Cytomolecular trends in Chamaecrista Moench (Caesalpinioideae, Leguminosae) diversification.

Chamaecrista is a Pantropical legume genus of the tribe Cassieae, which includes six other genera. In contrast to most of the other Cassieae genera, Chamaecrista shows significant variability in chromosome number (from 2n = 14 to 2n = 56), with small and morphologically similar chromosomes. Here, we performed a new cytomolecular analysis on chromosome number, genome size, and rDNA site distribution in a molecular phylogenetic perspective to interpret the karyotype trends of Chamaecrista and other two genera of Cassieae, seeking to understand their systematics and evolution. Our phylogenetic analysis revealed that Chamaecrista is monophyletic and can be divided into four major clades corresponding to the four sections of the genus. Chromosome numbers ranged from 2n = 14, 16 (section Chamaecrista) to 2n = 28 (sections Absus, Apoucouita, and Baseophyllum). The number of 5S and 35S rDNA sites varied between one and three pairs per karyotype, distributed on different chromosomes or in synteny, with no obvious phylogenetic significance. Our data allowed us to propose x = 7 as the basic chromosome number of Cassieae, which was changed by polyploidy generating x = 14 (sections Absus, Apoucouita, and Baseophyllum) and by ascending dysploidy to x = 8 (section Chamaecrista). The DNA content values supported this hypothesis, with the genomes of the putative tetraploids being larger than those of the putative diploids. We hypothesized that ascending dysploidy, polyploidy, and rDNA amplification/deamplification are the major events in the karyotypic diversification of Chamaecrista. The chromosomal marks characterized here may have cytotaxonomic potential in future studies.

Comparing the Predicted versus Realized Rate of Adaptation of Chamaecrista fasciculata to Climate Change.

AbstractFisher's fundamental theorem of natural selection (FTNS) can be used in a quantitative genetics framework to predict the rate of adaptation in populations. Here, we estimated the capacity for a wild population of the annual legume Chamaecrista fasciculata to adapt to future environments and compared predicted and realized rates of adaptation. We planted pedigreed seeds from one population into three prairie reconstructions along an east-to-west decreasing precipitation gradient. The FTNS predicted adaptation at all sites, but we found a response to selection that was smaller at the home and westernmost sites and maladaptive at the middle site because of changes in the selective environment between generations. However, mean fitness of the progeny generation at the home and westernmost sites exceeded population replacement, which suggests that the environment was sufficiently favorable to promote population persistence. More studies employing the FTNS are needed to clarify the degree to which predictions of the rate of adaptation are realized and its utility in the conservation of populations at risk of extinction from climate change.

Is there a latitudinal diversity gradient for symbiotic microbes? A case study with sensitive partridge peas.

Mutualism is thought to be more prevalent in the tropics than temperate zones and may therefore play an important role in generating and maintaining high species richness found at lower latitudes. However, results on the impact of mutualism on latitudinal diversity gradients are mixed, and few empirical studies sample both temperate and tropical regions. We investigated whether a latitudinal diversity gradient exists in the symbiotic microbial community associated with the legume Chamaecrista nictitans. We sampled bacteria DNA from nodules and the surrounding soil of plant roots across a latitudinal gradient (38.64-8.68 °N). Using 16S rRNA sequence data, we identified many non-rhizobial species within C. nictitans nodules that cannot form nodules or fix nitrogen. Species richness increased towards lower latitudes in the non-rhizobial portion of the nodule community but not in the rhizobial community. The microbe community in the soil did not effectively predict the non-rhizobia community inside nodules, indicating that host selection is important for structuring non-rhizobia communities in nodules. We next factorially manipulated the presence of three non-rhizobia strains in greenhouse experiments and found that co-inoculations of non-rhizobia strains with rhizobia had a marginal effect on nodule number and no effect on plant growth. Our results suggest that these non-rhizobia bacteria are likely commensals-species that benefit from associating with a host but are neutral for host fitness. Overall, our study suggests that temperate C. nictitans plants are more selective in their associations with the non-rhizobia community, potentially due to differences in soil nitrogen across latitude.

