Plastome phylogenomics and phylogenetic diversity of endangered and threatened grassland species (Poaceae) in a North American tallgrass prairie.

Native grasslands are one of the most endangered ecosystems in North America. In this study, we examined the ecological and evolutionary roles of endangered and threatened (e/t) grasses by establishing robust evolutionary relationships with other nonthreatened native and introduced grass species of the community. We hypothesized that the phylogenomic distribution of e/t species of grasses in Illinois would be phylogenetically clustered because closely related species would be vulnerable to the same threats and have similar requirements for survival. This study presents the first time a phylogeny based on complete plastome DNA of Poaceae was analyzed by phylogenetic diversity analysis. To avoid the disturbance of e/t populations, DNA was extracted from herbarium specimens. Next-generation sequencing (NGS) techniques were used to sequence DNA of plastid genomes (plastomes). The resulting phylogenomic tree was analyzed by phylogenetic diversity metrics. The extracted DNA successfully produced complete plastomes demonstrating that herbarium material is a practical source of DNA for genomic studies. The phylogenomic tree was strongly supported and defined Dichanthelium as a separate clade from Panicum. The phylogenetic metrics revealed phylogenetic clustering of e/t species, confirming our hypothesis.

Continued Adaptation of C4 Photosynthesis After an Initial Burst of Changes in the Andropogoneae Grasses.

C$_{4}$ photosynthesis is a complex trait that sustains fast growth and high productivity in tropical and subtropical conditions and evolved repeatedly in flowering plants. One of the major C$_{4}$ lineages is Andropogoneae, a group of $\sim $1200 grass species that includes some of the world's most important crops and species dominating tropical and some temperate grasslands. Previous efforts to understand C$_{4}$ evolution in the group have compared a few model C$_{4}$ plants to distantly related C$_{3}$ species so that changes directly responsible for the transition to C$_{4}$ could not be distinguished from those that preceded or followed it. In this study, we analyze the genomes of 66 grass species, capturing the earliest diversification within Andropogoneae as well as their C$_{3}$ relatives. Phylogenomics combined with molecular dating and analyses of protein evolution show that many changes linked to the evolution of C$_{4}$ photosynthesis in Andropogoneae happened in the Early Miocene, between 21 and 18 Ma, after the split from its C$_{3}$ sister lineage, and before the diversification of the group. This initial burst of changes was followed by an extended period of modifications to leaf anatomy and biochemistry during the diversification of Andropogoneae, so that a single C$_{4}$ origin gave birth to a diversity of C$_{4}$ phenotypes during 18 million years of speciation events and migration across geographic and ecological spaces. Our comprehensive approach and broad sampling of the diversity in the group reveals that one key transition can lead to a plethora of phenotypes following sustained adaptation of the ancestral state. [Adaptive evolution; complex traits; herbarium genomics; Jansenelleae; leaf anatomy; Poaceae; phylogenomics.].

Plastome phylogenomics and characterization of rare genomic changes as taxonomic markers in plastome groups 1 and 2 Poeae (Pooideae; Poaceae).

A phylogenomic analysis of 42 complete plastid genomes (plastomes), including 16 that were newly sequenced, was conducted. Plastomes were sampled from 19 subtribes of Pooideae, to investigate relationships within and between Chloroplast Group 1 (Aveneae) and Group 2 (Poeae) species. Two data partitions: complete plastomes, and a combined plastome and rare genomic change (RGC) data matrix, were analyzed. Overall, 156 non-ambiguous RGC were identified, of which homology was inferred for 38 RGC. Among the 38 RGC identified, six were synapomorphic among the Group 1 subtribes: Aveninae, Agrostidinae, and Anthoxanthinae, (Phalaridinae + Torreyochloinae), and 27 were synapomorphic among the Group 2 subtribes: Loliinae, (Ammochloinae + Parapholiinae + Dactylidinae), Parapholiinae, Dactylidinae, Poinae, and Coleanthinae. Four RGC were determined to be homoplasious in Groups 1 and 2. Two other RGC originated through intrastrand deletion events. The remaining RGC events likely originated through recombination given their size and lack of sequence evidence for other types of mutations. This study also determined that relationships between taxa, even those only weakly supported in previous studies, could be inferred with strong support when utilizing complete plastomes.

Leaf shape and size track habitat transitions across forest-grassland boundaries in the grass family (Poaceae).

