Phylogenetic signatures of ecological divergence and leapfrog adaptive radiation in Espeletia.

PREMISE: Events of accelerated species diversification represent one of Earth's most celebrated evolutionary outcomes. Northern Andean high-elevation ecosystems, or páramos, host some plant lineages that have experienced the fastest diversification rates, likely triggered by ecological opportunities created by mountain uplifts, local climate shifts, and key trait innovations. However, the mechanisms behind rapid speciation into the new adaptive zone provided by these opportunities have long remained unclear. METHODS: We address this issue by studying the Venezuelan clade of Espeletia, a species-rich group of páramo-endemics showing a dazzling ecological and morphological diversity. We performed several comparative analyses to study both lineage and trait diversification, using an updated molecular phylogeny of this plant group. RESULTS: We showed that sets of either vegetative or reproductive traits have conjointly diversified in Espeletia along different vegetation belts, leading to adaptive syndromes. Diversification in vegetative traits occurred earlier than in reproductive ones. The rate of species and morphological diversification showed a tendency to slow down over time, probably due to diversity dependence. We also found that closely related species exhibit significantly more overlap in their geographic distributions than distantly related taxa, suggesting that most events of ecological divergence occurred at close geographic proximity within páramos. CONCLUSIONS: These results provide compelling support for a scenario of small-scale ecological divergence along multiple ecological niche dimensions, possibly driven by competitive interactions between species, and acting sequentially over time in a leapfrog pattern.

Phylogenomic Analysis of the Explosive Adaptive Radiation of the Espeletia Complex (Asteraceae) in the Tropical Andes.

The subtribe Espeletiinae (Asteraceae), endemic to the high-elevations in the Northern Andes, exhibits an exceptional diversity of species, growth-forms, and reproductive strategies. This complex of 140 species includes large trees, dichotomous trees, shrubs and the extraordinary giant caulescent rosettes, considered as a classic example of adaptation in tropical high-elevation ecosystems. The subtribe has also long been recognized as a prominent case of adaptive radiation, but the understanding of its evolution has been hampered by a lack of phylogenetic resolution. Herein, we produce the first fully resolved phylogeny of all morphological groups of Espeletiinae, using whole plastomes and about a million nuclear nucleotides obtained with an original de novo assembly procedure without reference genome, and analyzed with traditional and coalescent-based approaches that consider the possible impact of incomplete lineage sorting and hybridization on phylogenetic inference. We show that the diversification of Espeletiinae started from a rosette ancestor about 2.3 Ma, after the final uplift of the Northern Andes. This was followed by two independent radiations in the Colombian and Venezuelan Andes, with a few trans-cordilleran dispersal events among low-elevation tree lineages but none among high-elevation rosettes. We demonstrate complex scenarios of morphological change in Espeletiinae, usually implying the convergent evolution of growth-forms with frequent loss/gains of various traits. For instance, caulescent rosettes evolved independently in both countries, likely as convergent adaptations to life in tropical high-elevation habitats. Tree growth-forms evolved independently three times from the repeated colonization of lower elevations by high-elevation rosette ancestors. The rate of morphological diversification increased during the early phase of the radiation, after which it decreased steadily towards the present. On the other hand, the rate of species diversification in the best-sampled Venezuelan radiation was on average very high (3.1 spp/My), with significant rate variation among growth-forms (much higher in polycarpic caulescent rosettes). Our results point out a scenario where both adaptive morphological evolution and geographical isolation due to Pleistocene climatic oscillations triggered an exceptionally rapid radiation for a continental plant group.

Keep your feet warm? A cryptic refugium of trees linked to a geothermal spring in an ocean of glaciers.

