Francisco Sepúlveda-Espinoza

Ariana Bertin-Benavides

Rodrigo Hasbún

Oscar Toro-Núñez

Antonio Varas-Myrik

Diego Alarcón

Marie-Laure Guillemin
email: marielaure.guillemin@gmail.com.

Impact of Pleistocene glaciations and environmental gradients on Embothrium coccineum genetic structure

Keywords:

The South American temperate forests have been subjected to drastic past topographic and climatic changes during the Pliocene -Pleistocene linked to Andean orogeny and glacial cycles. Such changes are regarded standard drivers of genetic structure and adaptation for the local biota of this area.

Embothrium coccineum, a member of the Proteaceae family and an emblematic tree of the South American temperate forest, is assumed to show patterns of genetic variation strongly derived by these historical drivers. Interestingly, recently documented genetic structure inferred from ecotype variants suggest that adaptive genetic responses could also reflect similar patterns of genetic differentiation. The goal of this study was to investigate the effects of both historical and contemporary landscape features affecting the genetic diversity and connectivity of E. coccineum throughout its natural distribution.

Using more than 2,000 SNPs, two genetic groups (Northern and Central-Southern) that diverged some 2.8 million years ago were observed. The level of genetic structure was higher between populations within the Northern genetic group than within the Central-Southern group. We propose that these contrasting patterns are related to differences in pollinator's assemblage and evolutionary histories between genetic groups. Moreover, we observed a strong pattern of isolation by the environment in E. coccineum, suggesting that selection could have led to adaptive divergence among localities. We propose that, within the Chilean temperate forest, the patterns of genetic variation in E. coccineum reflect both a Quaternary phylogenetic imprint and the impact of selection on the solid environmental gradient.

Introduction

Due to their isolation and a high degree of endemism, South American temperate forests are considered of great ecological and evolutionary importance (Smith-Ramirez et al., 2007). In Chile, temperate forests stretch from the 35°S down to the 55°S, forming a continuous area that can be considered a "biogeographic island" separated by impassable barriers (deserts, mountains, and oceans) from the rest of the ancestral sources of its biota [START_REF] Villagrán | Historia de los bosques templados del sur de Chile durante el Tardiglacial y Postglacial[END_REF]. As a result, both climate and topography, which are distributed over a narrow strip (2,000 km long and 120 km wide) of Chilean temperate forests, define a highly heterogeneous landscape comprised of multiple forest and soil types, climatic conditions, and disturbance regimes [START_REF] Loguercio | Silviculture of temperate mixed forests from South America[END_REF]. Under such conditions, it is expected that different, and even complementary, mechanisms equally affect genetic and phenotypic diversity. Adaptive processes linked to persistence in a mosaic of heterogeneous habitats may have contributed to plant species diversification in the temperate forests of southern South America [START_REF] Johnson | Speciation and extinction in the Greater Cape Floristic Region. Fynbos: Ecology, Evolution, and Conservation of a Megadiverse Region[END_REF][START_REF] Prunier | Isolation by distance and isolation by environment contribute to population differentiation in Protea repens (Proteaceae L.), a widespread South African species[END_REF]. In the same way, the within-species genetic divergence of plants with extensive distribution under strong natural environmental gradients could also reflect the action of selection (Saldaña et al., 2007[START_REF] Zeppel | Impacts of extreme precipitation and seasonal changes in precipitation on plants[END_REF][START_REF] Grossiord | Genetic consequences of climatic oscillations in the Quaternary[END_REF]. Contrasting climatic and edaphic features on phenotypic and genetic diversity have been observed in temperate plants (Saldaña et al., 2007;[START_REF] Torres-Díaz | Genetic diversity in Nothofagus alessandrii (Fagaceae), an endangered endemic tree species of the Coastal Maulino Forest of Center Chile[END_REF].

For instance, distinct ecotypes have been linked to contrasting access to water and snow winter coverage in Embothrium coccineum J.R. Forts. & G. Forst (Fig. 1) [START_REF] Dimitri | Colección científica del Instituto Nacional de Tecnología Agropecuaria (INTA)[END_REF], a widespread tree that inhabits the temperate forest of South America [START_REF] Zegers | Bosques templados de Chile y Argentina[END_REF]. This species is an endemic tree of Gondwanan origin with a distribution range matching the extent of the temperate forest biome in Chile and Argentina, exhibiting different phenotypes throughout its distribution (Alberdi y Donoso, 2004; [START_REF] Chalcoff | Uncoupled geographical variation between leaves and flowers in a South-Andean Proteaceae[END_REF]. These ecotypes are coincidently distributed with observed pattern of genetic structure observed from isoenzymatic genetic markers [START_REF] Souto | Genetic variation in the widespread Embothrium coccineum (Proteaceae) endemic to Patagonia: effects of phylogeny and historical events[END_REF], suggesting that E. coccineum populations across the species range may be locally adapted to distinct environmental conditions [START_REF] Souto | Genetic variation in the widespread Embothrium coccineum (Proteaceae) endemic to Patagonia: effects of phylogeny and historical events[END_REF]Bertin-Benavides et al., 2020). ,000 years Before Present, BP), given to the presence of ice caps, a large expanse of temperate forest located between the 42 °S and the 56 °S disappeared [START_REF] Rabassa | Chronology of the Late Cenozoic Patagonian glaciations and their correlation with biostratigraphic units of the Pampean region (Argentina)[END_REF]. Glacial maxima triggered a rapid range shift, with most temperate South American species surviving only in glacial refugia located north of the 42°S [START_REF] Allnutt | Genetic variation in Fitzroya cupressoides (alerce), a threatened South American conifer[END_REF][START_REF] Premoli | Isozyme variation and recent biogeographical history of the long-lived conifer Fitzroya cupressoides[END_REF], Sérsic et al., 2011). Some cold-tolerant species, such as E. coccineum, were also able to survive in refugia embedded within the main glaciated area in localities where microclimate, topography or geothermic activity hindered the formation of the ice cap [START_REF] Allnutt | Genetic variation in Fitzroya cupressoides (alerce), a threatened South American conifer[END_REF][START_REF] Premoli | Isozyme variation and recent biogeographical history of the long-lived conifer Fitzroya cupressoides[END_REF], Sérsic et al., 2011). These areas could represent sources of regional diversity recovery after deglaciation [START_REF] Comps | Diverging trends between heterozygosity and allelic richness during postglacial colonization in the European beech[END_REF]. The presence of 11,000 years BP cold-tolerant plant pollen fossil in areas covered by ice during the LGM [START_REF] Fesq-Martin | Late-glacial and Holocene vegetation history of the Magellanic rain forest in southwestern Patagonia, Chile[END_REF][START_REF] Steubing | Seasonal changes of cold resistance of Proteaceae of the South Chilean laurel forest[END_REF], plus results of genetic studies based on sequences of chloroplast genes or genotyping of nuclear isoenzymatic markers (Vidal-Russel et al., 2011;[START_REF] Souto | Genetic variation in the widespread Embothrium coccineum (Proteaceae) endemic to Patagonia: effects of phylogeny and historical events[END_REF] suggest the existence of a complex glacial-interglacial history in this species.

Recent developments in high throughput sequencing technologies and population genomics have allowed us to distinguish the influence of past environmental changes and selection to the current environmental conditions, particularly on spatial patterns of genetic variation in both South American [START_REF] Wang | Isolation by environment[END_REF]. The correlation between genetic differentiation and environmental distances should be contrasted against the observed between genetic differentiation and geographic distances (isolation by distance; IBD; [START_REF] Slatkin | Isolation by distance in equilibrium and non-equilibrium populations[END_REF] to test the IBE. IBD could be considered a null model for which genetic differentiation increases with geographical distance, given the local accumulation of genetic differences when the dispersion between populations is geographically restricted. In E. coccineum, gene flow could be limited by constraints on pollen transport that is linked to the distribution and behaviour of pollinators (i.e., more than 20 species, including birds and insects, are reported as pollinators of E. coccineum; [START_REF] Chalcoff | Uncoupled geographical variation between leaves and flowers in a South-Andean Proteaceae[END_REF] and anemochorous seed dispersal [START_REF] Rovere | Dispersión asimétrica de semillas de Embothrium coccineum (Proteaceae) en el bosque templado de Chiloé, Chile[END_REF].

The main goal of this study was to evaluate the effects of historical and contemporary landscape features affecting the genetic diversity and connectivity of E. coccineum throughout its natural distribution using SNPs obtained by genotyping-by-sequencing (GBS). Patterns of genetic structure were evaluated independently using SNPs found in genomic regions that may be subject to selection (i.e., outlier loci) and all loci. We hypothesized that patterns of genetic variation in E. coccineum would reflect both the impact of historical processes (i.e., glacial/interglacial cycles during the Pleistocene) and contemporary landscape features, including the selective effect of environmental heterogeneity.

Materials and methods

Samples collection and DNA extraction

The locations were selected to cover an extensive range of climatic conditions across E. DNA samples were analyzed for their integrity by agarose gel electrophoresis, and the total amount of recovery was determined using a Qubit fluorometer (Invitrogen, USA).

Climatic data collection

To characterize patterns of spatial environmental variation among the locations, we used the historical values (1,970-2,000) of 19 environmental variables (bio1 -bio19) and elevation (Elv), which were obtained using the package raster v.

GBS data sets

To test the possible effects of parameter assembly and missing data on patterns of population genetic structure, distinct sets of parameter of populations module on Stack v.2.2 (-p: minimum number of populations where a locus must be present for it to be included; and -r: percentage of individuals in a population that must possess a particular locus for it to be included), were examined before choosing optimal filtering options for further analysis. Eight combinations of parameters -p and -r were tested (-r= 0.6 and 0.7; -p= 6, 7, 8 and 9). For each combination the % of missing data, % of heterozygosity and number of SNPs were estimated (data not shown). The minimum minor allele frequency (min-maf) was set to 0.02, and the maximum observed heterozygosity (max-obs-het) was set to 0.5. The "Population Genetic data set" (i.e., PG_dataset), was built using -p 9 and -r 0.7. These allowed us to obtain a final matrix with the largest SNPs and the lower number of missing data.

