Fresh and wet salt, sediment from under the salt and water, all sampled directly from salt production ponds, were collected from Aveiro saltern (40º38’50” N, 8º39’46” W) in September 2018 and from Olhão (37${}^{\circ }$1’35” N, 7${}^{\circ }$51’59” W) in August 2018, during salt production season. All samples were used for microbial isolation, aiming at obtaining members from different phyla, applying, therefore, different methodologies represented in Fig. 1 and specified below.

Each axenic isolate was cultivated in the corresponding broth medium of isolation and these cultures were used for DNA extraction. For isolates obtained in PDA medium, NZY Plant/Fungi gDNA Isolation kit (NZYTech, Lisbon, Portugal) was used, according to the manufacturer’s instructions. In the case of the isolates obtained in the remaining media, DNA was extracted using the E.Z.N.A. Bacterial DNA kit (Omega Bio-Tek, Norcross, Georgia, USA), according to the manufacturer’s instructions. DNA quantification was done by using the $\mu$DropTM platform (Thermo Fisher Scientific, Massachusetts, USA) and DNA quality was checked through a 30 min electrophoresis at 100 V in a 0.8% agarose gel with 1X Tris-acetate-EDTA (TAE) buffer (Bio-Rad, Hercules, California, USA) and stained with GreenSafe Premium (NZYTech).

Bacterial DNA was PCR-amplified with the universal primers 27F and 1492R [36], while Fungi DNA was PCR-amplified by using ITS1 and ITS4 primers [37].

For the isolates which strain designation starts with “C.”, the 16S rRNA gene primers, 27F and 1492R, were used and the PCR mixture consisted of 1 $\times$ Qiagen Multiplex PCR Master Mix (Qiagen, California, USA), 0.1 $\mu$M each primer and 25 ng of DNA template, in a final volume of 10 $\mu$L. The PCR reaction was carried out on the Veriti® Thermal Cycler (Applied Biosystems, Massachusetts, USA) using the following PCR program: initial denaturation for 5 min at 95 ${}^{\circ }$C, followed by 30 cycles of denaturation at 95 ${}^{\circ }$C for 45 s, annealing at 56 ${}^{\circ }$C for 45 s, and extension at 72 ${}^{\circ }$C for 1 min, and a final extension of 10 min at 72 ${}^{\circ }$C. PCR products were visualized through electrophoresis in a 0.8% agarose gel with 1 $\times$ TAE buffer, which was stained with SYBR Safe (Thermo Fisher Scientific). Purification and sequencing of these amplicons was carried at i3S (Porto, Portugal).

For the remaining isolates, including Fungi, the PCR mixture (25 $\mu$L) consisted of 1 $\times$ NZYTaq 2 $\times$ Green Master Mix (NZYTech), 0.1 $\mu$M each primer and 25 ng of DNA template. PCR program was carried out in a MyCycler™ Thermo Cycler (Bio-Rad) and, for 16S rRNA gene amplification, consisted in an initial denaturation step of 5 min at 95 ${}^{\circ }$C, followed by 30 cycles of 1 min at 95 ${}^{\circ }$C, 1 min at 56 ${}^{\circ }$C (annealing temperature) and 1.5 min at 72 ${}^{\circ }$C, and a final extension of 10 min at 72 ${}^{\circ }$C. For amplification of the ITS region, the PCR program was the same as previously described, with exception that an annealing temperature of 55 ${}^{\circ }$C was applied. PCR products were visualized through electrophoresis in a 0.8% agarose gel with 1 $\times$ TAE buffer, which was stained with GreenSafe Premium. These amplicons were purified using GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, United Kingdom) and sequenced at GATC Biotech (Constance, Germany).

Sequence analyses were carried out using Geneious Prime (Biomatters Ltd, Auckland, New Zealand). Curated sequences were matched in the EzBioCloud [38] database to determine their closest relatives. Phylogenetic trees were built using MEGA 7 (Pennsylvania State University, Pennsylvania, USA) software [39]. Briefly, all sequences, either from the isolates in this study and from the closest related strains determined on EzBioCloud, were grouped by phyla. Separately, phyla-grouped sequences were aligned using the ClustalW algorithm with a Gap Open Penality of 15.00 and a Gap Extension Penalty of 6.66. The resulting multiple alignments for each phylum were used to construct phylogenetic trees, by applying the Maximum Likelihood statistical method, the phylogeny test based on the Bootstrap method considering 1000 replicates, and the Tamura-Nei substitution model. Different strains were used as outgroup depending on the phyla.

