Multiple introgressions shape mitochondrial evolutionary history in Drosophila paulistorum and the Drosophila willistoni group.

Hybridization and the consequent introgression of genomic elements is an important source of genetic diversity for biological lineages. This is particularly evident in young clades in which hybrid incompatibilities are still incomplete and mixing between species is more likely to occur. Drosophila paulistorum, a representative of the Neotropical Drosophila willistoni subgroup, is a classic model of incipient speciation. The species is divided into six semispecies that show varying degrees of pre- and post-mating incompatibility with each other. In the present study, we investigate the mitochondrial evolutionary history of D. paulistorum and the willistoni subgroup. For that, we perform phylogenetic and comparative analyses of the complete mitochondrial genomes and draft nuclear assemblies of 25 Drosophila lines of the willistoni and saltans species groups. Our results show that the mitochondria of D. paulistorum are polyphyletic and form two non-sister clades that we name α and ß. Identification and analyses of nuclear mitochondrial insertions further reveal that the willistoni subgroup has an α-like mitochondrial ancestor and strongly suggest that both the α and ß mitochondria of D. paulistorum were acquired through introgression from unknown fly lineages of the willistoni subgroup. We also uncover multiple mitochondrial introgressions across D. paulistorum semispecies and generate novel insight into the evolution of the species.

Life and Death of Selfish Genes: Comparative Genomics Reveals the Dynamic Evolution of Cytoplasmic Incompatibility.

Cytoplasmic incompatibility is a selfish reproductive manipulation induced by the endosymbiont Wolbachia in arthropods. In males Wolbachia modifies sperm, leading to embryonic mortality in crosses with Wolbachia-free females. In females, Wolbachia rescues the cross and allows development to proceed normally. This provides a reproductive advantage to infected females, allowing the maternally transmitted symbiont to spread rapidly through host populations. We identified homologs of the genes underlying this phenotype, cifA and cifB, in 52 of 71 new and published Wolbachia genome sequences. They are strongly associated with cytoplasmic incompatibility. There are up to seven copies of the genes in each genome, and phylogenetic analysis shows that Wolbachia frequently acquires new copies due to pervasive horizontal transfer between strains. In many cases, the genes have subsequently acquired loss-of-function mutations to become pseudogenes. As predicted by theory, this tends to occur first in cifB, whose sole function is to modify sperm, and then in cifA, which is required to rescue the cross in females. Although cif genes recombine, recombination is largely restricted to closely related homologs. This is predicted under a model of coevolution between sperm modification and embryonic rescue, where recombination between distantly related pairs of genes would create a self-incompatible strain. Together, these patterns of gene gain, loss, and recombination support evolutionary models of cytoplasmic incompatibility.

Comparative genome sequencing reveals insights into the dynamics of Wolbachia in native and invasive cherry fruit flies.

Wolbachia is a maternally inherited obligate endosymbiont that can induce a wide spectrum of effects in its host, ranging from mutualism to reproductive parasitism. At the genomic level, recombination within and between strains, transposable elements, and horizontal transfer of strains between host species make Wolbachia an evolutionarily dynamic bacterial system. The invasive cherry fruit fly Rhagoletis cingulata arrived in Europe from North America ~40 years ago, where it now co-occurs with the native cherry pest R. cerasi. This shared distribution has been proposed to have led to the horizontal transfer of different Wolbachia strains between the two species. To better understand transmission dynamics, we performed a comparative genome study of the strain wCin2 in its native United States and invasive European populations of R. cingulata with wCer2 in European R. cerasi. Previous multilocus sequence genotyping (MLST) of six genes implied that the source of wCer2 in R. cerasi was wCin2 from R. cingulata. However, we report genomic evidence discounting the recent horizontal transfer hypothesis for the origin of wCer2. Despite near identical sequences for the MLST markers, substantial sequence differences for other loci were found between wCer2 and wCin2, as well as structural rearrangements, and differences in prophage, repetitive element, gene content, and cytoplasmic incompatibility inducing genes. Our study highlights the need for whole-genome sequencing rather than relying on MLST markers for resolving Wolbachia strains and assessing their evolutionary dynamics.

