Genetic diversity of the symbiotic fungus: Rhizophagus irregularis and its effect on a plant host and a plant community

Arbuscular mycorrhizal fungi (AMF) are endosymbionts of the vast majority of terrestrial plants species exchanging soil nutrient for plant carbohydrates. This symbiotic association evolved more than 600 Myrs ago and might have helped the colonization of land by plants. AMF have been shown to confer a large number of benefits to plants, such as resistance to drought, pathogens, salt and pollutants. They were also shown to be central in terrestrial ecosystems as AMF diversity was found to impact plant community diversity, structure and productivity. Up to now, AMF diversity was measured to around 348 virtual molecular taxa for approximately 200’000 host plants. It is surprising that during their long evolution, the AMF did not radiate in species number, as did their hosts. In the first part of the thesis, I thus investigated the possibility that AMF could hide more functional diversity than previously thought. A diversity that could have been hidden until now because traditional molecular methods used could have had a to low resolution. Therefore using double digested restriction-site associated DNA sequencing (ddRAD-seq) protocols on the model AMF, Rhizophagus irregularis, I found that ecologists probably underestimated the functional diversity of AMF. Indeed I found well-defined genetic groups within this model species with differential transcriptome expression and differences in phenotypic traits. I also confirmed the controversial results suggesting a low endemism of AMF. In the second part, I then tested the hypothesis that within species AMF diversity could be ecologically functional. The intra-specific diversity was then inoculated either as a single isolate inoculum or as a mix inoculum on simulated plant communities. I found that phylogenetically similar AMF tend to impact the plant community in the same way by more or less repressing the dominant plant of the community, thus, resulting in a change in resource partitioning among subordinate plants. In the third part I characterized the molecular interaction between within species functional AMF diversity and the globally important crop plant Manihot esculenta. By performing a dual-transcriptome sequencing experiment I was able to unravel important plant genes and fungal genes that could partly explain how the AM symbiosis could switch from one extreme to another along the symbiosis continuum, parasitic to mutualist. Finally I have also found that R. irregularis hosts a rare and probably parasitic endosymbotic bacterium adapted to the intracytoplasmic life. This thesis showed several plant and fungal factors explaining the variability in the outcome of this ecologically and agriculturally important symbiosis. -- Les champignons endosymbiotiques à arbuscules (CEA) forment une symbiose avec la vaste majorité des plantes terrestres en échangeant des nutriments du sol contre des sucres issus de la photosynthèse. Cette association à évolué il y a plus de 600 millions d’années et a pu être d’une grande aide pour la colonisation du milieu terrestre par les plantes. Il a été démontré que la symbiose avec CEA peut apporter de nombreux bienfait aux plantes, comme une meilleure résistance à la sécheresse, aux pathogènes, au sel ainsi qu’aux polluants. De plus, les CEA s’avèrent jouer un rôle central dans les écosystèmes de plantes de par le fait que la diversité des CEA influence leur diversité, leur structure ainsi que leur productivité. De nos jours, la diversité des CEA est évaluée à 348 espèces moléculaires, pour environ 200'000 plantes hôtes. Il est surprenant cependant, après une évolution aussi longue, que si peu d’espèces soient trouvées chez les CEA comparées à leurs plantes hôtes. La première partie de cette thèse m’a permis de tester l’hypothèse qu’il existe une diversité demeurée cachée chez les CEA, du fait que les méthodes moléculaires utilisées jusque là avaient une trop faible résolution. A travers l’utilisation d’un protocole ddRAD-seq sur l’espèce modèle des CEA, Rhizophagus irregularis, j’ai pu déterminer que les écologistes moléculaires sous-estiment très probablement leur diversité. En effet, j’ai identifié quatre groupes génétiques bien définis avec de fortes différences transcriptomiques ainsi que phénotypiques. Dans la foulée, ce travail confirme les résultats débattus sur le faible taux d’endémisme trouvé chez les CEA. Dans la deuxième partie de cette thèse j’ai voulu savoir si cette nouvelle diversité pouvait jouer un rôle écologiquement fonctionnel. J’ai donc inoculé cette diversité, soit sous la forme d’une seule souche soit sous la forme d’un mix de souches, sur des communautés de plantes simulées en serre. Il en résulte que des CEA phylogénétiquement similaires tendent à influencer dans le même sens les communautés de plantes en réprimant, plus au moins selon les clades, la dominance d’une plante et permettant ainsi un changement dans la répartition des ressources entre les plantes secondaires. Dans la troisième partie de cette thèse, je me suis intéressé à l’effet de cette diversité sur la réponse phénotypique et moléculaire d’une espèce de végétale, le manioc (Manihot esculenta), une plante agricole de grande importance alimentaire. Par le biais d’une expérience de double séquençage d’ARN, j’ai pu identifier des gènes de plantes et de champignons importants pour la symbiose qui pourraient expliquer comment cette symbiose passe de parasitique à mutualistique. Finalement, j’ai identifié une bactérie endosymbiotique complètement adaptée à la vie intracytoplasmique dans Rhizophagus irregularis. Cette thèse apporte de nombreux éléments permettant de mieux comprendre la variété d’interactions existant entre les CEA et les plantes ainsi que leurs communautés

