Discovery of a novel Wolbachia supergroup in Isoptera Seth Bordenstein* and Rebeca B. Rosengaus** * The Marine Biological Laboratory The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution 7 MBL Street Woods Hole, MA 02543 Email: sbordenstein@mbl.edu / Phone: 508-289-7220 / Fax: 508-457-4727 ** Northeastern University Department of Biology 134 Mugar Life Sciences Building 360 Huntington Avenue Boston, MA 02115-5000 Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY764275-AY764284. Running title: Wolbachia in termites Key Words: endosymbiont, Isoptera, supergroup, Wolbachia, Zootermopsis 1 Abstract. Wolbachia are one of the most abundant groups of bacterial endosymbionts in the biosphere. Interest in these heritable microbes has expanded with the discovery of wider genetic diversity in undersampled host species. Here, we report on the putative discovery of a new genetic lineage, denoted supergroup H, which infects the Isopteran species Zootermopsis angusticollis and Z. nevadensis. Evidence for this novel supergroup is based on portions of new Wolbachia gene sequences from each species spanning 3.5 Kilobases of DNA and the following genes: 16S rDNA, dnaA, gltA, groEL, ftsZ. Single gene and concatenated maximum likelihood phylogenies establish this new supergroup and validate the positioning of the other Wolbachia supergroups. This discovery is the first example of a termite Wolbachia that is highly divergent from the Isopteran Wolbachia previously described in supergroup F. This study highlights the importance of multilocus approaches to resolving Wolbachia supergroup relationships. It also suggests that surveys of Wolbachia in more earlier-originating (and undersampled) groups of arthropods are more apt to reveal novel genetic diversity. Wolbachia is a genus of obligate, intracellular bacteria in the Anaplasmatacea family of the -Proteobacteria [37], which also includes the intracellular parasites Ehrlichia, Anaplasma, Neorickettsia, and Rickettsia [14]. Despite the phylogenetic proximity to these mammalian pathogens, Wolbachia evolved a distinct endosymbiotic lifestyle within a broad Ecdyzoan host range that spans a diversity of taxa within the phylum Arthropoda [33] and filarial nematodes [3]. Because of the limitations of studying non-culturable microbes such as obligate bacterial endosymbionts, the majority of Wolbachia research has focused on Wolbachia distribution [20, 38], cytology [10, 23, 25, 34], and the phenotypic effects of infection throughout their hosts [9, 18]. Findings from these studies indicate that Wolbachia may be among the most widespread 2 obligate bacterial endosymbionts on the planet and that the symbiotic associations formed by these bacteria span the symbiosis spectrum from reproductive parasitism to mutualism. Much less is known about the phylogenomics and molecular evolution of this important endosymbiont. This gap in knowledge is evident by a lack of robust molecular markers for finescale strain typing and numerous sequence analyses of genetic drift to which other insect endosymbionts have been subjected [12, 22]. With two Wolbachia genome sequence complete [16, 40] and several others partial genomes published [28], it will become increasingly important to combine the perspectives of both phenotypic and molecular evolution approaches under a solid taxonomic framework. Recent efforts reveal an increasing level of taxonomic diversity. In the absence of a formal species identification system, the Wolbachia community currently refers to the different lineages as supergroups and today, the genus is divided into seven taxonomic supergroups (denoted A through G) based on phylogenetic studies of up to six chromosomal genes [21, 27]. These seven supergroups are labeled alphabetically and include A and B of various arthropods [39], C and D of filarial nematodes [1, 7], E of springtails [11, 21, 35], F of termites, weevils, true bugs, and filarial nematodes [21, 24], and G of Australian spiders [27]. Additional taxonomic diversity has recently been discovered in filarial nematodes and fleas (Casiraghi et al, unpublished data), though these have not officially been labeled supergroups. Supergroups are defined loosely in the Wolbachia literature as distinct clades that typically adhere to the criteria of greater than 3% divergence in 16S rDNA sequences [32]. However, it is becoming increasingly clear that 16S rDNA sequences do not provide an accurate measure of phylogenetic relationships and that multilocus approaches are needed to better resolve phylogenetic relationships in prokaryotes [26, 31]. We have taken a multilocus approach to characterize a putative new supergroup of Wolbachia from termites. 