Four New Choanoflagellate Species from Extreme Saline Environments

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European Journal of Protistology 66 (2018) 86–96

Four new choanoflagellate species from extreme saline environments: Indication for isolation-driven speciation exemplified by highly adapted Craspedida from salt flats in the Atacama Desert (Northern Chile) Sabine Schiwitza, Hartmut Arndt, Frank Nitsche∗

University of Cologne, Cologne Biocenter, Institute of Zoology, General Ecology, Zuelpicher Str. 47b, D-50674 Cologne, Germany

Received 25 June 2018; received in revised form 6 August 2018; accepted 8 August 2018 Available online 31 August 2018

Abstract

With this study we aim to extend the knowledge on the biogeography of craspedid choanoflagellates with additional data from extreme environments. Up to now, very little is known about choanoflagellates from extreme saline environments, as most studies have focused on marine and freshwater habitats. Though previously investigated high saline ice biota communities have indicated a possible adaptation to environments with high salt concentrations. Hypersaline endorheic basins, so-called salt flats or salares from the Atacama Desert in Northern Chile provide an intense environment regarding fluctuating and extreme salinities, which allow for studies on evolutionary adaptations of protists to hypersaline conditions. This study focused on choanoflagellate species isolated from different salt flats, their morphological characteristics using light and electron microscopy, molecular marker genes (SSU and LSU rDNA) and their salinity tolerance. Here, we described four new craspedid choanoflagellate species, highly adapted to the hypersaline environment of the Atacama Desert. This study extends our knowledge on choanoflagellate phylogeny and ecology and can become the basis for further molecular studies to understand the mechanisms of adaptations. Additionally, we emphasize the need of adding additional data such as autecological characteristics to amend species definitions, which is only possible from cultivated strains. This data would support the use of molecular data originating from metagenomic analyses also in an ecological context. © 2018 Elsevier GmbH. All rights reserved.

Keywords: Adaptation; Atacama Desert; Craspedida; Choanoﬂagellatea; Phylogeny; Salinity tolerance

Introduction within the desert. These salt flats are characterised by high variability regarding salinity - from low saline (less than The Atacama Desert in Northern Chile is one of the 15 practical salinity units (PSU)) to almost salt-saturated driest places on earth resulting in extreme environmental waters (Risacher et al., 1999). Varying abiotic conditions, as conditions, which demand a high degree of specialisation seasonal fluctuations of chemical and physical water compo- and adaptation from the residentiary unicellular eukary- sition (e.g. nutrients, temperature, salinity), are challenging otic organisms. Low precipitation and high temperatures for the biota and affect species assemblages and ecosys- have led to the formation of hypersaline endorheic basins tem stability (Márquez-García et al., 2009). In addition, high doses of UV radiation, toxins like arsenic and heavy metals and an anomaly of salt composition, significantly differ- ∗ ing from marine conditions, demand multiple adaptations. Corresponding author. E-mail address: [email protected] (F. Nitsche). Recent gene-fingerprinting studies have shown high ecolog- https://doi.org/10.1016/j.ejop.2018.08.001 0932-4739/© 2018 Elsevier GmbH. All rights reserved. S. Schiwitza et al. / European Journal of Protistology 66 (2018) 86–96 87

Table 1. Sampling site, original salinity (PSU) and NCBI accession numbers of sequences of the newly described species.

Species Coordinates Locality Original Accession Accession salinity number number [PSU] (NCBI) (NCBI) LSU SSU rDNA rDNA