Fast and Pervasive Transcriptomic Resilience and Acclimation of Extremely Heat-Tolerant Coral Holobionts from the Northern Red Sea

Corals from the northern Red Sea and Gulf of Aqaba exhibit extreme thermal tolerance. To examine the underlying gene expression dynamics, we exposed Stylophora pistillata from the Gulf of Aqaba to short-term (hours) and long-term (weeks) heat stress with peak seawater temperatures ranging from their maximum monthly mean of 27 °C (baseline) to 29.5 °C, 32 °C, and 34.5 °C. Corals were sampled at the end of the heat stress as well as after a recovery period at baseline temperature. Changes in coral host and symbiotic algal gene expression were determined via RNA-sequencing (RNA-Seq). Shifts in coral microbiome composition were detected by complementary DNA (cDNA)-based 16S ribosomal RNA (rRNA) gene sequencing. In all experiments up to 32 °C, RNA-Seq revealed fast and pervasive changes in gene expression, primarily in the coral host, followed by a return to baseline gene expression for the majority of coral (\u3e94%) and algal (\u3e71%) genes during recovery. At 34.5 °C, large differences in gene expression were observed with minimal recovery, high coral mortality, and a microbiome dominated by opportunistic bacteria (including Vibrio species), indicating that a lethal temperature threshold had been crossed. Our results show that the S. pistillata holobiont can mount a rapid and pervasive gene expression response contingent on the amplitude and duration of the thermal stress. We propose that the transcriptomic resilience and transcriptomic acclimation observed are key to the extraordinary thermal tolerance of this holobiont and, by inference, of other northern Red Sea coral holobionts, up to seawater temperatures of at least 32 °C, that is, 5 °C above their current maximum monthly mean

Spiral spin-liquid and the emergence of a vortex-like state in MnSc2_2S4_4

Spirals and helices are common motifs of long-range order in magnetic solids, and they may also be organized into more complex emergent structures such as magnetic skyrmions and vortices. A new type of spiral state, the spiral spin-liquid, in which spins fluctuate collectively as spirals, has recently been predicted to exist. Here, using neutron scattering techniques, we experimentally prove the existence of a spiral spin-liquid in MnSc$_2$S$_4$ by directly observing the 'spiral surface' - a continuous surface of spiral propagation vectors in reciprocal space. We elucidate the multi-step ordering behavior of the spiral spin-liquid, and discover a vortex-like triple-q phase on application of a magnetic field. Our results prove the effectiveness of the $J_1$-$J_2$ Hamiltonian on the diamond lattice as a model for the spiral spin-liquid state in MnSc$_2$S$_4$, and also demonstrate a new way to realize a magnetic vortex lattice.Comment: 10 pages, 11 figure

Experimental signatures of emergent quantum electrodynamics in Pr2_2Hf2_2O7_7

In a quantum spin liquid, the magnetic moments of the constituent electron spins evade classical long-range order to form an exotic state that is quantum entangled and coherent over macroscopic length scales [1-2]. Such phases offer promising perspectives for device applications in quantum information technologies, and their study can reveal fundamentally novel physics in quantum matter. Quantum spin ice is an appealing proposal of one such state, in which the fundamental ground state properties and excitations are described by an emergent U(1) lattice gauge theory [3-7]. This quantum-coherent regime has quasiparticles that are predicted to behave like magnetic and electric monopoles, along with a gauge boson playing the role of an artificial photon. However, this emergent lattice quantum electrodynamics has proved elusive in experiments. Here we report neutron scattering measurements of the rare-earth pyrochlore magnet Pr$_2$Hf$_2$O$_7$ that provide evidence for a quantum spin ice ground state. We find a quasi-elastic structure factor with pinch points - a signature of a classical spin ice - that are partially suppressed, as expected in the quantum-coherent regime of the lattice field theory at finite temperature. Our result allows an estimate for the speed of light associated with magnetic photon excitations. We also reveal a continuum of inelastic spin excitations, which resemble predictions for the fractionalized, topological excitations of a quantum spin ice. Taken together, these two signatures suggest that the low-energy physics of Pr$_2$Hf$_2$O$_7$ can be described by emergent quantum electrodynamics. If confirmed, the observation of a quantum spin ice ground state would constitute a concrete example of a three-dimensional quantum spin liquid - a topical state of matter which has so far mostly been explored in lower dimensionalities.Comment: 15 pages, 3 figure

