Structure-guided design of C3-branched swainsonine as potent and selective human Golgi α-mannosidase (GMII) inhibitor.

The human Golgi α-mannosidase, hGMII, removes two mannose residues from GlcNAc-Man5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor of all complex N-glycans including tumour-associated ones. The natural product GMII inhibitor, swainsonine, blocks processing of cancer-associated N-glycans, but also inhibits the four other human α-mannosidases, rendering it unsuitable for clinical use. Our previous structure-guided screening of iminosugar pyrrolidine and piperidine fragments identified two micromolar hGMII inhibitors occupying the enzyme active pockets in adjacent, partially overlapping sites. Here we demonstrate that fusing these fragments yields swainsonine-configured indolizidines featuring a C3-substituent that act as selective hGMII inhibitors. Our structure-guided GMII-selective inhibitor design complements a recent combinatorial approach that yielded similarly configured and substituted indolizidine GMII inhibitors, and holds promise for the potential future development of anti-cancer agents targeting Golgi N-glycan processing.

Increased intestinal permeability and lipopolysaccharide contribute to swainsonine-induced systemic inflammation.

Long-term consumption of swainsonine could be poisonous to livestock, including facilitating apoptosis by impairing lysosomal function and inhibiting autophagic degradation, leading to liver inflammation and even death in livestock. However, the mechanism by swainsonine induced systemic inflammatory responses remained unclear, especially the effects of swainsonine on intestinal permeability, lipopolysaccharide (LPS) level and oxidative stress response were unknown. In this study, swainsonine increased intestinal permeability as evidenced by the significant down-regulation of colonic goblet cells, Akkermansia muciniphila and intestinal tight junction protein Occludin, Claudin 1 and ZO-1, and the significant up-regulation of mRNA expression level of the intestinal permeability indicator protein tyrosine phosphatase receptor type H (Ptprh) in the ileum of mice. Simultaneously, the elevated LPS biosynthetic genes in intestinal microbiota and increased intestinal permeability facilitated more bacterial endotoxin LPS to enter the blood. High concentration of free-form LPS induced high levels of proinflammatory cytokines and oxidative stress response, thereby causing the systemic inflammation. These findings provided a new perspective on swainsonine-induced systemic inflammation, suggesting that intestinal permeability and free-form LPS level may be the potential trigger factors.

(5S)-5-Benzylswainsonines as potent and selective inhibitors of Golgi α-mannosidase II: synthesis, enzyme evaluation and molecular modelling.

Development of novel anti-cancer therapeutics based on Golgi α-mannosidase II (GMII) inhibition is considerably impeded by an undesired co-inhibition of lysosomal α-mannosidase leading to severe side-effects. In this contribution, we describe a fully stereoselective synthesis of (5S)-5-[4-(halo)benzyl]swainsonines as highly potent and selective inhibitors of GMII. The synthesis starts from a previously reported aldehyde readily available from l-ribose, and the key features include an intramolecular reductive amination with substrate-controlled stereoselectivity and a late-stage derivatisation of the benzyl group via ipso-substitution. These novel swainsonine analogues were found to be nanomolar inhibitors of the Golgi-type α-mannosidase AMAN-2 (Ki = 23-75 nM) with excellent selectivity (selectivity index = 205-870) over the lysosomal-type Jack bean α-mannosidase. Finally, molecular docking and pKa calculations were performed to provide more insight into the structure of the inhibitor:enzyme complexes, and a pair interaction energy analysis (FMO-PIEDA) was carried out to rationalise the observed potency and selectivity of the inhibitors.

Lisosomal storage disease caused by ingestion of Astragalus spp in llamas: an emergent concern.

Lysosomal storage diseases are inherited or acquired disorders characterized by dysfunctional lysosomes that lead to intracytoplasmic accumulation of undegraded substrates, causing impaired cellular function and death. Many acquired lysosomal storage diseases are produced by toxic plants, which have indolizidine alkaloids, including swainsonine, that inhibits lysosomal α-mannosidase and Golgi α-mannosidase II. Swainsonine-induced nervous disease associated with various plants has been reported, including species of the genus Astragalus, Sida, Oxitropis, Swainsona, and Ipomoea. Two species of Astragalus (i.e. Astragalus garbancillo and Astragalus punae) have been found to cause neurologic disease in llamas. In addition, A. garbancillo was also associated with malformations in the offspring, and possibly abortions and neonatal mortality in llamas. The diagnosis of Astragalus spp. intoxication is established based on clinical signs, microscopic and ultrastructural findings, lectin histochemistry, abundance of these plants in the grazing area and determination of swainsonine in plant specimens.