All sequences obtained on this study were submitted to the GenBank database [40] at National Centre for Biotechnology Information (NCBI) web platform under the following accession numbers: OK169439-OK169485, OK216020-OK216124, OK287059 and OK326927.

PAST 3.22 (University of Oslo, Oslo, Norway) [41] software was used to calculate the Fisher’s $\alpha$, Margalef’s and Simpson’s indexes, which allow the characterization of the microbial community in terms of diversity, richness, and evenness, respectively.

A sample rarefaction curve was estimated in PAST 3.22 by using the Mao’s Tau index [42]. Similarities among samples were calculated on PAST 3.22 by using the Sørensen coefficient and Bray-Curtis index. All results were computed taking into account a 95% confidence and 1000 iterations as bootstrap value.

The putative potential of all isolates obtained in this study to produce NPs was assessed through PCR. Briefly, isolates DNA was screened for the presence of NRPS and PKS-I genes. NRPS genes were amplified using primers MTF2 [5${}^{\prime }$- GCNGG(C/T)GG(C/T)GCNTA(C/T)GTNCC-3${}^{\prime }$ (AGGAYVP, core motif I)] and MTR [5${}^{\prime }$-CCNCG(AGT)AT(TC)TTNAC(T/C)TG-3${}^{\prime }$ (QVKIRG, core motif V)] [43], while $\beta$-ketosynthase (KS) domain fragments within the Type I polyketide synthase genes (PKS-I) was amplified using primers MDPQQRf (5${}^{\prime }$-RTRGAYCCNCAGCAICG-3${}^{\prime }$) and HGTGTr (5${}^{\prime }$-VGTNCCNGTGCCRTG-3${}^{\prime }$) [44]. PCR reactions of 25 $\mu$L containing 1 $\times$ NZYTaq 2 $\times$ Green Master Mix, 0.8 $\mu$M of each primer and 25 ng DNA template were prepared. The PCR protocol was carried out in a MyCycler™ Thermo Cycler and consisted of an initial denaturation at 95 ${}^{\circ }$C for 5 min, 11 cycles of denaturation at 95 ${}^{\circ }$C for 1 min, annealing at 60 ${}^{\circ }$C for 30 s and extension at 72 ${}^{\circ }$C for 1 min, with the annealing temperature being reduced 2 ${}^{\circ }$C every cycle; followed by 30 cycles of denaturation at 95 ${}^{\circ }$C for 1 min, annealing at 40 ${}^{\circ }$C for 30 s and extension at 72 ${}^{\circ }$C for 1 min, and a final extension of 10 min at 72 ${}^{\circ }$C [45]. Amplicons were visualized in a 1.2% agarose gel prepared with 1 $\times$ TAE buffer and stained with GreenSafe Premium. NRPS and PKS amplicons have an expected size of approximately 1000 bp [43] and 700 bp [44], respectively. The Planctomycete UC49.1 was used as positive control for amplification of both genes.

Aiming at a broader range of microbial isolation, 6 media with different nutritional compositions along with 2 pre-formulated media (Supplementary Table 1) were assayed. A medium selective for fungi, PDA, allowed the isolation of 45 fungal strains. The most effective media for bacteria were M600 (32 strains), R2A (32 strains) and M607 (31 strains). The remaining media used were less rich in nutrients and apparently less successful (14 strains altogether in 4 different media). Ultimately, the media used proved to be effective in the isolation of a highly heterogenous microbiota from salterns.

The microbial isolation screening carried out for samples from Aveiro and Olhão salterns allowed the isolation of 154 strains, of which 47 were affiliated with Fungi and 107 with Bacteria. Particularly, 26 fungal and 75 bacterial isolates were obtained from Aveiro, and 21 fungal and 32 bacterial isolates from Olhão. All these isolates have been putatively identified based on the ITS and 16S rRNA genes for Fungi and Bacteria, respectively. The isolates relatedness was examined by building phylogenetic trees (Supplementary Fig. 1).

