Rapid divergence in independent aspects of the compatibility phenotype in a Spiroplasma-Drosophila interaction.

Heritable symbionts represent important components of the biology, ecology and evolution of their arthropod hosts. Particular microbial taxa have become common across arthropods as a consequence of their ability to establish in new host species. For a host shift to occur, the symbiont must be exposed to a novel host and then be compatible: it must not cause excess pathology, must have good vertical transmission and must possess a drive phenotype that enables spread. Here we investigate the lability of compatibility to symbiosis with Spiroplasma. We used transinfection to establish the protective Spiroplasma symbiont from Drosophila hydei in two closely related novel hosts, Drosophila simulans and Drosophila melanogaster. The Spiroplasma had contrasting compatibility in the two species, exhibiting pathology and low vertical transmission but delivering protection from wasp attack in D. melanogaster but being asymptomatic and transmitted with high efficiency but with lower protection in D. simulans. Further work indicated that pathological interactions occurred in two other members of the melanogaster species group, such that D. simulans was unusual in being able to carry the symbiont without damage. The differing compatibility of the symbiont with these closely related host species emphasizes the rapidity with which host-symbiont compatibility evolves, despite compatibility itself not being subject to direct selection. Further, the requirement to fit three independent components of compatibility (pathology, transmission, protection) is probably to be a major feature limiting the rate of host shifts that will likely impact on the utility of Spiroplasma in pest and vector control. Moving forward, the variation between sibling species pairs provides an opportunity to identify the mechanisms behind variable compatibility, which will drive hypotheses as to the evolutionary drivers of compatibility variation.

Evidence for multiple colonisations and Wolbachia infections shaping the genetic structure of the widespread butterfly Polyommatus icarus in the British Isles.

The paradigm of isolation in southern refugia during glacial periods followed by expansions during interglacials, producing limited genetic differentiation in northern areas, dominates European phylogeography. However, the existence of complex structured populations in formerly glaciated areas, and islands connected to mainland areas during glacial maxima, call for alternative explanations. We reconstructed the mtDNA phylogeography of the widespread Polyommatus Icarus butterfly with an emphasis on the formerly glaciated and connected British Isles. We found distinct geographical structuring of CO1 haplogroups, with an ancient lineage restricted to the marginal European areas, including Northern Scotland and Outer Hebrides. Population genomic analyses, using ddRADSeq genomic markers, also reveal substantial genetic structuring within Britain. However, there is negligble mito-nuclear concordance consistent with independent demographic histories of mitochondrial versus nuclear DNA. While mtDNA-Wolbachia associations in northern Britain could account for the geographic structuring of mtDNA across most of the British Isles, for nuclear DNA markers (derived from ddRADseq data) butterflies from France cluster between northern and southern British populations - an observation consistent with a scenario of multiple recolonisation. Taken together our results suggest that contemporary mtDNA structuring in the British Isles (and potentially elsewhere in Europe) largely results from Wolbachia infections, however, nuclear genomic structuring suggests a history of at least two distinct colonisations. This two-stage colonisation scenario has previously been put forth to explain genetic diversity and structuring in other British flora and fauna. Additionally, we also present preliminary evidence for potential Wolbachia-induced feminization in the Outer Hebrides.

Evolution of mitochondrial gene order in Annelida.

Annelida is a highly diverse animal group with over 21,000 described species. As part of Lophotrochozoa, the vast majority of annelids are currently classified into two groups: Errantia and Sedentaria, together forming Pleistoannelida. Besides these taxa, Sipuncula, Amphinomidae, Chaetopteridae, Oweniidae and Magelonidae can be found branching at the base of the tree. Comparisons of mitochondrial genomes have been used to investigate phylogenetic relationship within animal taxa. Complete annelid mitochondrial genomes are available for some Sedentaria and Errantia and in most cases exhibit a highly conserved gene order. Only two complete genomes have been published from the basal branching lineages and these are restricted to Sipuncula. We describe the first complete mitochondrial genome sequences for all other basal branching annelid families: Owenia fusiformis (Oweniidae), Magelona mirabilis (Magelonidae), Eurythoe complanata (Amphinomidae), Chaetopterus variopedatus and Phyllochaetopterus sp. (Chaetopteridae). The mitochondrial gene order of all these taxa is substantially different from the pattern found in Pleistoannelida. Additionally, we report the first mitochondrial genomes in Annelida that encode genes on both strands. Our findings demonstrate that the supposedly highly conserved mitochondrial gene order suggested for Annelida is restricted to Pleistoannelida, representing the ground pattern of this group. All investigated basal branching annelid taxa show a completely different arrangement of genes than observed in Pleistoannelida. The gene order of protein coding and ribosomal genes in Magelona mirabilis differs only in two transposition events from a putative lophotrochozoan ground pattern and might be the closest to an ancestral annelid pattern. The mitochondrial genomes of Myzostomida show the conserved pattern of Pleistoannelida, thereby supporting their inclusion in this taxon.

