When ecological transitions are not so infrequent: independent colonizations of athalassohaline water bodies by Arcellidae (Arcellinida; Amoebozoa), with descriptions of four new species

Abstract The salinity and humidity barriers divide biodiversity and strongly influence the distribution of organisms. Crossing them opens the possibility for organisms to colonize new niches and diversify, but requires profound physiological adaptations and is supposed to happen rarely in evolutionary history. We tested the relative importance of each ecological barrier by building the phylogeny, based on mitochondrial cytochrome oxidase gene (COI) sequences, of a group of microorganisms common in freshwater and soils, the Arcellidae (Arcellinida; Amoebozoa). We explored the biodiversity of this family in the sediments of athalassohaline water bodies (i.e. of fluctuating salinity that have non-marine origins). We found three new aquatic species, which represent, to the best of our knowledge, the first reports of Arcellinida in these salt-impacted ecosystems, plus a fourth terrestrial one in bryophytes. Culturing experiments performed on Arcella euryhalina sp. nov. showed similar growth curves in pure freshwater and under 20 g/L salinity, and long-term survival at 50 g/L, displaying a halotolerant biology. Phylogenetic analyses showed that all three new athalassohaline species represent independent transition events through the salinity barrier by freshwater ancestor, in contrast to the terrestrial species, which are monophyletic and represent a unique ecological transition from freshwater to soil environments.


Introduction
The distribution of biodiversity at a global scale is structured by the major ecosystems, which are delimited by ecological barriers (Simpson 1944 ). Among these, the salinity barrier, which separates marine and continental saline systems from other environments, is by far the best studied. It di vides biodi versity at a global scale, because crossing it demands profound physiological and metabolic adaptations . T he efficiency of this barrier has been well documented in animals, whose internal or gans ar e separ ated fr om the envir onment by skin or an exoskeleton, which helps to regulate osmotic processes (Hutchinson 1975 ). Unicellular eukary otes (protists), ho w ever, are more permeable and require specialized organelles (i.e. contractile vacuoles) (Docampo et al. 2013 ) and/or metabolic pathways that enable osmoregulation (i.e. ionic pumps and transporters or compatible solutes) (Harding et al. 2017 ).
Ecological transitions between these ecosystems have been documented in se v er al clades (Lee and Bell 1999 ), but are considered to happen rarely in the evolutionary history of eukaryotes, microbial or not (Hutchinson 1975, Logares et al. 2009 ). Still, along with an increasing knowledge of protist systematics, a growing number of new instances of transitions through the salinity barrier are appearing, as in cryptophytes (Filker et al. 2017 ), diatoms (Alverson et al. 2007, Roberts et al. 2022, dinoflagellates (Annenk ov a et al. 2020 ) or testate amoebae (González-Miguéns, Soler-Zamor a, Fernando User os et al. 2022, González-Miguéns et al. 2023.
The salinity barrier has been tr aditionall y studied in marine gr oups that subsequentl y ada pted to fr eshwater (Lee and Bell 1999 ). Although less frequent and less studied, there are cases in the opposite dir ection, suc h as the marine Tric hopter a famil y Chathamidae (Riek 1977 ). Ne v ertheless, these studies are generally conducted in brackish environments with a clear connectivity between marine and freshwater. In this case, our aim was to study the transitions of a primarily freshwater group to saline and marine systems. For this reason, our study took place in athalassohaline systems, which, by definition, are those saline systems that hav e ne v er been connected to the sea and whose salinity le v el and composition ar e differ ent fr om seawater (Last 2002, Or en 2007, Safarpour et al. 2018. They typically occur in endorheic dr aina ge basins under arid or semiarid climates wher e e v a por ation exceeds pr ecipitation (Hammer 1986, O'leary and Glenn 1994, Wurtsbaugh et al. 2017. Envir onmental par ameters in these lakes often vary to extreme values. Salinity can fluctuate along the year and r eac h v alues m uc h higher than the av er a ge 35 g/L of seawater . T emper atur es and solar r adiation ar e usuall y ele v ated and water le v els fluctuate gr eatl y. Like in estuaries, the or ganisms that inhabit athalassohaline systems need important physiologi-cal adaptations to cope with these variations (Mesbah and Wiegel 2011, Plemenitaš and Gunde-Cimerman 2011, Harding et al. 2017. All these c har acteristics and their lac k of connection with the marine environment make them an interesting case, ideal for the study of ecological transitions from freshwater to saline environments. Similarly to the salinity barrier, the humidity barrier, which we define here as the separation between terrestrial and aquatic en vironments , also demands profound adaptations to withstand the constraints of living out of water, that is, desiccation, or underwater, that is, osmotic pr essur e and gas exchange . T he humidity barrier has structured much of vascular plants' evolutionary history and generated changes in their anatomy, physiology and r epr oductiv e biology (K enric k andCr ane 1997 , Niklas 2000 ). In animals, the most obvious examples are the colonization of land by amphibians (Sc hoc h 2014 ) and the ancestor of insects (Grimaldi et al. 2005, Rota-Stabelli et al. 2013. Transitions from terrestrial to aquatic systems can be witnessed in the evolutionary history of marine mammals (Fordyce and Barnes 1994 ) and also in many insect groups, such as Gyrinidae , Nepidae , Odonata, Tric hopter a or Ephemer opter a (Grimaldi et al. 2005 ). In protists, the colonization of terrestrial environments requires the capacity for forming desiccation-resistant structures (cysts, spores or sclerotia) (Geisen et al. 2018 ). These structures are widespread in the tree of eukaryotes, and although they have not been studied in depth in free-living forms, they are likely to not be homologous in all groups and to have been 'r einv ented' independently in many clades (Geisen et al. 2018 ). The humidity barrier has been r elativ el y less studied in protists than the salinity barrier (Geisen et al. 2017 ). A r ecent surv ey of protists global diversity in soil, freshwater and marine systems suggests nevertheless that aquatic (marine and freshwater) and terrestrial communities do differ (Singer et al. 2021 ), which shows that the humidity barrier is effective in protists. Ho w ever, which barrier is the most difficult to overcome for unicellular eukaryotes still remains to be determined.
In order to e v aluate the r elativ e importance of salinity and humidity in the evolutionary history of other eukaryotic micr oor ganisms, we took the family Arcellidae (Sphaerothecina; Arcellinida; Elardia; Amoebozoa) as a study group. The order Arcellinida, to which Arcellidae belong, occurs worldwide in different terrestrial, wetland and freshwater habitats (Smith et al. 2007 ), with very fe w species r ecorded in coastal salt marshes and br ac kish waters (Golemansky 1998, Charman et al. 2002, Gehrels et al. 2006, González-Miguéns et al. 2023. They are often ecological specialists with narrow tolerance to alterations of their environment (Singer et al. 2018 ), hence their use as bioindicators for environmental health Patterson 2014 , Creevy et al. 2018 , Kosak y an and Lara 2019 ). The family Arcellidae are not typically found in saline en vironments , although they ha ve been episodically found in coastal marshes (Golemansky 1991, 1998, Charman et al. 2002, Gehrels et al. 2006, anchialine cenotes with subterranean seawater connections (van Hengstum et al. 2008 ) and coastal peatlands exposed to salt spray (Whittle et al. 2018 ). While the original habitat of the common ancestor of all Arcellinida r emains unclear, famil y Arcellidae most pr obabl y a ppear ed in aquatic habitats (González-Miguéns , Todoro v et al. 2022 ). Indeed, onl y certain deriv ed species fr om genus Galeripora, suc h as those from the G. arenaria complex (González-Miguéns, Soler-Zamora, Villar-Depablo et al. 2022 ), are strictly terrestrial and can settle down e v en in arid envir onments, while stem groups are aquatic.
In this w ork, w e trace the ancestral habitat of the family Arcellidae in order to infer the r elativ e importance of these eco-logical barriers in the evolution of this family. First, we isolated single cells from different athalassohaline lakes, streams and surrounding soils ecosystems in central Spain. Then we inferred the ancestral habitat of the different lineages in the famil y Arcellidae (marine/fr eshwater/soil/ Sphagnum ) based on ancestr al tr ait r econstruction to e v aluate the r elativ e fr equency of ecological tr ansitions. Mor eov er, we inferr ed the salinity tolerance (euryhaline vs. stenohaline) for one species using a culturing a ppr oac h to measur e gr owth r ates under differ ent salt concentr ations. Finall y, we formall y described the ne w species encountered.