Up to now, the most widely accepted idea of the periglacial environment is that of treeless ecosystems such as the arctic or the alpine tundra, also called the tabula rasa paradigm. However, several palaeoecological studies have recently challenged this idea, that is, treeless environments in periglacial areas where all organisms would have been exterminated near the glacier formed during the Last Glacial Maximum, notably in the Scandinavian mountains. In the Alps, the issue of glacial refugia of trees remains unanswered. Advances in glacier reconstructions show that ice domes did not cover all upper massifs, but glaciers filled valleys. Here, we used fossils of plant and malacofauna from a travertine formation located in a high mountain region to demonstrate that trees (Pinus, Betula) grew with grasses during the Lateglacial-Holocene transition, while the glacier fronts were 200-300 m lower. The geothermal travertine started to accumulate more than 14,500 years ago, but became progressively more meteogene about 11,500 years ago due to a change in groundwater circulation. With trees, land snails (gastropods) associated to woody or open habitats and aquatic mollusc were also present at the onset of the current interglacial, namely the Holocene. The geothermal spring, due to warm water and soil, probably favoured woody glacial ecosystems. This new finding of early tree growth, combined with other scattered proofs of the tree presence before 11,000 years ago in the western Alps, changes our view of the tree distribution in periglacial environments, supporting the notion of tree refugia on nunataks in an ocean of glaciers. Therefore, the tabula rasa paradigm must be revisited because it has important consequences on the global changes, including postglacial plant migrations and biogeochemical cycles.

Plant functional traits reveal the relative contribution of habitat and food preferences to the diet of grasshoppers.

Food preferences and food availability are two major determinants of the diet of generalist herbivores and of their spatial distribution. How do these factors interact and eventually lead to diet differentiation in co-occurring herbivores? We quantified the diet of four grasshopper species co-occurring in subalpine grasslands using DNA barcoding of the plants contained in the faeces of individuals sampled in the field. The food preferences of each grasshopper species were assessed by a choice (cafeteria) experiment from among 24 plant species common in five grassland plots, in which the four grasshoppers were collected, while the habitat was described by the relative abundance of plant species in the grassland plots. Plant species were characterised by their leaf economics spectrum (LES), quantifying their nutrient vs. structural tissue content. The grasshoppers' diet, described by the mean LES of the plants eaten, could be explained by their plant preferences but not by the available plants in their habitat. The diet differed significantly across four grasshopper species pairs out of six, which validates food preferences assessed in standardised conditions as indicators for diet partitioning in nature. In contrast, variation of the functional diversity (FD) for LES in the diet was mostly correlated to the FD of the available plants in the habitat, suggesting that diet mixing depends on the environment and is not an intrinsic property of the grasshopper species. This study sheds light on the mechanisms determining the feeding niche of herbivores, showing that food preferences influence niche position whereas habitat diversity affects niche breadth.

Tracking genes of ecological relevance using a genome scan in two independent regional population samples of Arabis alpina.

Understanding the genetic basis of adaptation in response to environmental variation is fundamental as adaptation plays a key role in the extension of ecological niches to marginal habitats and in ecological speciation. Based on the assumption that some genomic markers are correlated to environmental variables, we aimed to detect loci of ecological relevance in the alpine plant Arabis alpina L. sampled in two regions, the French (99 locations) and the Swiss (109 locations) Alps. We used an unusually large genome scan [825 amplified fragment length polymorphism loci (AFLPs)] and four environmental variables related to temperature, precipitation and topography. We detected linkage disequilibrium among only 3.5% of the considered AFLP loci. A population structure analysis identified no admixture in the study regions, and the French and Swiss Alps were differentiated and therefore could be considered as two independent regions. We applied generalized estimating equations (GEE) to detect ecologically relevant loci separately in the French and Swiss Alps. We identified 78 loci of ecological relevance (9%), which were mainly related to mean annual minimum temperature. Only four of these loci were common across the French and Swiss Alps. Finally, we discuss that the genomic characterization of these ecologically relevant loci, as identified in this study, opens up new perspectives for studying functional ecology in A. alpina, its relatives and other alpine plant species.

Phosphate (Pi) starvation effect on the cytosolic Pi concentration and Pi exchanges across the tonoplast in plant cells: an in vivo 31P-nuclear magnetic resonance study using methylphosphonate as a Pi analog.

