Cultivation and characterization of halophilic archaea from Moroccan Atlantic salterns: insights into diversity and bioactive potential

Cultivation and characterization of halophilic archaea from Moroccan Atlantic salterns: insights into diversity and bioactive potential | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cultivation and characterization of halophilic archaea from Moroccan Atlantic salterns: insights into diversity and bioactive potential Imane Charroud, Naima Boum’handi, Mohamed Alouani, Altaf ElBlidi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8022563/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract This study provides the first culture-based survey of archaeal diversity in Moroccan Atlantic solar salterns (Oualidia and Khenifiss), using sediment samples collected across a gradient of moderate to extreme salinities (16.7–29.2% w/v) and slightly alkaline pH (7.4–7.5). A total of 67 representative strains were isolated, all belonging to the class Halobacteria. The community was dominated by the genus Halorubrum , with additional genera ( Haloarcula, Haloferax , Natrinema , Halobacterium , Halogeometricum , Halococcus , Halomicrobium , and Halostagnicola ) exhibiting site- and phase-specific distributions, likely influenced by salinity fluctuations, microhabitat availability, and harvesting stage. Phylogenetic analyses revealed several isolates with ≤ 98.65% 16S rRNA gene sequence similarity to known taxa, forming distinct clades and suggesting the presence of putatively novel species. Antagonism assays demonstrated widespread inhibitory activity among isolates, with Natrinema and Haloferax strains exhibiting the strongest antagonism against co-isolated haloarchaea, indicative of bioactive compound production (e.g., halocins, lanthipeptides, or halolysins). These results expand our understanding of haloarchaeal diversity in Atlantic salterns, underscoring their potential as reservoirs of extremophiles with biotechnological applications. Furthermore, this work highlights the necessity of integrating polyphasic and culture-independent approaches to resolve the taxonomy and chemical ecology of these dynamic hypersaline environments. Solar salterns Halophilic Archaea Halobacteria diversity Antimicrobial activity Atlantic Morocco Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Marine solar salterns represent model ecosystems for studying microbial adaptation and evolution under multiple physicochemical constraints (Oren, 2019 ; Carré-Mlouka, 2021 ; Song et al., 2025 ). Formed along tropical and subtropical coasts for sea salt production, these anthropogenic systems comprise sequential evaporation ponds where calcite (CaCO₃) and gypsum (CaSO₄) precipitate first, followed by NaCl crystallization around 35% salinity (Oren, 2002 , 2006 ; Oren et al., 2009 ; Ventosa et al., 2015 ). The resulting salinity gradient, along with variations in ionic composition, temperature, pH, and oxygen availability, imposes strong selective pressure, shaping highly specialized microbial communities. From salinities above 25%, these communities are typically dominated by members of the class Halobacteria , with Halorubrum and Haloquadratum (the latter being typical of Mediterranean salterns) representing the most abundant and cosmopolitan genera, (Naghoni et al., 2017 ; Mani et al., 2020 ; Cui and Dyall-Smith, 2021 ; Dindhoria et al., 2024 ; García-Roldán et al., 2024 ), followed by other genera such as Haloarcula, Halobacterium, Haloferax , and Natrinema . Less abundant yet ecologically significant taxa, including Halonotius, Halogeometricum, Halomicrobium, Halostagnicola, Halococcus, Natronomonas , and Haloplanus , reflect the remarkable phylogenetic and ecological diversification of Halobacteria under fluctuating physicochemical conditions (Ding et al., 2025 ; Gómez-Villegas et al., 2018 ; Hashemzahi et al., 2020 ; Menasria et al., 2018 ; Straková et al., 2024 ; Youssef et al., 2012 ; Zhu et al., 2021 ). Taxonomically, Halobacteria (phylum Methanobacteriota (Formerly Euryarchaeota ) (Göker and Oren, 2024 )) comprises two orders: Halobacteriales , encompassing nine families ( Haladaptataceae , Halalkalicoccaceae, Haloarculaceae, Halobacteriaceae , Halococcaceae , Haloferacaceae , Natrialbaceae , Natronoarchaeaceae , and Salinarchaeaceae ), 81 genera and 375 species, and Halorutilales represented by the single described species, Halorutilus salinus ; Recent (meta)genomic analyses suggest a far greater diversity, with approximately 1,033 Halobacteriales species (divided into 114 genera) and seven Halorutilales species (three genera) ( https://gtdb.ecogenomic.org/tree?r=f__UBA12382 , october 24, 2025). Although metagenomics provides global distribution patterns, culture-dependent studies remain crucial to uncover their physiology, ecological rules, and potential applications, as cultivable taxa represent only a small fraction of natural hypersaline diversity (Cui and Dyall-Smith, 2021 ). Beyond abiotic constraints, Halobacteria communities are also shaped by complex biotic interactions. Competition for limited nutrients and space drives the production of antimicrobial compounds, notably halocins, proteinaceous molecules reported in Haloferax , Halobacterium , Natrinema , Haloterrigena , Halorubrum , Haloarcula, Halogeometricum, and Halomicrobium (Atanasova et al., 2013 ; Besse et al., 2017 , 2015 ; De Castro et al., 2020 ; Ghanmi et al., 2020 ; Kumar and Tiwari, 2019 ). Halocins, classified as large (> 10 kDa, e.g., Halocin H1 and H4) or microhalocins (≤ 10 kDa, e.g., Halocin C8, Halocin S8), act by disrupting membranes, Na⁺/H⁺ antiporter inhibition, receptor blocking, or cell lysis, sometimes requiring activation by halolysins (Chen et al., 2024 ; Hao et al., 2024 ; Kumar et al., 2021 ; Martínez-Espinosa, 2024 ). Some have broad-spectrum activity, some inhibit other haloarchaea, halophilic bacteria and even certain eukaryotes (Atanasova et al., 2013 ; Besse et al., 2017 ; Ghanmi et al., 2020 ; Martínez-Espinosa, 2024 ; Shand and Leyva, 2008 ). Recent findings on lanthipeptides further indicate that Halobacteria possess additional narrow-spectrum antagonism against related taxa (Costa et al., 2023 ; Liang et al., 2023 ; Song et al., 2025 ). These stable and biocompatible molecules hold promising biotechnological potential (Kumar et al., 2021 ), and in vitro antagonism assays are key for identifying novel bioactive archaea and understanding their ecological role (Elshafey et al., 2023 ; Gómez-Villegas et al., 2018 ; Waditee-Sirisattha et al., 2016). Despite their ecological and applied relevance, the diversity of halophilic archaea and antimicrobial producing strains remains largely unexplored in Moroccan Atlantic salterns. These ecosystems, ranging from the semi-arid, groundwater-influenced Oualidia saltern to the hyperarid, ocean-fed Khenifiss saltern (Amimi et al., 2021 ; El Hamoumi, 2022 ), provide natural gradients in salinity, sediment composition, and organic content that likely influence the diversity and functional potential of resident Halobacteria . This study aims to fill this knowledge gap by exploring the cultivable diversity of halophilic archaea from Oualidia and Khenifiss sediments. Through environmental characterization, culture-dependent isolation, 16S rRNA-based phylogenetic identification, and in vitro antagonism assays, we characterize their taxonomic diversity and antimicrobial potential. By establishing the first comparative culture-based inventory of Halobacteria from Moroccan Atlantic salterns, this work provides insights into their taxonomic diversity and highlights their potential as sources of stress-adapted bioactive compounds. 2. Materials and methods 2.1. Sampling sites and sediment collection Sediment samples were collected from two Moroccan marine solar salterns (Fig. 1 ), including the Oualidia saltern (32°47'14.2"N 8°58'10.1"W), located 9.4 km northeast of the Oualidia lagoon on the Atlantic coast. This receives regular Atlantic seawater and significant submarine groundwater discharge (0.2–1.2 m³/s), supplying 30–50% of the lagoon’s water (Fakir et al., 2019 ). The groundwater originates mainly from deep Jurassic aquifers, which are highly mineralized and rich in sulfates and calcium, as shown by isotopic studies (El Meknassi et al., 2020 ; Somoue et al., 2020 ). In contrast, the khenifiss saltern located 17 km inland from the Atlantic Ocean (27°54'57.9"N 12°21'16.3"W) and upstream of the Khenifiss lagoon, this site relies solely on seawater for salt production practices, with no freshwater inputs. Strong, persistent winds (average 6.13 ± 0.41 m/s) (Tnoumi et al., 2020 ) drive intense evaporation. Recent geomorphological shifts, such as increased vegetation and sedimentation, have led to channel closure and greater salt accumulation upstream (El Behja et al., 2024 ). At both sites, about 50 g of sediment was collected from multiple points within at 5–10 cm depth (June 2022 for Oualidia and March 2023 for khenifiss). Samples were stored in sterile polyethylene bags, transported cooled, and processed within 24 hours. In situ measurements (pH, salinity, and temperature), were taken using a HANNA HI9828 multiparameter probe. For lab analyses, sediment was mixed with water (1:5 w/v). 2.2. Isolation and cultivation of halophilic archaea Two culture media were used to support the growth of different halophilic populations. The Halophilic Medium (HM) was prepared as described by Kis-Papo and Oren ( 2000 ) with the following composition (g/L): NaCl 158; MgCl₂·6H₂O 13; MgSO₄·7H₂O 20; KCl 4; CaCl₂·2H₂O 1; NaBr 0.5; NaHCO₃ 0.2; yeast extract 5; tryptone 8; glucose 1. The DSMZ Medium 97 contained (g/L): NaCl 250; MgSO₄·7H₂O 15; KCl 2; trisodium citrate 3; yeast extract 5; casamino acids 7.5; FeSO₄·7H₂O 0.05; MnSO₄·H₂O 0.20 mg/L. The pH of the media was adjusted to 7.5 using 1M NaOH, and agar (20 g/L) was added for solidification. For each sampling site, 1 g of sediment was homogenized in sterile saline solution (20% NaCl). Serial decimal dilutions (10⁻¹ to 10⁻⁶) were plated (100 µL/plate) onto HM media and DSMZ 97 medium. Plates were incubated in sealed bags at 37°C (HM media) and 40°C (DSMZ 97) for 4 to 8 weeks. Distinct colonies were purified by repeated streaking, and pure cultures were preserved in liquid medium containing 15–20% glycerol at − 80°C. Purified isolates were subsequently transferred onto HM agar containing 20% (w/v) NaCl and incubated under standardized conditions (pH 7.5, 40°C). Colony morphology, pigmentation, and growth dynamics were recorded. Growth was further examined across salinity (5–30% NaCl) at 40°C and temperatures (10–55°C) at 20% ranges. From the initial collection of 204 isolates, a representative set of phenotypically distinct strains was selected. Archaeal and bacterial affiliation was preliminarily assessed by antibiotic susceptibility testing using ampicillin (10 µg), ciprofloxacin (5 µg), streptomycin (30 µg), gentamicin (10 µg), nalidixic acid (30 µg), vancomycin (30 µg), and bacitracin (8 µg) (Dridi et al., 2011 ). 2.3. Molecular identification and phylogenetic analysis Genomic DNA was extracted from freshly grown colonies using osmotic cell lysis in 50 µL of sterile ultrapure water, following the protocol of Pfeiffer adapted for halophilic archaea, as described in the Halohandbook (Dyall-Smith, 2009 ). The resulting DNA served as a template for amplification of the 16S rRNA gene via PCR. PCR amplification was performed in a final volume of 50 µL, containing: 10 µL of 10x buffer, 2 µL of each universal primer A8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and R1492 (5′-ACGGCTACCTTGTTACGACTT-3′) (10 mM each), 1 µL of dNTPs (10 mM), 0.8 µL of MgCl₂, 2.5 U of Taq polymerase, 2 µL of DNA template, and nuclease-free water to volume. PCR conditions were as follows: initial denaturation at 95°C for 10 min; 30 cycles of denaturation at 95°C for 1 min; annealing at 48°C for 1.5 min; and extension at 72°C for 2 min; followed by a final extension at 72°C for 5 min and a hold at 12°C. Amplified products were verified by 1% agarose gels, purified and sequenced by the Sanger method (Genewiz GmbH, Leipzig, Germany). Chromatograms were inspected using Chromas v2.6.6 (Goodstadt and Ponting, 2001 ), and sequences were assembled in BioEdit ( http://www.mbio.ncsu.edu/bioedit ). Taxonomic affiliation was determined by comparison with reference sequences in the EzBioCloud (EzTaxon) database ( https://www.ezbiocloud.net/ ). Potential chimeras were screened using Pintail v1.0 (Ashelford et al., 2006 ). All 16S rRNA sequences obtained from isolates of both salterns were deposited in GenBank ( https://www.ncbi.nlm.nih.gov/submit/genbank/ ), and accession numbers are listed in Table 1 . Table 1 Overview of halophilic archaeal strains from Oualidia (O-S) and khenifiss (K-S) salterns, showing strain designation, GenBank accession numbers, and molecular identification based on 16S rRNA gene BLAST analysis. Strain Origin Genbank Accession No. Molecular identification Strain Origin Genbank Accession No. Molecular identification SDIO15 O-S PV248879 Natrinema pelliribrum JCM 10476 T (98.40%) TSD140 K-S PX282994 Haloarcula hispanica Y27 T (98.29%) SDI16 O-S PV248887 Natrinema pallidum BOL6-1 T (99.91%) TSDR102 K-S PX282998 Haloarcula mannanilytica MD130-1 T (97.65%) SDI39 O-S PV248881 Natrinema pallidum BOL6-1 T (99.26%) TSD100 K-S PX282993 Haloarcula mannanilytica MD130-1 T (98.43%) SDIO6 O-S PV248880 Natrinema altunense AJ2 T (98.65%) TSD50 K-S PX282992 Haloarcula marismortui 8966 T (98.09%) SDI23 O-S PV248885 Natrinema altunense AJ2 T (99.82%) TSD02 K-S PX283008 Halobacterium jilantaiense NG4 T (98.67%) SDI22 O-S PV248886 Natrinema altunense AJ2 T (99.81%) TSDP2 K-S PX283009 Halobacterium jilantaiense NG4 T (98.85%) SDI19 O-S PV248882 Natrinema altunense AJ2 T (99.63%) TSD56 K-S PX283007 Halobacterium rubrum TGN-42-S1 T (98.32%) SDI24 O-S PV248884 Natrinema altunense AJ2 T (99.32%) TSDP11 K-S PX283012 Haloferax larsenii ZJ206 T (99.24%) SDI14 O-S PV248883 Natrinema altunense AJ2 T (99.83%) TSD25 K-S PX283011 Haloferax mediterranei R-4 T (99.73%) SDI46 O-S PV248890 Halorubrum tebenquichense ALT6-92 T (99.58%) TSD102 K-S PX283030 Haloferax volcanii DS2 T (99.27%) SDIP4 O-S PV248895 Halorubrum persicum C49 T (99.92%) TSD52 K-S PX283015 Haloferax volcanii DS2 T (99.28%) SDI38 O-S PV248892 Halorubrum distributum I-M T (98.73%) TSD22 K-S PX283031 Haloferax volcanii DS2 T (99.54%) SDI41 O-S PV248896 Halorubrum depositum Y78 T (99,43%) TSD19 K-S PX283014 Haloferax volcanii DS2 T (99.83%) SDI03 O-S PV248893 Halorubrum coriense Ch2 T (99,57%) TSDP1 K-S PX283003 Halorbrum persicum C49 T (99. 