A novel miR160a-GmARF16-GmMYC2 module determines soybean salt tolerance and adaptation.

Salt stress is a major challenge that has a negative impact on soybean growth and productivity. Therefore, it is important to understand the regulatory mechanism of salt response to ensure soybean yield under such conditions. In this study, we identified and characterized a miR160a-GmARF16-GmMYC2 module and its regulation during the salt-stress response in soybean. miR160a promotes salt tolerance by cleaving GmARF16 transcripts, members of the Auxin Response Factor (ARF) family, which negatively regulates salt tolerance. In turn, GmARF16 activates GmMYC2, encoding a bHLH transcription factor that reduces salinity tolerance by down-regulating proline biosynthesis. Genomic analysis among wild and cultivated soybean accessions identified four distinct GmARF16 haplotypes. Among them, the GmARF16H3 haplotype is preferentially enriched in localities with relatively saline soils, suggesting GmARF16H3 was artificially selected to improve salt tolerance. Our findings therefore provide insights into the molecular mechanisms underlying salt response in soybean and provide valuable genetic targets for the molecular breeding of salt tolerance.

ChecMatE: A workflow package to automatically generate machine learning potentials and phase diagrams for semiconductor alloys.

Semiconductor alloy materials are highly versatile due to their adjustable properties; however, exploring their structural space is a challenging task that affects the control of their properties. Traditional methods rely on ad hoc design based on the understanding of known chemistry and crystallography, which have limitations in computational efficiency and search space. In this work, we present ChecMatE (Chemical Material Explorer), a software package that automatically generates machine learning potentials (MLPs) and uses global search algorithms to screen semiconductor alloy materials. Taking advantage of MLPs, ChecMatE enables a more efficient and cost-effective exploration of the structural space of materials and predicts their energy and relative stability with ab initio accuracy. We demonstrate the efficacy of ChecMatE through a case study of the InxGa1-xN system, where it accelerates structural exploration at reduced costs. Our automatic framework offers a promising solution to the challenging task of exploring the structural space of semiconductor alloy materials.

Time-series transcriptome comparison reveals the gene regulation network under salt stress in soybean (Glycine max) roots.

BACKGROUND: Soil salinity is a primary factor limiting soybean (Glycine max) productivity. Breeding soybean for tolerance to high salt conditions is therefore critical for increasing yield. To explore the molecular mechanism of soybean responses to salt stress, we performed a comparative transcriptome time-series analysis of root samples collected from two soybean cultivars with contrasting salt sensitivity. RESULTS: The salt-tolerant cultivar 'Qi Huang No.34' (QH34) showed more differential expression of genes than the salt-sensitive cultivar 'Dong Nong No.50' (DN50). We identified 17,477 genes responsive to salt stress, of which 6644 exhibited distinct expression differences between the two soybean cultivars. We constructed the corresponding co-expression network and performed Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. The results suggested that phytohormone signaling, oxidoreduction, phenylpropanoid biosynthesis, the mitogen-activated protein kinase pathway and ribosome metabolism may play crucial roles in response to salt stress. CONCLUSIONS: Our comparative analysis offers a comprehensive understanding of the genes involved in responding to salt stress and maintaining cell homeostasis in soybean. The regulatory gene networks constructed here also provide valuable molecular resources for future functional studies and breeding of soybean with improved tolerance to salinity.

Resolving the odd-even oscillation of water dissociation at rutile TiO2(110)-water interface by machine learning accelerated molecular dynamics.

