Ecotypic Adaptation Research Articles

Plastic and adaptive response of carbon allocation to temperature change in alpine treeline trees

Understanding how tree individuals allocate nonstructural carbohydrate (NSC) between storage and growth is key to reveal the mechanism of alpine treeline formation as well as its response to climate warming. Yet, little is known about whether carbon allocation of treeline trees responds to temperature change by means of phenotypic plasticity and/or ecotypic adaptation. Here, we employed a reciprocal transplant experiment and a growth chamber warming treatment to explore the effects of temperature change on functional traits and carbon allocation between NSC storage and growth in treeline species Larix chinensis. In the reciprocal transplant experiment, we observed that the individuals grown at high elevation always displayed relatively lower specific leaf area (SLA) than those grown at lower elevations irrespective of species provenance, demonstrating the phenotypic plasticity of functional traits in relation to elevation. Higher growth rate and late-season NSC concentration in the individuals at their provenance elevation than at the elevations away from home, indicated that trees were locally adapted to their home environments in terms of NSC storage and growth. Additionally, individuals from high-elevation provenance showed considerably less phenotypic plasticity in carbon allocation (i.e., the ratio of NSC storage to growth) in response to temperature change compared to those from low-elevation provenance. In the chamber experiment, warming greatly accelerated shoot growth but not accompanied by a down-regulation in NSC storage in cold-edge trees, resulting in a lower ratio of NSC storage to growth. It was noteworthy that there was a time lag in variations of carbon allocation, especially in variations of NSC storage when cold-edge trees were grown in warmed climates. Our findings demonstrate that the responses of NSC storage and growth to temperature change might be a combination of short-term plastic and long-term adaptive reactions, and suggest a limited response of cold-edge populations in carbon allocation to climate warming.

Changing patterns of genetic differentiation in the slender wild oat, Avena barbata

The slender wild oat (Avena barbata) was widely studied in California using allozymes in the 1970s and interpreted as a case of ecotypic adaptation to contrasting moisture environments. However, common garden studies suggested that the moist-associated ("mesic") ecotype had high fitness in both moist and dry habitats, thus predicting an adaptive spread into areas occupied by the dry associated ("xeric") ecotype. To test this prediction, we revisited 100 populations of A. barbata that were screened genetically 40 y ago. As expected, mesic allozyme and morphological markers are much more common than in the 1970s. The less-fit xeric ecotype, while still widespread, has declined markedly in range and frequency. Genotyping by sequencing of modern populations reveals striking genetic uniformity within each of the two ecotypes. In recombinants between the two ecotypes, the mesic allele at a major fitness quantitative trait locus (QTL) shows a high frequency but so do many other genomic regions not identified as fitness QTL. Additional introduced genotypes are diverse and more widespread than in the past, and our results show that these have spread into the former range of the xeric ecotype to an even greater extent than the mesic ecotype has. While these results confirm the prediction of contemporary evolution from common gardens, they also suggest that much of the change has been driven by additional waves of introduced genotypes.

Using plant diversity to reduce vulnerability and increase drought resilience of permanent and sown productive grasslands

AbstractClimate change associated with a greater variability of inter‐ and intra‐annual droughts and the occurrence of extreme events act in combination to challenge semi‐natural and sown productive grasslands in Europe. Successful plant strategies under drought strongly depend on stress intensity. Drought resistance to maintain leaf growth under moderate stress trades off with drought survival after growth cessation under life‐threatening drought. Substantial intra‐specific variability exists in key forage grasses originating from the Mediterranean to the cool‐temperate climates and represents a great potential for adaptation of future ecotypes and cultivars to a larger range of drought intensities. Plant species diversity offers an opportunity to stabilize forage production in two ways. First, growth reduction under stress is significantly smaller for diverse compared to simple plant communities because the former offers the opportunity to include drought‐resistant (or drought‐surviving) species. Second, positive interactions among species increase ecosystem functioning of more diverse plant communities under moderate drought, allowing them to compensate for drought‐induced yield reductions. Currently, available cultivars of perennial forage species adapted to dry climate are still rare and only a few forage species are used in productive systems. Thus, both intra‐ and inter‐specific plant diversity should be better valued to reduce vulnerability and increase resilience of productive grasslands.