“Salpingoeca” 23◦1716.6S Salar de 100 MH490945 – crinita sp. nov. 68◦1026.6W Atacama “Salpingoeca” 23◦5859.8S Salar de 40 MH490946 MH490947 prava sp. nov. 67◦4336.6W Talar “Salpingoeca” 18◦4943.0S Salar de 50 MH490948 MH490949 surira sp. nov. 69◦0340.5W Surire “Salpingoeca” 20◦1557.5S Salar de 288 MH490950 – huasca sp. nov. 68◦4916.0W Huasco ical diversity and novelty of protists in hypersaline inland flasks (Falcon, Durham, USA) in artificial seawater (Instant lakes, especially for choanoflagellates (Casamayor et al., Ocean, Aquarium Systems, Strasbourg, France) with salini- 2013; Triadó-Margarit and Casamayor, 2013). ties according to the original conditions. The cultures were Choanoflagellates have great ecological impact on micro- enriched with the bacterial strain Pseudomonas putida (sup- bial food webs as they are highly efficient filter feeders, plemented with cereal grass as carbon source) as food source forming occasionally up to 40% of the biomass of het- for heterotrophic flagellates and regularly monitored by light erotrophic flagellates in aquatic ecosystems (Arndt et al., microscopy (Zeiss Axiovert S 100) for choanoflagellate 2000; Boenigk and Arndt, 2002; Buck and Garrison, 1988), species. Mixed cultures containing choanoflagellates were living sedentary or freely suspended in the water column. The diluted using the liquid aliquot method (LAM) (Butler and morphology among choanoflagellates is highly conserved, Rogerson, 1995). characterised by a cell with a single flagellum surrounded by a collar of microvilli (James-Clark, 1867; Leadbeater, 2015). Video microscopy Traditional taxonomy based on morphological characteristics of the external cell covering has proved to be misleading Morphology was investigated by an inverted light micro- regarding a monophyletic classification within the Craspe- scope system as described by Jeuck et al. (2014). Clonal dida. Due to lack of data from the type specimen Salpingoeca cultures were transferred to modified Petri dishes (coverslips gracilis (James-Clark, 1867), recent higher level taxonomic as base to optimize the optics) and observed using a Zeiss revision including molecular techniques, has led to the amal- Axio Observer with a 100×/1.4 NA oil immersion objec- gamation of the former Codonosigidae and Salpingoecidae tive (differential interference contrast (DIC)) and a water to only one family, the Salpingoecidae sensu Nitsche et al. immersion condenser. Videos were taken using a black/white (2011). analogous Hamamatsu C6489 camera with noise reduction Here, we focus on the taxonomy, morphology and autecol- and contrast enhancement by the Allen Video Enhanced Con- ogy of four new craspedid choanoflagellate species highly trast (AVEC) system (Hamamatsu, Argus-20) (for a detailed adapted to the abiotic conditions of the salt flats from the illustration see Stoupin et al. (2012)). Atacama Desert. We aim to extend the knowledge on bio- Videos were analysed frame by frame with VirtualDub geography and taxonomic resolution of choanoflagellates by (www.virtualdub.org) and processed with ImageJ (Abràmoff including new craspedid species from this extreme saline et al., 2004). Cell measurements were performed using the habitat and to prepare the ground for future detailed genetic imaging software AxioVision (Zeiss). analyses of adaptive processes based on comparative tran- scriptome analyses. Electron microscopy

For scanning electron microscopy (basic method see Material and Methods Nitsche (2016)), all samples were fixed in 2.5% cacody- late buffered glutaraldehyde (final concentration) at 4 ◦C Sampling and cultivation for 120 min. Cultures remained in culture flasks for prepa- ration. After the treatment with 1% osmium tetroxide for Water samples were collected March 2017 from differ- 10 min, samples were dehydrated in an ascending ethanol ent salt flats (salares) of the Atacama Desert in Northern series of 30%, 50%, 70%, 80%, 90% and 96%. Samples were Chile (Table 1). Aliquots were cultured in 50 ml culture washed twice with the corresponding ethanol concentration 88 S. Schiwitza et al. / European Journal of Protistology 66 (2018) 86–96 and finally remained for 10 min in each solution. As a replace- GCC ATG C-3) for re-amplificated single cell SSU rDNA ment for critical point drying, a final dehydration using 50:50 and NLF184/21 + NLR2098/24 + NLF1105/22 (5-CCG hexamethyldisilazane (HMDS)-ethanol solution was applied AAG TTT CCC TCA GGA TAG C-3) + NLR1126R/22 for 10 min followed by two washing steps with pure HMDS (5-GCT ATC CTG AGG GAA ACT TCG G-3) for and 5 min incubation. Finally, the samples were allowed to the LSU rDNA. Sequences were assembled using dry. The bottom of each flask was cut out and stuck to a BioEdit (Hall, 1999). sample holder. Mounted samples were sputter coated with a 120 Å layer of gold and examined by SEM (FEI Quanta 250 Phylogenetic analysis FEG). SEM pictures were analysed for total flagellar length using the program MeasureMaster v.4.103. A concatenated, six-gene (SSU and LSU rDNA, hsp90, tubA, EFL and EF-1A), 9769 bp (manually corrected) align- Molecular biology ment (Carr et al., 2017) including latest published sequences (Nitsche, 2016; Nitsche et al., 2017) was used to analyse the phylogenetic position of the new sequences (accession For the molecular analysis of the SSU and LSU rDNA, numbers see Suppl. Table 1). Sequences were individu- single cells were isolated using a micromanipulator (Patch- ally integrated to the alignment using BioEdit (Hall, 1999). Man NP2 Eppendorf, Hamburg, Germany), transferred to