Furthermore, another data set was built to estimate the divergence time among the E. coccineum genetic groups, using the Bayesian multispecies coalescent model [START_REF] Stange | Bayesian Divergence-Time Estimation with Genome-Wide Single-Nucleotide Polymorphism Data of Sea Catfishes (Ariidae) Supports Miocene Closure of the Panamanian Isthmus[END_REF].

First, we selected a subset of samples representing the Northern (Nah and Cu), the Central (ChlN and ChlS) and the Southern (Coy and TP) locations of E. coccineum. Then, we selected 12 E. coccineum individuals (i.e., two per location) to maximize the SNPs available and minimize the amount of missing data. These 12 E. coccineum individuals were processed jointly with the samples of L. hirsuta, included as an outgroup. A new SNP calling was conducted following the same parameters as those used for the PG_dataset in Stacks v.2.2 pipeline [START_REF] Catchen | Stacks: an analysis tool set for population genomics[END_REF], eventually generating the "divergence time data set" (i.e., DT_dataset). In this case, optimal values for -p and -r parameters were chosen to decrease the percentage of missing data: only the loci present in all locations, including the outgroup (-p 7), and present in the least 80 % of the individuals (-r 0.8) were kept for further analyses. Putative outliers loci were removed from the DT_dataset. One SNP was randomly selected per locus for each data set to approximate unlinked loci variation.

Populations diversity statistics

To explore the distribution of genetic diversity, we calculated, for each location, the observed (Ho) and expected (He) levels of heterozygosity and the inbreeding coefficient FIS for each location with GenoDive v.3.02. The percentage of polymorphic loci and private alleles were estimated using the R package "hierfstat v. 0.04-22" [START_REF] Goudet | Hierfstat, a package for R to compute and test hierarchical F-statistics[END_REF].

Genetic structure

Four complimentary analyses were conducted to characterize the pattern of genetic structure within E. coccineum using the PG_dataset. First, we calculated the pairwise FST values among all sampling localities using the function stamppFst(), using 1,000 bootstrap replicates across loci and 95% confidence intervals with the R package StAMPP v.1.5.1 [START_REF] Pembleton | StAMPP: An R package for calculation of genetic differentiation and structure of mixed-ploidy level populations[END_REF]. Second, a principal component analysis (PCA) was performed using the glPCA() function with the R package adegenet v.2.1.1 [START_REF] Jombart | adegenet: a R package for the multivariate analysis of genetic markers[END_REF]. Only the first three components were retained to estimate genetic groups.

Third, a model-based STRUCTURE clustering analysis was performed using STRUCTURE v.2. Rate (FDR), set at 5%. Second, we implemented a Bayesian test to detect outlier loci using the BAYESCAN 2.1 software [START_REF] Foll | An approximate Bayesian computation approach to overcome biases that arise when using AFLP markers to study population structure[END_REF]. All simulations were performed using the default parameters with 20 pilot runs of 5,000 iterations followed by 50,000 sampling iterations and an FDR of 5%. Outlier loci detected by both PCAdapt and BAYESCAN were selected as candidate adaptive loci.

To search for candidate adaptive loci with allelic frequency potentially influenced by the environmental variation characterizing our study area (see Appendix I), a Gradient Forest (GF) analysis was implemented in the R package gradientForest [START_REF] Ellis | Gradient forests: calculating importance gradients on physical predictors[END_REF]. GF provides a ranked list of the relative predictive power (R2) of all environmental variables allowing identifying those that best explain the observed genetic variation. Allele frequency of candidate adaptive loci (i.e., detected in both PCAdapt and BAYESCAN) was used as a response variable. GF was fitted using 2,000 regression trees per SNP and a variable correlation of 0.5 [START_REF] Fitzpatrick | Ecological genomics meets community-level modelling of biodiversity: mapping the genomic landscape of current and future environmental adaptation[END_REF].

Isolation by distance and isolation by environment

To better understand the drivers potentially explaining the observed patterns of spatial genetic variation in E. coccineum, we performed distance-based redundancy analysis (db-RDA) and partial db-

RDA at the individual level on the complete PG_dataset and loci potentially influenced by selection.

RDA is a multiple linear regression method performed between a matrix of dependent variables and used as a co-ancestry variable. Prior to the analysis, all three independent matrices were scaled to a mean of zero and variance of one with the scale() function in R.

matrices
Among populations, variation in E. coccineum was partitioned into components explained by geographic distance (IBD), ecological gradients (IBE) and co-ancestry (IBA), or their combination (i.e., constrained by the effects of the remaining two independent matrices), using the vartpart() function within R package vegan v.2.5.7 [START_REF] Oksanen | The vegan package[END_REF]. The significance of each partition was tested with the anova.cca() function through 1,000 permutations on the R package vegan v.2.5.7 [START_REF] Oksanen | The vegan package[END_REF].

Divergence time among Embothrium coccineum genetics groups

We implemented a Bayesian multispecies coalescent model to estimate divergence times among

E. coccineum

Genetic diversity

The Expected and observed heterozygosity (He and Ho, respectively), inbreeding coefficient (FIS), the percentages of polymorphic loci (PPL) and the number of private alleles (PA) are reported in

Patterns of population genetic differentiation

The highest pairwise values of FST (Table 2) were observed between Northern and Southern locations (0.319 < FST < 0.424), followed by those calculated between Northern and Central locations (0.222 < FST < 0.429) and then by the ones calculated between Central and Southern locations (0.070 < FST < 0.193). Lower levels of divergence were observed between locations sampled in the same macro region (Northern: 0.089 < FST < 0.174; Central 0.010 < FST < 0.152; Southern: 0.043 < FST < 0.061) (Table 2). All FST values were significant. Regarding the distribution of E. coccineum, not all localities were under ice at LGM (Fig. 1a).

The PCA showed that E. coccineum was divided into two major genetic groups: Northern and Central-Southern (Fig. 2b). The Northern locations were retrieved separated from those corresponding to the Central-Southern along the first axis of the PCA (PC1, representing 21.54% of the genetic variance). In contrast, samples from the Central and the Southern were separated along the second axis of the PCA (PC2, representing 5.82% of the genetic variance). Results from the model-based Bayesian clustering approach showed a K = 2 (highest LnP(D) and ΔK) as optimal and K = 4 as a possible secondary level of substructure (Appendix I). The STRUCTURE clustering (K = 2) revealed that all individuals sampled from the Northern were assigned to the blue genetic cluster. In contrast, all individuals sampled from both Central and Southern locations were assigned to the orange genetic cluster (Fig. 3a). In both Northern and Southern samples, almost no admixture was observed. Individuals from PM, ChlN and ChlS (Central) were majorly assigned to the orange genetic cluster but exhibited low levels of admixture from the blue genetic cluster. The level of assignation to the blue genetic cluster tends to recede across latitudes (from 20% of the genome assigned to the blue genetic cluster in PM to less than 10% in ChlS; Fig. 3a). The STRUCTURE clustering using K = 4 showed two new genetic clusters within the Central-Southern part of the species range (Fig. 3b). Individuals from the Southern were assigned to the orange genetic cluster, individuals from Pumalín (PU, Central) mainly were assigned to the new purple genetic cluster, and the rest of the individuals from the Central (PM, ChlN and ChlS) were admixed between the orange genetic cluster and the newly detected green genetic cluster (Fig. 3b).

Genome scan for outlier loci detection

Within the 2,155 loci included in the PG_dataset, PCAdapt detected 132 outlier loci and BAYESCAN 89 outlier loci. The two methods concur with the detection of 59 loci (2.73%) that were then identified as potentially adaptive loci in E. coccineum.

The gradient forest (GF) analysis revealed four environmental factors with the highest impact on E. coccineum populations (i.e., estimated as the variables presenting the highest values of R2 weighted importance), which correspond to Precipitation of the driest month (bio14), Mean temperature of the wettest quarter (bio8), Elevation, and the Mean temperature of the driest quarter (bio9) (Fig. 5a). The value of the R2 weighted importance for bio 14 (R2 weighted importance > 0.05) was at least two times higher than those observed for the other variables.

In addition, GF also produced individual turnover functions for each potentially adaptive loci having an R2> 0 [START_REF] Fitzpatrick | Ecological genomics meets community-level modelling of biodiversity: mapping the genomic landscape of current and future environmental adaptation[END_REF]. This value allows listing the potentially adaptive loci showing the highest correlation of allelic frequencies with the four environmental factors of the significant impact on E. coccineum populations. Results show that allelic frequencies of outliers S92192_43 and S60272_8 are strongly correlated with Precipitation of the driest month (bio14; Fig. 5b: pink and blue lines, respectively). S60272_8 allelic frequency also exhibited a weak response to the Mean temperature of the driest quarter (bio9; Fig. 5b). Three outliers (S27842_67, S56778_42 and S83147_54) exhibited a significant and concurrent response to the Mean Temperature of the Wettest Quarter (bio8; Fig. 5b: green, calypso and purple lines, respectively). The allelic frequency of two outliers (S272842_67 and S56778_42) also shows a weak response to elevation and Mean Temperature of the Wettest Quarter (bio9) (Fig. 5b; light green and light blue lines, respectively).

Four outliers showed allelic frequency gradually changing with latitude (Figure 5b; S60272_8, S27842_67, S56778_42 and S83147_54). Allelic frequencies among locations sampled in the northern distribution (i.e., Ch, Nah, Cu) were similar. The locus S92192_43 showed the fixation of a private allele in the Curacautín (Cu) location. The sampling localities are ordered along a latitudinal gradient.

Isolation by distance and isolation by environment

The full RDA model using the PG_datset explained 59.4 % of the total genetic variation and supported an influence of environmental (Env), geographic (Geo) and coancestry (Anc) variables in shaping allelic variation (Env: adjusted R2 = 0.420, p < 0.001; Geo: adjusted R2 = 0.226, p < 0.001; Anc: adjusted R2 = 0.105, p < 0.001; Table 3). With the partial db_RDA, the contribution of the environmental variables to genetic divergence was much higher than the contribution of the geographic distance or the co-ancestry (Env | Geo+Anc: adjusted R2 = 0.288; Geo | Env+Anc: adjusted R2 = 0.118; Anc | Env+Geo: adjusted R2 = 0.011; Table 3). In total, 0.178 explained variation was confounded between the effects of IBE, IBD and IBA, and 0.405 of the variation remained unexplained (Table 3). Similar results were obtained for the subset of 59 loci potentially influenced by selection (i.e., outlier loci). The partial db-RDA highlights the important contribution of the environmental variables to potential local adaptation in E. coccineum (Env | Geo+Anc: adjusted R2 = 0.244; Table 3).

