Phylogeny, function, and evolution of the cupins, a structurally conserved, functionally diverse superfamily of proteins

The cupin superfamily is a group of functionally diverse proteins that are found in all three kingdoms of life, Archaea, Eubacteria, and Eukaryota. These proteins have a characteristic signature domain comprising two histidine- containing motifs separated by an intermotif region of variable length. This domain consists of six beta strands within a conserved beta barrel structure. Most cupins, such as microbial phosphomannose isomerases (PMIs), AraC- type transcriptional regulators, and cereal oxalate oxidases (OXOs), contain only a single domain, whereas others, such as seed storage proteins and oxalate decarboxylases (OXDCs), are bi-cupins with two pairs of motifs. Although some cupins have known functions and have been characterized at the biochemical level, the majority are known only from gene cloning or sequencing projects. In this study, phylogenetic analyses were conducted on the conserved domain to investigate the evolution and structure/function relationships of cupins, with an emphasis on single- domain plant germin-like proteins (GLPs). An unrooted phylogeny of cupins from a wide spectrum of evolutionary lineages identified three main clusters, microbial PMIs, OXDCs, and plant GLPs. The sister group to the plant GLPs in the global analysis was then used to root a phylogeny of all available plant GLPs. The resulting phylogeny contained three main clades, classifying the GLPs into distinct subfamilies. It is suggested that these subfamilies correlate with functional categories, one of which contains the bifunctional barley germin that has both OXO and superoxide dismutase (SOD) activity. It is proposed that GLPs function primarily as SODs, enzymes that protect plants from the effects of oxidative stress. Closer inspection of the DNA sequence encoding the intermotif region in plant GLPs showed global conservation of thymine in the second codon position, a character associated with hydrophobic residues. Since many of these proteins are multimeric and enzymatically inactive in their monomeric state, this conservation of hydrophobicity is thought to be associated with the need to maintain the various monomer- monomer interactions. The type of structure-based predictive analysis presented in this paper is an important approach for understanding gene function and evolution in an era when genomes from a wide range of organisms are being sequenced at a rapid rate.

Extinction processes in hotspots of avian biodiversity and the targeting of pre-emptive conservation action

Hot spots of endemism are regarded as important global sites for conservation as they are rich in threatened endemic species and currently experiencing extensive habitat loss. Targeting pre-emptive conservation action to sites that are currently relatively intact but which would be vulnerable to particular human activities if they occurred in the future is, however, also valuable but has received less attention. Here, we address this issue by using data on Endemic Bird Areas (EBAs). First, we identify the ecological factors that affect extinction risk in the face of particular human activities, and then use these insights to identify EBAs that should be priorities for pre-emptive conservation action. Threatened endemic species in EBAs are significantly more likely to be habitat specialists or relatively large-bodied than non-threatened species, when compared across avian families. Increasing habitat loss causes a significant increase in extinction risk among habitat specialists, but we found no evidence to suggest that the presence of alien species/human exploitation causes a significant increase in extinction risk among large-bodied species. This suggests that these particular human activities are contributing to high extinction risk among habitat specialists, but not among large-bodied species. Based on these analyses, we identify 39 EBAs containing 570 species (24% of the total in EBAs) that are not currently threatened with severe habitat loss, but would be ecologically vulnerable to future habitat loss should it occur. We show that these sites tend to be poorly represented in existing priority setting exercises involving hot spots, suggesting that vulnerability must be explicitly included within these exercises if such sites are to be adequately protected.

Phylogeny and the hierarchical organization of plant diversity

R. H. Whittaker's idea that plant diversity can be divided into a hierarchy of spatial components from alpha at the within-habitat scale through beta for the turnover of species between habitats to gamma along regional gradients implies the underlying existence of alpha, beta, and gamma niches. We explore the hypothesis that the evolution of a, (3, and gamma niches is also hierarchical, with traits that define the a niche being labile, while those defining a and 7 niches are conservative. At the a level we find support for the hypothesis in the lack of close significant phylogenetic relationship between meadow species that have similar a niches. In a second test, a niche overlap based on a variety of traits is compared between congeners and noncongeners in several communities; here, too, there is no evidence of a correlation between a niche and phylogeny. To test whether beta and gamma niches evolve conservatively, we reconstructed the evolution of relevant traits on evolutionary trees for 14 different clades. Tests against null models revealed a number of instances, including some in island radiations, in which habitat (beta niche) and elevational maximum (an aspect of the gamma niche) showed evolutionary conservatism.