Tracing horizontal Wolbachia movements among bees (Anthophila): a combined approach using multilocus sequence typing data and host phylogeny.

The endosymbiotic bacterium Wolbachia enhances its spread via vertical transmission by generating reproductive effects in its hosts, most notably cytoplasmic incompatibility (CI). Additionally, frequent interspecific horizontal transfer is evident from a lack of phylogenetic congruence between Wolbachia and its hosts. The mechanisms of this lateral transfer are largely unclear. To identify potential pathways of Wolbachia movements, we performed multilocus sequence typing of Wolbachia strains from bees (Anthophila). Using a host phylogeny and ecological data, we tested various models of horizontal endosymbiont transmission. In general, Wolbachia strains seem to be randomly distributed among bee hosts. Kleptoparasite-host associations among bees as well as other ecological links could not be supported as sole basis for the spread of Wolbachia. However, cophylogenetic analyses and divergence time estimations suggest that Wolbachia may persist within a host lineage over considerable timescales and that strictly vertical transmission and subsequent random loss of infections across lineages may have had a greater impact on Wolbachia strain distribution than previously estimated. Although general conclusions about Wolbachia movements among arthropod hosts cannot be made, we present a framework by which precise assumptions about shared evolutionary histories of Wolbachia and a host taxon can be modelled and tested.

Draft genome sequence of a Wolbachia endosymbiont from Syringophilopsis turdi (Fritsch, 1958) (Acari, Syringophilidae).

We present the draft genome of a Wolbachia endosymbiont from quill mites. This is the first representative of a recently discovered distinct Wolbachia lineage (supergroup P). We hope the genome will be a useful resource for comparative evolutionary and genomic studies across the globally distributed symbiont Wolbachia.

Microbial protection favors parasite tolerance and alters host-parasite coevolutionary dynamics.

Coevolution between hosts and parasites is a major driver of rapid evolutionary change1 and diversification.2,3 However, direct antagonistic interactions between hosts and parasites could be disrupted4 when host microbiota form a line of defense, a phenomenon widespread across animal and plant species.5,6 By suppressing parasite infection, protective microbiota could reduce the need for host-based defenses and favor host support for microbiota colonization,6 raising the possibility that the microbiota can alter host-parasite coevolutionary patterns and processes.7 Here, using an experimental evolution approach, we co-passaged populations of nematode host (Caenorhabditis elegans) and parasites (Staphylococcus aureus) when hosts were colonized (or not) by protective bacteria (Enterococcus faecalis). We found that microbial protection during coevolution resulted in the evolution of host mortality tolerance-higher survival following parasite infection-and in parasites adapting to microbial defenses. Compared to unprotected host-parasite coevolution, the protected treatment was associated with reduced dominance of fluctuating selection dynamics in host populations. No differences in host recombination rate or genetic diversity were detected. Genomic divergence was observed between parasite populations coevolved in protected and unprotected hosts. These findings indicate that protective host microbiota can determine the evolution of host defense strategies and shape host-parasite coevolutionary dynamics.

Host genotype and genetic diversity shape the evolution of a novel bacterial infection.

