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Genetic diversity of closely related free-living microorganisms is widespread and underpins ecosystem functioning, but most evolutionary theories predict that it destabilizes intimate mutualisms. Accordingly, strain diversity is assumed to be highly restricted in intracellular bacteria associated with animals. Here, we sequenced metagenomes and metatranscriptomes of 18 Bathymodiolus mussel individuals from four species, covering their known distribution range at deep-sea hydrothermal vents in the Atlantic. We show that as many as 16 strains of intracellular, sulfur-oxidizing symbionts coexist in individual Bathymodiolus mussels. Co-occurring symbiont strains differed extensively in key functions, such as the use of energy and nutrient sources, electron acceptors and viral defence mechanisms. Most strain-specific genes were expressed, highlighting their potential to affect fitness. We show that fine-scale diversity is pervasive in Bathymodiolus sulfur-oxidizing symbionts, and hypothesize that it may be widespread in low-cost symbioses where the environment, rather than the host, feeds the symbionts.

Eukaryotes are habitats for bacterial organisms where the host colonization and dispersal among individual hosts have consequences for the bacterial ecology and evolution. Vertical symbiont transmission leads to geographic isolation of the microbial population and consequently to genetic isolation of microbiotas from individual hosts. In contrast, the extent of geographic and genetic isolation of horizontally transmitted microbiota is poorly characterized. Here we show that chemosynthetic symbionts of individual Bathymodiolus brooksi mussels constitute genetically isolated populations. The reconstruction of core genome-wide strain sequences from high-resolution metagenomes revealed distinct phylogenetic clades. Nucleotide diversity and strain composition vary along the mussel lifespan and individual hosts show a high degree of genetic isolation. Our results suggest that the uptake of environmental bacteria is a restricted process in B. brooksi, where self-infection of the gill tissue results in serial founder effects during symbiont evolution. We conclude that bacterial colonization dynamics over the host life-cycle is thus an important determinant of population structure and genome evolution of horizontally transmitted symbionts.

Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain Methanoculleus bourgensis E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany. The virus consists of an icosahedral head 60 nm in diameter and a long non-contractile tail of 125 nm in length, which is consistent with the new isolate belonging to the Siphoviridae family. Electron microscopy revealed that Blf4 attaches to the vegetative cells of M. bourgensis E02.3 as well as to cellular appendages. Apart from M. bourgensis E02.3, none of the tested Methanoculleus strains were lysed by Blf4, indicating a narrow host range. The complete 37 kb dsDNA genome of Blf4 contains 63 open reading frames (ORFs), all organized in the same transcriptional direction. For most of the ORFs, potential functions were predicted. In addition, the genome of the host M. bourgensis E02.3 was sequenced and assembled, resulting in a 2.6 Mbp draft genome consisting of nine contigs. All genes required for a hydrogenotrophic lifestyle were predicted. A CRISPR/Cas system (type I-U) was identified with six spacers directed against Blf4, indicating that this defense system might not be very efficient in fending off invading Blf4 virus.

In the Middle Ages, European settlements of all sizes were repeatedly affected by outbreaks of infectious diseases. Some of the outbreaks reached epidemic proportions, the second plague pandemic being the most notorious of all. A Late Medieval mass-burial site next to the Heiligen-Geist-Hospital (HGH) in Lubeck, a city of the Hanseatic League, contained the skeletal remains of more than 800 individuals assumed to have died of an infectious disease, most probably of the plague. However, Lubeck was ravaged by at least six pestilences in the 14 th century alone. We investigated ancient DNA extracts of 92 individuals from the HGH site to determine whether viral or bacterial pathogenic DNA was present that would help identify the cause of death. Metagenomic screening revealed evidence of an infection with Mi>Salmonella enterica subsp. enterica serovar Paratyphi C, suggesting an epidemic outbreak of enteric paratyphoid fever. A full reconstruction was possible for three S. Paratyphi C genomes that showed a close similarity to another strain from 1200 CE Norway. Based on radiocarbon dates, we determined the enteric paratyphoid fever outbreak in Lubeck to have occurred between 1360 and 1400 CE, historical records indicating the year 1367 CE as the most probable date. Our results also showed that the disease victims were people of northern and eastern European descent, confirming that Hanseatic Lubeck was an important trading centre for the Baltic region.