Study area
Spain, due to its geomorphology and its generally dry climate, has se v er al important athalassohaline systems distributed in smallsize patches in different endorheic basins (Pardo 1948 , Comin andAlonso 1988 ). Samples for this study were collected from two main sites: Salobral de Ocaña (39º 59' N, 3º 36' W) and the 'Special Area of Conservation' of Petrola, Salobrejo and Corral Rubio saline lakes (38º 50' N, 1º 33' W).
We sampled the top millimeters of sediment at different points along thr ee str eams that e v entuall y joined into a single one during February and September 2020 and January 2021. The Special Area of Conservation of Petrola, Salobrejo and Corral Rubio salt lakes, included in the Red Natura 2000, is a wetland formed by more than 20 endorheic lakes, most of them temporary (Donate et al. 2004, Gómez-Alday et al. 2014. Lake Petrola is the largest and most permanent, as well as the best studied. Its ion composition is Mg 2 + -Na + -Cl − -SO4 2 − during summer and fall, and c hanges to Mg 2 + -Cl − -SO4 2in earl y spring (Ordóñez et al. 1973, Valiente et al. 2017. The lakes suffer se v er al anthr opogenic pr essur es due to a gricultur e and waste water disposal (Gómez-Alday et al. 2014 ). We collected se v er al sediment samples at the borders of the lakes of Horna, Petrola and Corral Rubio's Large lake during February and October 2020 and March 2021.

Microscopic observ a tions
Samples were transferred to a Petri dish to be observed under an inv erted micr oscope (Leica DMI8), up to 400x magnification DIC. A Leica MC170 HD camera with the Leica application suite (v. 4.12.0) softw are w as used to take photogr a phs.
Observed individuals were isolated using a small diameter pipette and tr ansferr ed to a dr op of sterile water, then further rinsed into another drop in order to get rid of other eukaryotic contaminants. Living cells were stored, individually or in groups of up to four cells, in Eppendorf tubes containing 100 μL of guanidine thioc y anate-based nucleic acids extraction buffer (Chomczynski and Sacchi 1987 ) for later DNA extraction.
Some individuals or empty tests were deposited, after washing in distilled water, on stubs for scanning electr on micr oscopy. Stubs were desiccated in a box with silica gel at least one day before metallization and observation. Then they were coated with 8-nm gold using a Balzers SCD 004 sputter coater and a tension of 15 kV. They were observed with a Hitachi S-3000 N and a JEOL JSM-5510 (operating at 10 kV) scanning electron microscope.

