The genome of Rhizobium leguminosarum has recognizable core and accessory components

Background: Rhizobium leguminosarum is an alpha-proteobacterial N-2-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841. Results: The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes ( Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were overrepresented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens. Conclusion: Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.

Predicting functional gene links from phylogenetic-statistical analyses of whole genomes

An important element of the developing field of proteomics is to understand protein-protein interactions and other functional links amongst genes. Across-species correlation methods for detecting functional links work on the premise that functionally linked proteins will tend to show a common pattern of presence and absence across a range of genomes. We describe a maximum likelihood statistical model for predicting functional gene linkages. The method detects independent instances of the correlated gain or loss of pairs of proteins on phylogenetic trees, reducing the high rates of false positives observed in conventional across-species methods that do not explicitly incorporate a phylogeny. We show, in a dataset of 10,551 protein pairs, that the phylogenetic method improves by up to 35% on across-species analyses at identifying known functionally linked proteins. The method shows that protein pairs with at least two to three correlated events of gain or loss are almost certainly functionally linked. Contingent evolution, in which one gene's presence or absence depends upon the presence of another, can also be detected phylogenetically, and may identify genes whose functional significance depends upon its interaction with other genes. Incorporating phylogenetic information improves the prediction of functional linkages. The improvement derives from having a lower rate of false positives and from detecting trends that across-species analyses miss. Phylogenetic methods can easily be incorporated into the screening of large-scale bioinformatics datasets to identify sets of protein links and to characterise gene networks.

A phylogenetic reappraisal of the Peltophorum group (Caesalpinieae: Leguminosae) based on the chloroplast trnL-F, rbcL and rps16 sequence data

The monophyly of the Peltophorum group, one of nine informal groups recognized by Polhill in the Caesalpinieae, was tested using sequence data from the trnL-F, rbcL, and rps16 regions of the chloroplast genome. Exemplars were included from all 16 genera of the Peltophorum group, and from 15 genera representing seven of the other eight informal groups in the tribe. The data were analyzed separately and in combined analyses using parsimony and Bayesian methods. The analysis method had little effect on the topology of well-supported relationships. The molecular data recovered a generally well-supported phylogeny with many intergeneric relationships resolved. Results show that the Peltophorum group as currently delimited is polyphyletic, but that eight genera plus one undescribed genus form a core Peltophorum group, which is referred to here as the Peltophorum group sensu stricto. These genera are Bussea, Conzattia, Colvillea, Delonix, Heteroflorum (inedit.), Lemuropisum, Parkinsonia, Peltophorum, and Schizolobium. The remaining eight genera of the Peltophorum group s.l. are distributed across the Caesalpinieae. Morphological support for the redelimited Peltophorum group and the other recovered clades was assessed, and no unique synapomorphy was found for the Peltophorum group s.s. A proposal for the reclassification of the Peltophorum group s.l. is presented.

Bayesian estimation of ancestral character states on phylogenies

Biologists frequently attempt to infer the character states at ancestral nodes of a phylogeny from the distribution of traits observed in contemporary organisms. Because phylogenies are normally inferences from data, it is desirable to account for the uncertainty in estimates of the tree and its branch lengths when making inferences about ancestral states or other comparative parameters. Here we present a general Bayesian approach for testing comparative hypotheses across statistically justified samples of phylogenies, focusing on the specific issue of reconstructing ancestral states. The method uses Markov chain Monte Carlo techniques for sampling phylogenetic trees and for investigating the parameters of a statistical model of trait evolution. We describe how to combine information about the uncertainty of the phylogeny with uncertainty in the estimate of the ancestral state. Our approach does not constrain the sample of trees only to those that contain the ancestral node or nodes of interest, and we show how to reconstruct ancestral states of uncertain nodes using a most-recent-common-ancestor approach. We illustrate the methods with data on ribonuclease evolution in the Artiodactyla. Software implementing the methods ( BayesMultiState) is available from the authors.

