Museomics unveil systematics, diversity and evolution of Australian cycad-pollinating weevils.

Weevils have been shown to play significant roles in the obligate pollination of Australian cycads. In this study, we apply museomics to produce a first molecular phylogeny estimate of the Australian cycad weevils, allowing an assessment of their monophyly, placement and relationships. Divergence dating suggests that the Australian cycad weevils originated from the Late Oligocene to the Middle Miocene and that the main radiation of the cycad-pollinating groups occurred from the Middle to the Late Miocene, which is congruent with the diversification of the Australian cycads, thus refuting any notion of an ancient ciophilous system in Australia. Taxonomic studies reveal the existence of 19 Australian cycad weevil species and that their associations with their hosts are mostly non-species-specific. Co-speciation analysis shows no extensive co-speciation events having occurred in the ciophilous system of Australian cycads. The distribution pattern suggests that geographical factors, rather than diversifying coevolution, constitute the overriding process shaping the Australian cycad weevil diversity. The synchronous radiation of cycads and weevil pollinators is suggested to be a result of the post-Oligocene diversification common in Australian organisms.

Composition of Sexual Fluids in Cycas revoluta Ovules During Pollination and Fertilization.

The composition of fluids that mediate fertilization in cycads is described for the first time. Using tandem mass spectrometry, proteomes of two stages of fluid production, megagametophyte fluid and archegonial chamber fluid production, are compared in Cycas revoluta. These were compared with the proteome of another sexual fluid produced by ovules, the pollination drop proteins. Cycad ovules produce complex liquids immediately prior fertilization. Compared with the pollination drops that mainly had few proteins in classes involved in defense and carbohydrate modification, megagametophyte fluid and archegonial chamber fluid had larger proteomes with many more protein classes, e.g. proteins involved in programmed cell death. Using high-performance liquid chromatography, megagametophyte fluid and archegonial chamber fluid were shown to have elevated concentrations of smaller molecular weight molecules including glucose, pectin and glutamic acid. Compared to megagametophyte fluid, archegonial chamber fluid had elevated pH as well as higher osmolality. Supplementary Information: The online version contains supplementary material available at 10.1007/s12229-021-09271-1.

A modified diet to support conservation of the Atala hairstreak butterfly (Eumaeus atala Poey).

Raising insects in a laboratory for release into the wild is a common conservation practice, but maintaining breeding colonies year-round can be limited by seasonal food availability. Food availability is particularly challenging for insects which depend on specific host plants. For example, our early efforts to rear the imperiled Atala hairstreak butterfly (Eumaeus atala Poey) resulted in colony failure during winter due to lack of food. To overcome this barrier, we developed a modified freeze-dried host plant diet to support the colony. The diet consisted of reconstituted freeze-dried leaves and stems from fresh-growth coontie (Zamia integrifolia), the host plant for the Atala butterflies. We fed larvae less than 9 mm on this freeze-dried diet and transferred them to live coontie plants after they were more than 9 mm. We reared a colony of Atala butterflies using these methods for 859 days, resulting in more than 3400 animals released into the wild. Comparing colony counts during that time period to the 548 days we reared them without modified freeze-dried diet showed a clear benefit in using freeze-dried diet. A growth trial (N = 40) of larvae fed on only freeze-dried diet compared to larvae fed on fresh coontie cuttings found no significant difference in larval or pupal development between groups (p = 0.71 and p = 0.47, respectively). We, therefore, conclude that the freeze-dried diet provided an appropriate alternative for Atala colonies when fresh growth from the host plant is unavailable, and we recommend use of this technique for raising other host plant-dependent insect species of conservation concern.

Cycas revoluta (sago cycad) exposures reported to Texas poison centers.