DN A extr action and amplifica tion
Single cell DNA extractions were performed on cells stored in guanidine thioc y anate buffer, as described in Duckert et al . ( 2018 ).
We amplified the COI region using the semi-nested protocol described in González-Miguéns, Soler-Zamora, Villar-Depablo et al. ( 2022 ); a first amplification was performed using the mitochondrial cytoc hr ome c oxidase subunit I universal primer pair LCO 1490 (5 GGTC AAC AAATC ATAAAGATATTGG 3 ) and HCO 2198 (5 TAAA CTTCA GGGTGA CCAAAAAATCA 3 ) (Folmer et al. 1994 ) with the following PCR pr ogr am: initial denatur ation at 96 • C for 5 min, follo w ed b y 40 c ycles at 94 • C for 15 s, 40 • C for 15 s and 72 • C for 90 s and a final extension step at 72 • C for 10 min. We used the product of this first PCR, usually diluted 1:20, as the base for a second amplification using the Arcellinida-specific primers Ar-COIF (5 GGT A TTYT AGCWCA TTCNRGTGG 3 ) (González-Miguéns, Soler -Zamora, Villar -Depablo et al. 2022 ) coupled with HCO, and its r e v erse and complementary ArCOIR coupled with LCO. The PCR profile for this step was an initial denaturation at 96 • C for 5 min, follo w ed b y 35 or 40 cycles at 94 • C for 15 s, 55 • C for 15 s and 72 • C for 90 s, and a final extension step at 72 • C for 10 min (González-Miguéns, Soler-Zamora, Villar-Depablo et al. 2022 ). After each amplification, 3 μL of the reaction was analyzed by electrophoresis on a 1% a gar ose gel to verify fragment size and check for contaminations. Final products with bands of expected size were run on a 1% a gar ose gel and the bands were excised and stored at 4ºC or −20ºC. The samples were sequenced using Sanger dideoxytechnology in both directions by Macrogen Inc. (Macrogen Europe, Madrid, Spain). Control quality of the raw sequences and assembling of both PCR products were carried out using the software GENEIOUS PRIME (v. 2019.0.4). Finally, we performed a blastn analysis (Altschul et al. 1990 ) against the GenBank database to ensure that our sequences belonged to Arcellinida.

Phylogenetic and ancestral habitat reconstruction analyses
We aligned our sequences with other Arcellidae sequences present in Genbank, as well as some Netzeliidae and Hyalospheniformes as outgroups. We also included Arcellidae environmental sequences from González-Miguéns et al. ( 2023 ), altogether resulting in a total of 76 sequences. A first alignment was performed using the MAFFT algorithm (Katoh et al. 2002 ), as implemented in GENEIOUS PRIME (v. 2019.0.4). This alignment was manually edited using Aliview (v. 1.27) (Larsson 2014 ). Phylogenetic trees were obtained using maximum likelihood and Bayesian inferences.
Maxim um likelihood anal yses wer e performed using the online version of IQ-TREE (Nguyen et al. 2015, Trifinopoulos et al. 2016, allowing it to search for the best substitution model with free rate heter ogeneity; 10'000 non-par ametric bootstr a ps wer e performed to assess node supports. Bayesian inference phylogenetic analysis was performed using MrBayes 3.2.7a (Huelsenbeck and Ronquist 2001 ) as implemented in CIPRES (Miller et al. 2011 ). We used the substitution parameters obtained in IQ-TREE as priors for the analysis. We performed two independent runs consisting of four MCMC chains of 10 7 generations. Tr ees wer e sampled e v ery 1000 gener ations, and the first 25% were discarded as burn-in.
We inferred the habitat of the ancestors of each clade by phylogenetic ancestral character reconstruction. We considered four habitats: freshw ater, saline w ater, terrestrial and Sphagnum (peat bog). Because it creates permanently wet microhabitats for associated microbes (coined the sphagnosphere , Jassey et al. 2011 ), Sphag-num was considered here as akin to aquatic (freshwater). We used the R pac ka ge phytools (Re v ell 2012 ) to trim the tr ee and join it to the database with the habitat trait. We fitted evolutionary models with equal, symmetric or all-different transition rates using the fitDiscrete function of the pac ka ge geiger v. 2.0.10 (Pennell et al. 2014 ). We used the equal-rates model, because it had the lo w est Akaike's information criterion, for the ancestral character estimation, using the ace function in the ape v. 5.6.2 package (Paradis and Schliep 2019 ).

Comparing growth at different salinities
Or ganisms fr om a str ain superficiall y looking lik e Ar cella intermedia were isolated in Petri dishes for the purpose of cultivation. We used autoclaved water initially collected from a highly saline stream situated in the Salobral de Ocaña (with a salinity of 75 g/L) as a medium that we diluted to r eac h the desir ed salinities. We also added autoclaved rice grains as the nutrient source for the environmental bacteria that would be co-inoculated with the amoebae, and that would serve as prey. Cultures were stored at 15ºC. The cultures were set at different salinities: freshwater (autoclaved), 20 g/L (the salinity of the sample from which individuals were isolated) and 50 g/L (the maximum salinity measured in the main body of Pétrola lake). Eight cells per culture were deposited in each Petri dish; we had eight culture replicates for treatments of 0 and 20 g/L, r espectiv el y, and four for the 50 g/L treatment. We then counted the number of living cells and dead individuals every 2-3 days during 20 da ys , in order to obtain a gr owth curv e for eac h tr eatment.
Statistical anal yses wer e performed using R softwar e v. 4.1.3 (R Core Team 2022 ) implemented in R studio v. 1.3.1093 (RStudio Team 2020 ). In order to test how far growth curves diverged from each other or not, we adjusted Poisson and quasi-Poisson general linear models using the glm function of the stats base R pac ka ge and a negative binomial model using the glm.nb function of the MASS pac ka ge (Venables and Ripley 2002 ). We tested for overdispersion using the gof function of the aods3 pac ka ge (Lesnoff and Lancelot 2022 ) and selected the model that adjusted better. We c hec ked for significant differences between the treatments using ANOVA type III as implemented in the pac ka ge car (Fox and Weisberg 2019 ).