In genome evolution, genetic variants are the source of diversity, which natural selection acts upon. Treatment of human tuberculosis (TB) induces a strong selection pressure for the emergence of antibiotic resistance-conferring variants in the infecting Mycobacterium tuberculosis (MTB) strains. MTB evolution in response to treatment has been intensively studied and mainly attributed to point substitutions. However, the frequency and contribution of insertions and deletions (indels) to MTB genome evolution remains poorly understood. Here, we analyzed a multi-drug resistant MTB outbreak for the presence of high-quality indels and substitutions. We find that indels are significantly enriched in genes conferring antibiotic resistance. Furthermore, we show that indels are inherited during the outbreak and follow a molecular clock with an evolutionary rate of 5.37e-9 indels/site/year, which is 23 times lower than the substitution rate. Inherited indels may co-occur with substitutions in genes along related biological pathways; examples are iron storage and resistance to second-line antibiotics. This suggests that epistatic interactions between indels and substitutions affect antibiotic resistance and compensatory evolution in MTB., Author summary Mycobacterium tuberculosis (MTB) is a human pathogen causing millions of deaths every year. Its genome evolution has been intensively characterized through point substitutions, i.e., nucleotide exchanges that are inherited. Additional mutations are insertions and deletions of nucleotides, termed indels, that can involve few nucleotides up to thousands and even more. Short indels in genes might change the reading frame and disrupt the gene product. Here we show that indels occur frequently in genes causing antibiotic resistance upon disruption suggesting that antibiotic treatment has a strong impact on indel evolution in an MTB outbreak. Furthermore, we show that the molecular clock, i.e., the temporal emergence of variants over time, holds for short indels in MTB genomes. Finally, we observe that indels may co-occur with substitutions in genes along related biological pathways. These results support the notion that indels are important contributors to MTB evolution. We anticipate that including indels in the analyses of MTB outbreaks will improve our understanding of antibiotic resistance evolution.

Cyanophages are characterized by vast genomic diversity and the formation of stable ecotypes over time. The evolution of phage diversity includes vertical processes, such as mutation, and horizontal processes, such as recombination and gene transfer. Here, we study the contribution of vertical and horizontal processes to short-term evolution of marine cyanophages. Analyzing time series data of Synechococcus-infecting Myoviridae ecotypes spanning up to 17 years, we found a high contribution of recombination relative to mutation (r/m) in all ecotypes. Additionally, we found a molecular clock of substitution and recombination in one ecotype, RIM8. The estimated RIM8 evolutionary rates are 2.2 genome-wide substitutions per year (1.275 × 10−5 substitutions/site/year) and 29 genome-wide nucleotide alterations due to recombination per year. We found 26 variable protein families, of which only two families have a predicted functional annotation, suggesting that they are auxiliary metabolic genes with bacterial homologs. A comparison of our rate estimates to other phage evolutionary rate estimates in the literature reveals a negative correlation of phage substitution rates with their genome size. A comparison to evolutionary rates in bacterial organisms further shows that phages have high rates of mutation and recombination compared to their bacterial hosts. We conclude that the increased recombination rate in phages likely contributes to their vast genomic diversity.

Phages are increasingly recognized as important members of host associated microbial communities. While recent studies have revealed vast genomic diversity in the virosphere, the new frontier is to understand how newly discovered phages may affect higher order processes, such as in the context of host-microbe interactions. Here, we aim to understand the tripartite interplay between phages, bacterial symbionts and marine sponges. In a viromics approach, we discover 491 novel viral clusters and show that sponges, as filter-feeding organisms, are distinct viral niches. By using a nested sampling design, we show that each sponge individual of the four species investigated harbours its own unique virome, regardless of the tissue investigated. We further discover a novel, symbiont phage-encoded ankyrin domain-containing protein which appears to be widely spread in phages of many host-associated contexts including human. The ankyrin protein (ANKp) modulates the eukaryotic immune response against bacteria as confirmed in macrophage infection assays. We predict that the role of ANKp in nature is to facilitate co-existence in the tripartite interplay between phages, symbionts and sponges and possibly in many other host-microbe associations.