The effect of Wolbachia on gene expression in Drosophila paulistorum and its implications for symbiont-induced host speciation.

BACKGROUND: The Neotropical fruit fly Drosophila paulistorum (Diptera: Drosophilidae) is a species complex in statu nascendi comprising six reproductively isolated semispecies, each harboring mutualistic Wolbachia strains. Although wild type flies of each semispecies are isolated from the others by both pre- and postmating incompatibilities, mating between semispecies and successful offspring development can be achieved once flies are treated with antibiotics to reduce Wolbachia titer. Here we use RNA-seq to study the impact of Wolbachia on D. paulistorum and investigate the hypothesis that the symbiont may play a role in host speciation. For that goal, we analyze samples of heads and abdomens of both sexes of the Amazonian, Centro American and Orinocan semispecies of D. paulistorum. RESULTS: We identify between 175 and 1192 differentially expressed genes associated with a variety of biological processes that respond either globally or according to tissue, sex or condition in the three semispecies. Some of the functions associated with differentially expressed genes are known to be affected by Wolbachia in other species, such as metabolism and immunity, whereas others represent putative novel phenotypes involving muscular functions, pheromone signaling, and visual perception. CONCLUSIONS: Our results show that Wolbachia affect a large number of biological functions in D. paulistorum, particularly when present in high titer. We suggest that the significant metabolic impact of the infection on the host may cause several of the other putative and observed phenotypes. We also speculate that the observed differential expression of genes associated with chemical communication and reproduction may be associated with the emergence of pre- and postmating barriers between semispecies, which supports a role for Wolbachia in the speciation of D. paulistorum.

The Norway spruce genome sequence and conifer genome evolution.

Conifers have dominated forests for more than 200 million years and are of huge ecological and economic importance. Here we present the draft assembly of the 20-gigabase genome of Norway spruce (Picea abies), the first available for any gymnosperm. The number of well-supported genes (28,354) is similar to the >100 times smaller genome of Arabidopsis thaliana, and there is no evidence of a recent whole-genome duplication in the gymnosperm lineage. Instead, the large genome size seems to result from the slow and steady accumulation of a diverse set of long-terminal repeat transposable elements, possibly owing to the lack of an efficient elimination mechanism. Comparative sequencing of Pinus sylvestris, Abies sibirica, Juniperus communis, Taxus baccata and Gnetum gnemon reveals that the transposable element diversity is shared among extant conifers. Expression of 24-nucleotide small RNAs, previously implicated in transposable element silencing, is tissue-specific and much lower than in other plants. We further identify numerous long (>10,000 base pairs) introns, gene-like fragments, uncharacterized long non-coding RNAs and short RNAs. This opens up new genomic avenues for conifer forestry and breeding.

Comparative genomics of Wolbachia and the bacterial species concept.

The importance of host-specialization to speciation processes in obligate host-associated bacteria is well known, as is also the ability of recombination to generate cohesion in bacterial populations. However, whether divergent strains of highly recombining intracellular bacteria, such as Wolbachia, can maintain their genetic distinctness when infecting the same host is not known. We first developed a protocol for the genome sequencing of uncultivable endosymbionts. Using this method, we have sequenced the complete genomes of the Wolbachia strains wHa and wNo, which occur as natural double infections in Drosophila simulans populations on the Seychelles and in New Caledonia. Taxonomically, wHa belong to supergroup A and wNo to supergroup B. A comparative genomics study including additional strains supported the supergroup classification scheme and revealed 24 and 33 group-specific genes, putatively involved in host-adaptation processes. Recombination frequencies were high for strains of the same supergroup despite different host-preference patterns, leading to genomic cohesion. The inferred recombination fragments for strains of different supergroups were of short sizes, and the genomes of the co-infecting Wolbachia strains wHa and wNo were not more similar to each other and did not share more genes than other A- and B-group strains that infect different hosts. We conclude that Wolbachia strains of supergroup A and B represent genetically distinct clades, and that strains of different supergroups can co-exist in the same arthropod host without converging into the same species. This suggests that the supergroups are irreversibly separated and that barriers other than host-specialization are able to maintain distinct clades in recombining endosymbiont populations. Acquiring a good knowledge of the barriers to genetic exchange in Wolbachia will advance our understanding of how endosymbiont communities are constructed from vertically and horizontally transmitted genes.

