Hypersaline environments (> 40 practical salinity units [PSU]) are among the most extreme conditions for all domains of life (Javor 1989). These environments, which include salt lakes, solar salterns, and deep-sea hypersaline anoxic basins, are globally widespread yet sparsely localized. They serve as habitats for a variety of halophilic and halotolerant bacteria, archaea, and protists (Harding and Simpson 2018; Ventosa et al. 2012). In particular, bacteria such as Salinibacter and archaea such as Haloarchaea have been the most extensively studied in hypersaline environments (Antón et al. 2000; Oren 2002; Ventosa et al. 2012). Among protists, the alga Dunaliella has been the subject of significant research for a long time (Javor 1989; Oren 2002). Since the 2000s, the isolation and identification of various protists from hypersaline environments sparked an increased research interest in halophilic protists (Harding and Simpson 2018). This burgeoning interest has led to numerous studies aimed at understanding the diversity (Filker et al. 2015; Lee et al. 2022a; Park and Simpson 2015; Toumi et al. 2005), ecological roles (Park et al. 2003; Pedrós-Alió et al. 2000), and adaptive mechanisms (Harding et al. 2016; Lee et al. 2022b) of these protists in extreme salinity conditions.

The taxon Heterolobosea Page and Blanton 1985 encompasses diverse protists that inhabit freshwater, marine, and various extreme environments (Harding and Simpson 2018; Lee and Park 2023; Pánek et al. 2017). This taxonomic group is particularly noteworthy for including numerous halophilic/halotolerant protists, making it a valuable model taxon for studying the evolutionary history and adaptive strategies of eukaryotic organisms in hypersaline environments (Aucher et al. 2020; Harding and Simpson 2018; Jhin and Park 2019; Lee et al. 2022b; Tikhonenkov et al. 2019). Among the members of Heterolobosea, the genus Pharyngomonas holds significant interest. Initially reported as Trichomastix salina by Entz (1904), the name was subsequently changed to Tetramitus salinus and then to Percolomonas salina (Kirby 1932; Larsen and Patterson 1990). The genus name Pharyngomonas was first introduced by Cavalier-Smith and Nikolaev (2008). Later, Park and Simpson (2011) and Harding et al. (2013) reconfirmed the flagellate and amoeba forms of Pharyngomonas, establishing its position as a deep-branching lineage within Heterolobosea. Except for the halotolerant Pharyngomonas turkanaensis, all species within the genus Pharyngomonas are obligate halophiles (Aucher et al. 2020; Cavalier-Smith and Nikolaev 2008; Harding et al. 2013; Park and Simpson 2011, 2015; Plotnikov et al. 2015). This trait makes the Pharyngomonas group particularly valuable for shedding light on the early adaptations and evolutionary trajectories of protists in hypersaline environments.

Halophilic protozoa have been thought to play a crucial role as grazers of bacteria and archaea in hypersaline environments (Park et al. 2003, 2006). Interestingly, the type of prokaryotic prey required for protozoan growth varies among different halophilic species. For instance, Halocafeteria readily consumes haloarchaea, which are prevalent in hypersaline environments; however, haloarchaea do not significantly influence the growth of Pharyngomonas, indicating a different ecological adaptation and dietary preference (Harding et al. 2016).

In the present study, we isolated two strains of amoeboflagellate from hypersaline water samples (~300 PSU) in the Republic of Korea. Based on light microscopic observations and molecular phylogenetic analysis, we identify the two strains as Pharyngomonas kirbyi. Furthermore, we determine the salinity (3 to 300 PSU) and temperature (5°C to 55°C) regimes required for the growth of these isolates. Based on the observed ranges of salinity and temperature for growth, we hypothesize that these two strains are true halophiles. Additionally, we propose that P. kirbyi is commonly found in hypersaline environments worldwide and plays an ecologically significant role as a grazer of prokaryotes or prey for higher trophic levels, contributing significantly to the ecological dynamics of these extreme environments.