In vivo (31)P-NMR analyses showed that the phosphate (Pi) concentration in the cytosol of sycamore (Acer pseudoplatanus) and Arabidopsis (Arabidopsis thaliana) cells was much lower than the cytoplasmic Pi concentrations usually considered (60-80 mum instead of >1 mm) and that it dropped very rapidly following the onset of Pi starvation. The Pi efflux from the vacuole was insufficient to compensate for the absence of external Pi supply, suggesting that the drop of cytosolic Pi might be the first endogenous signal triggering the Pi starvation rescue metabolism. Successive short sequences of Pi supply and deprivation showed that added Pi transiently accumulated in the cytosol, then in the stroma and matrix of organelles bounded by two membranes (plastids and mitochondria, respectively), and subsequently in the vacuole. The Pi analog methylphosphonate (MeP) was used to analyze Pi exchanges across the tonoplast. MeP incorporated into cells via the Pi carrier of the plasma membrane; it accumulated massively in the cytosol and prevented Pi efflux from the vacuole. This blocking of vacuolar Pi efflux was confirmed by in vitro assays with purified vacuoles. Subsequent incorporation of Pi into the cells triggered a massive transfer of MeP from the cytosol to the vacuole. Mechanisms for Pi exchanges across the tonoplast are discussed in the light of the low cytosolic Pi level, the cell response to Pi starvation, and the Pi/MeP interactive effects.

Effects of copper on the photosynthesis of intact chloroplasts: interaction with manganese.

Highly purified, intact chloroplasts were prepared from pea (Pisum sativum L.) and spinach (Spinacia oleracea L.) following an identical procedure, and were used to investigate the cupric cation inhibition on the photosynthetic activity. In both species, copper inhibition showed a similar inhibitor concentration that decreases the enzyme activity by 50% (IC(50) approximately 1.8 microM) and did not depend on the internal or external phosphate (Pi) concentration, indicating that copper did not interact with the Pi translocator. Fluorescence analysis suggested that the presence of copper did not facilitate photoinhibition, because there were no changes in maximal fluorescence (F(m)) nor in basal fluorescence (F(o)) of copper-treated samples. The electron transport through the photosystem II (PSII) was also not affected (operating efficiency of PSII-F'v/F'm similar in all conditions). Yet, under Cu(2+) stress, the proportion of open PSII reaction centers was dramatically decreased, and the first quinone acceptor (Q(A)) reoxidation was fully inhibited, as demonstrated by the constant photochemical quenching (q(P)) along experiment time. The quantum yield of PSII electron transport (Phi(PSII)) was also clearly affected by copper, and therefore reduced the photochemistry efficiency. Manganese, when added simultaneously with copper, delayed the inhibition, as measured by oxygen evolution and chlorophyll fluorescence, but neither reversed the copper effect when added to copper-inhibited plastids, nor prevented the inhibition of the Hill activity of isolated copper-treated thylakoids. Our results suggest that manganese competed with copper to penetrate the chloroplast envelope. This competition seems to be specific because other divalent cations e.g. magnesium and calcium, did not interfere with the copper action in intact chloroplasts. All results do suggest that, under these conditions, the stroma proteins, such as the Calvin-Benson cycle enzymes or others are the most probable first target for the Cu(2+) action, resulting in the total inhibition of chloroplast photosynthesis and in the consequent unbalanced rate of production and consumption of the reducing power.

13C and 15N allocations of two alpine species from early and late snowmelt locations reflect their different growth strategies.

Intense efforts are currently devoted to disentangling the relationships between plant carbon (C) allocation patterns and soil nitrogen (N) availability because of their consequences for growth and more generally for C sequestration. In cold ecosystems, only a few studies have addressed whole-plant C and/or N allocation along natural elevational or topographical gradients. (12)C/(13)C and (14)N/(15)N isotope techniques have been used to elucidate C and N partitioning in two alpine graminoids characterized by contrasted nutrient economies: a slow-growing species, Kobresia myosuroides (KM), and a fast-growing species, Carex foetida (CF), located in early and late snowmelt habitats, respectively, within the alpine tundra (French Alps). CF allocated higher labelling-related (13)C content belowground and produced more root biomass. Furthermore, assimilates transferred to the roots were preferentially used for growth rather than respiration and tended to favour N reduction in this compartment. Accordingly, this species had higher (15)N uptake efficiency than KM and a higher translocation of reduced (15)N to aboveground organs. These results suggest that at the whole-plant level, there is a compromise between N acquisition/reduction and C allocation patterns for optimized growth.