α-Amylase and α-glucosidase inhibitors from the aerial parts of Chamaecrista pumila (Lam.) K. Larsen.

One new compound, 4,7-dihydroxy-2-hydroxymethyl-5,6-dimethoxyanthraquinone (1), along with eight known compounds (2-9) were isolated from the methanol extracts of the aerial parts of Chamaecrista pumila (Lam.) K. Larsen. Their chemical structure was determined based on spectroscopic data interpretation and comparison with the reported data. The inhibitory effects of them on α-amylase and α-glucosidase were performed. The results showed that compounds 4, 6, 8, and 9 against potent α-glucosidase with the inhibition values of 98.14 ± 0.05, 98.19 ± 0.04, 97.01 ± 0.49, 84.43 ± 0.6% at 50 µM, respectively. Compounds 4 and 6 displayed significance against α-amylase at 200 µM with inhibition values of 22.35 ± 1.10 and 60.47 ± 0.91%.

Distribution and biodiversity of rhizobia nodulating Chamaecrista mimosoides in the Shandong peninsula of china.

Chamaecrista mimosoides is an annual herb legume widely distributed in tropical and subtropical Asia and Africa. It may have primitive and independently-evolved root nodule types but its rhizobia have not been systematically studied. Therefore, in order to learn the diversity and species affinity of its rhizobia, root nodules were sampled from C. mimosoides plants growing in seven geographical sites along the coast line of Shandong Peninsula, China. A total of 422 rhizobial isolates were obtained from nodules, and they were classified into 28 recA haplotypes. By using multilocus sequence analysis of the concatenated housekeeping genes dnaK, glnII, gyrB, recA and rpoB, the representative strains for these haplotypes were designated as eight defined and five candidate novel genospecies in the genus Bradyrhizobium. Bradyrhizobium elkanii and Bradyrhizobium ferriligni were predominant and universally distributed. The symbiotic genes nodC and nifH of the representative strains showed very similar topology in their phylogenetic trees indicating their co-evolution history. All the representative strains formed effective root nodules in nodulation tests. The correlation between genospecies and soil characteristics analyzed by CANOCO software indicated that available potassium (AK), organic carbon (OC) and available nitrogen (AN) in the soil samples were the main factors affecting the distribution of the symbionts involved in this current study. The study is the first systematic survey of Chamaecrista mimosoides-nodulating rhizobia, and it showed that Chamaecrista spp. were nodulated by bradyrhizobia in natural environments. In addition, the host spectrum of the corresponding rhizobial species was extended, and the study provided novel information on the biodiversity and biogeography of rhizobia.

Minimum size threshold of visiting bees of a buzz-pollinated plant species: consequences for pollination efficiency.

PREMISE: Flowering plants with poricidal anthers are commonly visited by buzzing bees, which vibrate flowers to extract pollen. However, not all flower visitors are in fact pollinators, and features such as body size and duration of flower visits are important factors in determining pollination effectiveness. We tested whether bee-to-flower size relationships predict the pollination effectiveness of flower visitors of a buzz-pollinated species (Chamaecrista ramosa, Fabaceae). METHODS: We sorted 13 bee taxa into three groups: smaller than, equivalent to ("fit-size"), and larger than flower herkogamy (spatial separation between anthers and stigma). We expected the latter two groups to touch the stigmas, which would be an indicator of pollination effectiveness, more frequently than the first group. To test this hypothesis, we assessed contact with stigmas, foraging behavior, and duration of visits for the three size groups of bees. RESULTS: Our data reveal that small bees scarcely touched the stigmas, while large and fit-size bees were the most efficient pollinators, achieving high stigma-touching rates, conducting much shorter flower visits, and visiting flowers and conspecific plants at high rates during foraging bouts. CONCLUSIONS: The results did not show size-matching among bees and flowers, as expected, but rather a minimum size threshold of efficient pollinators. The finding of such a threshold is a nonarbitrary approach to predicting pollination effectiveness of visitors to herkogamous flowers with poricidal anthers.

Lifetime Fitness through Female and Male Function: Influences of Genetically Effective Population Size and Density.