Sampling, isolation, and cultivation

Two amoeboflagellates were isolated from hypersaline water samples collected in June 2021 from two solar salterns in the Republic of Korea (Geumjeong solar saltern [GJ] and Naeyang solar saltern [NY]; Fig. 1). Protists in the samples were enriched by inoculating 10% of each sample into liquid media with salinities of 200 PSU and 150 PSU for GJ and NY, respectively. These media were prepared by diluting Medium V (300 PSU; 272 g NaCl, 7.6 g KCl, 17.8 g MgCl₂, 1.8 g MgSO₄·7H₂O, and 1.3 g CaCl₂ per liter water; see Park 2012) with sterile double-distilled water and adding autoclaved barley grains. The barley grain supported the growth of prokaryotes, which were in turn prey for the protists. Monoprotistan cultures of amoeboflagellates were established using manual single-cell picking with a micropipette. Briefly, isolated single cells were inoculated into separate wells of a 24-well culture plate (30,024, SPL Life Sciences Co., Ltd., Pocheon, Korea) filled with the respective liquid media plus autoclaved barley grains and incubated at 35°C until they reached qualitatively sufficient abundance. Subsequently, each isolate was transferred to and maintained in 50 mL tissue flasks (70,125, SPL Life Sciences Co., Ltd.) containing 10 mL of the respective liquid media with autoclaved barley grains at 35°C. Subculturing was carried out every month.

Figure 1. Map of sampling locations (GJ and NY). GJ: Geumjeong solar saltern; NY: Naeyang solar saltern; PSU: practical salinity units.

Microscopy

Live trophozoites, flagellates, and cysts of each amoeboflagellate were imaged using differential interference contrast and phase contrast optics on a Leica DM5500B microscope (Leica, Wetzlar, Germany). Images were taken with a Leica DFC550 camera, and dimensions were measured using FIJI v2.14.0 (Schindelin et al. 2012).

Molecular sequencing and phylogeny

Nucleic acids from both amoeboflagellate cultures were extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. 18S ribosomal RNA (18S rRNA) gene sequences were obtained by PCR amplification using the primer set EukA/EukB (EukA 5´-AACCTGGTTGATCCTGCCAGT-3´; EukB 5´-TGATCCTTCTGCAGGTTCACCTAC-3´, Medlin et al. 1988). PCR amplification was conducted using TaKaRa Taq^TM polymerase (TaKaRa Bio Inc., Shiga, Japan) in a Biometra TRIO thermal cycler (Analytik Jena, Jena, Germany). The PCR steps were as follows: an initial denaturing step at 94°C for 5 minutes, followed by 35 cycles of 45 s at 94°C, 1 minute of annealing at 55˚C, and 3 minutes of extension at 72°C, with a final extension step at 72°C for 20 minutes. Each 18S rRNA gene sequence from the two amoeboflagellates were deposited in GenBank as accessions PP905598 and PP905599.

The 18S rRNA gene sequences from 71 representative heterolobosean species, including our two isolates, plus sequences from 16 representative non-heterolobosean species selected as outgroups, were used for phylogenetic analysis, based on a seed alignment originating from Lee et al. (2022b). The dataset was aligned using MAFFT v.7 (Katoh and Standley 2013) and masked by eye, with 1,407 sites retained. Maximum likelihood trees were estimated using IQ-TREE v.1.6.12. The GTR + F + I + G4 model of sequence evolution was selected using the best-fit model test option (−m TEST, Flouri et al. 2015; Minh et al. 2013; Nguyen et al. 2015). Statistical support was assessed using ultrafast bootstrapping with 1,000 replicates. Bayesian analysis was conducted using MrBayes 3.2.7a (Ronquist et al. 2012) with two independent runs, each with four chains running for 2,000,000 generations, with the default heating parameter (0.1) and sampling frequency (0.01). A conservative 30% burn-in was used; the average standard deviation of split frequencies for the last 70% of generations was < 0.01.

Salinity and temperature ranges for growth

Growth experiments of the two isolates under various salinity and temperature conditions were conducted as reported by Lee et al. (2022b). All treatments were performed in triplicate.

The estimation of salinity ranges for the growth of each isolate was conducted using media with 10 different salinities ranging from 3 to 300 PSU. Specifically, 990 μL of Medium V with varying salinities (3–300 PSU) and a barley grain were inoculated with 10 μL of actively growing stock culture (totaling 1 mL) and incubated in the dark at 35°C for 7 days. Culture viability was assessed daily by observing actively moving cells in 10 μL of the culture using phase contrast microscopy. The salinity range tolerated for growth was confirmed by serial transfer into fresh media with the same salinity (990 μL of media and 10 μL of inoculum), followed by daily re-examination of the culture for active cells over another 7 days.

The optimal temperature conditions for the growth of each isolate were determined by inoculating culture tubes containing 990 μL of 75 PSU Medium V with 10 μL of actively growing stock culture, along with an autoclaved barley grain, and incubating them in the dark at various temperatures: 5, 15, 25, 35, 37, 40, 45, 50, and 55°C. Actively moving cells were monitored daily in 10 μL of the culture for 7 days. Growth was further confirmed by transferring a sample of each isolate into fresh media, incubating it at the same temperature, and performing microscopic observations as described above.