75%) SDI36 O-S PV248894 Halorubrum coriense Ch2 T (99,32%) TSD03 K-S PX283002 Halorubrum californiense SF3 213 T (99.23) SDI43 O-S PV248888 Halorubrum coriense Ch2 T (99.08%) TSD53 K-S PX282997 Halorubrum californiense SF3 213 T (99.22%) SDIE43 O-S PV248897 Halorubrum coriense Ch2 T (98.83%) TSD09 K-S PX282996 Halorubrum californiense SF3 213 T (99.40%) SDI81 O-S PV248891 Halorubrum coriense Ch2 T (98.67%) TSD07 K-S PX282995 Halorubrum californiense SF3 213 T (99.63%) SDI47 O-S PV248889 Halorubrum californiense SF3 213 T (99.43%) TSD12 K-S PX282999 Halorubrum distributum I-M T (99.22%) SDIB3 O-S PV248898 Halorubrum californiense SF3 213 T (98.83%) TSD04 K-S PX283001 Halorubrum kocurii BG-1 T (99.18%) SDI34 O-S PV248899 Halorubrum californiense SF3 213 T (99.09%) TSBK32 K-S PX283034 Halorubrum kocurii BG-1 T (99.23%) SDI01 O-S PV248900 Halomicrobium mukohatatei arg-2 T (98.35%) TSD08 K-S PX283010 Halostagnicola larsenii XH-48 T (99.40%) SDI18 O-S PV248901 Halogeometricum rufum RO1-4 T (99,67%) TSD76 K-S PX283005 Natrinema altunense AJ2 T (98.84%) SDII35 O-S PV248902 Halogeometricum rufum RO1-4 T (98.86%) TSDOR2 K-S PX283006 Natrinema altunense AJ2 T (99.74%) SDI44 O-S PV248907 Haloferax volcanii DS2 T (99.45%) TSD31 K-S PX283004 Natrinema pellirubrum JCM 10476 T (99.64%) SDI50 O-S PV248905 Haloferax volcanii DS2 T (99.16%) SDI05 O-S PV248903 Haloferax sulfurifontis M6 T (98.56) SDIB5 O-S PV248904 Haloferax mediterranei R-4 T (99.12%) SDI17 O-S PV248906 Haloferax larsenii ZJ206 T (99.66%) SDI20 O-S PV248908 Halococcus saccharolyticus P-423 T (99.62%) SDI13 O-S PV248909 Halococcus saccharolyticus P-423 T (98.67%) SDI94 O-S PV248916 Haloarcula salaria HST01-2R T (98.74%) SDI48 O-S PV248915 Haloarcula marismortui JCM 8966 T (99.60%) SDI30 O-S PV248913 Haloarcula marismortui JCM 8966 T (99.71%) SDI30A O-S PV248914 Haloarcula marismortui JCM 8966 T (99.24%) SDI02 O-S PV248912 Haloarcula hispanica Y27 T (99.05%) SDI04 O-S PV248910 Haloarcula hispanica Y27 T (99.02%) SDIR04 O-S PV248917 Haloarcula hispanica Y27 T (99.19%) SDI10 O-S PV248911 Haloarcula hispanica Y27 T (98.49%) SDI31 O-S PV248918 Haloarcula hispanica Y27 T (98.37%) SDI35 O-S PV248919 Haloarcula argentiensis arg-1 T (99.77%) SDI45 O-S PV248920 Haloarcula quadrata JCM 11048 T (99.58%) Phylogenetic relationships were inferred from aligned sequences generated using MUSCLE (Edgar, 2004 ) implemented in Seaview v5.1 (Gouy et al., 2021 ). Phylogenetic trees were constructed with the Maximum Likelihood (ML) algorithm (Felsenstein, 1981 ), and node support was evaluated by 1000 bootstrap replicates (Felsenstein, 1985 ). Reference sequences of closely related taxa retrieved from NCBI were included to accurately position the isolates within their respective phylogenetic lineages. 2.4. Antagonistic interactions between isolated strains Purified isolates were cultured to the early stationary phase to ensure active metabolism and maximal secondary metabolite production. Both producer and target strains were standardized and reinoculated into fresh HM medium at an initial optical density of OD₆₀₀=0.001. Target strains were previously spread uniformly onto HM agar plates containing 20% (w/v) NaCl. Sterile paper discs (6 mm diameter) were impregnated with 15 µL of the producer culture and placed onto the surface of pre-inoculated plates. Plates were incubated at 40°C for 5–10 days in sealed plastic bags to prevent desiccation. Antimicrobial activity was evaluated by measuring the radius of the inhibition zones formed around the discs. Discs impregnated with sterile, uninoculated HM medium served as negative controls. Each assay was performed in triplicate, and inhibition zone radius (mm) are reported as mean standard deviation from three independent experiments. 3. Results and discussion 3.1. Physicochemical characteristics of the studied salterns Soils from both salterns were slightly alkaline (pH 7.40–7.49) consistent with industrial salterns (Ventosa et al., 2008 ). Salinity levels were high (16.7–29.2%, w/v), suitable for extremely halophilic microorganisms. Temperatures ranged from 27.5 and 36.1°C, peaking at Oualidia in June due to its semi-arid coastal climate, characterized by mild winters, dry summers, and low annual precipitation (< 230 mm). Khenifiss, sampled in early spring, showed lower temperatures, reflecting its arid southern Moroccan coastal desert environment, marked by persistent winds, intense evaporation, and minimal rainfall (< 80mm) (Tnoumi et al., 2020 ) (Table S1 ). 3.2. Culture dependent recovery of Halobacteria along a salinity gradient A culture-dependent approach applied to sediments from two Moroccan salterns, Oualidia and Khenifiss, yielded 115 and 89 isolates respectively. The higher number of isolates from Oualidia suggests greater cultivability under intermediate salinities, which typically support a brorder diversity of halophilic microorganisms. In contrast, extreme salinities exert strong selective pressures, favoring only highly specialized taxa capable of key biogeochemical functions under osmotic stress (Gao et al., 2024 ). From these isolates, 42 from Oualidia and 25 from Khenifiss were selected based on distinct colony morphology, pigmentation, and physiological responses to variations in salinity, temperature, and pH (Table S2, S3). Pigmentation among the isolates ranged from pale pink to bright red (Fig. S1 ). Notably, all isolates exhibited resistance to a panel of bacterial-targeting antibiotics (ampicillin, ciprofloxacin, streptomycin, gentamicin, nalidixic acid, and vancomycin) but remained sensitive to bacitracin (8 µg). This antibiotic susceptibility profile is characteristic of the class Halobacteria , as bacitracin disrupts the dephosphorylation of bactoprenol, a critical membrane carrier molecule, thereby inhibiting lipid biosynthesis (Kumar and Tiwari, 2019 ; Oren, 2015a ). The isolates demonstrated broad environmental tolerance, growing across a temperature range of 20–60°C, pH 5–9.5, and NaCl concentrations from 7.5 to 30%. Optimal growth was observed at 15–25% NaCl, although several strains thrived even at 30%, confirming their classification as extreme halophiles and their affiliation with the class Halobacteria (Amoozegar et al., 2017 ). While Halobacteria can tolerate NaCl concentrations from 10% up to saturation (Cui and Dyall-Smith, 2021 ), they are generally more abundant in salinities above 21% and less prevalent below 13–19% (Oren, 2015a ). The recovery of numerous Halobacteria strains from the moderately saline Oualidia saltern may be attributed to the clay-rich sediment matrix (Maanan et al., 2014 ), which enhances Na⁺ and K⁺ retention and maintains microenvironments conducive to osmotic balance (Fukushima et al., 2007 ; Rozic, 2000 ). Similar patterns have been documented in other salterns. For example, in the Siridao saltern (India), Halobacteria constitute up to 85% of the microbial community in clayey sediments with intermediate salinities (Mani et al., 2020 ). Metagenomic surveys of the Atlantic Isla Cristina saltern (Spain), which shares a thalassohaline water source with the Moroccan sites, revealed that Halobacteria accounted for approximately 85% of the community at 21% salinity. A comparable fraction was observed in the Mediterranean Santa Pola saltern at 33% salinity, whereas a pond at 19% salinity within the same site contained only 47% Halobacteria (Fernández et al., 2014 ; García-Roldán et al., 2024 ; Ventosa et al., 2014 ). These finding underscore that, in addition to salinity, sediment texture, hydrological connectivity, and oceanic origin play pivotal roles in shaping the composition and resilience of halophilic archaeal communities. 3.3. Phylogenetic resolution and species delineation of halophilic archaeal isolates using 16S rRNA analysis The 16S rRNA gene was sequenced to determine the species affiliation of the isolates (Table 1 ). However, several strains exhibited high sequence similarity to multiple closely related species, illustrating the well-documented limitations of 16S rRNA for precise species delineation. For example, strain SDI44 shared 99.45% identity with Haloferax volcanii DS2 T , with only slightly lower similarity to H. gibbonsii , H. sulfurifontis , H. chudinovii , and H. massiliensis , all exceeding the 98.56% threshold commonly used for species delineation (Kim et al., 2014 ). Similar ambiguities were observed among several Halorubrum and Haloarcula isolates, as well as for Halococcus strains SDI13 and SDI20. Notably, SDI20 exhibited 100% identity with both H. saccharolyticus and H. agarilyticus , while SDI13 reached 98.67% similarity with the same species. In contrast, isolate SDIR04 was confidently assigned to Haloarcula hispanica (99.19% similarity), with a clear separation from the next closest species (< 98.65%). Several isolates, including SDI01, SDI15, TSD56, and all Haloarcula strains from Khenifiss, exhibited < 98.65% 16S rRNA gene similarity to any described species, suggesting the presence of potential novel taxa (Table 1 ). Phylogenetic analysis, incorporating these isolates alongside closely related type species above the 98.56% threshold, confirmed their placement within the respective genera. However, some formed distinct branches, indicating evolutionary divergence between populations from Khenifiss and Oualidia (Fig. 2 ). Within the genus Haloferax , most isolates clustered with H. sulfurifontis and were clearly separated from H. volcanii , despite the latter showing the highest BLAST similarity (Table 1 , Fig. 2 ). Haloarcula isolates either formed independent clusters or grouped with H. salaria , while Natrinema isolates mostly branched separately, except for TSD31, which shared a common ancestor with several type species (Fig. 2 ). Interestingly, some isolates with identical 16S rRNA sequences exhibited distinct phenotypic traits under identical culture conditions, including differences in colony and cell morphology, as well as variable responses to NaCl concentration, pH, and temperature (Tables 1 , S2 and S3). These observations suggest that, despite genetic similarity, the isolates represent distinct strains with divergent physiological adaptations. These results highlight the limited resolution of 16S rRNA for species-level assignment, consistent with previous studies advocating the use of more variable markers, such as rpob’ , for refined classification within closely related haloarchaeal taxa (Minegishi et al., 2010 ; Walsh, 2004 ). For definitive species delineation, a polyphasic, approach integrating genomic and phenotypic analyses against reference species under standardized conditions, is essential, in accordance with current standards for describing novel taxa within the class Halobacteria (Cui et al., 2024 ) 3.4. Taxonomic diversity and site-specific distribution of Halobacteria isolates Isolates from the Moroccan salterns were assigned to five families: Haloferacaceae ( Halorubrum, Haloferax , and Halogeometricum ), Haloarculaceae ( Haloarcula, Halomicrobium ), Natrialbaceae (Natrinema, Halostagnicola ), Halococcaceae ( Halococcus ), and Halobacteriaceae ( Halobacterium ) (Table 1 , Fig. 2 ). Among these, Halorubrum , Haloarcula , Natrinema , and Haloferax were the most frequently isolated genera at both sites, with Halorubrum emerging as the dominant taxon. 3.4.1. Dominant genera and their ecological adaptations The predominance of Halorubrum in both salterns is likely due to its broad adaptability to a wide range of NaCl concentrations (1.0–5.2 M), minimal magnesium requirements (0.005–0.6 M), rapid growth rates, and high adaptability under laboratory conditions, which enable it to colonize dynamic environments and outcompete slower-growing taxa (Ding et al., 2025 ; Oren, 2015b ; Yujie Tao et al., 2025). This ecological success is consistent with its widespread distribution in diverse hypersaline environments, including solar salterns, salt lakes, saline soils, and salted foods worldwide (Baati et al., 2008 ; Burns et al., 2004 ; Çınar and Mutlu, 2016 ; Fernández et al., 2014 ; Kambourova et al., 2016 ; Naghoni et al., 2017 ; Oren, 2020 ). Haloarcula was co-dominant with Halorubrum in the Oualidia saltern and also detected in khenifiss. This pattern mirrors its frequent co-occurrence with Halorubrum in Indian and Turkish salterns (Akpolat et al., 2021 ; Mani et al., 2020 ), as well as its widespread detection in salterns from China, Spain, Algeria, Tunisia, Pakistan, and Germany (Cycil et al., 2020 ; Ding et al., 2025 ; Hassani et al., 2023 ; Ochsenreiter et al., 2002 ; Straková et al., 2024 ; Ventosa et al., 2014 ; Verma et al., 2020 ). The ecological versatility of Haloarcula is attributed to its rapid colonization following salinity fluctuations, tolerance to low Mg²⁺ concentrations, and metabolic flexibility (aerobic respiration, rhodopsin-mediated phototrophy, anaerobic nitrate reduction). In addition, its high carotenoid content provides protection against oxidative stress and intense solar radiation, particularly relevant under the sun-exposed conditions of Oualidia and Khenifiss salterns (Dillon et al., 2013 ; Mani et al., 2020 ; Ochsenreiter et al., 2002 ; Straková et al., 2025 , 2024 ; Ventosa et al., 2014 ). Haloferax , isolated from both salterns, frequently co-occurred with Halorubrum and Haloarcula , consistent with its cosmopolitan distribution across solar salterns in Australia, Algeria, California, Tunisia, India, Spain, and South Africa (Baati et al., 2008 ; Bidle et al., 2005 ; Burns et al., 2004 ; Ding et al., 2025 ; Mani et al., 2012 ; McDuff et al., 2016 ; Mizuno et al., 2019 ; Najjari et al., 2021 ; Zafrilla et al., 2010 ). Its ecological success is linked to rapid growth, pronounced metabolic versatility, including carotenoid production, extremophilic enzymes, the ability to grow on diverse substrates, and broad salinity tolerance (0.34–5.2 M) (Elshahed et al., 2004b , 2004a ; Griffiths et al., 2025 ). These traits likely explain its success in both Moroccan salterns despite differences in salinity, temperature, and sediment composition. Finally, Natrinema species, previously reported to dominate Indian saltern sediments through culture-independent studies (Manikandan et al., 2009 ), have also been reported in Slovenian and Algerian salterns (Imadalou-Idres et al., 2013 ; Pašić et al., 2007). Their adaptation to extreme salinities (up to 30% NaCl), variable temperatures (25–55°C), UV exposure, and heavy metals aligns with the environmental characteristics of Oualidia and Khenifiss, where sediments are enriched in metals such as nickel and cadmium (Kim et al., 2018 ; Straková et al., 2025 )(Mejjad et al., 2025 ; Tnoumi et al., 2020 ), supporting their persistence in these habitats. 