The evolutionary genomics of adaptation to stress in wild rhizobium bacteria.

Microbiota comprise the bulk of life's diversity, yet we know little about how populations of microbes accumulate adaptive diversity across natural landscapes. Adaptation to stressful soil conditions in plants provides seminal examples of adaptation in response to natural selection via allelic substitution. For microbes symbiotic with plants however, horizontal gene transfer allows for adaptation via gene gain and loss, which could generate fundamentally different evolutionary dynamics. We use comparative genomics and genetics to elucidate the evolutionary mechanisms of adaptation to physiologically stressful serpentine soils in rhizobial bacteria in western North American grasslands. In vitro experiments demonstrate that the presence of a locus of major effect, the nre operon, is necessary and sufficient to confer adaptation to nickel, a heavy metal enriched to toxic levels in serpentine soil, and a major axis of environmental soil chemistry variation. We find discordance between inferred evolutionary histories of the core genome and nreAXY genes, which often reside in putative genomic islands. This suggests that the evolutionary history of this adaptive variant is marked by frequent losses, and/or gains via horizontal acquisition across divergent rhizobium clades. However, different nre alleles confer distinct levels of nickel resistance, suggesting allelic substitution could also play a role in rhizobium adaptation to serpentine soil. These results illustrate that the interplay between evolution via gene gain and loss and evolution via allelic substitution may underlie adaptation in wild soil microbiota. Both processes are important to consider for understanding adaptive diversity in microbes and improving stress-adapted microbial inocula for human use.

Serpentine environment prevails over geographic distribution in shaping the genetic diversity of Medicago lupulina L.

Edaphic conditions of serpentine soils, naturally rich in heavy metals, act as a strong selection pressure that shapes specific metal-tolerant ecotypes. Medicago lupulina L. (black medick) is not only a widespread plant species that prefers calcareous and dry soil types but also grows at the borders of serpentine formations. It can also be found in waste and disturbed habitats. This is a species with reported phytoremediation potential, however, there is no published data regarding the impact of the environment on the genetic distribution of this species. The aim of our research was to explore how selection pressure of serpentine soils affects genetic diversity of M. lupulina and to test heavy-metal accumulation capacity of this species. Specimens of 11 M. lupulina populations were collected from serpentine outcrops located in Central and Eastern Bosnia as well as from non-serpentine sites. Soil and plant samples were analyzed for the total contents of heavy metals using air-acetylene flame atomic absorption spectroscopy. Genetic diversity was analyzed using AFLP (Amplified Fragment Length Polymorphism) markers. Serpentine soils showed high nickel, cobalt, chromium and iron concentrations. Nickel and manganese concentrations in soil samples and plant material showed statistically significant correlation. Although plants in two populations show the ability to extract Ni, M. lupulina does not show hyperaccumulating properties. Despite severe selective pressure, genetic diversity in serpentine populations is not reduced. Analyses of intrapopulation and interpopulation genetic diversity showed significant genetic differentiation among populations which is not related to their geographic distance. Population from non-metalliferous soil showed clear separation from all other populations. Diversity data suggest that serpentine populations maintain genetic diversity by undetected mechanisms and that edaphic factors rather than geography influence genetic structure analyzed M. lupulina populations.

Unravelling the ultramafic rock-driven serpentine soil formation leading to the geo-accumulation of heavy metals: An impact on the resident microbiome, biogeochemical cycling and acclimatized eco-physiological profiles.

