Ryoko Oono

Multiple evolutionary origins of legume traits leading to extreme rhizobial differentiation

SUMMARY *When rhizobia differentiate inside legume host nodules to become nitrogen-fixing bacteroids, they undergo a physiological as well as a morphological transformation. These transformations are more extreme in some legume species than others, leading to fundamental differences in rhizobial life history and evolution. Here, we analysed the distribution of different bacteroid morphologies over a legume phylogeny to understand the evolutionary history of this host-influenced differentiation. *Using existing electron micrographs and new flow cytometric analyses, bacteroid morphologies were categorized as swollen or nonswollen for 40 legume species in the subfamily Papilionoideae. Maximum likelihood and Bayesian frameworks were used to reconstruct ancestral states at the bases of all major subclades within the papilionoids. *Extreme bacteroid differentiation leading to swelling was found in five out of the six major papilionoid subclades. The inferred ancestral state for the Papilionoideae was hosting nonswollen bacteroids, indicating at least five independent origins of host traits leading to swollen bacteroids. *Repeated evolution of host traits causing bacteroid swelling indicates a possible fitness benefit to the plant. Furthermore, as bacteroid swelling is often correlated with loss of reproductive viability, the evolution of bacteroid cooperation or cheating strategies could be fundamentally different between the two bacteroid morphologies.

Controlling the reproductive fate of rhizobia: how universal are legume sanctions?

When a single host plant is infected by more than one strain of rhizobia, they face a tragedy of the commons. Although these rhizobia benefit collectively from nitrogen fixation, which increases host-plant photosynthesis, each strain might nonetheless increase its own reproduction, relative to competing strains, by diverting resources away from nitrogen fixation. Host sanctions can limit the evolutionary success of such rhizobial cheaters (strains that would otherwise benefit by fixing less nitrogen). Host sanctions have been shown in soybean (Glycine max) nodules, where the next generation of symbiotic rhizobia is descended from bacteroids (the differentiated cells that can fix nitrogen). Evidence for sanctions is less clear in legume species that induce rhizobial dimorphism inside their nodules. There, bacteroids are swollen and cannot reproduce regardless of how much nitrogen they fix, but sanctions could reduce reproduction of their undifferentiated clonemates within the same nodule. This rhizobial dimorphism can affect rhizobial evolution, including cheating options, in ways that may affect future generations of legumes. Both the importance of sanctions to hosts and possible physiological mechanisms for sanctions may depend on whether bacteroids are potentially reproductive.

Failure to fix nitrogen by non-reproductive symbiotic rhizobia triggers host sanctions that reduce fitness of their reproductive clonemates

The legume–rhizobia symbiosis is a classical mutualism where fixed carbon and nitrogen are exchanged between the species. Nonetheless, the plant carbon that fuels nitrogen (N2) fixation could be diverted to rhizobial reproduction by ‘cheaters’—rhizobial strains that fix less N2 but potentially gain the benefit of fixation by other rhizobia. Host sanctions can decrease the relative fitness of less-beneficial reproductive bacteroids and prevent cheaters from breaking down the mutualism. However, in certain legume species, only undifferentiated rhizobia reproduce, while only terminally differentiated rhizobial bacteroids fix nitrogen. Sanctions were, therefore, tested in two legume species that host non-reproductive bacteroids. We demonstrate that even legume species that host non-reproductive bacteroids, specifically pea and alfalfa, can severely sanction undifferentiated rhizobia when bacteroids within the same nodule fail to fix N2. Hence, host sanctions by a diverse set of legumes play a role in maintaining N2 fixation.

Comparing Symbiotic Efficiency between Swollen versus Nonswollen Rhizobial Bacteroids

Symbiotic rhizobia differentiate physiologically and morphologically into nitrogen-fixing bacteroids inside legume host nodules. The differentiation is apparently terminal in some legume species, such as peas (Pisum sativum) and peanuts (Arachis hypogaea), likely due to extreme cell swelling induced by the host. In other legume species, such as beans (Phaseolus vulgaris) and cowpeas (Vigna unguiculata), differentiation into bacteroids, which are similar in size and shape to free-living rhizobia, is reversible. Bacteroid modification by plants may affect the effectiveness of the symbiosis. Here, we compare symbiotic efficiency of rhizobia in two different hosts where the rhizobia differentiate into swollen nonreproductive bacteroids in one host and remain nonswollen and reproductive in the other. Two such dual-host strains were tested: Rhizobium leguminosarum A34 in peas and beans and Bradyrhizobium sp. 32H1 in peanuts and cowpeas. In both comparisons, swollen bacteroids conferred more net host benefit by two measures: return on nodule construction cost (plant growth per gram nodule growth) and nitrogen fixation efficiency (H2 production by nitrogenase per CO2 respired). Terminal bacteroid differentiation among legume species has evolved independently multiple times, perhaps due to the increased host fitness benefits observed in this study.