Autophagic-lysosomal damage induced by swainsonine is protected by trehalose through activation of TFEB-regulated pathway in renal tubular epithelial cells.

Swainsonine (SW) is the main toxic component of locoweed. Previous studies have shown that kidney damage is an early pathologic change in locoweed poisoning in animals. Trehalose induces autophagy and alleviates lysosomal damage, while its protective effect and mechanism against the toxic injury induced by SW is not clear. Based on the published literature, we hypothesize that transcription factor EB(TFEB) -regulated is targeted by SW and activating TFEB by trehalose would reverse the toxic effects. In this study, we investigate the mechanism of protective effects of trehalose using renal tubular epithelial cells. The results showed that SW induced an increase in the expression level of microtubule-associated protein light chain 3-II and p62 proteins and a decrease in the expression level of ATPase H+ transporting V1 Subunit A, Cathepsin B, Cathepsin D, lysosome-associated membrane protein 2 and TFEB proteins in renal tubular epithelial cells in a time and dose-dependent manner suggesting TFEB-regulated lysosomal pathway is adversely affected by SW. Conversely, treatment with trehalose, a known activator of TFEB promote TFEB nuclear translocation suggesting that TFEB plays an important role in protection against SW toxicity. We demonstrated in lysosome staining that SW reduced the number of lysosomes and increased the luminal pH, while trehalose could counteract these SW-induced effects. In summary, our results demonstrated for the first time that trehalose could alleviate the autophagy degradation disorder and lysosomal damage induced by SW. Our results provide an interesting method for reversion of SW-induced toxicity in farm animals and furthermore, activation of TFEB by trehalose suggesting novel mechanism of treating lysosomal storage diseases.

Identification and distribution of a fungal endosymbiotic Alternaria species (Alternaria section Undifilum sp.) in Astragalus garbancillo tissues.

Plants belonging to the genera Astragalus, Oxytropis, Ipomoea, Sida, and Swainsona often contain the toxin swainsonine (SW) produced by an associated fungal symbiont. Consumption of SW-containing plants causes a serious neurological disorder in livestock, which can be fatal. In this study, a fungal endophyte, Alternaria section Undifilum, was identified in Astragalus garbancillo seeds, using polymerase chain reaction (PCR) followed by direct sequencing. In seeds, the SW concentrations were about 4 times higher than in other parts of the plant. Furthermore, microscopic examination demonstrated that the fungus mycelium grows inside the petioles and stems, on the outer surface and inside the mesocarp of the fruit, in the mesotesta and endotesta layers of the seed coat, and inside the endosperm of the seeds. Our results support the notion that the SW-producing fungus is vertically transmitted in the host plant A. garbancillo.

Multiomics Reveals Mechanisms of Alternaria oxytropis Inhibiting Pathogenic Fungi in Oxytropis ochrocephala.

Endophytic fungi can benefit the host plant and increase the plant resistance. Now, there is no in-depth study of how Alternaria oxytropis (A. oxytropis) is enhancing the ability of inhibiting pathogenic fungi in Oxytropis ochrocephala (O. ochrocephala). In this study, the fungal community and metabolites associated with endophyte-infected (EI) and endophyte-free (EF) O. ochrocephala were compared by multiomics. The fungal community indicated that there was more A. oxytropis, less phylum Ascomycota, and less genera Leptosphaeria, Colletotrichum, and Comoclathris in the EI group. As metabolic biomarkers, the levels of swainsonine and apigenin-7-O-glucoside-4-O-rutinoside were significantly increased in the EI group. Through in vitro validation experiments, swainsonine and apigenin-7-O-glucoside-4-O-rutinoside can dramatically suppress the growth of pathogenic fungi Leptosphaeria sclerotioides and Colletotrichum americae-borealis by increasing the level of oxidative stress. This work suggested that O. ochrocephala containing A. oxytropis could increase the resistance to fungal diseases by markedly enhancing the content of metabolites inhibiting pathogenic fungi.

Ipomoea carnea alkaloid extract vs swainsonine: A comparative study on cytotoxic activity against glial cells.