Pathogens continue to emerge from increased contact with novel host species. Whilst these hosts can represent distinct environments for pathogens, the impacts of host genetic background on how a pathogen evolves post-emergence are unclear. In a novel interaction, we experimentally evolved a pathogen (Staphylococcus aureus) in populations of wild nematodes (Caenorhabditis elegans) to test whether host genotype and genetic diversity affect pathogen evolution. After ten rounds of selection, we found that pathogen virulence evolved to vary across host genotypes, with differences in host metal ion acquisition detected as a possible driver of increased host exploitation. Diverse host populations selected for the highest levels of pathogen virulence, but infectivity was constrained, unlike in host monocultures. We hypothesise that population heterogeneity might pool together individuals that contribute disproportionately to the spread of infection or to enhanced virulence. The genomes of evolved populations were sequenced, and it was revealed that pathogens selected in distantly-related host genotypes diverged more than those in closely-related host genotypes. S. aureus nevertheless maintained a broad host range. Our study provides unique empirical insight into the evolutionary dynamics that could occur in other novel infections of wildlife and humans.

Rapid molecular evolution of Spiroplasma symbionts of Drosophila.

Spiroplasma is a genus of Mollicutes whose members include plant pathogens, insect pathogens and endosymbionts of animals. Spiroplasma phenotypes have been repeatedly observed to be spontaneously lost in Drosophila cultures, and several studies have documented a high genomic turnover in Spiroplasma symbionts and plant pathogens. These observations suggest that Spiroplasma evolves quickly in comparison to other insect symbionts. Here, we systematically assess evolutionary rates and patterns of Spiroplasma poulsonii, a natural symbiont of Drosophila. We analysed genomic evolution of sHy within flies, and sMel within in vitro culture over several years. We observed that S. poulsonii substitution rates are among the highest reported for any bacteria, and around two orders of magnitude higher compared with other inherited arthropod endosymbionts. The absence of mismatch repair loci mutS and mutL is conserved across Spiroplasma, and likely contributes to elevated substitution rates. Further, the closely related strains sMel and sHy (>99.5 % sequence identity in shared loci) show extensive structural genomic differences, which potentially indicates a higher degree of host adaptation in sHy, a protective symbiont of Drosophila hydei. Finally, comparison across diverse Spiroplasma lineages confirms previous reports of dynamic evolution of toxins, and identifies loci similar to the male-killing toxin Spaid in several Spiroplasma lineages and other endosymbionts. Overall, our results highlight the peculiar nature of Spiroplasma genome evolution, which may explain unusual features of its evolutionary ecology.

Microbial composition of enigmatic bird parasites: Wolbachia and Spiroplasma are the most important bacterial associates of quill mites (Acariformes: Syringophilidae).

BACKGROUND: The microbiome is an integral component of many animal species, potentially affecting behavior, physiology, and other biological properties. Despite this importance, bacterial communities remain vastly understudied in many groups of invertebrates, including mites. Quill mites (Acariformes: Syringophilidae) are a poorly known group of permanent bird ectoparasites that occupy quills of feathers and feed on bird subcutaneous tissue and fluids. Most of the known species have strongly female-biased sex ratio, and it was hypothesized that this is caused by endosymbiotic bacteria. Previously, Anaplasma phagocytophilum (Foggie) and a high diversity of Wolbachia strains were detected in quill mites via targeted PCR screens. Here, we use an unbiased 16S rRNA gene amplicon sequencing approach to determine other bacteria that potentially impact quill mite biology. RESULTS: We performed 16S rRNA gene amplicon sequencing of 126 quill mite individuals from eleven species parasitizing twelve species (four families) of passeriform birds. In addition to Wolbachia, we found Spiroplasma as potential symbiont of quill mites. Consistently, high Spiroplasma titers were only found in individuals of two mite species associated with finches of the genus Carduelis, suggesting a history of horizontal transfers of Spiroplasma via the bird host. Furthermore, there was evidence for Spiroplasma negatively affecting Wolbachia titers. We found no evidence for the previously reported Anaplasma in quill mites, but detected sequences of high similarity to the potential pathogens Brucella and Bartonella at low abundances. Other amplicon sequence variants (ASVs) could be assigned to a diverse number of bacterial taxa, including several that were previously isolated from bird skin. Further, many frequently found ASVs were assigned to taxa that show a very broad distribution with no strong prior evidence for symbiotic association with animals. We interpret these findings as evidence for a scarcity of resident microbial associates (other than inherited symbionts) in quill mites.

'Multi-SpaM': a maximum-likelihood approach to phylogeny reconstruction using multiple spaced-word matches and quartet trees.

