A relic of the past: current genetic patterns of the palaeoendemic tree Nothofagus macrocarpa were shaped by climatic oscillations in central Chile.

BACKGROUND AND AIMS: The Mediterranean-type forest of central Chile is considered a 'biodiversity hotspot' and a relic of a wider ancient distribution produced by past climatic oscillations. Nothofagus macrocarpa, commonly known as 'roble de Santiago', is a threatened palaeoendemic of this forest, poorly represented in the protected area system. This tree has been repeatedly misidentified as the sister species N. obliqua, which has affected its recognition and protection. Only a few populations of N. macrocarpa remain within a matrix of intensive land use that has been affected by recent forest fires. We tested the hypothesis that current populations of N. macrocarpa are a relic state of a previously widespread range, with the aim of contributing to its identification, its biogeographical history and the design of conservation measures using genetic information. METHODS: We analysed remnant N. macrocarpa forests using nuclear (nDNA) and chloroplast DNA (cpDNA) sequences, conducted phylogenetic and phylogeographical analyses to reconstruct its biogeographical history, and assessed microsatellites [simple sequence repeats (SSRs)] to determine contemporary patters of diversity within and among all remnant populations. We also examined the degree of past, current and potential future isolation of N. macrocarpa populations using ecological niche models (ENMs). KEY RESULTS: The species N. macrocarpa was confirmed by nDNA sequences, as previously suggested by chromosomal analysis. Small isolated populations of N. macrocarpa exhibited moderate to high genetic diversity according to SSRs. cpDNA analysis revealed a marked past latitudinal geographical structure, whereas analysis of SSRs did not find such current structure. ENM analyses revealed local expansion-contraction of the N. macrocarpa range during warmer periods, particularly in the northern and central ranges where basal-most cpDNA haplotypes were detected, and recent expansion to the south of the distribution. CONCLUSIONS: Genetic patterns confirm that N. macrocarpa is a distinct species and suggest a marked latitudinal relic structure in at least two evolutionarily significant units, despite contemporary among-population gene flow. This information must be considered when choosing individuals (seeds and/or propagules) for restoration purposes, to avoid the admixture of divergent genetic stocks.

Population Genetic Structure of the Giant Cactus Echinopsis terscheckii in Northwestern Argentina Is Shaped by Patterns of Vegetation Cover.

Species inhabiting drylands commonly depend on the surrounding vegetation for recruitment under stress, while competition may affect populations in moister environments. Our objective was to analyze how different climates and vegetation affect the fine-scale spatial genetic structure (SGS) of the columnar cactus Echinopsis terscheckii. At 4 sites, we estimated vegetation cover by digitized patches and the normalized difference vegetation index (NDVI). We mapped 30 individuals per population and collected tissue for isozyme electrophoresis using 15 putative loci. Spatial autocorrelation between all possible genotype pairs and the number of genetically homogeneous groups and families were calculated for each population. Greater cover (66%) and average NDVI values were detected in the most humid habitat that consisted of fewer, larger, and more dispersed vegetation patches. All populations were genetically diverse and showed significant SGS. Positive correlations were found between the distance at which maximum autocorrelation and kinship values were reached and vegetation area and patch size. Also higher NDVI values were associated with lower number of patches. Populations exposed to higher precipitation and vegetation cover consisted of sparse individuals that clustered at larger distances whereas vegetation patches in arid climates produced groups of closely related genotypes at small distances. These results support the stress-gradient genetic hypothesis. Under water stress, facilitation promotes establishment underneath patchy vegetation resulting in fine-scale family structure. In moister xerophilous forests, competition for resources, that is, light, results in sparse individuals and thus coarse-scale neighborhoods. This information can guide conservation and/or restoration efforts, such as the spatial scale to be considered in germplasm collection.

Living on the edge: adaptive and plastic responses of the tree Nothofagus pumilio to a long-term transplant experiment predict rear-edge upward expansion.

