The symmetry spectrum in a hybridising, tropical group of rhododendrons.

Many diverse plant clades possess bilaterally symmetrical flowers and specialised pollination syndromes, suggesting that these traits may promote diversification. We examined the evolution of diverse floral morphologies in a species-rich tropical radiation of Rhododendron. We used restriction-site associated DNA sequencing on 114 taxa from Rhododendron sect. Schistanthe to reconstruct phylogenetic relationships and examine hybridisation. We then captured and quantified floral variation using geometric morphometric analyses, which we interpreted in a phylogenetic context. We uncovered phylogenetic conflict and uncertainty caused by introgression within and between clades. Morphometric analyses revealed flower symmetry to be a morphological continuum without clear transitions between radial and bilateral symmetry. Tropical Rhododendron species that began diversifying into New Guinea c. 6 million years ago expanded into novel floral morphological space. Our results showed that the evolution of tropical Rhododendron is characterised by recent speciation, recurrent hybridisation and the origin of floral novelty. Floral variation evolved via changes to multiple components of the corolla that are only recognised in geometric morphometrics with both front and side views of flowers.

Gene Duplication and Differential Expression of Flower Symmetry Genes in Rhododendron (Ericaceae).

Bilaterally symmetric flowers have evolved over a hundred times in angiosperms, yet orthologs of the transcription factors CYCLOIDEA (CYC), RADIALIS (RAD), and DIVARICATA (DIV) are repeatedly implicated in floral symmetry changes. We examined these candidate genes to elucidate the genetic underpinnings of floral symmetry changes in florally diverse Rhododendron, reconstructing gene trees and comparing gene expression across floral organs in representative species with radial and bilateral flower symmetries. Radially symmetric R. taxifolium Merr. and bilaterally symmetric R. beyerinckianum Koord. had four and five CYC orthologs, respectively, from shared tandem duplications. CYC orthologs were expressed in the longer dorsal petals and stamens and highly expressed in R. beyerinckianum pistils, whereas they were either ubiquitously expressed, lost from the genome, or weakly expressed in R. taxifolium. Both species had two RAD and DIV orthologs uniformly expressed across all floral organs. Differences in gene structure and expression of Rhododendron RAD compared to other asterids suggest that these genes may not be regulated by CYC orthologs. Our evidence supports CYC orthologs as the primary regulators of differential organ growth in Rhododendron flowers, while also suggesting certain deviations from the typical asterid gene regulatory network for flower symmetry.

Effects of historical demography and ecological context on spatial patterns of genetic diversity within foxtail pine (Pinus balfouriana; Pinaceae) stands located in the Klamath Mountains, California.

The density and dispersion of individuals, nonequilibrium demographics, and habitat fragmentation all affect the magnitude and extent of spatial genetic structure within forest tree populations. Here, we investigate the link between historical demography and spatial genetic structure within ecologically contrasting stands of foxtail pine (Pinus balfouriana) in the Klamath Mountains of northern California. We defined two stand types a priori, based largely on differences in foxtail pine density and basal area, and for each type we sampled two stands. Population expansions, likely from Pleistocene bottlenecks, were detected in three of the four stands. The magnitude and extent of spatial autocorrelation among genotypes at five nuclear microsatellites differed dramatically among stands, with those having lower foxtail pine density exhibiting strong patterns of isolation by distance. Moran's I statistics were 7-fold higher for the first distance class (<25 m) in these stands relative to those observed in stands with higher foxtail pine density (I(25) = 0.14 vs. 0.02). We conclude that differences in spatial genetic structure between stand types are due to differences in ecological attributes that affected expansion from inferred bottlenecks.

A phylogeographical analysis of the range disjunction for foxtail pine (Pinus balfouriana, Pinaceae): the role of Pleistocene glaciation.

