Introduction
Due to their isolation and 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 (Villagrán 1991). As a result, the climate and topography characterizing the narrow strip (2000 km long and 120 km wide) of Chilean temperate forests generate high heterogeneity of forest and soil types, climatic conditions, and disturbance regimes (Loguercio et al., 2018). 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 (Johnson et al., 2014; Prunier et al., 2017). In the same way, 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, Zeppel et al., 2014; Grossiord et al., 2004). The effect of contrasting climatic and edaphic features on phenotypic and genetic diversity has been observed in various temperate plants (Saldaña et al., 2007; Torres-Díaz et al., 2007). For instance, the existence of distinct ecotypes has been linked to contrasting access to water and snow winter coverage in Embothrium coccineum J.R. Forts. & G. Forst (Dimitri, 1972), a widespread tree that inhabits the temperate forest of South America (Zegers 1994). 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, which exhibits different phenotypes throughout its distribution (Alberdi y Donoso, 2004; Chalcoff et al., 2008). The distribution of these ecotypes is concordant with the pattern of genetic structure observed using isoenzymatic genetic markers (Souto and Premoli, 2007), suggesting thatE. coccineum populations across the species range may be locally adapted to distinct environmental conditions (Souto and Premoli, 2007).
Besides the possible effect of current selective regimes, climate shifts during the Pleistocene have also strongly altered the spatial patterns of genetic variation of many southern South American taxa (Vidal-Russell et al., 2011; Turchetto-Zolet et al., 2013). During the Last Glacial Maximum (LGM, 25.000 years Before Present, BP), due to the presence of ice caps, a large expanse of temperate forest located between the 42 °S and the 56 °S disappeared (Rabassa et al., 2005). Glacial maxima triggered a rapid range shift, with most temperate South American species surviving only in glacial refugia located north of the 42°S (Allnutt et al., 1999; Premoli et al., 2000, Sérsic et al., 2011). Some cold tolerant species, 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 (Allnutt et al., 1999; Premoli et al., 2000, Sérsic et al., 2011). These areas could represent sources of regional diversity recovery after deglaciation (Comps et al., 2001). The presence of 11.000 years BP cold tolerant plants pollen fossil in areas covered by ice during the LGM (Fesq-Martin et al., 2004, Steubing et al., 1983) and results of genetic studies based on sequences of chloroplast genes or genotyping of nuclear isoenzymatic markers (Vidal-Russel et al., 2011; Souto and Premoli, 2007) suggest a complex glacial-interglacial history in this species.
Recent developments in high throughput sequencing technologies and population genomics have allowed to distinguish the influence of past environmental changes and selection to the current environmental conditions on the spatial patterns of genetic variation in both South American 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 and Bradburd, 2014) should be expected. Indeed, when selection against immigrant limits gene flow among distinct environments, genetic differentiation increases with environmental differences (Wang and Summer, 2010; Wang and Bradburd, 2014). To test for the existence of IBE, the level of correlation between genetic differentiation and environmental distances should be contrasted against the one observed between genetic differentiation and geographic distances (isolation by distance; IBD; Slatkin, 1993). IBD could be considered as 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 behavior of pollinators (i.e., more than 20 species including birds and insects are reported as pollinators of E. coccineum ; Chalcoff et al., 2008) and anemochorous seed dispersal (Rovere and Premoli, 2005).
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 inE. coccineum will 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.