␮ ◦ The alignment was analysed using Maximum likelihood

10 l ddH O and kept frozen at −20 C until PCR treat- 2 (ML) and Bayesian inference (BI) methods. ML analy- ment. The SSU rDNA fragments were ampliﬁed with the sis was computed by RAxML v.8.2.10 (Stamatakis, 2014) primer pair 42F (5 -CTC AAR GAY TAA GCC ATG CA- using the GAMMA + P-Invar Model of rate heterogene- 3 ) and 18S-Rev-1 (5 -ACC TAC GGA AAC CTT GTT ity as suggested by the author with 1000 replicates for

ACG-3 ) at a concentration of 0.1 ␮M, using a PCR Mas- the bootstrap analysis. The Bayesian analysis was per- termix (2×) (VWR Life Science, Red Taq DNA Polymerase, formed by MrBayes v.3.2.6 (Ronquist et al., 2012) running Hassrode, Belgium). The PCR program started with a denat-

◦ a GTR+I+ model and a four-category gamma distribu- uration step at 98 C for 2 min, followed by 34 cycles ◦ ◦ ◦ tion to correct for among site rate variation. The search of 96 C for 30 s, 48 C for 30 s, 52 C for 30 s (due to ◦ consisted of two parallel chain sets run at default tem- primers) and 72 C for 2.30 min and a final extension step ◦ peratures with a sample frequency of 10 and run until by 7 min at 72 C. A 5 ␮l subsample of the first PCR was the average standard deviation of split frequencies dropped used as template for a reamplification (same PCR program) below 0.01. The analysis consisted of 1,750,000 genera- with the primer pair 82F (5 -GAA ACT GCG AAT GGC tions, with a burnin of 437,500 before calculating posterior TC-3 ) and 1630R (5 -CGA CGG GCG GTG TGT ACA probabilities. The choanoflagellate phylogeny was rooted A-3 ). The LSU rDNA fragment was amplified with the with a two-taxa ichthyosporean clade and an eight-taxa primer pair Rev-1-For-ITS (5 -CGT AAC AAG GTT TCC metazoan clade. The uncorrected pairwise distances (only GTA GGT-3 ) and a mixed reverse primer combination of SSU rDNA) were estimated using MEGA 7 (Kumar et al., NLR2571/22 (5 -CTC AAC AGG GTC TTC TTT CCC 2016). The alignment is available from the author upon C-3 ) + NLR2781/19 (5 - CG CCC CAG YCA AAC TCC request. C-3 ) + NLR3535/22 (5 -MRG GCT KAA TCT CAR YRG

ATC G-3 ) (primer concentration 0.1 ␮M). The PCR program started with a denaturation step at 98 ◦C for 2 min, Autecology followed by 30 cycles of 96 ◦C for 30 s, 52 ◦C for 30 s, 54 ◦C for 30 s (due to primers) and 72 ◦C for 4.20 min, finished To investigate the autecology of the new species, salin- ◦ by 11.11 min at 72 C. A 5 ␮l subsample of the first PCR ity tolerance experiments were performed. The cultures was used as template for a reamplification with the primer remained at least for three weeks in artificial seawater with pair NLF184/21 (5-ACC CGC TGA AYT TAA GCA TAT- the most suitable salinity for culture growing (tested before) 3) and NLR2098/24 (5-AGC CAA TCC TTW TCC CGA (“Salpingoeca” crinita sp. nov. and “Salpingoeca” surira AGT TAC-3) at a PCR program with a denaturation step sp. nov. at 90 PSU, “Salpingoeca” huasca sp. nov. and at 96 ◦C for 5.01 min, followed by 34 cycles of 96 ◦C for “Salpingoeca” prava sp. nov. at 35 PSU). A gradient of 30 s, 52 ◦C for 30 s, 55 ◦C for 30 s (due to primers) and different salinities (0, 1, 3, 5, 12, 20, 35, 60, 90 and 150 72 ◦C for 2.40 min and a final extension step by 7.21 min PSU) with three replicates of each treatment and one con- at 72 ◦C. trol (culture conditions) was established in culture flasks All PCR products were purified using the PCR Purifica- at room temperature to test for species’ survival. Salinity tion Kit (Bioscience, Jena, Germany). The sequencing was raised and decreased from culture conditions step- of rDNA amplicons was done using the primers wise (as described above) after an acclimatisation of 48 h. 82F + 1630R + 590F (5-CGG TAA TTC CAG CTC The bacterial strain P. putida (enriched with cereal grass CAA TAG C-3) + 600 R (5-GCT ATT GGA GCT GGA as carbon source) was added to each culture to exclude a ATT ACC G-3) + 1300R (5-CAC CAA CTA AGA ACG food limitation due to the effect of salinity changes on the S. Schiwitza et al. / European Journal of Protistology 66 (2018) 86–96 89