Cyclamen: time, sea and speciation biogeography using a temporally calibrated phylogeny

Aim The Mediterranean region is a species-rich area with a complex geographical history. Geographical barriers have been removed and restored due to sea level changes and local climatic change. Such barriers have been proposed as a plausible mechanism driving the high levels of speciation and endemism in the Mediterranean basin. This raises the fundamental question: is allopatric isolation the mechanism by which speciation occurs? This study explores the potential driving influence of palaeo-geographical events on the speciation of Cyclamen (Myrsinaceae), a group with most species endemic to the Mediterranean region. Cyclamen species have been shown experimentally to have few genetic barriers to hybridization. Location The Mediterranean region, including northern Africa, extending eastwards to the Black Sea coast. Methods A generic level molecular phylogeny of Myrsinaceae and Primulaceae is constructed, using Bayesian approximation, to produce a secondary age estimate for the stem lineage of Cyclamen. This estimate is used to calibrate temporally an infrageneric phylogeny of Cyclamen, built with nrDNA ITS, cpDNA trnL-F and cpDNA rps16 sequences. A biogeographical analysis of Cyclamen is performed using dispersal-vicariance analysis. Results The emergence of the Cyclamen stem lineage is estimated at 30.1-29.2 Ma, and the crown divergence at 12.9-12.2 Ma. The average age of Cyclamen species is 3.7 Myr. Every pair of sister species have mutually exclusive, allopatric distributions relative to each other. This pattern appears typical of divergence events throughout the evolutionary history of the genus. Main conclusions Geographical barriers, such as the varying levels of the Mediterranean Sea, are the most plausible explanation for speciation events throughout the phylogenetic history of Cyclamen. The genus demonstrates distributional patterns congruent with the temporally reticulate palaeogeography of the Mediterranean region.

Phylogeny, biogeography, and ecology of Ficus section Malvanthera (Moraceae)

We conducted the first molecular phylogenetic study of Ficus section Malvanthera (Moraceae; subgenus Urostigma) based on 32 Malvanthera accessions and seven outgroups representing other sections of Ficus subgenus Urostigma. We used DNA sequences from the nuclear ribosomal internal and external transcribed spacers (ITS and ETS), and the glyceraldehyde-3-phosphate dehydrogenase (G3pdh) region. Phylogenetic analysis using maximum parsimony, maximum likelihood and Bayesian methods recovered a monophyletic section Malvanthera to the exclusion of the rubber fig, Ficus elastica. The results of the phylogenetic analyses do not conform to any previously proposed taxonomic subdivision of the section and characters used for previous classification are homoplasious. Geographic distribution, however, is highly conserved and Melanesian Malvanthera are monophyletic. A new subdivision of section Malvanthera reflecting phylogenetic relationships is presented. Section Malvanthera likely diversified during a period of isolation in Australia and subsequently colonized New Guinea. Two Australian series are consistent with a pattern of dispersal out of rainforest habitat into drier habitats accompanied by a reduction in plant height during the transition from hemi-epiphytic trees to lithophytic trees and shrubs. In contradiction with a previous study of Pleistodontes phylogeny suggesting multiple changes in pollination behaviour, reconstruction of changes in pollination behaviour on Malvanthera, suggests only one or a few gains of active pollination within the section. (C) 2008 Elsevier Inc. All rights reserved.

Probing the receptor interactions ofan H5 avian influenza virus using a baculovirus expression system and functionalised poly(acrylic acid) ligands

Influenza viruses attach to host cells by binding to terminal sialic acid (Neu5Ac) on glycoproteins or glycolipids. Both the linkage of Neu5Ac and the identity of other carbohydrates within the oligosaccharide are thought to play roles in restricting the host range of the virus. In this study, the receptor specificity of an H5 avian influenza virus haemagglutinin protein that has recently infected man (influenza strain A/Vietnam/1194/04) has been probed using carbohydrate functionalised poly(acrylic acid) polymers. A baculovirus expression system that allows facile and safe analysis of the Neu5Ac binding specificity of mutants of H5 HA engineered at sites that are predicted to effect a switch in host range has also been developed. (C) 2007 Elsevier Ltd. All rights reserved.

NS1 proteins of avian influenza A viruses can act as antagonists of the human alpha/beta interferon response

Many viruses, including human influenza A virus, have developed strategies for counteracting the host type I interferon (IFN) response. We have explored whether avian influenza viruses were less capable of combating the type I IFN response in mammalian cells, as this might be a determinant of host range restriction. A panel of avian influenza viruses isolated between 1927 and 1997 was assembled. The selected viruses showed variation in their ability to activate the expression of a reporter gene under the control of the IFN-beta promoter and in the levels of IFN induced in mammalian cells. Surprisingly, the avian NS1 proteins expressed alone or in the genetic background of a human influenza virus controlled IFN-beta induction in a manner similar to the NS1 protein of human strains. There was no direct correlation between the IFN-beta induction and replication of avian influenza viruses in human A549 cells. Nevertheless, human cells deficient in the type I IFN system showed enhanced replication of the avian viruses studied, implying that the human type I IFN response limits avian influenza viruses and can contribute to host range restriction.