The consumption of Ipomoea carnea produces a neurological syndrome in animals. The toxic principles of I. carnea are the alkaloids swainsonine (SW) and calystegines B1, B2, B3 and C1. In this study, we investigated the cytotoxicity of an alkaloid extract of Ipomoea carnea (AEE) and natural swainsonine (SW) isolated from Astragalus lentiginosus (25-1000 µM of SW) for 48 h in a glioma cell line. Although the natural SW did not induce any changes in cell viability, the AEE exhibited a dose dependent cytotoxic effect and release of lactate dehydrogenase (LDH) indicative of cytolysis. In order to evaluate the morphological changes involved, cells were examined using phase contrast and fluorescence microscopy with acridine orange-ethidium bromide staining. The AEE caused a cell death compatible with necrosis, whereas exposure to 1000 µM of SW resulted in cytoplasmic vacuolation. Immunocytochemical studies revealed that astrocytes treated with 150 µM of AEE from I. carnea or 1000 µM of SW exhibited morphological characteristics of cell activation. These findings suggest that swainsonine would not be the only component present in the AEE of I. carnea responsible for in vitro cytotoxicity. Calystegines might also play a role in acting synergistically and triggering cell death through necrosis.

Swainsonine inhibits autophagic degradation and causes cytotoxicity by reducing CTSD O-GlcNAcylation.

Swainsonine (SW) is the primary toxin in locoweed, a poisonous plant. SW can cause animal poisoning, affect the quality and safety of meat products and threaten human health, but the mechanism of its toxicity is little defined. Here, we identified 159 differentially expressed proteins, many of which are involved in autophagy and glycosylation modification processes, using proteomics sequencing analysis. O-linked-N-acetylglucosamylation (O-GlcNAcylation) is a glycosylation modification widely involved in various biological processes. Our results show that SW toxicity is related to O-GlcNAcylation. In addition, increased O-GlcNAcylation with the O-GlcNAcase (OGA) inhibitor TMG promoted autophagy, while decreased O-GlcNAcylation with the O-GlcNAc transferase (OGT) inhibitor OSMI inhibited autophagy. Further analysis by Immunoprecipitation (IP) showed that SW could change the O-GlcNAcylation of Cathepsin D (CTSD), reducing the expression of mature CTSD (m-CTSD). In summary, these findings suggest that SW inhibits the O-GlcNAcylation of CTSD, affecting its maturation and leading to the impairment of lysosome function. Consequently, it inhibits autophagy degradation, and causes cytotoxicity, providing a new theoretical basis for SW toxicological mechanism.

Alternaria gansuense, a Plant Systematic Fungal Pathogen Producing Swainsonine in Vivo and in Vitro.

Astragalus adsurgens (A. adsurgens), which is considered a forage in China, grows widely in Eurasia and North America. However, Alternaria gansuense (A. gansuense) (synonym: Embellisia astragali) systematically infects A. adsurgens, producing swainsonine (SW), which poisons domesticated animals. In this study, we hypothesized that the A. gansuense SW-producing fungus is morphologically and molecularly related to the locoweed endophyte, Alternaria oxytropis (A. oxytropis), which systematically grows in host plants. Therefore, pure cultures of the fungi from diseased plants or endophytic interactions were collected from fields and assayed for SW via high-performance liquid chromatography linked to mass spectroscopy (HPLC-MS). The production of SW was also detected in A. adsurgens, A. oxytropis and diseased plants by assaying for the presence of the ß-ketoacyl synthase (KS) gene, which is required for SW synthesis. Diseased A. adsurgens and pure cultures of A. gansuense have SW and the healthy-looking A. adsurgens plants also contained SW, probably because they were infected with A. gansuense. Therefore, A. adsurgens-infected A. gansuense are not safe for livestock consumption.

Phylogenetic patterns of bioactive secondary metabolites produced by fungal endosymbionts in morning glories (Ipomoeeae, Convolvulaceae).

Heritable fungal endosymbiosis is underinvestigated in plant biology and documented in only three plant families (Convolvulaceae, Fabaceae, and Poaceae). An estimated 40% of morning glory species in the tribe Ipomoeeae (Convolvulaceae) have associations with one of two distinct heritable, endosymbiotic fungi (Periglandula and Chaetothyriales) that produce the bioactive metabolites ergot alkaloids, indole diterpene alkaloids, and swainsonine, which have been of interest for their toxic effects on animals and potential medical applications. Here, we report the occurrence of ergot alkaloids, indole diterpene alkaloids, and swainsonine in the Convolvulaceae; and the fungi that produce them based on synthesis of previous studies and new indole diterpene alkaloid data from 27 additional species in a phylogenetic, geographic, and life-history context. We find that individual morning glory species host no more than one metabolite-producing fungal endosymbiont (with one possible exception), possibly due to costs to the host and overlapping functions of the alkaloids. The symbiotic morning glory lineages occur in distinct phylogenetic clades, and host species have significantly larger seed size than nonsymbiotic species. The distinct and widely distributed endosymbiotic relationships in the morning glory family and their alkaloids provide an accessible study system for understanding heritable plant-fungal symbiosis evolution and their potential functions for host plants.

