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Abstract Acquisition of mobile genetic elements can confer novel traits to bacteria. Some integrative and conjugative elements confer upon members of Bradyrhizobium the capacity to fix nitrogen in symbiosis with legumes. These so-called symbiosis integrative conjugative elements (symICEs) can be extremely large and vary as monopartite and polypartite configurations within chromosomes of related strains. These features are predicted to impose fitness costs and have defied explanation. Here, we show that chromosome architecture is largely conserved despite diversity in genome composition, variations in locations of attachment sites recognized by integrases of symICEs, and differences in large-scale chromosomal changes that occur upon integration. Conversely, many simulated nonnative chromosome–symICE combinations are predicted to result in lethal deletions or disruptions to architecture. Findings suggest that there is compatibility between chromosomes and symICEs. We hypothesize that the size and structural flexibility of symICEs are important for generating combinations that maintain chromosome architecture across a genus of nitrogen-fixing bacteria with diverse and dynamic genomes. 

ABSTRACT Root nodulating rhizobia are nearly ubiquitous in soils and provide the critical service of nitrogen fixation to thousands of legume species, including staple crops. However, the magnitude of fixed nitrogen provided to hosts varies markedly among rhizobia strains, despite host legumes having mechanisms to selectively reward beneficial strains and to punish ones that do not fix sufficient nitrogen. Variation in the services of microbial mutualists is considered paradoxical given host mechanisms to select beneficial genotypes. Moreover, the recurrent evolution of non-fixing symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. Here, we deconstructed hundreds of genome sequences from genotypically and phenotypically diverse Bradyrhizobium strains and revealed mechanisms that generate variation in symbiotic nitrogen fixation. We show that this trait is conferred by a modular system consisting of many extremely large integrative conjugative elements and few conjugative plasmids. Their transmissibility and propensity to reshuffle genes generate new combinations that lead to uncooperative genotypes and make individual partnerships unstable. We also demonstrate that these same properties extend beneficial associations to diverse host species and transfer symbiotic capacity among diverse strains. Hence, symbiotic nitrogen fixation is underpinned by modularity, which engenders flexibility, a feature that reconciles evolutionary robustness and instability. These results provide new insights into mechanisms driving the evolution of mobile genetic elements. Moreover, they yield a new predictive model on the evolution of rhizobial symbioses, one that informs on the health of organisms and ecosystems that are hosts to symbionts and that helps resolve the long-standing paradox. IMPORTANCE Genetic variation is fundamental to evolution yet is paradoxical in symbiosis. Symbionts exhibit extensive variation in the magnitude of services they provide despite hosts having mechanisms to select and increase the abundance of beneficial genotypes. Additionally, evolution of uncooperative symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. We analyzed genome sequences of Bradyrhizobium, bacteria that in symbioses with legume hosts, fix nitrogen, a nutrient essential for ecosystems. We show that genes for symbiotic nitrogen fixation are within elements that can move between bacteria and reshuffle gene combinations that change host range and quality of symbiosis services. Consequently, nitrogen fixation is evolutionarily unstable for individual partnerships, but is evolutionarily stable for legume- Bradyrhizobium symbioses in general. We developed a holistic model of symbiosis evolution that reconciles robustness and instability of symbiosis and informs on applications of rhizobia in agricultural settings. 