In the present study, we have underpinned the serpentine rock, serpentinized ultramafic soil and rhizosphere's microbial communities, signifying their heavy metals-exposed taxa signatures and functional repertoires in comparison to non-serpentine soils. The results revealed that the serpentine rock embedded soil highlighted the geo-accumulation of higher amount of Cr and Ni impacting soil microbial diversity negatively by metal stress-driven selection. Biolog Ecoplate CLPP defined a restricted spectrum of C-utilization in the higher heavy metal-containing serpentine samples compared to non-serpentine. The linear discriminant analysis (LDA) score identified a higher abundance of Desulfobacterota, Opitutales, and Bacteroidales in low Cr and Ni-stressed non-serpentine-exposed samples. Whereas the abundance of Propionibacteriales and Actinobacteriota were significantly enriched in the serpentine niche. Further, the C, N, S, Fe, and methane biogeochemical cycles linked functional members were identified, and showing higher functional diversity in low Cr and Ni concentration-containing rhizosphere JS-soils. The Pearson correlation coefficient (r) value confirmed the abundance of functional members linked to specific biogeochemical cycle, positively correlated with relevant pathway enrichment. Ultimately, this study highlighted the heavy metal stress within a serpentine setting that could limit the resident microbial community's metabolic diversity and further select the bacteria that could thrive in the serpentine-associated heavy metal-stressed soils. These acclimatized microbes could pave the way for the future applications in the soil conservation and management.

Ni, Cr and Co Phytoremediations by Alyssum murale Grown in the Serpentine Soils Around Guleman Cr Deposits, Elazig Turkey.

Serpentine soils containing high levels of nickel and other metals are particularly preferred by some plants that accumulate nickel in their bodies. In this study, the Ni, Co, and Cr accumulation capacities of A. murale grown in Guleman's serpentine soils were measured. In this respect, 12 A. murale and their soils were collected from the mining site and surroundings. Afterwards, the collected samples were measured in order to evaluate the translocation and accumulation amounts of Ni, Cr, and Co. For that, soil and plant samples were analyzed using inductively coupled plasma mass spectroscopy (ICP-MS). The mean Ni concentrations in the soil, roots, and shoots of A. murale were measured as 2475, 7384, and 7694 mg/kg, respectively. The mean Cr concentrations in the soil, roots, and shoots of A. murale were measured as 742, 33, and 8.4 mg/kg while the mean Co concentrations of A. murale in the soil, roots, and shoots were 166, 10.2, and 23.5 mg/kg, respectively. Then, ECR and ECS values were calculated for Ni, Co, and Cr. The results indicated that A. murale grown in Guleman's serpentine soils may be helpful for the rehabilitation studies of mining soils contaminated by Ni and can be utilized for phytoextraction.

Chromate tolerance and removal of bacterial strains isolated from uncontaminated and chromium-polluted environments.

Investigation of bacterial chromate tolerance has mostly focused on strains originating from polluted sites. In the present study, we isolated 33 chromate tolerant strains from diverse environments harbouring varying concentrations of chromium (Cr). All of these strains were able to grow on minimal media with at least 2 mM hexavalent chromium (Cr(VI)) and their classification revealed that they belonged to 12 different species and 8 genera, with a majority (n = 20) being affiliated to the Bacillus cereus group. Selected B. cereus group strains were further characterised for their chromate tolerance level and the ability to remove toxic Cr(VI) from solution. A similar level of chromate tolerance was observed in isolates originating from environments harbouring high or low Cr. Reference B. cereus strains exhibited the same Cr(VI) tolerance which indicates that a high chromate tolerance could be an intrinsic group characteristic. Cr(VI) removal varied from 22.9% (strain PCr2a) to 98.5% (strain NCr4). Strains NCr1a and PCr12 exhibited the ability to grow to the greatest extent in Cr(VI) containing media (maximum growth of 65.3% and 64.9% relative to that in the absence of Cr(VI), respectively) accompanied with high chromate removal activity (73.7% and 74.4%, respectively), making them prime candidates for the investigation of chromate tolerance mechanisms in Gram-positive bacteria and Cr(VI) bioremediation applications.

Cascading effects of soil type on assemblage size and structure in a diverse herbivore community.

