SarCTAB: an efficient and cost-effective DNA isolation protocol from geophytes.

Geophytes are herbaceous plants that grow anew from underground buds and are excellent models to study storage organ formation. However, molecular studies involving geophytes are constrained due to the presence of a wide spectrum of polysaccharides and polyphenols that contaminate the genomic DNA. At present, several protocols exist for the extraction of genomic DNA from different plant species; however, isolating high-quality DNA from geophytes is challenging. Such challenges are further complexed by longer incubation time and multiple precipitation steps involved in existing DNA isolation methods. To overcome such problems, we aimed to establish a DNA extraction method (SarCTAB) which is an economical, quick, and sustainable way of DNA isolation from geophytes. We improved the traditional CTAB method by optimizing key ingredients such as sarcosine, ß-mercaptoethanol, and high molar concentration of sodium chloride (NaCl), which resulted in high concentration and good-quality DNA with lesser polysaccharides, proteins, and polyphenols. This method was evaluated to extract DNA from storage organs of six different geophytes. The SarCTAB method provides an average yield of 1755 ng/µl of high-quality DNA from 100 mg of underground storage tissues with an average standard purity of 1.86 (260/280) and 1.42 (260/230). The isolated genomic DNA performed well with Inter-simple sequence repeat (ISSR) amplification, restriction digestion with EcoRI, and PCR amplification of plant barcode genes viz. matK and rbcL. Also, the cost involved in DNA isolation was low when compared to that with commercially available kits. Overall, SarCTAB method works effectively to isolate high-quality genomic DNA in a cost-effective manner from the underground storage tissues of geophytes, and can be applied for next-generation sequencing, DNA barcoding, and whole genome bisulfite sequencing.

Functional and ecological diversification of underground organs in Solanum.

The evolution of geophytes in response to different environmental stressors is poorly understood largely due to the great morphological variation in underground plant organs, which includes species with rhizomatous structures or underground storage organs (USOs). Here we compare the evolution and ecological niche patterns of different geophytic organs in Solanum L., classified based on a functional definition and using a clade-based approach with an expert-verified specimen occurrence dataset. Results from PERMANOVA and Phylogenetic ANOVAs indicate that geophytic species occupy drier areas, with rhizomatous species found in the hottest areas whereas species with USOs are restricted to cooler areas in the montane tropics. In addition, rhizomatous species appear to be adapted to fire-driven disturbance, in contrast to species with USOs that appear to be adapted to prolonged climatic disturbance such as unfavorable growing conditions due to drought and cold. We also show that the evolution of rhizome-like structures leads to changes in the relationship between range size and niche breadth. Ancestral state reconstruction shows that in Solanum rhizomatous species are evolutionarily more labile compared to species with USOs. Our results suggest that underground organs enable plants to shift their niches towards distinct extreme environmental conditions and have different evolutionary constraints.

Metabolic Profiling in Tuberous Roots of Ranunculus asiaticus L. as Influenced by Vernalization Procedure.

Ranunculus asiaticus L. is an ornamental geophyte. In commercial practice, it is mainly propagated by rehydrated tuberous roots. Vernalization before planting is a common practice to overcome the natural dormancy of tuberous roots; however, little is known about the mechanisms underlying the plant's response to low temperatures. We investigated the influence of three preparation procedures of tuberous roots, only rehydration (control, C), and rehydration plus vernalization at 3.5 °C for 2 weeks (V2) and for 4 weeks (V4), on plant growth, leaf photosynthesis, flowering, and metabolism in plants of two hybrids, MBO (early flowering, pale orange flower) and MDR (medium earliness, bright orange flower), grown in pots in an unheated greenhouse. We reported the responses observed in the aerial part in a previous article in this journal. In this paper, we show changes in the underground organs in carbohydrate, amino acids, polyphenols, and protein levels throughout the growing cycle in the different plant stages: pre-planting, vegetative growth, and flowering. The metabolic profile revealed that the two hybrids had different responses to the root preparation procedure. In particular, MBO synthesized GABA and alanine after 2 weeks and sucrose after 4 weeks of vernalization. In contrast, MDR was more sensitive to vernalization; in fact, a higher synthesis of polyphenols was observed. However, both hybrids synthesized metabolites that could withstand exposure to low temperatures.