Genetic diversity of closely-related free-living microbes is widespread and underpins ecosystem functioning, but most evolutionary theories predict that it destabilizes intimate mutualisms. Indeed, symbiont strain diversity has long assumed to be restricted in intracellular bacteria associated with animals. Here, we sequenced the metagenomes and metatranscriptomes of 18Bathymodiolusmussel individuals from four species, covering their known distribution range at deep-sea hydrothermal vents in the Atlantic. We show that as many as 16 strains of intracellular, sulfur-oxidizing symbionts coexist in individualBathymodiolusmussels. Co-occurring symbiont strains differed extensively in key metabolic functions, such as the use of energy and nutrient sources, electron acceptors and viral defense mechanisms. Most strain-specific genes were expressed, highlighting their adaptive potential. We show that fine-scale diversity is pervasive inBathymodiolussymbionts, and hypothesize that it may be widespread in low-cost symbioses where the environment, not the host, feeds the symbionts.

Viruses are ubiquitous in the biosphere and greatly affect the hosts they infect. It is generally accepted that members of every microbial taxon are susceptible to at least one virus, and a plethora of bacterial viruses are known. In contrast, knowledge of the archaeal virosphere is still limited. Here, a novel lytic archaeal virus is described, designated “Drs3”, as well as its host, Methanobacterium formicicum strain Khl10. This hydrogenotrophic methanogenic archaeon and its virus were isolated from the anaerobic digester of an experimental biogas plant in Germany. The tailed virus has an icosahedral head with a diameter of approximately 60 nm and a long non-contractile tail of approximately 230 nm. These structural observations suggest that the new isolate belongs to the family Siphoviridae, but it could not be assigned to an existing genus. Lysis of the host Khl10 was observed 40-44 h after infection. Lysis of the type strain Methanobacterium formicicum DSMZ 1535 was not observed in the presence of Drs3, pointing towards resistance in the type strain or a rather narrow host range of this newly isolated archaeal virus. The complete 37-kb linear dsDNA genome of Drs3 contains 39 open reading frames, only 12 of which show similarity to genes with predicted functions.

The evolution of asexual organisms is driven not only by the inheritance of genetic modification but also by the acquisition of foreign DNA. The contribution of vertical and horizontal processes to genome evolution depends on their rates per year and is quantified by the ratio of recombination to mutation. These rates have been estimated for bacteria; however, no estimates have been reported for phages. Here, we delineate the contribution of mutation and recombination to dsDNA phage genome evolution. We analyzed 34 isolates of the 936 group of Siphoviridae phages using a Lactococcus lactis strain from a single dairy over 29 years. We estimate a constant substitution rate of 1.9 × 10−4 substitutions per site per year due to mutation that is within the range of estimates for eukaryotic RNA and DNA viruses. The reconstruction of recombination events reveals a constant rate of five recombination events per year and 4.5 × 10−3 nucleotide alterations due to recombination per site per year. Thus, the recombination rate exceeds the substitution rate, resulting in a relative effect of recombination to mutation (r/m) of ∼24 that is homogenous over time. Especially in the early transcriptional region, we detect frequent gene loss and regain due to recombination with phages of the 936 group, demonstrating the role of the 936 group pangenome as a reservoir of genetic variation. The observed substitution rate homogeneity conforms to the neutral theory of evolution; hence, the neutral theory can be applied to phage genome evolution and also to genetic variation brought about by recombination.

CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial immune system against foreign DNA. Recognition sequences (spacers) encoded within the CRISPR array mediate the immune reaction in a sequence-specific manner. The known mechanisms for the evolution of CRISPR arrays include spacer acquisition from foreign DNA elements at the time of invasion and array erosion through spacer deletion. Here, we consider the contribution of genetic recombination between homologous CRISPR arrays to the evolution of spacer repertoire. Acquisition of spacers from exogenic arrays via recombination may confer the recipient with immunity against unencountered antagonists. For this purpose, we develop a novel method for the detection of recombination in CRISPR arrays by modeling the spacer order in arrays from multiple strains from the same species. Because the evolutionary signal of spacer recombination may be similar to that of pervasive spacer deletions or independent spacer acquisition, our method entails a robustness analysis of the recombination inference by a statistical comparison to resampled and perturbed data sets. We analyze CRISPR data sets from four bacterial species: two Gammaproteobacteria species harboring CRISPR type I and two Streptococcus species harboring CRISPR type II loci. We find that CRISPR array evolution in Escherichia coli and Streptococcus agalactiae can be explained solely by vertical inheritance and differential spacer deletion. In Pseudomonas aeruginosa, we find an excess of single spacers potentially incorporated into the CRISPR locus during independent acquisition events. In Streptococcus thermophilus, evidence for spacer acquisition by recombination is present in 5 out of 70 strains. Genetic recombination has been proposed to accelerate adaptation by combining beneficial mutations that arose in independent lineages. However, for most species under study, we find that CRISPR evolution is shaped mainly by spacer acquisition and loss rather than recombination. Since the evolution of spacer content is characterized by a rapid turnover, it is likely that recombination is not beneficial for improving phage resistance in the strains under study, or that it cannot be detected in the resolution of intraspecies comparisons.

A novel archaeal lytic virus targeting species of the genus Methanosarcina was isolated using Methanosarcina mazei strain Gö1 as the host. Due to its spherical morphology, the virus was designated Met hanosarcina s pherical v irus (MetSV). Molecular analysis demonstrated that MetSV contains double-stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs), all oriented in the same direction. Functions were predicted for some of these ORFs, i.e., such as DNA polymerase, ATPase, and DNA-binding protein as well as envelope (structural) protein. MetSV-derived spacers in CRISPR loci were detected in several published Methanosarcina draft genomes using bioinformatic tools, revealing a potential protospacer-adjacent motif (PAM) motif (TTA/T). Transcription and expression of several predicted viral ORFs were validated by reverse transcription-PCR (RT-PCR), PAGE analysis, and liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Analysis of core lipids by atmospheric pressure chemical ionization (APCI) mass spectrometry showed that MetSV and Methanosarcina mazei both contain archaeol and glycerol dialkyl glycerol tetraether without a cyclopentane moiety (GDGT-0). The MetSV host range is limited to Methanosarcina strains growing as single cells ( M. mazei , Methanosarcina barkeri and Methanosarcina soligelidi ). In contrast, strains growing as sarcina-like aggregates were apparently protected from infection. Heterogeneity related to morphology phases in M. mazei cultures allowed acquisition of resistance to MetSV after challenge by growing cultures as sarcina-like aggregates. CRISPR/Cas-mediated resistance was excluded since neither of the two CRISPR arrays showed MetSV-derived spacer acquisition. Based on these findings, we propose that changing the morphology from single cells to sarcina-like aggregates upon rearrangement of the envelope structure prevents infection and subsequent lysis by MetSV. IMPORTANCE Methanoarchaea are among the most abundant organisms on the planet since they are present in high numbers in major anaerobic environments. They convert various carbon sources, e.g., acetate, methylamines, or methanol, to methane and carbon dioxide; thus, they have a significant impact on the emission of major greenhouse gases. Today, very little is known about viruses specifically infecting methanoarchaea that most probably impact the abundance of methanoarchaea in microbial consortia. Here, we characterize the first identified Methanosarcina -infecting virus (MetSV) and show a mechanism for acquiring resistance against MetSV. Based on our results, we propose that growth as sarcina-like aggregates prevents infection and subsequent lysis. These findings allow new insights into the virus-host relationship in methanogenic community structures, their dynamics, and their phase heterogeneity. Moreover, the availability of a specific virus provides new possibilities to deepen our knowledge of the defense mechanisms of potential hosts and offers tools for genetic manipulation.