Morphological characteristics

The morphology of P. kirbyi strains GJ1 (Fig. 2) and NY2 (Fig. 3) observed under the light microscope was similar. The amoeboid stages of strains GJ1 and NY2 were flattened and exhibited three distinct shapes: flabellate (Figs. 2A and 3A), ovoid (Figs. 2B and 3B), and rectangular (Figs. 2C and 3C). These shapes continuously changed among the three while the amoebae were in motion. The amoebae had a crenulated anterior hyaline area and moved slowly (Figs. 2A-F and F and 3A-E). They possessed short conical subpseudopodia (Figs. 2C and 3C) and long finger-like subpseudopodia (Figs. 2D and 3D). The mean lengths of the strains GJ1 and NY2 were 20.7 μm (range: 14.1–27.7 μm; n = 30) and 19.3 μm (range: 14.7–27.8 μm; n = 30), and the mean widths were 14.0 μm (range: 9.3–20.9 μm; n = 30) and 13.0 μm (range: 9.5–18.1 μm; n = 30), respectively (Table 1). The average ratios of length-to-width were 1.5 (range: 1.2–2.2) and 1.5 (range: 1.1–2.2), respectively (Table 1). The posterior ends of the amoebae displayed uroidal structures with root-like extensions (Figs. 2B and 3E). The single nucleus was usually located in the middle of the cell. Amoebae with two nuclei were often observed, indicating cells in the process of division (Fig. 2F). Cells of both strains contained one or more contractile vacuoles in the granuloplasm (Fig. 2C).

Table 1 . Morphological characteristics of Pharyngomonas kirbyi strains.

Species	Strain	Amoeba				Flagellate			Cyst max. diameter (µm)	References
		Length (µm)	Width (µm)	Length-to-width ratio		Length (µm)	Width (µm)	Length-to-width ratio
Pharyngomonas kirbyi	GJ1	20.7 (14.1–27.7)	14.0 (9.3–20.9)	1.5 (1.2–2.2)		13.6 (7.3–18.7)	5.2 (4.3–7.0)	2.6 (1.4–3.9)	10.2 (8.1–11.8)	This study
	NY2	19.3 (14.7–27.8)	13.0 (9.5–18.1)	1.5 (1.1–2.2)		13.7 (10.1–17.5)	6.5 (4.7–8.5)	2.1 (1.7–2.8)	8.7 (7.3–10.3)	This study
	AS12B	NR (12–33.5)	NR (12.5–30)	NR		15.0 (11–18)	5.3 (3–8)	2.9 (1.7–3.8)	NR (9–12.5)	Park and Simpson (2011); Harding et al. (2013)
	SD1A	NR (12–30.5)	NR (12–30)	NR		11.6 (9–14)	4.6 (3–6)	2.6 (1.9–4.1)	NR (10.5–15)	Park and Simpson (2011); Harding et al. (2013)

Values are presented as mean (range).

NR: not reported.

Figure 2. Differential interference contrast micrographs (A-I, K-N) and phase contrast micrograph (J) of Pharyngomonas kirbyi strain GJ1. (A-F) Amoebae (trophozoites) showing size, shape, and pseudopod formation. (G-J) Flagellate phase, with four flagella. (K-N) Cysts with a spherical shape and smooth surface. Scale bar = 10 µm.

Figure 3. Differential interference contrast micrographs of Pharyngomonas kirbyi strain NY2. (A-E) Amoebae (trophozoites) showing size, shape, and pseudopod formation. (F-I) Cysts with a spherical shape and smooth surface. (J-N) Flagellate phase, with four flagella. Scale bar = 10 µm.