Soil type is understudied as a driver of herbivore community size and structure across host plants. This study extends predictions of resource availability hypotheses to understand how soil types of different resource levels alter plant resistance and structure of herbivore assemblages. In this 2-yr study we use seven dominant chaparral shrub species that grow across a natural mosaic of low and high resource soils to explore effects of soil type on plant resistance, and relate these soil-based differences in resistance to the abundance and diversity of the larval lepidopteran community. We show that growing on low-resource soils increases plant resistance, as measured by herbivore performance, both within and across host plant species, and that resistance may be driven by variation in plant nutritive and defensive traits. We then show that more resistant plants on low-resource soils host less abundant and less diverse herbivore assemblages across a natural soil mosaic in the field.

A metaproteomic approach dissecting major bacterial functions in the rhizosphere of plants living in serpentine soil.

A metaproteomic approach, based on liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis, was followed to map the major bacterial metabolic functions associated with the rhizosphere of metal-tolerant and metal hyperaccumulator plants, growing in a serpentine soil naturally contaminated by heavy metals such as Ni, Co and Cr. In particular, an "in-house" bacterial protein database was built based on the genera recognised by 16S rDNA profiling, then it was used for protein identification from LC-MS data. The combination of the information arising from three different extraction protocols, applied to each soil sample, permitted the identification of almost 800 proteins, corresponding to functions assigned to proper Gene Ontology categories. Mainly proteins involved in response to stimulus or in transport of metals and nutrients revealed variability of bacteria responses to microenvironment conditions. As for taxonomy, Phyllobacterium, Microbacterium oxidans, Pseudomonas oryzihabitans, Stenotrophomonas rhizophila and Bacillus methylotrophicus bacterial species were more represented in the rhizosphere samples of the metal-tolerant Biscutella laevigata and of the Ni hyperaccumulator Noccaea caerulescens with respect to bulk soil. Combining 16S rRNA gene-based sequencing and metaproteomic analysis, we get insights into microbial community functions and their interaction with plants colonising the stressful environment of serpentine soils.

Leaching potential of geogenic nickel in serpentine soils from Taiwan and Austria.

Serpentine soils may be natural sources of metal leaching and pollution. In this study, two contrasting serpentine soils from Taiwan and Austria were selected to evaluate the leaching potential of geogenic nickel (Ni). We applied selective sequential extractions and dissolution kinetics with three inorganic acids (HCl, HNO3, and H2SO4) and three organic acids (citric, acetic, and oxalic acids) in concentrations ranging from 0.05 to 10 mM to determine the release rate of Ni in the soils with respect to pH and acid types. Chlorite and serpentine were the major Ni-bearing minerals in the studied soils. Ni was dominantly bound in unavailable forms within these silicate minerals, but smaller fractions of acid-soluble, Fe-Mn oxide-bound, and organically bound Ni represented more labile Ni sources in the soils. The release rate of Ni from the soils increased with decreasing pH in all acids. However, the organic acids caused stronger pH dependences than the inorganic acids, likely because of ligand-promoted dissolution. The maximum total rate of Ni dissolution occurred with citric acid in both soils. The dissolution of Ni strongly increased when the ionic strength in the background solutions increased. We observed marked differences in dissolution rates and ligand effects between the Austrian and Taiwanese soils, which reflect differences in labile Ni pools, especially in the organically bound fraction. Our results demonstrate that labile fractions control the leaching potential of Ni in serpentine soils and that Ni associated with soil organic matter may contribute to leaching at moderately acidic pH levels.

Plant communities on infertile soils are less sensitive to climate change.

BACKGROUND AND AIMS: Much evidence suggests that plant communities on infertile soils are relatively insensitive to increased water deficit caused by increasing temperature and/or decreasing precipitation. However, a multi-decadal study of community change in the western USA does not support this conclusion. This paper tests explanations related to macroclimatic differences, overstorey effects on microclimate, variation in soil texture and plant functional traits. METHODS: A re-analysis was undertaken of the changes in the multi-decadal study, which concerned forest understorey communities on infertile (serpentine) and fertile soils in an aridifying climate (southern Oregan) from 1949-1951 to 2007-2008. Macroclimatic variables, overstorey cover and soil texture were used as new covariates. As an alternative measure of climate-related change, the community mean value of specific leaf area was used, a functional trait measuring drought tolerance. We investigated whether these revised analyses supported the prediction of lesser sensitivity to climate change in understorey communities on infertile serpentine soils. KEY RESULTS: Overstorey cover, but not macroclimate or soil texture, was a significant covariate of community change over time. It strongly buffered understorey temperatures, was correlated with less change and averaged >50 % lower on serpentine soils, thereby counteracting the lower climate sensitivity of understorey herbs on these soils. Community mean specific leaf area showed the predicted pattern of less change over time in serpentine than non-serpentine communities. CONCLUSIONS: Based on the current balance of evidence, plant communities on infertile serpentine soils are less sensitive to changes in the climatic water balance than communities on more fertile soils. However, this advantage may in some cases be lessened by their sparser overstorey cover.

