Paramoeba aparasomata n. sp., a symbiont-free species, and its relative Paramoeba karteshi n. sp. (Amoebozoa, Dactylopodida)

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ScienceDirect European Journal of Protistology 71 (2019) 125630

Paramoeba aparasomata n. sp., a symbiont-free species, and its relative Paramoeba karteshi n. sp. (Amoebozoa, Dactylopodida) Ekaterina Volkovaa,c,∗ , Eckhard Völckerb , Steffen Claußb , Natalya Bondarenkoa , Alexander Kudryavtseva,c a

Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, St. Petersburg, Russia Penard Labs, Cape Town, South Africa c Laboratory of Cellular and Molecular Protistology, Zoological Institute, Russian Academy of Science, St. Petersburg, Russia b

Received 19 November 2018; received in revised form 17 June 2019; accepted 18 June 2019 Available online 29 August 2019

Abstract Two brackish water amoebae have been isolated and studied from the benthic biotopes of the Chupa Inlet (Kandalaksha Bay, northwestern Russia). Both strains can be identified as new species of the genus Paramoeba (Amoebozoa, Dactylopodida, Paramoebidae) based on light microscopical characters, structure of microscales on the cell surface and molecular evidence based on the analyses of two genes, nuclear SSU rRNA and mitochondrial cytochrome c oxidase subunit 1 (COI). Paramoeba aparasomata n. sp. is of particular interest because this amoeba is permanently lacking a symbiotic Perkinsela-like organism (PLO) present in other species of Paramoeba and Neoparamoeba. The results obtained show that scaly dactylopodial amoebae lacking PLO are not necessarily members of Korotnevella. In particular, we suggest that Korotnevella nivo Smirnov, 1997, with microscales very similar to those of Paramoeba eilhardi and the species studied here in structure, may be in fact a member of Paramoeba. Molecular data on K. nivo have to be obtained and analysed to test this hypothesis. Based on our new results we emend the diagnosis of the genus Paramoeba to make it more fit to the current phylogenetic conception. © 2019 Published by Elsevier GmbH. Keywords: Amoebae; Paramoeba; Perkinsela-like organism; Phylogeny; Scales; Scanning electron microscopy

Introduction The genus Paramoeba Schaudinn, 1896 comprises marine amoebae belonging to the family Paramoebidae Poche, 1913 (order Dactylopodida). These amoebae, as well as members of the related genus Neoparamoeba Page, 1987, possess an intracellular eukaryotic symbiont – a Perkinsela-like organism (the PLO, formerly known as the ‘parasome’) belonging ∗ Corresponding

author at: Laboratory of Cellular and Molecular Protistology, Zoological Institute, Russian Academy of Science, Universitetskaya nab., 1 199034 St. Petersburg, Russia. E-mail address: [email protected] (E. Volkova). https://doi.org/10.1016/j.ejop.2019.125630 0932-4739/© 2019 Published by Elsevier GmbH.

to the Kinetoplastida (Dyková et al. 2003). Besides the presence of a PLO, the genus Paramoeba is characterized by the dactylopodial morphotype and microscales on the plasma membrane surface. The brief historical overview of the PLO-containing genera Paramoeba, Neoparamoeba and Janickina Chatton, 1953 was presented in our previous paper and elsewhere (e.g. Nowak and Archibald 2018; Volkova and Kudryavtsev 2017). Here, we only have to add a rarely mentioned fact that Grassi (1881), who described Amoeba pigmentifera and Amoeba chaetognathi (both later transferred into the genus Janickina; Chatton 1953) parasitizing adult individuals of two species of chaetognaths, saw the PLO in the former species only. Janicki (1912) demonstrated the

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PLO in both species using cytochemical techniques and suggested that Grassi failed to observe the PLO in Janickina chaetognathi because of masking by cytoplasmic inclusions. Hamon (1957) carefully studied the nature of the PLO in Janickina pigmentifera. She noticed the stages of regression of the PLO down to a complete loss. However, she came to a wrong conclusion about the nature of the PLO as a phagosome where host spermatids were digested (Hamon 1957). The study we present here is mainly focused on the genus Paramoeba. There are two species of this genus described in detail: the type species, Paramoeba eilhardi Schaudinn, 1896, and P. atlantica Kudryavtsev et al., 2011. Several previously described species, P. schaudinni De Faria et al., 1922, P. perniciosa Sprague et al., 1969, and P. invadens Jones, 1985 cannot be included in this genus for various reasons. In particular, the description of the first species was not detailed enough to distinguish it from P. eilhardi, and no reinvestigation was performed since its original description (De Faria et al. 1922). The latter two species should have been included in the genus Neoparamoeba after it was established by Page (1987) due to the absence of microscales on their cell surface (Jones 1985; Sprague et al. 1969). Microscales of P. atlantica and P. eilhardi are boat-shaped; they have solid lateral walls in the former species while in the latter one there are four apertures in every wall of a microscale (Grell and Benwitz 1966; Kudryavtsev et al. 2011). In spite of the ultrastructural similarities, both species never form a single clade in a molecular phylogenetic tree (Feehan et al. 2013; Kudryavtsev et al. 2011; Volkova and Kudryavtsev 2017). For a long time the PLO was considered to be one of the most important characteristics justifying the assignment of a species to the genera Neoparamoeba, Paramoeba or Janickina (Hollande 1980; Page 1987). Some aspects of the lifecycle of the PLO in P. eilhardi were described by Grell (1961). In particular, he demonstrated a division of the PLO within the host cell independent of the host cell division, as well as the synchronous division of the PLO and the host cell (Grell 1961). The presence of the PLO was one of the characters distinguishing Neoparamoeba and Paramoeba from other paramoebids (Page 1987). However, two symbiontfree amoebae with very similar morphology to P. eilhardi were studied previously. In 1997 Korotnevella nivo isolated from a brackish sediment sample was described (Smirnov 1997). The scale structure of this amoeba was very similar to P. eilhardi but it possessed no PLO that favored its assignment to the genus Korotnevella. The second morphologically similar amoeba was isolated from the filaments of a colonial blue–green marine alga and had surface microscales also very similar to those of P. eilhardi. The symbiont has never been observed in amoebae of this strain (Anderson 1977). No molecular data were obtained for both strains, therefore, in spite of the similarity in microscale structure they were not included in the genus Paramoeba mainly due to the absence of a PLO.