3.4.2. Less abundant but ecologically significant genera Halococcus and Halomicrobium were detected at low abundances in the Oualidia saltern, reflecting their sensitivity to salinity fluctuations linked to salt production cycles. In particular, Halococcus , which generally constitutes a minor fraction of haloarchaeal communities (around 5% in Iranian salterns and < 1% in Egyptian and Indian salterns), (Hashemzahi et al., 2020 ; Mani et al., 2020 ), was observed too alongside Halorubrum , Haloferax , and Haloarcula during the early, lower-salinity phase of salt crystallization. It often becomes transiently dominant during the initial stages of salt production, when salinity remains moderate, before its proportion declines as other genera outcompete it under increasing salinity (Elshafey et al., 2023 ; Imadalou-Idres et al., 2013 ; Mani et al., 2020 , 2012 ). Halomicrobium , typically rare (1–3%) in salterns of Turkey, India, Argentina, and China (Akpolat et al., 2021 ; Oren, 2002 ; Yang and Cui, 2012 ; Youssef et al., 2012 ), preferentially colonizes organic-rich, low-Mg sediments. Its abundance increases after harvesting and declines during initial crystallization stages. This pattern is consistent with its low proportional occurrence in Oualidia during sampling in the evaporation ponds at the beginning of the salt-harvesting phase, a period that did not coincide too with the March sampling in Khenifiss (Castelán-Sánchez et al., 2019 ; Mani et al., 2020 ; Youssef et al., 2012 ). Halogeometricum , isolated from Oualidia, was found at low abundances alongside Haloferax , Halorubrum , Haloarcula and Halococcus in Indian solar salterns (Manikandan et al., 2009 ). Its ability to combine salt-in/ salt-out strategies, enables persistence under fluctuating salinity (Straková et al., 2024 ). However, it was absent from Khenifiss, possibly due to micro-niche availability or competitive exclusion (Bowers and Wiegel, 2011 ). Halostagnicola , recovered as a single isolate from the Khenifiss saltern, is a globally rare haloarchaeon, previously representing ~ 5% of cultivated communities in Iranian salterns (Hashemzahi et al., 2020 ) and reported from Thai saltern soils (Niyasom and Mamimin, 2023 ). Its association with halophyte rhizospheres suggests that local vegetation at Khenifiss may provide suitable microhabitats (Tnoumi et al., 2020 ). In the same site, where salinity remains both high and remarkably stable throughout the year, Halobacterium , adapted to persistently high salinity (2.5–5.2 M NaCl), elevated Mg²⁺concentrations, and anoxic, organic-rich sediments, was the only other genus detected (Bidle et al., 2005 ; Tnoumi et al., 2020 ; Youssef et al., 2012 ). 3.4.3. Drivers of spatial variability The spatial variability observed between the Oualidia and Khenifiss archaeal assemblages likely results from both biotic and abiotic factors. Migratory birds that frequent these Ramsar-listed ecosystems may facilitate long-distance microbial dispersal, acting as biological vectors connecting geographically isolated hypersaline environments (Amimi et al., 2021 ; Tnoumi et al., 2020 ; Yim et al., 2015 ). In parallel, local physicochemical parameters, such as salinity stability, pH, temperature, sediment structure, organic content, and vegetation, act as selective pressures shaping site-specific archaeal communities (Elshafey et al., 2023 ; Kipnyargis et al., 2024 ; Zhu et al., 2021 ). In addition, evidence of metabolic cooperation exists. Some taxa synthesize and share essential compounds (e.g., biotin, carotenoids) with others lacking these pathways, supporting coexistence and reducing the likelihood of strict competitive exclusion. This "altruistic" cooperation can stabilize community structure in resource-limited, extreme environments (García-Roldán et al., 2024 ). 3.5. Antimicrobial and antagonistic potential of cultivable haloarchaeal isolates Antagonism assays conducted under standardized conditions (22% NaCl, 40°C, pH 7.0), corresponding to the average growth optima of the isolates, revealed a complex network of intra- and intercommunity interactions among haloarchaeal isolates from the Oualidia and Khenifiss salterns (Fig. 3 , Fig. 5 ). Inhibition zones, ranging from 2 to 27 mm in radius, indicated substantial functional heterogeneity, likely due to differences in metabolite production or sensitivity among the isolates. 3.5.1. Patterns of Antagonistic Activity At Oualidia, isolates of Natrinema (SDI14, SDI16, SDI19, SDI22, SDI24, and SDI39) and Haloferax (SDI44, SDI50) exhibited broad-spectrum inhibitory activities. Notably, Haloferax sp. SDI17 displayed the strongest antagonistic activity (27 mm radius) against Halorubrum sp. SDI81, yet was itself inhibited by other Haloferax , Natrinema , and Halococcus isolates (Fig. 4 ). This reciprocal inhibition suggests the coexistence of multiple bioactive compounds within the community. Similarly, at Khenifiss, Halorubrum sp. SDI81 was inhibited by isolates from multiple genera (Fig. 5 ), indicating that antagonistic traits are conserved across geographically separated salterns. The apparent inactivity of some isolates under standardized conditions may reflect dependence on specific growth parameters or physiological states (Besse et al., 2015 ). 3.5.2. Putative antimicrobial compounds: Halocins and beyond While the chemical nature of the inhibitory compounds was not directly characterized in this study, the genera exhibiting pronounced antagonism ( Halorubrum , Halomicrobium , Haloferax , Halobacterium , Natrinema , Halococcus , and Halogeometricum ) have been previously reported as halocin producers (Besse et al., 2017 ; Ghanmi et al., 2020 ). Halocins are ribosomally synthesized antimicrobial peptides or proteins produced by Halobacteria, capable of acting both within and cross genera (Atanasova et al., 2013 ; Besse et al., 2015 ; Kumar et al., 2021 ; Najjari et al., 2015 ). Many active isolates are known to produce of C8-like halocins, often associated with (halI) immunity clusters that confer self protection, potentially explaining the observed reciprocal inhibition (Atanasova et al., 2013 ; De Castro et al., 2020 ; Song et al., 2025 ). As reported by Ghanmi et al. ( 2020 ), a single strain can produce multiple halocins simultaneously, thereby enhancing its overall antimicrobial potential. The exclusive detection of halocin S8 in Haloferax and Natrinema may account for their pronounced inhibitory activities. Additional halocins, such as H4, may also contribute to the observed antagonism particularly in Halomicrobium , Halogeometricum , Haloferax , and Natrinema (Besse et al., 2015 ; De Castro et al., 2020 ; Makarova et al., 2019 ; Song et al., 2025 ). 3.5.3. Ecological relevance and alternative mechanisms Despite their detectability under laboratory conditions, the ecological significance of halocins remains debated. Kis-Papo and Oren, ( 2000 ) reported no antimicrobial activity in ultrafiltered cell-free brines from Eilat saltern, even when concentrated 53.5-fold to match the density of a fully grown culture (~ 10⁹ cells ml⁻¹), suggesting rapid inactivation or insufficient accumulation in situ. However, subsequent studies have shown that halocins are often membrane-associated or transported via extracellular vesicles, indicating alternative stabilization and delivery mechanisms in natural habitats (Besse et al., 2015 ) Beyond halocins, lanthipeptides (lantibiotics), ribosomally synthesized and post-translationally modified by LanM enzymes, may contribute too to the observed antagonistic interactions. These compounds are widely distributed among Halobacteriales (40%), Haloferacales (38%), Natrialbales (17%), and unclassified lineages (5%), where they disrupt membranes or cell wall biosynthesis and modulate cellular motility through archaellin gene expression ( flgA1 ), thereby promoting competitive fitness and environmental adaptation (Chakraborty et al., 2019 ; Song et al., 2025 , 2024 ). The first archaeal lantipeptide, Archalan α, characterized in Haloferax mediterranei (Liang et al., 2023 ), exhibits narrow-spectrum activity against phylogenetically related strains. Furthermore, studies on Hfx. Mediterranei have demonstrated that antimicrobial activity persists even after deletion of halocin and lantipeptide genes, implicating halolysins, extracellular proteases capable of activating halocins or directly lysing target cells (Chen et al., 2021 , 2024 , 2019 ; Costa et al., 2023 ). 3.6. Biotechnological potential of halophilic Archaea Collectively, these finding demonstrate that Moroccan salterns serve as untapped reservoirs of archaeal bioactive compounds. While detectable through antagonism assays, the full biotechnological potential of these compounds remains largely unexplored. This dual perspective (ecological and applied) oepns new avenues for both understanding microbial dynamics in extreme environments and discovering novel antimicrobial peptides stable under harsh conditions. From a biotechnological standpoint, halocins stand out due to their exceptional stability under high temperatures, salinity, and pH fluctuations, as well as their resistance to organic solvents, properties rarely found in conventional antimicrobials (Dini et al., 2022 ; Kumar et al., 2021 ). These characteristics make halocins promising candidates as natural preservatives, particularly in extreme environments such as salted food systems, where traditional preservatives often fail. Their mechanism of action, which involves membrane pore formation and inhibition of ion transporters, offers an attractive alternative to conventional antibiotics, especially against multidrug-resistant pathogens (Bucataru and Ciobanasu, 2024 ; Zhang et al., 2021 ). Emerging research even suggests potential cardioprotective properties for certain halocin-like peptides (Kumar et al., 2021 ). Halolysins exhibit remarkable robustness, remaining active across a broad range of conditions: 40–60°C, pH 8–9, and up to 4 M NaCl. They also retain functionality in the presence of solvents, metals, or surfactants (Hao et al., 2024 ; Hou et al., 2024 , 2020 ). These properties support their potential use in industrial biotechnology, including leather tanning, marine product fermentation, and the degradation of hypersaline proteinaceous waste. Finally, the structural and enzymatic diversity of LanM systems represents a valuable resource for bioengineering, enabling the design of tailor-made therapeutic or industrial peptides. Beyond, their applied potential, these systems play a crucial ecological role in shaping microbial community structure, modulating coexistence and competition among haloarchaea in hypersaline ecosystems (Fu et al., 2023 ; Song et al., 2025 , 2024 ). 4. Conclusions This work provides the first comprehensive evidence that Moroccan Atlantic salterns represent a valuable yet largely untapped reservoir of cultivable extreme halophilic archaea with significant biotechnological potential. Through an integrated approach combining environmental characterization and culture-dependent isolation, we successfully recovered 67 archaeal strains from Oualidia and Khenifiss salterns. These isolates span five families and nine genera within the class Halobacteria . Despite exhibiting over 98.56% 16S rRNA gene sequence identity, several strains displayed marked phenotypic and metabolic diversity, underscoring both the limitations of 16S rRNA-based species delineation and the importance of polyphasic taxonomic strategies for accurate archaeal classification. The broad-spectrum antimicrobial activity detected among our isolates further emphasizes the salterns as promising sources of bioactive compounds naturally adapted to extreme conditions. Notably, some strains exhibited strong inhibitory effects against closely related haloarchaea, reflecting the long-recognised but poorly understood antagonistic interactions within hypersaline ecosystems. These findings support the hypothesis that haloarchaea employ diverse antagonistic mechanisms, potentially involving halocins, lantipeptides or halolysins, to compete for limited resources in such challenging environments. Collectively, this work establishes a solid foundation for several future research. First, it highlights the need to implement a comprehensive polyphasic taxonomic framework, integrating whole-genome sequencing, comparative proteomics, and metabolic profiling, to formally describe novel archaeal taxa identified here. Second, it opens perspectives for systematic exploration of halophilic archaea as sources of antimicrobial, and secondary metabolites with industrial and pharmaceutical relevance, particularly those functioning under high salinity, elevated temperature, or fluctuating pH. Finally, this study reinforces the importance of including Moroccan Atlantic salterns within global initiatives focused on extremophile biodiversity and bioprospecting, ensuring these unique ecosystems are recognized not only for their ecological value but also as strategic reservoirs of biotechnological innovation. By demonstrating the feasibility of isolating and functionally characterizing halophilic archaea from Moroccan salterns, and establishing a reproducible workflow for their screening, this study provides a robust platform for future efforts aimed at novel species discovery, metabolic pathway elucidation, and the development of bioactive compounds with applications across biotechnology and medicine. Declarations CRediT authorship contribution statement Imane Charroud (IC) : Data curation, Formal analysis, Visualization, Writing – review & editing, Investigation, Validation, Methodology, Writing – original draft. Naima Boum’handi (NB) : Data curation, Formal analysis, Writing – review & editing, Investigation, Validation, Methodology. Mohamed Alouani (MA) : Investigation, Validation, Methodology, Supervision, Funding acquisition. Altaf El Blidi (AE) : Investigation, Methodology, Supervision, Funding acquisition. Yannick Fleury (YF) : Methodology, Validation, Writing – review & editing and Supervision, Funding acquisition. Mohamed Jebbar (MJ) : Conceptualization, Methodology, Investigation, Formal analysis, Writing – review & editing, Supervision and funding acquisition. Acknowledgements This work was supported by TOUBKAL program, a Franco-Moroccan Hubert Curien Partnership (PHC), funded by the Ministry of Higher Education, Scientific Research, and Innovation (MESRSI) in Morocco, and the Ministry for Europe and Foreign Affairs (MEAE) and the Ministry of Higher Education, Research, and Innovation (MESRI) in France. It was also supported by the ISblue project, Interdisciplinary Graduate School for the Blue Planet (ANR-17-EURE-0015), co-funded by a grant from the French government under the “Investissements d’Avenir” program and embedded in France 2030. Declaration of generative AI and AI-assisted technologies in the writing process During the preparation of this work the authors used Mistral AI in order to rephrase some words and improve the readability and language of the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article. 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09:28:32","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":39923,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8022563/v1/09c0857855ee791094aeaa23.png"},{"id":96917591,"identity":"66eae9ac-1b2d-43f7-bbe4-d91e2cd59677","added_by":"auto","created_at":"2025-11-27 14:10:11","extension":"xml","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":268397,"visible":true,"origin":"","legend":"","description":"","filename":"1c073d56c20343d9bea88af5208c3a631structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8022563/v1/951923de8f4f1ebcb1a01784.xml"},{"id":96808388,"identity":"620a75dd-0c7c-4a87-a77a-88bd42795569","added_by":"auto","created_at":"2025-11-26 09:28:33","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":280731,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8022563/v1/849b9d22af39cd920df500d7.html"},{"id":96808363,"identity":"85d5bcd2-90ee-4b40-9c93-778d86c03178","added_by":"auto","created_at":"2025-11-26 09:28:32","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":637951,"visible":true,"origin":"","legend":"\u003cp\u003eHydrological Schematic of the Oualidia and Khenifiss Saltern Systems, Moroccan Atlantic Coast: Seawater Circulation (Blue) and Continental Water Sources (Red), Including Field Photographs of Saltern Ponds (Scale Bars: 1 km)\u003c/p\u003e","description":"","filename":"FIg1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022563/v1/86eaa2eb896f6562c2ccdd4f.jpeg"},{"id":96808361,"identity":"d06d9821-9295-477f-bcb1-30b5a4a4fbac","added_by":"auto","created_at":"2025-11-26 09:28:32","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":459652,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum Likelihood Tree of 16S rRNA Gene Sequences Illustrating the Phylogenetic Placement of Haloarchaeal Isolates from Oualidia (●) and Khenifiss (■) Salterns \u003cem\u003eBootstrap support (\u0026gt;50%) from 1000 replicates is shown at nodes. Methanospirillum hungatei JF-1^T was included as the outgroup. Scale bar: 0.1 substitutions per site.