Effects of floral metal accumulation on floral visitor communities: introducing the elemental filter hypothesis.

UNLABELLED: • PREMISE OF THE STUDY: For plant species that occur in heavy-metal-rich soil, floral metal accumulation may produce an "elemental filter" that reduces pollinator visitation rate and species richness and changes pollinator species composition relative to closely related species growing on normal soils. Consequently, metal hyperaccumulation may contribute to pollinator-mediated reproductive isolation between closely related plant species that differ in metal accumulation.• METHODS: To test these ideas, we characterized plant-pollinator interactions in a sympatric pair of species that differ in metal accumulation (Streptanthus polygaloides, a nickel (Ni) hyperaccumulator, and S. tortuosus, a nonaccumulator). To test the elemental filter hypothesis, we presented arrays of S. polygaloides that were grown in either Ni-treated or control soils to insects at both S. polygaloides and S. tortuosus sites and recorded visitation.• KEY RESULTS: Naturally occurring S. polygaloides hyperaccumulated Ni in anthers and accumulated Ni in nectar, while S. tortuosus did not. Floral visitation rates in natural populations were higher to S. tortuosus than S. polygaloides. In addition, while floral visitor richness was similar, few pollinator taxa were shared between the two plant species. Nickel-treatment of S. polygaloides reduced visits by bees, but only for arrays presented at S. tortuosus sites.• CONCLUSIONS: We show that the Ni hyperaccumulator S. polygaloides hosts a distinct floral visitor community, indicating that metal accumulation creates a filter for pollinators, similar to that documented for herbivores. Our study highlights a novel mechanism by which the abiotic environment can alter plant-pollinator interactions, and consequently plant reproduction and speciation.

Habitat type and interannual variation shape unique fungal pathogen communities on a California native bunchgrass.

The role of infectious disease in regulating host populations is increasingly recognized, but how environmental conditions affect pathogen communities and infection levels remains poorly understood. Over 3 y, we compared foliar disease burden, fungal pathogen community composition, and foliar chemistry in the perennial bunchgrass Stipa pulchra occurring in adjacent serpentine and nonserpentine grassland habitats with distinct soil types and plant communities. We found that serpentine and nonserpentine S. pulchra experienced consistent, low disease pressure associated with distinct fungal pathogen communities with high interannual species turnover. Additionally, plant chemistry differed with habitat type. The results indicate that this species experiences minimal foliar disease associated with diverse fungal communities that are structured across landscapes by spatially and temporally variable conditions. Distinct fungal communities associated with different growing conditions may shield S. pulchra from large disease outbreaks, contributing to the low disease burden observed on this and other Mediterranean grassland species.

Arbuscular mycorrhizal fungi and sewage sludge enhance growth and adaptation of Metrosideros laurifolia on ultramafic soil in New Caledonia: A field experiment.