The results we present in this paper justify the description of two new scaled species of the genus Paramoeba, one of which is permanently aposymbiotic. We also perform further analyses of the coevolution of Paramoeba/Neoparamoeba and their symbionts by including more PLO SSU rRNA gene sequences from the scale-bearing Paramoeba spp. in the molecular phylogenetic dataset.

Material and Methods Isolation of amoebae, culturing, light and electron microscopy Two strains of dactylopodial amoebae originated from Kandalaksha Bay of The White Sea (northwestern Russia). The first strain, Paramoeba aparasomata n. sp. (strain WS13.21T) was isolated from a sample of sublittoral soft bottom sediments collected by a scuba diver in July, 2013 off the beach of Luda Cheremshikha Island (66.321344N, 33.85184E; depth 6 m). Paramoeba karteshi n. sp. (strain WS14KH1) was isolated from the body material of a marine sponge Halisarca dujardini collected on August 3, 2014 from the sublittoral of Levaya Bay near the Marine Biological station “Kartesh” of the Zoological Institute of the Russian Academy of Science (66.3369944N, 33.6598806E; depth 5 m). The salinity in both biotopes was 24–27 ppt. The collected material was inoculated in Petri dishes, 90 mm in diameter and filled with 25 ppt seawater sterilized by filtration through a 0.2-␮m pore-sized filter with the addition of three autoclaved wheat grains per dish. Samples were incubated for a week. Isolation, cloning, and maintenance of the amoebae were performed as described by Kudryavtsev et al. (2011). Light microscopic observations, photo and video recording were performed on living amoebae from the clonal cultures using the inverted microscope Leica DMI3000 with phase contrast and integrated modulation contrast (IMC) optics, and the upright microscope Leica DM2500 with differential interference contrast (DIC) and phase contrast optics. To demonstrate the presence or absence of a PLO, the amoebae were fixed and stained with DAPI as described in Kudryavtsev et al. (2011) followed by observations and photography with the epifluorescent upright microscope Leica DM2500. For scanning electron microscopy the amoebae were allowed to settle overnight on glass coverslips and fixed with 2.5% glutaraldehyde solution in 25 ppt seawater (final concentration) for 30 min followed by a 5-min rinse with seawater and postfixation in 1% solution of osmium tetroxide in seawater for 30 min. After postfixation, the cells were sequentially washed with five portions of seawater (5 min each) diluted with double-distilled water to decrease the seawater concentration, followed by a quick rinse in double-distilled water before dehydration in a graded ethanol series of increasing concentration from 30 to 100% in 20% steps. Further, specimens were critical-point dried in liquid carbon dioxide

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using a K850 critical point drier and sputter-coated with platinum in a Q150R S sputter coater (Quorum Technologies). The specimens were observed using a Zeiss Sigma FE-SEM microscope operating at 1 kV.

DNA isolation, sequencing and phylogenetic analyses Total genomic DNA was isolated from the densely growing cultures using the guanidine isothiocyanate method (Maniatis et al. 1982). Amplification, PCR product purification, cloning and sequencing of the small-subunit (SSU) rRNA genes of amoebae were performed essentially as described in Kudryavtsev and Pawlowski (2015); universal eukaryotic primers RibA and RibB with annealing temperature +50 ◦ C were used for amplification (Medlin et al. 1988). Primers Kineto14F and Kineto2026R (von der Heyden and Cavalier-Smith 2005) were used to amplify the SSU rRNA gene of the PLO in P. karteshi. For molecular phylogeny reconstruction based on the cytochrome C oxidase subunit 1 (COI) gene partial sequences of the mitochondrial genome of P. aparasomata were used (Bondarenko et al., 2019). The corresponding contig was aligned with the publicly available mitochondrial genome sequence of Neoparamoeba (deposited as Paramoeba) pemaquidensis, GenBank accession number NC 031417. Based on this alignment the sequences of specific primers were obtained and further used to amplify fragments of COI gene in P. karteshi n. sp. and P. eilhardi (strain CCAP 1560/2) (Supplementary Table S1). For phylogenetic analyses the sequences were aligned with our databases of SSU rRNA genes of dactylopodid amoebae and PLOs as well as the COI genes of Amoebozoa using Seaview v. 4 (Gouy et al. 2010). The identity of the new strains was evaluated using preliminary trees which included all available sequences of Neoparamoeba, Paramoeba and several sequences as an outgroup. Poorly aligned sites and gaps were manually excluded from the final alignments which consisted of 104 SSU rRNA gene sequences of amoebae with 1903 well aligned nucleotide positions, 49 SSU rRNA gene sequences of Kinetoplastida (including 46 sequences of PLO and three sequences of Ichthyobodo spp. as outgroup) with 1411 well aligned nucleotide positions, and 37 COI gene sequences with 552 well aligned nucleotide positions. In addition, a concatenated alignment of amoebae SSU rRNA (1667 nucleotide positions) and COI (554 nucleotide positions) genes was constructed using Seaview and analyzed as a single partition. Phylogenetic trees were reconstructed using the maximum likelihood algorithm in RaxML v.8.2.10 (Stamatakis 2014) with 100 initial parallel searches based on independent starting trees and nonparametric bootstrapping of the best tree with 1000 pseudo-replicates. The GTRGAMMAI model with default parameters was used. Bayesian inference was performed

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using MrBayes v. 3.2.6 (Ronquist et al. 2012) with GTR model and gamma model for among-site rate variation (4 rate categories) including proportion of invariable sites. Markov chain Monte Carlo analysis was performed in two runs of four simultaneous chains sampled for 10,000,000 generations for SSU rRNA datasets and 30,000,000 generations for COI dataset; 25% of the sampled trees were discarded as a burnin. Analyses were run at CIPRES portal (Miller et al. 2010).