Extensive duplication of the Wolbachia DNA in chromosome four of Drosophila ananassae.

BACKGROUND: Lateral gene transfer (LGT) from bacterial Wolbachia endosymbionts has been detected in ~20% of arthropod and nematode genome sequencing projects. Many of these transfers are large and contain a substantial part of the Wolbachia genome. RESULTS: Here, we re-sequenced three D. ananassae genomes from Asia and the Pacific that contain large LGTs from Wolbachia. We find that multiple copies of the Wolbachia genome are transferred to the Drosophila nuclear genome in all three lines. In the D. ananassae line from Indonesia, the copies of Wolbachia DNA in the nuclear genome are nearly identical in size and sequence yielding an even coverage of mapped reads over the Wolbachia genome. In contrast, the D. ananassae lines from Hawaii and India show an uneven coverage of mapped reads over the Wolbachia genome suggesting that different parts of these LGTs are present in different copy numbers. In the Hawaii line, we find that this LGT is underrepresented in third instar larvae indicative of being heterochromatic. Fluorescence in situ hybridization of mitotic chromosomes confirms that the LGT in the Hawaii line is heterochromatic and represents ~20% of the sequence on chromosome 4 (dot chromosome, Muller element F). CONCLUSIONS: This collection of related lines contain large lateral gene transfers composed of multiple Wolbachia genomes that constitute >2% of the D. ananassae genome (~5 Mbp) and partially explain the abnormally large size of chromosome 4 in D. ananassae.

More than fishing in the dark: PCR of a dispersed sequence produces simple but ultrasensitive Wolbachia detection.

BACKGROUND: Detecting intracellular bacterial symbionts can be challenging when they persist at very low densities. Wolbachia, a widespread bacterial endosymbiont of invertebrates, is particularly challenging. Although it persists at high titers in many species, in others its densities are far below the detection limit of classic end-point Polymerase Chain Reaction (PCR). These low-titer infections can be reliably detected by combining PCR with DNA hybridization, but less elaborate strategies based on end-point PCR alone have proven less sensitive or less general. RESULTS: We introduce a multicopy PCR target that allows fast and reliable detection of A-supergroup Wolbachia--even at low infection titers--with standard end-point PCR. The target is a multicopy motif (designated ARM: A-supergroup repeat motif) discovered in the genome of wMel (the Wolbachia in Drosophila melanogaster). ARM is found in at least seven other Wolbachia A-supergroup strains infecting various Drosophila, the wasp Muscidifurax and the tsetse fly Glossina. We demonstrate that end-point PCR targeting ARM can reliably detect both high- and low-titer Wolbachia infections in Drosophila, Glossina and interspecific hybrids. CONCLUSIONS: Simple end-point PCR of ARM facilitates detection of low-titer Wolbachia A-supergroup infections. Detecting these infections previously required more elaborate procedures. Our ARM target seems to be a general feature of Wolbachia A-supergroup genomes, unlike other multicopy markers such as insertion sequences (IS).

Research on small genomes: implications for synthetic biology.