The ecological restauration of nickel mining-degraded areas in New Caledonia is strongly limited by soil mineral nutrient deficiencies, heavy metal toxicity and slow growth rate of the native plant species. The improvement of revegetation technics needs multidisciplinary knowledge. A field experiment with relocated topsoil was assessed to test plant inoculation with a mix of three selected arbuscular mycorrhizal fungi (AMF) combined with sewage sludge amendment of the soil. Metrosideros laurifolia seedlings, an endemic Myrtaceae, were inoculated with the mixed AMF isolates and grown in a nursery for 18 months before being planted. 528 days after plantation, the dry weight of inoculated plants was 4 times higher than non-inoculated ones. AMF inoculated plants growing in sewage sludge amended soil showed a dry weight more than seven times higher than control plants. These differences were positively correlated with mycorrhizal colonization. However at this stage, AMF inoculated isolates were reduced in roots of M. laurifolia and replaced by several AMF indigenous species. This AMF diversity was higher in inoculated plants and in non-amended plots. Inoculated plants were characterized by a better mineral nutrition, a higher Ca/Mg ratio and a lower heavy metal translocation. In conclusion, this study showed that AMF inoculation combined with sewage sludge amendment of soil can improve ecological restoration of ultramafic mine-degraded areas.

Epigenetic Differentiation of Natural Populations of Lilium bosniacum Associated with Contrasting Habitat Conditions.

Epigenetic variation in natural populations with contrasting habitats might be an important element, in addition to the genetic variation, in plant adaptation to environmental stress. Here, we assessed genetic, epigenetic, and cytogenetic structure of the three Lilium bosniacum populations growing on distinct habitats. One population was growing under habitual ecological conditions for this species and the other two were growing under stress associated with high altitude and serpentine soil. Amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses revealed that the three populations did not differentiate genetically, but were clearly separated in three distinct clusters according to DNA methylation profiles. Principal coordinate analysis showed that overall epigenetic variation was closely related to habitat conditions. A new methylation-sensitive amplification polymorphism scoring approach allowed identification of mainly unmethylated (φST = 0.190) and fully CpG methylated (φST = 0.118) subepiloci playing a role in overall population differentiation, in comparison with hemimethylated sites (φST = 0.073). In addition, unusual rDNA repatterning and the presence of B chromosomes bearing 5S rDNA loci were recorded in the population growing on serpentine soil, suggesting dynamic chromosome rearrangements probably linked to global genome demethylation, which might have reactivated some mobile elements. We discuss our results considering our earlier data on morphology and leaf anatomy of several L. bosniacum populations, and suggest a possible role of epigenetics as a key element in population differentiation associated with environmental stress in these particular lily populations.

Combined endophytic inoculants enhance nickel phytoextraction from serpentine soil in the hyperaccumulator Noccaea caerulescens.

This study assesses the effects of specific bacterial endophytes on the phytoextraction capacity of the Ni-hyperaccumulator Noccaea caerulescens, spontaneously growing in a serpentine soil environment. Five metal-tolerant endophytes had already been selected for their high Ni tolerance (6 mM) and plant growth promoting ability. Here we demonstrate that individual bacterial inoculation is ineffective in enhancing Ni translocation and growth of N. caerulescens in serpentine soil, except for specific strains Ncr-1 and Ncr-8, belonging to the Arthrobacter and Microbacterium genera, which showed the highest indole acetic acid production and 1-aminocyclopropane-1-carboxylic acid-deaminase activity. Ncr-1 and Ncr-8 co-inoculation was even more efficient in promoting plant growth, soil Ni removal, and translocation of Ni, together with that of Fe, Co, and Cu. Bacteria of both strains densely colonized the root surfaces and intercellular spaces of leaf epidermal tissue. These two bacterial strains also turned out to stimulate root length, shoot biomass, and Ni uptake in Arabidopsis thaliana grown in MS agar medium supplemented with Ni. It is concluded that adaptation of N. caerulescens in highly Ni-contaminated serpentine soil can be enhanced by an integrated community of bacterial endophytes rather than by single strains; of the former, Arthrobacter and Microbacterium may be useful candidates for future phytoremediation trials in multiple metal-contaminated sites, with possible extension to non-hyperaccumulator plants.

Nickel accumulation in leaves, floral organs and rewards varies by serpentine soil affinity.