Novel roles of HSFs and HSPs, other than relating to heat stress, in temperature-mediated flowering.

The thermotolerant ability of heat shock factors (HSFs) and heat shock proteins (HSPs) in plants has been shown. Recently, focus has been on their function in plant growth and development under non-stress conditions. Their role in flowering has been suggested given that lower levels of HSF/HSPs resulted in altered flowering in Arabidopsis. Genetic and molecular studies of Arabidopsis HSF/HSP mutants advocated an association with temperature-mediated regulation of flowering, but the fundamental genetic mechanism behind this phenomenon remains obscure. Here we outline plausible integration between HSFs/HSPs and temperature-dependent pathways in plants regulating flowering. Moreover, we discuss how similar pathways can be present in thermoperiodic geophytic plants that require ambient high temperatures for flowering induction.

Vernalization Procedure of Tuberous Roots Affects Growth, Photosynthesis and Metabolic Profile of Ranunculus asiaticus L.

In Ranunculus asiaticus L., vernalization of propagation material is a common practice for the production scheduling of cut flowers, however little is known about the plant physiology and metabolism of this species as affected by cold treatments. We investigated the influence of two hybrids, MBO and MDR, and three preparation procedures of tuberous roots, only rehydration (control, C), and rehydration plus vernalization at 3.5 °C for 2 weeks (V2) and for 4 weeks (V4), on plant growth and flowering, leaf photosynthesis, and leaf metabolic profile in plants grown in pot in a cold greenhouse. Net photosynthesis (NP) was higher in MDR than in MBO. In the two genotypes, the NP did not change in V2 and increased in V4 compared to C in MBO, while was unaffected by vernalization in MDR. Quantum yield of PSII electron transport (ΦPSII), linear electron transport rate (ETR) and non-photochemical quenching (NPQ) did not differ in the two hybrids, whereas maximal PSII photochemical efficiency (Fv/Fm) was higher in MBO than in MDR. Fluorescence indexes were unaffected by the preparation procedure, except for ETR, which decreased in V2 compared to C and V4 in MDR. A significant interaction between genotype and preparation procedure was found in plant leaf area, which was reduced only in V4 in MBO, while decreased in both the vernalization procedures in MDR. In Control plants, flowering started in 65 days in MBO and 69 days in MDR. Compared to controls, both the vernalization treatments anticipated flowering in MDR, while they were detrimental or only slightly efficient in promoting flowering in MBO. Vernalization always reduced the quality of flower stems in both the hybrids.

Genome size and endoreplication in two pairs of cytogenetically contrasting species of Pulmonaria (Boraginaceae) in Central Europe.