BACKGROUND: Cycas revoluta (sago cycad or palm) is a popular ornamental plant in the United States. All parts of the plant contain toxins such cycasin and beta-methylamino-l-alanine, the ingestion of which can be harmful to humans and animals. The objective of this study was to characterize C. revoluta exposures reported to poison centers. METHODS: Cases were C. revoluta exposures reported to Texas poison centers during 2000-2018. The distribution of cases by selected variables was determined. RESULTS: Of 192 total C. revoluta exposures, the most common exposure routes were ingestion (55.7%) and dermal (34.4%). The patient age distribution was 28.1% 5 years or less, 15.1% 6-12 years, 4.7% 13-19 years, and 50.5% 20 years or more; 55.2% were male. The exposure was unintentional in 92.2% of the cases and occurred at the patient's own residence in 94.8%. The patient was managed on site in 78.6% of the cases, already at/en route to a healthcare facility in 12.0%, and referred to a healthcare facility in 8.9%. The most common reported clinical effects were dermal (23.4%), particularly puncture/wound (16.1%), dermal irritation/pain (14.6%), and edema (8.9%), followed by gastrointestinal (13.0%), particularly vomiting (8.9%) and nausea (7.8%). CONCLUSION: Most of the C. revoluta exposures tended to be unintentional and occurred at home. Although most of the C. revoluta exposures involved ingestion, 35% were dermal, and the most frequently reported clinical effects were dermal followed by gastrointestinal. Most of the exposures were not serious and were managed outside of a healthcare facility.

First cycad seedling foliage from the fossil record and inferences for the Cenozoic evolution of cycads.

The morphology of the early ontogenetic stages of cycad foliage may help resolve the relationships between extinct to extant cycad lineages. However, prior to this study, fossil evidence of cycad seedlings was not known. We describe a compression fossil of cycad eophylls with co-occurring fully developed leaves of adult specimens from the early Palaeocene ( ca 63.8 Ma) Castle Rock flora from the Denver Basin, CO, USA and assign it to the fossil genus Dioonopsis (Cycadales) based on leaf morphology and anatomy. The new fossil seedling foliage is particularly important because fully differentiated pinnate leaves of adult plants and the eophylls belong to the same species based on shared epidermal micromorphology, therefore, increasing the number of morphological characteristics that can be used to place Dioonopsis phylogenetically. Significantly, the seedling fossil has a basic foliage structure that is very similar to seedlings of extant cycads, which is consistent with a cycadalean affinity of Dioonopsis. Nevertheless, the set of morphological characters in the seedling and adult specimens of Dioonopsis suggests a distant relationship between Dioonopsis and extant Dioon. This indicates that extinct lineages of cycads were present and widespread during the early Cenozoic (Palaeogene) coupled with the subordinate role of extant genera in the Palaeogene fossil record of cycads.

Shifting Quaternary migration patterns in the Bahamian archipelago: Evidence from the Zamia pumila complex at the northern limits of the Caribbean island biodiversity hotspot.

PREMISE OF THE STUDY: The Bahamas archipelago is formed by young, tectonically stable carbonate banks that harbor direct geological evidence of global ice-volume changes. We sought to detect signatures of major changes on gene flow patterns and reconstruct the phylogeographic history of the monophyletic Zamia pumila complex across the Bahamas. METHODS: Nuclear molecular markers with both high and low mutation rates were used to capture two different time scale signatures and test several gene flow and demographic hypotheses. KEY RESULTS: Single-copy nuclear genes unveiled apparent ancestral admixture on Andros, suggesting a significant role of this island as main hub of diversity of the archipelago. We detected demographic and spatial expansion of the Zamia pumila complex on both paleo-provinces around the Piacenzian (Pliocene)/Gelasian (Pleistocene). Populations evidenced signatures of different migration models that have occurred at two different times. Populations on Long Island (Z. lucayana) may either represent a secondary colonization of the Bahamas by Zamia or a rapid and early-divergence event of at least one population on the Bahamas. CONCLUSIONS: Despite changes in migration patterns with global climate, expected heterozygosity with both marker systems remains within the range reported for cycads, but with significant levels of increased inbreeding detected by the microsatellites. This finding is likely associated with reduced gene flow between and within paleo-provinces, accompanied by genetic drift, as rising seas enforced isolation. Our study highlights the importance of the maintenance of the predominant direction of genetic exchange and the role of overseas dispersion among the islands during climate oscillations.

Tau pathology involves protein phosphatase 2A in parkinsonism-dementia of Guam.