Morphometric analysis
We took the following measurements: shell diameter, aperture diameter and pr ea pertur al ar ea, and av er a ged them. When possible, we also measured the height of the test and the pr ea pertural depth. We also included in our dataset the measurements of shell and a pertur e diameter fr om González-Miguéns, Soler-Zamora, Villar-Depablo et al. ( 2022 ), in order to detect possible similarities or differences with those other species of Arcellidae.
Statistical anal yses wer e performed using R softwar e v. 4.1.3 (R Core Team 2022 ) implemented in R studio v. 1.3.1093 (RStudio Team 2020 ). We plotted the distribution of each variable by morphospecies and sample location to c hec k for different clusters.
We also performed a PCA, using the core package stats, for this purpose. We also conducted linear discriminant analysis (LDA) using the pac ka ge MASS v. 7.3.55 (Venables and Ripley 2002 ), which identifies the combination of morphological variables that could be used to delimitate the clades. We used 75% of the dataset as a training set for the LDA, and 25% as a testing set. The ggplot2 pac ka ge (Wic kham et al. 2016 ) was used to gr a phicall y r epr esent the results.

Abiotic parameters on the sampling sites
We observ ed lar ge fluctuations in salinity between different sampling points at the same site and between different sampling times (i.e. in Pétrola lake, salinities ranged from 2.7 to 37 g/L in March, but in October they increased from 48 to 142 g/L). The total salinity ranges of each locality are shown in Table 1 (more data ar e pr ovided in the Supplementar y mater ial ). The salinity values at which living specimens of each species were found are indicated in Table 2 .

Ne w str ains isola ted and corresponding morphometric analyses
We isolated four differ ent str ains fr om saline aquatic envir onments and identified them based on their test morphology: (1) a str ain fr om the Galeripora discoides group , in sediments of the Pétrola lake, that we will describe as Galeripora marichusae sp. nov.; (2) a strain resembling Arcella intermedia , also from Pétrola lake, that will be described as Arcella euryhalina sp. nov.; (3) a strain also resembling Arcella intermedia from a saline stream in Salobral de Ocaña, Arcella salobris sp. nov.; and (4) a strain from the Galeripora arenaria species complex in terrestrial mosses growing on saline soils, in Salobral de Ocaña, Galeripora halaurula sp. nov.
There is some ov erla p between species morphometry in the PC A and LDA. T he LDA was able to corr ectl y assign the specimens with 75.5% accuracy. Galeripora marichusae sp. nov. has some overlap with Galeripora naiadis, although it is gener all y smaller; Galeripora halaurula sp. nov. with G. sitiens and, to a lo w er extent, G. balari ; Arcella euryhalina sp. nov. and Arcella salobris sp. nov. have some ov erla pping between them (Fig. 1 ), but in Arcella euryhalina sp. nov. the pr ea pertur al ar ea is mor e clearl y delimited than in Arcella salobris sp. nov. Another species isolated in Brazil, A. uspiensis (according to the measurements in Porfírio-Sousa et al. ( 2017 ) and Ribeiro et al . (in pr epar ation)), also has a marked pr ea pertur al ar ea, but the lip surrounding the aperture is much thinner. Table 3 indicates the Genbank r efer ence numbers for all the different obtained sequences, as well as how many isolates we tried to sequence and how many were successfully sequenced.

Barcoding, phylogenetic analysis and ancestral character reconstruction
The monophyly of Arcellidae is recovered with a Bayesian posterior probability (PP) of 1 and a maximum likelihood bootstrap (ML) of 100. Genus Galeripora is r ecov er ed with PP = 0.62 and ML = 87. Ho w e v er, Arcella euryhalina , whic h has the mor phological c haracteristics of Arcella , br anc hes as a sister clade to all Galeripora instead of within Arcella (PP = 0.98 and ML = 95).
The individuals obtained from the sampled athalassic saline envir onments a ppear in differ ent parts of the tr ee. Arcella salobris appears to be genetically closely related to the freshwater species A. uspiensis , forming a str ongl y supported clade (PP = 1, ML = 100). Arcella euryhalina , in turn, forms a clade with an environmental sequence (ON651640) from freshwater Lake Oromocto (Canada) (PP = 0.93, ML = 82). Galeripora maric husae a ppears well nested within the aquatic Galeripora discoides clade (PP = 0.97, ML = 1), without any clear relationship to the other barcoded species, G . naiadis, G . bathystoma or G . pol ypora . Finall y, Galeripora halaurula , found in terr estrial br own mosses in the area of Salobral de Ocaña, appears within the terrestrial clade of Galeripora (PP = 1, ML = 98), gr ouped with se v er al envir onmental sequences fr om br own mosses samples (PP = 1, ML = 100), most of them from coastal ar eas that r eceiv e salt spr ay; one sequence also came from an inland, non-saline location (González-Miguéns et al. 2023 ). The closest described sister species would be G. sitiens (PP = 1, ML = 82) .
The reconstruction of the ancestral habitat recovered freshwater environments as the most likely ecosystem for the ancestor of the family Arcellidae . T he independent ecological transitions through the salinity barrier in Arcellidae can be seen (Fig. 2 ), corresponding to three of the new species found in this study (see Taxonomic actions and species accounts). There is also one ecological transition in family Netzeliidae corresponding to environmental sequences from subtidal marine sediment samples, obtained by metabarcoding in González-Miguéns et al. ( 2023 ). The humidity barrier has only been crossed once, in the Galeripora arenaria species complex group, being a monophyletic group of terr estrial envir onments.