Aqueous rutile TiO2(110) is the most widely studied water-oxide interface, and yet questions about water dissociation are still controversial. Theoretical studies have systematically investigated the influence of the slab thickness on water dissociation energy (Ediss) at 1 monolayer coverage using static density functional theory calculation and found that Ediss exhibits odd-even oscillation with respect to the TiO2 slab thickness. However, less studies have accounted for the full solvation of an aqueous phase using ab initio molecular dynamics due to high computational costs in which only three, four, and five trilayer models of rutile(110)-water interfaces have been simulated. Here, we report Machine Learning accelerated Molecular Dynamics (MLMD) simulations of defect-free rutile(110)-water interfaces, which allows for a systematic study of the slab thickness ranging from 3 to 17 trilayers with much lower costs while keeping ab initio accuracy. Our MLMD simulations show that the dissociation degree of surface water (α) oscillates with the slab thickness and converges to ∼2% as the TiO2 slab becomes thicker. Converting α into dissociation free energy (ΔAdiss) and comparing with dissociation total energy Ediss calculated with a single monolayer of water, we find that the full solvation of the interfaces suppresses surface water from dissociating. It is interesting to note that the machine learning potential trained from the dataset containing exclusively the five trilayer TiO2 model exhibits excellent transferability to other slab thicknesses and further captures the oscillating behavior of surface water dissociation. Detailed analyses indicate that the central plane in odd trilayer slabs modulates the interaction between double trilayers and, thus, the bonding strength between terminal Ti and water, which affects pKa of surface water and water dissociation degree.

Uncertainty estimation for molecular dynamics and sampling.

Machine-learning models have emerged as a very effective strategy to sidestep time-consuming electronic-structure calculations, enabling accurate simulations of greater size, time scale, and complexity. Given the interpolative nature of these models, the reliability of predictions depends on the position in phase space, and it is crucial to obtain an estimate of the error that derives from the finite number of reference structures included during model training. When using a machine-learning potential to sample a finite-temperature ensemble, the uncertainty on individual configurations translates into an error on thermodynamic averages and leads to a loss of accuracy when the simulation enters a previously unexplored region. Here, we discuss how uncertainty quantification can be used, together with a baseline energy model, or a more robust but less accurate interatomic potential, to obtain more resilient simulations and to support active-learning strategies. Furthermore, we introduce an on-the-fly reweighing scheme that makes it possible to estimate the uncertainty in thermodynamic averages extracted from long trajectories. We present examples covering different types of structural and thermodynamic properties and systems as diverse as water and liquid gallium.

Evolutionary and ecological drivers of plant flavonoids across a large latitudinal gradient.

Flavonoids are important secondary metabolites that play an integral role in protecting plants against UV radiation and other forms of environmental stress. Given widespread impacts of environmental effects associated with latitude on a multitude of biological systems and a well-documented increase in solar radiation towards the equator, plant flavonoid production is expected to increase as a response to factors associated with decreasing latitude. Using data from a Neotropical genus (Ruellia) that spans an exceptionally broad latitudinal gradient, we tested a hypothesis of a positive latitudinal gradient in flavonoid concentration and assessed other factors that influence flavonoid production including habitat type (xeric vs. wet), altitude, phylogenetic relatedness, and pleiotropic effects. Two flavones with peak absorbance in ultraviolet wavelengths, apigenin and luteolin, were detected across all species. Transcriptome data confirm high expression of the gene required for flavone biosynthesis, flavone synthase (FNS). Contrary to our prediction, data revealed a positive correlation between flavone concentration and higher latitudes. Further, we recovered strong impacts of xeric habitat, pleiotropy, and phylogenetic relatedness on flavone concentrations. This study documents a complex interplay of ecological, historical, phylogenetic relatedness, and pleiotropic factors driving plant flavonoid production.

Genome-scale transcriptional study of hybrid effects and regulatory divergence in an F1 hybrid Ruellia (Wild Petunias: Acanthaceae) and its parents.

BACKGROUND: New combinations of divergent genomes can give rise to novel genetic functions in resulting hybrid progeny. Such functions may yield opportunities for ecological divergence, contributing ultimately to reproductive isolation and evolutionary longevity of nascent hybrid lineages. In plants, the degree to which transgressive genotypes contribute to floral novelty remains a question of key interest. Here, we generated an F1 hybrid plant between the red-flowered Ruellia elegans and yellow flowered R. speciosa. RNA-seq technology was used to explore differential gene expression between the hybrid and its two parents, with emphasis on genetic elements involved in the production of floral anthocyanin pigments. RESULTS: The hybrid was purple flowered and produced novel floral delphinidin pigments not manufactured by either parent. We found that nearly a fifth of all 86,475 unigenes expressed were unique to the hybrid. The majority of hybrid unigenes (80.97%) showed a pattern of complete dominance to one parent or the other although this ratio was uneven, suggesting asymmetrical influence of parental genomes on the progeny transcriptome. However, 8.87% of all transcripts within the hybrid were expressed at significantly higher or lower mean levels than observed for either parent. A total of 28 unigenes coding putatively for eight core enzymes in the anthocyanin pathway were recovered, along with three candidate MYBs involved in anthocyanin regulation. CONCLUSION: Our results suggest that models of gene evolution that explain phenotypic novelty and hybrid establishment in plants may need to include transgressive effects. Additionally, our results lend insight into the potential for floral novelty that derives from unions of divergent genomes. These findings serve as a starting point to further investigate molecular mechanisms involved in flower color transitions in Ruellia.

