Classification of Wolbachia (Alphaproteobacteria, Rickettsiales): No evidence for a distinct supergroup in cave spiders

Wolbachia are intracellular, inherited Alphaproteobacteria present in a large proportion of arthropod species and in filarial nematodes. Wolbachia-host interactions are many-faceted and include, but are not limited to reproductive manipulations (Werren et al. 2008), nutritional mutualism (Nikoh et al. 2014), and protection from pathogens (Hedges et al. 2008, Teixeira et al. 2008). Although there is just a single Wolbachia species described, the genus is highly diverse with regard to its distribution, phenotypes induced in the host, and genomic architecture. The generally accepted classification scheme currently in use differentiates among genetically distinct, monophyletic lineages named 9supergroups9 (Lindsey et al. 2016). To date, 16 of these Wolbachia lineages that - based on multiple genetic markers - are clearly distinct from another were described (supergroups A-F and H-Q, Glowska et al. 2015). 
 In a recent study, Wang et al. (2016) reported the discovery of a novel, 17th Wolbachia supergroup (9R9) from cave spiders (Telema ssp.). The authors base this conclusion on phylogenetic analyses of sequences from three protein coding genes (ftsZ, coxA, groEL) and the 16S rRNA gene. Here, I re-analyse these data and show that Wolbachia from Telema spiders clusters with supergroup A strains and thus, the creation of a novel Wolbachia supergroup for these strains is without justification.


To the editor
Wolbachia are intracellular, inherited Alphaproteobacteria present in a large proportion of arthropod species and in filarial nematodes. Wolbachia-host interactions are many-faceted and include, but are not limited to reproductive manipulations (Werren et al. 2008), nutritional mutualism (Nikoh et al. 2014), and protection from pathogens (Hedges et al. 2008, Teixeira et al. 2008. Although there is just a single Wolbachia species described, the genus is highly diverse with regard to its distribution, phenotypes induced in the host, and genomic architecture. The generally accepted classification scheme currently in use differentiates among genetically distinct, monophyletic lineages named "supergroups" (Lindsey et al. 2016). To date, 16 of these Wolbachia lineages thatbased on multiple genetic markers -are clearly distinct from another were described (supergroups A-F and H-Q, Glowska et al. 2015).
In a recent study, Wang et al. (2016, from here on "Wang et al.") reported the discovery of a novel, 17 th Wolbachia supergroup ("R") from cave spiders (Telema ssp.). The authors base this conclusion on phylogenetic analyses of sequences from three protein coding genes (ftsZ, coxA, groEL) and the 16S rRNA gene. Here, I re-analyse these data and show that Wolbachia from Telema spiders clusters with supergroup A strains and thus, the creation of a novel Wolbachia supergroup for these strains is without justification.
Using the sequence data generated by Wang et al., I first performed an online BLAST search of all ftsZ, coxA, groEL, and 16S sequences (2, 6, 1, and 5 haplotypes, respectively) against the nucleotide database of NCBI GenBank. Strikingly, all of the sequences match to known supergroup A strains with an identity greater than 99%. Next, using Mafft version 7.245 (Katoh and Standley 2013) with automatic parameter settings, I aligned all novel sequences with the corresponding orthologs extracted from the Wolbachia genomes wRi, wHa, wMel (all supergroup A), wPip & wNo (supergroup B), and wCle (supergroup F). Using Aliview version 1.17.1 (Larsson 2014), the datasets were then trimmed manually to include only positions that were sequenced by Wang et al. CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted March 30, 2016. ; https://doi.org/10.1101/046169 doi: bioRxiv preprint All alignments are available under https://github.com/gerthmicha/supergroup_R. From these alignments, raw genetic distances were calculated using the "ape" package within the R statistical environment (Paradis et al. 2004, R Core Team 2016. All Wolbachia loci from Telema hosts are very similar to each other and to the reference supergroup A loci (~0-1% distance, Fig. S1). In general, the distances within supergroup A (including Telema strains) are almost one order of magnitude smaller compared to the distances between supergroups (Fig. S1). The distance measures are therefore in line with the conclusion that Wolbachia from cave spiders are supergroup A strains.
Finally, maximum likelihood trees were created for all single gene alignments and a supermatrix of all concatenated genes using IQ-TREE multicore version 1.4.1 (Nguyen et al. 2015) with automated model selection ("-m TEST") and 1000 ultrafast bootstrap replicates. All of the single gene trees and the tree based on the concatenated genes strongly support an association of Wolbachia strains from Telema ssp. with supergroup A (Fig. 1). This becomes especially clear when comparing the genetic distances within supergroup A and the distances between supergroups A, B, and F ( Fig. 1).
Visual comparisons of the trees presented in Fig. 1  CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted March 30, 2016. ; https://doi.org/10.1101/046169 doi: bioRxiv preprint Wang et al. and led them to conclude that a single locus is not sufficient to resolve phylogenetic relationships within Wolbachia. Whilst this is certainly true, the observation that each single gene analysis consistently places Wolbachia from Telema ssp. within (or closely associated to) supergroup A questions the validity of the supermatrix analysis. Further to this, the fact that In summary, the here presented similarity searches, genetic distance measures, and phylogenetic analyses are all consistent and provide strong evidence for the association of Wolbachia strains from Telema with supergroup A. Their placement within a novel supergroup, as proposed by Wang et al., lacks any support and is likely artefactual. In order to minimize confusion, it may be helpful not to assign the name "supergroup R" to any novel Wolbachia lineage potentially to be discovered in the future.

Acknowledgements
I am grateful to Guan-Hong Wang for providing sequence data and for discussions. I thank  not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made