Fig. 1. Concatenated six-gene bayesian inference phylogeny of choanoflagellates based on a manual corrected alignment of SSU and LSU rDNA, hsp90, tubA, EFL and EF-1A (9769 nt). The number of nucleotide substitutions per site is proportionally defined in the scale bar at the lower left. Support values are given for BI/RAxML at each branch. 1.00 BI posterior probabilities (biPP) and 100% RAxML bootstrap percentage support (mlBP) are denoted by a *, biPP and mlBP values are otherwise given above and below branches, respectively. Support values below 0.7 biPP and 50% mlBP are indicated by a -. The newly described species are marked by bold letters. environmental bacteria community. As tested in previous Results studies, this bacterial strain is suitable as food source for choanoflagellates and tolerant regarding changes in salin- In this study, we cultivated and described four new cras- ity (Nitsche, 2014). Culture flasks were monitored every pedid choanoflagellate species. The description is based on 24 h with cell counts (MoxiZ, ORFLO, Ketchum, USA) for molecular data (SSU and LSU rDNA) (Fig. 1), a detailed mor- 5 days. phological approach including light and electron microscopy 90 S. Schiwitza et al. / European Journal of Protistology 66 (2018) 86–96

Fig. 2. Morphology of “Salpingoeca” crinita sp. nov. and “Salpingoeca” prava sp. nov. (A–F) “Salpingoeca” crinita sp. nov.: (A) Schematic illustration of the specimen; (B) collared cell; (C) flagellum; (D) cell overview with stalk; (E) EM micrograph of whole cell; (F) enlargement of the flagellum showing small filaments in an opposite arrangement. (G–L) “Salpingoeca” prava sp. nov.: (G) Schematic illustration of collared specimen; (H) Schematic illustration of specimen in life cycle stage with pseudopodia; (I&J) collar bearing cells; (K) cell with

pseudopodia; (L) EM micrograph of whole cell. Scalebar: 2 ␮m. Abbreviations: b — Bacterium; ﬂ — Flagellum; m — Microvilli; n — Nucleus; p — Pseudopodium; st — Stalk; th — Theca.

(Fig. 2, Fig. 3), as well as autecological experiments regard- the uncertain taxonomy of this genus which is not mono- ing the salinity tolerance (Fig. 4). We were able to maintain phyletic (Fig. 1). Until molecular data on the type species, all species as monocultures. All new species were denomi- Salpingoeca gracilis (James-Clark, 1867; Thomsen, 1977), nated in quotation marks as “Salpingoeca” species, reﬂecting is available, we refrained from generating artiﬁcial genera S. Schiwitza et al. / European Journal of Protistology 66 (2018) 86–96 91

Fig. 3. Morphology of “Salpingoeca” surira sp. nov. and “Salpingoeca” huasca sp. nov. (A–G) “Salpingoeca” surira sp. nov.: (A) Schematic illustration of the specimen; (B) cell body with collar; (C) “fast swimmer” with shortened collar and pseudopodia; (D) EM micrograph of whole cell; (E) thecate cell with stalk; (F&G) cell with pseudopodia. (H-O) “Salpingoeca” huasca sp. nov.: (H) Schematic illustration of the specimen; (I) collared cell; (J) cell with pseudopodia; (K) “fast swimmer” with shortened collar; (L) theca; (M) collar; (N) EM cell

overview; (O) “fast swimmer” with shortened collar. Scalebar: 2 ␮m. Abbreviations: b — Bacterium; ﬂ — Flagellum; m — Microvilli; p — Pseudopodium; st — Stalk; th — Theca. 92 S. Schiwitza et al. / European Journal of Protistology 66 (2018) 86–96

