Macroevolutionary Dynamics in Micro-organisms: Generalists Give Rise to Specialists Across Biomes in the Ubiquitous Bacterial Phylum Myxococcota.

Prokaryotes dominate the Tree of Life, but our understanding of the macroevolutionary processes generating this diversity is still limited. Habitat transitions are thought to be a key driver of prokaryote diversity. However, relatively little is known about how prokaryotes successfully transition and persist across environments, and how these processes might vary between biomes and lineages. Here, we investigate biome transitions and specialization in natural populations of a focal bacterial phylum, the Myxococcota, sampled across a range of replicated soils and freshwater and marine sediments in Cornwall (UK). By targeted deep sequencing of the protein-coding gene rpoB, we found >2,000 unique Myxococcota lineages, with the majority (77%) classified as biome specialists and with only <5% of lineages distributed across the salt barrier. Discrete character evolution models revealed that specialists in one biome rarely transitioned into specialists in another biome. Instead, evolved generalism mediated transitions between biome specialists. State-dependent diversification models found variation in speciation rates across the tree, but this variation was independent of biome association or specialization. Our findings were robust to phylogenetic uncertainty, different levels of species delineation, and different assumed amounts of unsampled diversity resulting in an incomplete phylogeny. Overall, our results are consistent with a "jack-of-all-trades" tradeoff where generalists suffer a cost in any individual environment, resulting in rapid evolution of niche specialists and shed light on how bacteria could transition between biomes.

Copper reduces the virulence of bacterial communities at environmentally relevant concentrations.

Increasing environmental concentrations of metals as a result of anthropogenic pollution are significantly changing many microbial communities. While there is evidence metal pollution can result in increased antibiotic resistance, the effects of metal pollution on the virulence of bacterial communities remains largely undetermined. Here, we experimentally test whether metal stress alters the virulence of bacterial communities. We do this by incubating three wastewater influent communities under different environmentally relevant copper concentrations for three days. We then quantify the virulence of the community phenotypically using the Galleria mellonella infection model, and test if differences are due to changes in the rate of biomass accumulation (productivity), copper resistance, or community composition (quantified using 16S amplicon sequencing). The virulence of the communities was found to be reduced by the highest copper concentration, but not to be affected by the lower concentration. As well as reduced virulence, communities exposed to the highest copper concentration were less diverse and had lower productivity. This work highlights that metal pollution may decrease virulence in bacterial communities, but at a cost to diversity and productivity.

Quantitative Polymerase Chain Reaction for the estimation of toxigenic microalgae abundance in shellfish production waters.

Certain species of marine microalgae produce potent biotoxins that pose a risk to human health if contaminated seafood is consumed, particularly filter feeding bivalve shellfish. In regions where this is likely to occur water and seafood produce are regularly monitored for the presence of harmful algal cells and their associated toxins, but the current approach is flawed by a lengthy delay before results are available to local authorities. Quantitative Polymerase Chain Reaction (qPCR) can be used to measure phytoplankton DNA sequences in a shorter timeframe, however it is not currently used in official testing practices. In this study, samples were collected almost weekly over six months from three sites within a known HAB hotspot, St Austell Bay in Cornwall, England. The abundance of algal cells in water was measured using microscopy and qPCR, and lipophilic toxins were quantified in mussel flesh using LC-MS/MS, focusing on the okadaic acid group. An increase in algal cell abundance occurred alongside an increase in the concentration of okadaic acid group toxins in mussel tissue at all three study sites, during September and October 2021. This event corresponded to an increase in the measured levels of Dinophysis accuminata DNA, measured using qPCR. In the following spring, the qPCR detected an increase in D. accuminata DNA levels in water samples, which was not detected by microscopy. Harmful algal species belonging to Alexandrium spp. and Pseudo-nitzschia spp. were also measured using qPCR, finding a similar increase in abundance in Autumn and Spring. The results are discussed with consideration of the potential merits and limitations of the qPCR technique versus conventional microscopy analysis, and its potential future role in phytoplankton surveillance under the Official Controls Regulations pertaining to shellfish.

Bacterial colonisation dynamics of household plastics in a coastal environment.