Sequence-based classification and identification of Fungi

Abstract Fungal taxonomy and ecology have been revolutionized by the application of molecular methods and both have increasing connections to genomics and functional biology. However, data streams from traditional specimen- and culture-based systematics are not yet fully integrated with those from metagenomic and metatranscriptomic studies, which limits understanding of the taxonomic diversity and metabolic properties of fungal communities. This article reviews current resources, needs, and opportunities for sequence-based classification and identification (SBCI) in fungi as well as related efforts in prokaryotes. To realize the full potential of fungal SBCI it will be necessary to make advances in multiple areas. Improvements in sequencing methods, including long-read and single-cell technologies, will empower fungal molecular ecologists to look beyond ITS and current shotgun metagenomics approaches. Data quality and accessibility will be enhanced by attention to data and metadata standards and rigorous enforcement of policies for deposition of data and workflows. Taxonomic communities will need to develop best practices for molecular characterization in their focal clades, while also contributing to globally useful datasets including ITS. Changes to nomenclatural rules are needed to enable validPUBLICation of sequence-based taxon descriptions. Finally, cultural shifts are necessary to promote adoption of SBCI and to accord professional credit to individuals who contribute to community resources.

A comparison of the community diversity of foliar fungal endophytes between seedling and adult loblolly pines (Pinus taeda)

Fungal endophytes represent one of the most ubiquitous plant symbionts on Earth and are phylogenetically diverse. The structure and diversity of endophyte communities have been shown to depend on host taxa and climate, but there have been relatively few studies exploring endophyte communities throughout host maturity. We compared foliar fungal endophyte communities between seedlings and adult trees of loblolly pines (Pinus taeda) at the same seasons and locations by culturing and culture-independent methods. We sequenced the internal transcribed spacer region and adjacent partial large subunit nuclear ribosomal RNA gene (ITS-LSU amplicon) to delimit operational taxonomic units and phylogenetically characterize the communities. Despite the lower infection frequency in seedlings compared to adult trees, seedling needles were receptive to a more diverse community of fungal endophytes. Culture-free method confirmed the presence of commonly cultured OTUs from adult needles but revealed several new OTUs from seedling needles that were not found with culturing methods. The two most commonly cultured OTUs in adults were rarely cultured from seedlings, suggesting that host age is correlated with a selective enrichment for specific endophytes. This shift in endophyte species dominance may be indicative of a functional change between these fungi and their loblolly pine hosts.

Genetic variation in horizontally transmitted fungal endophytes of pine needles reveals population structure in cryptic species

UNLABELLED • PREMISE OF THE STUDY Fungal endophytes comprise one of the most ubiquitous groups of plant symbionts, inhabiting healthy leaves and stems of all major lineages of plants. Together, they comprise immense species richness, but little is known about the fundamental processes that generate their diversity. Exploration of their population structure is needed, especially with regard to geographic distributions and host affiliations.• METHODS We take a multilocus approach to examine genetic variation within and among populations of Lophodermium australe, an endophytic fungus commonly associated with healthy foliage of pines in the southeastern United States. Sampling focused on two pine species ranging from montane to coastal regions of North Carolina and Virginia.• KEY RESULTS Our sampling revealed two genetically distinct groups within Lophodermium australe. Our analysis detected less than one migrant per generation between them, indicating that they are distinct species. The species comprising the majority of isolates (major species) demonstrated a panmictic structure, whereas the species comprising the minority of isolates (cryptic species) demonstrated isolation by distance. Distantly related pine species hosted the same Lophodermium species, and host species did not influence genetic structure.• CONCLUSIONS We present the first evidence for isolation by distance in a foliar fungal endophyte that is horizontally transmitted. Cryptic species may be common among microbial symbionts and are important to delimit when exploring their genetic structure and microevolutionary processes. The hyperdiversity of endophytic fungi may be explained in part by cryptic species without apparent ecological and morphological differences as well as genetic diversification within rare fungal species across large spatial scales.