Genome size is species-specific feature and commonly constant in an organism. In various plants, DNA content in cell nucleus is commonly increased in process of endoreplication, cellular-specific multiplication of DNA content without mitosis. This leads to the endopolyploidy, the presence of multiplied chromosome sets in a subset of cells. The relationship of endopolyploidy to species-specific genome size is rarely analysed and is not fully understood. While negative correlation between genome size and endopolyploidy level is supposed, this is species- and lineage-specific. In the present study, we shed light on this topic, exploring both genome size and endoreplication-induced DNA content variation in two pairs of morphologically similar species of Pulmonaria, P. obscura-P. officinalis and P. mollis-P. murinii. We aim (i) to characterize genome size and chromosome numbers in these species using cytogenetic, root-tip squashing and flow cytometry (FCM) techniques; (ii) to investigate the degree of endopolyploidy in various plant organs, including the root, stem, leaf, calyx and corolla using FCM; and (iii) to comprehensively characterize and compare the level of endopolyploidy and DNA content in various organs of all four species in relation to species systematic relationships and genome size variation. We have confirmed the diploid-dysploid nature of chromosome complements, and divergent genome sizes for Pulmonaria species: P. murinii with 2n = 2x = 14, 2.31 pg/2C, P. obscura 2n = 2x = 14, 2.69 pg/2C, P. officinalis 2n = 2x = 16, 2.96 pg/2C and P. mollis 2n = 2x = 18, 3.18 pg/2C. Endopolyploidy varies between species and organs, and we have documented 4C-8C in all four organs and up to 32C (64C) endopolyploid nuclei in stems at least in some species. Two species with lower genome sizes tend to have higher endopolyploidy levels than their closest relatives. Endoreplication-generated tissue-specific mean DNA content is increased and more balanced among species in all four organs compared to genome size. Our results argue for the narrow relationship between genome size and endopolyploidy in the present plant group within the genus Pulmonaria, and endopolyploidization seems to play a compensatory developmental role in organs of related morphologically similar species.

Phylogenetic endemism of the orchids of Megamexico reveals complementary areas for conservation.

Orchid diversity provides a unique opportunity to further our understanding of biotic and abiotic factors linked to patterns of richness, endemism, and phylogenetic endemism in many regions. However, orchid diversity is consistently threatened by illegal trade and habitat transformation. Here, we identified areas critical for orchid conservation in the biogeographic province of Megamexico. For this purpose, we evaluated orchid endemism, phylogenetic diversity, and phylogenetic endemism within Megamexico and characterized orchid life forms. Our results indicate that the majority of the regions with the highest estimates of endemism and phylogenetic endemism are in southern Mexico and northern Central America, mostly located on the Pacific side of Megamexico. Among the most important orchid lineages, several belong to epiphytic lineages such as Pleurothallidinae, Laeliinae and Oncidiinae. We also found that species from diverse and distantly related lineages converge in montane forests where suitable substrates for epiphytes abound. Furthermore, the southernmost areas of phylogenetic diversity and endemism of Megamexico are in unprotected areas. Thus, we conclude that the most critical areas for orchid conservation in Megamexico are located in southern Mexico and northern Central America. We recommend that these areas should be given priority by the Mexican system of natural protected areas as complementary conservation areas.

Negative effects of winter and spring warming on the regeneration of forest spring geophytes.

The climate is changing rapidly, provoking species to shift their ranges poleward and upslope. We currently lack a mechanistic understanding of the effect of warmer temperatures on plants, especially for seasonally distinct patterns. Spring geophytes are emblematic forest plants that have a short aboveground lifecycle in the first half of the year and are thus particularly sensitive to winter and spring warming. We set up a warming experiment with separate and combined winter and spring warming on seedlings of three European spring geophytes: Anemone nemorosa, Hyacinthoides non-scripta and Ornithogalum pyrenaicum. Seedling emergence and plant height were recorded at the end of winter and spring treatment, when also biomass of the root, shoot and storage organ was determined. We found negative effects of combined winter and spring warming on seedling emergence. The weight of the storage organ proved to be the best indicator of seedling performance and was negatively affected by separate winter warming in Anemone and by spring warming in Hyacinthoides. Successful seedling emergence was jeopardized by the absence of a cold period, while seedling performance seemed to be negatively influenced directly by higher temperatures through a phenological shift. Our findings confirm that warmer winter and spring temperatures could hamper regeneration of spring geophytes.

The cost of cooking for foragers.