Synthetic genomics is a new field of research in which small DNA pieces are assembled in a series of steps into whole genomes. The highly reduced genomes of host-associated bacteria are now being used as models for de novo synthesis of small genomes in the laboratory. Bacteria with the smallest genomes identified in nature provide nutrients to their hosts, such as amino acids, co-factors and vitamins. Comparative genomics of these bacteria enables predictions to be made about the gene sets required for core cellular functions and the associated metabolic network for the biosynthesis of host-selected compounds. Synthetic biology may ultimately enable researchers to make customized cell-specific organelles for the production and delivery of drugs to humans and domestic animals. Synthetic genomics may also become the method of choice for functional analyses of genes and genomes from bacteria that cannot be cultivated in the laboratory.

The mosaic genome structure of the Wolbachia wRi strain infecting Drosophila simulans.

The obligate intracellular bacterium Wolbachia pipientis infects around 20% of all insect species. It is maternally inherited and induces reproductive alterations of insect populations by male killing, feminization, parthenogenesis, or cytoplasmic incompatibility. Here, we present the 1,445,873-bp genome of W. pipientis strain wRi that induces very strong cytoplasmic incompatibility in its natural host Drosophila simulans. A comparison with the previously sequenced genome of W. pipientis strain wMel from Drosophila melanogaster identified 35 breakpoints associated with mobile elements and repeated sequences that are stable in Drosophila lines transinfected with wRi. Additionally, 450 genes with orthologs in wRi and wMel were sequenced from the W. pipientis strain wUni, responsible for the induction of parthenogenesis in the parasitoid wasp Muscidifurax uniraptor. The comparison of these A-group Wolbachia strains uncovered the most highly recombining intracellular bacterial genomes known to date. This was manifested in a 500-fold variation in sequence divergences at synonymous sites, with different genes and gene segments supporting different strain relationships. The substitution-frequency profile resembled that of Neisseria meningitidis, which is characterized by rampant intraspecies recombination, rather than that of Rickettsia, where genes mostly diverge by nucleotide substitutions. The data further revealed diversification of ankyrin repeat genes by short tandem duplications and provided examples of horizontal gene transfer across A- and B-group strains that infect D. simulans. These results suggest that the transmission dynamics of Wolbachia and the opportunity for coinfections have created a freely recombining intracellular bacterial community with mosaic genomes.

Comparative Genomics Reveals Factors Associated with Phenotypic Expression of Wolbachia.

Wolbachia is a widespread, vertically transmitted bacterial endosymbiont known for manipulating arthropod reproduction. Its most common form of reproductive manipulation is cytoplasmic incompatibility (CI), observed when a modification in the male sperm leads to embryonic lethality unless a compatible rescue factor is present in the female egg. CI attracts scientific attention due to its implications for host speciation and in the use of Wolbachia for controlling vector-borne diseases. However, our understanding of CI is complicated by the complexity of the phenotype, whose expression depends on both symbiont and host factors. In the present study, we perform a comparative analysis of nine complete Wolbachia genomes with known CI properties in the same genetic host background, Drosophila simulans STC. We describe genetic differences between closely related strains and uncover evidence that phages and other mobile elements contribute to the rapid evolution of both genomes and phenotypes of Wolbachia. Additionally, we identify both known and novel genes associated with the modification and rescue functions of CI. We combine our observations with published phenotypic information and discuss how variability in cif genes, novel CI-associated genes, and Wolbachia titer might contribute to poorly understood aspects of CI such as strength and bidirectional incompatibility. We speculate that high titer CI strains could be better at invading new hosts already infected with a CI Wolbachia, due to a higher rescue potential, and suggest that titer might thus be a relevant parameter to consider for future strategies using CI Wolbachia in biological control.

Parallel Sequencing of Wolbachia wCer2 from Donor and Novel Hosts Reveals Multiple Incompatibility Factors and Genome Stability after Host Transfers.