Gro wth a t different salinities
Cultures of the Arcella euryhalina specimens from Laguna de Pétr ola gr e w both in fr eshw ater and in w ater with a salinity of 20 g/L without significant differences ( P = 0.676), r eac hing, after 20 da ys , 55.4 ± 43.6 and 60 ± 16.8 alive individuals, r espectiv el y (Fig. 3 ). Cultures at a salinity of 50 g/L behaved significantly differ entl y fr om the other tr eatments ( P = 1.1 x 10 −13 ): we saw little for a ging activity and no divisions during the time of the experiment. The number of individuals slowly decreased from the eight initial individuals to 4.8 ± 1 after 20 da ys . Ne v ertheless, the source culture at 50 g/L remained alive for more than 4 months after the experiment, and we e v en saw instances of cell division, e v en although these e v ents wer e v ery uncommon.

Taxonomic actions and species accounts
We follo w ed an integr ativ e a ppr oac h based on ecological, molecular and morphological results to make taxonomic decisions on the ne w mitoc hondrial linea ges obtained in this study. The taxonomic decisions were taken in accordance with the rules and recommendations of the International Code of Zoological Nomenclatur e (ICZN, 1999), whic h a ppl y to testate amoebae (Lahr et al. 2012, Adl et al. 2019.
Family Round test, circular in oral and aboral view, hemispherical in later al vie w. Round borders, no ridges or depr essions . T he a pertur e is circular and inv a ginated, surr ounded by a thin collar. The borders of the pr ea pertur al ar ea ar e gener all y clear and well marked. There is a very large amount of cement between the building units of the cell, which covers the majority of the test surface.
Diagnosis with closely related species: Arcella euryhalina can be diagnosed by its specific mtDNA (COI) sequence. It is morphologicall y v ery similar to A. salobris , but the pr ea pertur al ar ea is gener all y wider and with a more marked margin in A. euryhalina. It also has a thicker cement layer between the building units.
Habita t: Aquatic , variable salinity (4 to 36 g/L). In the sediment of an athalassohaline lake. In culture, these organisms grew optimally in pure freshwater, and survived at 50 g/L.
Deriv a tio nominis: T he epithet euryhalina refers to the fact that is an organism able to live in a wide range of salinities.
Round test with hemispherical shape and round borders . T hin lip surrounding the aperture . T he preapertural area is not clearly marked, its mar gins ar e gener all y smooth. Because of this, the pr ea pertur al ar ea is often difficult to differentiate in the a pertur al view. The test is formed by building units surrounded by relatively thic k cement. Por es can be observ ed in the cement.
Diagnosis with closely related species: Arcella salobris can be diagnosed by its specific mtDNA (COI) sequence and its phylogenetic placement. It is mor phologicall y v ery similar to A. euryhalina , but the pr ea pertur al ar ea has smooth borders and is not so clearly visible in A. salobris. Arcella salobris also tends to have a more hemispherical shape in the lateral view. It is very similar to both A. hemisphaerica and A. rotundata , but those species do not hav e thic k cement between their building units, according to the images in Lahr and Lopes ( 2009 ), and have not been recorded in athalassohaline en vironments .
Habita t: Aquatic , v ariable salinity (observ ed as activ e between 5-13 g/L; given the characteristics of the sampling site, the tolerance range may be wider). In the sediment of an athalassohaline little stream. Deriv a tio nomini: salobris means 'with salt'; the epithet also refers to the place where they were collected: 'Salobral de Ocaña'.
Genus Galeripora González-Miguéns, Soler -Zamora, Villar -deP ablo , Todorov & Lara Galeripora marichusae sp. nov. (Fig. 5 ) Zoobank registr a tion : 7E1B03D6-7359-4D86-AE67-857B3C74 E41A Holotype : MA-Amoeba 11269. Ro y al Botanical Gar den, Madrid, Spain. Table 3. COI sequences obtained for each species. N i indicates the number of tubes with isolated individuals from that species and N s how many of those were successfully barcoded.   Smooth test, without ridges or hollows and with a discoid, flattened shape. Composed of small building units surrounded by an organic cement. The aperture is large and round, invaginated and bordered with a lip, with several small pores surrounding it.