Parkinsonism-dementia (PD) of Guam is a neurodegenerative disease with parkinsonism and early-onset Alzheimer-like dementia associated with neurofibrillary tangles composed of hyperphosphorylated microtubule-associated protein, tau. ß-N-methylamino-l-alanine (BMAA) has been suspected of being involved in the etiology of PD, but the mechanism by which BMAA leads to tau hyperphosphorylation is not known. We found a decrease in protein phosphatase 2A (PP2A) activity associated with an increase in inhibitory phosphorylation of its catalytic subunit PP2Ac at Tyr(307) and abnormal hyperphosphorylation of tau in brains of patients who had Guam PD. To test the possible involvement of BMAA in the etiopathogenesis of PD, we studied the effect of this environmental neurotoxin on PP2A activity and tau hyperphosphorylation in mouse primary neuronal cultures and metabolically active rat brain slices. BMAA treatment significantly decreased PP2A activity, with a concomitant increase in tau kinase activity resulting in elevated tau hyperphosphorylation at PP2A favorable sites. Moreover, we found an increase in the phosphorylation of PP2Ac at Tyr(307) in BMAA-treated rat brains. Pretreatment with metabotropic glutamate receptor 5 (mGluR5) and Src antagonists blocked the BMAA-induced inhibition of PP2A and the abnormal hyperphosphorylation of tau, indicating the involvement of an Src-dependent PP2A pathway. Coimmunoprecipitation experiments showed that BMAA treatment dissociated PP2Ac from mGluR5, making it available for phosphorylation at Tyr(307). These findings suggest a scenario in which BMAA can lead to tau pathology by inhibiting PP2A through the activation of mGluR5, the consequent release of PP2Ac from the mGluR5-PP2A complex, and its phosphorylation at Tyr(307) by Src.

Crystal structures and inhibitor binding properties of plant class V chitinases: the cycad enzyme exhibits unique structural and functional features.

A class V (glycoside hydrolase family 18) chitinase from the cycad Cycas revoluta (CrChiA) is a plant chitinase that has been reported to possess efficient transglycosylation (TG) activity. We solved the crystal structure of CrChiA, and compared it with those of class V chitinases from Nicotiana tabacum (NtChiV) and Arabidopsis thaliana (AtChiC), which do not efficiently catalyze the TG reaction. All three chitinases had a similar (α/ß)8 barrel fold with an (α + ß) insertion domain. In the acceptor binding site (+1, +2 and +3) of CrChiA, the Trp168 side chain was found to stack face-to-face with the +3 sugar. However, this interaction was not found in the identical regions of NtChiV and AtChiC. In the DxDxE motif, which is essential for catalysis, the carboxyl group of the middle Asp (Asp117) was always oriented toward the catalytic acid Glu119 in CrChiA, whereas the corresponding Asp in NtChiV and AtChiC was oriented toward the first Asp. These structural features of CrChiA appear to be responsible for the efficient TG activity. When binding of the inhibitor allosamidin was evaluated using isothermal titration calorimetry, the changes in binding free energy of the three chitinases were found to be similar to each other, i.e. between -9.5 and -9.8 kcal mol(-1) . However, solvation and conformational entropy changes in CrChiA were markedly different from those in NtChiV and AtChiC, but similar to those of chitinase A from Serratia marcescens (SmChiA), which also exhibits significant TG activity. These results provide insight into the molecular mechanism underlying the TG reaction and the molecular evolution from bacterial chitinases to plant class V chitinases.

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction.

Exposure to ß-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Cycads and their association with certain neurodegenerative diseases.

INTRODUCTION: Cycads are ornamental plants that in some parts of the world are used as fresh food or raw material for producing flour with a high nutritional value. However, they also contain active compounds, including methylazoxymethanol, ß-methylamino-L-alanine, ß-alanine-L-oxalylamino and cycasin, which may produce neurotoxic effects. Some studies have associated consuming cycads and their derivatives with neurodegenerative diseases such as amyotrophic lateral sclerosis/Parkinsonism dementia complex, and other diseases characterised by motor impairment. Therefore, we must not forget that any product, no matter how natural, may present health risks or benefits depending on the chemical compounds it contains and the susceptibility of those who consume it. DEVELOPMENT: We completed a literature analysis to evaluate the neurotoxic properties of cycads and their association with neurological diseases in order to provide structured scientific information that may contribute to preventing health problems in people who use these plants. CONCLUSION: Cycads contain neurotoxic compounds that may contribute to the development of neurological diseases when ingested improperly. We must be mindful of the fact that while some plants have a high nutritional value and may fill the food gap for vulnerable populations, they can also be toxic and have a negative impact on health.

