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MG-RAST (http://metagenomics.anl.gov) is an open-submission data portal for processing, analyzing, sharing and disseminating metagenomic datasets. The system currently hosts over 200,000 datasets and is continuously updated. The volume of submissions has increased 4-fold over the past 24 months, now averaging 4 terabasepairs per month. In addition to several new features, we report changes to the analysis workflow and the technologies used to scale the pipeline up to the required throughput levels. To show possible uses for the data from MG-RAST, we present several examples integrating data and analyses from MG-RAST into popular third-party analysis tools or sequence alignment tools.

As technologies change, MG-RAST is adapting. Newly available software is being included to improve accuracy and performance. As a computational service constantly running large volume scientific workflows, MG-RAST is the right location to perform benchmarking and implement algorithmic or platform improvements, in many cases involving trade-offs between specificity, sensitivity and run-time cost. The work in [Glass EM, Dribinsky Y, Yilmaz P, et al. ISME J 2014;8:1–3] is an example; we use existing well-studied data sets as gold standards representing different environments and different technologies to evaluate any changes to the pipeline. Currently, we use well-understood data sets in MG-RAST as platform for benchmarking. The use of artificial data sets for pipeline performance optimization has not added value, as these data sets are not presenting the same challenges as real-world data sets. In addition, the MG-RAST team welcomes suggestions for improvements of the workflow. We are currently working on versions 4.02 and 4.1, both of which contain significant input from the community and our partners that will enable double barcoding, stronger inferences supported by longer-read technologies, and will increase throughput while maintaining sensitivity by using Diamond and SortMeRNA. On the technical platform side, the MG-RAST team intends to support the Common Workflow Language as a standard to specify bioinformatics workflows, both to facilitate development and efficient high-performance implementation of the community’s data analysis tasks.

Summary We reconstructed the complete 2.4 Mb-long genome of a previously uncultivated epsilonproteobacterium, Candidatus Sulfuricurvum sp. RIFRC-1, via assembly of short-read shotgun metagenomic data using a complexity reduction approach. Genome-based comparisons indicate the bacterium is a novel species within the Sulfuricurvum genus, which contains one cultivated representative, S. kujiense. Divergence between the species appears due in part to extensive genomic rearrangements, gene loss and chromosomal versus plasmid encoding of certain (respiratory) genes by RIFRC-1. Deoxyribonucleic acid for the genome was obtained from terrestrial aquifer sediment, in which RIFRC-1 comprised ∼ 47% of the bacterial community. Genomic evidence suggests RIFRC-1 is a chemolithoautotrophic diazotroph capable of deriving energy for growth by microaerobic or nitrate-/nitric oxide-dependent oxidation of S0, sulfide or sulfite or H2 oxidation. Carbon may be fixed via the reductive tricarboxylic acid cycle. Consistent with these physiological attributes, the local aquifer was microoxic with small concentrations of available nitrate, small but elevated concentrations of reduced sulfur and NH4+/NH3-limited. Additionally, various mechanisms for heavy metal and metalloid tolerance and virulence point to a lifestyle well-adapted for metal(loid)-rich environments and a shared evolutionary past with pathogenic Epsilonproteobacteria. Results expand upon recent findings highlighting the potential importance of sulfur and hydrogen metabolism in the terrestrial subsurface.

A novel type of Baeyer-Villiger monooxygenase (BVMO) has been found in a marine strain of Stenotrophomonas maltophila strain PML168 that was isolated from a temperate intertidal zone. The enzyme is able to use NADH as the source of reducing power necessary to accept the atom of diatomic oxygen not incorporated into the oxyfunctionalized substrate. Growth studies have establish that the enzyme is inducible, appears to serve a catabolic role, and is specifically induced by one or more unidentified components of seawater as well as various anthropogenic xenobiotic compounds. A blast search of the primary sequence of the enzyme, recovered from the genomic sequence of the isolate, has placed this atypical BVMO in the context of the several hundred known members of the flavoprotein monooxygenase superfamily. A particular feature of this BVMO lies in its truncated C-terminal domain, which results in a relatively small protein (357 amino acids; 38.4 kDa). In addition, metagenomic screening has been conducted on DNA recovered from an extensive range of marine environmental samples to gauge the relative abundance and distribution of similar enzymes within the global marine microbial community. Although low, abundance was detected in samples from many marine provinces, confirming the potential for biodiscovery in marine microorganisms.

