Halophilic archaea, mostly classified within the class Halobacteria of the domain Archaea, represent a unique group of microorganisms specifically adapted to thrive in hypersaline environments (Cui and Dyall-Smith, 2021). As of July 2025, taxonomic classification recognizes 2 orders, 10 families, 86 genera, and 406 species within this class. While most of these organisms are neutrophiles, 54 species have been identified as alkaliphiles. The remarkable survival capabilities of halophilic archaea in high-salt conditions are primarily attributed to their sophisticated salt adaptation mechanisms, which include both the “salt-in” strategy and the compatible-solute strategy (Oren, 2008; Grant, 2004). They regulate intracellular osmotic pressure by accumulating inorganic ions (such as potassium and chloride ions) and compatible organic solutes (such as trehalose and glycine betaine) to prevent cellular dehydration (Oren, 1999; Goh et al., 2011; Youssef et al., 2014). Moreover, it has been shown that some enzymes derived from halophilic archaea exhibit desirable properties such as high salt tolerance, extreme pH resistance, and thermal stability (Hou et al., 2024a; Hou et al., 2025). These characteristics make these enzymes highly promising for a wide range of biotechnological applications.

In China, the diverse hypersaline environments offer unique and favorable conditions for the survival and proliferation of halophilic archaea. Spanning vast geographical regions and encompassing a variety of environmental types, these high-salinity ecosystems range from coastal intertidal salt marshes to inland salt lakes. These environments host a multitude of unique microbial communities. In this study, four strains of DTA7T, DTA28T, FCH18aT, and WLHS9T were isolated from coastal saline-alkali land in Jiangsu Province and from two saline lakes of Feicui Salt Lake in Qinghai and Wulanhushao Salt Lake in Inner Mongolia. Phylogenetic analyses revealed that they share a close relationship with the genus Natronorubrum.

The genus Natronorubrum was established in 1999, with its type strain Natronorubrum bangense A33T isolated from a saline-alkaline lake in Tibet, China (Xu et al., 1999). To date, this genus comprises eight species with validly published names and two effectively published species (https://lpsn.dsmz.de/genus/natronorubrum). Halophilic archaea of Natronorubrum exhibit spherical or pleomorphic cellular morphology and typically form orange, pink, or red pigmented colonies on agar media. Among them, eight species, Natronorubrum bangense, Natronorubrum daqingense, “Natronorubrum halalkaliphilum”, Natronorubrum sediminis, Natronorubrum sulfidifaciens, Natronorubrum texcoconense, “Natronorubrum thiooxidans”, and Natronorubrum tibetense are alkaliphilic halophilic archaea, with an optimal growth pH range of 9–10 (Xu et al., 1999; Wang et al., 2010; Xue et al., 2021; Gutiérrez et al., 2010; Cui et al., 2007; Ruiz-Romero et al., 2013; Sorokin et al., 2005). In contrast, Natronorubrum aibiense and Natronorubrum halophilum are neutrophilic halophilic archaea, with an optimal growth pH range of around 7 (Cui et al., 2006; Tao et al., 2020). The genome sizes of the current species of the genus Natronorubrum range from 34.60 to 44.93 Mbp, with GC content varying between 61.0 % and 63.6 %.

In this study, phylogenetic, phylogenomic, and comparative genomic analyses were conducted to identify these four novel strains, DTA7T, DTA28T, FCH18aT, and WLHS9T. The results revealed that these four strains represent distinct novel species within the genus Natronorubrum. Furthermore, culture-independent analyses were performed to assess the diversity and ecological distribution of these novel species.