A new distribution and host record for the rare moth, Callioratis millari (Lepidoptera: Geometridae), and some ecological observations.

Callioratis millari Hampson (Lepidoptera: Geometridae) is a Critically Endangered moth endemic to South Africa. Despite extensive searches, it was previously known only from the Entumeni Nature Reserve in KwaZulu-Natal, where its larvae exclusively feed on the cycad Stangeria eriopus (Kunze) Baill (Cycadales: Stangeriaceae). In July 2022, a new population of C. millari was discovered in the Kabouga section of Addo Elephant National Park in the Eastern Cape. Larvae of C. millari were feeding on the cycad Encephalartos caffer (Thunb.) Lehm (Cycadales: Zamiaceae), which also constitutes a new host record. In June 2023, we determined larval incidence and herbivory at this new locality, offering insights into the ecological requirements of C. millari. Known C. millari localities, although ecologically different, share low altitudes (700-950 m a.s.l.), moderate to high rainfall, and grassy habitats with sparse woody cover. A total of 59 larvae were counted in Kabouga, mostly in the fifth and sixth (final) instars. Herbivory incidence was lower on smaller plants and those covered by other vegetation. The flight period of adult C. millari likely occurs between mid-March and April in Kabouga, but further investigation is needed to clarify this. The peak period of larval occurrence in Kabouga occurs during the driest and coldest months of the year (May-July). Considering limited habitat availability, host plant poaching, and the risk of untimely fires, the species should be considered highly threatened. This study adds to our understanding of the biology of C. millari and provides information on its ecological requirements and may contribute to making informed management decisions.

Two draft genomes of fungal leaf endophytes from tropical gymnosperms.

Two ascomycetes, Neofusicoccum sp. and Xylaria sp., were isolated from healthy leaves of the tropical gymnosperms Zamia pseudoparasitica (Z2) and Zamia nana (Z50) from Panama. The two draft genomes possess a broad predicted repertoire of carbohydrate-degrading CAZymes, peptidases, and secondary metabolites, with more secondary metabolite clusters in the Xylaria isolate.

Phylometagenomics of cycad coralloid roots reveals shared symbiotic signals.

Cycads are known to host symbiotic cyanobacteria, including Nostocales species, as well as other sympatric bacterial taxa within their specialized coralloid roots. Yet, it is unknown if these bacteria share a phylogenetic origin and/or common genomic functions that allow them to engage in facultative symbiosis with cycad roots. To address this, we obtained metagenomic sequences from 39 coralloid roots sampled from diverse cycad species and origins in Australia and Mexico. Culture-independent shotgun metagenomic sequencing was used to validate sub-community co-cultures as an efficient approach for functional and taxonomic analysis. Our metanalysis shows a host-independent microbiome core consisting of seven bacterial orders with high species diversity within the identified taxa. Moreover, we recovered 43 cyanobacterial metagenome-assembled genomes, and in addition to Nostoc spp., symbiotic cyanobacteria of the genus Aulosira were identified for the first time. Using this robust dataset, we used phylometagenomic analysis to reveal three monophyletic cyanobiont clades, two host-generalist and one cycad-specific that includes Aulosira spp. Although the symbiotic clades have independently arisen, they are enriched in certain functional genes, such as those related to secondary metabolism. Furthermore, the taxonomic composition of associated sympatric bacterial taxa remained constant. Our research quadruples the number of cycad cyanobiont genomes and provides a robust framework to decipher cyanobacterial symbioses, with the potential of improving our understanding of symbiotic communities. This study lays a solid foundation to harness cyanobionts for agriculture and bioprospection, and assist in conservation of critically endangered cycads.

Seed dispersal of the Australian cycad Macrozamia miquelii (Zamiaceae): are cycads megafauna-dispersed "grove forming" plants?