Microscopic Stages of North Atlantic Laminaria digitata (Phaeophyceae) Exhibit Trait-Dependent Thermal Adaptation Along Latitudes

Kelp forests in the North Atlantic are at risk of decline at their warm temperature distribution margins due to anthropogenic temperature rise and more frequent marine heat waves. To investigate the thermal adaptation of the cold-temperate kelpLaminaria digitata, we sampled six populations, from the Arctic to Brittany (Spitsbergen, Tromsø, Bodø [all Norway], Helgoland [Germany], Roscoff and Quiberon [both France]), across the species’ entire distribution range, spanning 31.5° latitude and 12-13°C difference in mean summer sea surface temperature. We used pooled vegetative gametophytes derived from several sporophytes to approximate the genetic diversity of each location. Gametophytes were exposed to (sub-) lethal high (20-25°C) and (sub-) optimal low (0-15°C) temperature gradients in two full-factorial, common-garden experiments, subjecting subsets of populations from different origins to the same conditions. We assessed survival of gametophytes, their ability to develop microscopic sporophytes, and subsequent growth. We hypothesized that the thermal performance of gametophytes and microscopic sporophytes corresponds to their local long-term thermal history. Integrated gametophyte survival revealed a uniform upper survival temperature (UST) of 24°C among five tested populations (Tromsø to Quiberon). In contrast, following two weeks of thermal priming of gametophytes at 20-22°C, sporophyte formation at 15°C was significantly higher in southern populations (Quiberon and Roscoff) compared to the high-latitude population of Tromsø. Between 0-15°C, survival of the Arctic population (Spitsbergen) was negatively correlated with increasing temperatures, while the southern-most population (Quiberon) showed the opposite. Thus, responses of survival at low, and sporophyte formation at high temperatures, support the concept of local adaption. On the other hand, sporophyte formation between 0-15°C peaked at 6-9°C in the Quiberon and at 9-12°C in the Spitsbergen population. Sporophyte growth rates (GR) both in length and width were similar for Spitsbergen, Tromsø and Quiberon; all had maximum GRs at 12-15°C and low GRs at 0-6°C. Therefore, responses of sporophyte formation and growth at low temperatures do not reflect ecotypic adaptation. We conclude thatL. digitatapopulations display trait-dependent adaptation, partly corresponding to their local temperature histories and partly manifesting uniform or unpredictable responses. This suggests differential selection pressures on the ontogenetic development of kelps such asL. digitata.

Landscape Genomics Provides Evidence of Ecotypic Adaptation and a Barrier to Gene Flow at Treeline for the Arctic Foundation Species Eriophorum vaginatum.

Global climate change has resulted in geographic range shifts of flora and fauna at a global scale. Extreme environments, like the Arctic, are seeing some of the most pronounced changes. This region covers 14% of the Earth’s land area, and while many arctic species are widespread, understanding ecotypic variation at the genomic level will be important for elucidating how range shifts will affect ecological processes. Tussock cottongrass (Eriophorum vaginatum L.) is a foundation species of the moist acidic tundra, whose potential decline due to competition from shrubs may affect ecosystem stability in the Arctic. We used double-digest Restriction Site-Associated DNA sequencing to identify genomic variation in 273 individuals of E. vaginatum from 17 sites along a latitudinal gradient in north central Alaska. These sites have been part of 30 + years of ecological research and are inclusive of a region that was part of the Beringian refugium. The data analyses included genomic population structure, demographic models, and genotype by environment association. Genome-wide SNP investigation revealed environmentally associated variation and population structure across the sampled range of E. vaginatum, including a genetic break between populations north and south of treeline. This structure is likely the result of subrefugial isolation, contemporary isolation by resistance, and adaptation. Forty-five candidate loci were identified with genotype-environment association (GEA) analyses, with most identified genes related to abiotic stress. Our results support a hypothesis of limited gene flow based on spatial and environmental factors for E. vaginatum, which in combination with life history traits could limit range expansion of southern ecotypes northward as the tundra warms. This has implications for lower competitive attributes of northern plants of this foundation species likely resulting in changes in ecosystem productivity.

Editorial 2022

Editorial 2022

Genomic Variation Shaped by Environmental and Geographical Factors in Prairie Cordgrass Natural Populations Collected across Its Native Range in the USA.