Word-based or 'alignment-free' methods for phylogeny inference have become popular in recent years. These methods are much faster than traditional, alignment-based approaches, but they are generally less accurate. Most alignment-free methods calculate 'pairwise' distances between nucleic-acid or protein sequences; these distance values can then be used as input for tree-reconstruction programs such as neighbor-joining. In this paper, we propose the first word-based phylogeny approach that is based on 'multiple' sequence comparison and 'maximum likelihood'. Our algorithm first samples small, gap-free alignments involving four taxa each. For each of these alignments, it then calculates a quartet tree and, finally, the program 'Quartet MaxCut' is used to infer a super tree for the full set of input taxa from the calculated quartet trees. Experimental results show that trees produced with our approach are of high quality.

Infection Patterns and Fitness Effects of Rickettsia and Sodalis Symbionts in the Green Lacewing Chrysoperla carnea.

Endosymbionts are widely distributed in insects and can strongly affect their host ecology. The common green lacewing (Chrysoperla carnea) is a neuropteran insect which is widely used in biological pest control. However, their endosymbionts and their interactions with their hosts have not been very well studied. Therefore, we screened for endosymbionts in natural and laboratory populations of Ch. carnea using diagnostic PCR amplicons. We found the endosymbiont Rickettsia to be very common in all screened natural and laboratory populations, while a hitherto uncharacterized Sodalis strain was found only in laboratory populations. By establishing lacewing lines with no, single or co-infections of Sodalis and Rickettsia, we found a high vertical transmission rate for both endosymbionts (>89%). However, we were only able to estimate these numbers for co-infected lacewings. Sodalis negatively affected the reproductive success in single and co-infected Ch. carnea, while Rickettsia showed no effect. We hypothesize that the fitness costs accrued by Sodalis infections might be more tolerable in the laboratory than in natural populations, as the latter are also prone to fluctuating environmental conditions and natural enemies. The economic and ecological importance of lacewings in biological pest control warrants a more profound understanding of its biology, which might be influenced by symbionts.

Is Anopheles gambiae a Natural Host of Wolbachia?

Wolbachia (Alphaproteobacteria, Rickettsiales) is an intraovarially transmitted symbiont of insects able to exert striking phenotypes, including reproductive manipulations and pathogen blocking. These phenotypes make Wolbachia a promising tool to combat mosquito-borne diseases. Although Wolbachia is present in the majority of terrestrial arthropods, including many disease vectors, it was considered absent from Anopheles gambiae mosquitos, the main vectors of malaria in sub-Saharan Africa. In 2014, Wolbachia sequences were detected in A. gambiae samples collected in Burkina Faso. Subsequently, similar evidence came from collections all over Africa, revealing a high Wolbachia 16S rRNA sequence diversity, low abundance, and a lack of congruence between host and symbiont phylogenies. Here, we reanalyze and discuss recent evidence on the presence of Wolbachia sequences in A. gambiae. We find that although detected at increasing frequencies, the unusual properties of these Wolbachia sequences render them insufficient to diagnose natural infections in A. gambiae Future studies should focus on uncovering the origin of Wolbachia sequence variants in Anopheles and seeking sequence-independent evidence for this new symbiosis. Understanding the ecology of Anopheles mosquitos and their interactions with Wolbachia will be key in designing successful, integrative approaches to limit malaria spread. Although the prospect of using Wolbachia to fight malaria is intriguing, the newly discovered strains do not bring it closer to realization.IMPORTANCEAnopheles gambiae mosquitos are the main vectors of malaria, threatening around half of the world's population. The bacterial symbiont Wolbachia can interfere with disease transmission by other important insect vectors, but until recently, it was thought to be absent from natural A. gambiae populations. Here, we critically analyze the genomic, metagenomic, PCR, imaging, and phenotypic data presented in support of the presence of natural Wolbachia infections in A. gambiae We find that they are insufficient to diagnose Wolbachia infections and argue for the need of obtaining robust data confirming basic Wolbachia characteristics in this system. Determining the Wolbachia infection status of Anopheles is critical due to its potential to influence Anopheles population structure and Plasmodium transmission.

Prot-SpaM: fast alignment-free phylogeny reconstruction based on whole-proteome sequences.