Permanent electric dipole moments (EDMs) in atoms or molecules are a signature of time-reversal and parity violation and represent an important window onto physics beyond the Standard Model. We are developing a next generation EDM search based on laser-cooled and trapped {sup 225}Ra atoms. Due to octupole deformation of the nucleus, {sup 225}Ra is predicted to be two to three orders of magnitude more sensitive to T-violating interactions than {sup 199}Hg, which currently sets the most stringent limits in the nuclear sector. We will discuss progress toward realizing a first EDM measurement for {sup 225}Ra.

Metagenomics, shotgun metagenomics in particular, is a field that has disproportionately benefited from advances in high-throughput sequencing technologies. This chapter discusses the choice of sample number, depth, and sequencing technology, the shearing, size-selection, and multiplexing options on Illumina platforms, and outlines the steps in the bioinformatic analysis and sharing of metagenomic data.

Although plasmids and other episomes are recognized as key players in horizontal gene transfer among microbes, their diversity and dynamics among ecologically structured host populations in the wild remain poorly understood. Here, we show that natural populations of marine Vibrionaceae bacteria host large numbers of families of episomes, consisting of plasmids and a surprisingly high fraction of plasmid-like temperate phages. Episomes are unevenly distributed among host populations, and contrary to the notion that high-density communities in biofilms act as hot spots of gene transfer, we identified a strong bias for episomes to occur in free-living as opposed to particle-attached cells. Mapping of episomal families onto host phylogeny shows that, with the exception of all phage and a few plasmid families, most are of recent evolutionary origin and appear to have spread rapidly by horizontal transfer. Such high eco-evolutionary turnover is particularly surprising for plasmids that are, based on previously suggested categorization, putatively nontransmissible, indicating that this type of plasmid is indeed frequently transferred by currently unknown mechanisms. Finally, analysis of recent gene transfer among plasmids reveals a network of extensive exchange connecting nearly all episomes. Genes functioning in plasmid transfer and maintenance are frequently exchanged, suggesting that plasmids can be rapidly transformed from one category to another. The broad distribution of episomes among distantly related hosts and the observed promiscuous recombination patterns show how episomes can offer their hosts rapid assembly and dissemination of novel functions., IMPORTANCE Plasmids and other episomes are an integral part of bacterial biology in all environments, yet their study is heavily biased toward their role as vectors for antibiotic resistance genes. This study presents a comprehensive analysis of all episomes within several coexisting bacterial populations of Vibrionaceae from the coastal ocean and represents the largest-yet genomic survey of episomes from a single bacterial family. The host population framework allows analysis of the eco-evolutionary dynamics at unprecedented resolution, yielding several unexpected results. These include (i) discovery of novel, nonintegrative temperate phages, (ii) revision of a class of episomes, previously termed “nontransmissible,” as highly transmissible, and (iii) surprisingly high evolutionary turnover of episomes, manifest as frequent birth, spread, and loss.

The heavy neutron-rich isotopes area is the least explored region of the nuclear landscape. Although sensitive techniques exist to gather the required information on these isotopes, they just have not been made available in sufficient quantity and with the right properties for many of the most basic studies. Recent measurements at the Canadian Penning trap (CPT) mass spectrometer, using isotopes produced from the fission of Cf 252 stopped in the CPT gas catcher system, have allowed the mass of a number of neutron-rich isotopes to be determined. This approach is being further pursued in the CARIBU project with the installation of a new dedicated heavily shielded source and gas catcher that will yield four orders of magnitude higher neutron-rich isotope yield at low energy for mass measurements and Coulomb barrier energy for nuclear structure studies.

The processes responsible for the creation of elements more massive than iron are not well understood. Possible production mechanisms involve the rapid capture of protons (rp-process) or the rapid capture of neutrons (r-process), which are thought to occur in explosive astrophysical events such as novae, x-ray bursts, and supernovae. Mass measurements of the nuclides involved with uncertainties on the order of 100 keV or better are critical to determine the process ‘paths’, the energy output of the events, and the resulting nuclide abundances. Particularly important are the masses of ‘waiting-point’ nuclides along the rp-process path where the process stalls until the subsequent β decay of the nuclides. This paper will discuss the precise mass measurements made of isotopes along the rp-process and r-process paths using the Canadian Penning Trap mass spectrometer, including the mass of the critical waiting-point nuclide 68 Se.