Biogeographical patterns within the California Floristic Province have been greatly affected by geological and climatic events. Here, we investigate the phylogeography of foxtail pine (Pinus balfouriana) in an effort to date its range disjunction using molecular data and to further our understanding of phylogeographical patterns for plants within the California Floristic Province. The distribution of foxtail pine is characterized by a 500-km disjunction separating populations located in the Klamath Mountains from those in the southern Sierra Nevada. Previous authors suggested that this disjunction occurred approximately 4000-8000 years ago during the Holocene Xerotherm when western North America became warmer and drier. Those dates, however, are inconsistent with the morphological differences that separate regional populations into formally recognized subspecies. Using the coalescent-based isolation with migration model and DNA sequence data from the chloroplast, mitochondrial, and nuclear genomes, we evaluate several hypotheses addressing the timing of this range disjunction and its effects on subsequent patterns of gene flow. Results from all three genomes are largely consistent with Middle to Early Pleistocene divergence dates. Those dates correspond to the Sherwin glaciation, which was the largest Pleistocene glacial episode in the Sierra Nevada. Gene flow, moreover, was only documented using data from the chloroplast genome, suggesting that low levels of long-distance pollen dispersal (N(e)m < 0.5) have occurred since this divergence event. These results are extended to a discussion of the biogeographical development of subalpine forests in California.

Duplication and paralog sorting of RPB2 and RPB1 genes in core eudicots.

RPB1 and RPB2, which encode the largest and second largest subunits of RNA polymerase II, respectively, are essential single copy genes in fungi, animals and most plants. Two paralogs of the RPB2 gene have been found in some groups of angioperms [Oxelman, B., Yoshikawa, N., McConaughy, B.L., Luo, J., Denton, A.L., Hall, B.D., 2004. RPB2 gene phylogeny in flowering plants, with particular emphasis on asterids. Mol. Phylogenet. Evol. 32, 462-479]. Here, we report the results of experiments designed to identify the evolutionary origin of the RPB2 duplicate copies. Through careful sampling and phylogenetic analysis, we were able to construct the RPB2 gene tree in angiosperms and infer the phylogenetic positions of the gene duplication and gene loss events that occurred. Our study shows that an RPB2 gene duplication occurred early in core eudicot evolution, at or near the time of the Buxaceae/Trochodendraceae divergence. Subsequently, multiple gene duplication and paralog sorting events happened independently in different core eudicot taxa. Differential expression of the two RPB2 gene paralogs may explain the preservation of both paralogs in the asterids. One gene (RPB2-i) accounts for most of the RPB2 mRNA made in the flower organs while the other gene (RPB2-d) is predominantly used in the vegetative tissues. We also found two paralogs of the RPB1 gene in some core eudicot species. The RPB1 gene duplication occurred before core eudicot divergence, around the time of RPB2 gene duplication. Several independent RPB1 paralog sorting events happened in different core eudicot taxa; their occurrence was independent of the RPB2 paralog sorting events. Our results suggest that a polyploidization event happened at or near the time of the Buxaceae/Trochodendraceae divergence. We propose that this polyploidization and the partial diploidization processes thereafter may have been the driving force of core eudicot radiation.

A multistep process gave rise to RNA polymerase IV of land plants.

Since their discovery in Metazoa, the three nuclear RNA polymerases (RNAPs) have been found in fungi, plants, and diverse protists. In all eukaryotes studied to date, RNAPs I, II, and III collectively transcribe all major RNAs made in the nucleus. We have found genes for the largest subunit (RPD1/RPE1) of a new DNA-dependent RNAP, RNAP IV, in all major land plant taxa and in closely related green algae. Genes for the second-largest subunit (RPD2) of this enzyme were found in all land plants. Phylogenetic study indicates that RNAP IV genes are sister to the corresponding RNAP II genes. Our results show the genesis of RNAP IV to be a multistep process in which the largest and second-largest subunit genes evolved by independent duplication events in the ancestors of Charales and land plants. These findings provide insights into evolutionary mechanisms that can explain the origin of multiple RNAPs in the eukaryotic nucleus.

Loss of the flagellum happened only once in the fungal lineage: phylogenetic structure of kingdom Fungi inferred from RNA polymerase II subunit genes.