AbstractAn individual's lifetime fitness and patterns of mating between individuals are interdependent features of sexual organisms. Mating systems (outcrossing vs. selfing or mating between close relatives) can affect the distribution of offspring fitness, which generally declines with inbreeding, which in turn is related to a population's genetically effective size (Ne). Fitness and mating patterns are also expected to vary with proximity of mates (i.e., population density). Consequently, density and Ne may influence demographic and genetic changes over generations and interact in their effects. Here, we report an experiment designed to assess the influence of these two population-level properties on mating system and lifetime fitness. In experimental arrays under quasi-natural conditions, we varied the density and Ne of the hermaphroditic annual legume Chamaecrista fasciculata. We recorded components of fitness for each individual and employed microsatellite markers to estimate outcrossing and assign paternity. We used aster analyses to estimate lifetime fitness for genetic families using female (seeds set) and male (seeds sired) reproduction as fitness measures. With estimates from these analyses, we assessed the evidence for a trade-off between fitness attained through female versus male function, but we found none. Lifetime fitness increased with density, especially under high Ne. Outcrossing rates increased with density under high Ne but declined modestly with density under low Ne. Our results show that density and Ne have strong direct effects on fitness and mating systems, with negative fitness effects of low Ne limiting the positive effects of increasing density. These findings highlight the importance of the interactive effects of density and Ne on lifetime fitness.

Hepatogenous photosensitization in ruminants and horses caused by the ingestion of Chamaecrista serpens in Brazil.

This study aimed to describe the first reports of outbreaks of hepatogenous photosensitization in cattle, sheep, and horses caused by spontaneous ingestion of Chamaecrista serpens, as well as to reproduce poisoning in sheep experimentally. Eleven photodermatitis outbreaks of unknown cause occurred in cattle, sheep and horses on nine farms in the semiarid region of Bahia, northeastern Brazil, between July 2017 and July 2020. Cutaneous lesions of photosensitization initiated until one week after the animals were introduced in paddocks invaded by the plant at the beginning of the rainy season. The photosensitive skin lesions were progressive and consisted of hyperemia, edema, ulcerative-crusted lesions with necrosis, especially in non-pigmented skin areas. The lesions in young animals were more severe. The animals avoided the sun and exhibited hyporexia, weight loss, restlessness, irritability, and severe itching. An experimental study was made using seven sheep, and resulted in photodermatitis, similar to that observed in the natural poisoning, seven days after the beginning of plant ingestion. Two sheep were reserved for the control group. Serum biochemistry changes indicated liver injury caused by the plant. Skin biopsies and liver biopsy guided by ultrasound were performed. The one sheep that had more pronounced skin lesions was euthanized and necropsied. At the necropsy, the liver was enlarged, diffusely pale, and firm, with an evident lobular pattern and an empty gallbladder. Histopathology revealed similar skin and liver lesions in samples from biopsies and the necropsy. There was a marked disorganization of the cords of hepatocytes associated with degenerative necrotic changes on the liver. The cutaneous injuries included orthokeratotic hyperkeratosis, hypergranulosis, acanthosis, and extensive areas of epidermic necrosis and ulceration. Three sheep were protected from sunlight and the lesions regressed within 45 days after the plant's consumption ceased. In conclusion, C. serpens causes hepatogenous photosensitization in ruminants and horses, and should be included in the list of differential diagnoses in cases of photosensitive dermatitis.

Estimating the capacity of Chamaecrista fasciculata for adaptation to change in precipitation.

Adaptation through natural selection may be the only means by which small and fragmented plant populations will persist through present day environmental change. A population's additive genetic variance for fitness (VA (W)) represents its immediate capacity to adapt to the environment in which it exists. We evaluated this property for a population of the annual legume Chamaecrista fasciculata through a quantitative genetic experiment in the tallgrass prairie region of the Midwestern United States, where changing climate is predicted to include more variability in rainfall. To reduce incident rainfall, relative to controls receiving ambient rain, we deployed rain exclusion shelters. We found significant VA (W) in both treatments. We also detected a significant genotype-by-treatment interaction for fitness, which suggests that the genetic basis of the response to natural selection will differ depending on precipitation. For the trait-specific leaf area, we detected maladaptive phenotypic plasticity and an interaction between genotype and environment. Selection for thicker leaves was detected with increased precipitation. These results indicate capacity of this population of C. fasciculata to adapt in situ to environmental change.