Results Morphology and ultrastructure of Paramoeba karteshi n. sp. strain WS14KH1 (Figs. 1, 2, Table 1) During locomotion, cells of Paramoeba karteshi n. sp. (strain WS14KH1) were flattened and adopted three different shapes: (a) with a somewhat thickened, ovoid posterior part of the cell body (Figs. 1A, 2 A), (b) elongated, equally broad along its whole length (Figs. 1A, 2 B) and (c) irregularly triangular, tapering towards the posterior end (Figs. 1A, 2 C). The cytoplasm was separated into the anterior hyaline area occupying a quarter to a half of the cell and the posterior area consisting of granuloplasm (Fig. 1A). The value of the length to breadth ratio was 1:1–4:1, on average 2.2:1. The frontal edge of the anterior hyaline area produced three types of subpseudopodia which varied in length: (1), 1–3 ␮m long; (2), 3–10 ␮m long and (3), 10–37 ␮m long (Figs. 1A, 2 A–C). Some of the cells produced dorsal ridges with the length 33–50% of the total length of the cell body which turned to the subseudopodia of the (2)-type or the (3)-type (not shown). The posterior end of moving amoebae was smooth without any adhesive uroidal filaments (Fig. 1A). The average rate of movement in a Petri dish at +22 ◦ C was 27 ␮m min−1 . During non-directed movement the amoebae produced short (1–2 ␮m long) conical subpseudopodia in all directions (not shown). The amoebae adopted floating forms immediately after detachment from the substrate. Floating forms consisted of a spherical central mass of granuloplasm, 10–15 ␮m in diameter, with 5–8 long hyaline pseudopodia (25–38 ␮m long), sometimes bent close to their tips and radiating from the central mass (Fig. 1B). In the cultures, between a quarter and a half of the cells were in the floating state. The amoebae possessed a spherical, vesicular nucleus with a single nucleolus located centrally. The nucleus of the rapidly moving cells was located in the central part of the cell body or slightly shifted towards the anterior part (Fig. 1D, E). The single PLO was seen near the nucleus (Fig. 1D, E). It had a shape of an oval body and consisted of the granulose central part and two smooth side parts (Fig. 1C, D). Measurement results of amoebae and PLO are provided in Table 1. Cells were covered with boat-shaped bilaterally symmetrical microscales (Fig. 1F–H). Every microscale consisted of

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Fig. 1. A–H. Morphology and ultrastructure of Paramoeba karteshi n. sp. in vivo (A–B, phase contrast, C, differential interference contrast), after DAPI-staining (D, E), and in the SEM (F–H). (A) Locomotive forms, arrows show the direction of movement. (B) Floating form. (C) PLO. (D, E) Nucleus and PLO visualized by DAPI staining (D) and a corresponding phase contrast image of the same cell (E). (F) Boat-shaped microscales on the plasma membrane surface. (G) Subpseudopodia covered with microscales. (H) The whole surface of the cell covered with microscales. b – bacteria adhered to the layer of microscales, Nu – nucleus, PLO – Perkinsela-like organism. Scale bars = 20 ␮m in A, 10 ␮m in B–E ␮m, 100 nm in F, 400 nm in G, 2 ␮m in H. Table 1. Measurements of the Paramoeba karteshi n. sp. cells and its PLOs. Locomotive form Length Breadth Length:breadth ratio Length of subpseudopodia Rate of locomotion 29–75 (47) n = 44 13–41 (22.5) n = 44 1:1–3.7:1 (2.2:1) n = 44 1–37 (10) n = 157 18–36 (27) n = 3 Nucleus diameter Nucleolus diameter Nucleus/nucleolus PLO length PLO breadth PLO L:B 5–9 (7) n = 15 3–5 (4) n = 15 1.7:1–2.2:1 (1.87:1) n = 15 4–6 (5) n = 15 3–4 (3) n = 15 1.15:1–1.82:1 (1.44:1) n = 15 Microscales Length Breadth Length:breadth ratio Marginal columns Central columns Marginal: central columns 295–389 (352) n = 66 124–203 (160) n = 66 1.6:1–2.8:1 (2.2:1) n = 66 112–129 (120) n = 6 77–91 (82) n = 6 1.4:1–1.6:1 (1.5:1) n = 6 Light microscopic measurements in ␮m; measurements of microscales in nm; locomotive rates in ␮m min−1 ). Range (min-max) is followed by an average value in brackets where appropriate.

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Fig. 2. A–E. Line drawings of trophic amoebae and a generalized scheme of a scale structure. (A–C) Three types of locomotive forms of Paramoeba karteshi: (A) – oval cell (B) – elongated cell (C) – triangular cell. (D) Floating form typical for P. karteshi and P. aparasomata. (E) Generalized scheme of a microscale typical for P. karteshi and P. aparasomata.

a proximal flat base with an oval outline and distal oval rim connected with the periphery of the base by eight upright columns (Fig. 2E). These columns differed in length. Two longest columns were located at the “stern” and the “bow” of a boat-shaped microscale. Other six columns were 1.5 times shorter and located by three columns on each “board” of the microscales. All columns within a “board” of a scale were equidistant from each other. Measurement results of microscales are presented in the Table 1.