The application of Wolbachia in insect pest and vector control requires the establishment of genotypically stable host associations. The cytoplasmic incompatibility (CI) inducing Wolbachia strain wCer2 naturally occurs in the cherry fruit fly Rhagoletis cerasi as co-infection with other strains and was transferred to other fruit fly species by embryonic microinjections. We obtained wCer2 genome data from its native and three novel hosts, Drosophila simulans, Drosophila melanogaster, and Ceratitis capitata and assessed its genome stability, characteristics, and CI factor (cif) genes. De novo assembly was successful from Wolbachia cell-enriched singly infected D. simulans embryos, with minimal host and other bacterial genome traces. The low yield of Wolbachia sequence reads from total genomic extracts of one multiply infected R. cerasi pupa and one singly infected C. capitata adult limited de novo assemblies but was sufficient for comparative analyses. Across hosts wCer2 was stable in genome synteny and content. Polymorphic nucleotide sites were found in wCer2 of each host; however, only one nucleotide was different between R. cerasi and C. capitata, and none between replicated D. simulans lines. The wCer2 genome is highly similar to wAu (D. simulans), wMel (D. melanogaster), and wRec (Drosophila recens). In contrast to wMel and wRec (each with one cif gene pair) and wAu (without any cif genes), wCer2 has three pairs of Type I cif genes, and one Type V cifB gene without a cifA complement. This may explain previously reported CI patterns of wCer2, including incomplete rescue of its own CI modification in three novel host species.

Horizontal gene transfer between Wolbachia and the mosquito Aedes aegypti.

BACKGROUND: The evolutionary importance of horizontal gene transfer (HGT) from Wolbachia endosymbiotic bacteria to their eukaryotic hosts is a topic of considerable interest and debate. Recent transfers of genome fragments from Wolbachia into insect chromosomes have been reported, but it has been argued that these fragments may be on an evolutionary trajectory to degradation and loss. RESULTS: We have discovered a case of HGT, involving two adjacent genes, between the genomes of Wolbachia and the currently Wolbachia-uninfected mosquito Aedes aegypti, an important human disease vector. The lower level of sequence identity between Wolbachia and insect, the transcription of all the genes involved, and the fact that we have identified homologs of the two genes in another Aedes species (Ae. mascarensis), suggest that these genes are being expressed after an extended evolutionary period since horizontal transfer, and therefore that the transfer has functional significance. The association of these genes with Wolbachia prophage regions also provides a mechanism for the transfer. CONCLUSION: The data support the argument that HGT between Wolbachia endosymbiotic bacteria and their hosts has produced evolutionary innovation.

Genome evolution of Wolbachia strain wPip from the Culex pipiens group.

The obligate intracellular bacterium Wolbachia pipientis strain wPip induces cytoplasmic incompatibility (CI), patterns of crossing sterility, in the Culex pipiens group of mosquitoes. The complete sequence is presented of the 1.48-Mbp genome of wPip which encodes 1386 coding sequences (CDSs), representing the first genome sequence of a B-supergroup Wolbachia. Comparisons were made with the smaller genomes of Wolbachia strains wMel of Drosophila melanogaster, an A-supergroup Wolbachia that is also a CI inducer, and wBm, a mutualist of Brugia malayi nematodes that belongs to the D-supergroup of Wolbachia. Despite extensive gene order rearrangement, a core set of Wolbachia genes shared between the 3 genomes can be identified and contrasts with a flexible gene pool where rapid evolution has taken place. There are much more extensive prophage and ankyrin repeat encoding (ANK) gene components of the wPip genome compared with wMel and wBm, and both are likely to be of considerable importance in wPip biology. Five WO-B-like prophage regions are present and contain some genes that are identical or highly similar in multiple prophage copies, whereas other genes are unique, and it is likely that extensive recombination, duplication, and insertion have occurred between copies. A much larger number of genes encode ankyrin repeat (ANK) proteins in wPip, with 60 present compared with 23 in wMel, many of which are within or close to the prophage regions. It is likely that this pattern is partly a result of expansions in the wPip lineage, due for example to gene duplication, but their presence is in some cases more ancient. The wPip genome underlines the considerable evolutionary flexibility of Wolbachia, providing clear evidence for the rapid evolution of ANK-encoding genes and of prophage regions. This host-Wolbachia system, with its complex patterns of sterility induced between populations, now provides an excellent model for unraveling the molecular systems underlying host reproductive manipulation.

