Phylogeny and PCR–based classification of Wolbachia strains using wsp gene sequences

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Abstract
Wolbachia are a group of intracellular inherited bacteria that infect a wide range of arthropods. They are associated with a number of different reproductive phenotypes in their hosts, such as cytoplasmic incompatibility, parthenogenesis and feminization. While it is known that the bacterial strains responsible for these different host phenotypes form a single clade within the α–Proteobacteria, until now it has not been possible to resolve the evolutionary relationships between differentWolbachia strains. To address this issue we have cloned and sequenced a gene encoding a surface protein ofWolbachia (wsp) from a representative sample of 28Wolbachia strains. The sequences from this gene were highly variable and could be used to resolve the phylogenetic relationships of differentWolbachia strains. Based on the sequence of thewsp gene from differentWolbachia isolates we propose that theWolbachia pipientis clade be initially divided into 12 groups. As more sequence information becomes available we expect the number of such groups to increase. In addition, we present a method ofWolbachia classification based on the use of group–specificwsp PCR primers which will allowWolbachia isolates to be typed without the need to clone and sequence individualWolbachia genes. This system should facilitate future studies investigating the distribution and biology ofWolbachia strains from large samples of different host species.