Grass leaf shape is a strong indicator of their habitat with linear leaves predominating in open areas and ovate leaves distinguishing forest-associated grasses. This pattern among extant species suggests that ancestral shifts between forest and open habitats may have coincided with changes in leaf shape or size. We tested relationships between habitat, climate, photosynthetic pathway, and leaf shape and size in a phylogenetic framework to evaluate drivers of leaf shape and size variation over the evolutionary history of the family. We also estimated the ancestral habitat of Poaceae and tested whether forest margins served as transitional zones for shifts between forests and grasslands. We found that grass leaf shape is converging toward different shape optima in the forest understory, forest margins, and open habitats. Leaf size also varies with habitat. Grasses have smaller leaves in open and drier areas, and in areas with high solar irradiance. Direct transitions between linear and ovate leaves are rare as are direct shifts between forest and open habitats. The most likely ancestral habitat of the family was the forest understory and forest margins along with an intermediate leaf shape served as important transitional habitat and morphology, respectively, for subsequent shifts across forest-grassland biome boundaries.

Investigation of mitochondrial-derived plastome sequences in the Paspalum lineage (Panicoideae; Poaceae).

BACKGROUND: The grass family (Poaceae), ca. 12,075 species, is a focal point of many recent studies that aim to use complete plastomes to reveal and strengthen relationships within the family. The use of Next Generation Sequencing technology has revealed intricate details in many Poaceae plastomes; specifically the trnI - trnL intergenic spacer region. This study investigates this region and the putative mitochondrial inserts within it in complete plastomes of Paspalum and other Poaceae. RESULTS: Nine newly sequenced plastomes, seven of which contain an insert within the trnI - trnL intergenic spacer, were combined into plastome phylogenomic and divergence date analyses with 52 other species. A robust Paspalum topology was recovered, originating at 10.6 Ma, with the insert arising at 8.7 Ma. The alignment of the insert across Paspalum reveals 21 subregions with pairwise homology in 19. In an analysis of emergent self-organizing maps of tetranucleotide frequencies, the Paspalum insert grouped with mitochondrial DNA. CONCLUSIONS: A hypothetical ancestral insert, 17,685 bp in size, was found in the trnI - trnL intergenic spacer for the Paspalum lineage. A different insert, 2808 bp, was found in the same region for Paraneurachne muelleri. Seven different intrastrand deletion events were found within the Paspalum lineage, suggesting selective pressures to remove large portions of noncoding DNA. Finally, a tetranucleotide frequency analysis was used to determine that the origin of the insert in the Paspalum lineage is mitochondrial DNA.

A 250 plastome phylogeny of the grass family (Poaceae): topological support under different data partitions.

The systematics of grasses has advanced through applications of plastome phylogenomics, although studies have been largely limited to subfamilies or other subgroups of Poaceae. Here we present a plastome phylogenomic analysis of 250 complete plastomes (179 genera) sampled from 44 of the 52 tribes of Poaceae. Plastome sequences were determined from high throughput sequencing libraries and the assemblies represent over 28.7 Mbases of sequence data. Phylogenetic signal was characterized in 14 partitions, including (1) complete plastomes; (2) protein coding regions; (3) noncoding regions; and (4) three loci commonly used in single and multi-gene studies of grasses. Each of the four main partitions was further refined, alternatively including or excluding positively selected codons and also the gaps introduced by the alignment. All 76 protein coding plastome loci were found to be predominantly under purifying selection, but specific codons were found to be under positive selection in 65 loci. The loci that have been widely used in multi-gene phylogenetic studies had among the highest proportions of positively selected codons, suggesting caution in the interpretation of these earlier results. Plastome phylogenomic analyses confirmed the backbone topology for Poaceae with maximum bootstrap support (BP). Among the 14 analyses, 82 clades out of 309 resolved were maximally supported in all trees. Analyses of newly sequenced plastomes were in agreement with current classifications. Five of seven partitions in which alignment gaps were removed retrieved Panicoideae as sister to the remaining PACMAD subfamilies. Alternative topologies were recovered in trees from partitions that included alignment gaps. This suggests that ambiguities in aligning these uncertain regions might introduce a false signal. Resolution of these and other critical branch points in the phylogeny of Poaceae will help to better understand the selective forces that drove the radiation of the BOP and PACMAD clades comprising more than 99.9% of grass diversity.

Grass plastomes reveal unexpected paraphyly with endemic species of Micrairoideae from India and new haplotype markers in Arundinoideae.