Despite the high abundance of salterns in Portugal, their microbiota has been poorly explored. The existent research reports comprises (i) the description of new strains detected by metagenomics approaches of Tavira saltern (Algarve salt flats) microbiota [46], (ii) metagenomics studies of Algarve salt flats [22, 23, 47], (iii) characterization of novel isolated species from Tavira saltern [26] and from Tejo salt flats [48], (iv) an archaeal isolate genome announcement from Figueira da Foz salt flats [49] and (v) extensive isolation approaches targeting Aveiro saltern and a saltern from Olhão (Algarve salt flats) focusing on the overall bacterial microbiota [24]. In this last study, the direct comparison between the microbiota of both salterns was not possible since the nature of samples, the methodology applied and the time of sampling were different between both salterns. None of these studies have targeted the fungal community, most likely because the prevalence of fungi in marine and aquatic environments, including hypersaline ones, was overlooked for many years [50, 51, 52]. In the last decades, Fungi members have been reported in every aquatic environment explored, including marine and hypersaline ones [50, 51, 52]. The first description of fungi in salterns was reported by Gunde-Cimerman and collaborators [53], which aroused a fascinating interest in the scientific community [12, 54, 55]. Since this, saltern fungi have been studied worldwide, however the Portuguese salterns mycobiota remains unknown. In this study, the microbial diversity was assessed at genera (Fig. 2) and phyla (Fig. 3) levels. The fungal isolates obtained were phylogenetically related with the phylum Ascomycota (Fig. 3), with the exception of isolate AW17 that was affiliated to the genus Sporobolomyces (Fig. 2), specifically to the species Sporobolomyces ruberrimus, a yeast within the phylum Basidiomycota (Fig. 3).

Colour scheme representation by phylum: grey for Ascomycota, black for Basidiomycota, blue for Pseudomonadota, orange for Actinomycetota, purple for Bacillota, brown for Bacteroidota, green for Planctomycetota, yellow for Rhodothermota and red for Gemmatimonadota. Full filled with colour represent isolates common to both salterns and filled with pattern represents exclusivity to a specific saltern. (*) represents isolates identified only to the family level.

Overall information of the environmental prevalence of strains phylogenetically closely related with the isolates obtained with the present study is compiled in Table 1 (Ref. [1, 5, 9, 11, 24, 26, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107]), where presence or absence in salterns environments was highlighted.

Within Fungi, members of genus Penicillium were the only ones detected in both salterns and the most abundant ones (Figs. 2 and 3; Supplementary Table 2).

The Pseudomonadota genera Rhodovibrio and Microbulbifer were the only ones common to both saltern (Fig. 2). The similarity score of all 4 Microbulbifer isolates with M. halophilus (98.50% in the 16S rRNA gene) revealed a distant relatedness, suggesting that these isolates can be members of a novel taxon (Fig. 4, Ref. [39]). Two other species of Pseudomonadota were detected as singletons in Olhão saltern, which were closely affiliated with Salinicola zeshunii and Aureimonas glaciistagni. Neither of these two species have been associated specifically with microbiota of salterns (Table 1). Furthermore, one Pseudomonadota isolate was affiliated with strains of Brevundimonas not yet related with halophilic environments (Table 1), but instead associated with humanized habitats, namely washing machines [108] and space laboratory [109]. Additionally, the Roseibacterium isolate AW9 obtained on this study showed a low similarity (97.53% in the 16S rRNA gene) with the closest related species, R. elongatum, which might be indicative of a novel species (Fig. 4). Also from Aveiro saltern, 4 strains of phylum Pseudomonadota phylogenetically related to uncultured members of Aquichromatiaceae were isolated, but showing low levels of similarity (98.08 up to 98.30% in the 16S rRNA gene), so highlighting the novelty of these strains (Fig. 4).

Within the phylum Bacillota, Mesobacillus and Rossellomorea were the only two Bacillota genera that were detect at both Aveiro and Olhão salterns. Despite the geographical distance, the 2 strains (AW18 and OW14) closely affiliated with the novel established genus Rossellomorea [110] showed a perfect similarity between their 16S rRNA gene sequences and a close phylogenetic relationship with the isolate Rossellomorea sp. es.034 (PDIY01000001). Therefore, these strains are strong candidates for the description of a novel species (Fig. 4). Moreover, 2 isolates (AW3 and AW5) showed a similarity of up to 98.66% in the 16S rRNA gene with Bacillus sinesaloumensis, and other 2 isolates (OSBR114 and OW16) showed similarity scores of up to 98.4% in the 16S rRNA gene with Metabacillus litoralis. All of them may be representatives of novel taxa (Fig. 4).