The flagellate forms of strains GJ1 and NY2 were frequently observed in cultures that had been maintained for an extended period (Figs. 2G-J and J and 3J-N). The flagellates of both strains had a spindle shape, with the posterior end being more pointed than the anterior end and the dorsal side more convex than the ventral side (Figs. 2G-I and I and 3J-L), and four flagella (Figs. 2J and 3M). The mean lengths of strains GJ1 and NY2 were 13.6 μm (range: 7.3–18.7 μm; n = 30) and 13.7 μm (range: 10.1–17.5 μm; n = 30), and the mean widths were 5.2 μm (range: 4.3–7.0 μm; n = 30) and 6.5 μm (range: 4.7–8.5 μm; n = 30), respectively (Table 1). The average ratios of length-to-width were 2.6 (range: 1.4–3.9) and 2.1 (range: 1.7–2.8), respectively (Table 1). Flagellates of both strains were mononucleate, with the nucleus positioned in the anterior portion of the cell (Figs. 2G and 3K). A ventral groove appeared at the anterior of the cell and extended to the posterior end (Figs. 2G and 3K), while a curved cytopharynx extended inward from below the nucleus (Figs. 2I and 3J, L, L). Numerous vacuoles, including food vacuoles, were widely distributed from near the center to the posterior end of the cell. Of the four flagella, two were directed anteriorly and two posteriorly (Figs. 2J and 3M). The anterior flagella were similar in length to the cell body, whereas the posterior flagella were often longer than the cell. The cells swam slowly while rotating. Several dividing cells were also observed (Fig. 3N).

Cysts of strains GJ1 and NY2 had a spherical shape with a smooth surface (Figs. 2K-N and N and 3F-I). The mean diameters of the cysts were 10.2 μm (range: 8.1–11.8 μm; n = 30) for strain GJ1 and 8.7 μm (range: 7.3–10.3 μm; n = 30) for strain NY2. The cells of both strains did not have a cyst pore and sometimes formed a thick cyst wall, which was distinctly divided into endocyst and ectocyst (Figs. 2M and 3H). Some cysts had no cytoplasm, presumably due to excystment (Figs. 2N and 3I).

Molecular phylogeny

The 18S rRNA gene sequences obtained from strains GJ1 and NY2 were 3,233 bp and 3,240 bp long, respectively. The sequences were 99.9% identical to each other and 99.5% similar to P. kirbyi (AF011465, listed as Macropharyngomonas halophila), the highest match returned by a BLASTN search of GenBank. The 18S rRNA gene sequences included three group I introns, of 409 bp (positions 539–947 in GJ1 and 538–946 in NY2), 381 bp (positions 1907–2287 in GJ1 and 1906–2286 in NY2), and 360 bp (positions 2503–2862 in GJ1 and 2502–2861 in NY2). The first intron in both strains was located at the same positions as the introns in Pharyngomonas sp. isolate DD1 (MN969060). The second intron matched the position of the intron in P. kirbyi AS12B (HQ898857) and the third intron was positioned similarly to the introns in P. kirbyi AS12B and SD1A (HQ898857 and HQ898858, respectively). Like other Pharyngomonas strains, the helix 17_1 was absent in sequences of both strains.

Phylogenetic analysis of 18S rDNA sequences placed both strains in the Pharyngomonada, a basal clade in Heterolobosea, with maximum ultrafast bootstrap support (UFBS) and a Bayesian posterior probability (PP) of 1 (Fig. 4). Specifically, they grouped with the halophilic P. kirbyi sequences (AF011465, HQ898857, and HQ898858) with good UFBS (98%) and a Bayesian PP of 1 (Fig. 4).

Figure 4. Maximum likelihood phylogenetic tree of 18S rRNA gene sequences showing the phylogenetic position of Pharyngomonas kirbyi strains GJ1 and NY2 relative to 69 heterolobosean taxa and 16 species outgroups (i.e., Euglenozoa, Jakobida, and Tsukubamonas globosa). Ultrafast bootstrap support values (≥ 95%) are shown at the nodes. Solid circles represent a Bayesian posterior probability of 1 (< 0.95 not shown).

Growth properties: salinity and temperature

Strains GJ1 and NY2 were able to grow in salinities ranging from 75–200 PSU at 35°C (Fig. 5). The optimal salinity for the growth of both strains in liquid culture was 75–100 PSU (Fig. 5). Cultures of strains GJ and NY did not exhibit positive net growth at 50 or 250 PSU, but the cells remained viable at these salinities for at least 7 days (data not shown). Strains GJ1 and NY2 grew well at 25°C, 35°C, and 37°C, and 35°C and 37°C, respectively, in a liquid culture of 75 PSU, with the highest density observed at 35°C. The cultures died after a 7-day incubation period at temperatures of 25°C or lower, or 40°C or higher.

Figure 5. Salinity range for growth of halophilic/halotolerant heteroloboseans, including Pharyngomonas kirbyi. Yellow asterisks represent relatively high densities of living cells.