Distance decay relationships in foliar fungal endophytes are driven by rare taxa

Foliar fungal endophytes represent a diverse and species-rich plant microbiome. Their biogeography provides essential clues to their cryptic relationship with hosts and the environment in which they disperse. We present species composition, diversity, and dispersal patterns of endophytic fungi associated with needles of Pinus taeda trees across regional scales in the absence of strong environmental gradients as well as within individual trees. An empirical designation of rare and abundant taxa enlightens us on the structure of endophyte communities. We report multiple distance-decay patterns consistent with effects of dispersal limitation, largely driven by community changes in rare taxa, those taxonomic units that made up less than 0.31% of reads per sample on average. Distance-decay rates and community structure also depended on specific classes of fungi and were predominantly influenced by rare members of Dothideomycetes. Communities separated by urban areas also revealed stronger effects of distance on community similarity, confirming that host density and diversity plays an important role in symbiont biogeography, which may ultimately lead to a mosaic of functional diversity as well as rare species diversity across landscapes.

Dimensions of Host Specificity in Foliar Fungal Endophytes

Foliar fungal endophytes (FFE) colonized the phyllosphere at least 400 million years ago and have since diversified across every terrestrial ecosystem that supports plant life. Understanding how these complex symbiotic associations are generated, distributed and maintained is a challenging task that requires an understanding of host specificity. We propose a conceptual framework that outlines four ‘dimensions’ of host specificity that account for the geographic, phylogenetic or sampling scale under consideration. These ‘dimensions’ quantify FFE abundance and evenness (structural specificity), interaction strength (network specificity), evolutionary relationships (phylogenetic specificity) and the spatial or temporal consistency of the interaction (beta-specificity). We present one case study that quantifies and compares structural, network and phylogenetic specificity across FFE communities partitioned by taxonomy (Ascomycota vs. Basidiomycota). We focus on the effects of rare FFE species, approximated as Operational Taxonomic Units (OTUs), as a key methodological consideration for communities surveyed with next-generation sequencing (NGS) because the statistical nature of rarity confounds the quantification of host specificity. The exclusion of rare FFE OTUs consistently changed ecological inference by decreasing host specificity averages and increasing variances within FFE phyla. To evaluate the degree to which rare FFE OTUs affect statistical power, we compared our empirical community to that of randomized communities. Excluding rare FFE OTUs (>10% of total sequences in the case community removed) may lead to spurious host specificity metrics that are not statistically significant from that of randomized communities. Therefore, rare FFE OTU removal should be done with explicit rationale. We propose conceptualizing FFE host specificity with a multidimensional framework that will allow future studies to use quantitative, comparable and theory-driven metrics that can scale towards more meaningful estimates of global fungal biodiversity.

Two new endophytic Atractiellomycetes, Atractidochium hillariae and Proceropycnis hameedii

ABSTRACT Sterile fungal isolates are often recovered in leaf and root endophytic studies, although these seldom play a significant role in downstream analyses. The authors sought to identify and characterize two such endophytes—one representing the most commonly recovered fungal isolate in recent studies of needle endophytes of Pinus taeda and the other representing a rarely isolated root endophyte of Populus trichocarpa. Both are shown by DNA sequencing to be undescribed species of Atractiellomycetes (Pucciniomycotina, Basidiomycota), a poorly characterized class of mostly plant-associated and presumably saprobic microfungi. The authors describe the new genus and species Atractidochium hillariae (Phleogenaceae) and the new species Proceropycnis hameedii (Hoehnelomycetaceae), both in the Atractiellales, to accommodate these unusual isolates. Following incubations of 1–2 mo, A. hillariae produces minute white sporodochia, similar to those produced by several other members of Atractiellales, whereas Pr. hameedii forms conidia singly or in chains in a manner similar to its sister species Pr. pinicola. Additionally, we provide a taxonomic revision of Atractiellomycetes based on multilocus analyses and propose the new genera Neogloea (Helicogloeaceae) and Bourdotigloea (Phleogenaceae) to accommodate ex-Helicogloea species that are not congeneric with the type H. lagerheimii. Atractiellomycetes consists of a single order, Atractiellales, and three families, Hoehnelomycetaceae, Phleogenaceae, and Helicogloeaceae. Accumulated evidence suggests that Atractiellomycetes species are common but infrequently isolated members of plant foliar and root endobiomes.