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022563/v1/7b37abce6bf1ebc30c84796a.jpeg"},{"id":96917718,"identity":"7ce69886-48f7-4bae-8a22-eb6d0744060a","added_by":"auto","created_at":"2025-11-27 14:10:27","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1102892,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative Heatmap of Antimicrobial Activity in Halophilic Strains \u003cem\u003eInhibition zone radii (mm) are mapped for producer (column) vs. target (row) interactions. Darker colors denote stronger activity; grey cells are self-comparisons. The map highlights broad-spectrum inhibitors (e.g., SDI50) and susceptible strains (e.g., SDI81).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022563/v1/f9c3ab49d24fc49187c61e9a.jpeg"},{"id":96917504,"identity":"272b478d-febc-4919-bff3-0094dddb167f","added_by":"auto","created_at":"2025-11-27 14:09:54","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":366072,"visible":true,"origin":"","legend":"\u003cp\u003eAntagonistic Interactions in Halophilic Archaea from Oualidia Salterns (Morocco), Visualized by Cross-Streak Assay \u003cem\u003e(a) SDI50 vs. SDI81 inhibition halo. (b) Haloferax sp. SDI17 vs. SDI81 (Ø = 54 mm). (c) Inhibition of SDI17 by other isolates, demonstrating diverse antimicrobial profiles.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022563/v1/6ad53fcc2b78325eb64da25a.jpeg"},{"id":96808365,"identity":"d47da64a-3ad7-465a-89f2-2a452e610a73","added_by":"auto","created_at":"2025-11-26 09:28:32","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":202070,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative Antimicrobial Activity of Khenifiss Saltern Haloarchaeal Isolates Against SDI81.\u003cem\u003e Inhibition zone diameters (mm) are shown for each isolate.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022563/v1/38edcf5d927eeeb0c483f0fe.jpeg"},{"id":96922874,"identity":"b32a1d9d-d749-4aa1-aa4f-0ca2f850311f","added_by":"auto","created_at":"2025-11-27 14:20:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4521482,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8022563/v1/c947e60f-f0cf-4e30-b3f5-46cb8ff9534f.pdf"},{"id":96917194,"identity":"ade3ea6e-6093-4b81-92cc-afccd7aec90f","added_by":"auto","created_at":"2025-11-27 14:09:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1282645,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemetaryinformationsVF.docx","url":"https://assets-eu.researchsquare.com/files/rs-8022563/v1/88ec7f0119934d562d5b9a63.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cultivation and characterization of halophilic archaea from Moroccan Atlantic salterns: insights into diversity and bioactive potential","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMarine solar salterns represent model ecosystems for studying microbial adaptation and evolution under multiple physicochemical constraints (Oren, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Carr\u0026eacute;-Mlouka, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Formed along tropical and subtropical coasts for sea salt production, these anthropogenic systems comprise sequential evaporation ponds where calcite (CaCO₃) and gypsum (CaSO₄) precipitate first, followed by NaCl crystallization around 35% salinity (Oren, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Oren et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ventosa et al., \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The resulting salinity gradient, along with variations in ionic composition, temperature, pH, and oxygen availability, imposes strong selective pressure, shaping highly specialized microbial communities. From salinities above 25%, these communities are typically dominated by members of the class \u003cem\u003eHalobacteria\u003c/em\u003e, with \u003cem\u003eHalorubrum\u003c/em\u003e and \u003cem\u003eHaloquadratum\u003c/em\u003e (the latter being typical of Mediterranean salterns) representing the most abundant and cosmopolitan genera, (Naghoni et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mani et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cui and Dyall-Smith, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dindhoria et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Garc\u0026iacute;a-Rold\u0026aacute;n et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), followed by other genera such as \u003cem\u003eHaloarcula, Halobacterium, Haloferax\u003c/em\u003e, and \u003cem\u003eNatrinema\u003c/em\u003e. Less abundant yet ecologically significant taxa, including \u003cem\u003eHalonotius, Halogeometricum, Halomicrobium, Halostagnicola, Halococcus, Natronomonas\u003c/em\u003e, and \u003cem\u003eHaloplanus\u003c/em\u003e, reflect the remarkable phylogenetic and ecological diversification of \u003cem\u003eHalobacteria\u003c/em\u003e under fluctuating physicochemical conditions (Ding et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; G\u0026oacute;mez-Villegas et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hashemzahi et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Menasria et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Strakov\u0026aacute; et al., \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Youssef et al., \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTaxonomically, \u003cem\u003eHalobacteria\u003c/em\u003e (phylum \u003cem\u003eMethanobacteriota\u003c/em\u003e (Formerly \u003cem\u003eEuryarchaeota\u003c/em\u003e) (G\u0026ouml;ker and Oren, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)) comprises two orders: \u003cem\u003eHalobacteriales\u003c/em\u003e, encompassing nine families (\u003cem\u003eHaladaptataceae\u003c/em\u003e, \u003cem\u003eHalalkalicoccaceae, Haloarculaceae, Halobacteriaceae\u003c/em\u003e, \u003cem\u003eHalococcaceae\u003c/em\u003e, \u003cem\u003eHaloferacaceae\u003c/em\u003e, \u003cem\u003eNatrialbaceae\u003c/em\u003e, \u003cem\u003eNatronoarchaeaceae\u003c/em\u003e, and \u003cem\u003eSalinarchaeaceae\u003c/em\u003e), 81 genera and 375 species, and \u003cem\u003eHalorutilales\u003c/em\u003e represented by the single described species, \u003cem\u003eHalorutilus salinus\u003c/em\u003e; Recent (meta)genomic analyses suggest a far greater diversity, with approximately 1,033 \u003cem\u003eHalobacteriales\u003c/em\u003e species (divided into 114 genera) and seven \u003cem\u003eHalorutilales\u003c/em\u003e species (three genera) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gtdb.ecogenomic.org/tree?r=f__UBA12382\u003c/span\u003e\u003cspan address=\"https://gtdb.ecogenomic.org/tree?r=f__UBA12382\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, october 24, 2025). Although metagenomics provides global distribution patterns, culture-dependent studies remain crucial to uncover their physiology, ecological rules, and potential applications, as cultivable taxa represent only a small fraction of natural hypersaline diversity (Cui and Dyall-Smith, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBeyond abiotic constraints, \u003cem\u003eHalobacteria\u003c/em\u003e communities are also shaped by complex biotic interactions. Competition for limited nutrients and space drives the production of antimicrobial compounds, notably halocins, proteinaceous molecules reported in \u003cem\u003eHaloferax\u003c/em\u003e, \u003cem\u003eHalobacterium\u003c/em\u003e, \u003cem\u003eNatrinema\u003c/em\u003e, \u003cem\u003eHaloterrigena\u003c/em\u003e, \u003cem\u003eHalorubrum\u003c/em\u003e, \u003cem\u003eHaloarcula, Halogeometricum, and Halomicrobium\u003c/em\u003e (Atanasova et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Besse et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; De Castro et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ghanmi et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kumar and Tiwari, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Halocins, classified as large (\u0026gt;\u0026thinsp;10 kDa, e.g., Halocin H1 and H4) or microhalocins (\u0026le;\u0026thinsp;10 kDa, e.g., Halocin C8, Halocin S8), act by disrupting membranes, Na⁺/H⁺ antiporter inhibition, receptor blocking, or cell lysis, sometimes requiring activation by halolysins (Chen et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Hao et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mart\u0026iacute;nez-Espinosa, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Some have broad-spectrum activity, some inhibit other haloarchaea, halophilic bacteria and even certain eukaryotes (Atanasova et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Besse et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ghanmi et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mart\u0026iacute;nez-Espinosa, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Shand and Leyva, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Recent findings on lanthipeptides further indicate that \u003cem\u003eHalobacteria\u003c/em\u003e possess additional narrow-spectrum antagonism against related taxa (Costa et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liang et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These stable and biocompatible molecules hold promising biotechnological potential (Kumar et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and in vitro antagonism assays are key for identifying novel bioactive archaea and understanding their ecological role (Elshafey et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; G\u0026oacute;mez-Villegas et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Waditee-Sirisattha et al., 2016).\u003c/p\u003e\u003cp\u003eDespite their ecological and applied relevance, the diversity of halophilic archaea and antimicrobial producing strains remains largely unexplored in Moroccan Atlantic salterns. These ecosystems, ranging from the semi-arid, groundwater-influenced Oualidia saltern to the hyperarid, ocean-fed Khenifiss saltern (Amimi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; El Hamoumi, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), provide natural gradients in salinity, sediment composition, and organic content that likely influence the diversity and functional potential of resident \u003cem\u003eHalobacteria\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThis study aims to fill this knowledge gap by exploring the cultivable diversity of halophilic archaea from Oualidia and Khenifiss sediments. Through environmental characterization, culture-dependent isolation, 16S rRNA-based phylogenetic identification, and in vitro antagonism assays, we characterize their taxonomic diversity and antimicrobial potential. By establishing the first comparative culture-based inventory of \u003cem\u003eHalobacteria\u003c/em\u003e from Moroccan Atlantic salterns, this work provides insights into their taxonomic diversity and highlights their potential as sources of stress-adapted bioactive compounds.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Sampling sites and sediment collection\u003c/h2\u003e\u003cp\u003eSediment samples were collected from two Moroccan marine solar salterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), including the Oualidia saltern (32\u0026deg;47'14.2\"N 8\u0026deg;58'10.1\"W), located 9.4 km northeast of the Oualidia lagoon on the Atlantic coast. This receives regular Atlantic seawater and significant submarine groundwater discharge (0.2\u0026ndash;1.2 m\u0026sup3;/s), supplying 30\u0026ndash;50% of the lagoon\u0026rsquo;s water (Fakir et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The groundwater originates mainly from deep Jurassic aquifers, which are highly mineralized and rich in sulfates and calcium, as shown by isotopic studies (El Meknassi et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Somoue et al., \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, the khenifiss saltern located 17 km inland from the Atlantic Ocean (27\u0026deg;54'57.9\"N 12\u0026deg;21'16.3\"W) and upstream of the Khenifiss lagoon, this site relies solely on seawater for salt production practices, with no freshwater inputs. Strong, persistent winds (average 6.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 m/s) (Tnoumi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) drive intense evaporation. Recent geomorphological shifts, such as increased vegetation and sedimentation, have led to channel closure and greater salt accumulation upstream (El Behja et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAt both sites, about 50 g of sediment was collected from multiple points within at 5\u0026ndash;10 cm depth (June 2022 for Oualidia and March 2023 for khenifiss). Samples were stored in sterile polyethylene bags, transported cooled, and processed within 24 hours. In situ measurements (pH, salinity, and temperature), were taken using a HANNA HI9828 multiparameter probe. For lab analyses, sediment was mixed with water (1:5 w/v).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Isolation and cultivation of halophilic archaea\u003c/h2\u003e\u003cp\u003eTwo culture media were used to support the growth of different halophilic populations. The Halophilic Medium (HM) was prepared as described by Kis-Papo and Oren (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) with the following composition (g/L): NaCl 158; MgCl₂\u0026middot;6H₂O 13; MgSO₄\u0026middot;7H₂O 20; KCl 4; CaCl₂\u0026middot;2H₂O 1; NaBr 0.5; NaHCO₃ 0.2; yeast extract 5; tryptone 8; glucose 1.\u003c/p\u003e\u003cp\u003eThe DSMZ Medium 97 contained (g/L): NaCl 250; MgSO₄\u0026middot;7H₂O 15; KCl 2; trisodium citrate 3; yeast extract 5; casamino acids 7.5; FeSO₄\u0026middot;7H₂O 0.05; MnSO₄\u0026middot;H₂O 0.20 mg/L. The pH of the media was adjusted to 7.5 using 1M NaOH, and agar (20 g/L) was added for solidification.