Polyphasic taxonomic description of Frankia abietis sp. nov., Frankia limosa sp. nov., Frankia palustris sp. nov. and Frankia luti sp. nov. isolated from Alnus spp.

Frankia strain AiPa1 ^T was isolated from root nodules of Alnus incana induced from soil under Picea abies in Finland. S trains AgKG’84/4 ^T , AgB32 ^T and AgB1.8 ^T were isolated from Alnus glutinosa nodules from Germany. AiPa1 ^T , AgB32 ^T and AgKG’84/4 ^T are infective on A. glutinosa and fix nitrogen in symbiotic nodules and pure culture, and strain AgB1.8 ^T is infective on A. glutinosa but does not fix nitrogen and does not have the nif genes. Colonies of the four strains are white, and they form hyphae and sporangia. N ₂ -fixing vesicles are formed by AiPa1 ^T , AgB32 ^T and AgKG’84/4 ^T in NH4 ⁺ -free medium but not by AgB1.8 ^T . Comparative 16S rRNA gene sequence analyses showed AiPa1 ^T , AgB32 ^T and AgKG’84/4 ^T were in cluster 1 (Fig. S1), while strain AgB1.8 ^T was in cluster 4. A whole-genome phylogeny confirmed this positioning. Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the four strains and all validly named Frankia species were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 64 and 71%, respectively. Strains AiPa1 ^T , AgB32 ^T and AgKG’84/4 ^T have genome sizes of 7.00, 6.40 and 6.71 Mb, respectively, while the genome size of AgB1.8 ^T is 9.82 Mb. Phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain AiPa1 ^T (=DSM 114742 ^T =LMG 32596 ^T ) to a novel species, for which the name Frankia abietis sp. nov. is proposed. Strain AgKG’84/4 ^T (=DSM 114736 ^T =LMG 32597 ^T ) is assigned to a novel species, for which the name Frankia limosa sp. nov. is proposed. Strain AgB32 ^T (=DSM 114735 ^T =LMG 32598 ^T ) is assigned to a novel species, with AgB32 ^T as the type strain, for which the name Frankia palustris sp. nov. is proposed. Strain AgB1.8 ^T (=DSM 114738 ^T =LMG 33088 ^T ) is assigned to a novel species, for which the name Frankia luti sp. nov. is proposed.