High diversity, high insular endemism and recent origin in the lichen genus Sticta (lichenized Ascomycota, Peltigerales) in Madagascar and the Mascarenes.

Lichen biodiversity and its generative evolutionary processes are practically unknown in the MIOI (Madagascar and Indian Ocean Islands) biodiversity hotspot. We sought to test the hypothesis that lichenized fungi in this region have undergone a rapid radiation, following a single colonization event, giving rise to narrow endemics, as is characteristic of other lineages of plants. We extensively sampled specimens of the lichen genus Sticta in the Mascarene archipelago (mainly Réunion) and in Madagascar, mainly in the northern range (Amber Mt and Marojejy Mt) and produced the fungal ITS barcode sequence for 148 thalli. We further produced a four-loci data matrix for 68 of them, representing the diversity and geographical distribution of ITS haplotypes. We reconstructed the phylogenetic relationships within this group, established species boundaries with morphological context, and estimated the date of the most recent common ancestor. Our inferences resolve a robust clade comprising 31 endemic species of Sticta that arose from the diversification following a single recent (c. 11 Mya) colonization event. All but three species have a very restricted range, endemic to either the Mascarene archipelago or a single massif in Madagascar. The first genus of lichens to be studied with molecular data in this region underwent a recent radiation, exhibits micro-endemism, and thus exemplifies the biodiversity characteristics found in other taxa in Madagascar and the Mascarenes.

Playing with extremes: Origins and evolution of exaggerated female forelegs in South African Rediviva bees.

Despite close ecological interactions between plants and their pollinators, only some highly specialised pollinators adapt to a specific host plant trait by evolving a bizarre morphology. Here we investigated the evolution of extremely elongated forelegs in females of the South African bee genus Rediviva (Hymenoptera: Melittidae), in which long forelegs are hypothesised to be an adaptation for collecting oils from the extended spurs of their Diascia host flowers. We first reconstructed the phylogeny of the genus Rediviva using seven genes and inferred an origin of Rediviva at around 29MYA (95% HPD=19.2-40.5), concurrent with the origin and radiation of the Succulent Karoo flora. The common ancestor of Rediviva was inferred to be a short-legged species that did not visit Diascia. Interestingly, all our analyses strongly supported at least two independent origins of long legs within Rediviva. Leg length was not correlated with any variable we tested (ecological specialisation, Diascia visitation, geographic distribution, pilosity type) but seems to have evolved very rapidly. Overall, our results indicate that foreleg length is an evolutionary highly labile, rapidly evolving trait that might enable Rediviva bees to respond quickly to changing floral resource availability.

Crystal structure of RNA polymerase II from Komagataella pastoris.

RNA polymerase II (Pol II) is a 12-subunit protein complex that conducts the transcription of mRNA and some small RNAs. In this work, the crystal structure of Pol II from the methylotropic yeast Komagataella pastoris (Pichia pastoris) was determined. While the structure is highly homologous to that of Pol II from the budding yeast Saccharomyces cerevisiae, the stalk and clamp modules of the K. pastoris Pol II displayed large inward rotations, closing the central cleft to a greater extent than in the known S. cerevisiae Pol II structures. The conformational differences reflect the inherent flexibilities of the stalk and the clamp, as additional low-resolution structures of K. pastoris Pol II in different crystal forms revealed diverse stalk and clamp orientations. Comparisons with other eukaryotic/archaeal RNA polymerase structures in the Protein Data Bank revealed the distributions of the stalk and clamp orientations. The conformational plasticity should be essential for transcriptional functions and binding various regulatory factors.

Molecular evidence of RNA polymerase II gene reveals the origin of worldwide cultivated barley.

The origin and domestication of cultivated barley have long been under debate. A population-based resequencing and phylogenetic analysis of the single copy of RPB2 gene was used to address barley domestication, to explore genetic differentiation of barley populations on the worldwide scale, and to understand gene-pool exchanges during the spread and subsequent development of barley cultivation. Our results revealed significant genetic differentiation among three geographically distinct wild barley populations. Differences in haplotype composition among populations from different geographical regions revealed that modern cultivated barley originated from two major wild barley populations: one from the Near East Fertile Crescent and the other from the Tibetan Plateau, supporting polyphyletic origin of cultivated barley. The results of haplotype frequencies supported multiple domestications coupled with widespread introgression events that generated genetic admixture between divergent barley gene pools. Our results not only provide important insight into the domestication and evolution of cultivated barley, but also enhance our understanding of introgression and distinct selection pressures in different environments on shaping the genetic diversity of worldwide barley populations, thus further facilitating the effective use of the wild barley germplasm.

Selective constraint and the evolution of the RNA polymerase II C-Terminal Domain.