PREMISE OF THE STUDY: We investigated the little-studied Arundinoideae/Micrairoideae clade of grasses with an innovative plastome phylogenomic approach. This method gives robust results for taxa of uncertain phylogenetic placement. Arundinoideae comprise ∼45 species, although historically was much larger. Arundinoideae is notable for the widely invasive Phragmites australis. Micrairoideae comprise nine genera and ∼200 species. Some are threatened with extinction, including Hubbardia, some Isachne spp., and Limnopoa. Two micrairoid genera, Eriachne and Pheidochloa, exhibit C4 photosynthesis in this otherwise C3 subfamily and represent an independent origin of the C4 pathway among grasses. METHODS: Five new plastomes were sequenced with next-generation sequencing-by-synthesis methods. Plastomes were assembled by de novo methods and phylogenetically analyzed with eight other recently published arundinoid or micrairoid plastomes and 11 outgroup species. Stable carbon isotope ratios were determined for micrairoid and arundinoid species to investigate ambiguities in the proxy evidence for C4 photosynthesis. KEY RESULTS: Phylogenomic analyses showed strong support for ingroup nodes in the Arundinoideae/Micrairoideae subtree, including a paraphyletic clade of Hubbardieae with Isachneae. Anatomical, biochemical, and positively selected sites data are ambiguous with regard to the photosynthetic pathways in Micrairoideae. Species of Hubbardia, Isachne, and Limnopoa were definitively shown by δ13C measurements to be C3 and Eriachne to be C4. CONCLUSIONS: Our plastome phylogenomic analyses for Micrairoideae are the first phylogenetic results to indicate paraphyly between Isachneae and Hubbardieae. The definitive δ13C data for four genera of Micrairoideae indicates the breadth of variation possible in the proxy evidence for photosynthetic pathways of both C3 and C4 taxa.

Plastid phylogenomic study of species within the genus Zea: rates and patterns of three classes of microstructural changes.

This project examines the relationships within the genus Zea using complete plastid genomes (plastomes). While Zea mays has been well studied, congeneric species have yet to be as thoroughly examined. For this study four complete plastomes and a fifth nearly complete plastome were sequenced in the five species (Zea diploperennis, Zea perennis, Zea luxurians, Zea nicaraguensis, and Zea mays subsp. huehuetenangensis) by Sanger or next-generation methods. An analysis of the microstructural changes, such as inversions, insertion or deletion mutations (indels) and determination of their frequencies were performed for the complete plastomes. It was determined that 193 indels and 15 inversions occurred across the examined plastomes of Zea. Tandem repeat indels were the most common type of microstructural change observed. Divergence times were estimated using a noncorrelated relaxed clock method. Divergence dates for specific nodes relative to Zea were calculated to fall between 38,000 years before present (YBP) for the subspecies included in this study and 23,000 YBP for section Luxuriantes included in this study. The stem lineage of all Zea species was calculated to have diverged at 176,000 YBP. The calculated mutation rates for the genus fell within the range of 1.7E-8 to 3.5E-8 microstructural changes per site per year. These rates of change are not uniform, despite the close relationships of taxa in this study. Phylogenomic analyses using full plastome alignments were also conducted to compare tree topologies from different types of mutations. In most cases, the previous work examining Zea mitochondrial and nuclear data was confirmed.

Correction: The First Complete Plastid Genome from Joinvilleaceae (J. ascendens; Poales) Shows Unique and Unpredicted Rearrangements.

[This corrects the article DOI: 10.1371/journal.pone.0163218.].

The First Complete Plastid Genome from Joinvilleaceae (J. ascendens; Poales) Shows Unique and Unpredicted Rearrangements.

Joinvilleaceae is a family of tropical grass-like monocots that comprises only the genus Joinvillea. Previous studies have placed Joinvilleaceae in close phylogenetic proximity to the well-studied grass family. A full plastome sequence was determined and characterized for J. ascendens. The plastome was sequenced with next generation methods, fully assembled de novo and annotated. The assembly revealed two novel inversions specific to the Joinvilleaceae lineage and at least one novel plastid inversion in the Joinvilleaceae-Poaceae lineage. Two previously documented inversions in the Joinvilleaceae-Poaceae lineage and one previously documented inversion in the Poaceae lineage were also verified. Inversion events were identified visually and verified computationally by simulation mutations. Additionally, the loss and subsequent degradation of the accD gene in order Poales was explored extensively in Poaceae and J. ascendens. The two novel inversions along with changes in gene composition between families better delimited lineages in the Poales. The presence of large inversions and subsequent reversals in this small family suggested a high potential for large-scale rearrangements to occur in plastid genomes.