Development of a reverse genetics system enabling the rescue of recombinant avian influenza virus A/Turkey/England/50-92/91 (H5N1)

We previously described the use of an established reverse genetics system for the generation of recombinant human influenza A viruses from cloned cDNAs. Here, we have assembled a set of plasmids to allow recovery of the avian H5N1 influenza virus A/Turkey/England/50-92/91 entirely from cDNA. This system enables us to introduce mutations or truncations into the cDNAs to create mutant viruses altered specifically in a chosen gene. These mutant viruses can then be used in future pathogenesis studies in chickens and in studies to understand the host range restrictions of avian influenza viruses in humans.

Diversity, phylogeny and distribution of bean rhizobia in salt-affected soils of North-West Morocco

Different molecular methods: BOX-PCR fingerprinting, R-FLP-PCR and sequencing of the 16S rDNA as well as the symbiotic genes nodC and nifH, were used to study the genetic diversity within a collection of nodulating bean rhizobia isolated from five soils of North-West Morocco. BOX fingerprints analysis of 241 isolates revealed 19 different BOX profiles. According to the PFLP-PCR and sequencing of 16S rDNA carried out on 45 representative isolates, 5 genotypes were obtained corresponding to the species Rhizobium etli, R. tropici, R. gallicum, R. leguminosarum and Sinorhizobium meliloti. The most abundant species were R. etli and R. tropici (61% and 24%, respectively). A high intraspecific diversity was observed among the R. etli isolates, while the R. tropici group was homogeneous. Most of the rhizobia studied belong to species known to nodulate common bean, while 2 species were unconventional microsymbionts: R. leguminosarum biovar viciae and S. meliloti. Our results, especially the nodulation promiscuity of common bean and the relation between the predominance of some species of rhizobia in particular soils and the salt content of these soils, indicate that there is a real need for a better understanding of the distribution of common bean rhizobia species in the soils of Morocco before any inoculation attempt.

Origin of avian genome size and structure in non-avian dinosaurs

Avian genomes are small and streamlined compared with those of other amniotes by virtue of having fewer repetitive elements and less non-coding DNA(1,2). This condition has been suggested to represent a key adaptation for flight in birds, by reducing the metabolic costs associated with having large genome and cell sizes(3,4). However, the evolution of genome architecture in birds, or any other lineage, is difficult to study because genomic information is often absent for long-extinct relatives. Here we use a novel bayesian comparative method to show that bone-cell size correlates well with genome size in extant vertebrates, and hence use this relationship to estimate the genome sizes of 31 species of extinct dinosaur, including several species of extinct birds. Our results indicate that the small genomes typically associated with avian flight evolved in the saurischian dinosaur lineage between 230 and 250 million years ago, long before this lineage gave rise to the first birds. By comparison, ornithischian dinosaurs are inferred to have had much larger genomes, which were probably typical for ancestral Dinosauria. Using comparative genomic data, we estimate that genome-wide interspersed mobile elements, a class of repetitive DNA, comprised 5 - 12% of the total genome size in the saurischian dinosaur lineage, but was 7 - 19% of total genome size in ornithischian dinosaurs, suggesting that repetitive elements became less active in the saurischian lineage. These genomic characteristics should be added to the list of attributes previously considered avian but now thought to have arisen in non-avian dinosaurs, such as feathers(5), pulmonary innovations 6, and parental care and nesting

Using phylogeny to investigate the origins of the Cape flora: the importance of taxonomic, gene and genome sampling strategies

Phylogenetic methods hold great promise for the reconstruction of the transition from precursor to modern flora and the identification of underlying factors which drive the process. The phylogenetic methods presently used to address the question of the origin of the Cape flora of South Africa are considered here. The sampling requirements of each of these methods, which include dating of diversifications using calibrated molecular trees, sister pair comparisons, lineage through time plots and biogeographical optimizations are reviewed. Sampling of genes, genomes and species are considered. Although increased higher-level studies and increased sampling are required for robust interpretation, it is clear that much progress is already made. It is argued that despite the remarkable richness of the flora, the Cape flora is a valuable model system to demonstrate the utility of phylogenetic methods in determining the history of a modern flora.

