mL to 60 ml and thereafter by 20 mL-stepwise increments. At the end of each distension the patient was asked to determine a pain score on a 5 levels scale with 0 represented no pain, 1 a moderate but discontinuous pain, 2 a moderate but continuous pain, 3 an intense but tolerable pain and 4 an intolerable intense pain. If the level 4 was reported during the distension, the balloon was immediately deflated. The moderate pain threshold (level 2) was first determined and then we started tracking around this volume with 5 random distensions above or under the volume according to reported sensations using a randomised tracking paradigm. . At the end of 15 16 the distending protocol, the mean volume inducing moderate pain was calculated and patients were classified into two groups: IBS with rectal hypersensitivity if the mean volume was 140 mL, IBS with normal rectal sensitivity if the mean volume was > 140 mL. The 140 ≤ mL threshold was previously defined in a group of healthy subjects and represents the volume threshold to identify rectal hypersensitivity during rectal manometry with the latex balloon.

Statistical analysis

Results are expressed as mean SE. First, the assessment of severity was determined using the irritable bowel syndrome severity symptoms.

Rectal distension

The mean rectal volume for moderate pain threshold (level 2) was 127.1 64.6 mL and was not significantly different from the ± moderate pain threshold determined after 5 random distensions using the tracking method which was 133.7 66.6 mL. Forty five patients ± (66,2 ) had rectal hypersensitivity (mean moderate pain threshold < 140 mL). The percentage of patients with rectal hypersensitivity was

IBS severity scoring and FDDQL

The mean severity score during the last 10 days was 306 76 (range: 66 458 in hypersensitive patients, but differences were not significant.

Relationship between pain threshold, and severity score and FDDQL

The mean volume at the moderate pain threshold was not significantly related neither to overall IBS intensity (r 0.66, 0.62,

= - P =
) nor to the FDDQL score (r 0.30, 0.14, figure 3). Similarly, rectal pain threshold was not related neither to intensity of pain figure 2 = P = neither to the others components of IBS intensity score ( ). Results were similar when patients were classified as hypersensitive or Table 1 normosensitive to rectal distension (results not shown). Among the 8 domains explored by the FDDQL ( ), only anxiety (r 0.30, table 2 = P 0.01, Figure 3) and coping (r 0.31, 0.009, Figure 4) were significantly related to the mean volume at the moderate pain threshold.

= = P =
Finally, a significant relationship could be evidenced between the severity score and the global score of the FDDQL (r 0,54; < = -P 0,0001).

Specific profiles associated with visceral hypersensitivity (Logistic regression)

Beyond subscores and global scores of IBS symptoms severity score and FDDQL score, only coping with disease (OR 0.928, = P = 0.011) was significantly associated with visceral hypersensitivity. There was a trend for anxiety (OR 0.944, 0.0515).

= P =

DISCUSSION

This study aimed to explore the possible associations between IBS intensity and the presence of rectal hypersensitivity. Our data suggest that rectal hypersensitivity is frequent in IBS patients but that IBS intensity measured by a validated specific questionnaire could not distinguish hypersensitive patients from normosensitive ones. Similarly, we could not evidence a significant relationship between rectal sensitivity and the global score of quality of life but only with coping. We confirmed that intensity of symptoms and alteration in QoL were significantly correlated.

During the last 15 years, several studies have clearly demonstrated that rectal hypersensitivity was prevalent in IBS patients. Some authors suggested this could be considered as the biological marker of IBS which varied with symptom intensity. Another study has 3

reported that a pharmacological intervention with an antidepressant, amitriptyline, induced a decrease in symptom intensity and an increase in sensory thresholds, while a psychological intervention did not modify rectal sensitivity whereas symptoms decreased.4

Although it could have a direct clinical consequence for patient s management, the question of a relationship between rectal sensitivity and ' IBS symptoms intensity remains poorly explored and only in single centre studies. Recently, Posserud et al could demonstrate that altered rectal perception was associated with GI symptom severity in general, and pain and bloating more specifically. Other studies reported 8

relationships between visceral hypersensitivity and some, but not all, of the hallmark symptoms in IBS . . whereas the degree of 5-7 10 18 basal hypersensitivity was not correlated to symptoms in another study and only to the presence of severe abdominal pain in a last one. 4 9 The assessment of symptoms severity in IBS patients is still a matter of debate. Only few questionnaires like the symptoms severity scoring system, that we used, or the gastro-intestinal symptom rating scale-IBS . have been validated in the literature. Most of these 8 9

studies previous did not use such validated questionnaire and their main objective was to distinguish sensitivity between C-IBS and D-IBS patients. . In the present study, rectal distensions were not performed by an electronic barostat but by a latex balloon slowly inflated 5-7 10 by air with a syringe. This was a deliberated choice because we planned a pragmatic study that could be performed, in case of positive results, during daily clinical practice as it has been proposed to identify patients with defecation disorders. However, to decrease the 19 potential bias related to the balloon choice and the technique of inflation, we used a tracking method with five randomised distensions above or below the threshold for moderate pain as it is recommended to avoid perceptual response bias. . The frequency of two thirds of 20 patient with abnormal pain threshold is in accordance with previous studies . but higher than the 39 of patients with lowered

9

between patients with and without rectal hypersensitivity. Concerning this last point, contradictory results have been published, rectal hypersensitivity being found more frequent in D-IBS patients by some authors . but not by others. . One explanation for our negative 6 10 8 9

results could be that we explored IBS patients recruited in tertiary centre, with long-standing disease duration and associated comorbidities. These patients are known to have more severe IBS. In accordance with this hypothesis, logistic regression found that 22 coping with disease was significantly correlated with rectal sensitivity.