Narcissistic chiral self-sorting of molecular face-rotating polyhedra.

Narcissistic chiral self-sorting prevailed in the assembly of molecular face-rotating polyhedra from a C3h building block 5,5,10,10,15,15-hexabutyl-truxene-2,7,12-tricarbaldehyde and racemic mixtures of 1,2-diamines. Out of 124 possible stereoisomers, a pair of racemic polyhedra dominated, wherein (1R,2R)-diamines were segregated in AAAA polyhedra and (1S,2S)-diamines in CCCC polyhedra. This chiral self-sorting process is regulated by facial non-covalent interactions in the polyhedra. In contrast, D3h facial building blocks 1,3,5-tris-(4-formyl-phenyl)triazine and racemic mixtures of 1,2-diamines assembled into polyhedra without facial interactions, and their assembly process did not undergo apparent chiral self-sorting.

Airway Progenitor Clone Formation Is Enhanced by Y-27632-Dependent Changes in the Transcriptome.

The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more wide-spread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 × 10(10) cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Whole-transcriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632-treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell-cell junctions, and cell-extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell-cell and cell-ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.

Identification of functional genic components of major fusarium head blight resistance quantitative trait loci in wheat cultivar Sumai 3.

Fusarium head blight (FHB) is a devastating disease worldwide, affecting wheat and other small grains. To identify key wheat genes involved in FHB pathogenesis, 406 FHB-related wheat expressed sequence tags functionally identified in Sumai 3 were investigated for their association with FHB-resistance quantitative trait loci (QTL) Fhb1 and Fhb_6BL in 2010 and 2011. A total of 47 candidate genes were identified by bulk analysis, near-isogenic screening and expression QTL mapping, and were finally mapped to their carrier chromosomes with Chinese Spring nulli-tetra deficiency lines. One gene, designated WFhb1_c1 (wheat Fhb1 candidate gene 1), was both functionally associated with and physically located within Fhb1 and was found to be weakly similar (E = 5e+0) to an Arabidopsis gene encoding pectin methyl esterase inhibitor. Two other genes, designated WFI_6BL1 and WFI_6BL2 (wheat-Fusarium interaction genes 6BL1 and 6BL2), were functionally associated with Fhb_6BL but physically mapped on chromosomes 7D and 5A, respectively. WFI_6BL1 was annotated as a 13- lipoxygenase gene and WFI_6BL2 might encode a PR-4-like protein. Our data suggested that i) Fhb1 seems to contribute to FHB resistance by reducing susceptibility in the first 60 h, ii) Fhb_6BL makes its contribution via the jasmonate-mediated pathways, and iii) wheat seems to activate its defense mechanism in the biotrophic phase of FHB pathogenesis.

Co-expression network analyses of anthocyanin biosynthesis genes in Ruellia (Wild Petunias; Acanthaceae).

