Stygamoeba cauta n. sp. (Amoebozoa, Discosea) – a new brackish-water species from Nivå Bay (Baltic Sea, The Sound)

Journal Pre-proof Stygamoeba cauta n. sp. (Amoebozoa, Discosea) – a new brackish-water species from Niva˚ Bay (Baltic Sea, The Sound) Kirill Lotonin, Alexey Smirnov

PII:

S0932-4739(19)30097-5

DOI:

https://doi.org/10.1016/j.ejop.2019.125660

Reference:

EJOP 125660

To appear in:

European Journal of Protistology

Received Date:

3 October 2019

Revised Date:

21 November 2019

Accepted Date:

26 November 2019

Please cite this article as: Lotonin K, Smirnov A, Stygamoeba cauta n. sp. (Amoebozoa, Discosea) – a new brackish-water species from Niva˚ Bay (Baltic Sea, The Sound), European Journal of Protistology (2019), doi: https://doi.org/10.1016/j.ejop.2019.125660

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Stygamoeba cauta n. sp. (Amoebozoa, Discosea) – a new brackishwater species from Nivå Bay (Baltic Sea, The Sound) Kirill Lotonin and Alexey Smirnov Department of Invertebrate Zoology, Faculty of Biology, Saint Petersburg State University, 199034 Universitetskaya nab. 7/9, Saint Petersburg, Russia Corresponding author: Kirill Lotonin ([email protected])

Abstract

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Several evolutionary lineages of Amoebozoa are characterized by unusual morphological and ultrastructural features that impede resolving of their position in the phylogenetic tree. Among them is the genus Stygamoeba, not yet reliably placed on the phylogenetic tree even by a phylogenomic analysis. Only two species of Stygamoeba are known at present, and molecular data exists on one species only. Here, we present a description of the mesohaline species Stygamoeba cauta n. sp. isolated from the bottom sediments of Nivå Bay (Baltic Sea, The Sound). This stick-like, flattened amoeba morphologically resembles the previously described species Stygamoeba regulata Smirnov, 1996. However, the molecular analysis based on the 18S rRNA gene sequences and differences in cell behavior and pattern of locomotion provide strong support for establishing a new species.

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Keywords: Amoebozoa; Discosea; phylogeny; Stygamoeba; systematics

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Introduction

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The genus Stygamoeba was established by Sawyer (1975) with the type species S. polymorpha Sawyer, 1975 from the seawater of Chincoteague Bay (Atlantic Ocean) as a single species. The cells were described as slender, stick-like and actively branching during locomotion. They could also transform to leaf-like forms and the cytoplasm moved by very slow, almost invisible steady flow. Basing on this set of morphological features, the genus was assigned to the family Stereomyxidae Grell, 1966 (class Lobosea) that included some other branching marine amoebae (Corallomyxa, Stereomyxa) (Bovee and Sawyer 1979; Page 1983; Sawyer 1975). Since then, evidence of Stygamoeba polymorpha inhabiting various sea waters was recorded in several ecological and faunistic research works. For instance, it was isolated from the polluted sea bottom during a long-term study of marine amoebae from the Atlantic Ocean and Gulf of Mexico (Sawyer 1980). The species was also discovered in Georgia coastal surface waters in the vicinity of Sapelo Island (Munson 1992). Also, distribution of Stygamoeba polymorpha on the macroalgae from the intertidal zone of the Clyde Sea was noted and its role as a component of the grazing protozoan population was estimated (Rogerson 1991). Additionally, the species was characterized as ubiquitous in marine sediments of the Clyde Sea after numerous findings throughout the whole year during research of benthic naked amoebae (Butler and Rogerson 2000). The results of these works prove wide distribution of S. polymorpha in the sea waters of both New and Old World and probably testify its tolerance to environmental pollution. The second mesohaline species, Stygamoeba regulata Smirnov, 1996, was isolated from the bottom sediments of Nivå Bay (Baltic Sea, The Sound), a location, where a number of studies dedicated specifically to benthic protozoan ecosystems were carried out previously (Fenchel 1969; 1987; Fenchel et al. 1977; Fenchel and Finlay 1995; Smirnov 1999; 2001; Smirnov and