Ankyrin repeat domain-encoding genes in the wPip strain of Wolbachia from the Culex pipiens group.

BACKGROUND: Wolbachia are obligate endosymbiotic bacteria maternally transmitted through the egg cytoplasm that are responsible for several reproductive disorders in their insect hosts, such as cytoplasmic incompatibility (CI) in infected mosquitoes. Species in the Culex pipiens complex display an unusually high number of Wolbachia-induced crossing types, and based on present data, only the wPip strain is present. RESULTS: The sequencing of the wPip strain of Wolbachia revealed the presence of 60 ankyrin repeat domain (ANK) encoding genes and expression studies of these genes were carried out in adult mosquitoes. One of these ANK genes, pk2, is shown to be part of an operon of three prophage-associated genes with sex-specific expression, and is present in two identical copies in the genome. Another homolog of pk2 is also present that is differentially expressed in different Cx. pipiens group strains. A further two ANK genes showed sex-specific regulation in wPip-infected Cx. pipiens group adults. CONCLUSION: The high number, variability and differential expression of ANK genes in wPip suggest an important role in Wolbachia biology, and the gene family provides both markers and promising candidates for the study of reproductive manipulation.

The Complexities and Nuances of Analyzing the Genome of Drosophila ananassae and Its Wolbachia Endosymbiont.

In "Retrotransposons Are the Major Contributors to the Expansion of the Drosophila ananassae Muller F Element," Leung et al. (2017) improved contigs attributed to the Muller F element from the original CAF1 assembly, and used them to conclude that most of the sequence expansion of the fourth chromosome of D. ananassae is due to a higher transposon load than previously thought, but is not due to Wolbachia DNA integrations. While we do not disagree with the first conclusion, the authors base their second conclusion on the lack of homology detected between their improved CAF1 genome assembly attributed to D. ananassae and reference Wolbachia genomes. While the consensus CAF1 genome assembly lacks any sequence similarity to the reference genome of the Wolbachia endosymbiont of Drosophila melanogaster (wMel), numerous studies from multiple laboratories provide experimental support for a large lateral/horizontal gene transfer (LGT) of a Wolbachia genome into this D. ananassae line. As such, we strongly suspect that the original whole genome assembly was either constructed after the removal of all Wolbachia reads, or that Wolbachia sequences were directly removed from the contigs in the CAF1 assembly. Hence, Leung et al. (2017) could not have identified the Wolbachia LGT using the CAF1 assembly. This manuscript by Leung et al. (2017) highlights that an assembly of the Wolbachia sequence reads and their mate pairs was erroneously attributed solely to the Wolbachia endosymbiont, albeit before we understood the extent of LGT in D. ananassae As such, we recommend that the sequences deposited at the National Center for Biotechnology Information (NCBI) under PRJNA13365 should not be attributed to Wolbachia endosymbiont of D. ananassae, but should have their taxonomy reclassified by NCBI as "Unclassified sequences." As our knowledge about genome biology improves, we need to reconsider and reanalyze earlier genomes removing the prejudice introduced from now defunct paradigms.

Microbial genome evolution: sources of variability.

Comparative genome analyses of close relatives have yielded exciting insight into the sources of microbial genome variability with respect to gene content, gene order and evolution of genes with unknown functions. The genomes of free-living bacteria often carry phages and repetitive sequences that mediate genomic rearrangements in contrast to the small genomes of obligate host-associated bacteria. This suggests that genomic stability correlates with the genomic content of repeated sequences and movable genetic elements, and thereby with bacterial lifestyle. Genes with unknown functions present in a single species tend to be shorter than conserved, functional genes, indicating that the fraction of unique genes in microbial genomes has been overestimated.

Evolution of minimal-gene-sets in host-dependent bacteria.