Evolution in novel environments: Do restored prairie populations experience strong selection?

When populations colonize new habitats, they are likely to experience novel environmental conditions, and as a consequence may experience strong selection. While selection and the resulting evolutionary responses may have important implications for establishment success in colonizing populations, few studies have estimated selection in such scenarios. Here we examined evidence of selection in recently established plant populations in two prairie restorations in close proximity (<15 km apart) using two approaches: (1) we tested for evidence of past selection on a suite of traits in two Chamaecrista fasciculata populations by comparing the restored populations to each other and their shared source population in common gardens to quantify evolutionary responses and (2) we measured selection in the field. We found evidence of past selection on flowering time, specific leaf area, and root nodule production in one of the populations, but detected contemporary selection on only one trait (plant height). Our findings demonstrate that while selection can occur in colonizing populations, resulting in significant trait differences between restored populations in fewer than six generations, evolutionary responses differ across even nearby populations sown with the same source population. Because contemporary measures of selection differed from evolutionary responses to past selection, our findings also suggest that selection likely differs over the early stages of succession that characterize young prairies.

Bradyrhizobium frederickii sp. nov., a nitrogen-fixing lineage isolated from nodules of the caesalpinioid species Chamaecrista fasciculata and characterized by tolerance to high temperature in vitro.

The symbioses between legumes and nitrogen-fixing rhizobia make the greatest contribution to the global nitrogen input via the process of biological nitrogen fixation (BNF). Bradyrhizobium stands out as the main genus nodulating basal Caesalpinioideae. We performed a polyphasic study with 11 strains isolated from root nodules of Chamaecristafasciculata, an annual multi-functional native legume of the USA. In the 16S rRNA gene phylogeny the strains were clustered in the Bradyrhizobium japonicumsuperclade. The results of analysis of the intergenic transcribed spacer (ITS) indicated less than 89.9 % similarity to other Bradyrhizobium species. Multilocus sequence analysis (MLSA) with four housekeeping genes (glnII, gyrB, recA and rpoB) confirmed the new group, sharing less than 95.2 % nucleotide identity with other species. The MLSA with 10 housekeeping genes (atpD, dnaK, gap, glnII, gltA, gyrB, pnp, recA, rpoB and thrC) indicated Bradyrhizobium daqingense as the closest species. Noteworthy, high genetic diversity among the strains was confirmed in the analyses of ITS, MLSA and BOX-PCR. Average nucleotide identity and digital DNA-DNA hybridization values were below the threshold of described Bradyrhizobium species, of 89.7 and 40 %, respectively. In the nifH and nodC phylogenies, the strains were grouped together, but with an indication of horizontal gene transfer, showing higher similarity to Bradyrhizobium arachidis and Bradyrhizobium forestalis. Other phenotypic, genotypic and symbiotic properties were evaluated, and the results altogether support the description of the CNPSo strains as representatives of the new species Bradyrhizobiumfrederickii sp. nov., with CNPSo 3426T (=USDA 10052T=U686T=CL 20T) as the type strain.

Bradyrhizobium niftali sp. nov., an effective nitrogen-fixing symbiont of partridge pea [Chamaecrista fasciculata (Michx.) Greene], a native caesalpinioid legume broadly distributed in the USA.