2 Thar 2003). The locomotive form of this amoeba was characterized as very regular, flattened, monopodial, and stick-like, possessing a bulbous uroid and never showing active branching (Smirnov 1996). The use of electron microscopy showed that S. regulata had an unusual set of ultrastructural features, among them was the presence of the cytoplasmic microtubule-organizing center (MTOC) with radiating microtubules, associated with the dictyosome. The second remarkable character was the flattened, ribbon-like shape of the mitochondrial cristae. At that time Stygamoeba was placed among Rhizopoda incertae sedis because of the absence of any clear morphological traits (Smirnov 1996).

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Some time later, an amoeba species resembling Stygamoeba spp. in light microscopy and ultrastructure - Vermistella antarctica was described from the sediments near the Ross Ice Shelf (Moran et al. 2007). Further, the second member of this genus, V. arctica was described from the littoral zone of Svalbard archipelago (Tyml et al. 2015). The analysis of the ultrastructure showed an outstanding similarity between Vermistella Moran, Anderson, Dennett, Caron et Gast, 2007 and Stygamoeba. The most important feature was the presence of a flattened mitochondrial cristae in both genera.

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The application of molecular methods indicated a strong affiliation of Stygamoeba and Vermistella to the class Discosea, first in 18S rRNA gene trees (Kudryavtsev et al. 2011; Lahr et al. 2011; Moran et al. 2007; Smirnov et al. 2011) and later – in multigene phylogenetic trees (Cavalier-Smith et al. 2016; Tekle et al. 2016; 2017). Nevertheless, the exact position of these enigmatic genera remained poorly resolved even in the tree inferred from the phylogenomic analysis (Kang et al. 2017). Moreover, these two genera, despite the high morphological similarity, did not group together in the phylogenetic tree (Cavalier-Smith et al. 2016; Lahr et al. 2011; Melton et al. 2019; Tyml et al. 2015). Nevertheless, Smirnov and Cavalier-Smith formed a paraphyletic order Stygamoebida to unify these two genera, basing on their high morphological similarity and the unique character among lobose amoebae – the presence of flattened mitochondrial cristae (Smirnov et al. 2011).

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In the present paper we describe a new mesohaline species, Stygamoeba cauta, from the bottom sediments of Nivå Bay (Baltic Sea, The Sound). This species has tiny differences in morphology to S. regulata, but significantly differs in SSU sequence. This finding provides further evidence for the presence of sibling species that could be reliably distinguished only on the level of gene sequences in various amoebae genera.

Material and Methods

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Sampling and cultivation In August 2018, samples containing sand with sulfur bacteria mats were collected from the upper layer of sandy bottom sediments in the brackish-water Nivå Bay (Baltic Sea, The Sound, 55°55'41.8"N 12°31'23.0"E). Small amounts (1-2 ml) of the sediments were inoculated onsite in 50 ml plastic flasks (Greiner), containing 20 ml of sterile (Millipore-filtered, 0.22 µm) artificial 15 ppt seawater and autoclaved wheat grains (one in each flask) as a source of nutrients for accompanying bacteria. The cultures were further maintained in 60 mm Petri dishes (Orange Bioscience) with 0.025% Cerophyl medium made on Millipore-filtered (0.22 µm) 15 ppt artificial seawater (Page 1983). Cultures were stored at 18°C under the room light. All attempts to clone amoebae using both liquid and agar media failed, so the species was maintained in mixed culture, containing few other eukaryotes, some unicellular algae and bacteria. All studied cells originated from the same flask and were identified as belonging to the same species; this was checked many times during the study. Light microscopy

3 Observations of amoebae in cultures, along with measurements of the cell dimensions were made with the use of an inverted microscope Leica DMI3000B equipped with phase contrast optics. The micrographs of amoebae moving across a glass object slide were taken with Leica DM2500 microscope under DIC optics with Nikon DS-Fi3 camera. The size of the nucleus was measured in amoebae placed on the object slide but not pressed with the coverslip.