Prairie cordgrass (Spartina pectinata Link) is a native perennial warm-season (C4) grass common in North American prairies. With its high biomass yield and abiotic stress tolerance, there is a high potential of developing prairie cordgrass for conservation practices and as a dedicated bioenergy crop for sustainable cellulosic biofuel production. However, as with many other undomesticated grass species, little information is known about the genetic diversity or population structure of prairie cordgrass natural populations as compared to their ecotypic and geographic adaptation in North America. In this study, we sampled and characterized a total of 96 prairie cordgrass natural populations with 9315 high quality SNPs from a genotyping-by-sequencing (GBS) approach. The natural populations were collected from putative remnant prairie sites throughout the Midwest and Eastern USA, which are the major habitats for prairie cordgrass. Partitioning of genetic variance using SNP marker data revealed significant variance among and within populations. Two potential gene pools were identified as being associated with ploidy levels, geographical separation, and climatic separation. Geographical factors such as longitude and altitude, and environmental factors such as annual temperature, annual precipitation, temperature of the warmest month, precipitation of the wettest month, precipitation of Spring, and precipitation of the wettest month are important in affecting the intraspecific distribution of prairie cordgrass. The divergence of prairie cordgrass natural populations also provides opportunities to increase breeding value of prairie cordgrass as a bioenergy and conservation crop.

Leaf epidermal anatomy of Cynodon dactylon (L.) Pers. in relation to ecotypic adaptation

Qualitative and quantitative studies were done on leaf epidermal characteristics with special reference to stomatal features to find out the impact of environmental condition on twenty four accessions of Cynodon dactylon (L.) Pers. collected from different ecological habitats of Bangladesh. The foliar epidermal peels from both surfaces of mature leaves were observed under microscope. The leaves were found to be amphistomatic and stomata were paracytic type. The epidermal cells in this study were found to be sinuous. Silica bodies were found to be saddle and cross shaped. Prickles angular were pointed at the tip. Macro-hairs were present in all the accessions, but no micro-hair was found both adaxially or abaxially. Stomatal frequency and stomatal index were found to vary from accession to accession on both adaxial and abaxial surface of leaves, and the differences were statistically significant in most of the cases.
 Bangladesh J. Plant Taxon. 28(1): 171-193, 2021 (June)

Environmental adaptation of the root microbiome in two rice ecotypes

The root microbiome plays a key role that can influence host plant growth and abiotic stress. While there has been extensive characterization of community structure, spatial compartmentalization, and the impact of drought stresses on the root microbiome in rice and other plants, there is relatively little known about the differences in root microbiome among rice ecotypes in natural upland and lowland fields. Herein, we used two rice ecotypes, upland and irrigated ecotype rice (two Indica and two Japonica genotypes), as a model to explore the responses of the root microbiome under different environmental conditions. We aimed to identify environment-induced adaptation in the root bacterial and fungal composition of rice ecotypes by high-throughput sequencing. Rice from lowland field or upland had significantly altered overall bacterial and fungal community compositions of the two ecotypes, with diversity of both ecotypes greatly decreased from lowland field to upland. The overall response of the root microbiome to upland conditions was taxonomically driven by the enrichment of family Enterobacteriaceae and genera Serratia, and phylum Ascomycota. Interestingly, rice ecotypes specifically enriched root microbes when they were transferred from their original environment, such as the enrichment of class Thermoleophilia and phylum Actinobacteria when the irrigated ecotype rice was moved from lowland to upland field. These results revealed that different environmental conditions and rice ecotypes resulted in a restructuring of root microbiome communities, and suggested the possibility that components responsible for the beneficial attributes in the altered root microbiome might contribute to the adaptation of different ecotypes in natural fields.

Massive haplotypes underlie ecotypic differentiation in sunflowers.

Species often include multiple ecotypes that are adapted to different environments1. However, it is unclear how ecotypes arise and how their distinctive combinations of adaptive alleles are maintained despite hybridization with non-adapted populations2-4. Here, by resequencing 1,506wild sunflowers from 3species (Helianthus annuus, Helianthus petiolaris and Helianthus argophyllus), we identify 37large (1-100Mbp in size), non-recombining haplotype blocks that are associated with numerous ecologically relevant traits, as well as soil and climate characteristics. Limited recombination in these haplotype blocks keeps adaptive alleles together, and these regions differentiate sunflower ecotypes. For example, haplotype blocks control a 77-day difference in flowering between ecotypes of the silverleaf sunflower H.argophyllus (probably through deletion of a homologue of FLOWERINGLOCUST (FT)), and are associated with seed size, flowering time and soil fertility in dune-adapted sunflowers. These haplotypes are highly divergent, frequently associated with structural variants and often appear to represent introgressions from other-possibly now-extinct-congeners. These results highlight a pervasive role of structural variation in ecotypic adaptation.

Local adaptation, genetic divergence, and experimental selection in a foundation grass across the US Great Plains' climate gradient.