\u003c/p\u003e\u003cp\u003eFor each sampling site, 1 g of sediment was homogenized in sterile saline solution (20% NaCl). Serial decimal dilutions (10⁻\u0026sup1; to 10⁻⁶) were plated (100 \u0026micro;L/plate) onto HM media and DSMZ 97 medium. Plates were incubated in sealed bags at 37\u0026deg;C (HM media) and 40\u0026deg;C (DSMZ 97) for 4 to 8 weeks. Distinct colonies were purified by repeated streaking, and pure cultures were preserved in liquid medium containing 15\u0026ndash;20% glycerol at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e\u003cp\u003ePurified isolates were subsequently transferred onto HM agar containing 20% (w/v) NaCl and incubated under standardized conditions (pH 7.5, 40\u0026deg;C). Colony morphology, pigmentation, and growth dynamics were recorded. Growth was further examined across salinity (5\u0026ndash;30% NaCl) at 40\u0026deg;C and temperatures (10\u0026ndash;55\u0026deg;C) at 20% ranges. From the initial collection of 204 isolates, a representative set of phenotypically distinct strains was selected. Archaeal and bacterial affiliation was preliminarily assessed by antibiotic susceptibility testing using ampicillin (10 \u0026micro;g), ciprofloxacin (5 \u0026micro;g), streptomycin (30 \u0026micro;g), gentamicin (10 \u0026micro;g), nalidixic acid (30 \u0026micro;g), vancomycin (30 \u0026micro;g), and bacitracin (8 \u0026micro;g) (Dridi et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Molecular identification and phylogenetic analysis\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted from freshly grown colonies using osmotic cell lysis in 50 \u0026micro;L of sterile ultrapure water, following the protocol of Pfeiffer adapted for halophilic archaea, as described in the Halohandbook (Dyall-Smith, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The resulting DNA served as a template for amplification of the 16S rRNA gene via PCR.\u003c/p\u003e\u003cp\u003ePCR amplification was performed in a final volume of 50 \u0026micro;L, containing: 10 \u0026micro;L of 10x buffer, 2 \u0026micro;L of each universal primer A8F (5\u0026prime;-AGAGTTTGATCCTGGCTCAG-3\u0026prime;) and R1492 (5\u0026prime;-ACGGCTACCTTGTTACGACTT-3\u0026prime;) (10 mM each), 1 \u0026micro;L of dNTPs (10 mM), 0.8 \u0026micro;L of MgCl₂, 2.5 U of Taq polymerase, 2 \u0026micro;L of DNA template, and nuclease-free water to volume. PCR conditions were as follows: initial denaturation at 95\u0026deg;C for 10 min; 30 cycles of denaturation at 95\u0026deg;C for 1 min; annealing at 48\u0026deg;C for 1.5 min; and extension at 72\u0026deg;C for 2 min; followed by a final extension at 72\u0026deg;C for 5 min and a hold at 12\u0026deg;C.\u003c/p\u003e\u003cp\u003eAmplified products were verified by 1% agarose gels, purified and sequenced by the Sanger method (Genewiz GmbH, Leipzig, Germany). Chromatograms were inspected using Chromas v2.6.6 (Goodstadt and Ponting, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and sequences were assembled in BioEdit (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.mbio.ncsu.edu/bioedit\u003c/span\u003e\u003cspan address=\"http://www.mbio.ncsu.edu/bioedit\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Taxonomic affiliation was determined by comparison with reference sequences in the EzBioCloud (EzTaxon) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ezbiocloud.net/\u003c/span\u003e\u003cspan address=\"https://www.ezbiocloud.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Potential chimeras were screened using Pintail v1.0 (Ashelford et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAll 16S rRNA sequences obtained from isolates of both salterns were deposited in GenBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/submit/genbank/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/submit/genbank/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and accession numbers are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOverview of halophilic archaeal strains from Oualidia (O-S) and khenifiss (K-S) salterns, showing strain designation, GenBank accession numbers, and molecular identification based on 16S rRNA gene BLAST analysis.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOrigin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGenbank Accession No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMolecular identification\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eOrigin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eGenbank Accession No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMolecular identification\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDIO15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248879\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNatrinema pelliribrum\u003c/em\u003e JCM 10476\u003csup\u003eT\u003c/sup\u003e (98.40%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD140\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX282994\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula hispanica\u003c/em\u003e Y27\u003csup\u003eT\u003c/sup\u003e (98.29%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248887\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNatrinema pallidum\u003c/em\u003e BOL6-1\u003csup\u003eT\u003c/sup\u003e (99.91%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSDR102\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX282998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula mannanilytica\u003c/em\u003e MD130-1\u003csup\u003eT\u003c/sup\u003e (97.65%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248881\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNatrinema pallidum\u003c/em\u003e BOL6-1\u003csup\u003eT\u003c/sup\u003e (99.26%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD100\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX282993\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula mannanilytica\u003c/em\u003e MD130-1\u003csup\u003eT\u003c/sup\u003e (98.43%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDIO6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248880\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNatrinema altunense\u003c/em\u003e AJ2\u003csup\u003eT\u003c/sup\u003e (98.65%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD50\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX282992\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula marismortui\u003c/em\u003e 8966\u003csup\u003eT\u003c/sup\u003e (98.09%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248885\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNatrinema altunense\u003c/em\u003e AJ2\u003csup\u003eT\u003c/sup\u003e (99.82%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHalobacterium jilantaiense\u003c/em\u003e NG4\u003csup\u003eT\u003c/sup\u003e (98.67%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248886\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNatrinema altunense\u003c/em\u003e AJ2\u003csup\u003eT\u003c/sup\u003e (99.81%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSDP2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHalobacterium jilantaiense\u003c/em\u003e NG4\u003csup\u003eT\u003c/sup\u003e (98.85%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248882\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNatrinema altunense\u003c/em\u003e AJ2\u003csup\u003eT\u003c/sup\u003e (99.63%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD56\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHalobacterium rubrum\u003c/em\u003e TGN-42-S1\u003csup\u003eT\u003c/sup\u003e (98.32%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248884\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNatrinema altunense\u003c/em\u003e AJ2\u003csup\u003eT\u003c/sup\u003e (99.32%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSDP11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHaloferax larsenii\u003c/em\u003e ZJ206\u003csup\u003eT\u003c/sup\u003e (99.24%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248883\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eNatrinema altunense\u003c/em\u003e AJ2\u003csup\u003eT\u003c/sup\u003e (99.83%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHaloferax mediterranei\u003c/em\u003e R-4\u003csup\u003eT\u003c/sup\u003e (99.73%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248890\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum tebenquichense\u003c/em\u003e ALT6-92\u003csup\u003eT\u003c/sup\u003e (99.58%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD102\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283030\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHaloferax volcanii\u003c/em\u003e DS2\u003csup\u003eT\u003c/sup\u003e (99.27%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDIP4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248895\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum persicum\u003c/em\u003e C49\u003csup\u003eT\u003c/sup\u003e (99.92%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD52\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHaloferax volcanii\u003c/em\u003e DS2\u003csup\u003eT\u003c/sup\u003e (99.28%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248892\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum distributum\u003c/em\u003e I-M\u003csup\u003eT\u003c/sup\u003e (98.73%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD22\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283031\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHaloferax volcanii\u003c/em\u003e DS2\u003csup\u003eT\u003c/sup\u003e (99.54%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248896\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum depositum\u003c/em\u003e Y78\u003csup\u003eT\u003c/sup\u003e (99,43%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD19\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHaloferax volcanii\u003c/em\u003e DS2\u003csup\u003eT\u003c/sup\u003e (99.83%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248893\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum coriense\u003c/em\u003e Ch2\u003csup\u003eT\u003c/sup\u003e (99,57%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSDP1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHalorbrum persicum\u003c/em\u003e C49\u003csup\u003eT\u003c/sup\u003e (99. 75%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248894\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum coriense\u003c/em\u003e Ch2\u003csup\u003eT\u003c/sup\u003e (99,32%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD03\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum californiense\u003c/em\u003e SF3 213\u003csup\u003eT\u003c/sup\u003e (99.23)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248888\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum coriense\u003c/em\u003e Ch2\u003csup\u003eT\u003c/sup\u003e (99.08%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD53\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX282997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum californiense\u003c/em\u003e SF3 213\u003csup\u003eT\u003c/sup\u003e (99.22%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDIE43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248897\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum coriense\u003c/em\u003e Ch2\u003csup\u003eT\u003c/sup\u003e (98.83%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD09\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX282996\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum californiense\u003c/em\u003e SF3 213\u003csup\u003eT\u003c/sup\u003e (99.40%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248891\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum coriense\u003c/em\u003e Ch2\u003csup\u003eT\u003c/sup\u003e (98.67%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD07\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX282995\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum californiense\u003c/em\u003e SF3 213\u003csup\u003eT\u003c/sup\u003e (99.63%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248889\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum californiense\u003c/em\u003e SF3\u0026nbsp;213\u003csup\u003eT\u003c/sup\u003e (99.43%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX282999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum distributum\u003c/em\u003e I-M\u003csup\u003eT\u003c/sup\u003e (99.22%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDIB3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248898\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum californiense\u003c/em\u003e SF3\u0026nbsp;213\u003csup\u003eT\u003c/sup\u003e (98.83%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD04\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum kocurii\u003c/em\u003e BG-1\u003csup\u003eT\u003c/sup\u003e (99.18%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248899\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum californiense\u003c/em\u003e SF3\u0026nbsp;213\u003csup\u003eT\u003c/sup\u003e (99.09%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSBK32\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283034\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum kocurii\u003c/em\u003e BG-1\u003csup\u003eT\u003c/sup\u003e (99.23%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248900\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalomicrobium mukohatatei\u003c/em\u003e arg-2\u003csup\u003eT\u003c/sup\u003e (98.35%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD08\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eHalostagnicola larsenii\u003c/em\u003e XH-48\u003csup\u003eT\u003c/sup\u003e (99.40%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248901\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalogeometricum rufum\u003c/em\u003e RO1-4\u003csup\u003eT\u003c/sup\u003e (99,67%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD76\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eNatrinema altunense\u003c/em\u003e AJ2\u003csup\u003eT\u003c/sup\u003e (98.84%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDII35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248902\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalogeometricum rufum\u003c/em\u003e RO1-4\u003csup\u003eT\u003c/sup\u003e (98.86%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSDOR2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eNatrinema altunense\u003c/em\u003e AJ2\u003csup\u003eT\u003c/sup\u003e (99.74%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248907\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloferax volcanii\u003c/em\u003e DS2\u003csup\u003eT\u003c/sup\u003e (99.45%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTSD31\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePX283004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eNatrinema pellirubrum\u003c/em\u003e JCM 10476\u003csup\u003eT\u003c/sup\u003e (99.64%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248905\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloferax volcanii\u003c/em\u003e DS2\u003csup\u003eT\u003c/sup\u003e (99.16%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248903\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloferax sulfurifontis\u003c/em\u003e M6\u003csup\u003eT\u003c/sup\u003e (98.56)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDIB5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248904\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloferax mediterranei\u003c/em\u003e R-4\u003csup\u003eT\u003c/sup\u003e (99.12%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248906\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloferax larsenii\u003c/em\u003e ZJ206\u003csup\u003eT\u003c/sup\u003e (99.66%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248908\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalococcus\u003c/em\u003e saccharolyticus P-423\u003csup\u003eT\u003c/sup\u003e (99.62%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248909\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHalococcus saccharolyticus\u003c/em\u003e P-423\u003csup\u003eT\u003c/sup\u003e (98.67%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248916\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula salaria\u003c/em\u003e HST01-2R\u003csup\u003eT\u003c/sup\u003e (98.74%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248915\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula marismortui\u003c/em\u003e JCM 8966\u003csup\u003eT\u003c/sup\u003e (99.60%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248913\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula marismortui\u003c/em\u003e JCM 8966\u003csup\u003eT\u003c/sup\u003e (99.71%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI30A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248914\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula marismortui\u003c/em\u003e JCM 8966\u003csup\u003eT\u003c/sup\u003e (99.24%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248912\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula hispanica\u003c/em\u003e Y27\u003csup\u003eT\u003c/sup\u003e (99.05%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248910\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula hispanica\u003c/em\u003e Y27\u003csup\u003eT\u003c/sup\u003e (99.02%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDIR04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248917\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula hispanica\u003c/em\u003e Y27\u003csup\u003eT\u003c/sup\u003e (99.19%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248911\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula hispanica\u003c/em\u003e Y27\u003csup\u003eT\u003c/sup\u003e (98.49%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248918\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula hispanica\u003c/em\u003e Y27\u003csup\u003eT\u003c/sup\u003e (98.37%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248919\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula argentiensis\u003c/em\u003e arg-1\u003csup\u003eT\u003c/sup\u003e (99.77%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSDI45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePV248920\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eHaloarcula quadrata\u003c/em\u003e JCM 11048\u003csup\u003eT\u003c/sup\u003e (99.58%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ePhylogenetic relationships were inferred from aligned sequences generated using MUSCLE (Edgar, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) implemented in Seaview v5.1 (Gouy et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Phylogenetic trees were constructed with the Maximum Likelihood (ML) algorithm (Felsenstein, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1981\u003c/span\u003e), and node support was evaluated by 1000 bootstrap replicates (Felsenstein, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Reference sequences of closely related taxa retrieved from NCBI were included to accurately position the isolates within their respective phylogenetic lineages.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Antagonistic interactions between isolated strains\u003c/h2\u003e\u003cp\u003ePurified isolates were cultured to the early stationary phase to ensure active metabolism and maximal secondary metabolite production. Both producer and target strains were standardized and reinoculated into fresh HM medium at an initial optical density of OD₆₀₀=0.001. Target strains were previously spread uniformly onto HM agar plates containing 20% (w/v) NaCl. Sterile paper discs (6 mm diameter) were impregnated with 15 \u0026micro;L of the producer culture and placed onto the surface of pre-inoculated plates.\u003c/p\u003e\u003cp\u003ePlates were incubated at 40\u0026deg;C for 5\u0026ndash;10 days in sealed plastic bags to prevent desiccation. Antimicrobial activity was evaluated by measuring the radius of the inhibition zones formed around the discs. Discs impregnated with sterile, uninoculated HM medium served as negative controls. Each assay was performed in triplicate, and inhibition zone radius (mm) are reported as mean standard deviation from three independent experiments.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Physicochemical characteristics of the studied salterns\u003c/h2\u003e\u003cp\u003eSoils from both salterns were slightly alkaline (pH 7.40\u0026ndash;7.49) consistent with industrial salterns (Ventosa et al., \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Salinity levels were high (16.7\u0026ndash;29.2%, w/v), suitable for extremely halophilic microorganisms. Temperatures ranged from 27.5 and 36.1\u0026deg;C, peaking at Oualidia in June due to its semi-arid coastal climate, characterized by mild winters, dry summers, and low annual precipitation (\u0026lt;\u0026thinsp;230 mm). Khenifiss, sampled in early spring, showed lower temperatures, reflecting its arid southern Moroccan coastal desert environment, marked by persistent winds, intense evaporation, and minimal rainfall (\u0026lt;\u0026thinsp;80mm) (Tnoumi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Culture dependent recovery of \u003cem\u003eHalobacteria\u003c/em\u003e along a salinity gradient\u003c/h2\u003e\u003cp\u003eA culture-dependent approach applied to sediments from two Moroccan salterns, Oualidia and Khenifiss, yielded 115 and 89 isolates respectively. The higher number of isolates from Oualidia suggests greater cultivability under intermediate salinities, which typically support a brorder diversity of halophilic microorganisms. In contrast, extreme salinities exert strong selective pressures, favoring only highly specialized taxa capable of key biogeochemical functions under osmotic stress (Gao et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). From these isolates, 42 from Oualidia and 25 from Khenifiss were selected based on distinct colony morphology, pigmentation, and physiological responses to variations in salinity, temperature, and pH (Table S2, S3). Pigmentation among the isolates ranged from pale pink to bright red (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Notably, all isolates exhibited resistance to a panel of bacterial-targeting antibiotics (ampicillin, ciprofloxacin, streptomycin, gentamicin, nalidixic acid, and vancomycin) but remained sensitive to bacitracin (8 \u0026micro;g). This antibiotic susceptibility profile is characteristic of the class \u003cem\u003eHalobacteria\u003c/em\u003e, as bacitracin disrupts the dephosphorylation of bactoprenol, a critical membrane carrier molecule, thereby inhibiting lipid biosynthesis (Kumar and Tiwari, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Oren, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe isolates demonstrated broad environmental tolerance, growing across a temperature range of 20\u0026ndash;60\u0026deg;C, pH 5\u0026ndash;9.5, and NaCl concentrations from 7.5 to 30%. Optimal growth was observed at 15\u0026ndash;25% NaCl, although several strains thrived even at 30%, confirming their classification as extreme halophiles and their affiliation with the class \u003cem\u003eHalobacteria\u003c/em\u003e (Amoozegar et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). While \u003cem\u003eHalobacteria\u003c/em\u003e can tolerate NaCl concentrations from 10% up to saturation (Cui and Dyall-Smith, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), they are generally more abundant in salinities above 21% and less prevalent below 13\u0026ndash;19% (Oren, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e). The recovery of numerous \u003cem\u003eHalobacteria\u003c/em\u003e strains from the moderately saline Oualidia saltern may be attributed to the clay-rich sediment matrix (Maanan et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), which enhances Na⁺ and K⁺ retention and maintains microenvironments conducive to osmotic balance (Fukushima et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Rozic, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Similar patterns have been documented in other salterns. For example, in the Siridao saltern (India), \u003cem\u003eHalobacteria\u003c/em\u003e constitute up to 85% of the microbial community in clayey sediments with intermediate salinities (Mani et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Metagenomic surveys of the Atlantic Isla Cristina saltern (Spain), which shares a thalassohaline water source with the Moroccan sites, revealed that \u003cem\u003eHalobacteria\u003c/em\u003e accounted for approximately 85% of the community at 21% salinity. A comparable fraction was observed in the Mediterranean Santa Pola saltern at 33% salinity, whereas a pond at 19% salinity within the same site contained only 47% \u003cem\u003eHalobacteria\u003c/em\u003e (Fern\u0026aacute;ndez et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Garc\u0026iacute;a-Rold\u0026aacute;n et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ventosa et al., \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These finding underscore that, in addition to salinity, sediment texture, hydrological connectivity, and oceanic origin play pivotal roles in shaping the composition and resilience of halophilic archaeal communities.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Phylogenetic resolution and species delineation of halophilic archaeal isolates using 16S rRNA analysis\u003c/h2\u003e\u003cp\u003eThe 16S rRNA gene was sequenced to determine the species affiliation of the isolates (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, several strains exhibited high sequence similarity to multiple closely related species, illustrating the well-documented limitations of 16S rRNA for precise species delineation. For example, strain SDI44 shared 99.45% identity with \u003cem\u003eHaloferax volcanii\u003c/em\u003e DS2\u003csup\u003eT\u003c/sup\u003e, with only slightly lower similarity to \u003cem\u003eH. gibbonsii\u003c/em\u003e, \u003cem\u003eH. sulfurifontis\u003c/em\u003e, \u003cem\u003eH. chudinovii\u003c/em\u003e, and \u003cem\u003eH. massiliensis\u003c/em\u003e, all exceeding the 98.56% threshold commonly used for species delineation (Kim et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Similar ambiguities were observed among several \u003cem\u003eHalorubrum\u003c/em\u003e and \u003cem\u003eHaloarcula\u003c/em\u003e isolates, as well as for \u003cem\u003eHalococcus\u003c/em\u003e strains SDI13 and SDI20. Notably, SDI20 exhibited 100% identity with both \u003cem\u003eH. saccharolyticus\u003c/em\u003e and \u003cem\u003eH. agarilyticus\u003c/em\u003e, while SDI13 reached 98.67% similarity with the same species. In contrast, isolate SDIR04 was confidently assigned to \u003cem\u003eHaloarcula hispanica\u003c/em\u003e (99.19% similarity), with a clear separation from the next closest species (\u0026lt;\u0026thinsp;98.65%).\u003c/p\u003e\u003cp\u003eSeveral isolates, including SDI01, SDI15, TSD56, and all \u003cem\u003eHaloarcula\u003c/em\u003e strains from Khenifiss, exhibited\u0026thinsp;\u0026lt;\u0026thinsp;98.65% 16S rRNA gene similarity to any described species, suggesting the presence of potential novel taxa (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Phylogenetic analysis, incorporating these isolates alongside closely related type species above the 98.56% threshold, confirmed their placement within the respective genera. However, some formed distinct branches, indicating evolutionary divergence between populations from Khenifiss and Oualidia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Within the genus \u003cem\u003eHaloferax\u003c/em\u003e, most isolates clustered with \u003cem\u003eH. sulfurifontis\u003c/em\u003e and were clearly separated from \u003cem\u003eH. volcanii\u003c/em\u003e, despite the latter showing the highest BLAST similarity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003eHaloarcula\u003c/em\u003e isolates either formed independent clusters or grouped with \u003cem\u003eH. salaria\u003c/em\u003e, while \u003cem\u003eNatrinema\u003c/em\u003e isolates mostly branched separately, except for TSD31, which shared a common ancestor with several type species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eInterestingly, some isolates with identical 16S rRNA sequences exhibited distinct phenotypic traits under identical culture conditions, including differences in colony and cell morphology, as well as variable responses to NaCl concentration, pH, and temperature (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, S2 and S3). These observations suggest that, despite genetic similarity, the isolates represent distinct strains with divergent physiological adaptations.\u003c/p\u003e\u003cp\u003eThese results highlight the limited resolution of 16S rRNA for species-level assignment, consistent with previous studies advocating the use of more variable markers, such as \u003cem\u003erpob\u0026rsquo;\u003c/em\u003e, for refined classification within closely related haloarchaeal taxa (Minegishi et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Walsh, \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). For definitive species delineation, a polyphasic, approach integrating genomic and phenotypic analyses against reference species under standardized conditions, is essential, in accordance with current standards for describing novel taxa within the class \u003cem\u003eHalobacteria\u003c/em\u003e (Cui et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Taxonomic diversity and site-specific distribution of \u003cem\u003eHalobacteria\u003c/em\u003e isolates\u003c/h2\u003e\u003cp\u003eIsolates from the Moroccan salterns were assigned to five families: \u003cem\u003eHaloferacaceae\u003c/em\u003e (\u003cem\u003eHalorubrum, Haloferax\u003c/em\u003e, and \u003cem\u003eHalogeometricum\u003c/em\u003e), \u003cem\u003eHaloarculaceae\u003c/em\u003e (\u003cem\u003eHaloarcula, Halomicrobium\u003c/em\u003e), \u003cem\u003eNatrialbaceae (Natrinema, Halostagnicola\u003c/em\u003e), \u003cem\u003eHalococcaceae\u003c/em\u003e (\u003cem\u003eHalococcus\u003c/em\u003e), and \u003cem\u003eHalobacteriaceae\u003c/em\u003e (\u003cem\u003eHalobacterium\u003c/em\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among these, \u003cem\u003eHalorubrum\u003c/em\u003e, \u003cem\u003eHaloarcula\u003c/em\u003e, \u003cem\u003eNatrinema\u003c/em\u003e, and \u003cem\u003eHaloferax\u003c/em\u003e were the most frequently isolated genera at both sites, with \u003cem\u003eHalorubrum\u003c/em\u003e emerging as the dominant taxon.