The kingdom of fungi provides model organisms for biotechnology, cell biology, genetics, and life sciences in general. Only when their phylogenetic relationships are stably resolved, can individual results from fungal research be integrated into a holistic picture of biology. However, and despite recent progress, many deep relationships within the fungi remain unclear. Here, we present the first phylogenomic study of an entire eukaryotic kingdom that uses a consistency criterion to strengthen phylogenetic conclusions. We reason that branches (splits) recovered with independent data and different tree reconstruction methods are likely to reflect true evolutionary relationships. Two complementary phylogenomic data sets based on 99 fungal genomes and 109 fungal expressed sequence tag (EST) sets analyzed with four different tree reconstruction methods shed light from different angles on the fungal tree of life. Eleven additional data sets address specifically the phylogenetic position of Blastocladiomycota, Ustilaginomycotina, and Dothideomycetes, respectively. The combined evidence from the resulting trees supports the deep-level stability of the fungal groups toward a comprehensive natural system of the fungi. In addition, our analysis reveals methodologically interesting aspects. Enrichment for EST encoded data-a common practice in phylogenomic analyses-introduces a strong bias toward slowly evolving and functionally correlated genes. Consequently, the generalization of phylogenomic data sets as collections of randomly selected genes cannot be taken for granted. A thorough characterization of the data to assess possible influences on the tree reconstruction should therefore become a standard in phylogenomic analyses.

Supertree methods are widely applied and give rise to new conclusions about phylogenies (e.g., BinindaEmonds et al. 2007). Although several desiderata for supertree methods exist (Wilkinson, Thorley, et al. 2004), only few of them have been studied in greater detail, examples include shape bias (Wilkinson et al. 2005) or pareto properties (Wilkinson et al. 2007). Here I look more closely at two matrix representation methods, matrix representation with compatibility (MRC) and matrix representation with parsimony (MRP). Different null models of random data are studied and the resulting tree shapes are investigated. Thereby I consider unrooted trees and a bias in tree shape is determined by a tree balance measure. The measure for unrooted trees is a modification of a tree balance measure for rooted trees. I observe that depending on the underlying null model of random data, the methods may resolve conflict in favor of more balanced tree shapes. The analyses refer only to trees with the same taxon set, also known as the consensus setting (e.g., Wilkinson et al. 2007), but I will be able to draw conclusions on how to deal with missing data.

Models of RNA secondary structure folding are widely used to study evolution in theory and simulation. However, systematic studies of the parameters involved are rare. In this paper, we study by simulation how RNA evolution is influenced by three different factors, namely the mutation rate, scaling of the fitness function, and distance measure. We found that for low mutation rates the qualitative evolutionary behavior is robust with respect to the scaling of the fitness function. For efficient mutation rates, which are close to the error threshold, scaling and distance measure have a strong influence on the evolutionary behavior. A global distance measure that takes sequence information additively into account lowers the error threshold. When using a local sequence–structure alignment for the distance, we observed a smother evolution of the fitness over time. Finally, in addition to the well known error threshold, we identify another threshold of the mutation rate, called divergence threshold, where the qualitative transient behavior changes from a localized to an exploratory search.

Background Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis of gene expression (SAGE) was used for its unbiased approach to quantitative acquisition of transcripts. Results We generated a HGMEC SAGE library consisting of 68,987 transcript tags. Then taking advantage of large public databases and advanced bioinformatics we compared the HGMEC SAGE library with a SAGE library of non-cultured ex vivo human glomeruli (44,334 tags) which contained endothelial cells. The 823 tags common to both which would have the potential to be expressed in vivo were subsequently checked against 822,008 tags from 16 non-glomerular endothelial SAGE libraries. This resulted in 268 transcript tags differentially overexpressed in HGMEC compared to non-glomerular endothelia. These tags were filtered using a set of criteria: never before shown in kidney or any type of endothelial cell, absent in all nephron regions except the glomerulus, more highly expressed than statistically expected in HGMEC. Neurogranin, a direct target of thyroid hormone action which had been thought to be brain specific and never shown in endothelial cells before, fulfilled these criteria. Its expression in glomerular endothelium in vitro and in vivo was then verified by real-time-PCR, sequencing and immunohistochemistry. Conclusions Our results represent an extensive molecular characterization of HGMEC beyond a mere database, underline the endothelial heterogeneity, and propose neurogranin as a potential link in the kidney-thyroid axis. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-725) contains supplementary material, which is available to authorized users.