First report of Pharyngomonas kirbyi in the Republic of Korea

Strains GJ1 and NY2, which exhibited the characteristic amoeboid and flagellate morphologies of the genus Pharyngomonas, were cultured and observed in the Republic of Korea for the first time (Entz 1904; Gunderson 1981; Harding et al. 2013; Kirby 1932; Park and Simpson 2011; Plotnikov et al. 2015; Post et al. 1983; Ruinen 1938). The amoebae displayed a flattened shape and continuously transitioned between flabellate, ovoid, and rectangular forms during movement, with crenulated hyaline anterior pseudopodia. The flagellates possessed four flagella, two anterior and two posterior, and were characterized by a ventral groove and a curved cytopharynx. Comparative analysis of the amoeboid form of P. kirbyi strains AS12B and SD1A, as reported by Harding et al. (2013), indicates that while average sizes for the amoeboid forms were not provided, making direct comparisons of means difficult, the maximum sizes of strains GJ1 and NY2 were smaller than those of strains AS12B and SD1A, although their size ranges overlapped (Table 1). The length of the flagellates was slightly less than that of strain AS12B but greater than that of strain SD1A (Table 1) (Park and Simpson 2011). The width of strain GJ1 was similar to that of strain AS12B, while strain NY2 exhibited the largest width among the compared strains (Table 1) (Park and Simpson 2011). Molecular phylogeny of the 18S rDNA sequences of strains GJ1 and NY2 places them sister to P. kirbyi sequences (AF011465, HQ898857, and HQ898858). Strains GJ1 and NY2 lacked helix 17_1 in their 18S rDNA, in which is unusual for Heterolobosea. This absence is consistent with all other species within the genus Pharyngomonas, which also do not possess helix 17_1 (Aucher et al. 2020; Cavalier- Smith and Nikolaev 2008; Park and Simpson 2011). Based on morphology and 18S rDNA sequences, then, we identify strains GJ1 and NY2 as the first reported P. kirbyi strains from the Republic of Korea.

Halophily and adaptive radiation of Pharyngomonas kirbyi

Among halophilic/halotolerant protists, a significant number are classified within the Heterolobosea (Aucher et al. 2020; Jhin and Park 2019; Kirby et al. 2015; Lee et al. 2022b; , 2012, 2009; Park and Simpson 2011, 2016; Plotnikov et al. 2015; Tikhonenkov et al. 2019). The definition of “halophiles” varies, but the most commonly accepted classification is Kushner’s (1978), categorizing them into extreme halophiles (growing best at 150–300 PSU), borderline extreme halophiles (growing best at 90–230 PSU), moderate halophiles (growing best at 30–150 PSU), slight halophiles (growing best at 10–30 PSU), and halotolerant organisms that can grow without the absolute necessity of salt. More recently, Oren (2008) defined halophiles as organisms that grow optimally at 50 PSU or higher, and can tolerate salinities of 100 PSU or higher. All previously reported species of the genus Pharyngomonas, except for P. turkanaensis, have an optimal salinity of 75 PSU or higher, categorizing them as obligate halophiles based on these criteria (Aucher et al. 2020; Park and Simpson 2011; Plotnikov et al. 2015). Similarly, strains GJ1 and NY2, which grow within a salinity range of 75–200 PSU and thrive best at 75–100 PSU, can also be considered true halophiles. Although strains GJ1 and NY2 exhibited identical ranges of salinity tolerance (75–200 PSU) and optimal salinity for growth (75–100 PSU), these parameters differed from those observed in strains AS12B (growth range: 75–200 PSU, optimal salinity: 100–150 PSU) and SD1A (growth range: 100–250 PSU, optimal salinity: 150–200 PSU), despite all being the same species (Fig. 5). These differences are likely attributable to variations in cultured prey bacteria or the ionic composition of the culture medium (Park and Simpson 2011).