Accumulation of plastics in the marine environment has widespread detrimental consequences for ecosystems and wildlife. Marine plastics are rapidly colonised by a wide diversity of bacteria, including human pathogens, posing potential risks to health. Here, we investigate the effect of polymer type, residence time and estuarine location on bacterial colonisation of common household plastics, including pathogenic bacteria. We submerged five main household plastic types: low-density PE (LDPE), high-density PE (HDPE), polypropylene (PP), polyvinyl chloride (PVC) and polyethylene terephthalate (PET) at an estuarine site in Cornwall (U.K.) and tracked bacterial colonisation dynamics. Using both culture-dependent and culture-independent approaches, we found that bacteria rapidly colonised plastics irrespective of polymer type, reaching culturable densities of up to 1000 cells cm3 after 7 weeks. Community composition of the biofilms changed over time, but not among polymer types. The presence of pathogenic bacteria, quantified using the insect model Galleria mellonella, increased dramatically over a five-week period, with Galleria mortality increasing from 4% in week one to 65% in week five. No consistent differences in virulence were observed between polymer types. Pathogens isolated from plastic biofilms using Galleria enrichment included Serratia and Enterococcus species and they harboured a wide range of antimicrobial resistance genes. Our findings show that plastics in coastal waters are rapidly colonised by a wide diversity of bacteria independent of polymer type. Further, our results show that marine plastic biofilms become increasingly associated with virulent bacteria over time.

Greater Phage Genotypic Diversity Constrains Arms-Race Coevolution.

Antagonistic coevolution between hosts and parasites, the reciprocal evolution of host resistance and parasite infectivity, has important implications in ecology and evolution. The dynamics of coevolution-notably whether host or parasite has an evolutionary advantage-is greatly affected by the relative amount of genetic variation in host resistance and parasite infectivity traits. While studies have manipulated genetic diversity during coevolution, such as by increasing mutation rates, it is unclear how starting genetic diversity affects host-parasite coevolution. Here, we (co)evolved the bacterium Pseudomonas fluorescens SBW25 and two bacteriophage genotypes of its lytic phage SBW25ɸ2 in isolation (one phage genotype) and together (two phage genotypes). Bacterial populations rapidly evolved phage resistance, and phage reciprocally increased their infectivity in response. When phage populations were evolved with bacteria in isolation, bacterial resistance and phage infectivity increased through time, indicative of arms-race coevolution. In contrast, when both phage genotypes were together, bacteria did not increase their resistance in response to increasing phage infectivity. This was likely due to bacteria being unable to evolve resistance to both phage via the same mutations. These results suggest that increasing initial parasite genotypic diversity can give parasites an evolutionary advantage that arrests long-term coevolution. This study has important implications for the applied use of phage in phage therapy and in understanding host-parasite dynamics in broader ecological and evolutionary theory.

Resource quality determines the evolution of resistance and its genetic basis.

Parasites impose strong selection on their hosts, but the level of any evolved resistance may be constrained by the availability of resources. However, studies identifying the genomic basis of such resource-mediated selection are rare, particularly in nonmodel organisms. Here, we investigated the role of nutrition in the evolution of resistance to a DNA virus (PiGV), and any associated trade-offs in a lepidopteran pest species (Plodia interpunctella). Through selection experiments and whole-genome resequencing, we identify genetic markers of resistance that vary between the nutritional environments during selection. We do not find consistent evolution of resistance in the presence of virus but rather see substantial variation among replicate populations. Resistance in a low-nutrition environment is negatively correlated with growth rate, consistent with an established trade-off between immunity and development, but this relationship is highly context dependent. Whole-genome resequencing of the host shows that resistance mechanisms are likely to be highly polygenic and although the underlying genetic architecture may differ between high and low-nutrition environments, similar mechanisms are commonly used. As a whole, our results emphasize the importance of the resource environment on influencing the evolution of resistance.

A simple procedure for directly obtaining haplotype sequences of diploid genomes.