Limpets break Dollo's law

A new molecular phylogeny of the limpet molluscs (Calyptraeidae) reveals that coiled shells have independently re-evolved at least once in this family, which is a violation of Dollo's Law that complex ancestral states, once lost, are never reacquired. Reacquisition of the coiled ancestral state is remarkable in that uncoiled shells have been the most recent ancestral state for 20 million-100 million years. Adult coiling might have reevolved by the mechanism of prolonging the period during which genes for coiling are expressed in larvae. This and other developmental mechanisms could provide general routes for maintaining the potential to produce traits lost in distant ancestors.

Phylogenomics of eukaryotes: Impact of missing data on large alignments

Resolving the relationships between Metazoa and other eukaryotic groups as well as between metazoan phyla is central to the understanding of the origin and evolution of animals. The current view is based on limited data sets, either a single gene with many species (e.g., ribosomal RNA) or many genes but with only a few species. Because a reliable phylogenetic inference simultaneously requires numerous genes and numerous species, we assembled a very large data set containing 129 orthologous proteins (similar to30,000 aligned amino acid positions) for 36 eukaryotic species. Included in the alignments are data from the choanoflagellate Monosiga ovata, obtained through the sequencing of about 1,000 cDNAs. We provide conclusive support for choanoflagellates as the closest relative of animals and for fungi as the second closest. The monophyly of Plantae and chromalveolates was recovered but without strong statistical support. Within animals, in contrast to the monophyly of Coelomata observed in several recent large-scale analyses, we recovered a paraphyletic Coelamata, with nematodes and platyhelminths nested within. To include a diverse sample of organisms, data from EST projects were used for several species, resulting in a large amount of missing data in our alignment (about 25%). By using different approaches, we verify that the inferred phylogeny is not sensitive to these missing data. Therefore, this large data set provides a reliable phylogenetic framework for studying eukaryotic and animal evolution and will be easily extendable when large amounts of sequence information become available from a broader taxonomic range.

Cross-species amplification and phylogenetic reconstruction using fragaria microsatellite primers

Twenty-eight microsatellite primer pairs developed from Fragaria vesca ‘Rügen’ were applied to sixteen accessions representing eight diploid Fragaria species. The number of alleles generated, the power of discrimination and the percentage of accessions where no PCR product could be amplified were calculated for each locus for the thirteen non-F. vesca accessions. A phylogeny was then generated for the species accessions sampled, using the presence or absence of alleles at the polymorphic loci as character states. Despite the problems inherent in phylogeny reconstruction from microsatellite data, the phylogeny showed some congruence with a previously published phylogeny of Fragaria, based on nucleotide sequence data. However, relationships inferred from microsatellite allele data were relatively unresolved and poorly supported. The genetic basis of allelic polymorphisms at specific loci was investigated through direct sequencing of the PCR products amplified by three primer pairs. The potential utility of sequence data generated from microsatellite loci in evolutionary studies of closely related species groups is briefly explored.

Comparative genome analysis provides insights into the evolution and adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum.

A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree.

Sex allocation and local mate competition in Old World non-pollinating fig wasps

The populations of many species are structured such that mating is not random and occurs between members of local patches. When patches are founded by a single female and all matings occur between siblings, brothers may compete with each other for matings with their sisters. This local mate competition (LMC) selects for a female-biased sex ratio, especially in species where females have control over offspring sex, as in the parasitic Hymenoptera. Two factors are predicted to decrease the degree of female bias: (1) an increase in the number of foundress females in the patch and (2) an increase in the fraction of individuals mating after dispersal from the natal patch. Pollinating fig wasps are well known as classic examples of species where all matings occur in the local patch. We studied non-pollinating fig wasps, which are more diverse than the pollinating fig wasps and also provide natural experimental groups of species with different male morphologies that are linked to different mating structures. In this group of wasps, species with wingless males mate in the local patch (i.e. the fig fruit) while winged male species mate after dispersal. Species with both kinds of male have a mixture of local and non-local mating. Data from 44 species show that sex ratios (defined as the proportion of males) are in accordance with theoretical predictions: wingless male species < wing-dimorphic male species < winged male species. These results are also supported by a formal comparative analysis that controls for phylogeny. The foundress number is difficult to estimate directly for non-pollinating fig wasps but a robust indirect method leads to the prediction that foundress number, and hence sex ratio, should increase with the proportion of patches occupied in a crop. This result is supported strongly across 19 species with wingless males, but not across 8 species with winged males. The mean sex ratios for species with winged males are not significantly different from 0.5, and the absence of the correlation observed across species with wingless males may reflect weak selection to adjust the sex ratio in species whose population mating structure tends not to be subdivided. The same relationship is also predicted to occur within species if individual females adjust their sex ratios facultatively. This final prediction was not supported by data from a wingless male species, a male wing-dimorphic species or a winged male species.