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DNA isolation For molecular studies, individual amoeba cells were collected with small amount (ca. 1 µl) of medium using the tapering glass pipettes and placed in 200 µl PCR tubes. In order to destroy the cell, tubes were placed consequently in cold isopropanol (-80°C) and warm water (+45°C) – approach advised to us by Dr. Matthew Brown. This cycle was repeated four times. For singlecell PCR the content of the tube was topped with 50 µl of the ready PCR mixture and immediately placed in the thermocycler. A fragment of 18S rRNA gene (738 bp) was amplified by PCR using S12.2 (forward) and S20R (reverse) primers (Pawlowski 2000). Thermal cycler parameters started with initial denaturation (10 min at 95°C) followed by 34 cycles of 30 s at 94°C, 60 s at 50°C and 120 s at 72°C, then final extension at 72°C for 10 min. Amplicons were purified using Cleanup Mini Purification Kit (Eurogene, Moscow) and cloned using InsTAclone PCR cloning kit (Thermo Fisher Scientific), clones were sequenced with ABI Prism 310 sequencer using the facilities of SPSU Science Park. Attempts to get longer PCR fragment using a wide set of other eukaryotic primers failed. To obtain a sequence of a longer fragment of 18S rRNA gene, a DNA from individual cells placed in PCR tubes as described above was extracted using Arcturus PicoPure DNA isolation kit (Thermo Fisher Scientific). Each sample was topped with 12 µl of proteinase K solution in manufacturer’s buffer. The whole genome amplification was carried out using the REPLI-g Single Cell Kit (QIAGEN) according to manufacturer’s instruction (4 h of incubation). Obtained DNA was sequenced using the high-throughput Illumina Hiseq 2500 platform using the facilities of SPSU Science Park. NGS data were trimmed, filtered and assembled as described by Bondarenko et al. (2018). The assembled fragment counted 1622 bp in length.

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Phylogeny reconstruction The 18S rRNA gene sequence of Stygamoeba regulata Nivå Bay strain was used in our phylogeny reconstruction. This is an original sequence, obtained in 2009 from the culture, reisolated from Nivå Bay by A. Smirnov in 2007 and identified as belonging to this species. About 400 floating cells were collected manually by tapering Pasteur pipette in an Eppendorf tube and settled down with gentle centrifugation (500 g). DNA was extracted from the cell sediment with the use of guanidinium thiocyanate buffer (Maniatis 1982). Partial sequence of 18S rRNA gene (1837 bp) was obtained using Rib A (forward) and S20R (reverse) primers (Pawlowski 2000). These primers were used both for amplification and sequencing. Sequences were aligned manually using SeaView 4.0 (Gouy et al. 2010). The alignment included all named sequences of Stygamoeba and Vermistella and representatives of major clades of Discosea. Species and environmental sequences were selected in a way to have a balanced representation of long- and short-branching sequences in the tree. The phylogenetic analysis was performed using using RAxML 8.1.11 (Stamatakis 2014) at CIPRES portal (Miller et al. 2010) GTR + γ model; 1537 sites were selected for the analysis, 1,000 bootstrap pseudoreplicates were performed. Bayesian analysis was performed on the same dataset using MrBayes 3.2.6 run at CIPRES portal (Miller et al. 2010), GTR model with γ - correction for intersite rate variation (8 categories) and the covarion model (Ronquist and Huelsenbeck 2003) were applied. Trees were run as two separate chains (default heating parameters) for 6 million generations, by which time they had ceased converging (final average standard deviation of the split frequencies was less than 0.01). The quality of chains was estimated using built-in MrBayes tools and additionally using the software Tracer 1.6 (Rambaut et al. 2014); based on the estimates by Tracer, the first 25% of generations were discarded for burn-in.