Adaptive radiation in halophilic organisms is primarily observed in archaea and bacteria (Oren 2002). All known halophilic archaea belong to the class Haloarchaea (formerly Halobacteria), representing a prime example of adaptive radiation in these environments (Oren 2002). Conversely, halophilic bacteria are more widely distributed across various taxonomic groups, with the family Halomonadaceae particularly exemplifying adaptive radiation among halophilic bacteria (Oren 2002). The adaptive radiation of halophilic eukaryotes was first proposed by Kirby et al. (2015) within the family Tulamoebidae of Heterolobosea, which encompasses various obligate halophilic organisms. This hypothesis was further substantiated by Jhin and Park (2019) with the identification of the fully halophilic Aurem hypersalina, forming a sister lineage to Tulamoebidae. The Heterolobosea exclusively encompasses various groups of halophilic and halotolerant eukaryotes, including the Percolomonas group, the Euplaesiobystra group, Selenaion, and the Pharyngomonas group (Aucher et al. 2020; Lee et al. 2022b; , 2012; Park and Simpson 2011; Plotnikov et al. 2015; Tikhonenkov et al. 2019). The Pharyngomonas group is of particular interest as it represents a deep-branching lineage within Heterolobosea, composed of halophilic and halotolerant species. Notably, all strains of P. kirbyi, which form distinct subgroups within this lineage, are obligate halophiles, exhibiting a clear instance of adaptive radiation. This adaptive radiation underscores the evolutionary processes that enable these protists to thrive in hypersaline environments. Thus, the more detailed evolutionary history of the adaptive radiation of halophilic eukaryotes will be understood through further studies of the genus Pharyngomonas.

Prevalence and ecological importance of Pharyngomonas in hypersaline environments

Pharyngomonas spp. have been identified and isolated from a variety of hypersaline environments, such as salt lakes, pools, puddles, and solar salterns, across multiple continents, including Europe, North America, Australia, and Africa (Aucher et al. 2020; Entz 1904; Kirby 1932; Park and Simpson 2011, 2016; Plotnikov et al. 2015; Post et al. 1983). A previous study using a culture-independent method, specifically metabarcoding with primers that amplify the V9 region of 18S rDNA, identified four sequences closely related to P. kirbyi from hypersaline waters (180–380 PSU) of the Eui-Seong solar saltern in the Republic of Korea (Lee et al. 2022a). In the present study, P. kirbyi was isolated and identified for the first time in the Republic of Korea from high salinity waters (~300 PSU) in various salterns. This finding suggests that Pharyngomonas spp. are common halophilic protists inhabiting hypersaline environments.

Halophilic protozoa are considered to play an ecologically significant role as prokaryote grazers in hypersaline environments (, 2006). Previous studies have indicated that P. kirbyi faces challenges in feeding on the dominant Haloarchaea in hypersaline environments (Harding et al. 2016; Harding and Simpson 2018). However, it has been shown to grow by consuming a variety of bacteria (Harding et al. 2016; Park and Simpson 2011), and in this study, the two strains also thrived by feeding on indigenous prokaryotes. Although the specific prey preferences of Pharyngomonas spp. remain unknown, these organisms are likely to occupy an important position in the food web as prokaryote grazers in such habitats. Furthermore, Pharyngomonas spp. can contribute to a prey population for higher trophic levels in hypersaline environments.

In this study, two strains of P. kirbyi were isolated and identified for the first time from hypersaline waters in solar salterns in the Republic of Korea. Like previously known P. kirbyi strains, these two strains are obligate halophiles with optimal growth at 75–100 PSU, providing robust evidence for the adaptive radiation of halophilic eukaryotes. Moreover, species within the genus Pharyngomonas, which includes only halophilic and halotolerant organisms, have been commonly found in hypersaline environments across various continents, including Europe, North America, Australia, Africa, and now Asia (this study). This emphasizes their widespread presence in hypersaline environments and underscores their ecological importance as grazers of prokaryotes or prey for higher trophic levels within these habitats. Future research should focus on the specific mechanisms of adaptation and the broader ecological impacts of these protists in hypersaline habitats, enhancing our knowledge of microbial ecology and evolution in such extreme conditions.

Supplementary information accompanies this paper at https://doi.org/10.5141/jee.24.073.

Taxonomy summary.

We would like to thank Dong Hyuk Jeong for his assistance with experiments, and Aaron A Heiss for editing and reviewing this manuscript for English language.

GJ: Geumjeong solar saltern

NY: Naeyang solar saltern

PSU: Practical salinity units

UFBS: Ultrafast bootstrap support

PP: Posterior probability

JSP supervised the study. HBL performed the majority of the experiments. HBL and JSP drafted the manuscript. Both authors read and approved the final manuscript.

This work was supported by the National Institute of Biological Resources of Korea (NIBR) under grant (NIBRE202405), funded by the Ministry of Environment (MOE) of the Republic of Korea; Korea Institute of Marine Science & Technology (KIMST) under grant (RS-2023-00256330), funded by Ministry of Oceans and Fisheries (MOF); and the National Research Foundation of Korea (NRF) grant to JSP funded by the Korean government (NRF-2022R1I1A2064117).

All data generated in this study are deposited in the related repositories. The names of the repositories and accession numbers are included in the article.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.