Cooked food provides more calories to a consumer than raw food. When our human ancestors adopted cooking, the result was an increase in the caloric value of the diet. Generating the heat to cook, however, requires fuel, and accessing fuel was and remains a common problem for humanity. Cooking also frequently requires monitoring, special technology and other investments. These cooking costs should vary greatly across multiple contexts. Here I explain how to quantify this cooking trade-off as the ratio of the energetic benefits of cooking to the increased cost in handling time and examine the implications for foragers, including the first of our ancestors to cook. Ethnographic and experimental return rates and nutritional analysis about important prey items exploited by ethnohistoric Numic foragers in the North American Great Basin provide a demonstration of how the costs of cooking impact different types of prey. Foragers should make choices about which prey to capture based on expectations about the costs involved to cook them. The results indicate that the caloric benefit achieved by cooking meat is quickly lost as the cost of cooking increases, whereas many plant foods are beneficially cooked across a range of cooking costs. These findings affirm the importance of plant foods, especially geophytes, among foragers, and are highly suggestive of their importance at the onset of cooking in the human lineage.

Underground carbohydrate stores and storage organs in fire-maintained longleaf pine savannas in Florida, USA.

PREMISE: Many perennial herbaceous plants develop underground storage organs (USOs) that store carbohydrates, water, and minerals. The resprouting ability of plants is influenced by the availability of these materials and by the type of underground organ and number of viable buds. In this study, we illustrate the diversity of longleaf pine savanna species and their nonstructural carbohydrate (NSC) pools and concentrations. We also determined whether NSC concentrations by USO are good predictors of NSC pools in species with different types of underground structures. METHODS: We excavated in their entirety 1-4 individuals of each of 100 ground-layer pine savanna species, classified their USO types, and measured their NSC concentrations and NSC pools. RESULTS: The NSC concentrations in underground organs varied widely among the 100 species sampled. Surprisingly, the fibrous roots of Pityopsis graminifolia stored higher concentrations of NSCs than many species with USOs. The relationship between NSC concentrations and NSC pools was strong after controlling for underground biomass. CONCLUSIONS: Our results revealed the high diversity of underground organs in pine savannas. It also showed that NSC concentrations in species with USOs reach high levels. Predictions of NSC pool sizes from NSC concentrations are interpretable, when corrections for underground biomass are considered. Research on underground organs would benefit from inclusion of morphological-anatomical analyses and phylogenetic controls to promote use of the data in broad-scale analyses.

Get the shovel: morphological and evolutionary complexities of belowground organs in geophytes.

Herbaceous plants collectively known as geophytes, which regrow from belowground buds, are distributed around the globe and throughout the land plant tree of life. The geophytic habit is an evolutionarily and ecologically important growth form in plants, permitting novel life history strategies, enabling the occupation of more seasonal climates, mediating interactions between plants and their water and nutrient resources, and influencing macroevolutionary patterns by enabling differential diversification and adaptation. These taxa are excellent study systems for understanding how convergence on a similar growth habit (i.e., geophytism) can occur via different morphological and developmental mechanisms. Despite the importance of belowground organs for characterizing whole-plant morphological diversity, the morphology and evolution of these organs have been vastly understudied with most research focusing on only a few crop systems. Here, we clarify the terminology commonly used (and sometimes misused) to describe geophytes and their underground organs and highlight key evolutionary patterns of the belowground morphology of geophytic plants. Additionally, we advocate for increasing resources for geophyte research and implementing standardized ontological definitions of geophytic organs to improve our understanding of the factors controlling, promoting, and maintaining geophyte diversity.

Comparative transcriptomics of a monocotyledonous geophyte reveals shared molecular mechanisms of underground storage organ formation.