Infection of human airway epithelium by human and avian strains of influenza a virus

We describe the characterization of influenza A virus infection of an established in vitro model of human pseudostratified mucociliary airway epithelium (HAE). Sialic acid receptors for both human and avian viruses, alpha-2,6- and alpha-2,3-linked sialic acids, respectively, were detected on the HAE cell surface, and their distribution accurately reflected that in human tracheobronchial tissue. Nonciliated cells present a higher proportion of alpha-2,6-linked sialic acid, while ciliated cells possess both sialic acid linkages. Although we found that human influenza viruses infected both ciliated and nonciliated cell types in the first round of infection, recent human H3N2 viruses infected a higher proportion of nonciliated cells in HAE than a 1968 pandemic-era human virus, which infected proportionally more ciliated cells. In contrast, avian influenza viruses exclusively infected ciliated cells. Although a broad-range neuraminidase abolished infection of HAE by human parainfluenza virus type 3, this treatment did not significantly affect infection by influenza viruses. All human viruses replicated efficiently in HAE, leading to accumulation of nascent virus released from the apical surface between 6 and 24 h postinfection with a low multiplicity of infection. Avian influenza A viruses also infected HAE, but spread was limited compared to that of human viruses. The nonciliated cell tropism of recent human H3N2 viruses reflects a preference for the sialic acid linkages displayed on these cell types and suggests a drift in the receptor binding phenotype of the H3 hemagglutinin protein as it evolves in humans away from its avian virus precursor.

Detecting the node-density artifact in phylogeny reconstruction

The node-density effect is an artifact of phylogeny reconstruction that can cause branch lengths to be underestimated in areas of the tree with fewer taxa. Webster, Payne, and Pagel (2003, Science 301:478) introduced a statistical procedure (the "delta" test) to detect this artifact, and here we report the results of computer simulations that examine the test's performance. In a sample of 50,000 random data sets, we find that the delta test detects the artifact in 94.4% of cases in which it is present. When the artifact is not present (n = 10,000 simulated data sets) the test showed a type I error rate of approximately 1.69%, incorrectly reporting the artifact in 169 data sets. Three measures of tree shape or "balance" failed to predict the size of the node-density effect. This may reflect the relative homogeneity of our randomly generated topologies, but emphasizes that nearly any topology can suffer from the artifact, the effect not being confined only to highly unevenly sampled or otherwise imbalanced trees. The ability to screen phylogenies for the node-density artifact is important for phylogenetic inference and for researchers using phylogenetic trees to infer evolutionary processes, including their use in molecular clock dating. [Delta test; molecular clock; molecular evolution; node-density effect; phylogenetic reconstruction; speciation; simulation.]

Post dispersal weed seed predation by avian and non-avian predators

Seed predation by avian and non-avian predators was quantified in the boundaries and cropped areas of cereal fields by presenting known quantities of seed with and without exclusion cages. Predator encounter-rates with the dishes exceeded 99%. Birds removed on average 6.7% seed from the dishes during the seven-day trials compared to 51% by non-avian predators. A comparison was made of the causal factors responsible for predation of Avena fatua, Chenopodium album and Cirsium arvense seeds. A. fatua seeds were preyed most heavily by both avian and non-avian predators. Seed removal by birds was greater in the cropped area than in the field boundary, non-avian predators being generally more active in the field boundary. Seed predation by birds was greater in spring than in any other season, whilst losses to other animals were greater during autumn and winter. Although, birds were not the main seed predators in cereal fields, they may contribute to weed seed depletion, of relevance to reduced-input farming systems where herbicides use is restricted.

Phylogeny of Pelargonium (Geraniaceae) based on DNA sequences from three genomes

Phylogenetic hypotheses for the largely South African genus Pelargonium L'Hér. (Geraniaceae) were derived based on DNA sequence data from nuclear, chloroplast and mitochondrial encoded regions. The datasets were unequally represented and comprised cpDNA trnL-F sequences for 152 taxa, nrDNA ITS sequences for 55 taxa, and mtDNA nad1 b/c exons for 51 taxa. Phylogenetic hypotheses derived from the separate three datasets were overall congruent. A single hypothesis synthesising the information in the three datasets was constructed following a total evidence approach and implementing dataset specific stepmatrices in order to correct for substitution biases. Pelargonium was found to consist of five main clades, some with contrasting evolutionary patterns with respect to biogeographic distributions, dispersal capacity, pollination biology and karyological diversification. The five main clades are structured in two (subgeneric) clades that correlate with chromosome size. One of these clades includes a "winter rainfall clade" containing more than 70% of all currently described Pelargonium species, and all restricted to the South African Cape winter rainfall region. Apart from (woody) shrubs and small herbaceous rosette subshrubs, this clade comprises a large "xerophytic" clade including geophytes, stem and leaf succulents, harbouring in total almost half of the genus. This clade is considered to be the result of in situ proliferation, possibly in response to late-Miocene and Pliocene aridification events. Nested within it is a radiation comprising c. 80 species from the geophytic Pelargonium section Hoarea, all characterised by the possession of (a series of) tunicate tubers.