Many prior studies have uncovered evidence for local adaptation using reciprocal transplant experiments. However, these studies are rarely conducted for a long enough time to observe succession and competitive dynamics in a community context, limiting inferences for long-lived species. Furthermore, the genetic basis of local adaptation and genetic associations with climate has rarely been identified. Here, we report on a long-term (6-year) experiment conducted under natural conditions focused on Andropogon gerardii, the dominant grass of the North American Great Plains tallgrass ecosystem. We focus on this foundation grass that comprises 80% of tallgrass prairie biomass and is widely used in 20,000km2 of restoration. Specifically, we asked the following questions: (a) Whether ecotypes are locally adapted to regional climate in realistic ecological communities. (b) Does adaptive genetic variation underpin divergent phenotypes across the climate gradient? (c) Is there evidence of local adaptation if the plants are exposed to competition among ecotypes in mixed ecotype plots? Finally, (d) are local adaptation and genetic divergence related to climate? Reciprocal gardens were planted with 3 regional ecotypes (originating from dry, mesic, wet climate sources) of Andropogon gerardii across a precipitation gradient (500-1,200mm/year) in the US Great Plains. We demonstrate local adaptation and differentiation of ecotypes in wet and dry environments. Surprisingly, the apparent generalist mesic ecotype performed comparably under all rainfall conditions. Ecotype performance was underpinned by differences in neutral diversity and candidate genes corroborating strong differences among ecotypes. Ecotype differentiation was related to climate, primarily rainfall. Without long-term studies, wrong conclusions would have been reached based on the first two years. Further, restoring prairies with climate-matched ecotypes is critical to future ecology, conservation, and sustainability under climate change.

Adaptation of ecotypes and cultivars of subterranean clover (Trifolium subterraneum L.) to German environmental conditions and its suitability as living mulch

Abstract A total of 320 accessions of subterranean clover (Trifolium subterranenum L.) were screened for adaptation to the environmental conditions in southern Germany, as well as their suitability as cover crops or living mulches. The accessions, derived from collections of the Department of Agriculture and Food Western Australia and the International Research Center for Agriculture in Dry Areas, were propagated in the greenhouse and grown in rows. Selected accessions were then tested in plots and as an intercrop in wheat, in order to test their suitability as living mulch. Moreover, a frost resistance screening was carried out in a climate chamber. Adaptation to southern German conditions and frost tolerance was generally better than expected, as most accessions survived winters with snow cover and transitory temperatures as low as \(-\,10\,^\circ \)C. Accessions with particularly high frost tolerance were identified and several accessions persisted over four generations of self-reseeding. Although there was a large range of morphological characteristics, no differences concerning their suitability as living mulch could be observed. The results suggest that subterranean clover is sufficiently winter hardy to be grown as an overwintering cover crop or living mulch in southern Germany. Commercially available cultivars appear suitable in the first instance, even though these have not been selected for cold winters. However, adaptation might be further improved by targeted selection among accessions.

Genetic and environmental influences on stomates of big bluestem (Andropogon gerardii)

Big bluestem (Andropogon gerardii) is a dominant C4 prairie grass that has wide distribution and several genetically distinct ecotypes. Many of the ecotypic adaptations are related to water availability in the native environment. Stomates facilitate photosynthetic gas exchange and regulate water loss from the plant. As such, stomatal size and density represent possible adaptations to conserve water. We hypothesized drought-tolerant ecotypes of big bluestem would have fewer or smaller stomates compared to more mesic ecotypes. Five ecotypes of big bluestem were planted in four common gardens from western Kansas to southern Illinois, USA to determine genetic and environmental influences on stomates. Leaves of all ecotypes of A. gerardii were largely hypostomatous and genetics was a greater influence than environment for stomatal size and density. The drought-tolerant Sand bluestem had larger stomates on abaxial surfaces of leaves, but a lower density compared to most other ecotypes. The most mesic Illinois ecotype and the Kaw cultivar had the greatest density of stomates on abaxial surfaces of leaves. Sand Bluestem had a greater density of stomates on adaxial surfaces of leaves compared to all other ecotypes. Gas exchange measures followed patterns of stomate distribution, where abaxial CO2 uptake rates were greater than adaxial CO2 uptake rates, although differences between leaf surfaces was more pronounced in stomatal density than in CO2 uptake. There were minor differences in size and density of stomates among sites that corresponded with precipitation, although these differences were minor, illustrating the genetic underpinnings of stomates in big bluestem. There is a genetic predisposition for drought-tolerant ecotypes to have fewer stomates, illustrating an evolutionary adaptation to drought tolerance in an important prairie species.

