Coevolution of symbiotic spirochete diversity in lower termites

The phylogenetic relationships of symbiotic spirochetes from five dry-wood feeding lower termites (Cryptotermes cavifrons, Heterotermes tenuis, Kalotermes flavicollis, Neotermes mona, and Reticulitermes grassei) was compared to those described in previous reports. The 16S rDNA bacterial genes were PCR-amplified from DNA isolated from intestinal samples using a spirochete-selective primer, and the 16S amplicons were cloned into Escherichia coli. Sequences of the cloned inserts were then used to determine closest relatives by comparison with published sequences. Clones sharing more than 97% sequence identity were grouped into the same phylotype. Forty-three new phylotypes were identified. These termite whole-gut-spirochetes fell into two previous defined clusters, designated as Treponema Clusters I and II, and one new Cluster III. Thirty-seven phylotypes were grouped in Cluster I. Cluster II comprised three phylotypes, two from Reticulitermes grassei (LJ029 and LJ012) and one from Heterotermes tenuis (LQ016). Three phylotypes, LK057, LK050 and LK028, were affiliated to Cluster III. Members of Cluster I showed the following characteristics: (i) spirochete phylotypes from a particular species of termite were more closely related to each other than to phylotypes of other termite species; (ii) spirochetes obtained from different genera of the same family, such as Cryptotermes sp., Kalotermes sp., and Neotermes sp., all from the family Kalotermitidae, were also related to each other. It was therefore concluded that spirochetes are specific symbionts that have coevolved with their respective species of termites, are stably harbored, and are closely related to members of the same termite family.