New sequencing technologies facilitate the generation of large-scale molecular data sets for constructing the plant tree of life. We describe a new probe set for target enrichment sequencing to generate nuclear sequence data to build phylogenetic trees with any flagellate land plants, including hornworts, liverworts, mosses, lycophytes, ferns, and all gymnosperms. METHODS: We leveraged existing transcriptome and genome sequence data to design the GoFlag 451 probes, a set of 56,989 probes for target enrichment sequencing of 451 exons that are found in 248 single-copy or low-copy nuclear genes across flagellate plant lineages. RESULTS: Our results indicate that target enrichment using the GoFlag451 probe set can provide large nuclear data sets that can be used to resolve relationships among both distantly and closely related taxa across the flagellate land plants. We also describe the GoFlag 408 probes, an optimized probe set covering 408 of the 451 exons from the GoFlag 451 probe set that is commercialized by RAPiD Genomics. CONCLUSIONS: A target enrichment approach using the new probe set provides a relatively low-cost solution to obtain large-scale nuclear sequence data for inferring phylogenetic relationships across flagellate land plants.

Confirmed dioecy in at least 15 solanums is distributed across the genus, with perhaps 6 independent origins, and with crypticity in the form of morphologically hermaphroditic, but functionally unisexual, flowers characterizing all species. Dioecy is not more strongly associated with islands. Inaperturate pollen in pistillate flowers characterizes almost all, but not the two dioecious species studied herein. Dioecy in both species indicates leakiness (rare hermaphroditic flowers on male plants) that helps explain island colonization and radiation. Leakiness allowed confirmation-usually impossible for dioecious species-of self-compatibility for S. polygamum, and thus support for the hypothesis that dioecy evolved to promote outcrossing.

36Premise of the study: New sequencing technologies enable the possibility of generating large-37 scale molecular datasets for constructing the plant tree of life. We describe a new probe set for 38 target enrichment sequencing to generate nuclear sequence data to build phylogenetic trees with 39 any flagellate plants, comprising hornworts, liverworts, mosses, lycophytes, ferns, and 40 gymnosperms. 41 Methods and Results: We leveraged existing transcriptome and genome sequence data to design 42 a set of 56,989 probes for target enrichment sequencing of 451 nuclear exons and non-coding 43 flanking regions across flagellate plant lineages. We describe the performance of target 44 enrichment using the probe set across flagellate plants and demonstrate the potential of the data 45 to resolve relationships among both ancient and closely related taxa. 46 Conclusions: A target enrichment approach using the new probe set provides a relatively low-47 cost solution to obtain large-scale nuclear sequence data for inferring phylogenetic relationships 48 across flagellate plants. 49 50

Citation: Barrington, D. S., N. R. Patel, and M. W. Southgate. 2020. Inferring the impacts of evolutionary history and ecological constraints on spore size and shape in the ferns. Applications in Plant Sciences 8(4): e11339. PREMISE:In the ferns, cell size has been explored with spores, which are largely uniform within species, produced in abundance, and durable. However, spore size and shape have been variously defined, and the relationship of these traits to genome size has not been well established. Here, we explore the variation in fern spore size and shape by ploidy level and genome size. METHODS:Measurements of spore dimensions for two study sets of ferns, Polystichum and Adiantum, both including diploid and tetraploid taxa, provided the basis for computing estimates of shape and size as defined here. These traits were compared between and within ploidy levels and regressed with genome size estimates from flow cytometry analysis. RESULTS:All size traits were strongly correlated with genome size; the shape trait was weakly correlated. Tetraploids were larger than diploids as expected; the spores of some closely related diploid species were also significantly different in size. DISCUSSION:Researchers with access to a student-grade compound microscope can develop a valid estimate of relative genome size for ferns. These estimates provide enough resolution to infer ploidy level and explore the relationship between genome size, habitat, and physiological constraints for species within ploidy level. KEY WORDS ferns; genome size; ploidy level; spore shape; spore size; spore volume; spores. Applications in Plant Sciences 2020 8(4): e11339 Barrington et al.-Fern spores and genome size • 2 of 10

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