Fig. 4. Salinity tolerance ranges of all newly described species. Bars in dark grey indicate growth, bars in light grey the sporadic presence of species. Dots show the measured original salinity of the salt ﬂat, average marine salinity given by dashed line. to avoid future renaming problems. All new sequences were extended the most distant branch within the Craspedida of deposited in GenBank (Table 1) and the uncorrected pairwise Salpingoeca tuba and Salpingoeca dolichothecata with mod- distances of the SSU rDNA are available from the author upon erate support (0.71 biPP; <50% mlBP). Pairwise distances request. showed phylogenetic distances of 9.2% to Salpingoeca tuba and 13.9% to Salpingoeca dolichothecata. Taxonomy Autecology Morphological data from light and scanning electron microscopy, e.g. the presence of an organic cell coating We were able to successfully cultivate all species in a broad (Fig. 2, Fig. 3), support the allocation of the species “Salp- range of salinities (Fig. 4). All species were able to tolerate ingoeca” crinita sp. nov., “Salpingoeca” prava sp. nov., the salinity decrease to low saline waters although originating “Salpingoeca” surira sp. nov. and “Salpingoeca” huasca from hypersaline habitats (Table 1). In particular, “Salpin- sp. nov. to the order of Craspedida (Carr et al., 2017; Nitsche goeca” crinita sp. nov. (original salinity: 100 PSU) showed et al., 2011). positive growth from 12 to 150 PSU, “Salpingoeca” prava sp. nov. (original salinity: 40 PSU) from 12 to 90 PSU and “Salpingoeca” surira sp. nov. (original salinity: 50 PSU) as Phylogenetic analysis well as “Salpingoeca” huasca sp. nov. (original salinity: 288 PSU) from 5 to 150 PSU. Partial SSU and LSU rDNA of the four species were sequenced (Table 1) and analysed in a concatenated phylogeny (Fig. 1), based on previous studies (Carr et al., 2017; Descriptions of the new species Nitsche et al., 2011, 2017). The 60 taxa of choanoﬂagellates, 8 taxa of Metazoa and 2 taxa of Ichthyosporea formed dis- For taxonomic description, species were morphologically tinct clades with strong support (1.00 biPP; 100% mlBP). As analysed regarding cell characteristics (measurements) and described previously, we found strong Bayesian inference life cycle stages (holotype and paratype) (Table 2). support (1.00 biPP) for the orders Craspedida and Acanthoe- Order: Craspedida (Cavalier-Smith, 1997). cida to be monophyletic. Within the Craspedida we recorded Family: Salpingoecidae (Saville-Kent, 1880 a high support (1.00 biPP, 100% mlBP) that the newly intro- emend. sensu Nitsche et al. (2011)). duced species “Salpingoeca” crinita sp. nov., “Salpingoeca” Genus: Salpingoeca. surira sp. nov. and “Salpingoeca” huasca sp. nov. do cluster Species: “Salpingoeca” crinita sp. nov. (Fig. 2A–F), type together forming a new clade close to Salpingoeca rosetta strain: Heterotrophic Flagellate Collection Cologne (HFCC) (Fig. 1). Uncorrected pairwise distances revealed phyloge- strain 1287.

netic distances of 14.7–15.7% to Salpingoeca rosetta and Diagnosis: Hypersaline Salpingoeca-like species, about

13.3–14.3% to Microstomoeca roanoka (only based on SSU 4 × 3 ␮m in size with a tight-encompassing theca. The rDNA). Our sequences of “Salpingoeca” prava sp. nov. species occurs as solitary cells attached to surfaces and S. Schiwitza et al. / European Journal of Protistology 66 (2018) 86–96 93

Table 2. Morphometric data of the new species based on light microscopy. All measurements in ␮m, average N = 18, SD = Standard deviation.