Species
Diagnosis with closely related species: Galeripora marichusae can be diagnosed by its specific mtDNA (COI) sequence and its phylogenetic placement, as well as its morphometric characteristics. Its flat profile and numerous apertural pores are typical for the G. discoides group  ). Its diameter is situated between G. naiadis and G. polypora , although with some degree of o verlapping. T his is the only species of the group found in athalassohaline environments to date.
Habita t: Aquatic , variable salinity (4 to 19 g/L). On the sediment of athalassohaline lakes. In culture, they could survive at a salinity of 50 g/L. Deriv a tio nominis: marichusae is in honour of Maria Jesús Dabad-Mor eno, who liv es in a village next to Pétr ola lake, wher e this species was found.
Shell composed of small building units, but there is fr equentl y a smooth organic matrix that covers them and prevents their observ ation, particularl y in the oral side. On the aboral side the building units give it a rough toad-like texture . T here is no cement between them.
The center of the oral side is concave, but with a smooth transition, without a clearly delimited preapertural area. The aperture is small in relation with the diameter of the test; it has a lip and is surr ounded by se v er al r elativ el y lar ge por es. In some individuals ther e wer e a fe w other isolated por es on the margins of the oral side. In the lateral view it looks like a trapezoid, but the border pr otrudes. It r esembles a hat. On the aboral side there are some smooth, radial ridges. In most dead individuals the test was flattened.
Diagnosis with closely related species: Galeripora halaurula can be diagnosed by its specific mtDNA (COI) sequence and its phylogenetic placement. It is mor phologicall y v ery similar to Galeripora arenaria , G. sitiens , G. bufonipellita and G. balari.
Habitat: Terr estrial, gr owing on aerophilic mosses in dry soil, with salt deposits . En vironmental metabarcodes showed its presence in mosses growing on sandy beaches, exposed to the sea breeze. It has also been found, ho w ever, aw ay from the sea or any salt source; this suggests that this species is also euryhaline.
Deriv a tio nominis: Halaurula deriv es fr om the Gr eek 'halo' for 'salt', and 'aurula' from Latin for 'gentle breeze', referring to the fact that we found this species in mosses in locations where they may be exposed to salt spray or pollution.

Phylogenetic placement of the isolated organisms
The phylogenetic placement of Arcella salobris , Galeripora marichusae and G. halaurula are concordant with their morphology according to the phylogen y pr esented in González-Miguéns, Soler -Zamora, Villar -Depablo et al. ( 2022 ). In turn, Arcella euryhalina br anc hes as a sister group to the whole genus Galeripora but lacks the synapomorphies of the group: pores surrounding the a pertur e and an or ganic matrix that cov ers at least partiall y the oral side (González-Miguéns, Soler -Zamora, Villar -Depablo et al. 2022 ). Arcella euryhalina has a similar ov er all shell morphology to A. salobris or A. uspiensis (Ribeiro et al . (in pr epar ation)) and should be considered as belonging to the genus Arcella. Genus Arcella then becomes par a phyletic, according to this tree. Family Arcellidae is morphologically very diverse (Deflandre 1928 ) and still r emains under explor ed molecularl y; incomplete taxon sampling may affect the topology of the actual phylogenetic tree. Ther efor e, we pr ovisionall y k ee p this taxon within genus Arcella until a more complete picture of family Arcellidae is a vailable .
The validity of granting a specific status to A. salobris can be discussed. This species is mor phologicall y v ery similar to A. uspiensis (Ribeiro et al . (in preparation)); it is slightly smaller but the dimensions ov erla p (i.e. shell diameter 64-76 μm in A. uspiensis vs. 59.6-77.9 μm in A. salobris ; a pertur e diameter 16-22.8 μm in A. uspiensis vs. 15-26.4 μm in A. salobris ). Genetically, both species share 96.2% sequence similarity in the COI fr a gment anal yzed here; this distance overcomes all distances encountered within species in Arcellidae, except for A. guadarramensis , which is suspected to also be a species complex (González-Miguéns, Soler-Zamora, Villar-Depablo et al. 2022 ). While both species are very similar to each other, they div er ge deepl y in their ecology. Indeed, A. uspiensis has been collected from freshwater sediments in Brazil (Ribeiro et al . (in preparation)) and experimental evidence sug-gests that it dies above 2 g/l NaCl (D. Singer (personal communication)), whereas A. salobris was found active at salinities of 5-13 g/L ( Table 2 ). All this suggests important ecological differ ences, whic h, together with their genetic distance and their remote respective geogr a phical origins, would indicate that these taxa r epr esent two independent evolutionary units, that is, two different species.

Inland saline lake populations are euryhaline
Our records would be the highest salinity in which active organisms, not onl y fr om famil y Arcellidae, but also the whole order Arcellinida, have been found. Indeed, these or ganisms wer e r eported in coastal salt marshes at a maximum salinity of 13 g/L (Charman et al. 2002, Gehrels et al. 2006 ), but more frequently at salinities below 5 g/L (Golemansky 1998, Gehrels et al. 2006 ). Our observations are also, to the best of our knowledge, the first records of Arcellinida living in athalassohaline en vironments .
Inland saline water bodies are characterized by fluctuating par ameters, notabl y salinity, whic h dr ops dr asticall y during the r ain y season and in some cases can r eac h satur ation after the dry season. Ther efor e, athalassohaline systems can be expected to host euryhaline species that can cope with the sudden changes. In line, all three aquatic species described here have been found active in salinities which range from a few grams per liter to a salinity comparable with the sea in the case of A. euryhalina (Table 2 ). Our experiments with A. euryhalina show that it grows equally well in freshwater and at 20 g/L. Furthermore, this species was capable of long-term survival in a salinity of 50 g/L. Under these conditions, cells were mostly inactive, but did not die and e v entuall y r epr oduced (although r ar el y). They are , thus , ca pable of ada pting to a wide range of environmental conditions.
A similar pattern has been seen in other organisms living in athalassohaline lakes. For example, the dinoflagellate Biecheleria tirezensis , found in the nearby saline lake of Tirez (Spain), has been cultured in salinites from 2 up to 56 g/L (Raho et al. 2018 ). Metabarcoding studies have also found some centrohelid species that are present in inland lakes with salinities of 1 g/L and up to 78 g/L (Ger asimov a et al. 2020 ). Ther e ar e other examples in rotifers (Fontaneto et al. 2006), diatoms (Nakov et al. 2020 or Aspergillus (Nazareth and Gonsalves 2014 ) . All these examples show a common feature for organisms in athalassohaline ecosystems; they m ust hav e a high toler ance to osmotic str esses thr ough metabolic changes, similar to what is observed in other euryhaline systems, the coastal zones (Lahlou et al. 1969, Nordlie and Haney 1993, Meng et al. 2013. The potential presence of euryhaline Arcellidae in limnoterrestrial systems still needs to be e v aluated. Mor e data are needed to assess their distribution across the different ecosystems and to e v aluate their realized niche.