Within Actinomycetota, 3 isolates were phylogenetically closely related with known opportunistic human pathogens (Rothia and Tsukamurella), that were obtained from Olhão saltern [74]. The presence of these species in Olhão saltern microbiota may be related with human activity, not only through salt exploitation but also through touristic related activities happening in Olhão salterns, like saltern baths (Table 1). Additionally, one isolate affiliated with Gordonia oryzae was obtained. This specie was recently described [75] and was not associated with extreme environments until this study. Furthermore, the Olhão saltern isolate OSBR104 was affiliated with the Actinomycetota family Iamiaceae, but the closest related strain described was Aquihabitans daechungensis [111] with 92.37% similarity in the 16S rRNA gene. Therefore, OSBR104 may represent a novel taxon (Fig. 4). Additionally, isolates related with Microbacterium amylolyticum (C.AS10 and C.AW5), Microbacteium ginsengiterrae (C.AW1), Micrococcus luteus (C.OS4) and Nocardioides salarius (OSBR100) showed low similarities with the mentioned strains (Supplementary Table 2) and may represent new taxa (Fig. 4).

Both classes of the phylum Planctomycetota, namely Planctomycetia and Phycispharae were isolated in this study. Planctomycetia isolates obtained were affiliated with genera Alienomonas (1 isolate), Rhodopirellula (2 isolates) and Maioricimonas (1 isolate) (Fig. 2). Four out of the 5 Planctomycetia isolates showed only up to 97.95% similarity in the 16S rRNA gene with the closest related described species (Fig. 4; Supplementary Table 2). These data are indicative of novel Planctomycetia taxa (Fig. 4). Within the less known Planctomycetota class Phycispharae, only one isolate (ASED31) was obtained, for which the species Algisphaera agarilytica was the most related one but in a far distant level (88.8% similarity in the 16S rRNA gene), being putatively indicative of a new family within Phycispharae (Fig. 4). Curiously, all planctomycetal isolates obtained in the present study were obtained from Aveiro saltern.

Within Rhodothermota, the family Balneolaceae was the only one recovered with 3 isolates (ASBR13, AS101 and OSBR103), that due to the phylogenetic distance, could not be related with any known Rhodothermota genus (Fig. 4).

Inside the phylum Gemmatimonadota, only one isolate (AW12) phylogenetically placed within the family Longimicrobiaceae was retrieved (Fig. 3). However, the 16S rRNA gene distant relatedness with the closest described species, represented by 85.82% similarity with Longimicrobium terrae, may be indicative of a novel family (Fig. 4).

The most frequent fungal genus was Penicillium, since 26.6% of the overall microbial isolates were classified within this genus (Supplementary Table 2). The most common bacterial genera retrieved from both salterns were the Actinomycetotal Streptomyces (8.4%) and the Pseudomonadota Rhodovibrio (7.1%), followed by the genus Sinomicrobium (6.5%) that is a member of phylum Bacteroidota (Supplementary Table 2).

Regarding overall phyla and genera, the isolated microorganisms associated with Aveiro saltern showed a higher diversity (Fisher’s $\alpha$ index) and richness (Margalef’s index), as well as a lower dominance (Simpson’s index) (Table 2). By running separated analyses of the genera diversity within the fungal kingdom and the genera diversity within the bacterial domain, one difference about these indexes was observed when compared to the overall analysis. Particularly, the diversity of bacterial genera was higher in Olhão saltern population (Table 2).

In Aveiro, the highest diversity and richness were registered within Planctomycetota and Pseudomonadota, while the lowest value of dominance was observed in Pseudomonadota, highlighting the heterogeneity of the Pseudomonadota isolates community (Table 2). At Olhão saltern, Actinomycetota and Pseudomonadota showed the highest diversity, while the lowest diversity was observed within Ascomycota and Rhodothermota (Table 2). Additionally, in Olhão, the highest values of diversity and richness along with the lowest value of dominance were observed within Actinomycetota (Table 2), demonstrating the high heterogeneity of Actinomycetotal isolates obtained.

Despite the high isolated diversity, the Mao’s Tau rarefaction curve presented a continuous rise without achieving an asymptote, confirming that not all microbial diversity was recovered (Fig. 5).

