Scot A. Kelchner

HAIRPINS CREATE MINUTE INVERSIONS IN NON-CODING REGIONS OF CHLOROPLAST DNA

Abstract Minute inversions (4 bp in length), associated with probable hairpin secondary structures, were inferred from comparative analysis of rpl16 intron sequences from the chloroplast genomes of Chusquea species and related bamboos (Poaceae). The inverted sequences, which appear to have arisen independently on several occasions, comprise entire loops of the putative hairpins. The process of inversion seems dependent upon the stem length of the hairpin and its estimated free energy of formation. A similar inversion was uncovered for other plants in a previously published data set for a different non-coding region of the chloroplast genome, suggesting that the inversional process may be a common feature of non-coding DNA evolution. Several implications for phylogenetic analysis are noted.

Networks: expanding evolutionary thinking

Networks allow the investigation of evolutionary relationships that do not fit a tree model. They are becoming a leading tool for describing the evolutionary relationships between organisms, given the comparative complexities among genomes.

Review of the systematics of Scrophulariaceae s.l. and their current disposition

Recent molecular phylogenetic studies in Lamiales have shown that the large group traditionally recognised as Scrophulariaceae is not monophyletic. Efforts to reconstruct the phylogeny of this large clade and to revise its classification to reflect that phylogeny have resulted in seven monophyletic groups, comprised mostly of members of Scrophulariaceae s.l., recognised as families in recent angiosperm classifications. These are Scrophulariaceae s.s., Orobanchaceae, Veronicaceae (cf. Plantaginaceae), Phrymaceae, Calceolariaceae, Linderniaceae, and Stilbaceae. Sampling completeness at the genus level varies from group to group, but is quite good for many. A few individual genera formerly assigned to Scrophulariaceae do not fit into any existing clade recognised at family rank and are left, at present, unassigned to family. In addition to the recognition of several clades comprised primarily of former members of Scrophulariaceae s.l., several groups previously recognised as families are now included within some of these clades. For example, Scrophulariaceae s.s. includes Buddlejaceae and Myoporaceae, and Veronicaceae includes Callitrichaceae, Globulariaceae, Hippuridaceae, and Plantaginaceae. The clades now recognised as families often are not easily diagnosed, but in many cases are more consistent with certain functional traits and geographical patterns. Examples include Orobanchaceae, which comprises all of the parasitic plants (hemiparasites and holoparasites) and Scrophulariaceae s.s., which is predominantly a southern hemisphere group.

Higher level phylogenetic relationships within the bamboos (Poaceae: Bambusoideae) based on five plastid markers

Bamboos are large perennial grasses of temperate and tropical forests worldwide. Two general growth forms exist: the economically and ecologically important woody bamboos (tribes Arundinarieae and Bambuseae), and the understory herbaceous bamboos (tribe Olyreae). Evolutionary relationships among the 1400+described species have been difficult to resolve with confidence. Comparative analysis of bamboo plastid (chloroplast) DNA has revealed three to five major lineages that show distinct biogeographic distributions. Taxon sampling across tribes and subtribes has been incomplete and most published data sets include a relatively small number of nucleotide characters. Branching order among lineages is often poorly supported, and in more than one study herbaceous bamboos form a clade within the woody bamboos. In this paper, the Bamboo Phylogeny Group presents the most complete phylogeny estimation to date of bamboo tribes and subtribes using 6.7 kb of coding and noncoding sequence data and 37 microstructural characters from the chloroplast genome. Quality of data is assessed, as is the possibility of long branch attraction, the degree of character conflict at key nodes in the tree, and the legitimacy of three alternative hypotheses of relationship. Four major plastid lineages are recognized: temperate woody, paleotropical woody, neotropical woody, and herbaceous bamboos. Woody bamboos are resolved as paraphyletic with respect to Olyreae but SH tests cannot reject monophyly of woody species (Arundinarieae+Bambuseae).

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.