BACKGROUND: Almost all genome sequencing projects neglect the fact that diploid organisms contain two genome copies and consequently what is published is a composite of the two. This means that the relationship between alternate alleles at two or more linked loci is lost. We have developed a simplified method of directly obtaining the haploid sequences of each genome copy from an individual organism. RESULTS: The diploid sequences of three groups of cattle samples were obtained using a simple sample preparation procedure requiring only a microscope and a haemocytometer. Samples were: 1) lymphocytes from a single Angus steer; 2) sperm cells from an Angus bull; 3) lymphocytes from East African Zebu (EAZ) cattle collected and processed in a field laboratory in Eastern Kenya. Haploid sequence from a fosmid library prepared from lymphocytes of an EAZ cow was used for comparison. Cells were serially diluted to a concentration of one cell per microlitre by counting with a haemocytometer at each dilution. One microlitre samples, each potentially containing a single cell, were lysed and divided into six aliquots (except for the sperm samples which were not divided into aliquots). Each aliquot was amplified with phi29 polymerase and sequenced. Contigs were obtained by mapping to the bovine UMD3.1 reference genome assembly and scaffolds were assembled by joining adjacent contigs that were within a threshold distance of each other. Scaffolds that appeared to contain artefacts of CNV or repeats were filtered out leaving scaffolds with an N50 length of 27-133 kb and a 88-98 % genome coverage. SNP haplotypes were assembled with the Single Individual Haplotyper program to generate an N50 size of 97-201 kb but only ~27-68 % genome coverage. This method can be used in any laboratory with no special equipment at only slightly higher costs than conventional diploid genome sequencing. A substantial body of software for analysis and workflow management was written and is available as supplementary data. CONCLUSIONS: We have developed a set of laboratory protocols and software tools that will enable any laboratory to obtain haplotype sequences at only modestly greater cost than traditional mixed diploid sequences.

Whole-genome sequencing of Trypanosoma brucei reveals introgression between subspecies that is associated with virulence.

UNLABELLED: Human African trypanosomiasis is caused by two subspecies of Trypanosoma brucei. Trypanosoma brucei rhodesiense is found in East Africa and frequently causes acute disease, while Trypanosoma brucei gambiense is found in West Africa and is associated with chronic disease. Samples taken from a single focus of a Ugandan outbreak of T. b. rhodesiense in the 1980s were associated with either chronic or acute disease. We sequenced the whole genomes of two of these isolates, which showed that they are genetically distinct from each other. Analysis of single nucleotide polymorphism markers in a panel of 31 Ugandan isolates plus 32 controls revealed a mixture of East African and West African haplotypes, and some of these haplotypes were associated with the different virulence phenotypes. It has been shown recently that T. b. brucei and T. b. rhodesiense populations undergo genetic exchange in natural populations. Our analysis showed that these strains from the Ugandan epidemic were intermediate between the reference genome sequences of T. b. gambiense and T. b. brucei and contained haplotypes that were present in both subspecies. This suggests that the human-infective subspecies of T. brucei are not genetically isolated, and our data are consistent with genomic introgression between East African and West African T. b. brucei subspecies. This has implications for the control of the parasite, the spread of drug resistance, and understanding the variation in virulence and the emergence of human infectivity. IMPORTANCE: We present a genetic study of the acute form of "sleeping sickness" caused by the protozoan parasite Trypanosoma brucei rhodesiense from a single outbreak in Uganda. This represents an advance in our understanding of the relationship between the T. b. rhodesiense and Trypanosoma brucei gambiense subspecies that have previously been considered geographically distinct. Our data suggest that introgression of West African-derived T. brucei haplotypes may be associated with differences in disease presentation in the East African disease. These findings are not only of scientific interest but also important for parasite control, as they suggest that the human-infective T. brucei subspecies are not genetically isolated.

Analysis of the bread wheat genome using whole-genome shotgun sequencing.

Bread wheat (Triticum aestivum) is a globally important crop, accounting for 20 per cent of the calories consumed by humans. Major efforts are underway worldwide to increase wheat production by extending genetic diversity and analysing key traits, and genomic resources can accelerate progress. But so far the very large size and polyploid complexity of the bread wheat genome have been substantial barriers to genome analysis. Here we report the sequencing of its large, 17-gigabase-pair, hexaploid genome using 454 pyrosequencing, and comparison of this with the sequences of diploid ancestral and progenitor genomes. We identified between 94,000 and 96,000 genes, and assigned two-thirds to the three component genomes (A, B and D) of hexaploid wheat. High-resolution synteny maps identified many small disruptions to conserved gene order. We show that the hexaploid genome is highly dynamic, with significant loss of gene family members on polyploidization and domestication, and an abundance of gene fragments. Several classes of genes involved in energy harvesting, metabolism and growth are among expanded gene families that could be associated with crop productivity. Our analyses, coupled with the identification of extensive genetic variation, provide a resource for accelerating gene discovery and improving this major crop.

Novel SNP Discovery in African Buffalo, Syncerus caffer, using high-throughput Sequencing.