Divergence time

The divergence time between the Northern and Central-Southern genetic groups was estimated at The divergence time was inferred only for the nodes showing high support (posterior probability > 0.99). Sampling location codes are as in Appendix I; coloured symbols for each sampling location match Fig. 2a.

Discussion

In the present study, samples of E. coccineum from its distribution range along the Chilean temperate forest were genotyped through GBS to investigate patterns of genetic variation and their possible drivers using genome-wide data in this emblematic Chilean tree. The thousands of loci successfully genotyped provided a much better resolution and new knowledge of E. coccineum Our data showed the existence of two main lineages, Northern and Central-Southern. These genetic groups were already reported in Souto and Premoli ( 2007), but the GBS data set also uncovered fine-scale genetic differentiation, especially in the Northern distribution. Inference of divergence time between these two genetic groups suggested that intraspecific divergence occurred some 2.8Myr ago, at the beginning of the Pleistocene, driven by climatic changes linked to glacial cycles. Moreover, the heterogeneous environment characterizing the South American temperate forests has been recognized as a significant driver explaining the actual genetic makeup of various plants and animals [START_REF] Premoli | Isozyme variation and recent biogeographical history of the long-lived conifer Fitzroya cupressoides[END_REF]. It could also have led to adaptive divergence among locations in E. coccineum. The strong IBE pattern detected in E. coccineum could reflect the impact of an actual gradient of temperature, precipitation and elevation, characteristic of the species distribution range, on its genetic diversity.

These results suggest that selection linked to the strong environmental gradient could have led to adaptive divergence among localities. At present, one-third of the woody genera in southern South America are related to ancient paleo flora that occupied southern South America in pre-Quaternary times [START_REF] Villagrán | History of the forests of southern South America II: phytogeographical analysis[END_REF] Therefore, we hypothesize that the population structure differences between localities within the Northern and the Central-Southern genetic groups could be, in part, linked to the existence of a more restricted gene flow in the northern distribution.

Genetic differentiation within

Plant gene flow may occur via pollen and/or seed dispersal and is directly affected by the species' reproductive system [START_REF] Martins | Estrutura populacional, fluxo gênico e conservação "in situ[END_REF]. Seed dispersal decreases competition in areas close to the parent plant while allowing colonization of new sites [START_REF] Rovere | Dispersión asimétrica de semillas de Embothrium coccineum (Proteaceae) en el bosque templado de Chiloé, Chile[END_REF]. It also influences the genetic structure of populations since seed transport favours gene flow between and within populations [START_REF] Hamrick | Consequences of dispersal in plants[END_REF] while imposing restrictions on the potential for intraspecific differentiation into ecotypes and subpopulations [START_REF] Willson | Avian communities of fragmented south-temperate rainforests in Chile[END_REF]. E. coccineum has an anemochory seed dispersal and the density of dispersed seeds declines with distance from the tree: 95% of the seeds are dispersed 5 m around the mother tree, but a distance of 20 m has been detected [START_REF] Rovere | Dispersión asimétrica de semillas de Embothrium coccineum (Proteaceae) en el bosque templado de Chiloé, Chile[END_REF]). These short dispersal distances limit the distribution of the seeds to the surroundings of the parent trees, causing a restricted movement in the population. [START_REF] Chalcoff | Variación geográfica en los rasgos florales de Embothrium coccineum (Proteaceae) y su relación con los ensambles de polinizadores a ambos lados de los Andes australes[END_REF]. We propose that differences between Northern and Central-Southern E. coccineum pollinators' distribution and behaviour could lead to the observed genetic structure, with a lower dispersal capacity in the Northern locations, leading to a higher level of structure. However, other differences between regions, such as the level of landscape fragmentation, could also generate differences in gene flow due to the different flowering times detected between the localities of E. coccineum (Rovere & Chalcof, 2010). Differences in climatic conditions through the distribution range affect E. coccineum flowering period, with a flowering period beginning later (i.e., November -December) in populations located at high latitude and altitude when compared to the rest of the distribution (i.e., September-October) [START_REF] Hoffmann | Flora silvestre de Chile. Zona araucana. Arboles, arbustos y enredaderas[END_REF][START_REF] Chalcoff | Variación geográfica en los rasgos florales de Embothrium coccineum (Proteaceae) y su relación con los ensambles de polinizadores a ambos lados de los Andes australes[END_REF]Rovere & Chalcof, 2010). Limited gene flow between populations presenting differences in flowering phenology could also lead to the observed pattern of divergence between and within genetic groups by limiting exchange between close populations located at contrasting altitudes.

Signatures of local adaptation in Embothrium coccineum

On the other hand, we sought to elucidate the effects of local adaptation and isolation by environment (IBE) from neutral processes, such as isolation by distance (IBD) or co-ancestry linked to the species' glacial history (IBA), in shaping among-population genetic differentiation of E. from the redundancy analysis (RDA) show that IBE was a significant driver of population structure and explained the most considerable among-population variation in E. coccineum, even after controlling for IBD and IBA. These results were corroborated by the much higher divergence detected between the Northern and Central-Southern genetic groups in the NJ tree based on the genetic distance calculated using only the outlier loci putatively under selection than the one observed when using all the loci genotyped.

Our gradient forest analysis identifies the Precipitation of the driest month as the most critical

Conclusion and prospects

Known patterns of morphological variations coupled with new genetic diversity and structure results obtained during the present study revealed a long history of geographic isolation and local adaptation in E. coccineum in South American temperate forests. Here, we observed a robust genetic structure in E. coccineum that could be linked to local adaptation, especially differential access to water during the driest months. These results support previous studies that have reported the existence of distinct ecotypes along with the natural distribution. Identifying and quantifying the environmental variables structuring population genetic variation could inform management decisions for conservation, restoration, or reforestation purposes. For example, knowledge about the association between genotype and environment is essential for selecting the proper seed when tree populations are locally adapted, as is the case of E. coccineum. However, as the environment changes, nonlocal seed sources may be considered for restoration or reforestation purposes based on their match to the novel environment.

Therefore, seed transfer guidelines can benefit from the knowledge of factors structuring the landscape of genetic variation. Many conservation efforts rely on delineating distinct populations for management but often ignore the continuous nature of landscape variation and its potential relationship with local adaptation or other processes that lead to genotype-environment associations. The present results advocate for seed transfer guidelines that should be restricted within each ecological zone concerning land management decisions. The strong genetic structure also offers an opportunity to explore local adaptation among groups further and exploit these for agroforestry.

Fig. 1 .

 1 Fig. 1. Embothrium coccineum morphology tree. A) Mature tree, B) Leave, C) Flower, D) Root, E) Follicle and F) Seed.

  plants and animals (Turchetto-Zolet et al., 2013; Hasbún et al., 2016; Varas-Myrik et al., 2021). If divergent natural selection has led to genetic differentiation between populations of E. coccineum along environmental gradients characterizing South American temperate forests, a pattern of isolation by the environment (IBE; Wang & Bradburd, 2014) should be expected. Indeed, when selection against migration limits gene flow among distinct environments, genetic differentiation increases with environmental differences (Wang & Summer, 2010;

  coccineum natural distribution: i) three locations (Chillán, Nahuelbuta and Curacautín) representing the northern distribution, ii) four locations (Puerto Montt, Chiloé Norte, Chiloé Sur and Pumalín) the central distribution, and iii) three locations (Coyhaique, Chile Chico and Torres del Paine) the southern distribution (Fig. 2a and Appendix I). These three regions are subsequently named Northern, Central and Southern locations. Three to six individuals were sampled within each location and five mature leaves were collected per tree. A total of 42 trees were sampled. To calibrate divergence time among E. coccineum genetic groups, two individuals of Lomatia hirsuta Diels, a Proteaceae species from Embothrieae tribe (Sauquet et al., 2009), were included as an outgroup. The selecting mature leaves were deposited and transported to laboratory in a cooler. In the Laboratory, the samples were frozen with liquid nitrogen and stored at -80° C until the DNA extraction. Genomic DNA was obtained from tissue (50 mg) of mature leaves powdered in Precellys24 homogenizer (Precellys, USA) using the Qiagen DNeasy Plant kit (Qiagen Inc., USA) following the manufacturer's instructions.

3 . 4 .(

 34 [START_REF] Falush | Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies[END_REF]. Ten independent simulations allowing admixture were run for each K (K=1-10), each with 200,000 Markov chain Monte Carlo replicates (MCMC) and 100,000 samples discarded as burn-in. STRUCTURE HARVESTER (http://taylor0.biology.ucla.edu/structureHarvester/) was used to determine the optimum K based on L(K) and ΔK parameters (Zhang et al., 2018). Fourth, to describe the relationships between individuals and genetic groups among E. coccineum, the Nei's genetic distance between individuals was calculated within the R package StAMPP v.1.5.1 (Pembleton et al., 2013). Unrooted Neighbor-Joining (NJ) distances trees were constructed using the individual Nei's distance matrix, as implemented by the R package ape v.5.3 (Paradis & Schliep, 2018).Outlier detection and association of allele frequency with environmental variablesTwo complementary approaches were used to identify candidate loci influenced by selection within E. coccineum PG_dataset.. First, we used a Principal Component Analysis (PCA) to generate a "model-free" null distribution of individual genetic distances to detect outlier loci. This analysis was conducted using the R packages PCAdapt v.4.0.3[START_REF] Luu | pcadapt: an R package to perform genome scans for selection based on principal component analysis[END_REF]) and q-value v.2.16.0[START_REF] Storey | Strong control, conservative point estimation and simultaneous conservative consistency of false discovery rates: a unified approach[END_REF] with default parameters and a search with K=10. The detection of outliers was confirmed by plotting the histograms of p-values, and the Mahalanobis (D2) test statistic, with a False Discovery

Figure 2 :

 2 Figure 2: Multivariate analysis to genetic diversity in E. coccineum. a) Sampling locations of E. coccineum, the distribution area is highlighted in light blue and hatched lines represent ice limits from LGM. The colour symbols represent the locations. Circles, triangles and diamonds represent samples from the Northern, Central and Southern distributions. b) Principal component analysis (PCA) based on a 2,155 SNPs data set of E. coccineum samples.