Morphology and ultrastructure of Paramoeba aparasomata n. sp. strain WS13.21T (Figs. 2D, E, 3, Table 2) During locomotion cells of Paramoeba aparasomata n. sp. (strain WS13.21T) were flattened and rounded in outline with the length equal to breadth, or had an oval shape with the length to breadth ratio of 0.61:1–2.8:1, average 1.4:1 (Fig. 3A, Table 2). The locomotive forms with the length to breadth ratio below 1 had an oval shape with the long axis perpendicular to the direction of the movement (Fig. 3A). The posterior end was rounded and smooth or produced short hyaline projections (Fig. 3A). The cytoplasm was separated into an anterior hyaline area occupying 20–33% of the cell body and a posterior granular area (Fig. 3A). The anterior edge of the hyaloplasm produced short subpseudopodia 2–5 ␮m long as well as longer ones (up to 19 ␮m). Amoebae with numerous short subpseudopodia occurred in this strain more frequently than cells with longer subpseudopodia. The average rate of movement in the Petri dish at +22 ◦ C was ca. 22 ␮m min−1 . During non-directed movement the amoebae produced short (1–2 ␮m long) conical subpseudopodia in dif-

ferent directions (not shown). The amoebae adopted floating forms when detached form the substrate. The floating form had a spherical central body (15–26 ␮m in diameter), producing 7–9 hyaline pseudopodia 7–35 ␮m long (Fig. 3B). The amoebae possessed a spherical vesicular nucleus with a single nucleolus located centrally (Fig. 3C–E). In the moving cells the nucleus was located in the central part of the cell body or slightly shifted towards the posterior part of the cell. In some cells the nucleus was located very close to one of the lateral margins of the cell body (Fig. 3C). No PLO was observed in the cytoplasm of these amoebae. The examination of 45 cells stained with DAPI always revealed a single permanent DNAcontaining structure – the nucleus (Fig. 3D–E). Measurement results of amoebae are provided in Table 2. The amoebae were covered with a layer of boat-shaped microscales which were morphologically identical to those of P. karteshi (strain WS14KH1) (Figs. 2E, 3 F–H). However, these microscales were slightly bigger than those of P. karteshi (strain WS14KH1) (Table 2).

Small-subunit ribosomal RNA gene of the two new amoeba species (Fig. 4, Supplementary tree S1, Supplementary Table S2) The length of the SSU rRNA amplicon in Paramoeba karteshi n. sp. (strain WS14KH1) was 2122–2127 bp; in P. aparasomata n. sp. (strain WS13.21T) it was 2112–2124 bp. The G + C nucleotide content in the two strains was 43.7–44% (average 43.8%) and 44.2–44.3% (average 44.3%), respectively. Variation between the different molecular clones of the same amplicon was in 0.9–2.8% and 0.5–2.8% of the positions, respectively (Supplementary Table S2).

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Fig. 3. A–H. Morphology and ultrastructure of Paramoeba aparasomata n. sp. in vivo (A–B, phase contrast, C, differential interference contrast), after DAPI-stainig (D, E) and in the SEM (F–H). (A) Locomotive forms, arrows show the direction of movement. (B) Floating form. (C) Nucleus. (D, E) Phase contrast image (D) and corresponding fluorescent image of the nucleus visualized by DAPI staining (E). (F) Image of the whole surface of the cell covered with microscales. (G) Side view of the microscales showing lateral columns (arrows). (H) Basket-shaped microscales on the plasma membrane surface. b – bacteria attached to the layer of microscales, Nu – nucleus. Scale bars = 20 ␮m in A, 10 ␮m in B–E, 2 ␮m in F, 100 nm in G, H. Table 2. Measurements of the Paramoeba aparasomata n. sp. cells. Locomotive form Length

Breadth

Length:breadth ratio

Length of short subpseudopodia

Rate of locomotion

14–46 (26) n = 45

11–30 (18) n = 45

0.61:1–2.8:1 (1.4:1) n = 45

2–19 (7) ncells = 15, nsubpseudopodia per cell = 5–10

12–40 (22) n = 3

Nucleus Nucleus diameter

Nucleolus diameter

Nucleus/nucleolus

6–9 (7) n = 14

2–4 (3) n = 14

1.8:1–2.8:1 (2.4:1) n = 14

Microscales Length

Breadth

Length:breadth ratio

Marginal columns

Central columns

Marginal: central columns

303–390 (329) n = 27 118–168 (145) n = 27 1.8:1–2.7:1 (2.3:1) n = 27 105–136 (128) n = 14 66–101 (85) n = 17 1.1:1–1.8:1 (1.5:1) Light microscopic measurements in ␮m; measurements of microscales in nm; locomotive rates in ␮m min−1 . Range (min–max) is followed by an average value in brackets where appropriate.

Pairwise comparison of all SSU rRNA gene sequences obtained showed that variation between Paramoeba karteshi (strain WS14KH1) and P. eilhardi (strain CCAP1560/2) was 5.0–5.8%, between P. karteshi (strain WS14KH1) and P. aparasomata (strain WS13.21T), 3.4–4.2%, and between P. eilhardi (strain CCAP 1560/2) and P. aparasomata (strain WS13.21T), 5.3–6.7% (Supplementary Table S2).