A Broadscale Phylogenetic Analysis of Group II Intron RNAs and Intron-Encoded Reverse Transcriptases

Group II introns are self-splicing RNAs that are frequently assumed to be the ancestors of spliceosomal introns. They are widely distributed in bacteria and are also found in organelles of plants, fungi, and protists. In this study, we present a broadscale phylogenetic analysis of group II introns using sequence data from both the conserved RNA structure and the intron-encoded reverse transcriptase (RT). Two similar phylogenies are estimated for the RT open reading frame (ORF), based on either amino acid or nucleotide sequence, whereas one phylogeny is produced for the RNA. In making these estimates, we confronted nearly all the classic challenges to phylogenetic inference, including positional saturation, base composition heterogeneity, short internodes with low support, and sensitivity to taxon sampling. Although the major lineages are well-defined, robust resolution of topology is not possible between these lineages. The approximately unbiased (AU) and Shimodaira-Hasegawa topology tests indicated that the RT ORF and RNA ribozyme data sets are in significant conflict under a variety of models, revealing the possibility of imperfect coevolution between group II introns and their intron-encoded ORFs. The high level of sequence divergence, large timescale, and limited number of alignable characters in our study are representative of many RTs and group I introns, and our results suggest that phylogenetic analyses of any of these sequences could suffer from the same sources of error and instability identified in this study.

Conservation of Selection on matK Following an Ancient Loss of its Flanking Intron

The chloroplast gene trnK and its associated group II intron appear to be absent in a large and ancient clade that includes nearly 90% of fern species. However, the maturase protein encoded within the intron (matK) is still present and located on the boundary of a large-scale inversion. We surveyed the chloroplast genome sequence of clade-member Adiantum capillus-veneris for evidence of a still present but fragmented trnK intron. Lack of signature structural domains and sequence motifs in the genome indicate loss of the trnK intron through degradation in an ancestor of the clade. In plants, matK preferentially catalyzes splicing of the trnK intron, but may also have a generalist function, splicing other group II introns in the chloroplast genome. We therefore tested whether a shift in selective constraint has occurred after loss of the trnK intron. Using previously unavailable sequences for several ferns, we compared matK sequences of the intron-less fern clade to sequences from seed plants and ferns with the intron and found no significant differences in selection among lineages using multiple methods. We conclude that matK in ferns has maintained its apparently ancient and generalized function in chloroplasts, even after the loss of its co-evolved group II intron. Finally, we also present primers that will allow amplification and nucleotide sequencing of the phylogenetically useful matK gene in additional fern taxa.

Plastid phylogenomics of the cool-season grass subfamily: clarification of relationships among early-diverging tribes

Whole plastid genomes (plastomes) are being sequenced rapidly from across the green plant tree of life, and phylogenetic analyses of these are increasing resolution and support for relationships that were unresolved in earlier studies. The cool-season grass subfamily, Pooideae, includes important temperate cereals, turf grasses and forage species, yet some aspects of deep phylogeny in the lineage are unresolved. We newly sequenced 25 Pooideae plastomes, and conducted phylogenomic analyses of these and 20 existing plastomes from the subfamily. Most aspects of deep relationship in Pooideae are maximally supported in our analyses, including those among early-diverging tribes.

Resolving deep relationships of PACMAD grasses: a phylogenomic approach

BackgroundPlastome sequences for 18 species of the PACMAD grasses (subfamilies Panicoideae, Aristidoideae, Chloridoideae, Micrairoideae, Arundinoideae, Danthonioideae) were analyzed phylogenomically. Next generation sequencing methods were used to provide complete plastome sequences for 12 species. Sanger sequencing was performed to determine the plastome of one species, Hakonechloa macra, to provide a reference for annotation. These analyses were conducted to resolve deep subfamilial relationships within the clade. Divergence estimates were assessed to determine potential factors that led to the rapid radiation of this lineage and its dominance of warmer open habitats.ResultsNew plastomes were completely sequenced and characterized for 13 PACMAD species. An autapomorphic ~1140xa0bp deletion was found in Hakonechloa macra putatively pseudogenizing rpl14 and eliminating rpl16 from this plastome. Phylogenomic analyses support Panicoideae as the sister group to the ACMAD clade. Complete plastome sequences provide greater support at deep nodes within the PACMAD clade. The initial diversification of PACMAD subfamilies was estimated to occur at 32.4 mya.ConclusionsPhylogenomic analyses of complete plastomes provides resolution for deep relationships of PACMAD grasses. The divergence estimate of 32.4 mya at the crown node of the PACMAD clade coincides with the Eocene-Oligocene Transition (EOT). The Eocene was a period of global cooling and drying, which led to forest fragmentation and the expansion of open habitats now dominated by these grasses. Understanding how these grasses are related and determining a cause for their rapid radiation allows for future predictions of grassland distribution in the face of a changing global climate.

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.