4 The obtained 18S rRNA gene sequences were deposited with GenBank under the numbers MN547354, MN547355 and MN547356 (Stygamoeba cauta) and MN547357 (Stygamoeba regulata Nivå Bay strain). Identity and similarity of sequences was estimated using “Ident and Sim” online tool (https://www.bioinformatics.org/sms2/ident_sim.html).

Results

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Microscopy observations During locomotion amoebae were elongated, stick-shaped, almost equally wide along the entire length of the cell (Fig. 1A, B). They perfectly fitted the definition of spineolate morphotype (Smirnov and Goodkov 1999). The cell could form leaf-like lateral protrusions, mainly in the middle part of the cell. They were especially evident when the cell changed the direction of movement (Fig. 1D, I). The frontal hyaline area always was well-pronounced and occupied about 1/3 of the total cell length. There were no wrinkles or longitudinal folds on the dorsal cell surface. While changing the direction of movement, the cell formed a bifurcation in the anterior hyaline zone (Fig. 1C, D, I). One of these hyaline extensions formed a leading pseudopodium that could draw the cell even at right angle to its earlier direction (Fig. 1F, G, J, K). However, the cell could also change the direction by means of bending the entire body, without splitting it into discrete pseudopodia. Slowly moving amoebae might sometimes become branched (Fig. 1L). The average length of the cell was 26 µm (range 17.5-37.5 µm, n = 50) and the average width was 4 µm (range 2.5-7.5 µm, n = 50). The average Length/Width ratio was 6.6 (range 3-11, n = 50). The posterior end of the cell possessed a bulbous uroid, which was often detached from the substratum or demonstrated a smooth outline (Fig. 1B). The movement of cells was steady, resembling gliding and it was hard to observe cytoplasmic flows in moving cell. The nucleus was ellipsoid, of vesicular type, with rounded central nucleolus. Its largest dimension varied from 2 to 4 µm (average 3 µm, n = 50). The size of the nucleolus was ca. 1 µm, which was too small for reliable measurements. In the process of locomotion, S. cauta often protruded a thin waving pseudopodium (one or two) that “palpated” the substratum in front of the cell. Later this pseudopodium got adhered to it. Soon after this, its central part expanded and the cell mass flowed into the pseudopodium. (Fig. 1E-H). Stationary cells lacked discrete pseudopodia (Fig. 1M-O). Floating cells were observed seldom in cultures. In developed floating forms, the main cell mass was rounded; it consisted of the granuloplasm and produced up to three thin hyaline pseudopodia. Apart from the nucleus, the granuloplasm contained food vacuoles, refractile inclusions and granules. Cysts were not detected in our cultures; also, they might not be recognized because of the mixed nature of cultures.

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Phylogenetic analysis Our phylogenetic tree shows that Stygamoeba cauta groups with both strains identified as Stygamoeba regulata with full support (Fig. 2). The genus Stygamoeba in our tree is related to the genus Vermistella, hence this grouping is not supported statistically. In total, Stygamoebida formed a sister group to the assemblage, consisting of Thecamoebida, Dermamoebida and Acanthopodida, however to resolve the position of Stygamoebida in the global-scale phylogeny was not the aim of the present SSU tree, containing limited amount of amoebozoan taxa. Grouping of other taxa in the tree was usual, already known from previous studies, all clades corresponding to the groupings of the taxonomic level of order (except Stygamoebida) were fully or highly supported. According to the analysis of sequence identity level between Stygamoeba strains, Stygamoeba cauta shows maximum 84.5% identity with S. regulata Nivå Bay strain and 83.4% identity with S. regulata ATCC 50892 strain (the length of the shared fragment for analysis was 1606 bp).