Figure 3 :

 3 Figure 3: Embothrium coccineum genetic structure. Analyses for K = 2 (a) and K = 4 (b) of 38 E. coccineum individuals based on a 2,155 SNPs data set. Each individual is displayed from north to south, represented by a vertical bar. Sampling location codes are as in Appendix II. The Colour corresponds to the genetic group, and the proportion of the genome assigned to each genetic group is given along the Y-axis

Figure 4 :

 4 Figure 4: NJ tree reconstructed based on Nei´s distance between Embothrium coccineum individuals. NJ reconstruction based on (a) the PG_dataset (2,155 SNPs), and (b) the 59 outlier loci putatively under selection. Sampling location codes are as in Appendix I. Nodes with high support (> 0. 95) are filled in black.

Figure 5 :

 5 Figure 5: Environmental factors with the highest impact on Embothrium coccineum populations and allelic frequency. a) The R2 weighted importance of environmental variables with a low covariance explains the genetic variation across sampling locations obtained from GF analysis. Environmental variables used in the analyses are the same as in Appendix I; bio14, Precipitation of the driest month (mm); bio8, Mean temperature of the wettest quarter (°C); Elevation (masl); bio9, Mean temperature of the driest quarter (°C); bio4, Temperature seasonality (°C); bio3, Isothermality (%); bio13, Precipitation of the wettest month (mm). b) Change in allele frequency between sampling localities for the five potentially adaptive loci showing major response to environmental variables. (Loci: S92192_43, S60272_8, S27842_67, S56778_42 and S83147_54).

2. 81

 81 My (1.64 -4.17 My), near the end of the Pliocene and the beginning of the Pleistocene. However, the locations within the Northern genetic group (Nah and Cu) or within the Central-Southern genetic group (ChlN and Coy, TP, ChlS) seem to have diverged much more recently towards the end of the Pleistocene (0.33 / 0.37 My ago; Fig. 6).

Figure 6 :

 6 Figure 6: Estimation of divergence time in Embothrium coccineum based on Bayesian coalescent analysis using SNAPP. Nodes with high support (posterior probability > 0.99) are filled in black. Median ages are provided above nodes with 95% highest posterior densities (HDP) below.

  population genetic structure compared to previous studies. While these studies were based on allozymes genotypes or chloroplast sequences (Mathiasen et al., 2007; Souto and Premoli, 2007; Vidal-Russell et al., 2011; Souto and Smouse, 2014), their limited variability and genome coverage have not provided resolutive evidence to delineate clear genetic limits and clarify the underlying processes shaping them.

  Embothrium coccineum: two genetic groups separated since the early PleistoceneOur result confirms previous studies in E. coccineum documenting the presence of two genetic groups, one in the Northern and one in the Southern distribution[START_REF] Souto | Genetic variation in the widespread Embothrium coccineum (Proteaceae) endemic to Patagonia: effects of phylogeny and historical events[END_REF][START_REF] Vidal-Russell | Multiple Pleistocene refugia in the widespread Patagonian tree Embothrium coccineum (Proteaceae)[END_REF]; yet these datasets exhibit different levels of spatial resolution. Alloenzymes revealed only a weak geographic structure, with some Northern and Southern locations clustered together[START_REF] Vidal-Russell | Multiple Pleistocene refugia in the widespread Patagonian tree Embothrium coccineum (Proteaceae)[END_REF]. The authors explained this pattern by proposing a possible effect of glacial survival in multiple refugia followed by recolonization and/or the existence of actual gene flow, blurring the trace of the historical geographic structure. Contrastingly, chloroplast markers revealed a distinct Southern genetic group characterized by a very low genetic variation (i.e., only a few haplotypes were observed in the Southern). In this last case, two hypotheses could explain this pattern:1) the existence of both northern and southern glacial refugia and recent colonization from the southern to the northern part of the country, or 2) a unique refugium in the north and a recent recolonization to the south[START_REF] Souto | Genetic variation in the widespread Embothrium coccineum (Proteaceae) endemic to Patagonia: effects of phylogeny and historical events[END_REF].Given the higher resolution provided by genomic SNPs, the present study unambiguously supports the two proposed genetic groups and shows that individuals from the Central distribution are more genetically like those from the Southern. Furthermore, a divergence time of 2.8Myr was estimated between the Northern and Central-Southern genetic groups, corresponding to the Late Pliocene and Early Pleistocene. In contrast, the divergence between populations into Northern and Central-Southern groups was much more recent (i.e., some 0.35Myr ago). These results emphasize the possible role of the glacial cycles and associated environmental changes as a possible driver of divergence in E. coccineum. Our study provides a refined picture of the possible divergence time between the North and Central-Southern genetic groups. Most phylogeographic studies have shown that populations located at southern latitudes and impacted by glaciations have experienced a recent and rapid expansion after glacial retreat, whereas northern populations have remained more stable(Koszak et al., 2006;[START_REF] Fontanella | Phylogeography of Diadophis punctatus: extensive lineage diversity and repeated patterns of historical demography in a trans-continental snake[END_REF]. Indeed, various studies (e.g.,[START_REF] Markgraf | Late and postglacial vegetational and paleoclimatic changes in subantarctic, temperate, and arid environments in Argentina[END_REF][START_REF] Vidal | Intraspecific variation in morphology and sexual dimorphism in Liolaemus tenuis (Tropiduridae)[END_REF] propose the existence of refugia Northern of the area heavily impacted by ice (i.e., ranging between 36°S -40°S and 54°S).In this respect, it has been reported that during the Pleistocene glaciation, and its consequent climate change, dramatically altered the landscape of southern South America (Ramos and Ghiglione, 2008; Martínez and Kutscher, 2011;[START_REF] Ponce | Paleogeographic evolution of the Atlantic coast of Pampa and Patagonia since the Last Glacial Maximum to the Middle Holocene[END_REF]. The numerous glacial advances and retreats have impacted the abundance and distribution of the local biota[START_REF] Rabassa | Late Cenozoic glaciations in Patagonia and Tierra del Fuego: an updated review[END_REF]. As a result, southern south American forests are composed of paleo-flora with variable origins and histories[START_REF] Villagrán | History of the forests of southern South America II: phytogeographical analysis[END_REF] Segovia et al., 2013). Fossil records indicate that during the Paleocene, this region supported a widespread tropical flora[START_REF] Hinojosa | History of the forests of southern South America I: paleobotanical, geological and climatical background on Tertiary of southern South America[END_REF]. Then, lineages preadapted to cool temperate conditions, widely distributed in Gondwana[START_REF] Poole | Valdivian ecosystems in the Late Cretaceous and Early Tertiary of Antarctica: further evidence from myrtaceous and eucryphiaceous fossil wood[END_REF], joined in this area during the mid-Eocene, associated with a northward displacement of vegetational belts[START_REF] Hinojosa | Are Chilean coastal forests pre-Pleistocene relicts? Evidence from foliar physiognomy, palaeoclimate, and phytogeography[END_REF]. Finally, during the late Eocene and early Miocene, a mixed paleo-flora with similar Neotropical, Australasian and Australantarctic elements was established in response to colder and drier conditions from Antarctic glaciation and the drop in global temperature[START_REF] Zachos | Trends, rhythms, and aberrations in global climate 65 Ma to present[END_REF].

  coccineum. Our results show that the observed pattern of genetic variation is, at least in part, the result of adaptation to the solid environmental gradients characterizing the species distribution range (Danield & Veblen, 2000; Souto & Premoli, 2007; Souto et al., 2009; Souto and Smouse, 2013). Indeed, results

  explanatory environmental variable explaining the E. coccineum pattern of genetic variation. The level of precipitation of the driest month distinguishes the northern neighbouring Mediterranean biome and the Patagonian steepe to the east, both with deficient summer precipitation, from the temperate biome inhabited by E. coccineum[START_REF] Escobar | Embothrium coccineum[END_REF]. The second explanatory environmental variable was the mean temperature of the wettest quarter. In addition, GF also produced individual turnover functions for each potentially adaptive loci having a R2 > 0[START_REF] Fitzpatrick | Ecological genomics meets community-level modelling of biodiversity: mapping the genomic landscape of current and future environmental adaptation[END_REF]. This allows listing the potentially adaptive loci showing the highest correlation of allelic frequencies with the four environmental factors of the significant impact on E. coccineum populations. For 5 of the 59 outlier loci putatively under selection, a correlation between allelic frequency and Precipitation of the driest month, Mean temperature of the driest quarter, Mean Temperature of the Wettest Quarter, and Mean Temperature of Wettest Quarter was revealed. The covariation between latitude and these environmental variables translates into four outliers (i.e., S60272_8, S27842_67, S56778_42, and S83147_54), showing allelic frequency-changing gradually with latitude. These results showed that genetic differentiation in E. coccineum mainly was associated with environmental differences in summer rainfall and winter temperature. This result is consistent with studies focused on other Proteaceae; with summer rainfall associated with differences in functional traits in P. rapens[START_REF] Carlson | Intraspecific variation in stomatal traits, leaf traits and physiology reflects adaptation along aridity gradients in a South African shrub[END_REF] and in Protea section Exsertae (i.e., a group of six Protea species; Carlson et al., 2011; Prunier et al., 2012, Prunier et al., 2017). Differences in morphology associated with environmental gradients have already been reported in E. coccineum (Chalcof, 2008; Souto & Premoli, 2007), supporting the idea of possible local adaptation in the species. Access to water seems vital in determining the ecotypes observed in each region, with individuals from the Northern distribution growing in the driest climate and developing as short plants, of 0.5-2.5 m in height, with small leaves. In contrast, the individuals in a wet temperate forest of the Central distribution can reach more than 10 m in height and develop longer leaves (Souto et al., 2009; Souto & Smouse, 2013). Plants that develop in areas characterized by steady and high rainfall may be less heat and water-stressed, favouring the production of more extensive and broader leaves and maximizing the photosynthetic area when risks of overheating and drought stress are low (Givnish, 1987; Hallik et al., 2009). Indeed, a reduction in leaf size has been proposed as one of the critical traits allowing plants to withstand water deficit (Baldocchi and Xu, 2007, Peguero-Pina et al., 2014). For example, a reduction in Quercus faginea leaf size has been proposed as one of the critical traits allowing Mediterranean oaks to withstand the water deficit characteristic of the region (Baldocchi and Xu, 2007, Peguero-Pina et al., 2014).

