Sean V. Burke

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

UNLABELLED 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.

A multi-step comparison of short-read full plastome sequence assembly methods in grasses

Technological advances have allowed phylogenomic studies of plants, such as full chloroplast genome (plastome) analysis, to become increasingly popular and economically feasible. Although next-generation short-read sequencing allows for full plastomes to be sequenced relatively rapidly, it requires additional attention using software to assemble these reads into comprehensive sequences. Here we compare the use of three de novo assemblers combined with three contig assembly methods. Seven plastome sequences were analyzed. Three of these were Sanger-sequenced. The other four were assembled from short, single-end read files generated from next-generation libraries. These plastomes represented a total of six grass species (Poaceae), one of which was sequenced in duplicate by the two methods to allow direct comparisons for accuracy. Enumeration of missing sequence and ambiguities allowed for assessments of completeness and efficiency. All methods that used de Bruijn-based de novo assemblers were shown to produce assemblies comparable to the Sanger-sequenced plastomes but were not equally efficient. Contig assembly methods that utilized automatable and repeatable processes were generally more efficient and advantageous when applied to larger scale projects with many full plastomes. However, contig assembly methods that were less automatable and required more manual attention did show utility in determining plastomes with lower read depth that were not able to be assembled when automatable procedures were implemented. Although the methods here were used exclusively to generate grass plastomes, these could be applied to other taxonomic groups if previously sequenced plastomes were available. In addition to comparing sequencing methods, a supplemental guide for short-read plastome assembly and applicable scripts were generated for this study.

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.

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

BackgroundPanicoideae 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.Results35 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.ConclusionsThe 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 the Chloridoid Grasses (Chloridoideae: Poaceae)

Premise of research. Studies of complete plastomes have proven informative for our understanding of the molecular evolution and phylogenomics of grasses, but subfamily Chloridoideae has not been included in this research. In previous multilocus studies, specific deep branches, as in the large clade corresponding to Cynodonteae, are not uniformly well supported. Methodology. In this study, a plastome phylogenomic analysis sampled 14 species representing 4 tribes and 10 genera of Chloridoideae. One species was Sanger sequenced, and 14 other species, including outgroups, were sequenced with next-generation sequencing-by-synthesis methods. Plastomes from next-generation sequences were assembled by de novo methods, and the unambiguously aligned coding and noncoding sequences of the entire plastomes were analyzed phylogenetically. Pivotal results. Complete plastomes showed rare genomic changes in Distichlis, Centropodia, and Eragrostis tef that were of potential phylogenomic significance. Phylogenomic analyses showed uniformly strong support for all ingroup relationships except one node in Cynodonteae in which a short internal branch connected long terminal branches. Resolution within this clade was found to be taxon dependent and possibly subject to long-branch attraction artifacts. Conclusions. Our study indicates that the increase in phylogenetic information in sequences of entire plastomes well resolves and strongly supports relationships among tribes and genera of chloridoid grasses. Sampling more species, especially in the Centropodia + Ellisochloa clade and Cynodonteae, will further address relationships in these groups and clarify the evolutionary origins of the subfamily.

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.

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.

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.

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.