Frequency of ecological transitions across the salinity and humidity barriers
Tr ansitions acr oss the salinity barrier hav e al ways been consider ed infr equent in the e volutionary histories of the differ ent clades, especiall y of micr oor ganisms (Logar es et al. 2009 ). Howe v er, ther e is no precise definition of what can be called 'frequent' or 'infr equent', whic h r esults in subjectiv e a pplications of these terms . Moreo ver, this concept appears even less obvious in taxa where the systematics framework is far from complete and where only a part of the diversity is known, as is the case in most microbial groups.
In many cases there may also be a bias in the environmental sampling. Most r eported tr ansitions occur fr om marine to fr esh-water environments (Lee andBell 1999 , González-Miguéns, Soler-Zamor a, Fernando User os et al. 2022 ), with few cases in the opposite direction (Riek 1977 ). But marine ecosystems also have been better studied, at least for protists, than terrestrial and freshwater ecosystems (Geisen et al. 2017 ). In fact, Jamy et al. ( 2022 ) found equiv alent fr eshwater-to-marine and marine-to-fr eshwater tr ansition rates in their models across the whole eukaryotic tree based on environmental sequencing data, as well as hundreds of transitions. Within saline environments, marine ecosystems have been studied m uc h mor e than athalassohaline ones. And within athalassohaline envir onments ther e also seems to be a bias to w ar ds hypersaline, highl y extr eme habitats, r ather than subhaline or hypohaline lakes.
Furthermor e, gr oups that ar e tr aditionall y studied by the marine biologists community are hardly studied in freshwater en vironments . For example , foraminifera are a mainly marine group, but with a few species recorded from freshwater habitats (Siemensma et al. 2017 ). Ho w e v er, fr eshwater for aminifer a wer e lar gel y ignor ed during most of the 20th century (Holzmann et al. 2021 ). Inv ersel y, the tr aditionall y soil and fr eshwater gr oup Tr ebouxiophyceae hav e been bar el y studied in the ocean, although it is present (Metz et al. 2019 ). Drawing conclusions about the frequency or the directionality of the ecological transitions of the salinity barrier is ther efor e highl y c hallenging, given the limited extension of our knowledge of protist diversity and also the une v en sampling effort between the different en vironments . This is also the case in the family Arcellidae, as well as in the whole class Arcellinida, where the great majority of studies focus on freshwater and soil ecosystems, particularly peatlands. A few studies found some halotolerant species in hypohaline coastal lagoons and marshes (Golemansky 1998, Charman et al. 2002, Gehrels et al. 2006. Ho w ever, they had never been studied in athalassohaline en vironments . After this study of Arcellidae from these en vironments , we ha ve found not only new biodiversity (i.e. four new species), but also three independent ecological tr ansitions fr om fr eshwater to saline ecosystems . T hese numbers ar e likel y to incr ease as mor e Arcellinida that inhabit athalassohaline lakes will be sampled, especially in hypo and mesohaline systems.
The fluctuating salinity of estuaries and coastal lagoons may have facilitated ecological transitions to colonize, respectively, marine or freshwater systems (Lee and Bell 1999 ). In athalassohaline ecosystems they may also have played the role of a stepping stone . T here , in absence of contact with the sea, colonization e v ents ar e expected to occur fr om fr eshwater to high salinity ecosystems (Beadle 1969, Fontaneto et al. 2006, Bayly and Boxshall 2009. In contrast to these three ecological transitions across the salinity barrier in Arcellidae, the phylogeny of the family reveals a single transition to w ar ds terrestrial en vironments , even although terr estrial envir onments hav e been historicall y mor e sampled than saline ones in this famil y. This tr ansition occurr ed at the emergence of the Galeripora arenaria species complex. This clade is mor phologicall y v ery homogeneous and obtains maximum support in molecular phylogenies (González-Miguéns, Soler-Zamora, Villar-Depablo et al. 2022 ). Wet bryophytes such as Sphagnum mosses, which is a highly humid, sometimes subaquatic environment (Booth 2008 ), may have played a k e y role as a transition environment in the preadaptation of freshwater species to soil ecosystems . T he position of Galeripora succelli and Galeripora sp. KJ544163, both isolated in Sphagnum , as sister group to the whole G. arenaria species complex (Fig. 2 ), corr obor ates this hypothesis.
Terr estrializations ar e also r ar er than salinity tr ansitions in other groups of eukaryotes: in Decapoda, Davis et al. ( 2022 ) report numer ous marine-to-fr eshwater tr ansitions in se v er al clades, but only four transitions to the terrestrial environment (one in Anom ur a and thr ee in Br ac hyur a). This pattern r epeats in molluscs (Aristide and Fernández 2022 ), annelids (Rousset et al. 2008 ) and nematodes (Holterman et al. 2019 ). Se v er al gr oups that actuall y cr ossed the salinity barrier wer e unsuccessful in settling terr estrial envir onments, like Gastr otric ha (Kolic ka et al. 2020 ), Tintinnid ciliates (He et al. 2022 ) or Caridean shrimps (Davis et al. 2018 ). In summary, when extending to all eukaryotes (microbial or not), the humidity barrier could play a role at least as important as the salinity barrier in the structuration of biodiversity worldwide.