Testing the congruence of trees is a major task in phylogenetic research. The comparison of different trees allows to assess the similarity of trees originated from different genes or of trees from the same data but reconstructed with different methods. Another important application of phylogenetic comparisons are cospeciation studies. Cospeciation refers to the simultaneous speciation of ecologically associated lineages, e.g. hosts and their parasites. Since cospeciation is not the only reason for congruent host and parasite trees, the task to assess the congruence is denoted as cophylogenetic

Fungi are abundant in the biosphere. They have fascinated mankind as far as written history goes and have considerably influenced our culture. In biotechnology, cell biology, genetics, and life sciences in general fungi constitute relevant model organisms. Once the phylogenetic relationships of fungi are stably resolved individual results from fungal research can be combined into a holistic picture of biology. However, and despite recent progress, the backbone of the fungal phylogeny is not yet fully resolved. Especially the early evolutionary history of fungi and the order or below-order relationships within the ascomycetes remain uncertain. Here we present the first phylogenomic study for a eukaryotic kingdom that merges all publicly available fungal genomes and expressed sequence tags (EST) to build a data set comprising 128 genes and 146 taxa. The resulting tree provides a stable phylogenetic backbone for the fungi. Moreover, we present the first formal supertree based on 161 fungal taxa and 128 gene trees. The combined evidences from the trees support the deep-level stability of the fungal groups towards a comprehensive natural system of the fungi. They indicate that the classification of the fungi, especially their alliance with the Microsporidia, requires careful revision. Our analysis is also an inventory of present day sequence information for the fungi. It provides insights into which phylogenenetic conclusions can and which cannot be drawn from the current data and may serve as a guide to direct further sequencing initiatives. Together with a comprehensive animal phylogeny, we provide the second of three pillars to understand the evolution of the multicellular eukaryotic kingdoms, fungi, metazoa, and plants, in the past 1.6 billion years.

Background Supertree methods combine overlapping input trees into a larger supertree. Here, I consider split-based supertree methods that first extract the split information of the input trees and subsequently combine this split information into a phylogeny. Well known split-based supertree methods are matrix representation with parsimony and matrix representation with compatibility. Combining input trees on the same taxon set, as in the consensus setting, is a well-studied task and it is thus desirable to generalize consensus methods to supertree methods. Results Here, three variants of majority-rule (MR) supertrees that generalize majority-rule consensus trees are investigated. I provide simple formulas for computing the respective score for bifurcating input- and supertrees. These score computations, together with a heuristic tree search minmizing the scores, were implemented in the python program PluMiST (Plus- and Minus SuperTrees) available from http://www.cibiv.at/software/plumist. The different MR methods were tested by simulation and on real data sets. The search heuristic was successful in combining compatible input trees. When combining incompatible input trees, especially one variant, MR(-) supertrees, performed well. Conclusions The presented framework allows for an efficient score computation of three majority-rule supertree variants and input trees. I combined the score computation with a heuristic search over the supertree space. The implementation was tested by simulation and on real data sets and showed promising results. Especially the MR(-) variant seems to be a reasonable score for supertree reconstruction. Generalizing these computations to multifurcating trees is an open problem, which may be tackled using this framework.

Background The availability of many gene alignments with overlapping taxon sets raises the question of which strategy is the best to infer species phylogenies from multiple gene information. Methods and programs abound that use the gene alignment in different ways to reconstruct the species tree. In particular, different methods combine the original data at different points along the way from the underlying sequences to the final tree. Accordingly, they are classified into superalignment, supertree and medium-level approaches. Here, we present a simulation study to compare different methods from each of these three approaches. Results We observe that superalignment methods usually outperform the other approaches over a wide range of parameters including sparse data and gene-specific evolutionary parameters. In the presence of high incongruency among gene trees, however, other combination methods show better performance than the superalignment approach. Surprisingly, some supertree and medium-level methods exhibit, on average, worse results than a single gene phylogeny with complete taxon information. Conclusions For some methods, using the reconstructed gene tree as an estimation of the species tree is superior to the combination of incomplete information. Superalignment usually performs best since it is less susceptible to stochastic error. Supertree methods can outperform superalignment in the presence of gene-tree conflict.