Evaluation of Stagonospora Nodorum Blotch Severity and Parastagonospora nodorum Population Structure and Genetic Diversity Across Multiple Locations and Wheat Varieties in Virginia.

Parastagonospora nodorum is a necrotrophic pathogen that causes Stagonospora nodorum blotch (SNB) in wheat. Wheat varieties grown in Virginia vary in susceptibility to SNB, and the severity of SNB varies across locations and years. However, the impacts of wheat genetic backgrounds and environments on SNB severity and the structure of P. nodorum populations in the region have not been well studied. Thus, a population genetic study was conducted utilizing P. nodorum isolates collected from different wheat varieties and locations in Virginia. A total of 320 isolates were collected at seven locations over 2 years from five wheat varieties. Isolates were genotyped using multilocus simple sequence repeat markers, and necrotrophic effector (NE) and mating type genes were amplified using gene-specific primers. Wheat varieties varied in susceptibility to SNB, but site-specific environmental conditions were the primary drivers of disease severity. Fungal populations were genetically diverse, but no genetic subdivision was observed among locations or varieties. The ratio of the two mating type idiomorphs was not significantly different from 1:1, consistent with the P. nodorum population undergoing sexual reproduction. Three major NE genes were detected within the P. nodorum population, but not with equal frequency. However, NE gene profiles were similar for groups of isolates originating from different varieties, suggesting that wheat genetic backgrounds do not differentially select for NEs. There was no evidence of population structure among P. nodorum populations in Virginia and, thus, no support for wheat genetic backgrounds shaping these populations. Finally, although varieties only exhibited moderate resistance to SNB, current levels of resistance are likely to be durable over time and remain a useful tool for integrated management of SNB in the region. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Inferring the Genetic Basis of Sex Determination from the Genome of a Dioecious Nightshade.

Dissecting the genetic mechanisms underlying dioecy (i.e., separate female and male individuals) is critical for understanding the evolution of this pervasive reproductive strategy. Nonetheless, the genetic basis of sex determination remains unclear in many cases, especially in systems where dioecy has arisen recently. Within the economically important plant genus Solanum (∼2,000 species), dioecy is thought to have evolved independently at least 4 times across roughly 20 species. Here, we generate the first genome sequence of a dioecious Solanum and use it to ascertain the genetic basis of sex determination in this species. We de novo assembled and annotated the genome of Solanum appendiculatum (assembly size: ∼750 Mb scaffold N50: 0.92 Mb; ∼35,000 genes), identified sex-specific sequences and their locations in the genome, and inferred that males in this species are the heterogametic sex. We also analyzed gene expression patterns in floral tissues of males and females, finding approximately 100 genes that are differentially expressed between the sexes. These analyses, together with observed patterns of gene-family evolution specific to S. appendiculatum, consistently implicate a suite of genes from the regulatory network controlling pectin degradation and modification in the expression of sex. Furthermore, the genome of a species with a relatively young sex-determination system provides the foundational resources for future studies on the independent evolution of dioecy in this clade.

Fall Applications of Ethephon Modulates Gene Networks Controlling Bud Development during Dormancy in Peach (Prunus Persica).

Ethephon (ET) is an ethylene-releasing plant growth regulator (PGR) that can delay the bloom time in Prunus, thus reducing the risk of spring frost, which is exacerbated by global climate change. However, the adoption of ET is hindered by its detrimental effects on tree health. Little knowledge is available regarding the mechanism of how ET shifts dormancy and flowering phenology in peach. This study aimed to further characterize the dormancy regulation network at the transcriptional level by profiling the gene expression of dormant peach buds from ET-treated and untreated trees using RNA-Seq data. The results revealed that ET triggered stress responses during endodormancy, delaying biological processes related to cell division and intercellular transportation, which are essential for the floral organ development. During ecodormancy, ET mainly impeded pathways related to antioxidants and cell wall formation, both of which are closely associated with dormancy release and budburst. In contrast, the expression of dormancy-associated MADS (DAM) genes remained relatively unaffected by ET, suggesting their conserved nature. The findings of this study signify the importance of floral organogenesis during dormancy and shed light on several key processes that are subject to the influence of ET, therefore opening up new avenues for the development of effective strategies to mitigate frost risks.

Draft Assembly of Phytophthora capsici from Long-Read Sequencing Uncovers Complexity.