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Manual analysis of sequences reveales numerous short (two or three bp) indels and shows considerable differences in the variable regions E23 and E43 (approximate positions 657-844 and 1492-1571, respectively, in S.regulata ATCC 50892 sequence). Especially remarkable is the presence of a long fragment in the region 411-573 in S. cauta sequence (corresponds to the positions 749-832 in S. regulata ATCC 50892 sequence), which is absent in both other Stygamoeba sequences. To check the stability of 18S rRNA gene sequence in S. cauta, we compared two molecular clones after the single-cell PCR (from the same amplicon) and the contig obtained as a result of NGS sequencing of another cell, sampled from the same culture at the other day. All were almost identical; we have found only one nucleotide difference in the position 976 in S. cauta NGS sequence (a->g). This position was identical in two molecular clones obtained by conventional PCR, so it may be an NGS assembly error. Interestingly, S. regulata Nivå Bay strain shows considerable difference (only 93.7% identity) with ATCC50892 strain when all three strains are aligned and 94.5% identity when both strains of Stygamoeba regulata are unambiguously aligned (length of the shared fragment in the latter case increased to 1843 bp). Manual examination of sequences indicates the presence of numerous indels and two-nucleotide reversions distributed along the entire length of the molecule and no less than six areas, 5-26 nucleotides in length each, showing differing structure or not represented in one of sequences.

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Discussion

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Stygamoeba cauta entirely fits the diagnosis of the genus Stygamoeba provided by Sawyer (1975). He defined this genus as “thin elongated amoebae which resemble toothpicks or splinters and may change to a forked or branched form” (Sawyer 1975, p. 85). He also noted that nondirected movement usually results in conversion of lateral parts to broad and leaf-like forms and mentioned “slow gliding motion without detectable locomotive activity”. All these characters are evident in our species as well. Careful observations allow distinguishing Stygamoeba cauta from congeners even at the lightmicroscopic level. Its dimensions (length and width) are twice bigger than those of S. polymorpha. It is also worth noting that the latter species was isolated from the Atlantic Ocean with the water salinity 33-37 ppt, while S. cauta is a brackish-water organism (15 ppt). The similarity between S. cauta and S. regulata is much higher, especially when amoebae are monopodial and expose a bulbous uroid. This resemblance is reinforced with the almost equal length and width of the locomotive cells. Nevertheless, S. regulata never appears to be branched or deeply bifurcated, it adopts an irregular shape (still not branched) only in non-directed locomotion (Smirnov 1996). Stygamoeba cauta demonstrates a morphological feature, which was not previously described for this genus. During locomotion, the cell produces an anterior waving subpseudopodium that detaches from substratum and later touches it with a tip. It seems that this pseudopodium defines the direction of movement. The same pattern of waving subpseudopodium was noted for the genus Pseudoparamoeba (Udalov 2016) and described for the genus Oscillosignum by Bovee (1953), also the validity of the latter one remains doubtful. However, nothing like this was noted for Stygamoeba regulata. The latter species can produce a short frontal pseudopodium, but it never gets that long and waving in the water. At the light microscopy level Stygamoeba regulata and Stygamoeba cauta may be considered as almost sibling species because of the high morphological and size similarity of trophozoites. They populate the same habitat and potentially may co-occur in samples, which makes their distinction even more problematic. For reliable identification it is necessarily to apply the molecular analysis. The description of S. cauta as new species is supported by the analysis of 18S rRNA gene sequence, which shows rather high level of divergence from Stygamoeba

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regulata. The comparative analysis of sequences shows that the sequence of S. cauta has significant nucleotide and structural differences from S. regulata sequences. The low identity level (84.5%) and structural differences, including the presence of a long unique sequence fragment in E23 region confirms the individual status of this species. It is remarkable that the difference between two sequences identified as Stygamoeba regulata is rather high and may raise a question on their co-specificity. The analysis of sequences shows that they demonstrate numerous tiny differences (mostly one or two nucleotides indels) along the entire molecule and certain differences in variable regions, also much lower than between S. regulata and S. cauta. This case requires more detailed analysis, ideally – including more genes. At present, we consider segregation of these strains to be premature. Finally, the lack of molecular data on Stygamoeba polymorpha constrains substantially the phylogeny reconstruction leaving re-isolation and re-description of this species still pending. The UK National Culture Collection (Oban, Scotland) still lists among available the strain CCAP 1580/1 named as Stygamoeba regulata and deposited by A. Smirnov in 1994 and nominally representing the type culture of this species. Note that this is not the type material; the holotype for S. regulata is a permanent stained preparation 1995:2:9:1 deposited with the natural History Museum, London, UK (Smirnov 1996). However, the analysis of records by A. Smirnov evidences that the strain 1580/1 was obtained by A. Smirnov from CCAP on 29/06/2007 and it contained not S. regulata, but a Labirinthula-like organism (to the certain extent similar because of its oblong cells sliding in the tubular network). This strain was replaced by A. Smirnov with the culture isolated in 2007 and used for DNA isolation in the present study and it was listed for some time as CCAP 1580/2 strain, but further lost. As a result, the CCAP 1580/1 strain of S. regulata should be considered with care.

