# Biodiversity of strains belonging to the freshwater genus Aquirufa in a riparian forest restoration area in Salzburg, Austria, with a focus on the description of Aquirufa salirivi sp. nov. and Aquirufa novilacunae sp. nov

## TL;DR

This study discovered two new species of freshwater bacteria, Aquirufa salirivi and Aquirufa novilacunae, in a riparian forest in Austria and analyzed their genetic and ecological characteristics.

## Contribution

The paper describes two new Aquirufa species and provides insights into their genomic and ecological traits, including their distribution in global water samples.

## Key findings

- Aquirufa salirivi sp. nov. was not detected in public metagenomes, suggesting it may be a rare or newly emerging species.
- Aquirufa novilacunae sp. nov. was found in 15 water samples globally, mainly in rivers from Asia, North America, and Australia.
- Genomic analyses confirmed the two new species based on average nucleotide identity thresholds and distinct gene content.

## Abstract

During a citizen science project, four freshwater habitats in a riparian forest restoration area in Salzburg, Austria, were sampled. The primary aim was to obtain bacterial strains of the genus Aquirufa, a group of typical and widespread freshwater bacteria. Numerous pure cultures of Aquirufa strains could be obtained, three of them originating from the river Salzach, a newly created pond and the lake Ausee represented new species. Strain 1-SAACH-A3T was characterized by a genome size of 3.2 Mbp and a G + C value of 38.4 mol% and encoded genes predicted for nitrate uptake and nitrous oxide utilization. Strains BAHN-186BT and 2-AUSEE-184A6 were characterized by a genome size of 2.4 Mbp and a G + C value of 42.4 and 42.2 mol%, respectively, and encoded genes predicted for the light-harvesting rhodopsin system. Calculated whole-genome average nucleotide identity values with Aquirufa type strains resulted in a maximum value of 93.65% for comparison of strain 1-SAACHT with the type strain of Aquirufa ecclesiirivi, which is slightly under the proposed threshold of species demarcation. The calculated gANI value comparing strains BAHN-186BT and 2-AUSEE-184A6 revealed 95.76%, thus a value slightly above the threshold. Further analyses revealed that the three new strains represent two new species, proposed here as Aquirufa salirivi sp. nov. with type strain 1-SAACH-A3T (= DSM 117800 T = JCM 37097 T) and Aquirufa novilacunae sp. nov. with type strain BAHN-186BT (= DSM 118143 T = JCM 37099 T). Analyses of 123 publicly available metagenomes and a metagenome of the lake Ausee resulted in no detection of A. salirivi sp. nov. In contrast, A. novilacunae sp. nov. could be detected in 15 water samples of rivers, mainly from Asia, but also from North America and Australia. The analyses suggested that the species occurs in most of these samples in low relative abundance, detections derived from metagenomes of water samples from the river Yangtze in the subtropical zone could be interpreted as occurrence in higher abundances.

The online version contains supplementary material available at 10.1007/s10123-025-00642-x.