Species [␮m] Length Width Length Length Width Length cell body cell body ﬂagellum collar collar stalk

“Salpingoeca” crinita sp. nov. Mean 4.2 3.3 18.9 4.8 5.4 9.9 SD 0.4 0.4 7.0 2.1 1.4 2.5 Min. 3.6 2.6 9.1 1.0 2.9 5.7 Max. 5.0 4.1 30.5 9.0 8.0 14.1 “Salpingoeca” prava sp. nov. Mean 3.6 2.6 7.9 3.5 3.2 – SD 0.4 0.3 2.6 1.3 1.3 – Min. 3.1 1.9 4.1 1.7 1.7 – Max. 4.6 3.1 12.6 5.2 5.6 – “Salpingoeca” surira sp. nov. Mean 4.9 3.8 12.9 4.3 4.1 6.4 SD 0.5 0.4 4.8 2.1 1.6 1.5 Min. 4.1 3.1 6.1 1.0 1.9 4.4 Max. 5.7 4.5 22.6 7.9 6.6 8.3 “Salpingoeca” huasca sp. nov. Mean 4.5 3.9 15.9 4.6 4.6 8.1 SD 0.4 0.5 5.9 2.7 1.1 1.9 Min. 3.7 3.0 6.6 0.7 2.7 6.5 Max. 5.4 5.4 24 9.3 6.7 10.4

exhibits a long stalk (about 10 ␮m) and a flagellum up to Etymology: prava (Latin, adjective, feminine) from Latin 2.5–6 times longer than the protoplast. The flagellum is fea- “bended” in reference to the body shape by light microscop- tured by the formation of small filaments in an opposite ical view. arrangement. Species can tolerate low saline to hypersaline Type locality: Salar de Talar, a salt flat in the Atacama conditions (12–150 PSU). Desert in Northern Chile (23◦5859.8S67◦4336.6W) Etymology: crinita (Latin, adjective, feminine) from Latin (Table 1). “hairy” in reference to the observed flagellum with small Holotype: Specimen illustrated in Fig. 2G,H.

ﬁlaments. Description: Salpingoeca-like species with a body length

Type locality: Salar de Atacama, a salt ﬂat in the Atacama of 3.1–4.6 ␮m (3.6 ± 0.4 ␮m on average, N = 27) and a body

◦ ◦

Desert in Northern Chile (23 17 16.6 S6810 26.6 W) width of 1.9–3.1 ␮m (2.6 ± 0.3 ␮m on average, N = 27). The

(Table 1). collar has a length of 1.7–5.2 ␮m (3.5 ± 1.3 ␮m on average,

Holotype: Specimen illustrated in Fig. 2A. N = 11) and a width of 1.7–5.6 ␮m (3.2 ± 1.3 ␮monaver-

Description: Salpingoeca-like species with a body length age, N = 11). The ﬂagellum has a length from 4.1–12.6 ␮m

␮ ± ␮

of 3.6–5.0 m (4.2 0.4 m on average, N = 22) and a body (7.9 ± 2.6 ␮m on average, N = 23).

width of 2.6–4.1 ␮m (3.3 ± 0.4 ␮m on average, N = 22). The Type sequence data: The SSU and LSU rDNA sequences

theca is close-encompassing to the protoplast. The collar has were deposited in GenBank database with the accession num-

a variable length of 1.0–9.0 ␮m (4.8 ± 2.1 ␮m on average, bers MH490946 (SSU) and MH490947 (LSU). The closest

N = 17) and a width of 2.9–8.0 ␮m (5.4 ± 1.4 ␮monaver- relative sequence on NCBI nucleotide BLAST results in

age, N = 17). The ﬂagellum has a length from 9.1–30.5 ␮m an uncultured eukaryote marine clone (FN690480) with a

(18.9 ± 7.0 ␮m on average, N = 14). The stalk is 5.7–14.1 ␮m maximum identity of 93%. long (9.9 ± 2.5 ␮m on average, N = 14). Species: “Salpingoeca” surira sp. nov. (Fig. 3A–G), type Type sequence data: The SSU rDNA sequence was strain: HFCC strain 1244.

deposited in GenBank database with the accession num- Diagnosis: Hypersaline Salpingoeca-like species, about ber MH490945. The closest relative sequence on NCBI 5 × 4 ␮m in size. Cells frequently occur in accumulations ® nucleotide BLAST results in Microstomoeca roanoka (colony-like) attached to surfaces with a stalk. Fast swim- (KT757502) with a maximum identity of 90%. ming cells with a shortened collar and pseudopodia present. Species: “Salpingoeca” prava sp. nov. (Fig. 2.G–L), type Species can tolerate low saline to hypersaline conditions strain: HFCC strain 1254. (5–150 PSU).