Swainsonine Induces Liver Inflammation in Mice via Disturbance of Gut Microbiota and Bile Acid Metabolism.

Swainsonine induced liver inflammation in livestock; however, the underlying mechanisms, especially the role of bile acids (BAs), in the pathogenesis remained elusive. Here, our results showed that swainsonine induced hepatic inflammation via changing BA metabolism and gut microbiota in mice. Swainsonine significantly upregulated the levels of deoxycholic acid (DCA) and taurine-ß-muricholic acid (T-ß-MCA) in the serum and liver of mice due to the markedly increased genus Clostridium and the decreased genus Lactobacillus in the gut. As antagonists of the farnesoid X receptor (FXR), elevated DCA and T-ß-MCA inhibited hepatic Fxr gene expression and thus suppressed FXR-SHP signaling and activated hepatic Cyp7a1 gene expression, which induced a significant upregulation of the total BA level in serum, contributing to liver inflammation. These findings offer new insights into the underlying mechanisms in which swainsonine induced liver inflammation in mice via the gut-liver axis and suggest that gut microbiota and its metabolite BAs may be underlying triggering factors.

swnk plays an important role in the biosynthesis of swainsonine in Metarhizium anisopliae.

OBJECTIVE: Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by multiple fungi. A key enzyme in the SW synthesis pathway is a hybrid swnk/nrps. To analyze the role of swnk in the SW biosynthesis pathway of Metarhizium anisopliae. RESULTS: The concentration of SW and the swnk expression in M. anisopliae fermentation from 1st to 7th day were determined using LC-MS and RT-qPCR, respectively. M. anisopliae had the highest SW content and swnk expression on the 5th day of fermentation; Mutant strain (MT) were obtained by PEG-mediated homologous recombination (HR) which knocked out swnk in the wild-type (WT) strain. Complemented-type (CT) strain were obtained by transforming a modified PUC19 complementation vector containing the geneticin (G418) resistance gene and swnK. SW was not detected in the MT strain and reverted to its original level in the CT strain; A Psilent-1 plasmid with Benomyl (ben)-resistant that was used interfered with swnk of WT strain. The level of SW was markedly diminished in the RNAi strain. RNAi of swnk affects the formation of the cell wall in M. anisopliae. CONCLUSION: These results indicate that swnk plays a crucial role in the SW biosynthesis of M. anisopliae.

Biochemical characteristics of point mutated Capra hircus lysosome α-mannosidase.

Locoweeds, a type of poisonous weedare, are widely distributed throughout the world and have a significant impact on the development of herbivore animal husbandry. Swainsonine (SW), the main toxin in locoweeds, can competitively inhibit lysosomes α-mannosidase (LAM) in animal cells, resulting in α-mannosidosis. However, the specifics of the interaction between SW and LAM are still unclear. Here, we used molecular docking to predicte the interaction points between SW and LAM, built mutated lysosomes α-mannosidase (LAMM), and analyzed its biochemical properties changes in presumption points. The Trp at the 28th position and the Tyr at the 599th position of the LAM were interaction point candidates, and the above two amino acids in Capra hircus LAM (chLAM), were successfully mutated to glycine by constructing recombinant yeast GS115/PIC9K- LAMM. The results showed that the sensitivity of Capra hircus LAMM (chLAMM), to SW decreased significantly compared with wild-type LAM, the enzyme activity of LAM decreased approximately threefold, the optimum temperature of LAMM decreased from 55°C to 50°C, the optimum pH value increased from 4.5 to 5.0, and the effects of Mn2+, Fe3+, Al3+, Co2+, Cr3+, and ethylenediaminetetraacetic acid (EDTA) on LAM enzyme activity before and after point mutation changed significantly. These findings help us better understanding the molecular mechanism of the interaction mechanism between SW and chLAM, and provide new reference for solving locoweeds poisoning.

Swainsonine-induced vacuolar degeneration is regulated by mTOR-mediated autophagy in HT22 cells.

