Jimmy K. Triplett

Reference Genome Sequence Of The Model Plant Setaria

We generated a high-quality reference genome sequence for foxtail millet (Setaria italica). The ∼400-Mb assembly covers ∼80% of the genome and >95% of the gene space. The assembly was anchored to a 992-locus genetic map and was annotated by comparison with >1.3 million expressed sequence tag reads. We produced more than 580 million RNA-Seq reads to facilitate expression analyses. We also sequenced Setaria viridis, the ancestral wild relative of S. italica, and identified regions of differential single-nucleotide polymorphism density, distribution of transposable elements, small RNA content, chromosomal rearrangement and segregation distortion. The genus Setaria includes natural and cultivated species that demonstrate a wide capacity for adaptation. The genetic basis of this adaptation was investigated by comparing five sequenced grass genomes. We also used the diploid Setaria genome to evaluate the ongoing genome assembly of a related polyploid, switchgrass (Panicum virgatum).

Phylogeny of the Temperate Bamboos (Poaceae: Bambusoideae: Bambuseae) with an Emphasis on Arundinaria and Allies

Abstract The temperate bamboos are a morphologically diverse grass lineage with a complex and problematic taxonomy. We present the first robust multilocus chloroplast phylogeny of the temperate bamboos, assess relationships among key genera with an emphasis on Arundinaria and its allies, and highlight the potential role of hybridization and reticulate evolution in this group. Utilizing a total of twelve plastid DNA regions (1 gene, 10 intergenic spacers, and 1 intron), the temperate clade was resolved to include six major lineages: Bergbamboes, the African alpine bamboos, Chimonocalamus, the Shibataea clade, the Phyllostachys clade, and the Arundinaria clade. Internal resolution varied among these six, in part reflecting sampling density and in part due to apparent evolutionary rate heterogeneity. The recovered phylogeny is largely incongruent with morphological classifications, rendering subtribes and many genera paraphyletic or polyphyletic. Some associations are consistent with existing hypotheses of intergeneric hybridization, while others may indicate convergent evolution, lineage sorting, or previously unsuspected cases of hybridization. Several robust lineages were identified within the Arundinaria clade, including the Medake subclade (Pleioblastus s. s. and allies), Sasa s. s., and the Sinicae subclade (comprising a subset of Chinese taxa currently classified in Acidosasa, Indosasa, Pleioblastus sect. Amari, and Pseudosasa subg. Sinicae). Our analyses also recovered a monophyletic Arundinaria s. s. in North America, and revealed substantial divergence between A. gigantea and A. tecta.

Five nuclear loci resolve the polyploid history of switchgrass (Panicum virgatum L.) and relatives.

Polyploidy poses challenges for phylogenetic reconstruction because of the need to identify and distinguish between homoeologous loci. This can be addressed by use of low copy nuclear markers. Panicum s.s. is a genus of about 100 species in the grass tribe Paniceae, subfamily Panicoideae, and is divided into five sections. Many of the species are known to be polyploids. The most well-known of the Panicum polyploids are switchgrass (Panicum virgatum) and common or Proso millet (P. miliaceum). Switchgrass is in section Virgata, along with P. tricholaenoides, P. amarum, and P. amarulum, whereas P. miliaceum is in sect. Panicum. We have generated sequence data from five low copy nuclear loci and two chloroplast loci and have clarified the origin of P. virgatum. We find that all members of sects. Virgata and Urvilleana are the result of diversification after a single allopolyploidy event. The closest diploid relatives of switchgrass are in sect. Rudgeana, native to Central and South America. Within sections Virgata and Urvilleana, P. tricholaenoides is sister to the remaining species. Panicum racemosum and P. urvilleanum form a clade, which may be sister to P. chloroleucum. Panicum amarum, P. amarulum, and the lowland and upland ecotypes of P. virgatum together form a clade, within which relationships are complex. Hexaploid and octoploid plants are likely allopolyploids, with P. amarum and P. amarulum sharing genomes with P. virgatum. Octoploid P. virgatum plants are formed via hybridization between disparate tetraploids. We show that polyploidy precedes diversification in a complex set of polyploids; our data thus suggest that polyploidy could provide the raw material for diversification. In addition, we show two rounds of allopolyploidization in the ancestry of switchgrass, and identify additional species that may be part of its broader gene pool. This may be relevant for development of the crop for biofuels.

