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.

Resolving deep relationships of PACMAD grasses: a phylogenomic approach.

BACKGROUND: Plastome 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. RESULTS: New plastomes were completely sequenced and characterized for 13 PACMAD species. An autapomorphic ~1140 bp 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. CONCLUSIONS: Phylogenomic 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.

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

Whole plastid genomes 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 have varied among or been unresolved in earlier single- and multi-gene studies. Pooideae, the cool-season grass lineage, is the largest of the 12 grass subfamilies and includes important temperate cereals, turf grasses and forage species. Although numerous studies of the phylogeny of the subfamily have been undertaken, relationships among some 'early-diverging' tribes conflict among studies, and some relationships among subtribes of Poeae have not yet been resolved. To address these issues, we newly sequenced 25 whole plastomes, which showed rearrangements typical of Poaceae. These plastomes represent 9 tribes and 11 subtribes of Pooideae, and were analysed with 20 existing plastomes for the subfamily. Maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) robustly resolve most deep relationships in the subfamily. Complete plastome data provide increased nodal support compared with protein-coding data alone at nodes that are not maximally supported. Following the divergence of Brachyelytrum, Phaenospermateae, Brylkinieae-Meliceae and Ampelodesmeae-Stipeae are the successive sister groups of the rest of the subfamily. Ampelodesmeae are nested within Stipeae in the plastome trees, consistent with its hybrid origin between a phaenospermatoid and a stipoid grass (the maternal parent). The core Pooideae are strongly supported and include Brachypodieae, a Bromeae-Triticeae clade and Poeae. Within Poeae, a novel sister group relationship between Phalaridinae and Torreyochloinae is found, and the relative branching order of this clade and Aveninae, with respect to an Agrostidinae-Brizinae clade, are discordant between MP and ML/BI trees. Maximum likelihood and Bayesian analyses strongly support Airinae and Holcinae as the successive sister groups of a Dactylidinae-Loliinae clade.

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).

Inference of molecular homology and sequence alignment by direct optimization.

That homologies exist and can be recognized and discovered is of central importance to comparative and evolutionary biology. Traditionally, homology assessment for both molecular and morphological data has been treated as a two-step process involving the generation of a proposition of homology and then the evaluation of that hypothesis through the test of congruence in a phylogenetic analysis. An alternative phylogenetic method, direct optimization, combines these into a one-step process in which positional homology and cladograms are co-estimated simultaneously. Here we examine the use of the term "homology" as it is applied to molecular data, and critique the concept of molecular homology one must accept if cladograms are to be interpreted as phylogenetic estimates. The test of congruence alone cannot be used to determine homologous (historically identical) features, and character analysis and the proposition of primary homology hypotheses is the only procedure that can identify correspondences between features justified as retaining phylogenetic information. The practical ramifications for phylogenetic analysis, such as data exclusion and the interpretation of tree-like diagrams as phylogenies, are discussed.

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.

The D4 set: primers that target highly variable intron loops in plant chloroplast genomes.

Chloroplast group II introns offer high-quality, rapidly evolving single-copy loci for comparative sequence analysis. These introns feature diagnostic secondary structures with loops that are among the least evolutionarily constrained sequence in plastomes. We exploited these structures to develop universal primers that amplify and sequence the large Domain IV (D4) loop in several angiosperm introns. With a single sequence read, we recover 300-600 nucleotides of highly variable sequence across angiosperms, with rates of change that are equal to or higher than many of the best known intergenic spacers in plant chloroplast genomes.

Model use in phylogenetics: nine key questions.

Models of character evolution underpin all phylogeny estimations, thus model adequacy remains a crucial issue for phylogenetics and its many applications. Although progress has been made in selecting appropriate models for phylogeny estimation, there is still concern about their purpose and proper use. How do we interpret models in a phylogenetic context? What are their effects on phylogeny estimation? How can we improve confidence in the models that we choose? That the phylogenetics community is asking such questions denotes an important stage in the use of explicit models. Here, we examine these and other common questions and draw conclusions about how the community is using and choosing models, and where this process will take us next.

Group II introns as phylogenetic tools: structure, function, and evolutionary constraints.

Group II introns comprise the majority of noncoding DNA in many plant chloroplast genomes and include the commonly sequenced regions trnK/matK, the rps16 intron, and the rpl16 intron. As demand increases for nucleotide characters at lower taxonomic levels, chloroplast introns may come to provide the bulk of plastome sequence data for assessment of evolutionary relationships in infrageneric, intergeneric, and interfamilial studies. Group II introns have many attractive properties for the molecular systematist: they are confined to organellar genomes in eukaryotes and the majority are single-copy; they share a well-defined and empirically tested secondary and tertiary structure; and many are easily amplified due to highly conserved sequence in flanking exons. However, structure-linked mutation patterns in group II intron sequences are more complex than generally supposed and have important implications for aligning nucleotides, assessing mutational biases in the data, and selecting appropriate models of character evolution for phylogenetic analysis. This paper presents a summary of group II intron function and structure, reviews the link between that structure and specific mutational constraints in group II intron sequences, and discusses strategies for accommodating the resulting complex mutational patterns in subsequent phylogenetic analyses.