Word-based or 'alignment-free' sequence comparison has become an active research area in bioinformatics. While previous word-frequency approaches calculated rough measures of sequence similarity or dissimilarity, some new alignment-free methods are able to accurately estimate phylogenetic distances between genomic sequences. One of these approaches is Filtered Spaced Word Matches. Here, we extend this approach to estimate evolutionary distances between complete or incomplete proteomes; our implementation of this approach is called Prot-SpaM. We compare the performance of Prot-SpaM to other alignment-free methods on simulated sequences and on various groups of eukaryotic and prokaryotic taxa. Prot-SpaM can be used to calculate high-quality phylogenetic trees for dozens of whole-proteome sequences in a matter of seconds or minutes and often outperforms other alignment-free approaches. The source code of our software is available through Github: https://github.com/jschellh/ProtSpaM.

A critical re-evaluation of multilocus sequence typing (MLST) efforts in Wolbachia.

Wolbachia (Alphaproteobacteria, Rickettsiales) is the most common, and arguably one of the most important inherited symbionts. Molecular differentiation of Wolbachia strains is routinely performed with a set of five multilocus sequence typing (MLST) markers. However, since its inception in 2006, the performance of MLST in Wolbachia strain typing has not been assessed objectively. Here, we evaluate the properties of Wolbachia MLST markers and compare it to 252 other single copy loci present in the genome of most Wolbachia strains. Specifically, we investigated how well MLST performs at strain differentiation, at reflecting genetic diversity of strains, and as phylogenetic marker. We find that MLST loci are outperformed by other loci at all tasks they are currently employed for, and thus that they do not reflect the properties of a Wolbachia strain very well. We argue that whole genome typing approaches should be used for Wolbachia typing in the future. Alternatively, if few loci approaches are necessary, we provide a characterisation of 252 single copy loci for a number a criteria, which may assist in designing specific typing systems or phylogenetic studies.

Symbiosis: Wolbachia Host Shifts in the Fast Lane.

The inherited bacterium Wolbachia is an important component of the biology of many arthropods. What makes it so common? An analysis of drosophilids revealed one strain host shifts at a surprisingly high rate, infecting eight species in under 30,000 years.

Short reads from honey bee (Apis sp.) sequencing projects reflect microbial associate diversity.

High throughput (or 'next generation') sequencing has transformed most areas of biological research and is now a standard method that underpins empirical study of organismal biology, and (through comparison of genomes), reveals patterns of evolution. For projects focused on animals, these sequencing methods do not discriminate between the primary target of sequencing (the animal genome) and 'contaminating' material, such as associated microbes. A common first step is to filter out these contaminants to allow better assembly of the animal genome or transcriptome. Here, we aimed to assess if these 'contaminations' provide information with regard to biologically important microorganisms associated with the individual. To achieve this, we examined whether the short read data from Apis retrieved elements of its well established microbiome. To this end, we screened almost 1,000 short read libraries of honey bee (Apis sp.) DNA sequencing project for the presence of microbial sequences, and find sequences from known honey bee microbial associates in at least 11% of them. Further to this, we screened ∼500 Apis RNA sequencing libraries for evidence of viral infections, which were found to be present in about half of them. We then used the data to reconstruct draft genomes of three Apis associated bacteria, as well as several viral strains de novo. We conclude that 'contamination' in short read sequencing libraries can provide useful genomic information on microbial taxa known to be associated with the target organisms, and may even lead to the discovery of novel associations. Finally, we demonstrate that RNAseq samples from experiments commonly carry uneven viral loads across libraries. We note variation in viral presence and load may be a confounding feature of differential gene expression analyses, and as such it should be incorporated as a random factor in analyses.

Green lacewings (Neuroptera: Chrysopidae) are commonly associated with a diversity of rickettsial endosymbionts.