BACKGROUND: At present, there is not a widely accepted consensus view regarding the phylogenetic structure of kingdom Fungi although two major phyla, Ascomycota and Basidiomycota, are clearly delineated. Regarding the lower fungi, Zygomycota and Chytridiomycota, a variety of proposals have been advanced. Microsporidia may or may not be fungi; the Glomales (vesicular-arbuscular mycorrhizal fungi) may or may not constitute a fifth fungal phylum, and the loss of the flagellum may have occurred either once or multiple times during fungal evolution. All of these issues are capable of being resolved by a molecular phylogenetic analysis which achieves strong statistical support for major branches. To date, no fungal phylogeny based upon molecular characters has satisfied this criterion. RESULTS: Using the translated amino acid sequences of the RPB1 and RPB2 genes, we have inferred a fungal phylogeny that consists largely of well-supported monophyletic phyla. Our major results, each with significant statistical support, are: (1) Microsporidia are sister to kingdom Fungi and are not members of Zygomycota; that is, Microsporidia and fungi originated from a common ancestor. (2) Chytridiomycota, the only fungal phylum having a developmental stage with a flagellum, is paraphyletic and is the basal lineage. (3) Zygomycota is monophyletic based upon sampling of Trichomycetes, Zygomycetes, and Glomales. (4) Zygomycota, Basidiomycota, and Ascomycota form a monophyletic group separate from Chytridiomycota. (5) Basidiomycota and Ascomycota are monophyletic sister groups. CONCLUSION: In general, this paper highlights the evolutionary position and significance of the lower fungi (Zygomycota and Chytridiomycota). Our results suggest that loss of the flagellum happened only once during early stages of fungal evolution; consequently, the majority of fungi, unlike plants and animals, are nonflagellated. The phylogeny we infer from gene sequences is the first one that is congruent with the widely accepted morphology-based classification of Fungi. We find that, contrary to what has been published elsewhere, the four morphologically defined phyla (Ascomycota, Basidiomycota, Zygomycota and Chytridiomycota) do not overlap with one another. Microsporidia are not included within kingdom Fungi; rather they are a sister-group to the Fungi. Our study demonstrates the applicability of protein sequences from large, slowly-evolving genes to the derivation of well-resolved and highly supported phylogenies across long evolutionary distances.

Phylogeny, historical biogeography, and patterns of diversification for Pinus (Pinaceae): phylogenetic tests of fossil-based hypotheses.

Pines comprise one of the largest coniferous genera, are distributed throughout the Northern Hemisphere, and have an abundant fossil record. Distributions of fossils have been used to derive a three-step hypothesis of early pine evolution, which postulates a Mesozoic origin for the genus, east-west expansions across Laurasia, and retraction into Eocene refugia. Here, we present phylogenetic tests of this hypothesis using chloroplast sequence data from four loci for 83 pine species. We used the fossil-based hypothesis to derive null expectations concerning monophyly of taxonomic groups, dates of cladogenesis, and patterns of diversification. Phylogenetic analyses using several algorithms subsequently provided rigorous tests of these expectations. Our inferred phylogenies illustrated broad congruence with taxonomic groups, but highlighted consistent problems within subgenus Strobus. Estimated minimum dates of divergence derived from relaxed clock methods were largely consistent with the fossil record and yielded a date for the ingroup node of Pinus of 128+/-4 mya, depending upon the calibration used for subgenus Pinus. Ancestral area reconstructions showed Pinus to have most likely originated in Eurasia. Major clades differed in biogeographic patterns, but were consistent with the fossil-based hypothesis. We found weak support, however, for a change in diversification rate in the Eocene as interpretations of fossil distributions would have predicted.

RPB2 gene phylogeny in flowering plants, with particular emphasis on asterids.

Two, apparently functional, paralogues of the RPB2 gene, which encodes the second largest subunit of RNA polymerase II, are shown to be present in two major groups of asterid plants. Although all other land plants surveyed so far have been found to have only one of these two copies, the RPB2 gene phylogeny inferred from the 3' half of the gene for 35 angiosperm taxa and six other land plants indicates that the duplication of the RPB2 gene occurred earlier than the time for origin of the asterid group, probably near the origin of "core eudicots." The d copy is present in all plants which are unambiguously assigned to the core eudicots, whereas the I copy is retained only in the lamiid clade, Ericales, and Escallonia, all belonging to the asterid group of plants. Both parsimony and likelihood analyses of sequences from the 3' half of the gene give strong bootstrap support for these conclusions. There is no support for monophyly of the taxa having both copies. Thus, numerous losses of one of the copies must be inferred. Structurally, both paralogues appear functional, and transcription is demonstrated for both copies. In the lamiid group, the d copy has lost introns 18-23. The well supported phylogenetic relationships implied by the RPB2 gene phylogeny are largely congruent with well supported phylogenetic hypotheses based on other sequence data. However, Ilex, usually assigned to the campanuliid clade, is instead supported as being a member of the lamiid clade, both from sequence data and the presence of an I copy as well as the loss of introns 18-23 in the d copy. Escallonia, supported as a member of the campanuliid clade both by RPB2-d-sequences and previously published DNA sequence data, has all the introns 18-23 in its d copy, as do all other members studied from the campanuliid group. We used the Markov Chain Monte Carlo (MCMC) approach of the MrBayes program to implement Maximum Likelihood bootstrapping. Under the same model of evolution, bootstrapping frequencies are significantly lower than the Bayesian posterior probabilities inferred from the MCMC chain.