Information about the symbionts of legumes of the Caesalpinioideae subfamily is still limited, and we performed a polyphasic approach with three Bradyrhizobium strains-CNPSo 3448T, CNPSo 3394 and CNPSo 3442-isolated from Chamaecrista fasciculata, a native legume broadly distributed in the USA. In the phylogenetic analysis of both the 16S rRNA gene and the intergenic transcribed spacer, the CNPSo strains were clustered within the Bradyrhizobium japonicumsuperclade. Multilocus sequence analysis with six housekeeping genes-glnII, gyrB, recA, rpoB, atpD and dnaK-indicated that Bradyrhizobium diazoefficiens is the closest species, with 83 % of nucleotide identity. In the genome analyses of CNPSo 3448T, average nucleotide identity and digital DNA-DNA hybridization results confirmed higher similarity with B. diazoefficiens, with values estimated of 93.35 and 51.50 %, respectively, both below the threshold of the same species, confirming that the CNPSo strains represent a new lineage. BOX-PCR profiles indicated high intraspecific genetic diversity between the CNPSo strains. In the analyses of the symbiotic genes nodC and nifH the CNPSo strains were clustered with Bradyrhizobium arachidis, Bradyrhizobium forestalis, Bradyrhizobium cajani, Bradyrhizobium kavangense and Bradyrhizobium vignae, indicating a different phylogenetic history compared to the conserved core genes. Other physiological (C utilization, tolerance to antibiotics and abiotic stresses), chemical (fatty acid profile) and symbiotic (nodulation host range) properties were evaluated and are described. The data from our study support the description of the CNPSo strains as the novel species Bradyrhizobiumniftali sp. nov., with CNPSo 3448T (=USDA 10051T=U687T=CL 40T) designated as the type strain.

Additive genetic variance for lifetime fitness and the capacity for adaptation in an annual plant.

The immediate capacity for adaptation under current environmental conditions is directly proportional to the additive genetic variance for fitness, VA (W). Mean absolute fitness, W¯ , is predicted to change at the rate VA(W)W¯ , according to Fisher's Fundamental Theorem of Natural Selection. Despite ample research evaluating degree of local adaptation, direct assessment of VA (W) and the capacity for ongoing adaptation is exceedingly rare. We estimated VA (W) and W¯ in three pedigreed populations of annual Chamaecrista fasciculata, over three years in the wild. Contrasting with common expectations, we found significant VA (W) in all populations and years, predicting increased mean fitness in subsequent generations (0.83 to 6.12 seeds per individual). Further, we detected two cases predicting "evolutionary rescue," where selection on standing VA (W) was expected to increase fitness of declining populations ( W¯ < 1.0) to levels consistent with population sustainability and growth. Within populations, inter-annual differences in genetic expression of fitness were striking. Significant genotype-by-year interactions reflected modest correlations between breeding values across years, indicating temporally variable selection at the genotypic level that could contribute to maintaining VA (W). By directly estimating VA (W) and total lifetime W¯ , our study presents an experimental approach for studies of adaptive capacity in the wild.

Genetic diversity of Chamaecrista fasciculata (Fabaceae) from the USDA germplasm collection.

OBJECTIVE: Chamaecrista fasciculata is a widespread annual legume across Eastern North America, with potential as a restoration planting, biofuel crop, and genetic model for non-papillinoid legumes. As a non-Papilinoid, C. fasciculata, belongs to the Caesalpiniod group in which nodulation likely arose independently of the nodulation in Papilinoid and Mimosoid legumes. Thus, C. fasciculata is an attractive model system for legume evolution. In this study, we describe population structure and genetic diversity among 32 USDA germplasm accessions of C. fasciculata using 317 AFLP markers developed from 12 primer pairs, to assess where geographically there is the most genetic variation. RESULTS: We found that the C. fasciculata germplasm collection fall into four clusters with admixture among them. After correcting for outliers, our analysis shows two primary groups across Eastern and Central North America. To better understand the population biology of this species, further sampling of the full range of this widespread species is needed across North America, as well as the development of a larger set of markers providing denser coverage of the genome. Further sampling will help clarify geographical relationships in this widespread temperate species.

Phenolic variation among Chamaecrista nictitans subspecies and varieties revealed through UPLC-ESI(-)-MS/MS chemical fingerprinting.