Genomic Admixture Between Locally Adapted Populations of Arabidopsis thaliana (mouse ear cress): Evidence of Optimal Genetic Outcrossing Distance.

Admixture can break up divergent genetic architectures between populations, resulting in phenotypic novelty and generating raw material for environmental selection. The contribution of admixture to progeny trait variation and fitness varies based on the degree of genetic isolation between the parental populations, for which most studies have used geographic distance as a proxy. A novel approach is to estimate optimal crossing distance using the adaptive genetic distance between mates estimated from loci that contribute directly to local adaptation. Here, we aim to understand the effect of admixture on disrupting local adaptation of ecotypes of Arabidopsis thaliana separated along gradients of geographic, background, and locally adaptive genetic distances. We created experimental F1 hybrids between ecotypes that vary in geographic distance and used SNP data to estimate background (putatively neutral) and adaptive genetic distance. Hybrids were grown under controlled conditions, and fitness, growth, and phenology traits were measured. The different traits measured showed a clear effect of adaptive genetic distance, but not geographic distance. The earliest bolting hybrids were intermediate in the adaptive genetic distance between their parents, and also had higher biomass and fitness in terms of fruit and seed production. Our results suggest that disruption of locally adaptive genomic loci decreases the performance of offspring between distantly related parents, but that crosses between very closely related parents also reduce performance, likely through the expression of deleterious recessive alleles. We conclude that during admixture, selection may have to balance the consequences of disrupting local adaption while also avoiding inbreeding depression.

Chromosome inversions and ecological plasticity in the main African malaria mosquitoes.

Chromosome inversions have fascinated the scientific community, mainly because of their role in the rapid adaption of different taxa to changing environments. However, the ecological traits linked to chromosome inversions have been poorly studied. Here, we investigated the roles played by 23 chromosome inversions in the adaptation of the four major African malaria mosquitoes to local environments in Africa. We studied their distribution patterns by using spatially explicit modeling and characterized the ecogeographical determinants of each inversion range. We then performed hierarchical clustering and constrained ordination analyses to assess the spatial and ecological similarities among inversions. Our results show that most inversions are environmentally structured, suggesting that they are actively involved in processes of local adaptation. Some inversions exhibited similar geographical patterns and ecological requirements among the four mosquito species, providing evidence for parallel evolution. Conversely, common inversion polymorphisms between sibling species displayed divergent ecological patterns, suggesting that they might have a different adaptive role in each species. These results are in agreement with the finding that chromosomal inversions play a role in Anopheles ecotypic adaptation. This study establishes a strong ecological basis for future genome-based analyses to elucidate the genetic mechanisms of local adaptation in these four mosquitoes.

Ecotypic adaptations in Bermuda grass (Cynodon dactylon) for altitudinal stress tolerance

AbstractThree ecotypes [foot hill (700 m), mid hill (1571 m) and top hill (2804 m)] of a Bermuda grass

Life cycle traits of Philodina roseola Ehrenberg, 1830 (Rotifera, Bdelloidea), a model organism for bioassays.

This paper describes experimental results on the life cycle of the rotifer Philodina roseola cultured in the laboratory. Detailed information on life-cycle parameters of a certain species provides a deep understanding and contributes to a better knowledge of the role of the species in the community, besides providing data that are basic to other ecological investigations such as secondary production estimates and knowledge for applications such as its utilization as test-organism in ecotoxicological studies. The average duration of embryonic development of P. roseola was 23.88 h, the age at maturity of primipara was 3.5 days and the maximum lifespan was 23 days. The average size of the rotifer neonate was 198.77 µm, the mean size of primipara was 395.56 µm and for adults 429.96 µm. The average fecundity was 1.22 eggs per female per day and the mean number of eggs produced per female during the entire life was 22.33. The deceleration of somatic growth from the start of the reproductive stage represents a trade-off between growth and reproduction that is often seen in micrometazoans. The life history of P. roseola follows the strategy of other bdelloid species characterized by a rapid pre-reproductive development and canalization of most assimilated energy to reproduction after reaching maturity. The differences observed in total fecundity and longevity between our P. roseola cultures and those from previous studies were probably due to differences of intrinsic adaptation of this species ecotypes to the conditions of their natural environments.