Preliminary phylogenetic analyses showed that both strains belonged to the Dactylopodida (Amoebozoa) and grouped together as a sister clade to the sequences of the strain CCAP 1560/2 of Paramoeba eilhardi. In the maximum likelihood and Bayesian trees all sequenced molecular clones of the PLO-containing P. karteshi (strain WS14KH1) and the PLO-free P. aparasomata (strain WS13.21T) formed

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Fig. 4. Maximum likelihood tree based on 104 SSU rRNA gene sequences of Dactylopodida showing position of Paramoeba karteshi n. sp. and Paramoeba aparasomata n. sp. (in bold). The tree shown was based on 1903 nucleotide positions. Numbers at nodes indicate Bayesian posterior probabilities/bootstrap values if above 0.5/50%. Thick branches = 1.0/100%. Dash indicates value of PP/BS below 0.5/50%, while asterisk, that the branch does not exist in the tree derived with Bayesian analysis. Scale bar: 0.02 substitutions/site.

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separate well supported branches (BS = 98% and 97%, respectively; PP = 1 for both branches) that were sister branches to each other with high support (BS = 91%; PP = 1). The whole clade including P. karteshi (strain WS14KH1) and P. aparasomata (strain WS13.21T) branched always separately and was a sister group with the clade of P. eilhardi (strain CCAP 1560/2) with high support (BS = 97%; PP = 1). The whole clade including P. aparasomata, P. karteshi and P. eilhardi branched as a sister lineage to the clade comprising all Neoparamoeba spp. (Fig. 4). By contrast to the maximum likelihood tree, in the Bayesian tree this clade branched as a sister group to the clade comprising sequences identified as N. pemaquidensis (Page, 1970) and N. aestuarina (Page, 1970) (Supplementary Tree S1).

Small-subunit ribosomal RNA gene of the PLO of Paramoeba karteshi n. sp. (strain WS14KH1) (Fig. 5, Supplementary Table S2, Supplementary Tree S2) For the PLO of Paramoeba karteshi (strain WS14KH1) three molecular clones of SSU rRNA gene 1918–1923 bp long were sequenced. The sequences of molecular clones varied in 0.9–2.2% of the nucleotide positions. Their C + G content was 54–54.3% (Supplementary Table S2). In the phylogenetic tree new sequences grouped together with full support (BS = 100%; PP = 1) and formed a single long sister branch to the PLO of the strain of P. eilhardi (strain CCAP 1560/2) (BS = 100%; PP = 1) (Fig. 5; Supplementary Tree S2). Comparison of the sequences of the PLO of Paramoeba karteshi with the sequences of the PLO of the P. eilhardi (strain CCAP 1560/2) showed that variation between these strains was within 5.1–5.9%.

Cytochrome C oxidase subunit 1 (COI) gene of the new species (Supplementary Figure S1, Supplementary Table S2, Supplementary Tree S3) Comparison of partial sequences of the mitochondrial genome of P. aparasomata (strain WS13.21T) (Bondarenko et al., 2019) with that of Neoparamoeba (deposited as Paramoeba) pemaquidensis (strain CCAP 1560/4) (Genbank accession number NC 031417) allowed a design of primers NeoCoxOF (TTTGCTATGTTTGSTGGTGT) and NeoCoxR (GTATTRAAATTTCTATCRGTTAAATA) specific for Paramoeba and Neoparamoeba. The new primer NeoCoxR, in combination with a universal primer LCO1490 (Folmer et al. 1994) was used to amplify a fragment of the COI gene of P. karteshi (strain WS14KH1) and the pair of primers NeoCoxOF and HCO2198 (Folmer et al. 1994) for P. eilhardi (strain CCAP 1560/2; deposited in GenBank under accession numbers MK168797–MK168799). The length of the amplified fragments was 567 bp for P. karteshi (strain WS14KH1) and 630 bp for P. eilhardi (strain CCAP 1560/2)

(Supplementary Table S2). Different molecular clones within one amplicon of P. karteshi (strain WS14KH1) varied within 0.7–0.9%, while four sequences of P. eilhardi (strain CCAP 1560/2) were identical (Supplementary Table S2). The G + C content was 31–31.5% (average 31.2%) for sequences of P. karteshi (strain WS14KH1) and 32.5% for sequences of P. eilhardi (strain CCAP 1560/2) (Supplementary Table S2). The partial sequence of the COI gene of P. aparasomata (strain WS13.21 T) obtained from the partial mitochondrial genome sequence of this strain was 552 bp long and its C + G content was 34%. Pairwise comparison of sequences showed that their variation between species was significantly higher than within-amplicon (Supplementary Table S3). The overlapping part of the gene in all three species corresponded to 184 amino acids (552 bp). The amino acid sequences varied within 0.6–2.2% of the positions between species. Amino acid sequences of P. aparasomata (strain WS13.21T) and P. eilhardi (strain CCAP 1560/2) were most similar to each other. The maximum likelihood phylogenetic analysis of the COI gene showed that the sequences of P. aparasomata (strain WS13.21T) and the sequences of P. eilhardi (strain CCAP 1560/2) grouped in a single fully supported clade. Meanwhile the sequences of P. karteshi were a sister group to this clade (Supplementary Figure S1; Supplementary Tree S3).

Analysis of concatenated alignment of the SSU rRNA and COI genes (Fig. 6) Phylogenetic analysis of the concatenated alignment comprising available SSU rRNA and COI gene sequences of Dactylopodida and Vannellida resulted in the tree with much higher resolution than yielded by any of these markers when analyzed separately. Despite the number of species with both markers available being still low, all known genera of the order Dactylopodida are sampled. Four out of five known genera of the order Vannellida can be used as outgroup. The topology of Paramoeba branches in the tree corresponded to that of the tree based on the SSU rDNA, i.e., P. aparasomata (strain WS13.21T) was sister to P. karteshi (strain WS14KH1) (PP = 1; BS = 100) and this branch was better supported than in the tree based on SSU rDNA. The branch comprising three species of the genus Paramoeba had full support. In contrast to the tree based on the SSU rRNA gene, two species of Neoparamoeba did not form a single clade in this tree (Fig. 6).