Paraphyly in the Bamboo Subtribe Chusqueinae (Poaceae: Bambusoideae) and a Revised Infrageneric Classification for Chusquea

Abstract A phylogenetic analysis of five coding and noncoding chloroplast loci, totaling 6.6 kb of aligned nucleotide and indel characters, suggests that the large neotropical bamboo genus Chusquea is embedded within the much smaller Andean genus Neurolepis. Monophyly of each taxon was anticipated due to the unique occurrence of dimorphic multiple buds in Chusquea and the lack of aerial branching in Neurolepis. We tested whether the unexpected placement of Chusquea might be the result of a biased analysis. Both Neurolepis and the outgroup taxa have long branches that could influence rooting and inferred ingroup relationships. A number of methods were employed to test for long-branch attraction and sampling effects in our topology. Alternative hypothesis testing using a conservative form of the Shimodaira-Hasegawa test indicated that paraphyly of Neurolepis is a significantly better explanation of the data than monophyly, even when models of character evolution are changed. Given the robustness of the topology, high support measures for clades on the tree, and the results of the Shimodaira-Hasegawa tests, we conclude that chloroplast genomes indicate probable paraphyly of Neurolepis with respect to Chusquea. The species of Neurolepis are therefore transferred to Chusquea, resulting in the following new combinations and names: Chusquea acuminatissima, C. angusta, C. asymmetrica, C. cylindrica, C. diversiglumis, C. elata, C. fimbriligulata, C. laegaardii, C. magnifolia, C. mollis, C. nana, C. nobilis, C. petiolata, C. rigida, C. silverstonei, C. spectabilis, C. steyermarkii, C. stuebelii, C. tovari and C. villosa. The names Neurolepis elata, N. stuebelii, N. weberbaueri, Planotia ingens, and P. tessellata are lectotypified.

Independent allopolyploidization events preceded speciation in the temperate and tropical woody bamboos

The objectives of the current study were to investigate the origin of polyploidy in the woody bamboos and examine putative hybrid relationships in one major lineage (the temperate woody bamboos, tribe Arundinarieae). Phylogenetic analyses were based on sequence data from three nuclear loci and 38 species in 27 genera. We identify six ancestral genome donors for contemporary bamboo lineages: temperate woody bamboos (tribe Arundinarieae) contain genomes A and B, tropical woody bamboos (tribe Bambuseae) contain genomes C and D, and herbaceous bamboos (tribe Olyreae) contain genome H; some hexaploid paleotropical bamboos contain genome E in addition to C and D. Molecular data indicate that allopolyploidy arose independently in temperate (AABB) and tropical woody lineages (CCDD and CCDDEE), and speciation occurred subsequent to polyploidization. Moreover, hybridization has played a surprising and recurrent role in bamboo evolution, generating allohexaploid species in the paleotropical clade and intergeneric hybrids among the allotetraploid temperate bamboos. We suggest this complex history of reticulate evolution is at least partially responsible for the taxonomic difficulty associated with the woody bamboos. This newly-resolved phylogenetic framework reflects a major step forward in our understanding of bamboo biodiversity and has important implications for the interpretation of bamboo phylogenomics.