DNA methylation and methylation polymorphism in ecotypes of Jatropha curcas L. using methylation-sensitive AFLP markers

We investigated DNA methylation and polymorphism in the methylated DNA using AFLP based methylation-sensitive amplification polymorphism (MS-AFLP) markers in ecotypes of Jatropha curcas L. growing in similar and different geo-ecological conditions. Three ecotypes growing in different geo-ecological conditions with environmental heterogeneity (Group-1) and five ecotypes growing in similar environmental conditions (Group-2) were assessed. In ecotypes growing in group-1, 44.32 % DNA was methylated and of which 93.59 % DNA was polymorphic. While in group-2, 32.27 % DNA was methylated, of which 51.64 % DNA was polymorphic. In site 1 and site 2 of group-1, overall methylation was 18.94 and 22.44 % respectively with difference of 3.5 %, while overall polymorphism was 41.14 and 39.23 % with a difference of 1.91 %. In site 1 and site 2 of group-2, overall methylation was 24.68 and 24.18 % respectively with difference of 0.5 %, while overall polymorphism was 12.19 and 12.65 % with a difference of 0.46 %. The difference of methylation percentage and percentage of methylation polymorphism throughout the genome of J. curcas at site 1 and 2 of group-1 is higher than that of J. curcas at site 1 and 2 of group-2. These results correlated the physico-chemical properties of soil at these sites. The variations of physico-chemical properties of soil at Chorwadla (site 1 in group-1 and site 2 in group-2) compared to the soil at Brahmapur (site 2 in group-1) is higher than that of soil at Neswad (site 1 in group-2). The study suggests that these homologous nucleotide sequences probably play important role in ecotype adaptation to environmental heterogeneity by creating epiallelic variations hence in evolution of ecotypes/clines or forms of species showing phenotypic/genotypic differences in different geographical areas.

Morpho-physiological and adaptive variation of Italian germplasm of sulla (Hedysarum coronarium L.)

Sulla is a biennial forage legume native to the central-western Mediterranean Basin and has increasing interest for regions with Mediterranean-climate. The Italian germplasm is a reservoir of variation for important agronomic traits. This study aimed to support breeding programs by investigating patterns of agronomic, adaptive and morpho-physiological variation among ecotypes collected from the three main Italian regions of species cultivation: central Italy, and the two islands of Sicily and Sardinia. Forage yield and morpho-physiological traits were evaluated at a site with Mediterranean climate in Sicily. Forage yield and cold tolerance in a cold-prone site of northern Italy were also assessed, to locate useful germplasm for widening crop resilience and climatic adaptability. Collection regions, and ecotypes within regions, differed for total forage yield and final plant survival, but their responses were subjected to interactions with test location. Specific adaptation dominated the adaptive responses of ecotypes and elite commercial varieties. Ecotype adaptation to cold winter was associated with latitude and, more specifically, the extent of cold stress in collecting sites. The ecotype collection region had a bearing also on morpho-physiological characteristics. A few ecotypes from central Italy performed outstandingly with respect to elite varieties, displaying wide adaptation across cold-prone and drought-prone environments.

Phytoremediation of cadmium using plant species of Athyrium wardii (Hook.)

Athyrium wardii (Hook.) is a promising herbaceous plant species for phytostabilization of cadmium (Cd)-contaminated sites with large biomass and fast growth rate. However, little information is available on its tolerance mechanisms toward Cd. To further understand the mechanisms involved in Cd migration, accumulation and detoxification, the present study investigated subcellular distribution and chemical forms of Cd in the mining ecotypes and corresponding non-mining ecotypes of A. wardii via greenhouse pot experiment. Subcellular fractionation of Cd-containing tissues demonstrated that the majority of the element was mainly located in soluble fraction in cell walls. This indicated that both the vacuoles and cell walls might be evolved the Cd tolerance mechanisms to protect metabolically active cellular compartments from toxic Cd concentrations. Meanwhile, Cd taken up by the plant existed in different chemical forms. Results showed that the majority of Cd in plant was in undissolved Cd–phosphate complexes (extracted by 2 % CH3COOH), followed by water-soluble Cd–organic acid complexes, Cd(H2PO4)2, pectates and protein form (extracted by deionized water and 1 M NaCl), whereas only small amount of Cd in roots was in inorganic form (extracted by 80 % ethanol), which suggests low capacity to be transported to aboveground tissues. It could be suggested that Cd integrated with undissolved Cd–phosphate complexes in cell wall or compartmentalization in vacuole might be responsible for the adaptation of the mining ecotypes of A. wardii to Cd stress.