  These include the use of a lognormal distribution for the the crown age of the Embothrieae tribe, which was estimated at 66 million years (Myr) (Mean = 66.6 Standard Deviation = 0.13, Mean In Real Space = TRUE) using a phylogeny of Proteaceae that included all the genera currently recognized in the family[START_REF] Sauquet | Contrasted patterns of hyperdiversification in Mediterranean hotspots[END_REF]). This estimate was used to aproximate the node age of the common ancestor of Embothrium coccineum in our dataset, which was derived from the sister relationship with Lomatia samples. Additionally, a generation time of 10 years per individual was set, following experimental greenhouse observations (Bertin-Benavides A., personal observation). Alpha and beta parameters were left with an uniform prior, with an arbitrary value of 1.0[START_REF] Stange | Bayesian Divergence-Time Estimation with Genome-Wide Single-Nucleotide Polymorphism Data of Sea Catfishes (Ariidae) Supports Miocene Closure of the Panamanian Isthmus[END_REF]). For additional scrutiny, the XML file is available in supplementary material.Species trees were inferred directly with no prior grouping assignment. We used SNAPP with four independent runs of 2,000,000 generations with a sampling every 200 generations each. The In order to evaluate the population structure, the divergence time among genetics groups and the local adaptation in E. coccineum, 38 trees were genotyped using GBS. A total of 116,606,122 reads were generated and de novo assembled into 1,303,750 loci, from which 473,461 SNPs were called. The application of filtering criteria resulted in a final PG_dataset of 2,155 SNPs. For the DT_dataset, composed by 12 individuals of E. coccineum and two individuals of L. hirsuta, a total of 320 SNPs were called.

	Results
	genetic groups (Rannala & Yang, 2003) using the DT_dataset. This analysis was
	conducted with the coalescent simulator SNAPP v.1.3.0 (Bryant et al., 2012) included in the BEAST
	v.2.4.5 package (Bouckaert et al., 2014). For this approach we followed a model accomodated to
	genome-wide SNP data, which implements a strict molecular clock and nodes calibrated with
	biogeographic constraints resulted from one or more divergence events (Stange et al. 2018). This
	approach assumes a symmetric substitution model and equal frequencies, plus the implementation of a

single parameter for the rate of the strict molecular clock, as this is the only model currently supported by SNAPP for this type of data

[START_REF] Stange | Bayesian Divergence-Time Estimation with Genome-Wide Single-Nucleotide Polymorphism Data of Sea Catfishes (Ariidae) Supports Miocene Closure of the Panamanian Isthmus[END_REF]

).

The set of parameters used exactly followed the "Divergence-time estimation with SNAPP" tutorial (https://github.com/mmatschiner/tutorials/tree/master/divergence_time_estimation_with_snp_data), except with a few exceptions. results (i.e., ESS > 300) were checked for mixing for all parameters with Tracer v1.7 (Rambaut et al., 2018) and convergence of split frequencies among runs (Appendix III) with the R package rwty

(Warren et al., 2019)

. The final trees from each run (corresponding to the 95% highest posterior density (HPD), after discarding a 10% burn-in of tree topologies) were combined and subsequently summarized using a maximum credibility tree with TreeSetAnalyser v.2.4.5. Finally, this species tree was visualized as a cloudgram with DensiTree v.2.2.7

[START_REF] Bouckaert | DensiTree: making sense of sets of phylogenetic trees[END_REF]

.

Table 1

 1 for each sampling locality. Except for the low value observed in Chile Chico (ChCh;

PPL=12.668 %), PPL was reasonably homogeneous throughout the species range, varying from 18.794% in Chillán (Ch) to 42.367% in Coyhaique (Coy) (Table

1

). Four sampling locations, Puerto Montt (PM), Chiloé Norte (ChlN), Chiloé Sur (ChlS) and Chile Chico (ChCh), presented a lower PA (PA≤30) than the rest of the sampled range (44<PA<83). Ch and Pumalín (Pu) were the only two locations with a negative FIS value, while four locations (i.e., PM, ChlN, ChlS and ChCh) showed positive values of FIS superior to 0.1.

Table 1 : Genetic diversity of Embothrium coccineum

 1

						PPL
	ID	n	He	Ho	FIS	(%)	PA
	Ch	3	0.1	0.105	-0.049	18.794 51
	Nah	4	0.138	0.132	0.037	32.715 65
	Cu	4	0.128	0.126	0.012	30.998 73
	PM	3	0.152	0.135	0.114	29.791 30
	ChlN	3	0.163	0.139	0.15	36.241 29
	ChlS	3	0.152	0.128	0.155	33.132 29
	Pu	3	0.134	0.154	-0.148	27.425 83
	Coy	6	0.143	0.137	0.043	42.367 62
	ChCh	3	0.124	0.106	0.146	12.668 10

. Locations ID are as in Appendix I. n, number of genotyped individuals; He, expected heterozygosity; Ho, Observed heterozygosity; FIS, inbreeding coefficient; PPL, percentage of polymorphic loci; PA, private alleles.

Table 2 : Average FST values for pairwise comparisons among sampling locations of Embothrium coccineum in Chile based on a set of 2,155 SNPs

 2

		Ch	Cu	Nah	PM	ChlN	ChlS	Pu	Coy	ChCh	TP
	Ch	-
	Cu	0.163	-
	Nah	0.174	0.089	-
	PM	0.312	0.263	0.222	-
	ChlN	0.309	0.260	0.225	0.024	-
	ChlS	0.345	0.292	0.257	0.041	0.010	-
	Pu	0.429	0.369	0.338	0.152	0.126	0.135	-
	Coy	0.382	0.340	0.319	0.117	0.083	0.071	0.168	-
	ChCh	0.424	0.362	0.341	0.133	0.089	0.070	0.193	0.061	-
	TP	0.384	0.345	0.322	0.133	0.094	0.085	0.185	0.061	0.043	-

. All p-values are significant (<0.05). Sampling location codes are as in Appendix I. Northern: Ch, Cu and Nah; Central: PM, ChlN, ChlS and Pu; Southern: Coy, ChCh and TP.

Table 3 : Redundancy analysis (RDA) partitioning among-population genetic variation in Embothrium coccineum into three components

 3

		All loci	Outlier loci		All loci	Outlier loci
	Combined	Adjusted	Adjusted	Individual	Adjusted	Adjusted R 2
	fractions	R 2	R 2	fractions	R 2
	Env	0.420	0.569	Env | Geo+Anc	0.288	0.244
	Geo	0.226	0.378	Geo | Env+Anc	0.118	0.111
	Anc	0.105	0.259	Anc | Env+Geo	0.011	0.002
	Env+Geo	0.582	0.719	Total confounded	0.178	0.360
	Env+Anc	0.476	0.606	Unexplained	0.405	0.283
	Geo+Anc	0.305	0.473
	Env + Geo + Anc	0.594	0.717

: environmental (Env), geographic (Geo) and Northern/Central-Southern coancestry (Anc). All p-values <0.001.

regional differences in pollen and seeds dispersal: effect on Embothrium coccineum gene flow

  ;[START_REF] Wardle | Comparison of the flora and vegetation of the southern Andes and New Zealand[END_REF][START_REF] Hinojosa | Are Chilean coastal forests pre-Pleistocene relicts? Evidence from foliar physiognomy, palaeoclimate, and phytogeography[END_REF]. Comparative phylogeographic studies in southern South America[START_REF] Bruno | Phylogeography of Cnesterodon decemmaculatus (Cyprinodontiformes: poeciilidae) in Southern Pampas, Argentina: ancient versus recent patterns in freshwater fishes[END_REF][START_REF] Perez | Comparative phylogeography of woody plant species from mediterranean and temperate forest of southern south america: testing the influence of plant functional strategies on the response of species to past climatic changes[END_REF] Sérsic et al., 2011) propose the existence of at least six areas where both terrestrial plant and animal species survived during glacial maxima. These areas show higher genetic diversity than areas where post-glacial colonization occurred, as in some parts of the southern Patagonian Steppe[START_REF] Hewitt | Inferring glacial refugia and historical migrations with molecular phylogenies[END_REF][START_REF] Pinceel | Population genetics and identity of an introduced terrestrial slug: Arion subfuscus sl in the north-east USA (Gastropoda, Pulmonata, Arionidae)[END_REF][START_REF] Huck | Range-wide phylogeography of the European temperate-montane herbaceous plant Meum athamanticum Jacq.: evidence for periglacial persistence[END_REF].Our study found relatively high genetic diversity and polymorphic loci in Nahuelbuta, Curacautín, Chiloe Norte, Coyhaique, and Torres del Paine, and the number of private alleles was also high in these exact locations.Vidal-Rusell et al. (2011), based on cpDNA haplotypes, suggested that southern populations of E. coccineum had been affected by glacial cycles, declining gradually in size since the Pleistocene (100ky -12ky ago) and then rapidly increasing after the LGM (). Hence, evidence of areas of local expansion in the north is supported, particularly from the existence of various microrefugia located in Nahuelbuta (37°S -72°W), Manzanar (38°S -71°W, nearby Curacautín), Lagüen Ñadi (41°S -73°W, nearby Chiloé) and Lago Verde (42°S -71°W, nearby Coyhaique), as suggested by these authors. Combined with the observed pattern of genetic structure, these results support the hypothesis of E. coccineum survival during glacial periods of the Pleistocene glacial cycles in various refugia, including at least two refugia located south of 42°S. The existence of southern local refugia in E. coccineum is supported by fossil records of cold-tolerant plant species characteristic of the austral forest reported south of 42°S (Villagran et al., 1986; Markgraf, 1995). High levels of population divergence and phylogeographic structure attributed to isolation in multiple refugia have been detected in other cold-tolerant native trees such as Fitzroya cupressoides (Allnutt et al., 1999; Premoli et al., 2000; Premoli et al., 2003), Pilgerodendron uviferum (Premoli et al., 2001; Premoli et al., 2002; Ausin et al., 2003), Araucaria araucana (Bekessy et al., 2002), Podocarpus nubigena (Quiroga & Premoli, 2010), Nothofagus alpina (Marchelli et al., 1998; Marchelli & Gallo, 2006), and Nothofagus pumilio (Premoli, 2004; Mathiasen & Premoli, 2010; Premoli et al., 2010).Our analysis of the genetic differentiation drivers in E. coccineum identified a significant pattern of isolation by distance (IBD). The existence of IBD indicates that gene flow is limited as geographic distance increases[START_REF] Wright | Isolation by distance[END_REF]. However, some level of connectivity was observed between nearby populations, mainly in the Central and Southern groups. In plants such as Dactylis glomerata (Sun et al., 2017) and Protea rapens[START_REF] Prunier | Isolation by distance and isolation by environment contribute to population differentiation in Protea repens (Proteaceae L.), a widespread South African species[END_REF], IBD patterns were detected similarly geographic scale, pointing out opportunities for increased allelic exchange between neighbouring populations. Moreover, our STRUCTURE, PCA, and NJ distance tree analyses, and estimation of pairwise-FST values, showed that populations from the Northern genetic group of E. coccineum are highly structured.