Phylogenetic relationships and natural hybridization among the North American woody bamboos (Poaceae: Bambusoideae: Arundinaria)

In spite of the ecological and economic importance of temperate bamboos, relatively little is known about their population biology or evolutionary history. Recently, hybridization has emerged as a potential source of diversity in this group, as well as an underlying cause of taxonomic problems. As part of a broader phylogenetic study of the temperate bamboos, we report the results of an analysis of the North American Arundinaria gigantea species complex, including estimates of genetic variation and molecular evidence of natural hybridization among A. gigantea, A. tecta, and A. appalachiana. The study involved a comparative analysis of amplified fragment length polymorphisms (AFLPs) and chloroplast DNA sequences representing diversity within and among all three species plus individuals with intermediate or unusual morphological characteristics (putative hybrids). Molecular results support the recognition of three species previously defined on the basis of morphology, anatomy, and ecology, with most of the molecular variance accounted for by among-species variation. Molecular evidence also demonstrates that A. tecta and A. appalachiana are sister species, forming a clade that is significantly divergent from A. gigantea. The role of hybridization in the phylogenetic history of Arundinaria is discussed along with implications for the evolution and taxonomy of the temperate woody bamboos.

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.

Allotetraploid origin and divergence in Eleusine (Chloridoideae, Poaceae): evidence from low-copy nuclear gene phylogenies and a plastid gene chronogram

BACKGROUND AND AIMS Eleusine (Poaceae) is a small genus of the subfamily Chloridoideae exhibiting considerable morphological and ecological diversity in East Africa and the Americas. The interspecific phylogenetic relationships of Eleusine are investigated in order to identify its allotetraploid origin, and a chronogram is estimated to infer temporal relationships between palaeoenvironment changes and divergence of Eleusine in East Africa. METHODS Two low-copy nuclear (LCN) markers, Pepc4 and EF-1α, were analysed using parsimony, likelihood and Bayesian approaches. A chronogram of Eleusine was inferred from a combined data set of six plastid DNA markers (ndhA intron, ndhF, rps16-trnK, rps16 intron, rps3, and rpl32-trnL) using the Bayesian dating method. KEY RESULTS The monophyly of Eleusine is strongly supported by sequence data from two LCN markers. In the cpDNA phylogeny, three tetraploid species (E. africana, E. coracana and E. kigeziensis) share a common ancestor with the E. indica-E. tristachya clade, which is considered a source of maternal parents for allotetraploids. Two homoeologous loci are isolated from three tetraploid species in the Pepc4 phylogeny, and the maternal parents receive further support. The A-type EF-1α sequences possess three characters, i.e. a large number of variations of intron 2; clade E-A distantly diverged from clade E-B and other diploid species; and seven deletions in intron 2, implying a possible derivation through a gene duplication event. The crown age of Eleusine and the allotetraploid lineage are 3·89 million years ago (mya) and 1·40 mya, respectively. CONCLUSIONS The molecular data support independent allotetraploid origins for E. kigeziensis and the E. africana-E. coracana clade. Both events may have involved diploids E. indica and E. tristachya as the maternal parents, but the paternal parents remain unidentified. The habitat-specific hypothesis is proposed to explain the divergence of Eleusine and its allotetraploid lineage.

Towards a Stable Nomenclature for the North American Temperate Bamboos: Epitypification of Arundo gigantea Walt. and Arundinaria macrosperma Michx. (Poaceae)

Abstract Arundinaria sensu stricto is a genus encompassing three species that are endemic to the Southeastern United States: A. appalachiana (hill cane), A. gigantea (river cane), and A. tecta (switch cane). The taxonomy of this group has been problematic due to a poor understanding of its phylogeny and genetic variation as well as ambiguity surrounding original protologues and designated nomenclatural type specimens. In particular, the neotype of Arundo gigantea (the basionym of Arundinaria gigantea) and the holotype of A. macrosperma (a synonym of A. gigantea) both provide insufficient morphological characters for species identification. Here we designate epitypes to assist with the correct application of the names of these taxa. A summary of diagnostic morphological features for the three species of Arundinaria and comments on their phylogenetic relationships based on recent molecular and morphological analyses are also provided.