Viola pubescens is a perennial, mixed breeding herb that produces both chasmogamous and cleistogamous flowers at different times of the season. Once bud type is specified, it does not convert from one form to the other. While temporal production of the two flowers is known to be influenced by environmental factors, the specific environmental cues that signal emergence of each flower type have not been empirically studied. To investigate the environmental parameters driving seasonal development of chasmogamous versus cleistogamous flowers, a native V. pubescens population was examined during the spring and summer of 2016 and 2017. Measurements of light quantity, canopy cover, photoperiod, temperature, soil moisture, soil pH, and the number of chasmogamous and cleistogamous buds were collected on either a weekly or biweekly basis. Independent zero-inflated negative binomial (ZINB) regressions were used to model the odds of bud production (0 versus 1 bud) and bud counts (� 1 bud) as a function of the environmental variables. Results of the ZINB models highlight key differences between the environmental variables that influence chasmogamous versus cleistogamous bud development and counts. In addition to the ZINB regressions, individual logistic regressions were fit to the bud data. The logistic models support results of the ZINB models and, more crucially, identify specific environmental thresholds at which each bud type is probable. Collectively, this work offers novel insight into how environmental variables shape temporal development of chasmogamous and cleistogamous flowers, suggests distinct threshold values that may aid in selectively inducing each flower type, and provides insight into how climatic change may impact mixed breeding species. OPEN ACCESS Citation: Sternberger AL, Ruhil AVS, Rosenthal DM, Ballard HE, Wyatt SE (2020) Environmental impact on the temporal production of chasmogamous and cleistogamous flowers in the mixed breeding system of Viola pubescens. PLoS ONE 15(3): e0229726. https://doi.org/10.Environmental impact on chasmogamous and cleistogamous flowering in Viola pubescens PLOS ONE | https://doi.org/10.1371/journal.pone.0229726 March 11, 2020 2 / 22are capable of delayed self-fertilization, most species including Viola pubescens promote their cross-pollination [8]. In comparison, cleistogamous flowers are obligate self-pollinators with vestigial or obsolete petals, no nectar, usually reduced numbers of underdeveloped stamens (though still capable of producing a few viable pollen grains), and a reduced and commonly coiled style to effect self-pollination [16][17][18][19]. Reduced cleistogamous flowers require less energy to produce than chasmogamous flowers, and the differential cost in production of the two flowers is believed to lead to chasmogamous flowers being developmentally favored in optimal growth conditions when a surplus of resources is available [20,21]. Therefore, mixed breeding species like V. pubescens can increase genetic diversity through cross-pollination of chasmogamous flo...

Viola is a large genus with worldwide distribution and many traits not currently exemplified in model plants including unique breeding systems and the production of cyclotides. Here we report de novo genome assembly and transcriptomic analyses of the non-model species Viola pubescens using short-read DNA sequencing data and RNA-Seq from eight diverse tissues. First, V. pubescens genome size was estimated through flow cytometry, resulting in an approximate haploid genome of 455 Mbp. Next, the draft V. pubescens genome was sequenced and assembled resulting in 264,035,065 read pairs and 161,038 contigs with an N50 length of 3,455 base pairs (bp). RNA-Seq data were then assembled into tissue-specific transcripts. Together, the DNA and transcript data generated 38,081 ab initio gene models which were functionally annotated based on homology to Arabidopsis thaliana genes and Pfam domains. Gene expression was visualized for each tissue via principal component analysis and hierarchical clustering, and gene co-expression analysis identified 20 modules of tissue-specific transcriptional networks. Some of these modules highlight genetic differences between chasmogamous and cleistogamous flowers and may provide insight into V. pubescens’ mixed breeding system. Orthologous clustering with the proteomes of A. thaliana and Populus trichocarpa revealed 8,531 sequences unique to V. pubescens , including 81 novel cyclotide precursor sequences. Cyclotides are plant peptides characterized by a stable, cyclic cystine knot motif, making them strong candidates for drug scaffolding and protein engineering. Analysis of the RNA-Seq data for these cyclotide transcripts revealed diverse expression patterns both between transcripts and tissues. The diversity of these cyclotides was also highlighted in a maximum likelihood protein cladogram containing V. pubescens cyclotides and published cyclotide sequences from other Violaceae and Rubiaceae species. Collectively, this work provides the most comprehensive sequence resource for Viola , offers valuable transcriptomic insight into V. pubescens , and will facilitate future functional genomics research in Viola and other diverse plant groups.

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