Cross tolerance to heavy-metal and cold-induced photoinhibiton in leaves of Pisum sativum acclimated to low temperature.

Under high light intensity, low temperatures as well as heavy metals induce photoinhibition of PSII and oxidative stress in leaves. Since cold acclimation of leaves ameliorates their capacity of antioxidative defence, cross tolerance between cold-induced and heavy metal-induced photoinhibition was investigated in pea leaves grown at either 22 °C or 6 °C. The experimental conditions were chosen to induce a uniform level of short-term photoinhibition at low temperature or in the presence of CuSO4 or CdCl2 in leaves grown at 22 °C. Under all conditions photoinhibition of PSII was lower in cold-acclimated (6°C-grown) than in non-acclimated (22°C-grown) pea leaves. In darkness PSII was not affected by all treatments. Other parameters like catalase activity, chlorophyll content and metabolite contents were most sensitive to CuSO4, but less affected by CdCl2 and low temperature treatments. Strong oxidation of ascorbate and concomitant loss of catalase activity showed the enhanced oxidative stress in CuSO4-treated leaves. Generally, all measured parameters were less affected in cold-acclimated leaves than in non-acclimated leaves under all experimental conditions. Cold-acclimated pea leaves contained higher levels of ascorbate and particularly of glutathione and a higher capacity to keep the primary electron acceptor of PSII more oxidised. Incubation with heavy metals caused a nearly complete loss of reduced glutathione. It is suggested that reduced glutathione served as a source for phytochelatin synthesis. The extraordinarily high glutathione content in cold-acclimated pea leaves might therefore increase their ability to chelate heavy metals and thus to protect leaves from heavy-metal induced damage.

Cold- and light-induced changes of metabolite and antioxidant levels in two high mountain plant species Soldanella alpina and Ranunculus glacialis and a lowland species Pisum sativum.

Leaves of the two cold-acclimated alpine plant species Ranunculus glacialis and Soldanella alpina and, for comparison, of the non-acclimated lowland species Pisum sativum were illuminated with high light intensity at low temperature. The light- and cold-induced changes of antioxidants and of the major carbon and phosphate metabolites were analysed to examine which metabolic pathways might be limiting in non-acclimated pea leaves and whether alpine plants are able to circumvent such limitation. During illumination at low temperature pea leaves accumulated high quantities of sucrose, glucose-6-phosphate, fructose-6-phosphate, mannose-6-phosphate and phosphoglycerate (PGA) whereas ATP/ADP-ratios decreased. Although the PGA content also increased in leaves of R. glacialis the other metabolites did not accumulate and ATP/ADP-ratios remained fairly constant in either alpine species. These data indicate a inorganic phosphate (Pi)-limitation in the chloroplasts of pea leaves but not in the alpine species. However, the total phosphate pool and the percentage of free Pi were highest in pea and did not change during illumination in cold. In contrast, free Pi contents declined markedly in R. glacialis leaves, suggesting that Pi is available for metabolism in this species. In S. alpina leaves contents of ascorbate and glutathione doubled in light and cold, while the contents of sugars did not increase. Obviously, S. alpina leaves can use assimilated carbon for ascorbate synthesis, rather than for the synthesis of sugars. A high capacity for ascorbate synthesis might prevent the accumulation of mannose-6-phosphate and Pi-limitation.

Reconstructing the origins of high-alpine niches and cushion life form in the genus Androsace S.L. (Primulaceae).