The Canadian Penning Trap (CPT) mass spectrometer located at the ATLAS facility of Argonne National Laboratory is an online Penning trap system used for mass measurements of high accuracy on short-lived isotopes. It uses a gas catcher as a novel way to transform radioactive ions efficiently from different sources into cooled beams which are injected into ion traps for further preparation and measurement. The CPT has recently been successfully used to make precise mass measurements on over 40 neutron-rich and neutron-deficient radioactive nuclides related to astrophysical processes.

A new approach to the production of low energy radioactive beams involves the stopping of fast beams produced by fragmentation or in-flight fission in a large gas catcher where the reaction products are thermalized in high-purity helium and extracted as singly charged ions for post-acceleration. This removes the limitation present in standard ISOL techniques for species that are difficult to extract from the target/ion source assembly. This approach will be implemented in the RIA facility. The demonstration of this new technology is a key R&D goal for RIA and a full scale prototype was constructed and tested, both off-line and on-line, at the ATLAS facility at Argonne. The basic operating principles, techniques used for the construction, and results obtained in off-line and on-line tests are presented. The large gas catcher prototype has now been transported to GSI, where fragmentation products at the full RIA energy are available, for its final test.

Recent advances in next-generation sequencing have enabled high-throughput determination of biological sequences in microbial communities, also known as microbiomes. The large volume of data now presents the challenge of how to extract knowledge—recognize patterns, find similarities, and find relationships—from complex mixtures of nucleic acid sequences currently being examined. In this chapter we review basic concepts as well as state-of-the-art techniques to analyze hundreds of samples which each contain millions of DNA and RNA sequences. We describe the general character of sequence data and describe some of the processing steps that prepare raw sequence data for inference. We then describe the process of extracting features from the data, assigning taxonomic and gene labels to the sequences. Then we review methods for cross-sample comparisons: (1) using similarity measures and ordination techniques to visualize and measure differences between samples and (2) feature selection and classification to select the most relevant features for discriminating between samples.

We reconstructed the complete 2.4 Mb-long genome of a previously uncultivated epsilonproteobacterium, Candidatus Sulfuricurvum sp. RIFRC-1, via assembly of short-read shotgun metagenomic data using a complexity reduction approach. Genome-based comparisons indicate the bacterium is a novel species within the Sulfuricurvum genus, which contains one cultivated representative, S. kujiense. Divergence between the species appears due in part to extensive genomic rearrangements, gene loss and chromosomal versus plasmid encoding of certain (respiratory) genes by RIFRC-1. Deoxyribonucleic acid for the genome was obtained from terrestrial aquifer sediment, in which RIFRC-1 comprised ∼ 47% of the bacterial community. Genomic evidence suggests RIFRC-1 is a chemolithoautotrophic diazotroph capable of deriving energy for growth by microaerobic or nitrate-/nitric oxide-dependent oxidation of S°, sulfide or sulfite or H₂oxidation. Carbon may be fixed via the reductive tricarboxylic acid cycle. Consistent with these physiological attributes, the local aquifer was microoxic with small concentrations of available nitrate, small but elevated concentrations of reduced sulfur and NH(4)(+) /NH₃-limited. Additionally, various mechanisms for heavy metal and metalloid tolerance and virulence point to a lifestyle well-adapted for metal(loid)-rich environments and a shared evolutionary past with pathogenic Epsilonproteobacteria. Results expand upon recent findings highlighting the potential importance of sulfur and hydrogen metabolism in the terrestrial subsurface.

The democratized world of sequencing is leading to numerous data analysis challenges; MG-RAST addresses many of these challenges for diverse datasets, including amplicon datasets, shotgun metagenomes, and metatranscriptomes. The changes from version 2 to version 3 include the addition of a dedicated gene calling stage using FragGenescan, clustering of predicted proteins at 90% identity, and the use of BLAT for the computation of similarities. Together with changes in the underlying software infrastructure, this has enabled the dramatic scaling up of pipeline throughput while remaining on a limited hardware budget. The Web-based service allows upload, fully automated analysis, and visualization of results. As a result of the plummeting cost of sequencing and the readily available analytical power of MG-RAST, over 78,000 metagenomic datasets have been analyzed, with over 12,000 of them publicly available in MG-RAST.