BACKGROUND: Bacterial symbionts transmitted from mothers to offspring are found in the majority of arthropods. Numerous studies have illustrated their wide impact on host biology, such as reproduction, behavior, and physiology One of the most common inherited symbionts is Rickettsia spp. (Alphaproteobacteria, Rickettsiales), which are found in about one-quarter of terrestrial arthropods, as well as in other invertebrates. In insect populations, Rickettsia spp. have been reported to cause reproductive modifications and fecundity-enhancing effects. Here, we investigated the incidence and genetic diversity of Rickettsia symbionts in green lacewings (Neuroptera, Chrysopidae), which are best known for their use as biological control agents against crop pests. RESULTS: We screened 18 species of green lacewings and allies for Rickettsia and found the symbiont in 10 species, infecting 20-100% of sampled individuals. Strain characterization based on multiple bacterial loci revealed an unprecedented diversity of Rickettsia associated with lacewings, suggesting multiple independent acquisitions. Further, the detected Rickettsia lineages are restricted to a specific lineage (i.e., species or genus) of investigated lacewings, and these associations are stable across multiple sampled locations and points in time. CONCLUSIONS: We conclude that Rickettsia-lacewing symbioses are common and evolutionarily stable. The role of these symbionts remains to be identified, but is potentially important to optimizing their use in biological pest control.

Comparative genomics provides a timeframe for Wolbachia evolution and exposes a recent biotin synthesis operon transfer.

The genus Wolbachia (Alphaproteobacteria) comprises the most abundant inherited intracellular bacteria1. Despite their relevance as manipulators of human pathogen transmission2 and arthropod reproduction3, many aspects of their evolutionary history are not well understood4. In arthropods, Wolbachia infections are typically transient on evolutionary timescales5,6 and co-divergence between hosts and Wolbachia is supposedly rare. Consequently, much of our knowledge of Wolbachia genome evolution derives from very recently diverged strains, and a timescale for Wolbachia is lacking. Here, we investigated the genomes of four Wolbachia strains that have persisted within and co-diverged with their host lineage for ∼2 million years. Although the genomes showed very little evolutionary change on a nucleotide level, we found evidence for a recent lateral transfer of a complete biotin synthesis operon that has the potential to transform Wolbachia-host relationships7. Furthermore, this evolutionary snapshot enabled us to calibrate the divergence times of the supergroup A and B Wolbachia lineages using genome-wide data sets and relaxed molecular clock models. We estimated the origin of Wolbachia supergroups A and B to be ∼200 million years ago (Ma), which is considerably older than previously appreciated. This age coincides with the diversification of many insect lineages8 that represent most of Wolbachia's host spectrum.

Characterization of the complete mitochondrial genomes from Polycladida (Platyhelminthes) using next-generation sequencing.

The complete mitochondrial genomes of three polycladids, the acotylean Hoploplana elisabelloi and the cotyleans Enchiridium sp. and Prosthiostomum siphunculus have been assembled with high coverage from Illumina sequencing data. The mt genomes contain 36 genes including 12 of the 13 protein-coding genes characteristic for metazoan mitochondrial genomes, two ribosomal RNA genes, and 22 transfer RNA genes. Gene annotation, gene order, genetic code, start and stop codons and codon bias have been identified. In comparison with the well investigated parasitic Neodermata, our analysis reveals a great diversity of gene orders within Polycladida and Platyhelminthes in general. By analyzing representative genomes of the main groups of Platyhelminthes we explored the phylogenetic relationships of this group. The phylogenetic analyses strongly supported the monophyly of Polycladida, and based on a small taxon sampling suggest the monophyly of Acotylea and Cotylea.

Ancient horizontal transfers of retrotransposons between birds and ancestors of human pathogenic nematodes.

Parasite host switches may trigger disease emergence, but prehistoric host ranges are often unknowable. Lymphatic filariasis and loiasis are major human diseases caused by the insect-borne filarial nematodes Brugia, Wuchereria and Loa. Here we show that the genomes of these nematodes and seven tropical bird lineages exclusively share a novel retrotransposon, AviRTE, resulting from horizontal transfer (HT). AviRTE subfamilies exhibit 83-99% nucleotide identity between genomes, and their phylogenetic distribution, paleobiogeography and invasion times suggest that HTs involved filarial nematodes. The HTs between bird and nematode genomes took place in two pantropical waves, >25-22 million years ago (Myr ago) involving the Brugia/Wuchereria lineage and >20-17 Myr ago involving the Loa lineage. Contrary to the expectation from the mammal-dominated host range of filarial nematodes, we hypothesize that these major human pathogens may have independently evolved from bird endoparasites that formerly infected the global breadth of avian biodiversity.