The indolizidine alkaloid, swainsonine (SW), is the main toxic component of locoweed, which can cause locoism in animals with characteristic neurological dysfunction. Pathological manifestations at cellular level include extensive vacuolar degeneration. Studies have shown that SW can induces autophagy, but the role and mechanism of autophagy in SW-induced vacuolar degeneration is unclear. In this study, we analyzed the role of autophagy in SW-induced cell injury in mouse hippocampal neurons cell line (HT22) using western blotting, qRT-PCR, transmission electron microscopy and immunofluorescence microscopy. The results showed that the expressions of LC3-II, ATG5, Beclin1 and p62 proteins and their mRNAs in HT22 cells were induced by SW treatment. The SW treatment increased the number of autophagosomes with enhanced fluorescence intensity of monodansylcadaverine (MDC) and LC3-II in a time-dose dependent manner. The results of lysosome staining showed that SW could increase the number of lysosomes, increase the intraluminal pH. Transmission electron microscopy results indicate that SW induced autophagosomes, and Baf A1 could effectively alleviate SW-induced vacuolar degeneration. At the molecular level, SW treatment inhibited the expression of p-PI3K, p-AKT, p-ERK, p-AMPK, p-mTOR, p-p70S6K and p-4EBP1 and promoted the expression of p53. Our results collectively suggest, PI3K/AKT/mTOR, ERK/mTOR and p53/mTOR signaling pathways are involved in the regulation of SW-induced autophagy in HT22 cells, while the AMPK/mTOR signaling pathway is not involved in this regulation. Inhibition of autophagic degradation can effectively alleviate SW-induced vacuolar degeneration.

Assembly of high-quality genomes of the locoweed Oxytropis ochrocephala and its endophyte Alternaria oxytropis provides new evidence for their symbiotic relationship and swainsonine biosynthesis.

Locoweeds are perennial forbs poisonous to livestock and cause extreme losses to animal husbandry. Locoweed toxicity is attributed to the symbiotic endophytes in Alternaria sect. Undifilum, which produce a mycotoxin swainsonine (SW). We performed a de novo whole genome sequencing of the most common locoweed in China, Oxytropis ochrocephala (2n = 16), and assembled a high-quality, chromosome-level reference genome. Its genome size is 958.83 Mb with 930.94 Mb (97.09%) anchored and oriented onto eight chromosomes, and 31,700 protein-coding genes were annotated. Phylogenetic and collinearity analysis showed it is closely related to Medicago truncatula with a pair of large interchromosomal rearrangements, and both species underwent a whole-genome duplication event. We also derived the genome of A. oxytropis at 74.48 Mb with a contig N50 of 8.87 Mb and 10,657 protein-coding genes, and refined the genes of SW biosynthesis. Multiple Alternaria species containing the swnK gene were grouped into a single clade, but in other genera, swnK's homologues are diverse. Resequencing of 41 A. oxytropis strains revealed one SNP in the SWN cluster causing changes in SW concentration. Comparing the transcriptomes of symbiotic and nonsymbiotic interactions identified differentially expressed genes (DEGs) linked to defence and secondary metabolism in the host. Within the endophyte DEGs were linked to cell wall degradation, fatty acids and nitrogen metabolism. Symbiosis induced the upregulation of most of the SW biosynthetic genes. These two genomes and relevant sequencing data should provide valuable genetic resources for the study of the evolution, interaction, and SW biosynthesis in the symbiont.

The locoweed endophyte Alternaria oxytropis affects root development in Arabidopsis in vitro through auxin signaling and polar transport.

Locoweeds are leguminous forbs known for their toxicity to livestock caused by the endophytic fungi Alternaria sect. Undifilum. Unlike the defensive mutualisms reported in many toxin-producing endophytes and their plant hosts, the benefits that A. sect. Undifilum can confer to it host plants remains unclear. Here, we conducted physiological and genetic analyses to show that A. (sect. Undifilum) oxytropis influences growth, especially root development, in its locoweed host Oxytropis ochrocephala and Arabidopsis. The presence of A. oxytropis significantly decreased primary root length while increasing the numbers of lateral roots and root hairs, and increasing plant leaf area and fresh weight. The fungus also increased the concentrations of plant endogenous auxin, and the expression of key genes for auxin biosynthesis, signaling, and transport. These effects on root development were abolished in mutants deficient in auxin signaling and polar transport. Alternaria oxytropis down-regulated expression of PIN1 but increased expression of PIN2, PIN7, and AUX1, which might reflect alterations in the spatial accumulation of auxin responsible for the changes in root architecture. Plant growth was insensitive to A. oxytropis when naphthylphthalamic acid was applied. Our findings indicate a function of A. oxytropis in promoting the growth and development of Arabidopsis via the regulation of auxin, which in turn suggests a possible role in benefiting its locoweed hosts via a process independent of its toxin production.