The democratized world of sequencing is leading to numerous data analysis challenges; MG-RAST addresses many of these challenges for diverse datasets, including amplicon datasets, shotgun metagenomes, and metatranscriptomes. The changes from version 2 to version 3 include the addition of a dedicated gene calling stage using FragGenescan, clustering of predicted proteins at 90% identity, and the use of BLAT for the computation of similarities. Together with changes in the underlying software infrastructure, this has enabled the dramatic scaling up of pipeline throughput while remaining on a limited hardware budget. The Web-based service allows upload, fully automated analysis, and visualization of results. As a result of the plummeting cost of sequencing and the readily available analytical power of MG-RAST, over 78,000 metagenomic datasets have been analyzed, with over 12,000 of them publicly available in MG-RAST.

We demonstrate a technique for transferring ${}^{226}$Ra atoms from a three-dimensional magneto-optical trap (MOT) into a standing wave optical dipole trap (ODT) in an adjacent chamber. The resulting small trapping volume (120 $\ensuremath{\mu}$m in diameter) allows for high control of the electric and magnetic fields applied to the atoms. The atoms are first transferred to a traveling-wave optical dipole trap, which is then translated 46 cm to a science chamber. The atoms are subsequently transferred into an orthogonal standing-wave ODT by application of a one-dimensional MOT along the traveling-wave axis. For each stage, transfer efficiencies exceeding 60$%$ are demonstrated.

Meta-omics approaches such as metagenomics, metatranscriptomics and metaproteogenomics have the potential to improve our understanding of how the human microbiome affects digestive health and disease.

Background Gene prediction algorithms (or gene callers) are an essential tool for analyzing shotgun nucleic acid sequence data. Gene prediction is a ubiquitous step in sequence analysis pipelines; it reduces the volume of data by identifying the most likely reading frame for a fragment, permitting the out-of-frame translations to be ignored. In this study we evaluate five widely used ab initio gene-calling algorithms—FragGeneScan, MetaGeneAnnotator, MetaGeneMark, Orphelia, and Prodigal—for accuracy on short (75–1000 bp) fragments containing sequence error from previously published artificial data and “real” metagenomic datasets. Results While gene prediction tools have similar accuracies predicting genes on error-free fragments, in the presence of sequencing errors considerable differences between tools become evident. For error-containing short reads, FragGeneScan finds more prokaryotic coding regions than does MetaGeneAnnotator, MetaGeneMark, Orphelia, or Prodigal. This improved detection of genes in error-containing fragments, however, comes at the cost of much lower (50%) specificity and overprediction of genes in noncoding regions. Conclusions Ab initio gene callers offer a significant reduction in the computational burden of annotating individual nucleic acid reads and are used in many metagenomic annotation systems. For predicting reading frames on raw reads, we find the hidden Markov model approach in FragGeneScan is more sensitive than other gene prediction tools, while Prodigal, MGA, and MGM are better suited for higher-quality sequences such as assembled contigs.

The most feared complication following intestinal resection is anastomotic leakage. In high risk areas (esophagus/rectum) where neoadjuvant chemoradiation is used, the incidence of anastomotic leaks remains unacceptably high (≈ 10%) even when performed by specialist surgeons in high volume centers. The aims of this study were to test the hypothesis that anastomotic leakage develops when pathogens colonizing anastomotic sites become in vivo transformed to express a tissue destroying phenotype. We developed a novel model of anastomotic leak in which rats were exposed to pre-operative radiation as in cancer surgery, underwent distal colon resection and then were intestinally inoculated with Pseudomonas aeruginosa, a common colonizer of the radiated intestine. Results demonstrated that intestinal tissues exposed to preoperative radiation developed a significant incidence of anastomotic leak (>60%; p

Halomonas strain GFAJ-1 was reported in Science magazine to be a remarkable microbe for which there was “arsenate in macromolecules that normally contain phosphate, most notably nucleic acids.” The draft genome of the bacterium was determined (NCBI accession numbers AHBC01000001 through AHBC01000103 ). It appears to be a typical gamma proteobacterium.

