Complete chloroplast genome comparisons for Pityopsis (Asteraceae).

Pityopsis includes several regionally and one federally endangered species of herbaceous perennials. Four species are highly localized, including the federally endangered P. ruthii. The genus includes several ploidy levels and interesting ecological traits such as drought tolerance and fire-dependent flowering. Results from previous cladistic analyses of morphology and from initial DNA sequence studies did not agree with one another or with the infrageneric taxonomic classification, with the result that infrageneric relationships remain unresolved. We sequenced, assembled, and compared the chloroplast (cp) genomes of 12 species or varieties of Pityopsis to better understand generic evolution. A reference cp genome 152,569 bp in length was assembled de novo from P. falcata. Reads from other sampled species were then aligned to the P. falcata reference and individual chloroplast genomes were assembled for each, with manual gapfilling and polishing. After removing the duplicated second inverted region, a multiple sequence alignment of the cp genomes was used to construct a maximum likelihood (ML) phylogeny for the twelve cp genomes. Additionally, we constructed a ML phylogeny from the nuclear ribosomal repeat region after mapping reads to the Helianthus annuus region. The chloroplast phylogeny supported two clades. Previously proposed clades and taxonomic sections within the genus were largely unsupported by both nuclear and chloroplast phylogenies. Our results provide tools for exploring hybridity and examining the physiological and genetic basis for drought tolerance and fire-dependent flowering. This study will inform breeding and conservation practices, and general knowledge of evolutionary history, hybridization, and speciation within Pityopsis.

Population Structure and Genetic Diversity Within the Endangered Species Pityopsis ruthii (Asteraceae).

Pityopsis ruthii (Ruth's golden aster) is a federally endangered herbaceous perennial endemic to the Hiwassee and Ocoee Rivers in southeastern Tennessee, United States. Comprehensive genetic studies providing novel information to conservationists for preservation of the species are lacking. Genetic variation and gene flow were evaluated for 814 individuals from 33 discrete locations using polymorphic microsatellites: seven chloroplast and twelve nuclear. A total of 198 alleles were detected with the nuclear loci and 79 alleles with the chloroplast loci. Gene flow was estimated, with the Hiwassee River (Nm = 2.16; FST = 0.15) showing higher levels of gene flow and lower levels of population differentiation than the Ocoee River (Nm = 1.28; FST = 0.19). Population structure was examined using Bayesian cluster analyses. Nuclear and chloroplast analyses were incongruent. From the chloroplast microsatellites, three clusters were identified; all were present in sampling sites at both rivers, indicating a lack of allele fixation along rivers. Nuclear markers revealed two clusters and separated by river. When the Hiwassee River locations were analyzed, four clusters were identified for both the chloroplast and nuclear microsatellites, though the individuals clustered differently. Analysis of the Ocoee River revealed two clusters for the chloroplast microsatellites and three for the nuclear microsatellites. We recommend P. ruthii be managed as four populations for the Hiwassee River and three populations for the Ocoee River. Our results provide critical genetic information for P. ruthii that can be used for species management decisions to drive future population augmentation/reintroduction and ex situ conservation efforts.

Stepwise Evolution of a Buried Inhibitor Peptide over 45 My.

The de novo evolution of genes and the novel proteins they encode has stimulated much interest in the contribution such innovations make to the diversity of life. Most research on this de novo evolution focuses on transcripts, so studies on the biochemical steps that can enable completely new proteins to evolve and the time required to do so have been lacking. Sunflower Preproalbumin with SFTI-1 (PawS1) is an unusual albumin precursor because in addition to producing albumin it also yields a potent, bicyclic protease-inhibitor called SunFlower Trypsin Inhibitor-1 (SFTI-1). Here, we show how this inhibitor peptide evolved stepwise over tens of millions of years. To trace the origin of the inhibitor peptide SFTI-1, we assembled seed transcriptomes for 110 sunflower relatives whose evolution could be resolved by a chronogram, which allowed dates to be estimated for the various stages of molecular evolution. A genetic insertion event in an albumin precursor gene ∼45 Ma introduced two additional cleavage sites for protein maturation and conferred duality upon PawS1-Like genes such that they also encode a small buried macrocycle. Expansion of this region, including two Cys residues, enlarged the peptide ∼34 Ma and made the buried peptides bicyclic. Functional specialization into a protease inhibitor occurred ∼23 Ma. These findings document the evolution of a novel peptide inside a benign region of a pre-existing protein. We illustrate how a novel peptide can evolve without de novo gene evolution and, critically, without affecting the function of what becomes the protein host.

Origins and recent radiation of Brazilian Eupatorieae (Asteraceae) in the eastern Cerrado and Atlantic Forest.