Resolving complex plant pathogen genomes is important for identifying the genomic shifts associated with rapid adaptation to selective agents such as hosts and fungicides, yet assembling these genomes remains challenging and expensive. Phytophthora capsici is an important, globally distributed plant pathogen that exhibits widespread fungicide resistance and a broad host range. As with other pathogenic oomycetes, P. capsici has a complex life history and a complex genome. Here, we leverage Oxford Nanopore Technologies and existing short-read resources to rapidly generate a low-cost, improved assembly. We generated 10 Gbp from a single MinION flow cell resulting in >1.25 million reads with an N50 of 13 kb. The resulting assembly is 95.2 Mbp in 424 scaffolds with an N50 length of 313 kb. This assembly is approximately 30 Mbp bigger than the current reference genome of 64 Mbp. We confirmed this larger genome size using flow cytometry, with an estimated size of 110 Mbp. BUSCO analysis identified 97.4% complete orthologs (19.2% duplicated). Evolutionary analysis supports a recent whole-genome duplication in this group. Our work provides a blueprint for rapidly integrating benchtop long-read sequencing with existing short-read data, to dramatically improve assembly quality and integrity of complex genomes and offer novel insights into pathogen genome function and evolution.

Phylogenomics Reveals Three Sources of Adaptive Variation during a Rapid Radiation.

Speciation events often occur in rapid bursts of diversification, but the ecological and genetic factors that promote these radiations are still much debated. Using whole transcriptomes from all 13 species in the ecologically and reproductively diverse wild tomato clade (Solanum sect. Lycopersicon), we infer the species phylogeny and patterns of genetic diversity in this group. Despite widespread phylogenetic discordance due to the sorting of ancestral variation, we date the origin of this radiation to approximately 2.5 million years ago and find evidence for at least three sources of adaptive genetic variation that fuel diversification. First, we detect introgression both historically between early-branching lineages and recently between individual populations, at specific loci whose functions indicate likely adaptive benefits. Second, we find evidence of lineage-specific de novo evolution for many genes, including loci involved in the production of red fruit color. Finally, using a "PhyloGWAS" approach, we detect environment-specific sorting of ancestral variation among populations that come from different species but share common environmental conditions. Estimated across the whole clade, small but substantial and approximately equal fractions of the euchromatic portion of the genome are inferred to contribute to each of these three sources of adaptive genetic variation. These results indicate that multiple genetic sources can promote rapid diversification and speciation in response to new ecological opportunity, in agreement with our emerging phylogenomic understanding of the complexity of both ancient and recent species radiations.

Gut passage and secondary metabolites alter the source of post-dispersal predation for bird-dispersed chili seeds.

Plants can influence the source and severity of seed predation through various mechanisms; the use of secondary metabolites for chemical defense, for example, is well documented. Gut passage by frugivores can also reduce mortality of animal-dispersed seeds, although this mechanism has gained far less attention than secondary metabolites. Apart from influencing the severity of seed predation, gut passage may also influence the source of seed predation. In Bolivia, we compared impacts of these two mechanisms, gut passage and secondary metabolites, on the source of seed predation in Capsicum chacoense, a wild chili species that is polymorphic for pungency (individual plants either produce fruits and seeds containing or lacking capsaicinoids). Using physical exclosures, we isolated seed removal by insects, mammals, and birds; seeds in the trials were from either pungent or non-pungent fruits and were either passed or not passed by seed-dispersing birds. Pungency had little influence on total short-term seed removal by animals, although prior work on this species indicates that capsaicin reduces mortality caused by fungi at longer time scales. Gut passage strongly reduced removal by insects, altering the relative impact of the three predator types. The weak impact of pungency on short-term predation contrasts with previous studies, highlighting the context dependence of secondary metabolites. The strong impact of gut passage demonstrates that this mechanism alone can influence which seed predators consume seeds, and that impacts of gut passage can be larger than those of secondary metabolites, which are more commonly acknowledged as a defense mechanism.

No evidence for phylogenetic constraint on natural defense evolution among wild tomatoes.