Taxonomic Summary

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Amoebozoa Lühe, 1913 Class Discosea Cavalier-Smith, 2004 Order Stygamoebida Smirnov et Cavalier-Smith, 2011 Family Stygamoebidae Smirnov et Cavalier-Smith, 2011 Genus Stygamoeba Sawyer, 1975 sensu Smirnov (1996)

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Stygamoeba cauta n. sp. Diagnosis. Anterior hyaloplasm occupies up to 1/3 of body length. Cells sometimes with slightly raised bulbous uroid. Locomotive form on average 26 x 4 µm (range 17.5-37.5 x 2.5-7.5 µm), length/width ratio 6.6 (range 3-11). A thin waving subpseudopodium is usually exposed over substratum, able to adhere to substrate and then transform to a leading part of the cell. Floating cell is round and has several (up to 3) thin hyaline protrusions. Nucleus ellipsoidal and vesicular, 2-4 µm (average 3 µm). No cysts known. Type material. The holotype for this species is a permanent haematoxylin-stained preparation № 1021 deposited with the Museum of preparations of the Laboratory of Cytology of Unicellular organisms of the Institute of Cytology RAS. The type cell is marked on the slide. Three sequences of the 18S rRNA gene of the same strain obtained with both NGS and Sanger sequencing are placed in GenBank under the accession numbers MN547354, MN547355 and MN547356. Also, video record of the moving amoeba is available in supplementary materials. ZooBank LSID for this species is: E4CA9F42-29B1-446E-8564-677F9A41AC9B Type locality. Nivå Bay (Baltic Sea, The Sound, 55°55'41.8"N 12°31'23.0"E), upper layer of the bottom sediments (0-1 cm). Etymology. “Cauta” (Latin “cautious”) describes its ability to “examine” the substratum with a subpseudopodium prior to choose the direction of movement.

Author contributions Both authors equally contributed to the study

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Acknowledgements Supported by RSF 17-14-01391 grant (concept, molecular studies and analysis) and RFBR 1904-01147 grant (isolation, cultivation and purification of cells). The present study utilized equipment of the Core facility centres "Development of molecular and cell technologies", “Biobank”, “Computing Centre SPSU” and "Culture Collection of Microorganisms" of the Research park of Saint Petersburg State University.

Appendicies Videorecord of Stygamoeba cauta.

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Format: MPEG-4 File size: 29,4 Mb Date: 12th April 2019

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Legends to the Figures

Fig. 1A-P. Morphology of Stygamoeba cauta (Differential interference contrast). A-D: Amoeba in active locomotion (arrow, B – bulbous uroid). E-H: Extension of a thin subpseudopodium (arrow, G – thin waving subpseudopodium). I-L: Change of the direction of movement. M-P: Amoeba recently attached to the cover slip, during non-directed locomotion (arrow, P - new leading pseudopodium). Scale bar: 10 µm.

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Fig. 2. Phylogenetic tree based on the 18S r RNA gene, showing the position of Stygamoeba cauta. 1537 sites are used for the analysis; GTR + γ was used for ML analysis and GTR + γ with covarion – for Bayesian analysis. Labelling of nodes: PP/ML support. Black circles are used to label fully supported nodes (1.0/100 support).