PREMISE OF THE STUDY: Plants that invest in large, heavy seeds and colorful, fleshy fruits or analogous structures seem adapted for dispersal by large vertebrates. Some such plants, like Australian cycads in the genus Macrozamia, do not disperse well, which could be explained by seed-dispersal relationships with megafauna that are rare or extinct in contemporary ecosystems. Such plants provide an opportunity to investigate the ecological consequences of low seed-dispersal distances. • METHODS: We investigated seed dispersal of Macrozamia miquelii in Central Queensland by tracking the fate of marked seeds, identifying the dispersal fauna and quantifying population demography and spatial structure. • KEY RESULTS: We found that 70-100% of marked seeds remained within 1 m of maternal females (cycads are dioecious). Of the 812 seeds recovered (from 840 originally marked) only 24 dispersed >1 m from maternal females, the greatest observed dispersal being 5 m. We found an average of 2.2 seedlings and 0.7 juveniles within 1.5 m of mature females, which suggests that most seeds that remain in the vicinity of maternal females perish. Within-stand densities ranged between 1000 and 5000 plants/ha. The brushtail possum Trichosurus vulpecula was the only animal observed to move the seeds. • CONCLUSIONS: Macrozamia are adapted for dispersal by megafauna that are rare or absent in contemporary ecosystems. We argue that Macrozamia are "grove forming" plants that derive ecological benefit from existing as high-density, spatially discrete populations, the function of megafaunal dispersal adaptations being the infrequent dispersal of seeds en masse to establish new such groves in the landscape.

Characterization of starch extracted from seeds of Cycas revoluta.

Introduction: Starch is major component in the big seeds of Cycas revoluta, however the characteristics of Cycas revoluta remain unknown. Methods: In this study, the physicochemical and structural properties of two starch samples extracted from Cycad revoluta seeds were systematically investigated, using various techniques. Results: The amylose contents of the two samples were 34.3 % and 35.5%, respectively. The spherical-truncated shaped starch granules possessed A-type crystallinity, and had an average diameter less than 15 µm. Compared to most commonly consumed cereal and potato starch, Cycad revoluta starch showed distinctive characteristics. For physicochemical properties, in the process of gelatinization, the Cycad revoluta starch showed similar viscosity profile to starches of some potato varieties, but Cycad revoluta starch had higher gelatinization temperature. Upon cooling, Cycad revoluta starch formed harder gels than rice starch. For structure, the molecular weight (indexed by Mw, Mn and Rz values), branching degree and the branch chain length distribution were determined. Discussion: The results suggested that Cycad revoluta starch were different in structure from the main-stream starches. Notable differences in some starch traits between the two samples were recorded, which could be attributed to environmental factors. In general, this study provides useful information on the utilization of Cycad revoluta starch in both food and non-food industries.

Ghost introgression facilitates genomic divergence of a sympatric cryptic lineage in Cycas revoluta.

A cryptic lineage is a genetically diverged but morphologically unrecognized variant of a known species. Clarifying cryptic lineage evolution is essential for quantifying species diversity. In sympatric cryptic lineage divergence compared with allopatric divergence, the forces of divergent selection and mating patterns override geographical isolation. Introgression, by supplying preadapted or neutral standing genetic variations, can promote sympatric cryptic lineage divergence via selection. However, most studies concentrated on extant species introgression, ignoring the genetic legacy of introgression from extinct or unsampled lineages ("ghost introgression"). Cycads are an ideal plant for studying the influence of ghost introgression because of their common interspecific gene flow and past high extinction rate. Here, we utilized reference-based ddRADseq to clarify the role of ghost introgression in the evolution of a previously identified sympatric cryptic lineage in Cycas revoluta. After re-evaluating the evolutionary independency of cryptic lineages, the group-wise diverged single-nucleotide polymorphisms among sympatric and allopatric lineages were compared and functionally annotated. Next, we employed an approximate Bayesian computation method for hypothesis testing to clarify the cryptic lineage evolution and ghost introgression effect. SNPs with the genomic signatures of ghost introgression were further annotated. Our results reconfirmed the evolutionary independency of cryptic lineage among C. revoluta and demonstrated that ghost introgression to the noncryptic lineage facilitated their divergence. Gene function related to heat stress and disease resistance implied ecological adaptation of the main extant populations of C. revoluta.