Plant defense traits can be shaped by evolutionary and physiological constraints, as well as local ecological selection. We assessed the relative importance of these factors in shaping defense trait variation across the wild tomato clade (a group of 13 closely related species) using an herbivore bioassay (Manduca sexta). With phylogenetic comparative methods, we evaluated patterns of constitutive and induced defense variation, and the extent of coupling between alternative defense strategies. We detected substantial variation among species and found no evidence for phylogenetic conservatism among defensive traits, unlike for two other ecologically relevant (reproductive) traits. In addition, constitutive and induced defense syndromes were unassociated. These data indicate that, in this group, there is no evidence for either phylogenetic conservatism of shared consumer guilds that shape defense traits, or for constraints on defense trait evolution, including mechanistic trade-offs between defense strategies. Our data suggest that defense trait variation in this clade instead results from rapid responses to local ecological conditions.

Merging ecology and genomics to dissect diversity in wild tomatoes and their relatives.

To understand the origin, history, and function, of natural biological variation, from nucleotide to community levels, is a fundamental promise of ecological genomics. The most fruitful systems for this work are those that possess both ecological and genomic resources. Such systems provide an opportunity to precisely dissect genetic and developmental mechanisms, and to connect genotypes to phenotypes, as well as to directly demonstrate the ecological and evolutionary relevance of this phenotypic variation. Here we synthesize findings emerging from our efforts to understand two fundamental evolutionary processes - speciation and adaptation - using ecological genomics approaches. Many of these studies have been in the wild tomato clade (Solanum section Lycopersicon), a group that has both exceptional diversity and genomic tools. We also highlight the expanding taxonomic reach of this work, especially in two genera - Capsicum and Jaltomata - that are closely related to Solanum. Parallel approaches in these ecologically and reproductively diverse clades enable us to examine novel questions and traits that are not captured within Solanum, while leveraging the power of comparative studies to understand shared ecological and evolutionary patterns. By synthesizing findings from phenotypic, ecophysiological, genetic, and comparative perspectives, our ultimate goal is to understand the complex mechanistic and evolutionary contributions to the formation of new traits and species diversity.

Comparative genomics of bacteria from amphibian skin associated with inhibition of an amphibian fungal pathogen, Batrachochytrium dendrobatidis.

Chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), is a skin disease associated with worldwide amphibian declines. Symbiotic microbes living on amphibian skin interact with Bd and may alter infection outcomes. We completed whole genome sequencing of 40 bacterial isolates cultured from the skin of four amphibian species in the Eastern US. Each isolate was tested in vitro for the ability to inhibit Bd growth. The aim of this study was to identify genomic differences among the isolates and generate hypotheses about the genomic underpinnings of Bd growth inhibition. We identified sixty-five gene families that were present in all 40 isolates. Screening for common biosynthetic gene clusters revealed that this set of isolates contained a wide variety of clusters; the two most abundant clusters with potential antifungal activity were siderophores (N=17 isolates) and Type III polyketide synthases (N=22 isolates). We then examined various subsets of the 22 isolates in the phylum Proteobacteria for genes encoding specific compounds that may inhibit fungal growth, including chitinase and violacein. We identified differences in Agrobacterium and Sphingomonas isolates in the chitinase genes that showed some association with anti-Bd activity, as well as variation in the violacein genes in the Janthinobacterium isolates. Using a comparative genomics approach, we generated several testable hypotheses about differences among bacterial isolates from amphibian skin communities that could contribute to variation in the ability to inhibit Bd growth. Further work is necessary to explore and uncover the various mechanisms utilized by amphibian skin bacterial isolates to inhibit Bd.

Characterization of prophages in bacterial genomes from the honey bee (Apis mellifera) gut microbiome.

The gut of the European honey bee (Apis mellifera) possesses a relatively simple bacterial community, but little is known about its community of prophages (temperate bacteriophages integrated into the bacterial genome). Although prophages may eventually begin replicating and kill their bacterial hosts, they can also sometimes be beneficial for their hosts by conferring protection from other phage infections or encoding genes in metabolic pathways and for toxins. In this study, we explored prophages in 17 species of core bacteria in the honey bee gut and two honey bee pathogens. Out of the 181 genomes examined, 431 putative prophage regions were predicted. Among core gut bacteria, the number of prophages per genome ranged from zero to seven and prophage composition (the compositional percentage of each bacterial genome attributable to prophages) ranged from 0 to 7%. Snodgrassella alvi and Gilliamella apicola had the highest median prophages per genome (3.0 ± 1.46; 3.0 ± 1.59), as well as the highest prophage composition (2.58% ± 1.4; 3.0% ± 1.59). The pathogen Paenibacillus larvae had a higher median number of prophages (8.0 ± 5.33) and prophage composition (6.40% ± 3.08) than the pathogen Melissococcus plutonius or any of the core bacteria. Prophage populations were highly specific to their bacterial host species, suggesting most prophages were acquired recently relative to the divergence of these bacterial groups. Furthermore, functional annotation of the predicted genes encoded within the prophage regions indicates that some prophages in the honey bee gut encode additional benefits to their bacterial hosts, such as genes in carbohydrate metabolism. Collectively, this survey suggests that prophages within the honey bee gut may contribute to the maintenance and stability of the honey bee gut microbiome and potentially modulate specific members of the bacterial community, particularly S. alvi and G. apicola.