Many species from across the vascular plant tree-of-life have modified standard plant tissues into tubers, bulbs, corms, and other underground storage organs (USOs), unique innovations which allow these plants to retreat underground. Our ability to understand the developmental and evolutionary forces that shape these morphologies is limited by a lack of studies on certain USOs and plant clades. We take a comparative transcriptomics approach to characterizing the molecular mechanisms of tuberous root formation in Bomarea multiflora (Alstroemeriaceae) and compare these mechanisms to those identified in other USOs across diverse plant lineages; B. multiflora fills a key gap in our understanding of USO molecular development as the first monocot with tuberous roots to be the focus of this kind of research. We sequenced transcriptomes from the growing tip of four tissue types (aerial shoot, rhizome, fibrous root, and root tuber) of three individuals of B. multiflora. We identified differentially expressed isoforms between tuberous and non-tuberous roots and tested the expression of a priori candidate genes implicated in underground storage in other taxa. We identify 271 genes that are differentially expressed in root tubers versus non-tuberous roots, including genes implicated in cell wall modification, defense response, and starch biosynthesis. We also identify a phosphatidylethanolamine-binding protein, which has been implicated in tuberization signalling in other taxa and, through gene-tree analysis, place this copy in a phylogenetic context. These findings suggest that some similar molecular processes underlie the formation of USOs across flowering plants despite the long evolutionary distances among taxa and non-homologous morphologies (e.g., bulbs vs. tubers). (Plant development, tuberous roots, comparative transcriptomics, geophytes).

Tracking biological footprints of climate change using flowering phenology of the geophytes: Pancratium tenuifolium and Scadoxus multiflorus.

Drought-adapted geophytes are responding to the effects of climate change in arid and semi-arid environments. In this study, herbarium and historical rainfall data were used to examine the impact of rainfall changes on flowering trends of Pancratium tenuifolium Hochst. ex A.Rich and Scadoxus multiflorus (Martyn) Raf. subsp. multiflorus. Flowering was delayed by approximately 7 days per decade for P. tenuifolium during the period 1930 to 2018 and by approximately 14 days per decade for S. multiflorus subsp. multiflorus during the period 1924 to 2008. Scadoxus multiflorus subsp. multiflorus delayed the day of flowering by approximately 0.3 days per millimetre increase of rainfall, with Pancratium tenuifolium showing a non-significant response to summer rainfall during the same period. Overall, a linear mixed-effects model revealed that the day of flowering was delayed by approximately 8 days per degree rise in latitude and advanced by approximately 9 days per degree rise in longitude. Additionally, summer rainfall had significant effects on the day of flowering with a 1-mm increase in summer rainfall delaying the day of flowering by approximately 0.16 days. These changes in flowering times may ultimately alter the distribution of geophytes in Namibia.

Is sustainable extensive green roof realizable without irrigation in a temperate monsoonal climate? A case study in Beijing.

A strategy to combat the adverse effects of urbanization involves the installation of green roofs under different climatic conditions. The design and maintenance of green roof systems need to be tailored to the local climate. However, there is a scarcity of reports on the performance of plants under temperate monsoonal climatic conditions. This study follows the growth pattern of 28 species (18 non-succulent forbs and 10 succulents) grown at three substrate depths (10, 15, and 20 cm) over three years on an unirrigated extensive green roof, located in Beijing, China. The results of this study revealed that sustainable extensive green roof was realizable without irrigation in Beijing. In terms of plant adaptive strategies, the most successful plants in this study were the stress-tolerant species, followed by the ruderal species. While deeper substrate could facilitate the survival and performance of plants, substrate moisture content was more significant for the survival of plants in the dry and cold winter in Beijing. This study recommended the use of a substrate depth, which was at least 15 cm deep for unirrigated green roofs in Beijing.

Expression of LhFT1, the Flowering Inducer of Asiatic Hybrid Lily, in the Bulb Scales.