Body plan evolution of ascomycetes, as inferred from an RNA polymerase II phylogeny.

The mode of evolution of the biologically diverse forms of ascomycetes is not well understood, largely because the descent relationships remain unresolved. By using sequences of the nuclear gene RPB2, we have inferred with considerable resolution the phylogenetic relationships between major groups within the phylum Ascomycota. These relationships allow us to deduce a historical pattern of body plan evolution. Within Taphrinomycotina, the most basal group, two simple body plans exist: uncovered asci with unicellular growth, or rudimentary ascoma with hyphal growth. Ancestral ascomycetes were filamentous; hyphal growth was lost independently in the yeast forms of Taphrinomycotina and Saccharomycotina. Pezizomycotina, the sister group to Saccharomycotina, retained mycelial growth while elaborating two basic ontogenetic pathways for ascoma formation and centrum development. The RPB2 phylogeny shows with significant statistical support that taxa in Pezizomycotina with ascohymenial ontogeny (ascoma generally forms after nuclear pairing) are ancestral and paraphyletic, whereas ascolocular fungi with fissitunicate asci are a clade derived from them. Ascolocular lichens are polyphyletic, whereas ascohymenial lichens comprise a monophyletic group that includes the Lecanorales. Our data are not consistent with a derived origin of Eurotiomycetes including Aspergillus and Trichophyton from within a lichen-forming ancestral group. For these reasons, the results of this study are considerably at variance with the conclusion that major fungal lineages are derived from lichensymbiotic ancestors. Interpretation of our results in the context of early work suggests that ascoma ontogeny and centrum characters are not in conflict with the molecular data.

Evolution of the RNA polymerase II C-terminal domain.

In recent years a great deal of biochemical and genetic research has focused on the C-terminal domain (CTD) of the largest subunit (RPB1) of DNA-dependent RNA polymerase II. This strongly conserved domain of tandemly repeated heptapeptides has been linked functionally to important steps in the initiation and processing of mRNA transcripts in both animals and fungi. Although they are absolutely required for viability in these organisms, C-terminal tandem repeats do not occur in RPB1 sequences from diverse eukaryotic taxa. Here we present phylogenetic analyses of RPB1 sequences showing that canonical CTD heptads are strongly conserved in only a subset of eukaryotic groups, all apparently descended from a single common ancestor. Moreover, eukaryotic groups in which the most complex patterns of ontogenetic development occur are descended from this CTD-containing ancestor. Consistent with the results of genetic and biochemical investigations of CTD function, these analyses suggest that the enhanced control over RNA polymerase II transcription conveyed by acquired CTD/protein interactions was an important step in the evolution of intricate patterns of gene expression that are a hallmark of large, developmentally complex eukaryotic organisms.

Using RPB1 sequences to improve phylogenetic inference among mushrooms (Inocybe, Agaricales).

An investigation of mushroom phylogeny using the largest subunit of RNA polymerase II gene sequences (RPB1) was conducted in comparison with nuclear ribosomal large subunit RNA gene sequences (nLSU) for the same set of taxa in the genus Inocybe (Agaricales, Basidiomycota). The two data sets, though not significantly incongruent, exhibit conflict among the placement of two taxa that exhibit long branches in the nLSU data set. In contrast, RPB1 terminal branch lengths are rather uniform. Bootstrap support is increased for clades in RPB1. Combined data sets increase the degree of confidence for several relationships. Overall, nLSU data do not yield a robust phylogeny when independently assessed by RPB1 sequences. This multigene study indicates that Inocybe is a monophyletic group composed of at least four distinct lineages-subgenus Mallocybe, section Cervicolores, section Rimosae, and subgenus Inocybe sensu Kühner, Kuyper, non Singer. Within subgenus Inocybe, two additional lineages, one composed of species with smooth basidiospores (clade I) and a second characterized by nodulose-spored species (clade II), are recovered by RPB1 and combined data. The nLSU data recover only clade I. The genera Astrosporina and Inocybella cannot be recognized phylogenetically. "Supersections" Cortinatae and Marginatae are not monophyletic groups.