Evolutionary relationships in Panicoid grasses based on plastome phylogenomics (Panicoideae; Poaceae).

BACKGROUND: Panicoideae are the second largest subfamily in Poaceae (grass family), with 212 genera and approximately 3316 species. Previous studies have begun to reveal relationships within the subfamily, but largely lack resolution and/or robust support for certain tribal and subtribal groups. This study aims to resolve these relationships, as well as characterize a putative mitochondrial insert in one linage. RESULTS: 35 newly sequenced Panicoideae plastomes were combined in a phylogenomic study with 37 other species: 15 Panicoideae and 22 from outgroups. A robust Panicoideae topology largely congruent with previous studies was obtained, but with some incongruences with previously reported subtribal relationships. A mitochondrial DNA (mtDNA) to plastid DNA (ptDNA) transfer was discovered in the Paspalum lineage. CONCLUSIONS: The phylogenomic analysis returned a topology that largely supports previous studies. Five previously recognized subtribes appear on the topology to be non-monophyletic. Additionally, evidence for mtDNA to ptDNA transfer was identified in both Paspalum fimbriatum and P. dilatatum, and suggests a single rare event that took place in a common progenitor. Finally, the framework from this study can guide larger whole plastome sampling to discern the relationships in Cyperochloeae, Steyermarkochloeae, Gynerieae, and other incertae sedis taxa that are weakly supported or unresolved.

Phylogenomics and Plastome Evolution of Tropical Forest Grasses (Leptaspis, Streptochaeta: Poaceae).

Studies of complete plastomes have proven informative for our understanding of the molecular evolution and phylogenomics of grasses. In this study, a plastome phylogenomic analysis sampled species from lineages of deeply diverging grasses including Streptochaeta spicata (Anomochlooideae), Leptaspis banksii, and L. zeylanica (both Pharoideae). Plastomes from next generation sequences for three species were assembled by de novo methods. The unambiguously aligned coding and non-coding sequences of the entire plastomes were aligned with those from 43 other grasses and the outgroup Joinvillea ascendens. Outgroup sampling of grasses has previously posed a challenge for plastome phylogenomic studies because of major rearrangements of the plastome. Here, over 81,000 bases of homologous sequence were aligned for phylogenomic and divergence estimation analyses. Rare genomic changes, including persistently long ψycf1 and ψycf2 loci, the loss of the rpoC1 intron, and a 21 base tandem repeat insert in the coding sequence for rps19 defined branch points in the grass phylogeny. Marked differences were seen in the topologies inferred from the complete plastome and two gene matrices, and mean maximum likelihood support values for the former were 10% higher. In the full plastome phylogenomic analyses, the two species of Anomochlooideae were monophyletic. Leptaspis and Pharus were found to be reciprocally monophyletic, with the estimated divergence of two Leptaspis species preceding those of Pharus by over 14 Ma, consistent with historical biogeography. Our estimates for deep divergences among grasses were older than previous such estimates, likely influenced by more complete taxonomic and molecular sampling and the use of recently available or previously unused fossil calibration points.

Incident learning in pursuit of high reliability: implementing a comprehensive, low-threshold reporting program in a large, multisite radiation oncology department.

BACKGROUND: Incident learning programs have been recognized as cornerstones of safety and quality assurance in so-called high reliability organizations in industries such as aviation and nuclear power. High reliability organizations are distinguished by their drive to continuously identify and proactively address a broad spectrum of latent safety issues. Many radiation oncology institutions have reported on their experience in tracking and analyzing adverse events and near misses but few have incorporated the principles of high reliability into their programs. Most programs have focused on the reporting and retrospective analysis of a relatively small number of significant adverse events and near misses. To advance a large, multisite radiation oncology department toward high reliability, a comprehensive, cost-effective, electronic condition reporting program was launched to enable the identification of a broad spectrum of latent system failures, which would then be addressed through a continuous quality improvement process. METHODS: A comprehensive program, including policies, work flows, and information system, was designed and implemented, with use of a low reporting threshold to focus on precursors to adverse events. RESULTS: In a 46-month period from March 2011 through December 2014, a total of 8,504 conditions (average, 185 per month, 1 per patient treated, 3.9 per 100 fractions [individual treatments]) were reported. Some 77.9% of clinical staff members reported at least 1 condition. Ninety-eight percent of conditions were classified in the lowest two of four severity levels, providing the opportunity to address conditions before they contribute to adverse events. CONCLUSIONS: Results after approximately four years show excellent employee engagement, a sustained rate of reporting, and a focus on low-level issues leading to proactive quality improvement interventions.