Adaptive constraints at the range edge of a widespread and expanding invasive plant.

Identifying the factors that facilitate and limit invasive species' range expansion has both practical and theoretical importance, especially at the range edges. Here, we used reciprocal common garden experiments spanning the North/South and East/West range that include the North American core, intermediate and range edges of the globally invasive plant, Johnsongrass (Sorghum halepense) to investigate the interplay of climate, biotic interactions (i.e. competition) and patterns of adaptation. Our results suggest that the rapid range expansion of Johnsongrass into diverse environments across wide geographies occurred largely without local adaptation, but that further range expansion may be restricted by a fitness trade-off that limits population growth at the range edge. Interestingly, plant competition strongly dampened Johnsongrass growth but did not change the rank order performance of populations within a garden, though this varied among gardens (climates). Our findings highlight the importance of including the range edge when studying the range dynamics of invasive species, especially as we try to understand how invasive species will respond to accelerating global changes.

Why are not all chilies hot? A trade-off limits pungency.

Evolutionary biologists increasingly recognize that evolution can be constrained by trade-offs, yet our understanding of how and when such constraints are manifested and whether they restrict adaptive divergence in populations remains limited. Here, we show that spatial heterogeneity in moisture maintains a polymorphism for pungency (heat) among natural populations of wild chilies (Capsicum chacoense) because traits influencing water-use efficiency are functionally integrated with traits controlling pungency (the production of capsaicinoids). Pungent and non-pungent chilies occur along a cline in moisture that spans their native range in Bolivia, and the proportion of pungent plants in populations increases with greater moisture availability. In high moisture environments, pungency is beneficial because capsaicinoids protect the fruit from pathogenic fungi, and is not costly because pungent and non-pungent chilies grown in well-watered conditions produce equal numbers of seeds. In low moisture environments, pungency is less beneficial as the risk of fungal infection is lower, and carries a significant cost because, under drought stress, seed production in pungent chilies is reduced by 50 per cent relative to non-pungent plants grown in identical conditions. This large difference in seed production under water-stressed (WS) conditions explains the existence of populations dominated by non-pungent plants, and appears to result from a genetic correlation between pungency and stomatal density: non-pungent plants, segregating from intra-population crosses, exhibit significantly lower stomatal density (p = 0.003), thereby reducing gas exchange under WS conditions. These results demonstrate the importance of trait integration in constraining adaptive divergence among populations.

Exploring transposable element-based markers to identify allelic variations underlying agronomic traits in rice.

Transposable elements (TEs) are a major force in the production of new alleles during domestication; nevertheless, their use in association studies has been limited because of their complexity. We have developed a TE genotyping pipeline (TEmarker) and applied it to whole-genome genome-wide association study (GWAS) data from 176 Oryza sativa subsp. japonica accessions to identify genetic elements associated with specific agronomic traits. TE markers recovered a large proportion (69%) of single-nucleotide polymorphism (SNP)-based GWAS peaks, and these TE peaks retained ca. 25% of the SNPs. The use of TEs in GWASs may reduce false positives associated with linkage disequilibrium (LD) among SNP markers. A genome scan revealed positive selection on TEs associated with agronomic traits. We found several cases of insertion and deletion variants that potentially resulted from the direct action of TEs, including an allele of LOC_Os11g08410 associated with plant height and panicle length traits. Together, these findings reveal the utility of TE markers for connecting genotype to phenotype and suggest a potential role for TEs in influencing phenotypic variations in rice that impact agronomic traits.

Genotyping-by-sequencing analysis of Orobanche crenata populations in Algeria reveals genetic differentiation.