Co-evolution of PLOs and their hosts (Fig. 7, Supplementary Trees S4, S5) The phylogenetic analysis of the SSU rRNA gene sequences of amoebae and their symbionts originating from the same strains showed that the tree of amoebae is split into seven well supported branches (Fig. 7B; Supplementary Tree S4). In the trees of amoebae and PLOs (Fig. 7A,

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Fig. 5. Maximum likelihood phylogenetic tree of 49 SSU rRNA gene sequences of Perkinsela-like symbionts of Neoparamoeba and Paramoeba spp., rooted with Ichtyobodo spp. (3 sequences). New sequences of Perkinsela-like symbionts of Paramoeba karteshi n. sp. and Paramoeba eilhardi (strain CCAP 1560/2) are in bold. The tree shown was based on 1411 nucleotide positions. Numbers at nodes indicate posterior probabilities/bootstrap values if above 0.5/50%. Thick branches = 1.0/100%. Dash indicates value of PP/BS below 0.5/50%, while asterisk, that the branch does not exist in the tree derived with Bayesian analysis. Scale bar: 0.02 substitutions/site.

B; Supplementary Trees S4, S5) terminal branches showed similar topology. For instance, groups of sequences identified as N. pemaquidensis and the sequences of the PLO of N. pemaquidensis are split into three subclades. The sequences of N. pemaquidensis clade and N. aestuarina formed sister branches the same way in both trees as well as sequences of N. branchiphila with sequences of N. invadens, and P. eilhardi with P. karteshi n. sp. (Fig. 7; Supplementary Trees S4, S5). In contrast to these terminal branches, the topology of deep nodes differed in the two trees. In the tree of amoebae (Fig. 7B) a branch of sequences comprising P. eilhardi and P. karteshi was sister to a clade N. pemaquidensis + N. aestuarina while in the tree based on SSU rDNA of the PLO (Fig. 7A) the branch including sequences of the PLO of P. eilhardi and P. karteshi grouped at the base of the clade containing all sequences except the sequence of the PLO of P. atlantica (similar to the topology of the Bayesian tree of amoebae SSU rRNA gene; Supplementary Tree S1). Also, two branches comprising sequences of Neoparamoeba branchiphila Dyková et al., 2005 and Neop-

aramoeba invadens (Jones, 1985), and the group of sequences identified as Neoparamoeba perurans had incongruent positions in these two trees (Fig. 7).

Discussion The new strains are new species of Paramoeba: morphological and molecular evidence Both of the studied strains should be assigned to the genus Paramoeba because they reliably grouped together with P. eilhardi (strain CCAP 1560/2) in a molecular phylogenetic tree as well as adopt a dactylopodoial morphotype during locomotion and possess microscales of a characteristic shape on the plasma membrane surface. The phylogenetic analysis of SSU rRNA and mitochondrial COI genes showed that sequences of both new strains always formed separate branches and grouped in a single clade with sequences of P. eilhardi (strain CCAP 1560/2) (Figs. 4, 6; Supplementary

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Fig. 6. Maximum likelihood tree of Dactylopodida, rooted with Vannellida, showing the position of Paramoeba eilhardi (strain CCAP 1560/2), Paramoeba karteshi n. sp. and Paramoeba aparasomata n. sp. (in bold). The tree is based on the concatenated alignment of SSU rRNA (1667 nucleotide positions) and COI (554 nucleotide positions) genes. Numbers at nodes indicate Bayesian posterior probabilities/bootstrap support values if above 0.5/50. Thick branches = 1.0/100. Scale bar = 0.1 substitutions/site.

Fig. 7. Comparison of two maximum likelihood phylogenetic trees of SSU rRNA gene sequences of the Perkinsela-like symbionts and their hosts. (A) Tree based on 46 SSU rRNA gene sequences (1538 nucleotide positions) of Perkinsela-like organism derived from the same strain as in B. (B) Tree based on 46 SSU rRNA gene sequences (1965 nucleotide positions) of Neoparamoeba and Paramoeba strains. Numbers at nodes indicate posterior probabilities/bootstrap values if above 0.5/50%. Thick branches = 1.0/100%. Dash indicates value of PP/BS below 0.5/50%, while asterisk, that the branch does not exist in the tree derived with Bayesian analysis. Scale bars: 0.02 substitutions/site.

Figure S1). The position of clades corresponding to the new strains depends on the molecular marker. They demonstrate different relationships with P. eilhardi (strain CCAP 1560/2) in the trees based on SSU rRNA and COI genes (Fig. 4, Supplementary Figure S1). These differences in topology may be caused by a still low number of available sequences of the COI gene in Neoparamoeba. Anyway, the best resolved tree with topology similar to the topology of the tree based on SSU rRNA gene sequences was reconstructed based on concatenated alignment of SSU rRNA and COI genes (Fig. 6). Thus, we suggest this topology is more reliable than the topology of the tree based on the COI gene alone (Supplementary Fig. S1). The presence of the PLO is the most important morphological feature of Neoparamoeba and Paramoeba which allows separating these genera from other paramoebids (Dyková

et al. 2005; Jones 1985; Kudryavtsev et al., 2011; Page 1987). Two species of amoebae morphologically similar to the amoebae studied here are known today: Korotnevella nivo and P. eilhardi (Schaudinn 1896; Smirnov 1997). However, the molecular data for Korotnevella nivo are not yet available. Thus, in this case only the morphological comparison can be provided for our new strains. The comparison of P. aparasomata (strain WS13.21T) with P. eilhardi (strain CCAP 1560/2) revealed the following morphological differences: (1) during locomotion, cells of P. eilhardi (strain CCAP 1560/2) are longer than cells of P. aparasomata (strain WS13.21T); (2) locomotive forms of P. aparasomata have a more rounded outline than locomotive forms of P. eilhardi (strain CCAP 1560/2), confirmed by length to breadth ratio which reaches the value of 2:1 for amoebae of P. eilhardi (strain CCAP 1560/2) and 1.5:1 for P.