3 The order Isoptera (termites) has been relatively under-sampled for Wolbachia endosymbionts in comparison to other major insect orders such as the Hymenoptera, Diptera, and Lepidoptera. The few reports of Isopteran Wolbachia infection are from Kalotermes flavicollis (family Kalotermitidae), Microcerotermes sp. (Termitidae), and Coptotermes (Rhinotermitidae) [2, 21], whose Wolbachia nucleotide sequences cluster together in supergroup F. While termites form intimate symbiotic relationships with diverse partners such as fungi [29], protozoans [19], and bacteria [5, 6], more comprehensive surveys for obligate bacterial endosymbionts are needed. Here we report on the detection of a new Wolbachia supergroup infecting the pacific dampwood termites Zootermopsis angusticollis and Z. nevadensis (family Termopsidae). Termopsidae represents one of the earlier-originating lineages within the order Isoptera [15, 30]. The geographical distribution of these two social insect species is in the Neartic region, specifically in the forested areas along the Pacific coast including the Cascade and Sierra Nevada Mountains of the USA [36]. Their mating behavior, social organization and ecology have striking similarities; and young colonies of both species can be found nesting in the same log. Materials and methods Isolation, amplification, and sequencing of genomic DNA. Genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen) from abdomens of single alate females of Zootermopsis angusticollis (collected in Huddart Park, San Mateo County, California) and Z. nevadensis (collected in Pebble Beach, Monterey, California, provided by Dr. Colin Brent). Wolbachia and host chromosomal DNA was initially amplified using PCR in a volume of 10l [2.5mM MgCl2 (Promega), 0.25mM of each dNTP (Invitrogen), 0.4 M of the forward and reverse primer, 0.2U Taq polymerase (Promega), and 1l DNA, 1l 10x PCR buffer (Promega), and 4.36l of water 4 (ICN Biomedicals)]. Primers and PCR product amplification are described in [21] for the Wolbachia16S rDNA and ftsZ sequences, and in [8] for dnaA sequences. gltA and groEL amplifications were obtained with forward (WgltAF1, 5’-TACGATCCAGGGTTTGTTTCTAC3’ and WgroF1, 5’-GGTGAGCAGTTGCAAGAAGC-3’) and reverse (WgltARev1, 5’CTCATTAGCTCCACCGTGTG-3’ and WgroRev1, 5’-AGATCTTCCATCTTGATTCC-3’) primers under the following conditions: 95C for 2min, followed by 95C for 30sec, 50C for 1min, and 72C for 1min. Sequencing was performed directly and bidirectionally using purified PCR products (Promega Wizard System) and appropriate primers on an ABI 3730 automated sequencer with Big Dye v3.0 (Applied Biosystems). All new sequences were deposited in Genbank under accession numbers AY764275-AY764284. Sequence nomenclature, alignments, and analyses. Sequences are identified by the name of the host species. Accession numbers from previously published sequences are shown adjacent to each taxon in brackets. Sequences were assembled in Sequencher 4.1.2, checked manually, and any ambiguous base calls were changed to N and were treated as missing data. Translated amino acid sequences were aligned in Clustal X, and the corresponding nucleotide sequences were manually edited in MacClade 4.05. Maximum likelihood (ML) inference methods based on nucleotide sequences were used to infer phylogenetic relationships. Prior to ML analyses, a DNA substitution model for each data set was selected using Modeltest v3.06 and the Akaike information criterion. The following models were selected for each of the single-gene and concatenated analyses: 16S (TrN+), concatenated (GTR+I+), dnaA (TIM+), ftsZ (TrN+I+), gltA (TIM+), and groEL (GTR+I+). ML heuristic searches were performed using 100 random taxon addition replicates with tree bisection and reconnection (TBR) branch swapping. ML 5 bootstrap support was determined using 100 bootstrap replicates, each using 10 random taxon addition replicates with TBR branch swapping, except for the concatenated data set. In this case, bootstrap support was determined using 500 bootstrap replicates, each using 20 random taxon addition replicates with TBR branch swapping. Searches were performed in parallel on a Beowulf cluster using a clusterpaup program and PAUP version 4.0b10. Results From 3,458 nucleotide base pairs spanning five Wolbachia genes per species of Zootermopsis, we describe a new taxonomic lineage that is highly divergent from the other Isopteran Wolbachia in supergroup F. We