Asiatic hybrid lily leaves emerge from their bulbs in spring, after cold exposure in winter, and the plant then blooms in early summer. We identified four FLOWERING LOCUS T (FT)-like genes, LhFT1, LhFT4, LhFT6, and LhFT8, from an Asiatic hybrid lily. Floral bud differentiation initiated within bulbs before the emergence of leaves. LhFT genes were mainly expressed in bulb scales, and hardly in leaves, in which the FT-like genes of many plants are expressed in response to environmental signals. LhFT1 was expressed in bulb scales after vernalization and was correlated to flower bud initiation in two cultivars with different flowering behaviors. LhFT8 was upregulated in bulb scales after cold exposure and three alternative splicing variants with a nonsense codon were simultaneously expressed. LhFT6 was upregulated in bulb scales after flower initiation, whereas LhFT4 was expressed constantly in all organs. LhFT1 overexpression complemented the late-flowering phenotype of Arabidopsis ft-10, whereas that of LhFT8 did so partly. LhFT4 and LhFT6 overexpression could not complement. Yeast two-hybrid and in vitro analyses showed that the LhFT1 protein interacted with the LhFD protein. LhFT6 and LhFT8 proteins also interacted with LhFD, as observed in AlphaScreen assay. Based on these results, we revealed that LhFT1 acts as a floral activator during floral bud initiation in Asiatic hybrid lilies. However, the biological functions of LhFT4, LhFT6, and LhFT8 remain unclear.

Metabolic Profile and Performance Responses of Ranunculus asiaticus L. Hybrids as Affected by Light Quality of Photoperiodic Lighting.

Ranunculus asiaticus is a quantitative long day plant grown for cut flowers and flowering potted plants production. We evaluated the influence of light spectrum of three light sources for end-of-day photoperiodic treatments, with different phytochrome photoequilibria (PPE) induced at plant level, on the metabolic profiling of two hybrids of R. asiaticus L., MBO and MDR, in plants from vernalized tuberous roots. The following treatments were compared with natural day length (NL): white fluorescence lamp (FL, PPE 0.84), light emitting diodes (LEDs) Red:Far Red light at 3:1 ratio (R:FR 3:1, PPE 0.84), and LEDs Red:Far Red light at 1:3 ratio (R:FR 1:3, PPE 0.63). Measurements were carried out to evaluate the time course of carbohydrate, amino acid, and protein levels throughout the growing cycle in tuberous roots and leaves, in relation to the different plant stages (pre-planting, vegetative phase, and flowering). The study of metabolic profiling suggested that the differences between the tuberous root reserves of the two R. asiaticus hybrids could be responsible for the capacity of MBO to exert an early flowering. In particular, the proton-consuming synthesis during the pre-planting of two amino acids, alanine and γ-aminobutyric acid (GABA), is able to buffer the cytoplasmic acidosis and pH altered by the vernalization process, and GABA itself can efficiently scavenge reactive oxygen species. This fast response to the stress caused by vernalization allows MBO plants to accelerate the process of vegetative development and flowering. Some other changes in metabolites profile were certainly related to the different responses to day length and photoperiodic light quality in the two hybrids, such as dose exerted by low R:FR lighting in both MBO and MDR. However, most of the responses are under a strict genetic control.

Intricate Distribution Patterns of Six Cytotypes of Allium oleraceum at a Continental Scale: Niche Expansion and Innovation Followed by Niche Contraction With Increasing Ploidy Level.

The establishment and success of polyploids are thought to often be facilitated by ecological niche differentiation from diploids. Unfortunately, most studies compared diploids and polyploids, ignoring variation in ploidy level in polyploids. To fill this gap, we performed a large-scale study of 11,163 samples from 1,283 populations of the polyploid perennial geophyte Allium oleraceum with reported mixed-ploidy populations, revealed distribution ranges of cytotypes, assessed their niches and explored the pattern of niche change with increasing ploidy level. Altogether, six ploidy levels (3x-8x) were identified. The most common were pentaploids (53.6%) followed by hexaploids (22.7%) and tetraploids (21.6%). Higher cytotype diversity was found at lower latitudes than at higher latitudes (>52° N), where only tetraploids and pentaploids occurred. We detected 17.4% of mixed-ploidy populations, usually as a combination of two, rarely of three, cytotypes. The majority of mixed-ploidy populations were found in zones of sympatry of the participating cytotypes, suggesting they have arisen through migration (secondary contact zone). Using coarse-grained variables (climate, soil), we found evidence of both niche expansion and innovation in tetraploids related to triploids, whereas higher ploidy levels showed almost zero niche expansion, but a trend of increased niche unfilling of tetraploids. Niche unfilling in higher ploidy levels was caused by a contraction of niche envelopes toward lower continentality of the climate and resulted in a gradual decrease of niche breadth and a gradual shift in niche optima. Field-recorded data indicated wide habitat breadth of tetraploids and pentaploids, but also a pattern of increasing synanthropy in higher ploidy levels. Wide niche breadth of tetra- and pentaploids might be related to their multiple origins from different environmental conditions, higher "age", and retained sexuality, which likely preserve their adaptive potential. In contrast, other cytotypes with narrower niches are mostly asexual, probably originating from a limited range of contrasting environments. Persistence of local ploidy mixtures could be enabled by the perenniality of A. oleraceum and its prevalence of vegetative reproduction, facilitating the establishment and decreasing exclusion of minority cytotype due to its reproductive costs. Vegetative reproduction might also significantly accelerate colonization of new areas, including recolonization of previously glaciated areas.