Current climate change affects the competitive ability and reproductive success of many species, leading to local extinctions, adjustment to novel local conditions by phenotypic plasticity or rapid adaptation, or tracking their optima through range shifts. However, many species have limited ability to expand to suitable areas. Altitudinal gradients, with abrupt changes in abiotic conditions over short distances, represent "natural experiments" for the evaluation of ecological and evolutionary responses under scenarios of climate change. Nothofagus pumilio is the tree species which dominates as pure stands the montane forests of Patagonia. We evaluated the adaptive value of variation in quantitative traits of N. pumilio under contrasting conditions of the altitudinal gradient with a long-term reciprocal transplant experimental design. While high-elevation plants show little response in plant, leaf, and phenological traits to the experimental trials, low-elevation ones show greater plasticity in their responses to changing environments, particularly at high elevation. Our results suggest a relatively reduced potential for evolutionary adaptation of high-elevation genotypes, and a greater evolutionary potential of low-elevation ones. Under global warming scenarios of forest upslope migration, high-elevation variants may be outperformed by low-elevation ones during this process, leading to the local extinction and/or replacement of these genotypes. These results challenge previous models and predictions expected under global warming for altitudinal gradients, on which the leading edge is considered to be the upper treeline forests.

Identifying Genetic Hotspots by Mapping Molecular Diversity of Widespread Trees: When Commonness Matters.

Conservation planning requires setting priorities at the same spatial scale at which decision-making processes are undertaken considering all levels of biodiversity, but current methods for identifying biodiversity hotspots ignore its genetic component. We developed a fine-scale approach based on the definition of genetic hotspots, which have high genetic diversity and unique variants that represent their evolutionary potential and evolutionary novelties. Our hypothesis is that wide-ranging taxa with similar ecological tolerances, yet of phylogenetically independent lineages, have been and currently are shaped by ecological and evolutionary forces that result in geographically concordant genetic patterns. We mapped previously published genetic diversity and unique variants of biparentally inherited markers and chloroplast sequences for 9 species from 188 and 275 populations, respectively, of the 4 woody dominant families of the austral temperate forest, an area considered a biodiversity hotspot. Spatial distribution patterns of genetic polymorphisms differed among taxa according to their ecological tolerances. Eight genetic hotspots were detected and we recommend conservation actions for some in the southern Coastal Range in Chile. Existing spatially explicit genetic data from multiple populations and species can help to identify biodiversity hotspots and guide conservation actions to establish science-based protected areas that will preserve the evolutionary potential of key habitats and species.

Fine-scale genetic structure of Nothofagus pumilio (lenga) at contrasting elevations of the altitudinal gradient.

Montane forests provide the natural framework to test for various ecological settings at distinct elevations as they may affect population demography, which in turn will affect the spatial genetic structure (SGS). We analyzed the fine-scale SGS of Nothofagus pumilio, which dominates mountain areas of Patagonia, in three pairs of sites at contrasting elevations (low- vs. high-elevation). Within a total area of 1 ha fresh leaf tissue from 90 individuals was collected at each of the six studied stands following a spatially explicit sampling design. Population genetic diversity parameters were analyzed for all sampled individuals using five polymorphic isozyme loci, and a subset of 50 individuals per stand were also screened for five microsatellite loci. The SGS was assessed on 50 individuals/stand, using the combined datasets of isozymes and microsatellites. Most low-elevation stands consisted of older individuals with complex age structures and genetically diverse plots. In contrast, high-elevation stands and one post-fire low-elevation population yielded even-aged structures with evidence of growth suppression, and were genetically homogeneous. All stands yielded significant SGS. Similarly to mature stands of the non-sprouter congener Nothofagus dombeyi, multi-age low-altitude N. pumilio yielded significant SGS weakened by competing species of the understory and the formation of seedling banks. Alike the sprouter Nothofagus antarctica, high-altitude stands produced significant SGS as a consequence of occasional seedling establishment reinforced by vegetative spread.

Phylogeographically concordant chloroplast DNA divergence in sympatric Nothofagus s.s. How deep can it be?