tentatively denote this lineage supergroup H and we verify previous findings of the other six supergroups by inferring the global Wolbachia phylogeny based on a concatenated data set of four protein coding genes positioned across the length of the chromosome. An initial screen of genomic DNA’s from queens, kings, pseudergates (false workers), and nymphs of Zootermopsis angusticollis (n=18) and a queen of Z. nevadensis (n=1) yielded polymerase chain reaction products of the correct band size for two different Wolbachia genes (16S rDNA and ftsZ). PCR products of genomic DNA from an alate female of each species were then directly sequenced for portions of five genes spanning the length of the endosymbiont chromosome: 16S rDNA (875 bp), dnaA (356 bp), gltA (636 bp), groEL (865), and ftsZ (718 bp). These genes were selected because of the abundant sequence information that already exists for these genes in GenBank. Maximum Likelihood (ML) analyses of the 16S rDNA sequences indicated that the Wolbachia from Zootermopsis are divergent from the published Wolbachia sequences of other Isopteran species (i.e., Kalotermes, Microcerotermes, Coptotermes) [21]. The lineage grouped 6 more closely to supergroup E (springtail hosts) than to supergroup F (termite hosts), as shown in the unrooted 16S rDNA tree of Figure 1. This is the first report of a termite Wolbachia sequence that is divergent from supergroup F. To confirm this evolutionary relationship, we sequenced portions of conserved orthologs from the four additional protein-coding genes listed above. ML analyses of these genes indicated the same pattern of divergence as the 16S rDNA analyses did (data not shown), with some incongruency for the positioning of supergroup H among the various single-gene phylogenies. Nonetheless, the branch is typically positioned close to supergroups A and E for all five genes. Incongruence between single-gene phylogenies are most often overcome by an analysis of concatenated single genes into one large data set [26]. This is especially useful for bacterial phylogenetics in which recombination and lateral gene transfer can cause conflicting phylogenetic signals among single-gene analyses. We inferred a maximum likelihood phylogeny based on the concatenated data set of four protein coding genes, spanning a total length of 2.6 Kb of the Wolbachia chromosome from each Zootermopsis species. Orthologs of the Anaplasmatacea relatives, Anaplasma marginale and Ehrlichia ruminantium, were also included as outgroups. As shown in Figure 2, the position of supergroup H in this analysis is between A (various arthropod hosts) and E (springtail hosts), and separate from F (other termite hosts). When taken together with the similar positioning in the 16S rDNA single gene analysis (Figure 1), it likely reflects the evolutionary relationship of this branch. ML bootstrap support is below 50% in the concatenated analysis because of the varied support for this branch in the single gene phylogenies, and resolving why the four different protein coding genes give conflicting bootstrap support for supergroup H should be a topic of future investigation. In general, ML bootstrap percentages are typically considered the most conservative estimates of branch support and as a 7 result, ML bootstrap analyses are less prone to supporting a false phylogenetic history than other methods such as Bayesian posterior probabilities [13]. Discussion The discovery of a novel lineage of Wolbachia from termites has two implications for the biology of Wolbachia. First, it expands the scope of known genetic diversity across the Wolbachia clade and raises future interest in comprehensively surveying the order Isoptera for obligate endosymbiont infections, especially in basal species that might be undersampled and host to novel phylotypes. We are currently engaged in a systematic sampling of Wolbachia in the order Isoptera. Second, it indicates for the first time that termites as a group, like other groups of arthropods [38], can tolerate infection by more than one supergroup of Wolbachia, though individual species can harbor only single infections. This finding provides some evidence for horizontal transmission of Wolbachia into the Isoptera since the infection of two divergent Wolbachia lineages in different termite species can be parsimoniously explained by independent acquisitions of these two lineages into termites, rather than a single ancient ancestral infection in Isoptera, with subsequent divergence and/or widespread loss. However, more sampling for Wolbachia infection in