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1. Dominant genera and their ecological adaptations\u003c/h2\u003e\u003cp\u003eThe predominance of \u003cem\u003eHalorubrum\u003c/em\u003e in both salterns is likely due to its broad adaptability to a wide range of NaCl concentrations (1.0\u0026ndash;5.2 M), minimal magnesium requirements (0.005\u0026ndash;0.6 M), rapid growth rates, and high adaptability under laboratory conditions, which enable it to colonize dynamic environments and outcompete slower-growing taxa (Ding et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Oren, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e; Yujie Tao et al., 2025). This ecological success is consistent with its widespread distribution in diverse hypersaline environments, including solar salterns, salt lakes, saline soils, and salted foods worldwide (Baati et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Burns et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; \u0026Ccedil;ınar and Mutlu, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Fern\u0026aacute;ndez et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kambourova et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Naghoni et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Oren, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eHaloarcula\u003c/em\u003e was co-dominant with \u003cem\u003eHalorubrum\u003c/em\u003e in the Oualidia saltern and also detected in khenifiss. This pattern mirrors its frequent co-occurrence with \u003cem\u003eHalorubrum\u003c/em\u003e in Indian and Turkish salterns (Akpolat et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mani et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), as well as its widespread detection in salterns from China, Spain, Algeria, Tunisia, Pakistan, and Germany (Cycil et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ding et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Hassani et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ochsenreiter et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Strakov\u0026aacute; et al., \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ventosa et al., \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Verma et al., \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The ecological versatility of \u003cem\u003eHaloarcula\u003c/em\u003e is attributed to its rapid colonization following salinity fluctuations, tolerance to low Mg\u0026sup2;⁺ concentrations, and metabolic flexibility (aerobic respiration, rhodopsin-mediated phototrophy, anaerobic nitrate reduction). In addition, its high carotenoid content provides protection against oxidative stress and intense solar radiation, particularly relevant under the sun-exposed conditions of Oualidia and Khenifiss salterns (Dillon et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mani et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ochsenreiter et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Strakov\u0026aacute; et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ventosa et al., \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eHaloferax\u003c/em\u003e, isolated from both salterns, frequently co-occurred with \u003cem\u003eHalorubrum\u003c/em\u003e and \u003cem\u003eHaloarcula\u003c/em\u003e, consistent with its cosmopolitan distribution across solar salterns in Australia, Algeria, California, Tunisia, India, Spain, and South Africa (Baati et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Bidle et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Burns et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Ding et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Mani et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; McDuff et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mizuno et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Najjari et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zafrilla et al., \u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Its ecological success is linked to rapid growth, pronounced metabolic versatility, including carotenoid production, extremophilic enzymes, the ability to grow on diverse substrates, and broad salinity tolerance (0.34\u0026ndash;5.2 M) (Elshahed et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004b\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e; Griffiths et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These traits likely explain its success in both Moroccan salterns despite differences in salinity, temperature, and sediment composition.\u003c/p\u003e\u003cp\u003eFinally, \u003cem\u003eNatrinema\u003c/em\u003e species, previously reported to dominate Indian saltern sediments through culture-independent studies (Manikandan et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), have also been reported in Slovenian and Algerian salterns (Imadalou-Idres et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Pašić et al., 2007). Their adaptation to extreme salinities (up to 30% NaCl), variable temperatures (25\u0026ndash;55\u0026deg;C), UV exposure, and heavy metals aligns with the environmental characteristics of Oualidia and Khenifiss, where sediments are enriched in metals such as nickel and cadmium (Kim et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Strakov\u0026aacute; et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2025\u003c/span\u003e)(Mejjad et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Tnoumi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), supporting their persistence in these habitats.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e3.4.2. Less abundant but ecologically significant genera\u003c/h2\u003e\u003cp\u003e\u003cem\u003eHalococcus\u003c/em\u003e and \u003cem\u003eHalomicrobium\u003c/em\u003e were detected at low abundances in the Oualidia saltern, reflecting their sensitivity to salinity fluctuations linked to salt production cycles. In particular, \u003cem\u003eHalococcus\u003c/em\u003e, which generally constitutes a minor fraction of haloarchaeal communities (around 5% in Iranian salterns and \u0026lt;\u0026thinsp;1% in Egyptian and Indian salterns), (Hashemzahi et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mani et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), was observed too alongside \u003cem\u003eHalorubrum\u003c/em\u003e, \u003cem\u003eHaloferax\u003c/em\u003e, and \u003cem\u003eHaloarcula\u003c/em\u003e during the early, lower-salinity phase of salt crystallization. It often becomes transiently dominant during the initial stages of salt production, when salinity remains moderate, before its proportion declines as other genera outcompete it under increasing salinity (Elshafey et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Imadalou-Idres et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mani et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eHalomicrobium\u003c/em\u003e, typically rare (1\u0026ndash;3%) in salterns of Turkey, India, Argentina, and China (Akpolat et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Oren, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Yang and Cui, \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Youssef et al., \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), preferentially colonizes organic-rich, low-Mg sediments. Its abundance increases after harvesting and declines during initial crystallization stages. This pattern is consistent with its low proportional occurrence in Oualidia during sampling in the evaporation ponds at the beginning of the salt-harvesting phase, a period that did not coincide too with the March sampling in Khenifiss (Castel\u0026aacute;n-S\u0026aacute;nchez et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mani et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Youssef et al., \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eHalogeometricum\u003c/em\u003e, isolated from Oualidia, was found at low abundances alongside \u003cem\u003eHaloferax\u003c/em\u003e, \u003cem\u003eHalorubrum\u003c/em\u003e, \u003cem\u003eHaloarcula\u003c/em\u003e and \u003cem\u003eHalococcus\u003c/em\u003e in Indian solar salterns (Manikandan et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Its ability to combine salt-in/ salt-out strategies, enables persistence under fluctuating salinity (Strakov\u0026aacute; et al., \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, it was absent from Khenifiss, possibly due to micro-niche availability or competitive exclusion (Bowers and Wiegel, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eHalostagnicola\u003c/em\u003e, recovered as a single isolate from the Khenifiss saltern, is a globally rare haloarchaeon, previously representing\u0026thinsp;~\u0026thinsp;5% of cultivated communities in Iranian salterns (Hashemzahi et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and reported from Thai saltern soils (Niyasom and Mamimin, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Its association with halophyte rhizospheres suggests that local vegetation at Khenifiss may provide suitable microhabitats (Tnoumi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the same site, where salinity remains both high and remarkably stable throughout the year, \u003cem\u003eHalobacterium\u003c/em\u003e, adapted to persistently high salinity (2.5\u0026ndash;5.2 M NaCl), elevated Mg\u0026sup2;⁺concentrations, and anoxic, organic-rich sediments, was the only other genus detected (Bidle et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Tnoumi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Youssef et al., \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.4.3. Drivers of spatial variability\u003c/h2\u003e\u003cp\u003eThe spatial variability observed between the Oualidia and Khenifiss archaeal assemblages likely results from both biotic and abiotic factors. Migratory birds that frequent these Ramsar-listed ecosystems may facilitate long-distance microbial dispersal, acting as biological vectors connecting geographically isolated hypersaline environments (Amimi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tnoumi et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yim et al., \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In parallel, local physicochemical parameters, such as salinity stability, pH, temperature, sediment structure, organic content, and vegetation, act as selective pressures shaping site-specific archaeal communities (Elshafey et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kipnyargis et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, evidence of metabolic cooperation exists. Some taxa synthesize and share essential compounds (e.g., biotin, carotenoids) with others lacking these pathways, supporting coexistence and reducing the likelihood of strict competitive exclusion. This \"altruistic\" cooperation can stabilize community structure in resource-limited, extreme environments (Garc\u0026iacute;a-Rold\u0026aacute;n et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Antimicrobial and antagonistic potential of cultivable haloarchaeal isolates\u003c/h2\u003e\u003cp\u003eAntagonism assays conducted under standardized conditions (22% NaCl, 40\u0026deg;C, pH 7.0), corresponding to the average growth optima of the isolates, revealed a complex network of intra- and intercommunity interactions among haloarchaeal isolates from the Oualidia and Khenifiss salterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Inhibition zones, ranging from 2 to 27 mm in radius, indicated substantial functional heterogeneity, likely due to differences in metabolite production or sensitivity among the isolates.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.5.1. Patterns of Antagonistic Activity\u003c/h2\u003e\u003cp\u003eAt Oualidia, isolates of \u003cem\u003eNatrinema\u003c/em\u003e (SDI14, SDI16, SDI19, SDI22, SDI24, and SDI39) and \u003cem\u003eHaloferax\u003c/em\u003e (SDI44, SDI50) exhibited broad-spectrum inhibitory activities. Notably, \u003cem\u003eHaloferax\u003c/em\u003e sp. SDI17 displayed the strongest antagonistic activity (27 mm radius) against \u003cem\u003eHalorubrum\u003c/em\u003e sp. SDI81, yet was itself inhibited by other \u003cem\u003eHaloferax\u003c/em\u003e, \u003cem\u003eNatrinema\u003c/em\u003e, and \u003cem\u003eHalococcus\u003c/em\u003e isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This reciprocal inhibition suggests the coexistence of multiple bioactive compounds within the community. Similarly, at Khenifiss, \u003cem\u003eHalorubrum\u003c/em\u003e sp. SDI81 was inhibited by isolates from multiple genera (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e), indicating that antagonistic traits are conserved across geographically separated salterns. The apparent inactivity of some isolates under standardized conditions may reflect dependence on specific growth parameters or physiological states (Besse et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.5.2. Putative antimicrobial compounds: Halocins and beyond\u003c/h2\u003e\u003cp\u003eWhile the chemical nature of the inhibitory compounds was not directly characterized in this study, the genera exhibiting pronounced antagonism (\u003cem\u003eHalorubrum\u003c/em\u003e, \u003cem\u003eHalomicrobium\u003c/em\u003e, \u003cem\u003eHaloferax\u003c/em\u003e, \u003cem\u003eHalobacterium\u003c/em\u003e, \u003cem\u003eNatrinema\u003c/em\u003e, \u003cem\u003eHalococcus\u003c/em\u003e, and \u003cem\u003eHalogeometricum\u003c/em\u003e) have been previously reported as halocin producers (Besse et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ghanmi et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Halocins are ribosomally synthesized antimicrobial peptides or proteins produced by Halobacteria, capable of acting both within and cross genera (Atanasova et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Besse et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Najjari et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Many active isolates are known to produce of C8-like halocins, often associated with (halI) immunity clusters that confer self protection, potentially explaining the observed reciprocal inhibition (Atanasova et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; De Castro et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). As reported by Ghanmi et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), a single strain can produce multiple halocins simultaneously, thereby enhancing its overall antimicrobial potential. The exclusive detection of halocin S8 in \u003cem\u003eHaloferax\u003c/em\u003e and \u003cem\u003eNatrinema\u003c/em\u003e may account for their pronounced inhibitory activities. Additional halocins, such as H4, may also contribute to the observed antagonism particularly in \u003cem\u003eHalomicrobium\u003c/em\u003e, \u003cem\u003eHalogeometricum\u003c/em\u003e, \u003cem\u003eHaloferax\u003c/em\u003e, and \u003cem\u003eNatrinema\u003c/em\u003e (Besse et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; De Castro et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Makarova et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.5.3. Ecological relevance and alternative mechanisms\u003c/h2\u003e\u003cp\u003eDespite their detectability under laboratory conditions, the ecological significance of halocins remains debated. Kis-Papo and Oren, (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) reported no antimicrobial activity in ultrafiltered cell-free