We provide a novel method, DRISEE (duplicate read inferred sequencing error estimation), to assess sequencing quality (alternatively referred to as “noise” or “error”) within and/or between sequencing samples. DRISEE provides positional error estimates that can be used to inform read trimming within a sample. It also provides global (whole sample) error estimates that can be used to identify samples with high or varying levels of sequencing error that may confound downstream analyses, particularly in the case of studies that utilize data from multiple sequencing samples. For shotgun metagenomic data, we believe that DRISEE provides estimates of sequencing error that are more accurate and less constrained by technical limitations than existing methods that rely on reference genomes or the use of scores (e.g. Phred). Here, DRISEE is applied to (non amplicon) data sets from both the 454 and Illumina platforms. The DRISEE error estimate is obtained by analyzing sets of artifactual duplicate reads (ADRs), a known by-product of both sequencing platforms. We present DRISEE as an open-source, platform-independent method to assess sequencing error in shotgun metagenomic data, and utilize it to discover previously uncharacterized error in de novo sequence data from the 454 and Illumina sequencing platforms., Author Summary Sequence quality (referred to alternatively as the level of sequencing error or noise) is a primary concern to all sequence-dependent investigations. This is particularly true in the field of metagenomics where automated tools (e.g. annotation pipelines like MG-RAST) rely on high fidelity sequence data to derive meaningful biological inferences, and is exacerbated by the capacity of next generation sequencing platforms that continue to expand at a rate greater than Moore's law. We demonstrate that the most commonly utilized means to assess sequencing error exhibit severe limitations with respect to analysis of metagenomic data. Furthermore, we introduce a method (DRISEE) that accounts for these limitations through the application of a novel approach to assess sequencing error. DRISEE-based analyses reveal previously unobserved levels of sequencing error. DRISEE provides a platform independent measure of sequencing error that objectively assesses the quality of entire sequence samples. This assessment can be used to exclude low quality samples from computationally expensive analyses (e.g. annotation). It can also be used to evaluate the relative fidelity of analyses after they have been performed (e.g. annotation of error prone samples is less reliable than that of samples with low levels of sequencing error).

The lifetime of the $7s6d$ ${^{1}D}_{2}$ state of atomic radium is determined to be $385(45)\text{ }\ensuremath{\mu}\text{s}$ using cold $^{226}\text{R}\text{a}$ atoms prepared in a magneto-optical trap. The ${^{1}D}_{2}$ state is populated from the decay of the ${^{1}P}_{1}$ state which is excited by a pulse of 483 nm light. The decay of the ${^{1}D}_{2}$ state is observed by detecting delayed fluorescence at 714 nm from the last step in the decay sequence ${^{1}P}_{1}\text{\ensuremath{-}}{^{1}D}_{2}\text{\ensuremath{-}}{^{3}P}_{1}\text{\ensuremath{-}}{^{1}S}_{0}$. The measured lifetime is compared to a number of theoretical calculations. An improved value of the $7s7p$ ${^{1}P}_{1}$ level of $20\text{ }715.598(6)\text{ }{\text{cm}}^{\ensuremath{-}1}$ is obtained.

We report progress on 115In+ and 137Ba+ single ion optical frequency standards using all solid-state sources. Both are free from quadrupole field shifts and together enable a search for drift in fundamental constants., 2 pages, 1 figure, submitted to IEEE/LEOS Summer 2005 Topicals conference proceedings

International audience; The Canadian Penning trap (CPT) mass spectrometer at the Argonne National Laboratory makes precise mass measurements of nuclides with short half-lives. Since the previous ENAM conference, many significant modifications to the apparatus were implemented to improve both the precision and efficiency of measurement, and now more than 60 radioactive isotopes have been measured with half-lives as short as one second and with a precision ( Δm/m) approaching 10-8. The CPT mass measurement program has concentrated so far on nuclides of importance to astrophysics. In particular, measurements have been obtained of isotopes along the rp-process path, in which energy is released from a series of rapid proton-capture reactions. An X-ray burst is one possible site for the rp-process mechanism which involves the accretion of hydrogen and helium from one star onto the surface of its neutron star binary companion. Mass measurements are required as key inputs to network calculations used to describe the rp-process in terms of the abundances of the nuclides produced, the light-curve profile of the X-ray bursts, and the energy produced. This paper will present the precise mass measurements made along the rp-process path with particular emphasis on the "waiting-point" nuclides 68Se and 64Ge.