Taxonomic treatments start with the creation of taxon-by-character matrices. Systematics authors recognized data ambiguity issues in published phenotypic characters and are willing to adopt an ontology-aware authoring tool (Cui et al. 2022). To promote interoperable and reusable taxonomic treatments, we have developed two research prototypes: a web-based application, Character Recorder (http://chrecorder.lusites.xyz/login), to faciliate the use and addition of ontology terms by Carex systematist authors while building their matrices, and a mobile application, Conflict Resolver (Android, https://tinyurl.com/5cfatrz8), to identify potential conflicts among the terms added by the authors and facilitate the resolution of the conflicts. We have completed two usability studies on Character Recorder. a web-based application, Character Recorder (http://chrecorder.lusites.xyz/login), to faciliate the use and addition of ontology terms by Carex systematist authors while building their matrices, and a mobile application, Conflict Resolver (Android, https://tinyurl.com/5cfatrz8), to identify potential conflicts among the terms added by the authors and facilitate the resolution of the conflicts. We have completed two usability studies on Character Recorder. In the one-hour Student Usabiilty Study, 16 third-year biology students with a general introduction to Carex used Character Recorder and Excel to record a set of 11 given characters for two samples (shape of sheath summits = U-shaped/U shaped). In the three-day Expert Usability Study, 7 established Carex systematists and 1 graduate student with expert-level knowledge used Character Recorder to record characters for 1 sample each of Carex canesens and Carex rostrata as they would in their professional life, using real mounted specimens, microscope, reticles, and rulers. Experts activities were not timed but they spent roughly 1.5 days on recording the characters and the rest of time discussing features and improvements. Features of Character Recorder have been reported in 2021 TDWG meeting and we included here only a few figures to highlight its interoperability and reusability features at the time of the usability studies (Fig. 1, Fig. 2, and Fig. 3). The Carex Ontology accompanying Character Recorder was created by extracting terms from Carex treatments of Flora of China and Flora of North America using Explorer of Taxon Concept (Cui et al. 2016) with subsequent manual edits. The design principle of Character Recorder is to encourage standardization and also leave the authors the freedom to do their work. While it took students an average of 6 minutes to recover all the given characters using Microsoft® Excel®, as opposed to 11 minutes using Character Recorder, the total number of unique meaning-bearing words used in their characters was 116 with Excel versus 30 with Character Recorder, showing the power of the latter in reducing synonyms and spelling variations. All students reported that they learned to use Character Recorder quickly and some even thought their use was as fast or faster than using Excel. All preferred Character Recorder to Excel for teaching students to record character data. Nearly all of the students found Character Recorder was more useful for recording clear and consistent data and all students agreed that participating in this study raised their awareness of data variation issues. The expert group consisted of 3, 2, 1, 3 experts in age ranges 20-49, 50-59, 60-69, and >69, respectively. They each recorded over 100 characters for two or more samples. Detailed analysis of their characters is pending, but we have noticed color characters have more variations than other characters (Fig. 4). All experts reported that they learned to use Character Recorder quickly, and 6 out of 8 believed they would not need a tutorial the next time they used it. One out of 8 experts somewhat disliked the feature of reusing others' values ("Use This" in Fig. 2) as it may undermine the objectivity and independence of an author. All experts used Recommended Set of Characters and they liked the term suggestion and illustration features shown in Figs 2, 3. All experts would recommend that their colleagues try Character Recorder and recommended that it be further developed and integrated into every taxonomist's toolbox. Student and expert responses to the National Aeronautics and Space Administration Task Load Index (NASA-TLX, Hart and Staveland 1988) are summarized in Fig. 5, which suggests that, while Character Recorder may incur in a slightly higher cost, the performance it supports outweighs its cost, especially for students. Every piece of the software prototypes and associated resources are open for anyone to access or further develop. We thank all student and expert participants and US National Science Foundation for their support in this research. We thank Harris & Harris and Presses de l'Université Laval for the permissions to use their phenotype illustrations in Character Recorder. 

Legumes preferentially associate with and reward beneficial rhizobia in root nodules, but the processes by which rhizobia evolve to provide benefits to novel hosts remain poorly understood. Using cycles of in planta and in vitro evolution, we experimentally simulated lifestyles where rhizobia repeatedly interact with novel plant genotypes with which they initially provide negligible benefits. Using a full-factorial replicated design, we independently evolved two rhizobia strains in associations with each of two Lotus japonicus genotypes that vary in regulation of nodule formation. We evaluated phenotypic evolution of rhizobia by quantifying fitness, growth effects and histological features on hosts, and molecular evolution via genome resequencing. Rhizobia evolved enhanced host benefits and caused changes in nodule development in one of the four host–symbiont combinations, that appeared to be driven by reduced costs during symbiosis, rather than increased nitrogen fixation. Descendant populations included genetic changes that could alter rhizobial infection or proliferation in host tissues, but lack of evidence for fixation of these mutations weakens the results. Evolution of enhanced rhizobial benefits occurred only in a subset of experiments, suggesting a role for host–symbiont genotype interactions in mediating the evolution of enhanced benefits from symbionts. 

Bacterial mutualists generate major fitness benefits for eukaryotes, reshaping the host phenotype and its interactions with the environment. Yet, microbial mutualist populations are predicted to generate mutants that defect from providing costly services to hosts while maintaining the capacity to exploit host resources. Here, we examined the mutualist service of symbiotic nitrogen fixation in a metapopulation of root-nodulating Bradyrhizobium spp . that associate with the native legume Acmispon strigosus . We quantified mutualism traits of 85 Bradyrhizobium isolates gathered from a 700 km transect in California spanning 10 sampled A. strigosus populations. We clonally inoculated each Bradyrhizobium isolate onto A. strigosus hosts and quantified nodulation capacity and net effects of infection, including host growth and isotopic nitrogen concentration. Six Bradyrhizobium isolates from five populations were categorized as ineffective because they formed nodules but did not enhance host growth via nitrogen fixation. Six additional isolates from three populations failed to form root nodules. Phylogenetic reconstruction inferred two types of mutualism breakdown, including three to four independent losses of effectiveness and five losses of nodulation capacity on A. strigosus . The evolutionary and genomic drivers of these mutualism breakdown events remain poorly understood.