Tunicate bulb size variation in monocots explained by temperature and phenology.

Plant bulbs are modified shoot systems comprised of short internodes with apical bud(s) surrounded by layers of leaf bases. Bulb diameters can vary greatly, with overall bulb size playing a role in flower formation and resource allocation. Despite the importance of bulb size to the overall fitness of an individual, evolutionary and ecological aspects of this trait have been almost completely neglected. Examining over 2,500 herbarium vouchers for 115 selected species, we analyzed monocot tunicate bulb size within a phylogenetic context in order to investigate its evolutionary significance. We recorded two bulb diameter optima and observed that as bulb size increases taxa inhabit warmer areas with less temperature seasonality. Furthermore, we found that hysteranthous taxa, a habit where leaves emerge separately from flowers, exhibit overall larger bulbs potentially due to reliance upon belowground stored resources to flower rather than on current environmental inputs. This work highlights the importance of including the belowground portion of plants into ecological and evolutionary studies in order to gain a more complete understanding of the evolution of plant forms and functions.

Nitrogen-fixing bacteria and Oxalis - evidence for a vertically inherited bacterial symbiosis.

BACKGROUND: Plant-endophyte symbioses often revolve around nitrogen metabolism, and involve varying degrees of intimacy. Although evidence for vertical inheritance of nitrogen-fixing endophytic bacteria is increasing, it is confined mostly to crop plants, and to date no such system has been reported for geophytes. METHODS: Bacterial endophytes associated with Oxalis, the most species-rich geophytic genus form the Cape Flora in southern Africa was studied. Culturable endophytes were isolated from surface-sterilized vegetative and reproductive plant organs for six host species at three locations. Colonies of microbes on various artificial media were morphotyped, enumerated and identified using sequence data. Filter exclusion experiments were conducted to determine if endophytes were vertically transmitted to seeds, determine if mucilage plays a role to actively attract microbes from the soil and to assess microbial richness isolated from the mucilage of Oxalis seedlings. Fluorescent microscopy was implemented in order to visualize endophytic bacteria in cryo-sectioned seeds. RESULTS: Evidence for a novel, vertically transmitted symbiosis was reported. Communities of nitrogen-fixing and plant growth-promoting Bacillus endophytes were found to associate with selected Oxalis hosts from nitrogen-deficient environments of the Cape. Bacillus endophytes were ubiquitous and diverse across species and plant bodies, and were prominent in seeds. Three common nitrogen-fixing Bacillus have known oxalotrophic properties and appear to be housed inside specialised cavities (containing oxalates) within the plant body and seeds. CONCLUSIONS: The discovery of vertical transmission and potential benefits to both host and endophyte suggest a particularly tight mutualism in the Oxalis-endophyte system. This discovery suggests unexpected ways in which geophytes might avoid nitrogen deficiency, and suggest that such symbioses are more common than previously expected.