	Possible

  Thus, could have significant genetic consequences as the local establishment of propagules results in a clustering of genetically related individuals in the vicinity of the seed tree, which would generate a marked genetic structure(Rovere &

	Premoli, 2005).
	On the other hand, Mathiasen et al. (2007) consider E. coccineum a species with low sensitivity to
	forest fragmentation due to a predominantly outcrossing breeding system, favouring pollen exchange
	among nearby fragments (Rovere & Chalcof, 2010). Indeed, genetic erosion by drift in forest patches
	could be counteracted by pollinator's movements, reducing inbreeding, and allowing the populations to
	retain sufficient heterozygosity through a continuous influx of alleles from locally distinct gene pools
	(Mathiasen et al., 2007). E. coccineum, gene flow is highly dependent on pollinating agents
	(Mathiasen, 2004; Rovere & Chalcof, 2010), and its flowers are visited by more than 20 species,
	including birds of the Orders Passeriformes and Apodiformes, and insects of the orders Hymenoptera,
	Diptera, Lepidoptera and Coleoptera (Chalcoff, 2008).
	The composition of the pollinator assemblage affects both pollen transport and seed production.

In regions with fewer E. coccineum pollinators, pollen limitation can be high, leading to low reproductive efficiency (i.e., few fruits produced to the available flowers)

[START_REF] Chalcoff | Variación geográfica en los rasgos florales de Embothrium coccineum (Proteaceae) y su relación con los ensambles de polinizadores a ambos lados de los Andes australes[END_REF]

, which would translate to populations presenting reduced genetic diversity and high genetic structure due to the lack of genetic exchange (Rovere and Chalcof 2010). Pollinating insects, generally characterized by low migration capacities, are distributed throughout the E. coccineum distribution range, even if they are abundant, and visit the plants more frequently in populations located in drier and sunnier climates (i.e., Northern locations) (Rovere and Chalcof, 2010). Contrastingly, birds, such as the hummingbird Sephanoides sephaniodes, are abundant in the Southern distribution, characterized by the coldest and most humid conditions that act as principal pollinators

ACKNOWLEDGEMENTS A.B.B: Financial support was provided by both ANID/FONDECYT N° 3160551 and N° 11221214. D.A. was supported by FONDECYT 3200675 grant. O.T.N. was funded by CONICYT PAI

DATA AVAILABILITY STATEMENT

The authors declare that all the work presented in this manuscript was done according to the standards of the Ecology and Evolution. Raw GBS data used in this study has been already published in ncbi-SRA database and is available at http://www.ncbi.nlm.nih.gov/sra/?term=PRJNA783610.

Subvencion a la instalacion en la Academia convocatoria 2019 77190055. We thank Mr Cesar Tascón and Dr Luis Corcuera for allowing the access and the collection of samples within the Fundo Niagara and the Katalapi Park. Our thanks to Ximena Steinberg, Cristian Muñoz, Francisco Valdebenito and Alex Fajardo for their support and help during samples collection in Northern and Southern locations.

The authors thank Catalina Mardones for her enthusiastic support in the samples processing.

CONFLICT OF INTEREST

On behalf of all authors, the corresponding author states that there is no conflict of interest.

AUTHOR CONTRIBUTIONS

All authors contributed to the design of the study. Material preparation, data collection and analysis were carried out by Francisco Sepúlveda-Espinoza, Ariana Bertin-Benavides, Rodrigo Hasbún, Oscar Toro-Núñez, Antonio Varas-Myrik, Diego Alarcón and Marie-Laure Guillemin. The first draft of the manuscript was written by Francisco Sepúlveda-Espinoza, Ariana Bertin-Benavides and Marie-Laure Guillemin and all authors commented on earlier versions of the manuscript. All authors read and approved the final manuscript.

Appendix I: Environmental characteristics of each sampling location of


Marie Peng

Chang-Sheng Wang

Pan-Pan Chen

Thierry Roisnel

Henri Doucet

K N Houk
email: houk@chem.ucla.edu

Jean-François Soulé
email: jean-francois.soule@chimieparistech.psl.eu

Merging C-H Bond Activation, Alkyne Insertion, and Rearrangements by Rh(III)-Catalysis: Oxindole Synthesis from Nitroarenes and Alkynes

Keywords:

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

Introduction

Nitrogen-containing heterocycles are essential to life science since they are abundant in nature, exist as subunits in several natural products and more than half of prescribed drugs contain an N-heterocycle backbone. [START_REF] Heravi | Prescribed Drugs Containing Nitrogen Heterocycles: an Overview[END_REF] Moreover, they also found diverse applications in materials sciences. [START_REF] Chen | Nitrogen Heterocycle-Containing Materials for Highly Efficient Phosphorescent OLEDs with Low Operating Voltage[END_REF] Therefore, their formations, manipulations, and diversifications represent a significant stake for chemical companies, and the discovery of novel synthetic approaches fulfilling modern reaction ideals of green chemistry is still an important challenge, especially starting from the low-functionalized materials. Since the pioneer works by Fagnou on Rh(III)-catalyzed C-H bond annulation of acetanilides with alkynes for the synthesis of indoles, [START_REF] Stuart | Indole Synthesis via Rhodium Catalyzed Oxidative Coupling of Acetanilides and Internal Alkynes[END_REF] several N-directed C-H bond annulation protocols have been developed for straightforward access to N-heterocycles. [START_REF] Su | Synthesis of 2-Pyridones and Iminoesters via Rh(III)-Catalyzed Oxidative Coupling between Acrylamides and Alkynes[END_REF] However, the conditions often required a stochiometric amount of oxidant. An external oxidant-free alternative consists of merging C-H bond functionalization with O-atom transfer (OAT) -proved that the directing group holds an N-O bondthen, the resulting intermediate can undergo rearrangements to roll N-heterocycles in a one-pot cascade process (Figure 1A). This strategy was initially reported by Li and co-workers using quinoline-N-oxides, albeit the reaction stopped after the O-atom transfer (Figure 1B). [START_REF] Zhang | Rhodium(III)-Catalyzed C-C and C-O Coupling of Quinoline N-Oxides with Alkynes: Combination of C-H Activation with O-Atom Transfer[END_REF] Shortly later, Cp*Rh(III)-catalyzed one-pot cascade of C-H bond functionalization -OAT -rearrangement was implemented to prepare indolyl scaffolds from arylnitroles (Figure 1C), [START_REF] Dateer | Selective Cyclization of Arylnitrones to Indolines under External Oxidant-Free Conditions: Dual Role of Rh(III) Catalyst in the C-H Activation and Oxygen Atom Transfer[END_REF] indoles from arylnitroles (Figure 1D), [START_REF] Yan | Rhodium-Catalyzed C-H Annulation of Nitrones with Alkynes: A Regiospecific Route to Unsymmetrical 2,3-Diaryl-Substituted Indoles[END_REF] or amine-N-oxides (Figure 1E). [START_REF] Li | Rhodium-Catalyzed Annulation of Tertiary Aniline N-Oxides to N-Alkylindoles: Regioselective C-H Activation, Oxygen-Atom Transfer, and N-Dealkylative Cyclization[END_REF]. Although the high efficiency of these methods for the direct synthesis of N-heterocycles, all require the pre-installation of the oxidizing directing group (N-O bond) using hazardous reagents. [START_REF] Palav | The m-CPBA-NH3(g) System: A Safe and Scalable Alternative for the Manufacture of (Substituted) Pyridine and Quinoline N-Oxides[END_REF]

Rearrangements

Nitroarenes constitute abundant feedstocks of N-containing molecules as they are readily prepared by nitration of arenes. [START_REF] Greish | Nitration of Aromatic Hydrocarbons over Heterogenous Catalysts[END_REF] Despite of a few examples of C-H bond functionalizations of nitroarenes, [START_REF] Caron | Palladium-Catalyzed Direct Arylation of Nitro-Substituted Aromatics with Aryl Halides[END_REF] NO2 group had never been considered as a suitable oxidizing directing group to develop redox-neutral coupling reactions yet. Therefore, we plan to use nitroarenes in Cp*Rh(III)-catalyzed one-pot cascade of C-H bond functionalization -OAT -rearrangement(s) (Figure 1F). The advantage of this approach is to employ naturally occurring NO2 as an oxidizing directing group. Moreover, the OAT should deliver

N + Z O - N + Z O Rh Cp* N + Z O R 2 R 2 Rh Cp* N Z R 2 R
H N R 2 O R 2 N O R 3 O R 2 N O R 2 R 3 N + O O - R 3 R 2 + + O R 4 O R 4 O O R 4 Rh(III) (cat.)

THIS WORK

4 1➜ 3 R 1 R 1 R 1 1. C-H Bond Activation R 1 R 1 R 1 R 1 R 1 R 1 R 1 (D) from nitrones to indoles 1➜ 4 1➜ 4 R 1 R 1 R 1 R 1
nitrosoarene intermediates, which are very reactive and undergo unpredictable rearrangements toward N-heterocycles. [START_REF] Jana | Promoting Reductive Tandem Reactions of Nitrostyrenes with Mo(CO)6 and a Palladium Catalyst To Produce 3H-Indoles[END_REF] In contrast to previous methods to generate nitrosoarenes which employ reductants, our strategy is redox-neutral. This cascade approach led us to discover a novel synthesis of oxindoles. In retrospect to the realization of this unpredictable endeavor, we also optimized the catalyst design, investigated the scope of the reaction, and thoughtfully studied the reaction mechanism.