termite species is needed to fully resolve this issue. The termite families of Kalotermes flavicollis (Kalotermitidae), Microcerotermes sp. (Termitidae), and Coptotermes (Rhinotermitidae) probably share a more recent common ancestor with each other than they do with the family of Zootermopsis (Termopsidae) [15], which is a pattern consistent with vertical transmission of Wolbachia through these termites. The fast pace of supergroup discovery in the Wolbachia clade raises several important evolutionary questions including: How much genetic diversity exists? What are the evolutionary relationships of the supergroups? In addition, in which hosts will new supergroups be 8 discovered? The recent surge in supergroup discovery from arthropods such as springtails [35], termites [2], and spiders [27] strongly suggests that the major insect orders (i.e., Hymenoptera, Diptera, Lepidoptera), which have long been the subject of Wolbachia studies, may offer diminishing returns for discoveries of novel genetic lineages. Rather, undersampled groups of arthropods like the aforementioned one will be more promising to reveal further Wolbachia genetic diversity for the simple reason that they have been far less studied than the other orders. Why is the discovery of new genetic lineages important? First, it may facilitate current phylogenetic challenges in determining the root of the Wolbachia tree. Most global trees are depicted as unrooted trees since the genetic diversity of the clade is too divergent from that of its Anaplasmatacea relatives (e.g., Ehrlichia, Anaplasma, Neorickettsia) to confidently place the outgroup taxa [21]. The discovery of the taxonomic ‘missing links’ on the branch between the Wolbachia clade and Anaplasmatacea relatives could allow a more reliable determination of the position of the root and answer important evolutionary questions. Second, with the publications of the Wolbachia wMel and wBm genome sequences [16, 40], comparative genomic studies of Wolbachia diversity are underway to assess the genomic factors that underlie phenotypic diversity and chromosomal plasticity of this endosymbiont [4]. Information on new taxonomic lineages is a particularly helpful resource for molecular evolution and comparative genomic analyses, such as comparing gene gain and loss events, the timing of those events during the clade’s radiation, and the association of those events with the appearance of new phenotypes. Finally, the discovery of new supergroups in this dynamic symbiosis will provide fertile ground for expanding the outlook of the field, and the scope and range of its research. Only a decade ago, Wolbachia was deemed a relatively obscure bacterium occurring in just a few insect species. 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Appendix (Optional) Maximum-likelihood phylogenetic trees of the dnaA, ftsZ, gltA, and groEL data sets are available at: XXXXX 14 ACKNOWLEDGMENTS We thank P. Degnan and A. Lazarus for technical assistance with sequencing. For help with Zootermopsis specimens, we appreciate Dr. Colin Brent’s shipment of Z. nevadensis individuals and we thank the administrators of the Huddart Park, San Mateo County, CA for allowing collection of Z. angusticollis colonies. This work was supported by grants from the NASA Astrobiology Institute (NNA04CC04A) and the Neal W. Cornell Endowed Research Fund. This work was performed while Seth Bordenstein held a National Research Council Research Associateship Award at the Marine Biological Laboratory. Figure legends Fig. 1. Maximum-likelihood (ML) phylogenetic tree constructed from portions of 16S rDNA nucleotide sequences of Wolbachia. The tree is unrooted and ML bootstrap values are shown. Names of the host arthropod species followed by an accession number denote the specific Wolbachia taxa. All supergroups (A-H) are represented by at least two strains and the divergent genetic lineages from nematodes (Dipetalonema gracile) and fleas (Ctenocephalides felis and Orchopeas leucopus) are shown. Taxa in bold lettering denote the Wolbachia lineages from termites. Fig. 2. Maximum-likelihood (ML) phylogenetic tree constructed from concatenated nucleotide sequences of four protein coding genes, spanning 2,594 base pairs. The tree is rooted with Ehrlichia and Anaplasma as outgroups; ML bootstrap values are shown. Names of the host arthropod species denote the specific Wolbachia taxa. Taxa representing each supergroup are included when at least two of the four orthologs are available. Taxa shaded in grey boxes denote the Wolbachia lineages from termites. 15