Diagnosis: Hypersaline Salpingoeca-like species, about Etymology: surira (adjective, feminine) in reference to the

3.5 × 2.5 ␮m in size with a protoplast almost ﬁlling the theca. type locality. Species present in both a collared life cycle stage and with- Type locality: Salar de Surire, a salt ﬂat in the Atacama ◦ ◦ out a visible collar, but many pseudopodia. Solitary cells are Desert in Northern Chile (18 49 43.0 S6903 40.5 W) strongly attached to surfaces. Species can tolerate low saline (Table 1). to hypersaline conditions (12–90 PSU). Holotype: Specimen illustrated in Fig. 3A. 94 S. Schiwitza et al. / European Journal of Protistology 66 (2018) 86–96

Description: Salpingoeca-like species with a body length or environmental studies such as Tara Ocean project and

of 4.1–5.7 ␮m (4.9 ± 0.5 ␮m on average, N = 23) and a body others, indicating their geographic restriction. We hypoth-

width of 3.1–4.5 ␮m (3.8 ± 0.4 ␮m on average, N = 24). The esise that at least two marine last common ancestors were

collar has a variable length of 1.0–7.9 ␮m (4.3 ± 2.1 ␮mon transported independently to the different salt ﬂats by birds

average, N = 14) and a width of 1.9–6.6 ␮m (4.1 ± 1.6 ␮m (ﬂamingos and other water birds migrating from ocean to the

on average, N = 14). The ﬂagellum has a length from salt ﬂats), evolving to the present species. The distinct geo-

6.1–22.6 ␮m (12.9 ± 4.8 ␮m on average, N = 15). The stalk graphical separation and isolation of the investigated salt ﬂats

␮

is 4.4–8.3 m long (6.4 ± 1.5 ␮m on average, N = 6). will be the basis for future studies to further elucidate protist Type sequence data: The SSU and LSU rDNA sequences speciation in these isolated habitats. were deposited in GenBank database with the accession num- Three of our described species, “Salpingoeca” crinita bers MH490948 (SSU) and MH490949 (LSU). The closest sp. nov., “Salpingoeca” huasca sp. nov. and “Salpingoeca” ® relative sequence on NCBI nucleotide BLAST results in surira sp. nov. extended the clade around the model organism Salpingoeca rosetta (EU011941) with a maximum identity Salpingoeca rosetta (Fig. 1). “Salpingoeca” prava sp. nov. of 93%. clustered with Salpingoeca tuba and Salpingoeca dolichoth- Species: “Salpingoeca” huasca sp. nov. (Fig. 3 H-O), type ecata, forming a basal group within the Craspedida (Fig. 1). strain: HFCC strain 1286. We found that just like in clade 1 and clade 2 (see Carr

Diagnosis: Hypersaline Salpingoeca-like species, about et al., 2017), there are no diagnostic morphological traits

4.5 × 4 ␮m in size. Solitary cells attached to surfaces with to define the phylogenetic clade 3, containing Salpingoeca a stalk present as well as fast swimming cells with a short- tuba and Salpingoeca dolichothecata, which is now extended ened collar. Species can tolerate low saline to hypersaline by “Salpingoeca” prava sp. nov. (Fig. 2). In addition to the conditions (5–150 PSU). phylogenetic analysis, our detailed morphological approach Etymology: huasca (adjective, feminine) in reference to showed polymorphism in several species representing dif- the type locality. ferent life cycle stages. Therefore, we support the inclusion Type locality: Salar de Huasco, a salt flat in the Atacama of all paratypes in an accurate species description to avoid Desert in Northern Chile (20◦1557.5S68◦4916.0W) future redundant descriptions (Jeuck et al., 2014; Stoupin (Table 1). et al., 2012). Scanning electron microscopy revealed small Holotype: Specimen illustrated in Fig. 3H. filaments attached to the flagellum of “Salpingoeca” crinita

Description: Salpingoeca-like species with a body length sp. nov. with yet unknown function (Fig. 2).

of 3.7–5.4 ␮m (4.5 ± 0.4 ␮m on average, N = 21) and a body Aside with the recent taxonomic revision, studies suggest

width of 3.0–5.4 ␮m (3.9 ± 0.5 ␮m on average, N = 23). The to amend morphological and molecular data with autecolog-

collar has a variable length of 0.7–9.3 ␮m (4.6 ± 2.7 ␮mon ical traits such as habitat preferences and salinity tolerance

average, N = 15) and a width of 2.7–6.7 ␮m (4.6 ± 1.1 ␮mon to increase the accuracy and stability of species deﬁnition

average, N = 15). The ﬂagellum has a length from 6.6–24 ␮m in an ecological context (Carr et al., 2017; Nitsche, 2014).