Results and Discussion

Reaction Development. We began our studies on Rh-catalyzed C-H bond activation by reacting nitrobenzene with 1diphenylacetylene in the presence of [Cp*RhCl2]2 in 1,2-dichloroethane at 120 ºC. Surprisingly, we observed the unexpected formation of oxindole 1 in 41% yield that was formed upon ortho-C-H bond annulation of nitrobenzene via multi-step reaction: twofold reduction of NO2, migration of alkyne phenyl group, and N-acylation (Table 1, entry 1). The structure of 1 was secured by X-ray analysis. This class of oxindoles containing an α-carbonyl quaternary center was widely recognized as valuable synthetic intermediates, forming the core of numerous natural products and drugs, often requiring several consecutive reactions. [START_REF] Overman | Asymmetric Heck Reactions via Neutral Intermediates: Enhanced Enantioselectivity with Halide Additives Gives Mechanistic Insights[END_REF] A solvent screen revealed 1,2-dichloroethane to be essential for the reaction outcomes, whereas most of the common additives employed in C-H bond functionalization had deleterious effects and did not suppress or decrease the oligomerization of alkyne as the side reaction. [14] Following the seminal work of Rovis, [START_REF] Piou | Correlating Reactivity and Selectivity to Cyclopentadienyl Ligand Properties in Rh(III)-Catalyzed C-H Activation Reactions: An Experimental and Computational Study[END_REF] Tanaka, [START_REF] Shibata | Catalytic [2+2+1] Cross-Cyclotrimerization of Silylacetylenes and Two Alkynyl Esters To Produce Substituted Silylfulvenes[END_REF] and Cramer [START_REF] Wodrich | Ligand-Controlled Regiodivergent Pathways of Rhodium(III)-Catalyzed Dihydroisoquinolone Synthesis: Experimental and Computational Studies of Different Cyclopentadienyl Ligands[END_REF] on the modification of Cp ligand to improve the selectivity and performance in Rh(III) catalysis in other reactions, we decided to evaluate an array of [Cp R RhCl2]2 catalyst precursors (Table 1). Electron-deficient [Cp E RhCl2]2 was inactive for this transformation (Table 1, entry 2). [18] Rh(III) complex surrounded by Cp iPr * ligand -having a similar disc shape and electronic nature than Cp*- [START_REF] Piou | Correlating Reactivity and Selectivity to Cyclopentadienyl Ligand Properties in Rh(III)-Catalyzed C-H Activation Reactions: An Experimental and Computational Study[END_REF] gave the desired product 1 in a similar yield (Table 1, entry 3). We next prepared a set of Rh(III) complexes holding C5Me4-Ar ligands exhibiting an elliptical shape. The presence of substituents at the para position on the aryl group affects the redox potentials predictably, decreasing in the order of [CF3 (-1.21), H (-1.24), and OMe (-1.25)],

which is consistent with an increase in electron density of the rhodium center. Increasing the electron density of the Cp ring resulted in an improvement of the yields [Cp TFT *Rh (10%)< Cp Ph *Rh (25%) < Cp MP *Rh (55%)] (Table 1, entries 4-6).

Subsequently, we prepared two novels [C5Me4-ArRhCl2]2 complexes with Ar = 3,4-dimethoxyphenyl (DMP) and 3,4,5trimethoxyphenyl (TMP). Incorporating additional methoxy substituents affects the electronic nature slightly, but their multiplication leads to a bulkier system. Both complexes displayed higher reactivity, and the best result was obtained with [Cp TMP *RhCl2]2, allowing the formation of 1 in 72% isolated yield (Table 1, entries 7 and8). No reaction occurred without Rh(III) catalyst (Entry 9). Similar to the C-H bond arylation of nitroarenes, [11a] the use of a large excess of nitrobenzene ( 10equivalents) is required (Entry 10), but most of the nitrobenzene (70-85%) can be recovered by distillation after the reaction. OMe

Table1. Optimization: Nature of Cp-Ligand a Entry [Cp R RhCl2]2 Conv. (%) b Yield 1 (%) b 1 [Cp*RhCl2]2 100 41 c 2 [Cp E RhCl2]2 0 0 c
Cp MP * (-1.25 # ) [Cp E RhCl 2 ] 2 (-0.84 # ) [Cp iPr *RhCl 2 ] 2 (-1.33 # ) Rh Cl Cl 2 Ar Rh Cl Cl 2 Rh Cl Cl 2 iPr # = E(Rh III /Rh II ) (V vs. Cp 2 Fe) ◼disc-shape ◼elliptical-shape 1 O O O O [Cp TFT *RhCl 2 ] 2 [Cp TMP *RhCl 2 ] 2 X-Ray structures
Scope of the reaction. With the optimized conditions in hand using [Cp TMP *RhCl2]2, the scope of nitroarene, alkyne, and carboxylic anhydride partners was evaluated. We first subjected 12 different nitroarenes, adorned with various functional groups with different substitution patterns, to this C-H bond alkenylation -OAT - [1,2]-aryl shift cascade reaction (Scheme 1A). Halides (i.e., F, Cl, Br, I) at the para-position of the nitro group were well tolerated and delivered the corresponding products in good to moderate yields (2-5). Para-or meta-nitrotoluene reacted to afford oxindoles 6 and 7 in 48% and 58% yield, respectively; while ortho-nitrotoluene did not react (9). The reaction was regioselective with meta-substituted nitroarenes (R = Me or Ph), affording the products resulting from activating the less hindered C6-H bond (7, 8). From 5 equivalents of methyl 2-methyl-5-nitrobenzoate, corresponding oxindole 10 was isolated in 64% yield. From disubstituted nitroarenes at para and meta-positions, the corresponding products were obtained in good yields as single regioisomers (11, 12). Interestingly, when the meta substituent is an F atom, the reaction occurs at its adjacent C-H bond (12). The electronically enhanced ortho-to-fluorine selectivity is a common feature observed in C-H bond functionalizations involving a CMD mechanism. [START_REF] Lafrance | Catalytic Intermolecular Direct Arylation of Perfluorobenzenes[END_REF] Reaction with 3,4,5-trifluoronitrobenzene gave an excellent yield (13). Propionic, valeric, and isovaleric anhydrides gave the corresponding products in good yields (14-16) (Scheme 1B). The use of AcOH instead of Ac2O affords the NH-free oxindole 17 in 45% yield. A broad range of 1,2-diaryl alkynes was readily converted to the corresponding oxindole products in modest to good yields (Scheme 1C). The reaction conditions were compatible with a variety of functional groups at the para-position, such as bromo (18, 19), chloro (20), fluoro (21), trifluoromethyl (22), and alkyl (23, 24).

The reaction outcomes were not affected by the substitution patterns of the aryl group as a fluorine-atom at the meta-position (25, 26) or a chlorine-atom at the ortho-position (27) were well tolerated. It should be noted that the [1,2]-aryl shift took place with complete retention of the regioselectivity of the migrated aryl group. We next explored this transformation with 1,2-diaryl alkynes containing two different aryl groups to form oxindoles with an α-carbonyl quaternary stereogenic center (Scheme 1D). Again, the reaction tolerated a wide range of functional groups such as F, Cl, I, CO2Et, Me, and OMe affording the oxindoles 28-33 in 50-65% yields. However, no reaction occurred with dialkyl alkynes or alkyl aryl alkynes, as we have observed the both starting materials. This lack of reactivity might be due to a change of an aryl group by an alkyl group that prevents the 1,2-migration step. Post-functionalizations. In addition, to further illustrate the synthetic value of this novel reaction, we investigated the reduction of amide groups (Scheme 2). In the presence of LiAlH4, the selective reduction of the acetyl group was observed, affording the indolin-2-one 17 in 97% yield. While compound 1 was fully reduced using borane-tetrahydrofuran to deliver 34 in 80% yield. These 3,3-disubstituted indolines -which are architectures embedded in many complex alkaloids or key building blocks-are now easy to prepare from nitrobenzene in only two steps, where formerly multi-step synthesis was necessary. [START_REF] Bariwal | Recent Advances in Spirocyclization of Indole Derivatives[END_REF]. Due to the strongly electron-withdrawing nature of the nitro group, the main limitation of our novel cascade reaction is incompatibility with electron-poor nitroarenes because of an unfavorable C-H bond activation. [START_REF] Caron | Palladium-Catalyzed Direct Arylation of Nitro-Substituted Aromatics with Aryl Halides[END_REF] To improve the diversity of structures accessible through this methodology, we performed several post-functionalizations of iodosubstituted oxindoles. Firstly a formyl group was introduced through Rh(III)-catalyzed hydroformylation, affording oxindole 35 in 75% yield. Pd-catalyzed cyanation with Zn(CN)2 as coupling reagent allowed to prepare of 5-nitrilesubstituted oxindole 36 in 70% yield. Finally, we also succeeded in preparing alkynyl-substituted oxindole 37 through Pd/Cu-catalyzed Sonogashira reaction with phenylacetylene in 85% yield. Experimental mechanistic investigations. In order to get insight into this unpredictable reaction, mechanistic experiments were conducted (Figure 2). [START_REF]Control experiments using other reduced intermediates of nitrobenzene (i.e., aniline, N-hydroxybenzenamine, nitrosobenzene, azoxybenzene[END_REF] Significant kinetic isotope effects (KIE 3.5 or 2.6) with both [Cp TMP *RhCl2]2 and [Cp*RhCl2] support that the C-H bond cleavage corresponds to the turnover determining step of the catalytic cycle (Figure 2A). Next, we decided to map the reaction by capturing potential rhodacycles and studying their reactivity (Figure 2B). Our attempts to isolate a cyclometalated Rh(III) intermediate from a stoichiometric reaction between nitrobenzene and [Cp TMP *RhCl2]2 in the presence or not of AgSbF6 and acetate sources failed. However, cyclometalated Rh(III) 38a has been prepared by transmetalation from 2-nitrophenylmercurial. [START_REF] Vicente | Synthesis, Reactivity, and X-Ray Crystal Structure of [Rh{C6H4N(O)O-2}(η-C5Me5)Cl][END_REF] The reaction between 38a and 1-diphenylacetylene with AgSbF6 in 1,2dichloroethane at RT afforded Rh(III) η 3 -benzyl complex 39a. This 18e -complex 39a was fully characterized by NMR spectroscopies, mainly, the exchange η 3 -benzyl unit was observed by NOESY experiments. [14] This intermediate 39a results