The C-Terminal Domain (CTD) of the large subunit (Rpb1) of RNA Polymerase II has a Tyrosine-Serine-Proline-Threonine-Serine-Proline-Serine repeat structure in many eukaryotes. Chemical modifications of these residues play a central role in the regulation and coordination of the events of transcription. However, substantial variability in the presence and regularity of repeat arrays exists between eukaryote taxa. Following a survey of CTD structure from diverse eukaryote species, two hypotheses were tested relating to repeat structure and the action of selection on the CTD. First, it was found that degenerated repeat structure is associated with lower serine and proline frequencies in some eukaryote taxa but not in others. Second, maximum likelihood models of the evolution of Rpb1 in a number of species groups found that purifying selection on the non-repetitive CTD of several Leishmania species was substantially lower than for the rest of Rpb1, whereas purifying selection in a number of species groups containing repeat arrays was usually as high or nearly as high as for the rest of Rpb1. Characterization of CTD structure for a larger number of species than has been completed previously also revealed a greater diversity of CTD structures in eukaryotes than previously known, along with loss of repeat structure in the animals and fungi, two taxa where it has not previously been known. These results suggest that loss of CTD repeat structure has been an important aspect of RNA Polymerase II evolution in diverse eukaryotes.

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.

Foraminifera and Cercozoa share a common origin according to RNA polymerase II phylogenies.

Phylogenetic analysis of small and large subunits of rDNA genes suggested that Foraminifera originated early in the evolution of eukaryotes, preceding the origin of other rhizopodial protists. This view was recently challenged by the analysis of actin and ubiquitin protein sequences, which revealed a close relationship between Foraminifera and Cercozoa, an assemblage of various filose amoebae and amoeboflagellates that branch in the so-called crown of the SSU rDNA tree of eukaryotes. To further test this hypothesis, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1) from five foraminiferans, two cercozoans and the testate filosean Gromia oviformis. Analysis of our data confirms a close relationship between Foraminifera and Cercozoa and points to Gromia as the closest relative of Foraminifera.

Phylogenetic analysis of arthropods using two nuclear protein-encoding genes supports a crustacean + hexapod clade.

Recent phylogenetic analyses using molecular data suggest that hexapods are more closely related to crustaceans than to myriapods, a result that conflicts with long-held morphology-based hypotheses. Here we contribute additional information to this debate by conducting phylogenetic analyses on two nuclear protein-encoding genes, elongation factor-1 alpha (EF-1 alpha) and the largest subunit of RNA polymerase II (Pol II), from an extensive sample of arthropod taxa. Results were obtained from two data sets. One data set comprised 1092 nucleotides (364 amino acids) of EF-1 alpha and 372 nucleotides (124 amino acids) of Pol II from 30 arthropods and three lobopods. The other data set contained the same EF-1 alpha fragment and an expanded 1038-nucleotide (346-amino-acid) sample of Pol II from 17 arthropod taxa. Results from maximum-parsimony and maximum-likelihood analyses strongly supported the existence of a Crustacea + Hexapoda clade (Pancrustacea) over a Myriapoda + Hexapoda clade (Atelocerata). The apparent incompatibility between the molecule-based Pancrustacea hypothesis and morphology-based Atelocerata hypothesis is discussed.

Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae.

Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10 alpha, Hs10 beta, Hs8, and Hs6, respectively), homologous to the ABC10 alpha, ABC10 beta, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10 alpha, Sc10 beta, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10 alpha and the corresponding Sp10 alpha of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-delta::HIS3) defective in Sc10 alpha. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10 beta, Sc10 beta, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGXnCCR motif presumably forms an atypical zinc-binding domain. Hs10 beta, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-delta 1::HIS3) lacking Sc10 beta. Hs8 complemented a yeast mutant (rpb8-delta 1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10 beta cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit.

Two species of chromatin-RNA polymerase II complex are commonly present in nuclei of various tissues of rats.

When rat liver nuclei were digested with nuclease, we found that the chromatin-bound RNA polymerase II was liberated as two distinct complexes, peak 1 and peak 2, which seemed to reflect different functional states in cell nuclei. We further examined their occurrence in nuclear digests of various tissues of rats and the following results were obtained. Upon digestion with micrococcal nuclease of nuclei from brain, spleen, testis and kidney, chromatin-bound RNA polymerase II was liberated as two distinct forms which sedimented differently in a sucrose density gradient. The sedimentation rate of peak 1 varied depending on the tissue nuclei examined. After high salt or RNase treatment of the nuclear digests, peak 1 from liver, brain, spleen and testis nuclei showed the same sedimentation rate as did kidney peak 1, the rate for which remained unchanged by these treatments. The results suggested that peak 1 complexes from various tissue nuclei had basically the same structural organization, and we confirmed this by electrophoretic studies on RNase-treated liver and kidney nuclear digests. Peak 2 from various tissue nuclei exhibited identical sedimentation rates. Thus, the chromatin-bound RNA polymerase II seems to exist commonly in two distinct states in cell nuclei of rats.