Cone humidity is a strong attractant in an obligate cycad pollination system.

Studies of pollination biology often focus on visual and olfactory aspects of attraction, with few studies addressing behavioral responses and morphological adaptation to primary metabolic attributes. As part of an in-depth study of obligate nursery pollination of cycads, we find that Rhopalotria furfuracea weevils show a strong physiological response and behavioral orientation to the cone humidity of the host plant Zamia furfuracea in an equally sensitive manner to their responses to Z. furfuracea-produced cone volatiles. Our results demonstrate that weevils can perceive fine-scale differences in relative humidity (RH) and that individuals exhibit a strong behavioral preference for higher RH in binary choice assays. Host plant Z. furfuracea produces a localized cloud of higher than ambient humidity around both pollen and ovulate cones, and R. furfuracea weevils preferentially land at the zone of maximum humidity on ovulate cones, i.e., the cracks between rows of megasporophylls that provide access to the ovules. Moreover, R. furfuracea weevils exhibit striking antennal morphological traits associated with RH perception, suggesting the importance of humidity sensing in the evolution of this insect lineage. Results from this study suggest that humidity functions in a signal-like fashion in this highly specialized pollination system and help to characterize a key pollination-mediating trait in an ancient plant lineage.

Taxonomic Revision of the Genus Miltotranes Zimmerman, 1994 (Coleoptera: Curculionidae: Molytinae), the Bowenia-Pollinating Cycad Weevils in Australia, with Description of a New Species and Implications for the Systematics of Bowenia.

The Australian endemic weevils of the genus Miltotranes Zimmerman, 1994 (Curculionidae: Molytinae: Tranes group), comprising two species, M. prosternalis (Lea, 1929) and M. subopacus (Lea, 1929), are highly host-specific and the only known pollinators of Bowenia cycads, which comprise two CITES-protected species restricted to Tropical Queensland in Australia. In the present study, the taxonomy of Miltotranes is reviewed, a lectotype for the name Tranes prosternalis Lea, 1929 is designated and a new species associated with the Bowenia population in the McIlwraith Range is described as M. wilsoni sp. n. The descriptions and diagnoses of all species are supplemented with illustrations of their habitus and salient structures, and an identification key to all species and a distribution map are provided. Potential implications of the new species and of the taxonomy and biogeography of Miltotranes overall on the systematics and conservation of Bowenia are discussed.

Are legumes different? Origins and consequences of evolving nitrogen fixing symbioses.

Nitrogen fixing symbioses between plants and bacteria are ancient and, while not numerous, are formed in diverse lineages of plants ranging from microalgae to angiosperms. One symbiosis stands out as the most widespread one is that between legumes and rhizobia, leading to the formation of nitrogen-fixing nodules. The legume family is one of the largest and most diverse group of plants and legumes have been used by humans since the beginning of agriculture, both as high nitrogen food, as well as pastures and rotation crops. One open question is whether their ability to form a nitrogen-fixing symbiosis has contributed to legumes' success, and whether legumes have any unique characteristics that have made them more diverse and widespread than other groups of plants. This review examines the evolutionary journey that has led to the diversification of legumes, in particular its nitrogen-fixing symbiosis, and asks four questions to investigate which legume traits might have contributed to their success: 1. In what ways do legumes differ from other plant groups that have evolved nitrogen-fixing symbioses? In order to answer this question, the characteristics of the symbioses, and efficiencies of nitrogen fixation are compared between different groups of nitrogen fixing plants. 2. Could certain unique features of legumes be a reason for their success? This section examines the manifestations and possible benefits of a nitrogen-rich 'lifestyle' in legumes. 3. If nitrogen fixation was a reason for such a success, why have some species lost the symbiosis? Formation of symbioses has trade-offs, and while these are less well known for non-legumes, there are known energetic and ecological reasons for loss of symbiotic potential in legumes. 4. What can we learn from the unique traits of legumes for future crop improvements? While exploiting some of the physiological properties of legumes could be used to improve legume breeding, our increasing molecular understanding of the essential regulators of root nodule symbioses raise hope of creating new nitrogen fixing symbioses in other crop species.