How Many Wolbachia Supergroups Exist?

Abstract

Obligate intracellular bacteria of the genus Wolbachia (Class Alphaproteobacteria, Order Rickettsiales) are currently divided into four taxonomic supergroups on the basis of clustering patterns in ftsZ-based phylogenetic trees (Werren, Zhang, and Guo 1995 ; Bandi et al. 1998 ). Supergroups A and B are found only in arthropods, whereas C and D are found only in filarial nematodes. The term supergroup has recently been employed to avoid confusion with designation of more closely related groups based on wsp sequences (Zhou, Rousset, and O'Neill 1998 ). Wolbachia have generated substantial interest in recent years (Zimmer 2001), primarily because of the effects they have on their arthropod hosts, which include induction of cytoplasmic incompatibility (CI), parthenogenesis, feminization, and male-killing (reviewed in Stouthammer, Breeuwer, and Hurst 1999 ). Estimation of the phylogenetic relationships within each supergroup has provided useful information about the evolution and biology of these bacteria. The phylogenies of both A and B members have been found to be incongruent with that of their hosts, strongly suggesting horizontal transfer (Werren, Zhang, and Guo 1995 ). Recently, direct evidence for this phenomenon was found for parasitic wasps sharing a common food source (Huigens et al. 2000). Unlike the case of arthropods, the phylogeny of each nematode Wolbachia supergroup (C and D) appears to match that of their hosts (Casiraghi et al. 2001a ), although further gene sequencing studies are required to confirm this. Such phylogenetic congruence would suggest a strictly dependent relationship, and this idea is supported by evidence that removal of Wolbachia using antibiotics has negative effects on the filariae they reside in (Bandi et al. 1999 ; Langworthy et al. 2000 ). Progress toward answering several remaining questions about Wolbachia evolution—such as which of their host effects are primitive and which are derived, the type of animals they first invaded, and how they were transferred between arthropods and nematodes—is currently hindered by a poor understanding of the relationships between the supergroups. An improved estimate of Wolbachia phylogeny at this level will require: (1) the inclusion of sequence information from diverse, possibly as-yet-unknown taxa, (2) an appropriate choice of genes and outgroups, and (3) the use of sound data analysis techniques which enable statistical assessment of results. In this report, we demonstrate the presence of two divergent Wolbachia lineages, which increases the number of recognized supergroups from four to six. Phylogenetic analyses have been performed using likelihood-based techniques, which are becoming increasingly accepted as the most reliable and efficient for phylogenetic inference (Whelan, Liò, and Goldman 2001 ). Whereas some recent Wolbachia studies have explicitly used likelihood methods to examine within-supergroup relationships (Von der Schulenberg et al. 2000 ; Jiggins et al. 2001 ), to our knowledge this study represents the first likelihood-based analysis of relationships between supergroups. A report by Vandekerchove et al. (1999) on endosymbionts from the springtail Folsomia candida (Collembola) proposed that these bacteria represent a fifth divergent lineage of Wolbachia (supergroup E). However, rather than using ftsZ—the gene originally used to describe supergroups A-D—the authors based their proposal on the relatively slow-evolving 16S rDNA. The evidence supporting the establishment of this new Wolbachia supergroup could be considered inconclusive for the following reasons: (1) the bootstrap value for the separation of the clade containing A + B members from the branch representing the Wolbachia from F. candida was marginal (55%), and (2) the divergence between the 16S rDNAs of Wolbachia from F. candida and those of A members was very low (∼1%) (Vandekerchove et al. 1999 ). In order to examine whether Wolbachia from F. candida do actually represent a divergent supergroup, we amplified and sequenced ftsZ from representatives of this strain. Additionally, ftsZ sequences from Wolbachia strains present in the two termite (Isoptera) species Kalotermes flavicollis and Microcerotermes sp. (Bandi et al. 1997 ) were obtained. Wolbachia from K. flavicollis had previously been shown to be divergent from other Wolbachia based on 16S rDNA sequences (Bandi et al. 1999 ). DNA was extracted from five pooled F. candida individuals and abdomen tissues from individual termites (Bandi et al. 1994 ). Initial PCR attempts with the commonly used primers ftsZf1 and ftsZr1 (Werren, Zhang, and Guo 1995 ) were met with failure, and thus two additional primers (ftsZunif 5′–3′ GG(CT)AA(AG)GGTGC(AG)GCAGAAGA; ftsZunir 5′–3′ ATC(AG)AT(AG)CCAGTTGCAAG) were designed based on an alignment of all arthropod and filarial WolbachiaftsZ sequences. These primers enabled amplification of a 775-bp fragment (737 bp excluding primers) from F. candida and each termite specimen. Bo