The paradox of the absence of Arcellinida in marine systems
The fact that multiple lineages of Arcellidae and Netzeliidae have been able to adapt to salinities comparable with seawater ( Fig. 2 ) suggests that they should have been physiologically able to colonize oceans. Ho w e v er, the r ecor ds of Ar cellidae found alive in the sea are scarce, and are mainly limited to brackish coastal marshes with low salinities (Golemansky 1998, Charman et al. 2002, Gehrels et al. 2006 ). This suggests that they are possibly absent from marine systems, despite the fact that abiotic parameters should not pr e v ent their pr esence in these envir onments. They can cross the salinity barrier, but not colonize the sea, or more saline coastal lagoons that are connected to the sea. A similar pattern can be found in Anostraca (Eng et al. 1990, Mura 1999 and the green alga Dunaliella (Assunção et al. 2012, González et al. 2019: they are typical inhabitants of athalassohaline ecosystems, but are absent from marine en vironments . Because salinity is not the barrier pr e v enting these clades from conquering the ocean, or at least not on its own, it can be hypothesized that biotic parameters, such as predation and/or competition, may be playing a role.

Ecological transitions are favored under low biotic pressure
Would-be colonizers are under a competiti ve disad v anta ge a gainst well-ada pted local species, hindering their possibilities of colonizing the ne w envir onment (Vermeij and Dudley 2000 ). In fishes, competition with extant species seems to be a factor limiting diversification after ecological transitions (Betancur-R. et al. 2012 ). In oceanic islands, where many predators are absent and interspecific competition is r educed, man y species colonize new nic hes fr om whic h they ar e usuall y excluded in the continent. For example, the four only known aquatic-to-terrestrial transitions in truncatellid gastropods all occurred on oceanic islands (Rosenberg 1996 , Vermeij andDudley 2000 ). Darwin's finches (Grant and Grant 1982 ) and Anolis lizards' conv er gent e volution (Kolbe et al. 2011 ) are also typical examples where colonists find empty niches in islands and, in the end, speciate . T hese lo w er biotic pr essur es ma y ha ve facilitated, or allo w ed, the surviv al of ecological tr ansitioners that otherwise would have been outcompeted. Lakes have been considered as analogues of islands in many biogeographical studies (Browne 1981, MacDonald et al. 2018. T hus , it could be expected that interspecific competition and predation in athalassohaline lakes is lo w er than in the sea, which would be analogous to 'the mainland'. This could have facilitated the adaptation to this niche of the colonizers coming fr om fr eshwater environments. Because sampling efforts have been more focused on marine-to-fr eshwater tr ansitions, our perception of how difficult it is to cross the salinity barrier may be biased. Further sampling of these athalassohaline ecosystems may r e v eal man y mor e tr ansitions across this barrier.

What could be excluding Arcellinida from the marine environment?
Predation and/or competition that could prevent Arcellidae from settling marine sediments may come from many benthic shelled protists with similar lifestyles like Gr omiids, Eugl yphids, small Metazoa or possibl y For aminifer ans. For aminifer ans ar e a mainly marine group, although there are also a soil and a few freshwater species (Holzmann et al. 2021 ) and some reported in athalassohaline lakes (Plaziat 1991 ). For aminifer a and Arcellinida tend to ov erla p in coastal lagoons and other br ac kish envir onments, but For aminifer a usuall y dominate when the salinity is higher, and vice versa (Charman et al. 2002, Vázquez Riv eir os et al. 2007, v an Hengstum et al. 2008 ). They could be a candidate group that could exclude Arcellidae from marine sediments (Whittle et al. 2018 ), although the putative competition should be empirically tested. Mesocosm experiments in seawater tanks inoculated with salttolerant Arcellidae and benthic For aminifer ans can be instrumental in testing this hypothesis.

Conclusion
Her e we r eport the pr esence of Arcellidae, a mainl y fr eshwater and terrestrial group, in athalassohaline en vironments , living and thriving at salinities as high as seawater and facing strong salinity fluctuations . T hese ne w species r epr esent thr ee independent freshw ater-to-saltw ater transitions, as opposed to one single aquatic-to-terrestrial transition within Arcellidae. Most probabl y, tr ansitions acr oss the salinity barrier are likely to occur frequently, as the new species we describe here have been obtained from a single region (Spain); further diversity will certainly be revealed when the sampled regions are expanded. This opens the door to further r esearc h exploring the diversity of Arcellidae and other protists in athalassohaline en vironments , the evolutionary history of these halotolerant or halophilic groups, their ecology and the physiolog ical, cytolog ical and molecular basis of salinity tolerance in the group.
Arcellidae are a primarily freshwater group with a high potential for colonizing saline systems, and less so for soil. Ho w e v er, adapting to a new ecosystem type also requires overcoming biotic constr aints suc h as pr edation or competition. These constr aints, and not only the abiotic ones, need to be taken into account when studying ecological transitions in all or ganisms, micr obial and multicellular alike.

Supplementary data
Supplementary data are available at FEMSEC Journal online.
Conflict of inter est. The authors declar e that they hav e no known competing financial interests or personal relationships that could hav e a ppear ed to influence the work r eported in this pa per.

Funding
This w ork w as funded b y the project MYXOTR OPICS VI aw ar ded b y Spanish Government PGC2018-094660-B-I00 (MCIU/AEI/FEDER, UE) whose r efer ence can be found at / https:// doi.org/ 10.13039 /501100011033/. It was also supported by the FPU fellowship FPU19/06077 from the Spanish Ministry of Science , Inno vation and Universities to F. Useros.