(15.9 ± 5.9 ␮m on average, N = 21). The stalk is 6.5–10.4 ␮m As the abiotic environmental parameters play a key role for

long (8.1 ± 1.9 ␮m on average, N = 9). species adaptation and speciation (Atkins et al., 2002; Filker Type sequence data: The SSU rDNA sequence was et al., 2013; Nitsche, 2014; Nitsche and Arndt, 2015), the deposited in GenBank database with the accession num- salinity tolerance of all species, as they originate from salt ber MH490950. The closest relative sequence on NCBI flats with variable salinities, was tested. The autecological ® nucleotide BLAST results in Microstomoeca roanoka approach revealed that our newly described species can cope (HQ026770) with a maximum identity of 90%. with a broad range of salinities, from low saline to hypersaline waters (Fig. 4) supporting a high adaptation to the original hypersaline environment. In general, the cultivation of choanoflagellates and other protists is mainly limited Discussion by the environmental bacterial diversity. It must be consid- ered that an environmental bacterial community shift due to The Atacama Desert provides a unique environment with salinity changes might have favoured microbes of low food challenging conditions as the different investigated salt flats quality or species producing toxic metabolites and hence an in this study showed a broad range of hypersaline water indirect effect of the salinity shift might have occurred. To bodies (Table 1) demanding specific adaptations from the res- exclude a possible food limitation, we chose the bacterial idential organisms. In this study, we were able to describe four strain P. putida as a supplemental food source. These bac- new craspedid species from these extreme saline environ- teria were tested previously as a suitable food source for ments. Our phylogenetic analysis points toward a high degree protists and are able to tolerate salinity changes (Nitsche, of adaptation as the new species clustered in distinct branches 2014). It should be mentioned that this bacterial strain may with high p-distances (9.2–15.7%) to all previous sequenced have posed a challenge for “Salpingoeca” prava sp. nov. as choanoflagellates. In addition, none of these sequences was this species is probably dependent on smaller bacteria due previously recorded from high throughput sequencing (HTS) to its small size (Table 2). To understand the abiotic condi- S. Schiwitza et al. / European Journal of Protistology 66 (2018) 86–96 95 tions of the investigated salt flats, the elementary composition high sulfide and metal concentrations potentially encountered at was analysed via inductively coupled plasma mass spec- deep-sea hydrothermal vents. Mar. Ecol. Prog. Ser. 226, 63–75. trometry (MS platform, Biocenter Cologne, data not shown). Boenigk, J., Arndt, H., 2002. Bacterivory by heterotrophic flagel- This preliminary investigation showed intense chemical vari- lates: community structure and feeding strategies. A. van Leeuw. ations on salt structuring components but also the deposit J. Microb. 81, 465–480. of toxic elements, such as arsenic. As arsenic is a highly Buck, K.R., Garrison, D.L., 1988. Distribution and abundance of choanoflagellates (Acanthoecidae) across the ice-edge zone in volatile component, more sophisticated ways of detection the Weddell Sea. Antarct. Mar. Biol. 98, 263–269. and quantification will be applied in cooperation with the Butler, H., Rogerson, A., 1995. Temporal and spatial abundance of Department of Geosciences, University of Cologne. As the naked amoebae (Gymnamoebae) in marine benthic sediments adaptive mechanisms to extreme conditions, like hypersaline of the Clyde Sea area Scotland. J. Eukaryot. Microbiol. 42, environments or the presence of arsenic, are well-studied 724–730. for prokaryotes (Dorador et al., 2008; Kartal et al., 2006; Carr, M., Richter, D.J., Fozouni, P., Smith, T.J., Jeuck, A., Lead- Lara et al., 2012; Padan et al., 1989; Steil et al., 2003), fur- beater, B.S.C., Nitsche, F., 2017. A six-gene phylogeny provides ther investigations on the highly adapted choanoflagellates new insights into choanoflagellate evolution. Mol. Phylogenet. described in this study, based on comparative transcriptomes, Evol. 107, 166–178. could give new insights for eukaryotic adaptation to extreme Casamayor, E.O., Triadó-Margarit, X., Castaneda,˜ C., 2013. Micro- conditions and specially about horizontal gene transfer from bial biodiversity in saline shallow lakes of the Monegros Desert, Spain. FEMS Microbiol. Ecol. 85, 503–518. prokaryotes to eukaryotes (Yue et al., 2013). Cavalier-Smith, T., 1997. 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