The African buffalo, Syncerus caffer, is one of the most abundant and ecologically important species of megafauna in the savannah ecosystem. It is an important prey species, as well as a host for a vast array of nematodes, pathogens and infectious diseases, such as bovine tuberculosis and corridor disease. Large-scale SNP discovery in this species would greatly facilitate further research into the area of host genetics and disease susceptibility, as well as provide a wealth of sequence information for other conservation and genomics studies. We sequenced pools of Cape buffalo DNA from a total of 9 animals, on an ABI SOLiD4 sequencer. The resulting short reads were mapped to the UMD3.1 Bos taurus genome assembly using both BWA and Bowtie software packages. A mean depth of 2.7× coverage over the mapped regions was obtained. Btau4 gene annotation was added to all SNPs identified within gene regions. Bowtie and BWA identified a maximum of 2,222,665 and 276,847 SNPs within the buffalo respectively, depending on analysis method. A panel of 173 SNPs was validated by fluorescent genotyping in 87 individuals. 27 SNPs failed to amplify, and of the remaining 146 SNPs, 43-54% of the Bowtie SNPs and 57-58% of the BWA SNPs were confirmed as polymorphic. dN/dS ratios found no evidence of positive selection, and although there were genes that appeared to be under negative selection, these were more likely to be slowly evolving house-keeping genes.

Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis.

The α-proteobacterium Wolbachia is probably the most prevalent, vertically transmitted symbiont on Earth. In contrast with its wide distribution in arthropods, Wolbachia is restricted to one family of animal-parasitic nematodes, the Onchocercidae. This includes filarial pathogens such as Onchocerca volvulus, the cause of human onchocerciasis, or river blindness. The symbiosis between filariae and Wolbachia is obligate, although the basis of this dependency is not fully understood. Previous studies suggested that Wolbachia may provision metabolites (e.g., haem, riboflavin, and nucleotides) and/or contribute to immune defense. Importantly, Wolbachia is restricted to somatic tissues in adult male worms, whereas females also harbor bacteria in the germline. We sought to characterize the nature of the symbiosis between Wolbachia and O. ochengi, a bovine parasite representing the closest relative of O. volvulus. First, we sequenced the complete genome of Wolbachia strain wOo, which revealed an inability to synthesize riboflavin de novo. Using RNA-seq, we also generated endobacterial transcriptomes from male soma and female germline. In the soma, transcripts for membrane transport and respiration were up-regulated, while the gonad exhibited enrichment for DNA replication and translation. The most abundant Wolbachia proteins, as determined by geLC-MS, included ligands for mammalian Toll-like receptors. Enzymes involved in nucleotide synthesis were dominant among metabolism-related proteins, whereas the haem biosynthetic pathway was poorly represented. We conclude that Wolbachia may have a mitochondrion-like function in the soma, generating ATP for its host. Moreover, the abundance of immunogenic proteins in wOo suggests a role in diverting the immune system toward an ineffective antibacterial response.

Genomic diversity of the human intestinal parasite Entamoeba histolytica.

BACKGROUND: Entamoeba histolytica is a significant cause of disease worldwide. However, little is known about the genetic diversity of the parasite. We re-sequenced the genomes of ten laboratory cultured lines of the eukaryotic pathogen Entamoeba histolytica in order to develop a picture of genetic diversity across the genome. RESULTS: The extreme nucleotide composition bias and repetitiveness of the E. histolytica genome provide a challenge for short-read mapping, yet we were able to define putative single nucleotide polymorphisms in a large portion of the genome. The results suggest a rather low level of single nucleotide diversity, although genes and gene families with putative roles in virulence are among the more polymorphic genes. We did observe large differences in coverage depth among genes, indicating differences in gene copy number between genomes. We found evidence indicating that recombination has occurred in the history of the sequenced genomes, suggesting that E. histolytica may reproduce sexually. CONCLUSIONS: E. histolytica displays a relatively low level of nucleotide diversity across its genome. However, large differences in gene family content and gene copy number are seen among the sequenced genomes. The pattern of polymorphism indicates that E. histolytica reproduces sexually, or has done so in the past, which has previously been suggested but not proven.

Full genome re-sequencing reveals a novel circadian clock mutation in Arabidopsis.

Map based cloning in Arabidopsis thaliana can be a difficult and time-consuming process, specifically if the phenotype is subtle and scoring labour intensive. Here, we have re-sequenced the 120-Mb genome of a novel Arabidopsis clock mutant early bird (ebi-1) in Wassilewskija (Ws-2). We demonstrate the utility of sequencing a backcrossed line in limiting the number of SNPs considered. We identify a SNP in the gene AtNFXL-2 as the likely cause of the ebi-1 phenotype.