BACKGROUND: Anthocyanins are major pigments contributing to flower coloration and as such knowledge of molecular architecture underlying the anthocyanin biosynthetic pathway (ABP) is key to understanding flower color diversification. To identify ABP structural genes and associated regulatory networks, we sequenced 16 transcriptomes generated from 10 species of Ruellia and then conducted co-expression analyses among resulting data. RESULTS: Complete coding sequences for 12 candidate structural loci representing eight genes plus nine candidate regulatory loci were assembled. Analysis of non-synonymous/synonymous (dn/ds) mutation rates indicated all identified loci are under purifying selection, suggesting overall selection to prevent the accumulation of deleterious mutations. Additionally, upstream enzymes have lower rates of molecular evolution compared to downstream enzymes. However, site-specific tests of selection yielded evidence for positive selection at several sites, including four in F3'H2 and five in DFR3, and these sites are located in protein binding regions. A species-level phylogenetic tree constructed using a newly implemented hybrid transcriptome-RADseq approach implicates several flower color transitions among the 10 species. We found evidence of both regulatory and structural mutations to F3'5'H in helping to explain the evolution of red flowers from purple-flowered ancestors. CONCLUSIONS: Sequence comparisons and co-expression analyses of ABP loci revealed that mutations in regulatory loci are likely to play a greater role in flower color transitions in Ruellia compared to mutations in underlying structural genes.

Expanding the gene pool for soybean improvement with its wild relatives.

Genetic diversity is a cornerstone of crop improvement, However, cultivated soybean (Glycine max) has undergone several genetic bottlenecks, including domestication in China, the introduction of landraces to other areas of the world and, latterly, selective breeding, leading to low genetic diversity the poses a major obstacle to soybean improvement. By contrast, there remains a relatively high level of genetic diversity in soybean's wild relatives, especially the perennial soybeans (Glycine subgenus Glycine), which could serve as potential gene pools for improving soybean cultivars. Wild soybeans are phylogenetically diversified and adapted to various habitats, harboring resistance to various biotic and abiotic stresses. Advances in genome and transcriptome sequencing enable alleles associated with desirable traits that were lost during domestication of soybean to be discovered in wild soybean. The collection and conservation of soybean wild relatives and the dissection of their genomic features will accelerate soybean breeding and facilitate sustainable agriculture and food production.

Phylogenomics of the genus Glycine sheds light on polyploid evolution and life-strategy transition.

Polyploidy and life-strategy transitions between annuality and perenniality often occur in flowering plants. However, the evolutionary propensities of polyploids and the genetic bases of such transitions remain elusive. We assembled chromosome-level genomes of representative perennial species across the genus Glycine including five diploids and a young allopolyploid, and constructed a Glycine super-pangenome framework by integrating 26 annual soybean genomes. These perennial diploids exhibit greater genome stability and possess fewer centromere repeats than the annuals. Biased subgenomic fractionation occurred in the allopolyploid, primarily by accumulation of small deletions in gene clusters through illegitimate recombination, which was associated with pre-existing local subgenomic differentiation. Two genes annotated to modulate vegetative-reproductive phase transition and lateral shoot outgrowth were postulated as candidates underlying the perenniality-annuality transition. Our study provides insights into polyploid genome evolution and lays a foundation for unleashing genetic potential from the perennial gene pool for soybean improvement.

Recent Progress toward Ab Initio Modeling of Electrocatalysis.

Electrode potential is the key factor for controlling electrocatalytic reactions at electrochemical interfaces, and moreover, it is also known that the pH and solutes (e.g., cations) of the solution have prominent effects on electrocatalysis. Understanding these effects requires microscopic information on the electrochemical interfaces, in which theoretical simulations can play an important role. This Perspective summarizes the recent progress in method development for modeling electrochemical interfaces, including different methods for describing the electrolytes at the interfaces and different schemes for charging up the electrode surfaces. In the final section, we provide an outlook for future development in modeling methods and their applications to electrocatalysis.

The reference genome of Miscanthus floridulus illuminates the evolution of Saccharinae.