N R 4 O O R 2 R 3 N + O O - R 2 R 3 R 4 O O R 4 O [Cp TMP *RhCl 2 ] 2 (3 mol%) AgSbF 6 ( 12
from the alkyne insertion into Rh-C bond followed by OAT. We tried to get some crystal for X-ray analysis, but 39a spontaneously evolved into 40. We, therefore, repeated the experiments with [Cp*RhCl2]2. Successfully, the Rh(III) η 3benzyl complex 39b was obtained as single crystals and analyzed by X-ray experiments, demonstrating that OAT has proceeded to give the formal hydration of alkyne among with coordinated nitrosobenzene. When this reaction was conducted at 80 ºC, Rh(III) complex 40 was isolated by crystallization in good yield and was fully characterized, including by X-ray structure. This intermediate 40 is formed via the N-Rh bond insertion into the carbonyl group, followed by a migration of the phenyl ring at the meso-position of the nitrobenzene ring. Finally, the reaction from 38a or 40 in the presence of Ac2O at 120 ºC furnished the desired product 1. Combining with the detection of 2-chloroacetaldehyde by GC-analysis, these results suggest that the second N-O bond cleavage occurred through O-alkylation with 1,2-dichloroethane followed by a hydride-shift (Scheme S11 and Figure S24-26). [START_REF] Lee | 1,2]-Anionic Rearrangements of Substituted N-Hydroxy-2-azetidinones and Applications to the Synthesis of Bicyclic Beta-Lactams[END_REF] In addition, it should be mentioned that all isolated Rh(III)-complexes (38a, 39a, and 40) catalyze the reaction indicating they are intermediates of the reaction (Table S5). Theoretical mechanistic investigations. The experimental investigations outlined above have elucidated the overall features of the reaction mechanism; however, mechanistic details remained unclear, specifically the following: 1) what is the energy barrier for formal C-H activation, 2) how does OAT take place, and 3) how to regenerate the active catalyst. To address these questions, a computational investigation of the reaction was undertaken at the PBE0-D3/def2-TZVPP-SMD(1,2-dichloroethane)//B3LYP-D3/def2-SVP-SMD(1,2-dichloroethane) level of theory. [24] The reaction between nitrobenzene and diphenylacetylene in the presence of acetic anhydride to afford product 1 using Cp*Rh(III) catalysts was used as the model reaction in the calculations. The free energy changes of the most favorable pathway of Rh(III)/Cp*-catalyzed oxindole formation are shown in Figures 3A and4. DFT-optimized structures of selected transition states are shown in Acetic acid dissociation gives INT5, which has been confirmed by NMR, HRMS, and X-ray studies. Then, with the association of alkyne b, a more stable species INT6 is produced, from which alkyne insertion into the Rh-C bond takes place via TS7 to give seven-membered rhodacycle INT8. The alternative pathway involving alkyne insertion into the Rh-O bond is not feasible (Figure S27). Next, OAT affords the isolable η 3 -benzyl nitroso complex INT14, which was verified by NMR, HRMS, and X-ray studies. Our computations indicate that this process proceeds through a reductive elimination (via TS9) affording Rh(I) complex Cp TMP OH 2

N Rh + O Ph R Cp 39a R Cp = Cp TMP *, 84% 39b R Cp = Cp * , 89%
40 75% X-ray structure of 40 [a] X-ray structure of 39b [a] Ph Ph

+ + AgSbF 6 O 38a R Cp = Cp TMP * 38b R Cp = Cp *
X-ray structure of 38a [a] crystallization coordination. Subsequent nucleophilic attack of the nitroso O on Rh via TS15 leads to the formation of INT16. In analogy to the semipinacol rearrangement, [START_REF] Delayre | Natural Product Synthesis Enabled by Domino Processes Incorporating a 1,2-Rearrangement Step[END_REF] a 1,2-aryl shift via TS17 affords the isolable N-oxido Rh(III)complex INT18. The helical shape of Cp TMP * might favor the nucleophilic attack since 39a is converted into 40 at RT, while 39b with disc-shape Cp* is stable.

N O Ph Ph Ac Ac 2 O, DCE 80
Finally, based on INT18, ligand exchange with AcO -to regenerate the active catalytic species (INT1) for the next cycle, along with the formation of INT19. In the presence of 1,2-dichloroethane, we propose that a nucleophilic attack of INT19 to 1,2-dichloroethane via TS20 produces INT21. [START_REF]Multicomponent Reactions of Pyridines To Give Ring-Fused Pyridiniums: In Situ Activation Strategy Using 1,2-Dichloroethane as a Vinyl Equivalent[END_REF] Nucleophilic attack with the involvement of Rh is less favorable (Figure S28). After that, INT21 evolves to acylated indolinone 1 [START_REF]coordination of the product (1) with the catalyst, and details are included in the Supporting Information[END_REF] with the assistance of Ac2O in a non-catalyzed pathway. [START_REF] Lee | 1,2]-Anionic Rearrangements of Substituted N-Hydroxy-2-azetidinones and Applications to the Synthesis of Bicyclic Beta-Lactams[END_REF] as the catalyst, the barrier of C-H bond activation is lowered by 1.9 kcal/mol compared to Rh(III)/Cp* case (TS25 v.s. TS3), in agreement with the experimental results. After careful examination of the structures of TS3 and TS25, we believe that the leading factor responsible for the energy difference is a hydrogen-bonding interaction, which is present in TS25, but absent in TS3 (Figure 5B). To support our hypothesis, TS3-model and TS25-model were constructed by replacing methyl and aryl groups with hydrogens, respectively. After removing the hydrogen bonding interaction from TS25, TS3-model and TS25-model have almost the same energy, in agreement with our hypothesis (Figure 5C).

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

Experimental procedures, characterization data (1H and 13C NMR, HRMS, FTIR) for all new compounds, coordination geometries for DFT optimized compounds (PDF)

FIDs

Figure 1 .

 1 Figure 1. N-Heterocycles Synthesis by Rh(III)-Catalyzed C-H Bond Activation, Alkyne Insertion, O-Atom Transfer and

✓ 1 1➜ 3 ( 4 (

 134 Identification of the final product ✓ Catalyst design & substrates scopes ✓ Formation of stereogenic quaternary carbon ✓ Isolation of 3 rhodacycle intermediates ✓ DFT calculation ✓ Reaction pathway H CHALLENGES ✓ Few examples of NO 2 direct C-H bond activation ✓ No example of O-atom transfer from NO 2 ✓ Unpredictable reactivity of nitrosoarenes nitroso intermediate a) (A) General concept of Rh(III)-catalyzed C-H bond functionalization with O-atom transfer for heterocycle synthesis R B) C-H alkylation of quinoline N-oxides 1➜ C) from nitrones to indolines (E) from aniline N-oxides to indoles (F) from nitroarenes to indolines (this work)

  R RhCl2]2 (3 mol%), AgSbF6 (12 mol%), nitrobenzene (5 mmol), diphenylacetylene (0.5 mmol), and acetic anhydride (2 mmol) in 1,2-dichloroethane (2 mL) at 120 ºC. b Determined by GC-analysis using n-dodecane as internal standard, conversion is based on diphenylacetylene consumption; isolated yield is shown in parentheses. c alkyne-oligomers are observed as side products by GC-MS analysis. d nitrobenzene (2.5 mmol).

Scheme 1 .

 1 Scheme 1. Scope of the Rh(III)-Catalyzed C-H Bond Activation with Migrations of Nitroarenes a

Scheme 2 .

 2 Scheme 2. Access to Molecular Diversity through Post-Functionalizations.

5 Figure 2 .

 52 Figure 2. Determination of the Mechanism. a) Hydrogen Atoms and SbF6 are Omitted for Clarity. Thermal Ellipsoids are at the 50% Probability Level. [Cp TMP *Rh + ] = [Cp TMP *Rh + ](SbF6), [Cp*Rh + ] = [Cp*Rh + ](SbF6)

Figure 3B&C .

 3B&C Figure 3B&C. Starting from the catalytically active species INT1, which is generated from [Cp*RhCl2]2 and AgSbF6 in the presence of Ac2O by chloride abstraction by Ag + and ligand exchange, nitrobenzene (a) coordination to the metal center occurs to form INT2. Subsequent concerted metalation-deprotonation (CMD) arises via TS3 to cleave the C-H bond, generating INT4.

  (INT10) followed by oxidative addition of the N-O bond (via TS12) to generate the O-bond Rh(III)-enolate intermediate INT13. This intermediate adopts the C-enolate form (INT14) to give a more stable η 3 -benzyl (B) Reaction mapping through Iisolation and reactivities of intermediates 1 25% (from 38a) & 68% (from 40)

Figure 3 .Figure 4 .Figure 4 ,Scheme 3 .

 3443 Figure 3. DFT-Computed Free Energy Changes (A) and DFT-Optimized Structures of Selected Transition States (B & C) of Rh(III)/Cp*-Catalyzed Oxindole Formation from Nitrobenzene and Diphenylacetylene (part I).

Figure 5 .

 5 Figure 5. Comparison of C-H Bond Activation Step with Different Catalysts.

  for NMR data (ZIP) CCDC 2108500 ([Cp TFT *RhCl2]2), CCDC 2108501 ([Cp TMP *RhCl2]2) CCDC 2108502 (1), CCDC 2108503 (33) CCDC 2108504 (38a), CCDC 2108506 (39b) and CCDC 2108507 (40) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif.

ACKNOWLEDGMENT

We thank Clement Orione (CRMPO) for NMR analysis and Jérôme Ollivier and Philippe Jéhan (CRMPO) for the HRMS analysis.

We are grateful to the National Science Foundation (CHE-1764328 to KNH) for financial support of this research, and Financial supports from the Rennes 1 University (Ph.D. grant to M. Peng), China Scholarship Council (Ph.D. grant to C.-S. Wang), the Centre National de la Recherche Scientifique (CNRS) are also gratefully acknowledged. Calculations were performed on the IDRE Hoffman2 cluster at the University of California, Los Angeles, and the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the National Science Foundation (OCI-1053575). We thank Umicore AG & Co. KG for their generous donation of rhodium(lll) chloride hydrate.