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Table 3. Morphological comparison of Paramoeba karteshi n. sp. and Paramoeba aparasomata n. sp. with Korotnevella nivo Smirnov, 1997 and Paramoeba eilhardi strain CCAP 1560/2 according to Grell and Benwitz (1966); Page (1973) and Kudryavtsev et al. (2011). K. nivo

P. eilhardi CCAP 1560/2 Grell

P. eilhardi CCAP 1560/2 Page

P. eilhardi CCAP 1560/2 Kudryavtsev

P. aparasomata

P. karteshi

Length Breadth L:B ratio Rate of locomotion Nucleus diameter Nucleolus diameter Number of PLOs PLO length PLO breadth Microscale length Microscale breadth

19–51 (34) 14–40 (21) 0.95:1–2.8:1 (1.9:1) – 2.2–3.8 1.2–2.2 Absent – – 400–514 170–230

45–100 – – >10 – 1–2 11 – 350–600 –

28–63 (45) 11–40 (26) 0.93:1–3.1:1 (1.8:1) 14–35 (24) 5–10 (8) 3–6 (4) Mainly 2 6–8 (7) 3–5 (4) 357–490 (415) 179–238 (212)

14–46 (26) 11–30 (18) 0.61:1–2.8:1 (1.4:1) 12–40 (22) 6–9 (7) 2–4 (3) Absent – – 303–390 (329) 118–168 (145)

29–75 (47) 13–41 (22.5) 1:1–3.7:1 (2.2:1) 18–36 (27) 5–9 (7) 3–5 (4) 1 4–6 (5) 3–4 (3) 295–389 (352) 124–203 (160)

Microscale height

Ca. 200

– – – – – – Unknown – – 330–370 150–160; 180–190 near the upper edge 150

–

107–200 (154)

105–136 (128)

112–129 (120)

Light microscopic measurements in ␮m; measurements of microscales in nm; locomotive rates in ␮m min−1 . Range (min–max) is followed by an average value in brackets where appropriate.

aparasomata (strain WS13.21T); (3) another distinction is the number of PLOs per cell: 75% of P. eilhardi (strain CCAP 1560/2) amoebae contained two PLOs (Kudryavtsev et al. 2011), while in amoebae of P. aparasomata (strain WS13.21T) PLO was not observed. Microscales of P. eilhardi (strain CCAP 1560/2) are slightly bigger than microscales of P. aparasomata (strain WS13.21T) (Table 3). Paramoeba karteshi (strain WS14KH1) and P. eilhardi (strain CCAP 1560/2) are very similar in morphology at the first sight. We found differences in only two morphological characters between these two strains. Amoebae of P. karteshi (strain WS14KH1) always contain one PLO, while amoebae of the P. eilhardi (strain CCAP 1560/2) mainly contain two PLOs per cell. Amoebae of P. eilhardi (strain CCAP 1560/2) and Paramoeba karteshi (strain WS14KH1) also differ in their length to breadth ratio: amoebae of the latter species had more elongated locomotive forms than those of the former one (Table 3). In addition, differences in the nucleotide sequences of the molecular markers of the studied species were always higher than within-amplicon differences of these molecular markers; this is additional evidence that both strains are separate species and different from P. eilhardi (Supplementary Table S3). Among other paramoebid species, P. aparasomata (strain WS13.21T) is comparable with Korotnevella nivo due to similar light microscopic morphology and the structure of surface microscales. A detailed comparison shows that locomotive forms of K. nivo were more elongated (L:B ratio approaches the value of 2:1) while locomotive forms of P. aparasomata (strain WS13.21T) are more rounded (L:B ratio approximately 1.5:1). The nucleus of K. nivo is two-four times smaller than the nucleus of P. aparasomata (strain WS13.21T) (Smirnov 1997). Moreover, the microscales of K.

nivo are the largest ones compared to microscales of known Paramoeba spp. (Table 3; Grell and Benwitz 1966; Smirnov 1997).

Korotnevella nivo Smirnov, 1997 may be an aparasomate Paramoeba Although no molecular data were collected for K. nivo Smirnov, 1997, we suggest, based on the findings presented here, that this species is actually a member of the genus Paramoeba and not a Korotnevella species. Although the microscales of K. nivo were almost identical to those of P. eilhardi, the permanent absence of the PLO was the most significant reason why this species was included in Korotnevella and not in Paramoeba (Smirnov 1997). The finding of PLO-free P. aparasomata which is clearly related to the PLOcontaining P. eilhardi and P. karteshi make us pay attention to the still unclear relationships between amoebae and the PLOs. It shows that aposymbiotic species of Paramoeba do exist in nature, and the absence of a PLO does not automatically make a scaly paramoebid amoeba belong to Korotnevella. Moreover, among species confirmed as members of Korotnevella based on the molecular data, none was shown to possess microscales like those described in Paramoeba spp. or K. nivo (O’Kelly et al. 2001; Udalov 2015, 2016; Udalov et al. 2016, 2017; Zlatogursky et al. 2016). In addition, a strain of unidentified amoeba without a PLO studied by Anderson (1977) had scales and cytoplasmic ultrastructure very similar to those observed in P. eilhardi (Grell and Benwitz 1966, 1970). As no culture of K. nivo is preserved, we suggest putting an effort on re-isolation of this species from its original habitat and testing whether it is related to Paramoeba using molecular evidence.