Multiple diversity measures to identify complementary conservation areas for the Baja California peninsular cacti

The Baja California Peninsula is home to 85 species of cacti, of which 54 are endemic, highlighting its importance as a cactus diverse region within Mexico. Many species are under threat due to collection pressure and habitat loss, but ensuring maximal protection of cacti species requires a better understanding of diversity patterns. We assessed species richness, endemism, and phylogenetic and morphological diversity using herbarium records and a molecular phylogeny for 82 species of cacti found in the peninsula. The four diversity measures were estimated for the existing nature reserve network and for 314 hexagrids of 726 km2. Using the hexagrid data, we surveyed our results for areas that best complement the current protected cacti diversity in the Baja California Peninsula. Currently, the natural reserve network in Baja shelters an important amount of the cacti diversity (74% of the species, 85.9% of the phylogenetic diversity, 76% of endemics and all the growth forms). While species richness produced several solutions to complement the diversity protected, by identifying priority species (endemic species with high contribution to overall PD) one best solution is reported. Three areas (San Matías, Magdalena and Margarita Islands and El Triunfo), selected using species richness, PD and endemism, best complement the diversity currently protected, increasing species richness to 89%, PD to 94% and endemism to 89%, and should be considered in future conservation plans. Two of these areas could be included within nature reserves already established.

Patterns of nucleotide substitution in angiosperm cpDNA trnL (UAA)-trnF (GAA) regions

Patterns of substitution in chloroplast encoded trnL_F regions were compared between species of Actaea (Ranunculales), Digitalis (Scrophulariales), Drosera (Caryophyllales), Panicoideae (Poales), the small chromosome species clade of Pelargonium (Geraniales), each representing a different order of flowering plants, and Huperzia (Lycopodiales). In total, the study included 265 taxa, each with > 900-bp sequences, totaling 0.24 Mb. Both pairwise and phylogeny-based comparisons were used to assess nucleotide substitution patterns. In all six groups, we found that transition/transversion ratios, as estimated by maximum likelihood on most-parsimonious trees, ranged between 0.8 and 1.0 for ingroups. These values occurred both at low sequence divergences, where substitutional saturation, i.e., multiple substitutions having occurred at the same (homologous) nucleotide position, was not expected, and at higher levels of divergence. This suggests that the angiosperm trnL-F regions evolve in a pattern different from that generally observed for nuclear and animal mtDNA (transitional/transversion ratio > or = 2). Transition/transversion ratios in the intron and the spacer region differed in all alignments compared, yet base compositions between the regions were highly similar in all six groups. A>-C transversions were significantly less frequent than the other four substitution types. This correlates with results from studies on fidelity mechanisms in DNA replication that predict A<->T and G<->C transversions to be least likely to occur. It therefore strengthens confidence in the link between mutation bias at the polymerase level and the actual fixation of substitutions as recorded on evolutionary trees, and concomitantly, in the neutrality of nucleotide substitutions as phylogenetic markers.

Comparisons of host mitochondrial, nuclear and endosymbiont bacterial genes reveal cryptic fig wasp species and the effects of Wolbachia on host mtDNA evolution and diversity