• Here, we performed phylogenetic analyses and estimated the divergence times on mostly sympatric populations of five species within subgenus Nothofagus. We aimed to investigate whether phylogenetic relationships by nuclear internal transcribed spacer (ITS) and phylogeographic patterns by chloroplast DNA (cpDNA) mirror an ancient evolutionary history that was not erased by glacial eras. Extant species are restricted to Patagonia and share a pollen type that was formerly widespread in all southern land masses. Weak reproductive barriers exist among them. • Fifteen cpDNA haplotypes resulted from the analysis of three noncoding regions on 330 individuals with a total alignment of 1794 bp. Nuclear ITS data consisted of 822 bp. We found a deep cpDNA divergence dated 32 Ma at mid-latitudes of Patagonia that predates the phylogenetic divergence of extant taxa. Other more recent breaks by cpDNA occurred towards the north. • Complex paleogeographic features explain the genetic discontinuities. Long-lasting paleobasins and marine ingressions have impeded transoceanic dispersal during range expansion towards lower latitudes under cooler trends since the Oligocene. • Cycles of hybridization-introgression among extant and extinct taxa have resulted in widespread chloroplast capture events. Our data suggest that Nothofagus biogeography will be resolved only if thorough phylogeographic analyses and molecular dating methods are applied using distinct genetic markers.

Out in the cold: genetic variation of Nothofagus pumilio (Nothofagaceae) provides evidence for latitudinally distinct evolutionary histories in austral South America.

Nothofagus pumilio is the dominant and almost ubiquitous tree species in mountainous environments of temperate South America. We used two types of molecular markers (cpDNA and isozymes) to evaluate the effects of the Paleogene paleogeography of Patagonia and more recent climatic oscillations of the Neogene on such cold-tolerant species' genetic makeup. Phylogeographic analysis on sequences of three cpDNA non-coding regions at 85 populations yielded two latitudinally disjunct monophyletic clades north and south of c. 42 degrees S containing 11 and three haplotypes, respectively. This indicates a long-lasting vicariant event due to the presence of an extended open paleobasin at mid latitudes of Patagonia. Also distribution patterns of cpDNA haplotypes suggest regional spread following stepping-stone models using pre-Cenozoic mountains as corridors. Comparable genetic diversity measured along 41 sampled populations using seven polymorphic isozyme loci provides evidence of local persistence and spread from multiple ice-free locations. In addition, significantly higher heterozygosity and allelic richness at high latitudes, i.e. in areas of larger glacial extent, suggest survival in large and isolated refugia. While, higher cpDNA diversity in lower latitudes reflects the complex orogeny that historically isolated northern populations, lower isozyme diversity and reduced F(ST) values provide evidence of local glacial survival in numerous small locales. Therefore, current genetic structure of N. pumilio is the result of regional processes which took place during the Tertiary that were enhanced by contemporary local effects of drift and isolation in response to Quaternary climatic cycles.

Genetic structure and early effects of inbreeding in fragmented temperate forests of a self-incompatible tree, Embothrium coccineum.

Deforestation of temperate forests has created landscapes of forest remnants in matrices of intense human use. We studied the genetic effects of fragmentation in southern Chile on Embothrium coccineum J.R. et G. Forster, an early colonizing, bird-pollinated tree. We tested the hypothesis that, because of its self-incompatibility and life-history strategy, E. coccineum is less strongly affected by fragmentation. We studied the effects of reduced population size and increased isolation on population genetic structure and early performance of progeny. Samples were collected from spatially isolated trees and six fragments of differing sizes (small, 1 ha; medium, 20 ha; large, >150 ha). Based on isozyme polymorphisms we estimated parameters of genetic diversity, divergence, and inbreeding for adults and greenhouse-grown progeny. We also measured germination, seedling growth, and outcrossing rates on progeny arrays. Genetic variation of adults did not correlate significantly with population size, as expected, given that fragmentation occurred relatively recently. Weak effects of fragmentation were measured on progeny. Only adults yielded significant inbreeding. Similar total genetic diversity was found in adults and progeny. Low but significant genetic differentiation existed among adult and progeny populations. Seedling growth correlated positively with the effective number of alleles, showing deleterious effects of inbreeding on progeny. Seeds from small fragments had the highest outcrossing rates and germination success, indicating that higher pollinator activity in such fragments reduced selfing, thereby buffering genetic erosion and maintaining adaptive variation. The effects of forest fragmentation were detectable in E. coccineum, but these effects will probably not be detrimental to the viability of remnant populations because small, fragmented populations demonstrated higher levels of gene flow and lower inbreeding than larger stands. Pioneer species that are insensitive to forest clearing may be crucial in recovery plans to facilitate the establishment of species intolerant to such disturbance.