Relatively, few species have been able to colonize extremely cold alpine environments. We investigate the role played by the cushion life form in the evolution of climatic niches in the plant genus Androsace s.l., which spreads across the mountain ranges of the Northern Hemisphere. Using robust methods that account for phylogenetic uncertainty, intraspecific variability of climatic requirements and different life-history evolution scenarios, we show that climatic niches of Androsace s.l. exhibit low phylogenetic signal and that they evolved relatively recently and punctually. Models of niche evolution fitted onto phylogenies show that the cushion life form has been a key innovation providing the opportunity to occupy extremely cold environments, thus contributing to rapid climatic niche diversification in the genus Androsace s.l. We then propose a plausible scenario for the adaptation of plants to alpine habitats.

Contrasting diversity patterns of crenarchaeal, bacterial and fungal soil communities in an alpine landscape.

BACKGROUND: The advent of molecular techniques in microbial ecology has aroused interest in gaining an understanding about the spatial distribution of regional pools of soil microbes and the main drivers responsible of these spatial patterns. Here, we assessed the distribution of crenarcheal, bacterial and fungal communities in an alpine landscape displaying high turnover in plant species over short distances. Our aim is to determine the relative contribution of plant species composition, environmental conditions, and geographic isolation on microbial community distribution. METHODOLOGY/PRINCIPAL FINDINGS: Eleven types of habitats that best represent the landscape heterogeneity were investigated. Crenarchaeal, bacterial and fungal communities were described by means of Single Strand Conformation Polymorphism. Relationships between microbial beta diversity patterns were examined by using Bray-Curtis dissimilarities and Principal Coordinate Analyses. Distance-based redundancy analyses and variation partitioning were used to estimate the relative contributions of different drivers on microbial beta diversity. Microbial communities tended to be habitat-specific and did not display significant spatial autocorrelation. Microbial beta diversity correlated with soil pH. Fungal beta-diversity was mainly related to soil organic matter. Though the effect of plant species composition was significant for all microbial groups, it was much stronger for Fungi. In contrast, geographic distances did not have any effect on microbial beta diversity. CONCLUSIONS/SIGNIFICANCE: Microbial communities exhibit non-random spatial patterns of diversity in alpine landscapes. Crenarcheal, bacterial and fungal community turnover is high and associated with plant species composition through different set of soil variables, but is not caused by geographical isolation.

Metabolic processes sustaining the reviviscence of lichen Xanthoria elegans (Link) in high mountain environments.

To survive in high mountain environments lichens must adapt themselves to alternating periods of desiccation and hydration. Respiration and photosynthesis of the foliaceous lichen, Xanthoria elegans, in the dehydrated state were below the threshold of CO2-detection by infrared gas analysis. Following hydration, respiration totally recovered within seconds and photosynthesis within minutes. In order to identify metabolic processes that may contribute to the quick and efficient reactivation of lichen physiological processes, we analysed the metabolite profile of lichen thalli step by step during hydration/dehydration cycles, using 31P- and 13C-NMR. It appeared that the recovery of respiration was prepared during dehydration by the accumulation of a reserve of gluconate 6-P (glcn-6-P) and by the preservation of nucleotide pools, whereas glycolytic and photosynthetic intermediates like glucose 6-P and ribulose 1,5-diphosphate were absent. The large pools of polyols present in both X. elegans photo- and mycobiont are likely to contribute to the protection of cell constituents like nucleotides, proteins, and membrane lipids, and to preserve the integrity of intracellular structures during desiccation. Our data indicate that glcn-6-P accumulated due to activation of the oxidative pentose phosphate pathway, in response to a need for reducing power (NADPH) during the dehydration-triggered down-regulation of cell metabolism. On the contrary, glcn-6-P was metabolised immediately after hydration, supplying respiration with substrates during the replenishment of pools of glycolytic and photosynthetic intermediates. Finally, the high net photosynthetic activity of wet X. elegans thalli at low temperature may help this alpine lichen to take advantage of brief hydration opportunities such as ice melting, thus favouring its growth in harsh high mountain climates.