Serpentine soils are edaphically stressful environments that host many endemic plant species. In particular, serpentine soils are high in several heavy metals (e.g. nickel, cobalt and chromium) and these high heavy metal concentrations are thought, in part, to lead to varying levels of plant adaptation and soil affinities (i.e. endemic vs. non-endemic plant species). It is unclear, however, whether serpentine endemics vs. non-endemics differ with respect to heavy metal uptake into either vegetative or reproductive organs. Here, we use nickel as a model to determine whether plant heavy metal uptake varies with the level of endemism in several non-hyperaccumulating species. Under controlled greenhouse conditions, we grew seven plant species from the Brassicaceae family that vary in their degrees of affinity to serpentine soil from low (indifferent) to medium (indicator) and high (endemic) in soil that was nickel supplemented or not. We quantified nickel concentrations in leaves, pistils, anthers, pollen and nectar. While nickel concentrations did not vary across organs or affinities when grown in control soils, under conditions of nickel supplementation endemic species had the lowest tissue concentrations of nickel, particularly when considering leaves and pistils, compared with indifferent/indicator species. Species indifferent to serpentines incorporated higher concentrations of nickel into reproductive organs relative to leaves, but this was not the case for indicator species and endemics where nickel concentration was similar in these organs. Our findings suggest that endemic species possess the ability to limit nickel uptake into above-ground tissues, particularly in reproductive organs where it may interfere with survival and reproduction. Indifferent species accumulated significantly more nickel into reproductive organs compared with leaves, which may limit their reproductive potential relative to endemic species when growing on serpentine soils. Additional work determining the fitness consequences of these differences will further our understanding of edaphic endemism.

Unveiling neotropical serpentine flora: a list of Brazilian tree species in an iron saturated environment in Bom Sucesso, Minas Gerais

Serpentine soils are those holding at least of 70% iron-magnesium compounds, which make life intolerable for many species. Although plant's adaptation to environmental toughness is widely studied in tropics, virtually nothing is known about Brazilian serpentine flora. Our aim was to bring up and characterize the serpentine flora in Bom Sucesso, Minas Gerais state, Brazil. We performed expeditions utilizing rapid survey sampling method to identify the arboreal compound in the area. Plants within circumference at breast high (CBH) up to 15,7 cm were included in our study. A specialist identified all the individuals to species level. We found 246 species located in 59 botanical families. Fabaceae, Myrtaceae and Melastomataceae were the most representative families in the area. Serpentine areas usually present a few species capable to survive to adverse conditions, contrasting the high number found in our study. To our knowledge, this is the first floristic survey in serpentine areas in the neotropics, reinforcing the need for more studies about plant diversity in those areas. It seems that serpentinites is not the key factor influencing plant diversity in the neotropics. The high diversity found in our study strengthens serpentine areas as a place for conservation concern.

A Ni hyperaccumulator and a congeneric non-accumulator reveal equally effective defenses against herbivory.

The defense hypothesis is commonly used to explain the adaptive role of metal hyperaccumulation. We tested this hypothesis using two Brassicaceae congeneric species: Alyssum pintodasilvae, a Ni hyperaccumulator, and the non-accumulator Alyssum simplex both growing on serpentine soils in Portugal. Artificial diet disks amended with powdered leaves from each plant species were used to compare the performance (mortality, biomass change) and feeding behavior of Tribolium castaneum in no-choice and choice tests. The performance of T. castaneum was not affected at several concentrations of A. pintodasilvae or A. simplex in no-choice tests. However, the consumption of plant-amended disks was significantly lower than that of control disks, irrespectively of the species fed. Accordingly, when insects were given an alternative food choice, disks of both plant species were significantly less consumed than control disks. Moreover, insects did not discriminate between disks in the combination "A. pintodasilvae+A. simplex". Contrary to our expectations, these results suggest that both plant species have equally effective defenses against herbivory. While Ni is believed to be part of the deterrence mechanism in the hyperaccumulator A. pintodasilvae, it seems likely that organic compounds, possibly glucosinolates, play an important role in the defense of A. simplex or in both species.