Peroxisomal defects in microglial cells induce a disease-associated microglial signature

Microglial cells ensure essential roles in brain homeostasis. In pathological condition, microglia adopt a common signature, called disease-associated microglial (DAM) signature, characterized by the loss of homeostatic genes and the induction of disease-associated genes. In X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disease, microglial defect has been shown to precede myelin degradation and may actively contribute to the neurodegenerative process. We previously established BV-2 microglial cell models bearing mutations in peroxisomal genes that recapitulate some of the hallmarks of the peroxisomal β-oxidation defects such as very long-chain fatty acid (VLCFA) accumulation. In these cell lines, we used RNA-sequencing and identified large-scale reprogramming for genes involved in lipid metabolism, immune response, cell signaling, lysosome and autophagy, as well as a DAM-like signature. We highlighted cholesterol accumulation in plasma membranes and observed autophagy patterns in the cell mutants. We confirmed the upregulation or downregulation at the protein level for a few selected genes that mostly corroborated our observations and clearly demonstrated increased expression and secretion of DAM proteins in the BV-2 mutant cells. In conclusion, the peroxisomal defects in microglial cells not only impact on VLCFA metabolism but also force microglial cells to adopt a pathological phenotype likely representing a key contributor to the pathogenesis of peroxisomal disorders

Immune response of BV-2 microglial cells is impacted by peroxisomal beta-oxidation

Microglia are crucial for brain homeostasis, and dysfunction of these cells is a key driver in most neurodegenerative diseases, including peroxisomal leukodystrophies. In X-linked adrenoleukodystrophy (X-ALD), a neuroinflammatory disorder, very long-chain fatty acid (VLCFA) accumulation due to impaired degradation within peroxisomes results in microglial defects, but the underlying mechanisms remain unclear. Using CRISPR/Cas9 gene editing of key genes in peroxisomal VLCFA breakdown (Abcd1, Abcd2, and Acox1), we recently established easily accessible microglial BV-2 cell models to study the impact of dysfunctional peroxisomal β-oxidation and revealed a disease-associated microglial-like signature in these cell lines. Transcriptomic analysis suggested consequences on the immune response. To clarify how impaired lipid degradation impacts the immune function of microglia, we here used RNA-sequencing and functional assays related to the immune response to compare wild-type and mutant BV-2 cell lines under basal conditions and upon pro-inflammatory lipopolysaccharide (LPS) activation. A majority of genes encoding proinflammatory cytokines, as well as genes involved in phagocytosis, antigen presentation, and co-stimulation of T lymphocytes, were found differentially overexpressed. The transcriptomic alterations were reflected by altered phagocytic capacity, inflammasome activation, increased release of inflammatory cytokines, including TNF, and upregulated response of T lymphocytes primed by mutant BV-2 cells presenting peptides. Together, the present study shows that peroxisomal β-oxidation defects resulting in lipid alterations, including VLCFA accumulation, directly reprogram the main cellular functions of microglia. The elucidation of this link between lipid metabolism and the immune response of microglia will help to better understand the pathogenesis of peroxisomal leukodystrophies

Phylogeography of Aegean green toads (Bufo viridis subgroup): continental hybrid swarm vs. insular diversification with discovery of a new island endemic

BACKGROUND: Debated aspects in speciation research concern the amount of gene flow between incipient species under secondary contact and the modes by which post-zygotic isolation accumulates. Secondary contact zones of allopatric lineages, involving varying levels of divergence, provide natural settings for comparative studies, for which the Aegean (Eastern Mediterranean) geography offers unique scenarios. In Palearctic green toads (Bufo viridis subgroup or Bufotes), Plio-Pleistocene (~ 2.6 Mya) diverged species show a sharp transition without contemporary gene flow, while younger lineages, diverged in the Lower-Pleistocene (~ 1.9 Mya), admix over tens of kilometers. Here, we conducted a fine-scale multilocus phylogeographic analysis of continental and insular green toads from the Aegean, where a third pair of taxa, involving Mid-Pleistocene diverged (~ 1.5 Mya) mitochondrial lineages, earlier tentatively named viridis and variabilis, (co-)occurs. RESULTS: We discovered a new lineage, endemic to Naxos (Central Cyclades), while coastal islands and Crete feature weak genetic differentiation from the continent. In continental Greece, both lineages, viridis and variabilis, form a hybrid swarm, involving massive mitochondrial and nuclear admixture over hundreds of kilometers, without obvious selection against hybrids. CONCLUSIONS: The genetic signatures of insular Aegean toads appear governed by bathymetry and Quaternary sea level changes, resulting in long-term isolation (Central Cyclades: Naxos) and recent land-bridges (coastal islands). Conversely, Crete has been isolated since the end of the Messinian salinity crisis (5.3 My) and Cretan populations thus likely result from human-mediated colonization, at least since Antiquity, from Peloponnese and Anatolia. Comparisons of green toad hybrid zones support the idea that post-zygotic hybrid incompatibilities accumulate gradually over the genome. In this radiation, only one million years of divergence separate a scenario of complete reproductive isolation, from a secondary contact resulting in near panmixia