INTRODUCTION: Comparative analysis of metabolic features of plants has a high potential for determination of quality control of active ingredients, ecological or chemotaxonomic purposes. Specifically, the development of efficient and rapid analytical tools that allow the differentiation among species, subspecies and varieties of plants is a relevant issue. Here we describe a multivariate model based on LC-MS/MS fingerprinting capable of discriminating between subspecies and varieties of the medicinal plant Chamaecrista nictitans, a rare distributed species in Costa Rica. METHODS: Determination of the chemical fingerprint was carried out on a LC-MS (ESI-QTOF) in negative ionization mode, main detected and putatively identified compounds included proanthocyanidin oligomers, several flavonoid C- and O-glycosides, and flavonoid acetates. Principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA) and cluster analysis of chemical profiles were performed. RESULTS: Our method showed a clear discrimination between the subspecies and varieties of Chamaecrista nictitans, separating the samples into four fair differentiated groups: M1 = C. nictitans ssp. patellaria; M2 = C. nictitans ssp. disadena; M3 = C. nictitans ssp. nictitans var. jaliscensis and M4 = C. nictitans ssp. disadena var. pilosa. LC-MS/MS fingerprint data was validated using both morphological characters and DNA barcoding with ITS2 region. The comparison of the morphological characters against the chemical profiles and DNA barcoding shows a 63% coincidence, evidencing the morphological similarity in C. nictitans. On the other hand, genetic data and chemical profiles grouped all samples in a similar pattern, validating the functionality of our metabolomic approach. CONCLUSION: The metabolomic method described in this study allows a reliably differentiation between subspecies and varieties of C. nictitans using a straightforward protocol that lacks extensive purification steps.

Selection of Bradyrhizobium or Ensifer symbionts by the native Indian caesalpinioid legume Chamaecrista pumila depends on soil pH and other edaphic and climatic factors.

Nodules of Chamaecrista pumila growing in several locations in India were sampled for anatomical studies and for characterization of their rhizobial microsymbionts. Regardless of their region of origin, the nodules were indeterminate with their bacteroids contained within symbiosomes which were surrounded by pectin. More than 150 strains were isolated from alkaline soils from the Thar Desert (Rajasthan), wet-acidic soils of Shillong (Meghalaya), and from trap experiments using soils from four other states with different agro-ecological regions. Molecular phylogenetic analysis based on five housekeeping (rrs, recA, glnII, dnaK andatpD) and two symbiotic (nodA and nifH) genes was performed for selected strains. Chamaecrista pumila was shown to be nodulated by niche-specific diverse strains of either Ensifer or Bradyrhizobium in alkaline (Thar Desert) to neutral (Tamil Nadu) soils and only Bradyrhizobium strains in acidic (Shillong) soils. Concatenated core gene phylogenies showed four novel Ensifer-MLSA types and nine Bradyrhizobium-MLSA types. Genetically diverse Ensifer strains harbored similar sym genes which were novel. In contrast, significant symbiotic diversity was observed in the Bradyrhizobium strains. The C. pumila strains cross-nodulated Vigna radiata and some wild papilionoid and mimosoid legumes. It is suggested that soil pH and moisture level played important roles in structuring the C. pumila microsymbiont community.

Expression of additive genetic variance for fitness in a population of partridge pea in two field sites.

Despite the importance of adaptation in shaping biological diversity over many generations, little is known about populations' capacities to adapt at any particular time. Theory predicts that a population's rate of ongoing adaptation is the ratio of its additive genetic variance for fitness, VA(W) , to its mean absolute fitness, W¯ . We conducted a transplant study to quantify W¯ and standing VA(W) for a population of the annual legume Chamaecrista fasciculata in one field site from which we initially sampled it and another site where it does not currently occur naturally. We also examined genotype-by-environment interactions, G × E, as well as its components, differences between sites in VA(W) and in rank of breeding values for fitness. The mean fitness indicated population persistence in both sites, and there was substantial VA(W) for ongoing adaptation at both sites. Statistically significant G × E indicated that the adaptive process would differ between sites. We found a positive correlation between fitness of genotypes in the "home" and "away" environments, and G × E was more pronounced as the life-cycle proceeds. This study exemplifies an approach to assessing whether there is sufficient VA(W) to support evolutionary rescue in populations that are declining.

Detrimental effects of rhizobial inoculum early in the life of partridge pea, Chamaecrista fasciculata.