The second objective of our study was to look for a correlation between rectal sensitivity and QoL using a validated questionnaire, the FDDQL. We did not find any significant relationship between pain threshold and the global score of FDDQL. We only found a 14 correlation between pain threshold and two of the eight domains of QoL questionnaire, i.e anxiety and coping with disease; patients with more anxiety and bad coping strategies having lower moderate pain thresholds. In their study, Posserud et al. found that clinical anxiety 8 was more frequent in patients with altered perception, but not in those with only lowered rectal pain thresholds. Others also demonstrated that illness-specific coping and GI symptom specific anxiety can influence perception of experimental pain stimulus and symptom reporting. Taken together with our results and with functional imaging studies data these associations could be an argument for a role 23 24 of psychological processes (CNS implication) in perceptual rating.25

Finally, Drossman et al. in a study performed to identify factors influencing symptom severity in patients with painful functional 18 bowel disorders, only found a trend for lower rectal sensation thresholds in patients with a severe disease but evidenced that such patients had a greater depression and psychological distress, poorer physical functioning and health-related quality of life, more maladaptive coping strategies. In a previous study conducted in a large cohort of IBS patients visiting gastroenterologists in a non-hospital setting we 12 also found a correlation between severity of IBS symptoms and QoL evaluated by the GIQLI questionnaire. In the present study, we could confirm this association in a smaller cohort of IBS patients by using a more specific questionnaire, the FDDQL.

In conclusion, in this group of IBS patients consulting in tertiary academic centres, hypersensitivity to rectal distension has been evidenced among 2/3 of the patients, a proportion similar to those previously reported. No significant relationship could be evidenced between rectal threshold and symptom intensity or alteration in QoL, suggesting that a single symptomatic analysis cannot detect patients with rectal hypersensitivity. This strategy could be of importance in order to identify patients potentially responders to a treatment acting on visceral sensitivity, such as antidepressant agents, tegaserod or pregabaline. . . Therefore, the development and the validation of 4 26 27 more specific questionnaires to measure IBS intensity, like that recently developed to identify patients with neuropathic pain, warrant 28 further studies.

Table 1

Relationship between moderate pain threshold and the different items of the IBS severity scoring system. 0.857 Score for each dimension of FDDQL ranged from 0 (poor quality of life) to 100 (good quality of life). For positive correlations (r>0), an increase in score is associated with an increase in threshold (volume, in mL) of moderate pain threshold (less sensitive).

Item of the IBS

%

  no significantly different according to bowel habit (62 of C-IBS vs. 67 of D-IBS and unclassified IBS, 0.94). The number of % % P= patients with rectal hypersensitivity was no significantly different according to centre, gender, disease duration, the presence of dyspeptic symptoms or GERD or the presence of extra-intestinal symptoms and fibromyalgia (results not shown).

Figure 1

 1 Figure 1Global and specific values of the FDDQL in the total IBS group, in the IBS hypersensitive group and in the IBS normosensitive group.Differences were not significant.

Figure 2

 2 Figure 2Relationship between Moderate pain threshold and the coping with disease domain of the FDDQL score. There was a significant correlation " "

  and the global score of QoL was calculated and for each dimension measured by the FDDQL questionnaire. The mean volume of distension inducing moderate pain threshold was also calculated. Relationships between the moderate pain threshold, IBS severity score and QoL was calculated by the Pearson or Spearman correlation test. The severity score and global QoL, was compared for each IBS subgroups according to rectal sensitivity (Mann-Whitney test). Then the relationships between the moderate pain threshold and the 5 items of the severity score and the 8 dimensions of QoL was determined by using Pearson correlation tests and compared in different IBS subgroups with or without rectal hypersensitivity (t-test). Multivariate analysis has been performed (factorial and logistic regression) to identify specific profiles associated with visceral hypersensitivity. A P value < 0.05 was considered to be significant. who fulfilled Rome II criteria were included in the study. The mean duration of symptoms was 13.8 12.6 years with a mean age of first

	RESULTS
	Patients
	Sixty eight patients mean age: 44.2 12.7 yrs, 45 women (66.2 ) in whom the main motive of consultation was IBS symptoms and [ ± % ]
	±
	symptoms being 30.4 12.6 yrs (1 5 year-duration: 26.4 ; 5 10 year-duration: 27.9 ; > 10 year-duration: 45,6 ). The mean number of ± -% -% %
	days with abdominal pain during the last 30 days was 21.2 8.8 days with 33.8 of patients having constant abdominal pain. According ± %
	to Rome II criteria, 13 patients (19.1 ) had constipation predominant IBS (C-IBS), 28 (41,2 ) had diarrhoea predominant IBS (D-IBS)

± scoring system % % while 27 (39,7 ) could not be classified. According to Rome II criteria for dyspepsia, 42 patients (61,2 ) had also mild dyspeptic % 17 % symptoms and 30 (44,1 ) mild GERD symptoms. Migraine was present in 28 patients (41.2 ), mild symptoms of fibromyalgia in 33 % % (48.5 ), disturbed sleep in 41 (60,3 ) and premenstrual syndrome in 10 women. Only 20 patients (29,4 ) had no extra-intestinal % % %

  in somatovisceral referral areas, the UCLA group and Posserud et al found respectively a prevalence 3 8 of 94 and 61 of IBS patient having altered rectal perception. Like Kuiken et al., we did not find any difference in bowel habits

	3 21

% thresholds in the recent Posserud s study. Using a wider definition based not only the rectal pain threshold but also perceived intensity of ' 8 unpleasantness and abnormalities % %

Table 2

 2 Relationship between moderate pain threshold and the 8 specific domains of FDDQL questionnaire.

	Domains of the FDDQL scales

Ackowledgements:

this study was supported by an unrestricted grant from Solvay Pharma France