Crenate broomrape (Orobanche crenata Forsk.) is a serious long-standing parasitic weed problem in Algeria, mainly affecting legumes but also vegetable crops. Unresolved questions for parasitic weeds revolve around the extent to which these plants undergo local adaptation, especially with respect to host specialization, which would be expected to be a strong selective factor for obligate parasitic plants. In the present study, the genotyping-by-sequencing (GBS) approach was used to analyze genetic diversity and population structure of 10 Northern Algerian O. crenata populations with different geographical origins and host species (faba bean, pea, chickpea, carrot, and tomato). In total, 8004 high-quality single-nucleotide polymorphisms (5% missingness) were obtained and used across the study. Genetic diversity and relationships of 95 individuals from 10 populations were studied using model-based ancestry analysis, principal components analysis, discriminant analysis of principal components, and phylogeny approaches. The genetic differentiation (F ST) between pairs of populations was lower between adjacent populations and higher between geographically separated ones, but no support was found for isolation by distance. Further analyses identified four genetic clusters and revealed evidence of structuring among populations and, although confounded with location, among hosts. In the clearest example, O. crenata growing on pea had a SNP profile that was distinct from other host/location combinations. These results illustrate the importance and potential of GBS to reveal the dynamics of parasitic weed dispersal and population structure.

Comparative genomics of Lactobacillaceae from the gut of honey bees, Apis mellifera, from the Eastern United States.

Lactobacillaceae are an important family of lactic acid bacteria that play key roles in the gut microbiome of many animal species. In the honey bee (Apis mellifera) gut microbiome, many species of Lactobacillaceae are found, and there is functionally important strain-level variation in the bacteria. In this study, we completed whole-genome sequencing of 3 unique Lactobacillaceae isolates collected from hives in Virginia, USA. Using 107 genomes of known bee-associated Lactobacillaceae and Limosilactobacillus reuteri as an outgroup, the phylogenetics of the 3 isolates was assessed, and these isolates were identified as novel strains of Apilactobacillus kunkeei, Lactobacillus kullabergensis, and Bombilactobacillus mellis. Genome rearrangements, conserved orthologous genes (COG) categories and potential prophage regions were identified across the 3 novel strains. The new A. kunkeei strain was enriched in genes related to replication, recombination and repair, the L. kullabergensis strain was enriched for carbohydrate transport, and the B. mellis strain was enriched in transcription or transcriptional regulation and in some genes with unknown functions. Prophage regions were identified in the A. kunkeei and L. kullabergensis isolates. These new bee-associated strains add to our growing knowledge of the honey bee gut microbiome, and to Lactobacillaceae genomics more broadly.

Evolutionary ecology of pungency in wild chilies.

The primary function of fruit is to attract animals that disperse viable seeds, but the nutritional rewards that attract beneficial consumers also attract consumers that kill seeds instead of dispersing them. Many of these unwanted consumers are microbes, and microbial defense is commonly invoked to explain the bitter, distasteful, occasionally toxic chemicals found in many ripe fruits. This explanation has been criticized, however, due to a lack of evidence that microbial consumers influence fruit chemistry in wild populations. In the present study, we use wild chilies to show that chemical defense of ripe fruit reflects variation in the risk of microbial attack. Capsaicinoids are the chemicals responsible for the well known pungency of chili fruits. Capsicum chacoense is naturally polymorphic for the production of capsaicinoids and displays geographic variation in the proportion of individual plants in a population that produce capsaicinoids. We show that this variation is directly linked to variation in the damage caused by a fungal pathogen of chili seeds. We find that Fusarium fungus is the primary cause of predispersal chili seed mortality, and we experimentally demonstrate that capsaicinoids protect chili seeds from Fusarium. Further, foraging by hemipteran insects facilitates the entry of Fusarium into fruits, and we show that variation in hemipteran foraging pressure among chili populations predicts the proportion of plants in a population producing capsaicinoids. These results suggest that the pungency in chilies may be an adaptive response to selection by a microbial pathogen, supporting the influence of microbial consumers on fruit chemistry.

Impacts of climate warming on terrestrial ectotherms across latitude.