The masses of the radioactive nuclei {sup 22}Mg and {sup 22}Na have been measured with the Canadian Penning trap on-line mass spectrometer to a precision of 3x10{sup -8} and 1x10{sup -8}, respectively. A Q{sub EC} value of 4124.39(73) keV for the superallowed {beta} decay of {sup 22}Mg is obtained from the difference of these two masses. With this precise Q value, the Ft value for this decay is determined with improved precision and is found to be consistent with the existing precision data set of superallowed Fermi emitters. This provides an important test of the isospin symmetry-breaking corrections. If the mass of {sup 22}Mg determined here is used in the calibration of a recent {sup 21}Na(p,{gamma}){sup 22}Mg measurement, part of the discrepancy observed in that measurement is removed.

Mass measurements of $^{68}\mathrm{Ge}$, $^{68}\mathrm{As}$, and $^{68}\mathrm{Se}$ have been obtained with the Canadian Penning Trap mass spectrometer. The results determine the mass excess of $^{68}\mathrm{Se}$ as $\ensuremath{-}54\text{ }232(19)\text{ }\text{ }\mathrm{keV}$, the first measurement with a precision and reliability sufficient to address the light-curve and energy output of x-ray bursts as well as the abundances of the elements synthesized. Under typical conditions used for modeling x-ray bursts, $^{68}\mathrm{Se}$ is found to cause a significant delay in the rp process nucleosynthesis.

Agrobacterium albertimagni strain AOL15 is an alphaproteobacterium isolated from arsenite-oxidizing biofilms whose draft genome contains 5.1 Mb in 55 contigs with 61.2% GC content and includes a 21-gene arsenic gene island. This is the first available genome for this species and the second Agrobacterium arsenic gene island.

We present the genomic sequence of the human pathogen Legionella pneumophila serogroup 12 strain 570-CO-H (ATCC 43290), a clinical isolate from the Colorado Department of Health, Denver, CO. This is the first example of a genome sequence of L. pneumophila from a serogroup other than serogroup 1. We highlight the similarities and differences relative to six genome sequences that have been reported for serogroup 1 strains.

Achromobacter piechaudii strain HLE is a betaproteobacterium (previously known as Alcaligenes faecalis ) that was an early isolate with arsenite oxidase activity. This draft genome of 6.89 Mb is the second available genome for this species in the opportunistic pathogen Alcaligenaceae family.

Alcaligenes faecalissubsp.faecalisNCIB 8687, the betaproteobacterium from which arsenite oxidase had its structure solved and the first “arsenate gene island” identified, provided a draft genome of 3.9 Mb in 186 contigs (with the largest 15 comprising 90% of the total) for this opportunistic pathogen species.

The field of metagenomics is transforming our ability to study the enormous biomass and diversity of microbial life around us. Understanding this microbial world will lead to advances and practical applications in a broad range of fields. Metagenomic sequencing, provides unprecedented access to the thousands (or even millions) of microbes in an environment. Unlike 16S SSU rRNA amplicon sequencing, metagenomic sequencing (whole shotgun sequencing) provides information on not only who is in a community but what they are doing, extending understanding of community structure towards interactions within an environment. This talk will discuss the MG-RAST analysis pipeline starting from quality control assessment to annotation and an overview the interactive tools for comparative analysis. MG-RAST has analyzed over 60,000 WGS and amplicon datasets equaling approximately 20 Tbp.

BackgroundThe numerous classes of repeats often impede the assembly of genome sequences from the short reads provided by new sequencing technologies. We demonstrate a simple and rapid means to ascertain the repeat structure and total size of a bacterial or archaeal genome without the need for assembly by directly analyzing the abundances of distinctk-mers among reads.ResultsThe sensitivity of this procedure to resolve variation within a bacterial species is demonstrated: genome sizes and repeat structure of five environmental strains ofE. colifrom shortIlluminareads were estimated by this method, and total genome sizes corresponded well with those obtained for the same strains by pulsed-field gel electrophoresis. In addition, this approach was applied to read-sets for completed genomes and shown to be accurate over a wide range of microbial genome sizes.ConclusionsApplication of these procedures, based solely onk-mer abundances in short read data sets, allows aspects of genome structure to be resolved that are not apparent from conventional short read assemblies. This knowledge of the repetitive content of genomes provides insights into genome evolution and diversity.