Genomic Evidence for Cryptic Speciation in Tree Frogs From the Apennine Peninsula, With Description of Hyla perrini sp. nov

Despite increasing appreciation of the speciation continuum, delimiting and describing new species is a major yet necessary challenge of modern phylogeography to help optimize conservation efforts. In amphibians, the lack of phenotypic differences between closely-related taxa, their complex, sometimes unresolved phylogenetic relationships, and their potential to hybridize all act to blur taxonomic boundaries. Here we implement a multi-disciplinary approach to evaluate the nature of two deeply-diverged mitochondrial lineages previously documented in Italian tree frogs (Hyla intermedia s. l.), distributed north and south of the Northern Apennine Mountains. Based on evidence from mitochondrial phylogenetics, nuclear phylogenomics, hybrid zone population genomics, niche modeling analyses, and biometric assessments, we propose that these lineages be considered distinct, cryptic species. Both mitochondrial and nuclear data affirm that they belong to two monophyletic clades of Pliocene divergence (~3.5 My), only admixing over a relatively narrow contact zone restricted to the southeast of the Po Plain (50–100 km). These characteristics are comparable to similarly-studied parapatric amphibians bearing a specific status. Inferred from their current geographic distribution, the two Italian tree frogs feature distinct ecological niches (<15% of niche overlap), raising questions regarding potential adaptive components contributing to their incipient speciation. However, we found no diagnostic morphological and bioacoustic differences between them. This system illustrates the speciation continuum of Western-Palearctic tree frogs and identifies additional cryptic lineages of similar divergence to be treated as separate species (H. cf. meridionalis). We recommend combined approaches using genomic data as applied here for the future taxonomic assessment of cryptic diversity in alloparapatric radiations of terrestrial vertebrates, especially in controversial taxa. Finally, we formally described the northern Italian tree frogs as a new species, Hyla perrini sp. nov

A population genomics approach shows widespread geographical distribution of cryptic genomic forms of the symbiotic fungus Rhizophagus irregularis.

Arbuscular mycorrhizal fungi (AMF; phylum Gomeromycota) associate with plants forming one of the most successful microbe-plant associations. The fungi promote plant diversity and have a potentially important role in global agriculture. Plant growth depends on both inter- and intra-specific variation in AMF. It was recently reported that an unusually large number of AMF taxa have an intercontinental distribution, suggesting long-distance gene flow for many AMF species, facilitated by either long-distance natural dispersal mechanisms or human-assisted dispersal. However, the intercontinental distribution of AMF species has been questioned because the use of very low-resolution markers may be unsuitable to detect genetic differences among geographically separated AMF, as seen with some other fungi. This has been untestable because of the lack of population genomic data, with high resolution, for any AMF taxa. Here we use phylogenetics and population genomics to test for intra-specific variation in Rhizophagus irregularis, an AMF species for which genome sequence information already exists. We used ddRAD sequencing to obtain thousands of markers distributed across the genomes of 81 R. irregularis isolates and related species. Based on 6 888 variable positions, we observed significant genetic divergence into four main genetic groups within R. irregularis, highlighting that previous studies have not captured underlying genetic variation. Despite considerable genetic divergence, surprisingly, the variation could not be explained by geographical origin, thus also supporting the hypothesis for at least one AMF species of widely dispersed AMF genotypes at an intercontinental scale. Such information is crucial for understanding AMF ecology, and how these fungi can be used in an environmentally safe way in distant locations

Slow and steady wins the race: contrasted phylogeographic signatures in two Alpine amphibians

A deeper phylogeographic structure is expected for slow-dispersing habitat specialists compared to widespread adaptable species, especially in topographically complex regions. We tested this classic assumption by comparing the genomic (RAD-sequencing) phylogeographies of two amphibians inhabiting the Swiss Alps: the mobile, cosmopolitan common frog (Rana temporaria) against the stationary, mountain endemic Alpine salamander (Salamandra atra). Our results ran opposite of predictions: the frog displayed significantly higher genetic divergences and lower within-population variation compared to the salamander. This implies a prominent role for their distinctive glacial histories in shaping intraspecific diversity and structure: diversification and recolonization from several circum-Alpine micro-refugia for the frog versus a single refugium for the salamander, potentially combined with better population connectivity and stability. These striking differences emphasize the great variability of phylogeographic responses to the Quaternary glaciations, hence the complexity to predict general patterns of genetic diversity at the regional scale, and the forces that underlie them