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3810

SummaryThe bacterial tree contains many deep‐rooting clades without any cultured representatives. One such clade is ‘Endomicrobia’, a class‐level lineage in the phylum Elusimicrobia represented so far only by intracellular symbionts of termite gut flagellates. Here, we report the isolation and characterization of the first free‐living member of this clade from sterile‐filtered gut homogenate of defaunated (starch‐fed) Reticulitermes santonensis. Strain Rsa215 is a strictly anaerobic ultramicrobacterium that grows exclusively on glucose, which is fermented to lactate, acetate, hydrogen and CO2. Ultrastructural analysis revealed a Gram‐negative cell envelope and a peculiar cell cycle. The genome contains a single set of nif genes that encode homologues of Group IV nitrogenases, which were so far considered to have functions other than nitrogen fixation. We documented nitrogenase activity and diazotrophic growth by measuring acetylene reduction activity and 15N2 incorporation into cell mass, and demonstrated that transcription of nifH and nitrogenase activity occur only in the absence of ammonium. Based on the ancestral relationship to ‘Candidatus Endomicrobium trichonymphae’ and other obligate endosymbionts, we propose the name ‘Endomicrobium proavitum’ gen. nov., sp. nov. for the first isolate of this lineage and the name ‘Endomicrobia’ class. nov. for the entire clade.

The Xiphinema americanum-group is a large species complex containing more than 50 nematode species. They are economically important because they are vectors of nepoviruses. The species differentiation of X. americanum-group is problematic because the species share similar morphological characters. In the present study we collected nematode samples from different locations in the USA, Italy and Russia. Six valid species, X. americanum s. str., X. brevicolle, X. californicum, X. pachtaicum, X. rivesi and X. simile, and four unidentified putative Xiphinema species were characterised by morphology and sequencing of D2-D3 of 28S rRNA, ITS1 rRNA and mitochondrial COI genes. New nematode sequences generated totalled 147. Phylogenetic relationships of the X. americanum-group species reconstructed by Bayesian inference for D2-D3 of 28S rRNA gene sequences did not provide clear species delimitation of the samples studied, although the mtDNA presented interspecific variations useful for demarcation among species. Xiphinema americanum s. str., X. californicum, X. pachtaicum, X. rivesi, and two unidentified Xiphinema species were found in 72 soil samples from California. We also reconstructed the phylogenetic relationships using partial 16S rRNA gene sequences within endosymbiotic bacteria of the genus Candidatus Xiphinematobacter and provided solid evidence for distinguishing 17 species of this genus based on the analysis of new and previously published sequences. Fifty-five new bacterial sequences were obtained in the present study and comparison of the bacterial 16S rRNA and nematode COI phylogenies revealed a high level of co-speciation events between host and symbiont.