The remarkable diversity of Eupatorieae in the Brazilian flora has received little study, despite the tribe's very high levels of endemism and importance in the threatened Cerrado and the Atlantic Forest biodiversity hotspots. Eupatorieae are one of the largest tribes in Asteraceae with 14 of 19 recognized subtribes occurring in Brazil. We constructed the largest phylogeny of Brazilian Eupatorieae to date that sampled the nrITS and ETS, chloroplast ndhI and ndhF genes, and the ndhI-ndhG intergenic spacer for 183 species representing 77 of the 85 Brazilian genera of the tribe. Maximum likelihood and Bayesian phylogenetic analyses showed that these species are not collectively monophyletic, so their distribution reflects multiple introductions into Brazil. A novel clade was found that includes 75% of the genera endemic to Brazil (Cerrado-Atlantic Forest Eupatorieae, "CAFE" clade). This radiation of at least 247 species concentrated in the Cerrado and Atlantic Forest biomes of central eastern Brazil is <7 my old and exhibits several ecologically diverse life forms. Eight subtribes of Brazilian Eupatorieae (Ageratinae, Alomiinae, Ayapaninae, Critoniinae, Disynaphiinae, Eupatoriinae, Gyptidinae and Hebecliniinae) and 16 genera (Ageratum, Agrianthus, Austroeupatorium, Bejaranoa, Chromolaena, Critonia, Disynaphia, Grazielia, Hatschbachiella, Heterocondylus, Koanophyllon, Lasiolaena, Neocabreria, Praxelis, Stylotrichium, and Symphyopappus) were found to be polyphyletic. We attribute incongruities between the molecular phylogenetic results and the current classification of the tribe mostly to convergent evolution of morphological characters traditionally used in the classification of the tribe. We used these phylogenetic results to suggest changes to the classification of some subtribes and genera of Eupatorieae that occur in Brazil.

Bricklebush (Brickellia) phylogeny reveals dimensions of the great Asteraceae radiation in Mexico.

Data from molecular phylogenetics were used to assess aspects of diversity and relationships in Brickellia, a large and widespread genus of Eupatorieae. The dataset included sequence data from nuclear ribosomal ITS, ETS, and plastid psbA-trnH regions. An initial question was to assess the monophyly of the genus and whether Barroetea, Phanerostylis, and Kuhnia should be recognized as separate from or included in Brickellia. The results supported the hypothesis that Brickellia is monophyletic, with the small (2-3 species) Pleurocoronis as the sister group and showed Barroetea, Phanerostylis, and Kuhnia all embedded within the genus. Results of a time calibrated phylogeny from a BEAST analysis gave an estimated origination time for Brickellia at about 9 million years ago (Ma), with the oldest split within the genus dated at about 7.5Ma. A BAMM analysis based on the time calibrated tree showed that Brickellia has one rate shift in diversification associated with its origin in the late Miocene. Some lineages within the genus have had an increase in the rate of diversification over the past 5Ma, whereas other lineages have had a decrease in net diversification during this period. The results also elucidated nine clades within Brickellia which are accepted as taxonomic sections, and that will form logical units for future detailed studies.

Evolutionary origins of a bioactive peptide buried within Preproalbumin.

The de novo evolution of proteins is now considered a frequented route for biological innovation, but the genetic and biochemical processes that lead to each newly created protein are often poorly documented. The common sunflower (Helianthus annuus) contains the unusual gene PawS1 (Preproalbumin with SFTI-1) that encodes a precursor for seed storage albumin; however, in a region usually discarded during albumin maturation, its sequence is matured into SFTI-1, a protease-inhibiting cyclic peptide with a motif homologous to unrelated inhibitors from legumes, cereals, and frogs. To understand how PawS1 acquired this additional peptide with novel biochemical functionality, we cloned PawS1 genes and showed that this dual destiny is over 18 million years old. This new family of mostly backbone-cyclic peptides is structurally diverse, but the protease-inhibitory motif was restricted to peptides from sunflower and close relatives from its subtribe. We describe a widely distributed, potential evolutionary intermediate PawS-Like1 (PawL1), which is matured into storage albumin, but makes no stable peptide despite possessing residues essential for processing and cyclization from within PawS1. Using sequences we cloned, we retrodict the likely stepwise creation of PawS1's additional destiny within a simple albumin precursor. We propose that relaxed selection enabled SFTI-1 to evolve its inhibitor function by converging upon a successful sequence and structure.

The evolution of angiosperm lianescence without vessels--climbing mode and wood structure-function in Tasmannia cordata (Winteraceae).

• The lack of extant lianescent vessel-less seed plants supports a hypothesis that liana evolution requires large-diameter xylem conduits. Here, we demonstrate an unusual example of a lianoid vessel-less angiosperm, Tasmannia cordata (Winteraceae), from New Guinea. • Wood mechanical, hydraulic and structural measurements were used to determine how T. cordata climbs and to test for ecophysiological shifts related to liana evolution vs 13 free-standing congeners. • The tracheid-based wood of T. cordata furnished low hydraulic capacity compared with that of vessel-bearing lianas. In comparison with most nonclimbing relatives, T. cordata possessed lower photosynthetic rates and leaf and stem hydraulic capacities. However, T. cordata exhibited a two- to five-fold greater wood elastic modulus than its relatives. • Tasmannia cordata provides an unusual example of angiosperm liana evolution uncoupled from xylem conduit gigantism, as well as high plasticity and cell type diversity in vascular development. Because T. cordata lacks vessels, our results suggest that a key limitation for a vessel-less liana is that strong and low hydraulically conductive wood is required to meet the mechanical demands of lianescence.