Miscanthus, a member of the Saccharinae subtribe that includes sorghum and sugarcane, has been widely studied as a feedstock for cellulosic biofuel production. Here, we report the sequencing and assembly of the Miscanthus floridulus genome by the integration of PacBio sequencing and Hi-C mapping, resulting in a chromosome-scale, high-quality reference genome of the genus Miscanthus. Comparisons among Saccharinae genomes suggest that Sorghum split first from the common ancestor of Saccharum and Miscanthus, which subsequently diverged from each other, with two successive whole-genome duplication events occurring independently in the Saccharum genus and one whole-genome duplication occurring in the Miscanthus genus. Fusion of two chromosomes occurred during rediploidization in M. floridulus and no significant subgenome dominance was observed. A survey of cellulose synthases (CesA) in M. floridulus revealed quite high expression of most CesA genes in growing stems, which is in agreement with the high cellulose content of this species. Resequencing and comparisons of 75 Miscanthus accessions suggest that M. lutarioriparius is genetically close to M. sacchariflorus and that M. floridulus is more distantly related to other species and is more genetically diverse. This study provides a valuable genomic resource for molecular breeding and improvement of Miscanthus and Saccharinae crops.

WFhb1-1 plays an important role in resistance against Fusarium head blight in wheat.

Fusarium head blight (FHB) is a severe disease of wheat (Triticum aestivum L.). Qfhb1 is the most important quantitative trait locus (QTL) for FHB resistance. We previously identified wheat gene WFhb1-1 (aka WFhb1-c1) as a candidate for FHB resistance gene. Here we report that WFhb1-1 has been cloned. The gene (GenBank # KU304333.1) consists of a single exon, encoding a putative membrane protein of 127 amino acids. WFhb1-1 protein produced in Pichia pastoris inhibits growth of both F. graminearum and P. pastoris in culture. Western Blotting with anti- WFhb1-1 antibody revealed a significant decrease (p < 0.01) in WFhb1-1 accumulation, 12 hours post Fusarium inoculation in non-Qfhb1-carrier wheat but not in Qfhb1-carrier wheat. Overexpressing WFhb1-1 in non-Qfhb1-carrier wheat led to a significant decrease (p < 0.01) in Fusarium-damaged rachis rate, Fusarium-diseased kernel rate and DON content in harvested kernels, while silencing WFhb1-1 in Qfhb1-carrier wheat resulted in a significant increase (p < 0.01) in FHB severity. Therefore, WFhb1-1 is an important FHB resistance gene with a potential antifungal function and probably a key functional component of Qfhb1 in wheat. A model regarding how WFhb1-1 functions in FHB resistance/susceptibility is hypothesized and discussed.

The Genetics and Genome-Wide Screening of Regrowth Loci, a Key Component of Perennialism in Zea diploperennis.

Perennialism is common among the higher plants, yet little is known about its inheritance. Previous genetic studies of the perennialism in Zea have yielded contradictory results. In this study, we take a reductionist approach by specifically focusing on one trait: regrowth (the plant's ability to restart a new life cycle after senescence on the same body). To address this, six hybrids were made by reciprocally crossing perennial Zea diploperennis Iltis, Doebley & R. Guzman with inbred lines B73 and Mo17 and Rhee Flint, a heirloom variety, of Zmays L. ssp. mays All the F1 plants demonstrated several cycles of growth, flowering, senescence and regrowth into normal flowering plants, indicating a dominant effect of the Z. diploperennis alleles. The regrowability (i.e., the plants' ability to regrow after senescence) was stably transmitted to progeny of the hybrids. Segregation ratios of regrowth in the F2 generations are consistent with the trait controlled by two dominant, complementary loci, but do not exclude the influence of other modifiers or environment. Genome-wide screening with genotyping-by-sequencing technology indicated two major regrowth loci, regrowth 1 (reg1) and regrowth 2 (reg2), were on chromosomes 2 and 7, respectively. These findings lay the foundation for further exploration of the molecular mechanism of regrowth in Z. diploperennis Importantly, our data indicate that there is no major barrier to transferring this trait into maize or other grass crops for perennial crop development with proper technology, which enhances sustainability of grain crop production in an environmentally friendly way.

A Quantitative Proteomics View on the Function of Qfhb1, a Major QTL for Fusarium Head Blight Resistance in Wheat.