Intoxication of llamas by Astragalus punae in Argentina.

Several plants that contain indolizidine alkaloids, including swainsonine, are toxic to livestock, causing dysfunctional lysosomes and storage disease. Swainsonine induces a neurovisceral disease, known as locoism, in sheep, goats, and cattle, which occurs in several parts of the world, including, but not limited to, the western United States, China, and parts of Australia. In South America, locoism has been described in the Andean region of Argentina affecting sheep, cattle, and llamas. Intoxication by consumption of Astragalus punae was suspected in 4 llamas in Jujuy Province, northwestern Argentina. The grazing area contained abundant specimens of A. punae. The clinical course was ~15 d, and included moderate ataxia, incoordination of hindlimbs, and progressive loss of body condition. Microscopically, fine cytoplasmic microvacuolation was observed in the proximal convoluted renal tubules. Ultrastructurally, these changes consisted of severely dilated lysosomes. Swainsonine was detected in stem and leaf samples of A. punae at a concentration of 0.06%. Based on clinical history and signs, histologic and ultrastructural changes, and plant analysis, a diagnosis of swainsonine toxicosis caused by consumption of A. punae was made, which has not been reported previously, to our knowledge.

Evaluation of the context of downstream N- and free N-glycomic alterations induced by swainsonine in HepG2 cells.

Swainsonine (SWA), a potent inhibitor of class II α-mannosidases, is present in a number of plant species worldwide and causes severe toxicosis in livestock grazing these plants. The mechanisms underlying SWA-induced animal poisoning are not fully understood. In this study, we analyzed the alterations that occur in N- and free N-glycomic upon addition of SWA to HepG2 cells to understand better SWA-induced glycomic alterations. After SWA addition, we observed the appearance of SWA-specific glycomic alterations, such as unique fucosylated hybrid-type and fucosylated M5 (M5F) N-glycans, and a remarkable increase in all classes of Gn1 FNGs. Further analysis of the context of these glycomic alterations showed that (fucosylated) hybrid type N-glycans were not the precursors of these Gn1 FNGs and vice versa. Time course analysis revealed the dynamic nature of glycomic alterations upon exposure of SWA and suggested that accumulation of free N-glycans occurred earlier than that of hybrid-type N-glycans. Hybrid-type N-glycans, of which most were uniquely core fucosylated, tended to increase slowly over time, as was observed for M5F N-glycans. Inhibition of swainsonine-induced unique fucosylation of hybrid N-glycans and M5 by coaddition of 2-fluorofucose caused significant increases in paucimannose- and fucosylated paucimannose-type N-glycans, as well as paucimannose-type free N-glycans. The results not only revealed the gross glycomic alterations in HepG2 cells induced by swainsonine, but also provide information on the global interrelationships between glycomic alterations.

Host-Species Variation and Environment Influence Endophyte Symbiosis and Mycotoxin Levels in Chinese Oxytropis Species.

Oxytropis plants are widely distributed in the grasslands in northern China. Some Oxytropis species have been reported to contain the mycotoxin swainsonine, an alkaloid which causes poisoning in livestock, referred to as locoism. Previous studies showed that endophytic fungi (Alternaria oxytropis) symbiotically associate with these Oxytropis species to produce swainsonine. However, the influence of variation within the Oxytropis genus on the fixation or loss of symbiosis and toxicity is poorly understood, as is the influence of environmental factors. Here we used a collection of 17 common Oxytropis species sampled in northern China to assess genetic diversity using genotyping by sequencing which was compared with the levels of the endophyte and swainsonine. Results showed that nine Oxytropis species have detectable A. oxytropis colonisation, and seven Oxytropis species contain sufficient swainsonine to be considered poisonous, whereas the rest may be non-toxic. Species variation rather than the genetic lineage was associated with the fixation or loss of endophyte and swainsonine production, which appears to have resulted from genetic drift. Genotype × Environment (G × E) effects were also found to influence endophyte and swainsonine levels amongst species of the Oxytropis genus. Our study will provide a better understanding about the evolutionary basis of A. oxytropis symbiosis and swainsonine biosynthesis in locoweeds.