Several attempts have been made to identify the minimal set of genes that is required for life using computational approaches or studies of deletion mutants. These experiments resemble those already performed by nature; a few hundred million years ago an ancestor of Escherichia coli was domesticated by aphids, which resulted in the elimination of 70-75% of the original bacterial genome. Amazingly, the small genomes of these imprisoned bacteria are more stable than those of their free-living relatives. Minimal-gene-sets that have evolved naturally are largely species-specific, with the exception of a small set of core genes that are required for information processing. Comparative genomics of host-dependent bacteria have shown that minimal-gene-sets can persist in nature for tens of millions of years provided that the environment is rich in nutrients, that the host population size is large and that there is a strong host-level selection for bacterial gene functions.

Distinctive Genome Reduction Rates Revealed by Genomic Analyses of Two Coxiella-Like Endosymbionts in Ticks.

Genome reduction is a hallmark of symbiotic genomes, and the rate and patterns of gene loss associated with this process have been investigated in several different symbiotic systems. However, in long-term host-associated coevolving symbiont clades, the genome size differences between strains are normally quite small and hence patterns of large-scale genome reduction can only be inferred from distant relatives. Here we present the complete genome of a Coxiella-like symbiont from Rhipicephalus turanicus ticks (CRt), and compare it with other genomes from the genus Coxiella in order to investigate the process of genome reduction in a genus consisting of intracellular host-associated bacteria with variable genome sizes. The 1.7-Mb CRt genome is larger than the genomes of most obligate mutualists but has a very low protein-coding content (48.5%) and an extremely high number of identifiable pseudogenes, indicating that it is currently undergoing genome reduction. Analysis of encoded functions suggests that CRt is an obligate tick mutualist, as indicated by the possible provisioning of the tick with biotin (B7), riboflavin (B2) and other cofactors, and by the loss of most genes involved in host cell interactions, such as secretion systems. Comparative analyses between CRt and the 2.5 times smaller genome of Coxiella from the lone star tick Amblyomma americanum (CLEAA) show that many of the same gene functions are lost and suggest that the large size difference might be due to a higher rate of genome evolution in CLEAA generated by the loss of the mismatch repair genes mutSL. Finally, sequence polymorphisms in the CRt population sampled from field collected ticks reveal up to one distinct strain variant per tick, and analyses of mutational patterns within the population suggest that selection might be acting on synonymous sites. The CRt genome is an extreme example of a symbiont genome caught in the act of genome reduction, and the comparison between CLEAA and CRt indicates that losses of particular genes early on in this process can potentially greatly influence the speed of this process.

Testing the reproducibility of multiple displacement amplification on genomes of clonal endosymbiont populations.

The multiple displacement amplification method has revolutionized genomic studies of uncultured bacteria, where the extraction of pure DNA in sufficient quantity for next-generation sequencing is challenging. However, the method is problematic in that it amplifies the target DNA unevenly, induces the formation of chimeric reads and also amplifies contaminating DNA. Here, we have tested the reproducibility of the multiple displacement amplification method using serial dilutions of extracted genomic DNA and intact cells from the cultured endosymbiont Bartonella australis. The amplified DNA was sequenced with the Illumina sequencing technology, and the results were compared to sequence data obtained from unamplified DNA in this study as well as from a previously published genome project. We show that artifacts such as the extent of the amplification bias, the percentage of chimeric reads and the relative fraction of contaminating DNA increase dramatically for the smallest amounts of template DNA. The pattern of read coverage was reproducibly obtained for samples with higher amounts of template DNA, suggesting that the bias is non-random and genome-specific. A re-analysis of previously published sequence data obtained after amplification from clonal endosymbiont populations confirmed these predictions. We conclude that many of the artifacts associated with the use of the multiple displacement amplification method can be alleviated or much reduced by using multiple cells as the template for the amplification. These findings should be particularly useful for researchers studying the genomes of endosymbionts and other uncultured bacteria, for which a small clonal population of cells can be isolated.