brines from Eilat saltern, even when concentrated 53.5-fold to match the density of a fully grown culture (~\u0026thinsp;10⁹ cells ml⁻\u0026sup1;), suggesting rapid inactivation or insufficient accumulation in situ. However, subsequent studies have shown that halocins are often membrane-associated or transported via extracellular vesicles, indicating alternative stabilization and delivery mechanisms in natural habitats (Besse et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eBeyond halocins, lanthipeptides (lantibiotics), ribosomally synthesized and post-translationally modified by LanM enzymes, may contribute too to the observed antagonistic interactions. These compounds are widely distributed among \u003cem\u003eHalobacteriales\u003c/em\u003e (40%), \u003cem\u003eHaloferacales\u003c/em\u003e (38%), \u003cem\u003eNatrialbales\u003c/em\u003e (17%), and unclassified lineages (5%), where they disrupt membranes or cell wall biosynthesis and modulate cellular motility through archaellin gene expression (\u003cem\u003eflgA1\u003c/em\u003e), thereby promoting competitive fitness and environmental adaptation (Chakraborty et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The first archaeal lantipeptide, Archalan α, characterized in \u003cem\u003eHaloferax mediterranei\u003c/em\u003e (Liang et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), exhibits narrow-spectrum activity against phylogenetically related strains. Furthermore, studies on \u003cem\u003eHfx. Mediterranei\u003c/em\u003e have demonstrated that antimicrobial activity persists even after deletion of halocin and lantipeptide genes, implicating halolysins, extracellular proteases capable of activating halocins or directly lysing target cells (Chen et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Costa et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Biotechnological potential of halophilic Archaea\u003c/h2\u003e\u003cp\u003eCollectively, these finding demonstrate that Moroccan salterns serve as untapped reservoirs of archaeal bioactive compounds. While detectable through antagonism assays, the full biotechnological potential of these compounds remains largely unexplored. This dual perspective (ecological and applied) oepns new avenues for both understanding microbial dynamics in extreme environments and discovering novel antimicrobial peptides stable under harsh conditions. From a biotechnological standpoint, halocins stand out due to their exceptional stability under high temperatures, salinity, and pH fluctuations, as well as their resistance to organic solvents, properties rarely found in conventional antimicrobials (Dini et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These characteristics make halocins promising candidates as natural preservatives, particularly in extreme environments such as salted food systems, where traditional preservatives often fail. Their mechanism of action, which involves membrane pore formation and inhibition of ion transporters, offers an attractive alternative to conventional antibiotics, especially against multidrug-resistant pathogens (Bucataru and Ciobanasu, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Emerging research even suggests potential cardioprotective properties for certain halocin-like peptides (Kumar et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHalolysins exhibit remarkable robustness, remaining active across a broad range of conditions: 40\u0026ndash;60\u0026deg;C, pH 8\u0026ndash;9, and up to 4 M NaCl. They also retain functionality in the presence of solvents, metals, or surfactants (Hao et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Hou et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These properties support their potential use in industrial biotechnology, including leather tanning, marine product fermentation, and the degradation of hypersaline proteinaceous waste. Finally, the structural and enzymatic diversity of LanM systems represents a valuable resource for bioengineering, enabling the design of tailor-made therapeutic or industrial peptides. Beyond, their applied potential, these systems play a crucial ecological role in shaping microbial community structure, modulating coexistence and competition among haloarchaea in hypersaline ecosystems (Fu et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis work provides the first comprehensive evidence that Moroccan Atlantic salterns represent a valuable yet largely untapped reservoir of cultivable extreme halophilic archaea with significant biotechnological potential. Through an integrated approach combining environmental characterization and culture-dependent isolation, we successfully recovered 67 archaeal strains from Oualidia and Khenifiss salterns. These isolates span five families and nine genera within the class \u003cem\u003eHalobacteria\u003c/em\u003e. Despite exhibiting over 98.56% 16S rRNA gene sequence identity, several strains displayed marked phenotypic and metabolic diversity, underscoring both the limitations of 16S rRNA-based species delineation and the importance of polyphasic taxonomic strategies for accurate archaeal classification.\u003c/p\u003e\n\u003cp\u003eThe broad-spectrum antimicrobial activity detected among our isolates further emphasizes the salterns as promising sources of bioactive compounds naturally adapted to extreme conditions. Notably, some strains exhibited strong inhibitory effects against closely related haloarchaea, reflecting the long-recognised but poorly understood antagonistic interactions within hypersaline ecosystems. These findings support the hypothesis that haloarchaea employ diverse antagonistic mechanisms, potentially involving halocins, lantipeptides or halolysins, to compete for limited resources in such challenging environments.\u003c/p\u003e\n\u003cp\u003eCollectively, this work establishes a solid foundation for several future research. First, it highlights the need to implement a comprehensive polyphasic taxonomic framework, integrating whole-genome sequencing, comparative proteomics, and metabolic profiling, to formally describe novel archaeal taxa identified here. Second, it opens perspectives for systematic exploration of halophilic archaea as sources of antimicrobial, and secondary metabolites with industrial and pharmaceutical relevance, particularly those functioning under high salinity, elevated temperature, or fluctuating pH. Finally, this study reinforces the importance of including Moroccan Atlantic salterns within global initiatives focused on extremophile biodiversity and bioprospecting, ensuring these unique ecosystems are recognized not only for their ecological value but also as strategic reservoirs of biotechnological innovation.\u003c/p\u003e\n\u003cp\u003eBy demonstrating the feasibility of isolating and functionally characterizing halophilic archaea from Moroccan salterns, and establishing a reproducible workflow for their screening, this study provides a robust platform for future efforts aimed at novel species discovery, metabolic pathway elucidation, and the development of bioactive compounds with applications across biotechnology and medicine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImane Charroud (IC)\u003c/strong\u003e: Data curation, Formal analysis, Visualization, Writing \u0026ndash; review \u0026amp; editing, Investigation, Validation, Methodology, Writing \u0026ndash; original draft. \u003cstrong\u003eNaima Boum\u0026rsquo;handi (NB)\u003c/strong\u003e: Data curation, Formal analysis, Writing \u0026ndash; review \u0026amp; editing, Investigation, Validation, Methodology. \u003cstrong\u003eMohamed Alouani (MA)\u003c/strong\u003e: Investigation, Validation, Methodology, Supervision, Funding acquisition. \u003cstrong\u003eAltaf El Blidi (AE)\u003c/strong\u003e: Investigation, Methodology, Supervision, Funding acquisition. \u003cstrong\u003eYannick Fleury (YF)\u003c/strong\u003e: Methodology, Validation, Writing \u0026ndash; review \u0026amp; editing and Supervision, Funding acquisition. \u003cstrong\u003eMohamed Jebbar (MJ)\u003c/strong\u003e: Conceptualization, Methodology, Investigation, Formal analysis, Writing \u0026ndash; review \u0026amp; editing, Supervision and funding acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by TOUBKAL program, a Franco-Moroccan Hubert Curien Partnership (PHC), funded by the Ministry of Higher Education, Scientific Research, and Innovation (MESRSI) in Morocco, and the Ministry for Europe and Foreign Affairs (MEAE) and the Ministry of Higher Education, Research, and Innovation (MESRI) in France. It was also supported by the ISblue project, Interdisciplinary Graduate School for the Blue Planet (ANR-17-EURE-0015), co-funded by a grant from the French government under the \u0026ldquo;Investissements d\u0026rsquo;Avenir\u0026rdquo; program and embedded in France 2030.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;During the preparation of this work the authors used Mistral AI in order to rephrase some words and improve the readability and language of the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA sequences reported in this study have been deposited in GenBank. Dataset identifiers are provided in Table 1. The isolates described in this manuscript are available upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkpolat, C., Fern\u0026aacute;ndez, A.B., Caglayan, P., Calli, B., Birbir, M., Ventosa, A., 2021. Prokaryotic Communities in the Thalassohaline Tuz Lake, Deep Zone, and Kayacik, Kaldirim and Yavsan Salterns (Turkey) Assessed by 16S rRNA Amplicon Sequencing. Microorganisms 9, 1525. https://doi.org/10.3390/microorganisms9071525\u003c/li\u003e\n\u003cli\u003eAmimi, T., Elbelrhiti, K., Adnani, M., Elbelrhiti, H., Chao, J., Oubbih, J., 2021. 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Sci. 11, 1421769. https://doi.org/10.3389/fmars.2024.1421769\u003c/li\u003e\n\u003cli\u003eTnoumi, A., Angelone, M., Armiento, G., Caprioli, R., Crovato, C., De Cassan, M., Montereali, M.R., Nardi, E., Parrella, L., Proposito, M., Spaziani, F., Zourarah, B., 2020. Assessment of Trace Metals in Sediments from Khnifiss Lagoon (Tarfaya, Morocco). Earth 2, 16\u0026ndash;31. https://doi.org/10.3390/earth2010002\u003c/li\u003e\n\u003cli\u003eVentosa, A., De La Haba, R.R., S\u0026aacute;nchez-Porro, C., Papke, R.T., 2015. Microbial diversity of hypersaline environments: a metagenomic approach. Current Opinion in Microbiology 25, 80\u0026ndash;87. https://doi.org/10.1016/j.mib.2015.05.002\u003c/li\u003e\n\u003cli\u003eVentosa, A., Fern\u0026aacute;ndez, A.B., Le\u0026oacute;n, M.J., S\u0026aacute;nchez-Porro, C., Rodriguez-Valera, F., 2014. The Santa Pola saltern as a model for studying the microbiota of hypersaline environments. 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Arch Microbiol 203. https://doi.org/10.1007/s00203-020-02181-7\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"extremophiles","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Extremophiles](https://link.springer.com/journal/792)","snPcode":"792","submissionUrl":"https://submission.springernature.com/new-submission/792/3","title":"Extremophiles","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Solar salterns, Halophilic Archaea, Halobacteria diversity, Antimicrobial activity, Atlantic Morocco","lastPublishedDoi":"10.21203/rs.3.rs-8022563/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8022563/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study provides the first culture-based survey of archaeal diversity in Moroccan Atlantic solar salterns (Oualidia and Khenifiss), using sediment samples collected across a gradient of moderate to extreme salinities (16.7\u0026ndash;29.2% w/v) and slightly alkaline pH (7.4\u0026ndash;7.5). A total of 67 representative strains were isolated, all belonging to the class Halobacteria. The community was dominated by the genus \u003cem\u003eHalorubrum\u003c/em\u003e, with additional genera (\u003cem\u003eHaloarcula, Haloferax\u003c/em\u003e, \u003cem\u003eNatrinema\u003c/em\u003e, \u003cem\u003eHalobacterium\u003c/em\u003e, \u003cem\u003eHalogeometricum\u003c/em\u003e, \u003cem\u003eHalococcus\u003c/em\u003e, \u003cem\u003eHalomicrobium\u003c/em\u003e, and \u003cem\u003eHalostagnicola\u003c/em\u003e) exhibiting site- and phase-specific distributions, likely influenced by salinity fluctuations, microhabitat availability, and harvesting stage. Phylogenetic analyses revealed several isolates with \u0026le;\u0026thinsp;98.65% 16S rRNA gene sequence similarity to known taxa, forming distinct clades and suggesting the presence of putatively novel species. Antagonism assays demonstrated widespread inhibitory activity among isolates, with \u003cem\u003eNatrinema\u003c/em\u003e and \u003cem\u003eHaloferax\u003c/em\u003e strains exhibiting the strongest antagonism against co-isolated haloarchaea, indicative of bioactive compound production (e.g., halocins, lanthipeptides, or halolysins). These results expand our understanding of haloarchaeal diversity in Atlantic salterns, underscoring their potential as reservoirs of extremophiles with biotechnological applications. Furthermore, this work highlights the necessity of integrating polyphasic and culture-independent approaches to resolve the taxonomy and chemical ecology of these dynamic hypersaline environments.\u003c/p\u003e","manuscriptTitle":"Cultivation and characterization of halophilic archaea from Moroccan Atlantic salterns: insights into diversity and bioactive potential","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 09:28:27","doi":"10.21203/rs.3.rs-8022563/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-26T04:58:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-22T17:20:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-05T11:48:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-02T07:28:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39813943985592535159844349027174563888","date":"2025-11-22T13:45:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58080478654361728102156235953823372933","date":"2025-11-22T04:10:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95747406340795383357877391301676249588","date":"2025-11-15T11:24:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-14T15:42:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-06T01:56:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-06T01:55:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Extremophiles","date":"2025-11-03T20:55:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"extremophiles","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Extremophiles](https://link.springer.com/journal/792)","snPcode":"792","submissionUrl":"https://submission.springernature.com/new-submission/792/3","title":"Extremophiles","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"980cc92c-9f4d-4204-b562-aed5bfed214d","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-24T13:09:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-26 09:28:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8022563","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8022563","identity":"rs-8022563","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}