Development of a professionalism committee approach to address unprofessional medical staff behavior at an academic medical center.

BACKGROUND: The Joint Commission Leadership standard on the need to create and maintain a culture of safety and quality and to develop a code of conduct was based on the rationale that unprofessional behavior undermines a culture of safety and can thereby be harmful to patient care. Few reports have described effective and successful approaches to defining and managing unprofessional behavior. The Professionalism Committee (PC)-based approach at the University of Pennsylvania Health System (UPHS) may serve as a model for other hospitals and health systems. METHODS: Each of the three large teaching hospitals within UPHS has a PC that reports to its respective Medical Executive Committee. The PCs serve as a resource for department chairs and hospital administrators to address unprofessional behavior among faculty. Key features of the PC include the PC chair as the first point of contact and the integration of psychiatry into the model by virtue of the Professionalism Committee chair's training and expertise in psychiatry. RESULTS: In the 2009 calendar year, the PC chair received contacts concerning behavior of only 2 physicians, which increased to 42 physicians in 2011 and 39 in 2012. Contacts involved referrals, management consults, interview screening, and the need for general advice. Of 79 resolved cases, 30 involved interpersonal issues, and 2 were associated with poor clinical outcomes. CONCLUSION: One key feature of the UPHS approach is early identification of the role of behavioral health issues in unprofessional behavior (as opposed to physical, cognitive, or systems issues) by virtue of the PC chair's professional training and expertise. Although aspects of the UPHS experience may not be generalizable, the PC structure and approach are replicable.

Biogeography and phylogenomics of New World Bambusoideae (Poaceae), revisited.

UNLABELLED: • PREMISE OF THE STUDY: New World Bambusoideae have only recently been studied in a phylogenomic context. Plastome sequences were determined and analyzed from Arundinaria appalachiana, A. tecta, and Olyra latifolia, to refine our knowledge of their evolution and historical biogeography. A correction is noted regarding an error in an earlier report on the biogeography of Cryptochloa• METHODS: Single-end DNA libraries were prepared and sequenced on the Illumina platform. Complete plastomes were assembled and analyzed with 13 other Poaceae.• KEY RESULTS: Complete sampling in Arundinaria and an additional species of Olyreae gave a more detailed picture of their evolution/historical biogeography. Phylogenomic analyses indicated that the first major divergence in Arundinaria occurred around 2.3 to 3.2 mya and that Arundinaria tecta and A appalachiana diverged from their common ancestor around 0.57 to 0.82 mya. Estimates of the divergence of Olyra latifolia from Cryptochloa strictiflora ranged from 14.6 to 20.7 mya. The age of the stem node of Olyreae ranged from an estimated 26.9 to 38.2 mya.• CONCLUSIONS: Estimates of divergences in Arundinaria can be correlated with paleoclimatic events including an early Pliocene warming, subsequent cooling, and North American glaciations. Discriminating between alternate evolutionary/biogeographic scenarios in Olyreae is challenging.

Plastome sequences of two New World bamboos--Arundinaria gigantea and Cryptochloa strictiflora (Poaceae)--extend phylogenomic understanding of Bambusoideae.

PREMISE OF THE STUDY: Two New World species of Bambusoideae, Arundinaria gigantea and Crytpochloa strictiflora, were investigated in a phylogenomic context. Complete plastome sequences have been previously determined and analyzed for nine bambusoid species that exclusively represent Old World lineages. The addition of New World species provides more complete information on relationships within Bambusoideae. • METHODS: Plastomes from A. gigantea and C. strictiflora were sequenced using Sanger methods. Phylogenomic and divergence estimate analyses were conducted on both species with 23 other Poaceae. • KEY RESULTS: Phylogenomic and divergence analyses suggested that A. gigantea diverged from within Arundinarieae between 1.94-3.92 mya and that C. strictiflora diverged as the sister to tropical woody species between 24.83 and 40.22 mya. These results are correlated with modern relative diversities in the two lineages. • CONCLUSIONS: The two New World bamboos show unique plastome features accumulated and maintained in biogeographic isolation from Old World taxa. The overall evidence for A. gigantea is consistent with recent dispersal, and that for C. strictiflora is consistent with vicariance.