Fusarium head blight (FHB) is a highly detrimental disease of wheat. A quantitative trait locus for FHB resistance, Qfhb1, is the most utilized source of resistance in wheat-breeding programs, but very little is known about its resistance mechanism. In this study, we elucidated a prospective FHB resistance mechanism by investigating the proteomic signatures of Qfhb1 in a pair of contrasting wheat near-isogenic lines (NIL) after 24 h of inoculation of wheat florets by Fusarium graminearum. Statistical comparisons of the abundances of protein spots on the 2D-DIGE gels of contrasting NILs (fhb1+ NIL = Qfhb1 present; fhb1- NIL = Qfhb1 absent) enabled us to select 80 high-ranking differentially accumulated protein (DAP) spots. An additional evaluation confirmed that the DAP spots were specific to the spikelet from fhb1- NIL (50 spots), and fhb1+ NIL (seven spots). The proteomic data also suggest that the absence of Qfhb1 makes the fhb1- NIL vulnerable to Fusarium attack by constitutively impairing several mechanisms including sucrose homeostasis by enhancing starch synthesis from sucrose. In the absence of Qfhb1, Fusarium inoculations severely damaged photosynthetic machinery; altered the metabolism of carbohydrates, nitrogen and phenylpropanoids; disrupted the balance of proton gradients across relevant membranes; disturbed the homeostasis of many important signaling molecules induced the mobility of cellular repair; and reduced translational activities. These changes in the fhb1- NIL led to strong defense responses centered on the hypersensitive response (HSR), resulting in infected cells suicide and the consequent initiation of FHB development. Therefore, the results of this study suggest that Qfhb1 largely functions to either alleviate HSR or to manipulate the host cells to not respond to Fusarium infection.

The draft genome of Ruellia speciosa (Beautiful Wild Petunia: Acanthaceae).

The genus Ruellia (Wild Petunias; Acanthaceae) is characterized by an enormous diversity of floral shapes and colours manifested among closely related species. Using Illumina platform, we reconstructed the draft genome of Ruellia speciosa, with a scaffold size of 1,021 Mb (or ∼1.02 Gb) and an N50 size of 17,908 bp, spanning ∼93% of the estimated genome (∼1.1 Gb). The draft assembly predicted 40,124 gene models and phylogenetic analyses of four key enzymes involved in anthocyanin colour production [flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), and dihydroflavonol 4-reductase (DFR)] found that most angiosperms here sampled harboured at least one copy of F3H, F3'H, and DFR. In contrast, fewer than one-half (but including R. speciosa) harboured a copy of F3'5'H, supporting observations that blue flowers and/or fruits, which this enzyme is required for, are less common among flowering plants. Ka/Ks analyses of duplicated copies of F3'H and DFR in R. speciosa suggested purifying selection in the former but detected evidence of positive selection in the latter. The genome sequence and annotation of R. speciosa represents only one of only four families sequenced in the large and important Asterid clade of flowering plants and, as such, will facilitate extensive future research on this diverse group, particularly with respect to floral evolution.

RADseq dataset with 90% missing data fully resolves recent radiation of Petalidium (Acanthaceae) in the ultra-arid deserts of Namibia.

Deserts, even those at tropical latitudes, often have strikingly low levels of plant diversity, particularly within genera. One remarkable exception to this pattern is the genus Petalidium (Acanthaceae), in which 37 of 40 named species occupy one of the driest environments on Earth, the Namib Desert of Namibia and neighboring Angola. To contribute to understanding this enigmatic diversity, we generated RADseq data for 47 accessions of Petalidium representing 22 species. We explored the impacts of 18 different combinations of assembly parameters in de novo assembly of the data across nine levels of missing data plus a best practice assembly using a reference Acanthaceae genome for a total of 171 sequence datasets assembled. RADseq data assembled at several thresholds of missing data, including 90% missing data, yielded phylogenetic hypotheses of Petalidium that were confidently and nearly fully resolved, which is notable given that divergence time analyses suggest a crown age for African species of 3.6-1.4 Ma. De novo assembly of our data yielded the most strongly supported and well-resolved topologies; in contrast, reference-based assembly performed poorly, perhaps due in part to moderate phylogenetic divergence between the reference genome, Ruellia speciosa, and the ingroup. Overall, we found that Petalidium, despite the harshness of the environment in which species occur, shows a net diversification rate (0.8-2.1 species per my) on par with those of diverse genera in tropical, Mediterranean, and alpine environments.