PREMISE OF THE STUDY: Mutualistic relationships with microbes may aid plants in overcoming environmental stressors and increase the range of abiotic environments where plants can persist. Rhizobia, nitrogen-fixing bacteria associated with legumes, often confer fitness benefits to their host plants by increasing access to nitrogen in nitrogen-limited soils, but effects of rhizobia on host fitness under other stresses, such as drought, remain unclear. METHODS: In this greenhouse study, we varied the application of rhizobia (Bradyrhizobium sp.) inoculum and drought to examine whether the fitness benefits of rhizobia to their host, partridge pea (Chamaecrista fasciculata), would differ between drought and well-watered conditions. Plants were harvested 9 weeks after seeds were sown. KEY RESULTS: Young C. fasciculata plants that had been inoculated had lower biomass, leaf relative growth rate, and stem relative growth rate compared to young uninoculated plants in both drought and well-watered environments. CONCLUSIONS: Under the conditions of this study, the rhizobial interaction imposed a net cost to their hosts early in development. Potential reasons for this cost include allocating more carbon to nodule and root development than to aboveground growth and a geographic mismatch between the source populations of host plants and rhizobia. If developing plants incur such costs from rhizobia in nature, they may suffer an early disadvantage relative to other plants, whether conspecifics lacking rhizobia or heterospecifics.

Extrafloral nectaries of four varieties of Chamaecrista ramosa (Vogel) H.S.Irwin & Barneby (Fabaceae): anatomy, chemical nature, mechanisms of nectar secretion, and elimination.

Considering the importance of extrafloral nectaries (EFNs) in Fabaceae, the objectives of this research were to analyze (1) the anatomical and histochemical characteristics of the EFNs of Chamaecrista ramosa var. ramosa, C. ramosa var. curvifoliola, C. ramosa var. parvifoliola, and C. ramosa var. lucida and (2) the ultrastructure of the EFNs of C. ramosa var. ramosa. Standard techniques in plant anatomy and transmission electron microscopy were used. The anatomical analyses confirmed the characteristics described for extrafloral nectaries, evidencing three well-defined regions: epidermis, nectariferous, and subnectariferous parenchymas. Carbohydrates, proteins, pectins/mucilages, and lipids were detected by histochemical analyzes in all varieties. The ultrastructure of the EFNs of C. ramosa var. ramosa allowed the observation of microchannels at the external periclinal cell walls of the epidermis covering the secretory region. The nectariferous and subnectariferous parenchyma cells have periplasmic spaces, large plastids containing starch grains and plastoglobules, mitochondria, developed endoplasmic reticulum, large vacuoles with electron-dense contents, and membrane residues may be associated with the vacuole, suggesting the occurrence of autophagic processes. The anatomical, histochemical, and ultrastructural patterns revealed characteristics that confirm the glands of C. ramosa as extrafloral nectaries and suggest the eccrine mechanism of secretion.

Effects of multiple mutualists on plants and their associated arthropod communities.

Although most studies of mutualisms focus on a single partner at a time, host species often associate with multiple mutualist partners simultaneously. Because of potential interactions between mutualists, only studying a single type of mutualism could lead to a biased perspective of mutualism benefit and how mutualisms may scale-up to affect communities. The legume Chamaecrista fasciculata engages in a resource mutualism with nitrogen-fixing rhizobia and also forms symbiotic interactions with ants by providing nectar in exchange for defense against herbivores. Although they provide very different benefits to the plant, both mutualists receive carbon resources from the plant. As a result, these two mutualists are likely to interact, potentially competing for carbon resources or mutually benefitting each other via their positive effects on plant hosts. In a full-factorial field experiment, we explored how rhizobia and ants influence one another, C. fasciculata fitness, and the associated arthropod community. Ants reduced plant allocation to rhizobia, but ants also increased rhizobia contamination of uninoculated plants, suggesting that ants may disperse rhizobia. In turn, rhizobia increased ant abundances, with ants preferentially tending plants with rhizobia. Chamaecrista fasciculata received substantial fitness benefits from rhizobia; in contrast, associating with ants reduced fitness. Additionally, the mutualists interacted to influence the abundance of other arthropods found on the plants. Rhizobia increased arthropod abundances, likely because more nitrogen-rich leaf tissue was more attractive to arthropod herbivores, but ants negated these increases. As these results illustrate, multiple mutualists may interact, influencing each other's abundance and the abundance of other community members.