Concerning the fungal biodiversity, 3 singletons were observed in Aveiro, while none was detected in Olhão. These singletons represent 6.4% of the whole fungal diversity (Supplementary Material). The overall bacterial diversity was comprised by 40 different genera, with 25 singletons, that represent 23.4% of the overall bacterial diversity (Supplementary Table 2). These may be rare members of the isolated bacterial communities of salterns. Additionally, from the 28 bacterial genera recovered from Aveiro saltern, 12 occurred as singletons, representing 16.0% of the overall diversity of isolates, while Olhão taxa biodiversity consisted in 20 different bacterial genera, where 13 occurred as singletons, representing 37.1% of the overall diversity of isolates (Supplementary Table 2). From all these singletons, only 2 were common to both salterns, namely, the Bacillota genera, Mesobacillus and Rossellomorea (Supplementary Table 2).

The Sørensen and Bray-Curtis indexes were determined between the microbial populations of both salterns as 0.33 and 0.43, respectively. These parameters are measures of the similarity between both microbial populations, therefore highlighting the dissimilarities between them. Taking into account that sampling dates were close and that all the procedures undertaken were the same for all samples, these differences between the two microbial populations studied may be a result of the different geographical locations of the two salterns (Aveiro at the North of Portugal vs. Olhão at the South of Portugal).

The biotechnological potential of all isolates obtained in this study was screened by the PCR analysis of PKS-I and NRPS genes (Table 3; Supplementary Table 2). From the 154 microorganisms isolated in this study, 51 (33.1%) did not present any PKS or NRPS genes and 80 (52%) presented one (64–41.6% PKS positive and 16–10.4% NRPS positive) or both genes (23 bacteria, 14.9%) (Table 3; Supplementary Table 2). Overall, this screening revealed that 66.9% of the isolates may present at least one of the genes, revealing their putative biotechnological potential (Table 3; Supplementary Table 2). Although microbial ability to produce bioactive metabolites is not strictly linked to PKS and NRPS genes, because many other genes have been associated with bioactive NPs production [112], the genomic presence of these genes already proved to be a good indicator of the production of antimicrobial compounds [113]. Furthermore, NPs derived from these genes, as polyketides (PK), nonribosomal peptides (NRP) or even PK/NRP hybrids, have demonstrated their high economical and pharmacological value [20, 114]. These gene clusters were previously detected in ascomycetes and in a high number, which may be associated with the production of several different NRPS- and PKS-derived NPs [115].

The high percentages and widespread distribution of NRPS and PKS genes observed in the phyla Pseudomonadota, Actinomycetota, and Bacillota was already reported by Wang et al. [115]. In contrast, both Bacteroidota and Planctomycetota are less explored phyla in what concerns their potential for the production of bioactive molecules, being research fields that are being launched with encouraging results obtained from a preliminary screening of NRPS and PKS genes [116, 117, 118]. Concerning Rhodothermota, since this phylum was recently branched out from Bacteroidota [8], no studies targeting their overall genomic potential for production of bioactive NPs are yet published. Regardless of the only 3 representatives obtained, since, up to the present moment, the phylum Rhodothermota has only 13 described species from 4 different families of only 1 class, the presence of at least one of these genes in all isolated members of this phylum pave the way for future deeper studies on this poorly known phylum. As detected in the present work, NRPS and PKS-I gene clusters were previously detected in ascomycetes and in a high number, which may be associated with the production of several different NRPS- and PKS-derived NPs [115]. No NRPS or PKS-I amplicons were detected either in Gemmatimonadota or Basidiomycota (Table 3; Supplementary Table 2). In fact, Gressler et al. [119] reported that PKSs and NRPSs are not the main contributors for basidiomycetes diversity of NPs. On the other hand, the poorly known phylum Gemmatimonadota was firstly described less than two decades ago and presently only has 6 described species. This scarce number of described species is due to the difficulties in successfully isolating Gemmatimonadota members in laboratory, despite being widely distributed in the environment [120]. Therefore, most of the research made on its bioactive potential relies on metagenomes, which demonstrated the presence of not only NRPS, PKS and hybrid PKS/NRPS gene clusters [121], but also a widespread and high prevalence of bacteriocin-related biosynthetic gene clusters [122].

Overall, the microbiota isolated from both salterns showed a great biotechnological potential. Curiously, although with lower number of isolates, the phyla that showed higher bioactive potential were the Rhodothermota, Bacteroidota and Planctomycetota, and not the well-known bioactive top producers, Actinomycetota [123].