The impact of anthropogenic climate change on terrestrial organisms is often predicted to increase with latitude, in parallel with the rate of warming. Yet the biological impact of rising temperatures also depends on the physiological sensitivity of organisms to temperature change. We integrate empirical fitness curves describing the thermal tolerance of terrestrial insects from around the world with the projected geographic distribution of climate change for the next century to estimate the direct impact of warming on insect fitness across latitude. The results show that warming in the tropics, although relatively small in magnitude, is likely to have the most deleterious consequences because tropical insects are relatively sensitive to temperature change and are currently living very close to their optimal temperature. In contrast, species at higher latitudes have broader thermal tolerance and are living in climates that are currently cooler than their physiological optima, so that warming may even enhance their fitness. Available thermal tolerance data for several vertebrate taxa exhibit similar patterns, suggesting that these results are general for terrestrial ectotherms. Our analyses imply that, in the absence of ameliorating factors such as migration and adaptation, the greatest extinction risks from global warming may be in the tropics, where biological diversity is also greatest.

Into the weeds: new insights in plant stress.

Weeds, plants that thrive in the face of disturbance, have eluded human's attempts at control for >12 000 years, positioning them as a unique group of extreme stress tolerators. The most successful weeds have a suite of traits that enable them to rapidly adapt to environments typified by stress, growing in hostile conditions or subject to massive destruction from agricultural practices. Through their ability to persist and adapt, weeds illuminate principles of evolution and provide insights into weed management and crop improvement. Here we highlight why the time is right to move beyond traditional model systems and leverage weeds to gain a deeper understanding of the mechanisms, adaptations, and genetic and physiological bases for stress tolerance.

Horsenettle (Solanum carolinense) fruit bacterial communities are not variable across fine spatial scales.

Fruit house microbial communities that are unique from the rest of the plant. While symbiotic microbial communities complete important functions for their hosts, the fruit microbiome is often understudied compared to other plant organs. Fruits are reproductive tissues that house, protect, and facilitate the dispersal of seeds, and thus they are directly tied to plant fitness. Fruit microbial communities may, therefore, also impact plant fitness. In this study, we assessed how bacterial communities associated with fruit of Solanum carolinense, a native herbaceous perennial weed, vary at fine spatial scales (<0.5 km). A majority of the studies conducted on plant microbial communities have been done at large spatial scales and have observed microbial community variation across these large spatial scales. However, both the environment and pollinators play a role in shaping plant microbial communities and likely have impacts on the plant microbiome at fine scales. We collected fruit samples from eight sampling locations, ranging from 2 to 450 m apart, and assessed the fruit bacterial communities using 16S rRNA gene amplicon sequencing. Overall, we found no differences in observed richness or microbial community composition among sampling locations. Bacterial community structure of fruits collected near one another were not more different than those that were farther apart at the scales we examined. These fine spatial scales are important to obligate out-crossing plant species such as S. carolinense because they are ecologically relevant to pollinators. Thus, our results could imply that pollinators serve to homogenize fruit bacterial communities across these smaller scales.

Assessing age, breeding stage, and mating activity as drivers of variation in the reproductive microbiome of female tree swallows.

Sexually transmitted microbes are hypothesized to influence the evolution of reproductive strategies. Though frequently discussed in this context, our understanding of the reproductive microbiome is quite nascent. Indeed, testing this hypothesis first requires establishing a baseline understanding of the temporal dynamics of the reproductive microbiome and of how individual variation in reproductive behavior and age influence the assembly and maintenance of the reproductive microbiome as a whole. Here, we ask how mating activity, breeding stage, and age influence the reproductive microbiome. We use observational and experimental approaches to explain variation in the cloacal microbiome of free-living, female tree swallows (Tachycineta bicolor). Using microsatellite-based parentage analyses, we determined the number of sires per brood (a proxy for female mating activity). We experimentally increased female sexual activity by administering exogenous 17ß-estradiol. Lastly, we used bacterial 16S rRNA amplicon sequencing to characterize the cloacal microbiome. Neither the number of sires per brood nor the increased sexual activity of females significantly influenced female cloacal microbiome richness or community structure. Female age, however, was positively correlated with cloacal microbiome richness and influenced overall community structure. A hypothesis to explain these patterns is that the effect of sexual activity and the number of mates on variation in the cloacal microbiome manifests over an individual's lifetime. Additionally, we found that cloacal microbiome alpha diversity (Shannon Index, Faith's phylogenetic distance) decreased and community structure shifted between breeding stages. This is one of few studies to document within-individual changes and age-related differences in the cloacal microbiome across successive breeding stages. More broadly, our results contribute to our understanding of the role that host life history and behavior play in shaping the cloacal microbiomes of wild birds.