Comparative Genomics of Wolbachia and the Bacterial Species Concept

Ellegaard, Kirsten; Klasson, Lisa; Näslund, Kristina; Bourtzis, Kostas; Andersson, Siv G. E.

doi:10.1371/journal.pgen.1003381

Cited by 162 publications

(194 citation statements)

References 100 publications

(109 reference statements)

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“…Many methodologies physically separated the W. pipientis DNA from D. melanogaster DNA using differential centrifugation and pulse-field gel separation, which requires 1000 live adult flies to obtain sufficient quantities of W. pipientis DNA for genomic sequencing (Sun et al 2001;Wu et al 2004), thus eliminating the possibility of acquiring W. pipientis genomes from individual flies. Very deep sequencing of flies (Richardson et al 2012) or physical isolation of target DNA are becoming more feasible with technological advances (Richardson et al 2012;Ellegaard et al 2013), although they are still inefficient in both cost and labor and are thus prohibitive for population-level studies. The SWGA method maximizes the amount of target microbial DNA sequenced to create an efficient and cost-effective method to pursue population genomic studies.…”

Section: Discussionmentioning

confidence: 99%

Selective Whole Genome Amplification for Resequencing Target Microbial Species from Complex Natural Samples

Leichty¹,

Brisson

2014

Genetics

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Population genomic analyses have demonstrated power to address major questions in evolutionary and molecular microbiology. Collecting populations of genomes is hindered in many microbial species by the absence of a cost effective and practical method to collect ample quantities of sufficiently pure genomic DNA for next-generation sequencing. Here we present a simple method to amplify genomes of a target microbial species present in a complex, natural sample. The selective whole genome amplification (SWGA) technique amplifies target genomes using nucleotide sequence motifs that are common in the target microbe genome, but rare in the background genomes, to prime the highly processive phi29 polymerase. SWGA thus selectively amplifies the target genome from samples in which it originally represented a minor fraction of the total DNA. The post-SWGA samples are enriched in target genomic DNA, which are ideal for population resequencing. We demonstrate the efficacy of SWGA using both laboratoryprepared mixtures of cultured microbes as well as a natural host-microbe association. Targeted amplification of Borrelia burgdorferi mixed with Escherichia coli at genome ratios of 1:2000 resulted in .10 5 -fold amplification of the target genomes with ,6.7-fold amplification of the background. SWGA-treated genomic extracts from Wolbachia pipientis-infected Drosophila melanogaster resulted in up to 70% of high-throughput resequencing reads mapping to the W. pipientis genome. By contrast, 2-9% of sequencing reads were derived from W. pipientis without prior amplification. The SWGA technique results in high sequencing coverage at a fraction of the sequencing effort, thus allowing population genomic studies at affordable costs. C LASSICAL population genetics, coupled with advances in coalescent modeling, has been foundational to studies of the evolutionary histories and ecological forces that shape natural populations (Rosenberg and Nordborg 2002;Hume et al. 2003;Wakeley 2004). However, detecting fine scale processes using population genetics and coalescent analyses is limited by the amount of available sequence data per sample. Datasets with substantially greater genetic information per sample, such as genomic data from population-level sampling, would be optimal to study biological processes at all relevant scales. The promise of population genomics for many microbial species is tempered, however, by the difficulty of isolating and preparing microbial genomes for nextgeneration sequencing. Currently, sequencing microbial genomes requires laboratory culture to isolate them from other organisms with which they are naturally associated to obtain the appropriate samples for sequencing-sufficient numbers of the target genome with limited contaminating DNA (Mardis 2008).Methodological issues in obtaining populations of genomes from microbial species occur both because the target microbial genomes often constitutes only a miniscule fraction of the DNA in complex, field-derived samples and because many important microbial specie...

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Section: Discussionmentioning

confidence: 99%

Selective Whole Genome Amplification for Resequencing Target Microbial Species from Complex Natural Samples

Leichty¹,

Brisson

2014

Genetics

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“…As a control, we performed the same search using the complete reference genomes for w Ri (Klasson et al., 2009), w Au (Sutton, Harris, Parkhill, & Sinkins, 2014), w Mel (Wu et al., 2004), w Ha, and w No (Ellegaard et al., 2013). …”

Section: Methodsmentioning

confidence: 99%

Genome comparisons indicate recent transfer ofwRi‐likeWolbachiabetween sister speciesDrosophila suzukiiandD. subpulchrella

Conner

Blaxter

Anfora

et al. 2017

Ecology and Evolution

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Wolbachia endosymbionts may be acquired by horizontal transfer, by introgression through hybridization between closely related species, or by cladogenic retention during speciation. All three modes of acquisition have been demonstrated, but their relative frequency is largely unknown. Drosophila suzukii and its sister species D. subpulchrella harbor Wolbachia, denoted wSuz and wSpc, very closely related to wRi, identified in California populations of D. simulans. However, these variants differ in their induced phenotypes: wRi causes significant cytoplasmic incompatibility (CI) in D. simulans, but CI has not been detected in D. suzukii or D. subpulchrella. Our draft genomes of wSuz and wSpc contain full‐length copies of 703 of the 734 single‐copy genes found in wRi. Over these coding sequences, wSuz and wSpc differ by only 0.004% (i.e., 28 of 704,883 bp); they are sisters relative to wRi, from which each differs by 0.014%–0.015%. Using published data from D. melanogaster, Nasonia wasps and Nomada bees to calibrate relative rates of Wolbachia versus host nuclear divergence, we conclude that wSuz and wSpc are too similar—by at least a factor of 100—to be plausible candidates for cladogenic transmission. These three wRi‐like Wolbachia, which differ in CI phenotype in their native hosts, have different numbers of orthologs of genes postulated to contribute to CI; and the CI loci differ at several nucleotides that may account for the CI difference. We discuss the general problem of distinguishing alternative modes of Wolbachia acquisition, focusing on the difficulties posed by limited knowledge of variation in absolute and relative rates of molecular evolution for host nuclear genomes, mitochondria, and Wolbachia.

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“…Many of the tests herald from medical microbiology introduced in the late nineteenth and early twentieth century but are still applied to environmental isolates. Among the many examples that might be given to illustrate that polyphasic taxonomy is failing in the description of biodiversity are the cases of Burkholderia (Vandamme and Peeters 2014), Wolbachia (Ellegaard et al 2013), and Pseudomonas (AlvarezPérez et al 2013). For Wolbachia, two genetically distinct and irreversibly separated clades were distinguished (Ellegaard et al 2013), but these cannot be described as species.…”

Section: The Failure Of Polyphasic Taxonomymentioning

confidence: 99%

“…Whereas monoculture experimental standards and rules have guided the description of bacterial and archaeal species in the past, several colleagues have stressed that the time has come to integrate genomics as a reliable and reproducible standard into the taxonomy of the bacteria and archaea (Lan and Reeves 2000;Doolittle and Papke 2006;Fraser et al 2009;Whitman 2009;Staley 2009;Klenk and Göker 2010;Zhi et al 2012;Ellegaard et al 2013;Chun and Rainey 2014). However, simply incorporating genome sequence data into polyphasic taxonomy as proposed by Ramasamy et al (2014) might not be sufficient.…”

Section: The Failure Of Polyphasic Taxonomymentioning

confidence: 99%