Background Figs and fig-pollinating wasp species usually display a highly specific one-to-one association. However, more and more studies have revealed that the "one-to-one" rule has been broken. Co-pollinators have been reported, but we do not yet know how they evolve. They may evolve from insect speciation induced or facilitated by Wolbachia which can manipulate host reproduction and induce reproductive isolation. In addition, Wolbachia can affect host mitochondrial DNA evolution, because of the linkage between Wolbachia and associated mitochondrial haplotypes, and thus confound host phylogeny based on mtDNA. Previous research has shown that fig wasps have the highest incidence of Wolbachia infection in all insect taxa, and Wolbachia may have great influence on fig wasp biology. Therefore, we look forward to understanding the influence of Wolbachia on mitochondrial DNA evolution and speciation in fig wasps. Results We surveyed 76 pollinator wasp specimens from nine Ficus microcarpa trees each growing at a different location in Hainan and Fujian Provinces, China. We found that all wasps were morphologically identified as Eupristina verticillata, but diverged into three clades with 4.22-5.28% mtDNA divergence and 2.29-20.72% nuclear gene divergence. We also found very strong concordance between E. verticillata clades and Wolbachia infection status, and the predicted effects of Wolbachia on both mtDNA diversity and evolution by decreasing mitochondrial haplotypes. Conclusions Our study reveals that the pollinating wasp E. verticillata on F. microcarpa has diverged into three cryptic species, and Wolbachia may have a role in this divergence. The results also indicate that Wolbachia strains infecting E. verticillata have likely resulted in selective sweeps on host mitochondrial DNA.

The ecology of lizard reproductive output

Aim  We provide a new quantitative analysis of lizard reproductive ecology. Comparative studies of lizard reproduction to date have usually considered life-history components separately. Instead, we examine the rate of production (productivity hereafter) calculated as the total mass of offspring produced in a year. We test whether productivity is influenced by proxies of adult mortality rates such as insularity and fossorial habits, by measures of temperature such as environmental and body temperatures, mode of reproduction and activity times, and by environmental productivity and diet. We further examine whether low productivity is linked to high extinction risk. Location  World-wide. Methods  We assembled a database containing 551 lizard species, their phylogenetic relationships and multiple life history and ecological variables from the literature. We use phylogenetically informed statistical models to estimate the factors related to lizard productivity. Results  Some, but not all, predictions of metabolic and life-history theories are supported. When analysed separately, clutch size, relative clutch mass and brood frequency are poorly correlated with body mass, but their product – productivity – is well correlated with mass. The allometry of productivity scales similarly to metabolic rate, suggesting that a constant fraction of assimilated energy is allocated to production irrespective of body size. Island species were less productive than continental species. Mass-specific productivity was positively correlated with environmental temperature, but not with body temperature. Viviparous lizards were less productive than egg-laying species. Diet and primary productivity were not associated with productivity in any model. Other effects, including lower productivity of fossorial, nocturnal and active foraging species were confounded with phylogeny. Productivity was not lower in species at risk of extinction. Main conclusions  Our analyses show the value of focusing on the rate of annual biomass production (productivity), and generally supported associations between productivity and environmental temperature, factors that affect mortality and the number of broods a lizard can produce in a year, but not with measures of body temperature, environmental productivity or diet.

Energetics, lifestyle, and reproduction in birds

Theoretical and empirical studies of life history aim to account for resource allocation to the different components of fitness: survival, growth, and reproduction. The pioneering evolutionary ecologist David Lack [(1968) Ecological Adaptations for Breeding in Birds (Methuen and Co.,London)] suggested that reproductive output in birds reflects adaptation to environmental factors such as availability of food and risk of predation, but subsequent studies have not always supported Lack’s interpretation. Here using a dataset for 980 bird species (Dataset S1), a phylogeny, and an explicit measure of reproductive productivity, we test predictions for how mass-specific productivity varies with body size, phylogeny,and lifestyle traits. We find that productivity varies negatively with body size and energetic demands of parental care and positively with extrinsic mortality. Specifically: (i) altricial species are 50% less productive than precocial species; (ii) species with female-only care of offspring are about 20% less productive than species with other methods of parental care; (iii) nonmigrants are 14% less productive than migrants; (iv) frugivores and nectarivores are about 20% less productive than those eating other foods; and (v) pelagic foragers are 40% less productive than those feeding in other habitats. A strong signal of phylogeny suggests that syndromes of similar life-history traits tend to be conservative within clades but also to have evolved independently in different clades. Our results generally support both Lack’s pioneering studies and subsequent research on avian life history.