Hybrid genera in Liatrinae (Asteraceae: Eupatorieae).

Liatrinae is a small subtribe of Eupatorieae that occurs in North America with a center of generic-level diversity in the southeastern United States. Molecular phylogenetic data were sought to assess whether two monotypic genera, Garberia and Hartwrightia, are accurately placed in the subtribe, and to resolve questions of the generic-level classification of Carphephorus. Phylogenetic analyses of nuclear ITS/ETS and plastid DNA data indicated that Garberia is the basalmost diverging lineage, and that Hartwrightia is phylogenetically embedded in the subtribe. There was significant incongruence between the ITS/ETS and plastid DNA datasets in the placement of Hartwrightia and another monotypic genus, Litrisa, suggesting that both are of original hybrid origin. The results also showed that Carphephorus s.l. is not monophyletic, and even after removal of the two species of Trilisa, it is still paraphyletic to Liatris. The apparent hybrid origin of Hartwrightia, which is morphologically transgressive relative to its inferred parental lineages, suggests that reticulation between phylogenetically distinct lineages may be a recurrent problem for phylogenetic estimation in Asteraceae.

Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III.

Although the chloroplast genome contains many noncoding regions, relatively few have been exploited for interspecific phylogenetic and intraspecific phylogeographic studies. In our recent evaluation of the phylogenetic utility of 21 noncoding chloroplast regions, we found the most widely used noncoding regions are among the least variable, but the more variable regions have rarely been employed. That study led us to conclude that there may be unexplored regions of the chloroplast genome that have even higher relative levels of variability. To explore the potential variability of previously unexplored regions, we compared three pairs of single-copy chloroplast genome sequences in three disparate angiosperm lineages: Atropa vs. Nicotiana (asterids); Lotus vs. Medicago (rosids); and Saccharum vs. Oryza (monocots). These three separate sequence alignments highlighted 13 mutational hotspots that may be more variable than the best regions of our former study. These 13 regions were then selected for a more detailed analysis. Here we show that nine of these newly explored regions (rpl32-trnL((UAG)), trnQ((UUG))-5'rps16, 3'trnV((UAC))-ndhC, ndhF-rpl32, psbD-trnT((GGU)), psbJ-petA, 3'rps16-5'trnK((UUU)), atpI-atpH, and petL-psbE) offer levels of variation better than the best regions identified in our earlier study and are therefore likely to be the best choices for molecular studies at low taxonomic levels.

Molecular confirmation of the hybrid origin of Eupatorium godfreyanum (Asteraceae).

Analysis of nuclear ribosomal ITS sequence data was used to assess the relationships of Eupatorium godfreyanum, an agamospermous polyploid species of putative hybrid origin. A data set of ITS sequences that included representatives of all but two of the North American species of Eupatorium was compiled from a combination of previously published and newly obtained results. Assessment of the data showed that each species was relatively distinctive, although the results from parsimony analysis suggested that there was little phylogenetic structure within the data beyond a basal split between members of the dog fennel group ("Traganthes") and the remainder of the genus. Cloning was required to obtain readable ITS sequence from E. godfreyanum, and analysis of individual clones produced sequences that matched closely those of either E. rotundifolium or E. sessilifolium. The ITS sequence data thus supported the hypothesis that Eupatorium godfreyanum is of hybrid origin from a combination of E. rotundifolium and E. sessilifolium.

The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis.

Chloroplast DNA sequences are a primary source of data for plant molecular systematic studies. A few key papers have provided the molecular systematics community with universal primer pairs for noncoding regions that have dominated the field, namely trnL-trnF and trnK/matK. These two regions have provided adequate information to resolve species relationships in some taxa, but often provide little resolution at low taxonomic levels. To obtain better phylogenetic resolution, sequence data from these regions are often coupled with other sequence data. Choosing an appropriate cpDNA region for phylogenetic investigation is difficult because of the scarcity of information about the tempo of evolutionary rates among different noncoding cpDNA regions. The focus of this investigation was to determine whether there is any predictable rate heterogeneity among 21 noncoding cpDNA regions identified as phylogenetically useful at low levels. To test for rate heterogeneity among the different cpDNA regions, we used three species from each of 10 groups representing eight major phylogenetic lineages of phanerogams. The results of this study clearly show that a survey using as few as three representative taxa can be predictive of the amount of phylogenetic information offered by a cpDNA region and that rate heterogeneity exists among noncoding cpDNA regions.