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Co-evolution of PLO and their host

Genus Paramoeba Schaudinn, 1896, emend

The co-evolution of PLOs with Neoparmoeba and Paramoeba hosts was demonstrated in the previous studies, and the congruence between the phylogenetic trees based on SSU rRNA gene was shown (Dyková et al. 2008; Sibbald et al. 2017; Volkova and Kudryavtsev 2017; Young et al. 2014). Yet, in one of the recent studies, Sibbald et al. (2017) suggested the possibility that this congruence may alter after the addition of new sequences of N. aestuarina and P. eilhardi. Recently we showed that the addition of new sequences of “scaled” species in phylogenetic analysis of amoebae and their symbionts changed the previous congruent topology of two trees (Volkova and Kudryavtsev 2017). The results of this study additionally confirm the previous findings. Inclusion of PLO and amoebae SSU rRNA gene sequences of scaled species in the dataset led to swapping of the major clades in both trees and resulted in the loss of their congruence (Fig. 7; Supplementary Trees S4, S5). Currently we cannot exclude several alternative explanations for the topologies observed. The main issue of the present trees is that the deep nodes of the tree of amoebae are poorly supported which means the tree topology may further change with the addition of more data. On the other hand, while currently sequenced symbiont-containing lineages likely had a monophyletic origin, the position of Janickina in the phylogenetic tree is not yet known. As this genus is very different in morphology from other PLO-containing taxa (Chatton 1953; Janicki 1912), we cannot exclude that some of the PLO-containing lineages of amoebae might have an independent origin that would also alter the evolutionary scenarios. Moreover, if we assume that the demonstrated topologies do correspond to the evolutionary relationships between species of amoebae, this could mean that some analogue to incomplete lineage sorting occurred in the evolution of PLOs. This means the modern lineages of PLO present in the trees might not all have a monophyletic origin and strictly follow a scenario congruent to their hosts. The possibility of the secondary loss of a symbiont shows that at some points in the evolutionary history several lineages of PLOs might have been co-existing in amoebae and evolving in parallel. Some of these lineages might be differentially lost later in different lineages of amoebae. This may explain the observed incongruence of the evolutionary scenarios in the deeper nodes of this clade.

Diagnosis: Amoebae of dactylopodial morphotype containing one or two PLOs adjacent to nucleus or do not contain a PLO at all, floating form spherical with fine, radiating pseudopodia, surface covered with boat-shaped microscales.

Taxonomic summary Diagnoses of new and emended taxa Position in the system according to Adl et al. (2019): Amorphea: Amoebozoa: Discosea: Flabellinia: Dactylopodida

Type species (by original designation): P. eilhardi Schaudinn, 1896. Remarks: According to this diagnosis, Korotnevella nivo Smirnov, 1997 apparently might be included in the genus Paramoeba.

Paramoeba karteshi n. sp Diagnosis: Locomotive forms mainly oval and elongated, long subpseudopodia can reach length of cell, smooth posterior end. Length of locomotive form 29–75 ␮m (average 47 ␮m), breadth, 13–41 ␮m (average 22.5 ␮m), length:breadth ratio 1–3.7 (average 2.2) (n = 44). Single vesicular nucleus 5–9 ␮m (average 7 ␮m) in diameter (n = 15). Always a single PLO adjacent to the nucleus, ovoid, 4–6 ␮m long (average 5 ␮m), 3–4 ␮m broad (average 3 ␮m) (n = 15). Cells covered with boat-like microscales; length of a microscale 295–389 nm (average 352 nm), breadth, 124–203 nm (average 160 nm), height 112–129 nm (average 120 nm) (n = 66). Type locality: Marine, pieces of a sponge Halisarca dujardini from sublittoral of the Levaya Bay, Chupa Inlet, Kandalaksha Bay, The White Sea (66.3369944N, 33.6598806E; depth 5 m; salinity 24–27 ppt). Type material: Type culture (accession No RC CCMAm0453) and purified DNA sample (accession No A524) are deposited with the Core Facility Centre “Culture Collection of Microorganisms” of the Saint-Petersburg State University Research Park. Molecular sequence data: GenBank accession numbers MK168787-MK168789 (SSU rRNA gene of amoeba), MK168794-MK168796 (SSU rRNA gene of PLO), MK168800-MK168802 (COI gene). ZooBank LSID: urn:lsid:zoobank.org:act:14981EA7E5AF-4D63-97C8-54834DCA1E36. Etymology: karteshi refers to the Cape Kartesh and the name of the White Sea biological station of the Zoological Institute RAS where the strain was initially collected.

Paramoeba aparasomata n. sp Diagnosis: Locomotive forms rounded or oval in outline, amoebae produce long and short subpseudopodia during locomotion. Length of locomotive form 14–46 ␮m (average 26 ␮m) breadth 11–30 ␮m (average 18 ␮m), length:breadth

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ratio 0.61–2.8 (average 1.4) (n = 45). Posterior end smooth or with small hyaline projections. Single vesicular nucleus 6–9 ␮m (average 7 ␮m) in diameter (n = 14); no PLO. Cells covered with boat-shaped microscales; length of a microscale 303–390 nm (average 329 nm), breadth 118–168 nm (average 145 nm), height of a microscale 107–200 nm (average 120 nm) (n = 27). Type locality: Marine, sublittoral soft bottom sediments off the beach of Luda Cheremshikha Island Chupa Inlet, Kandalaksha Bay, The White Sea (66.321344 N, 33,85184E; depth 6 m; salinity 24–27 ppt).

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of the Zoological Institute RAS. Part of the study was supported by the grants 18-34-00726-mol a from the Russian Foundation for Basic Research (molecular and partial morphological investigation of P. aparasomata and P. karteshi), 15-29-02749-ofi-m from the Russian Foundation for Basic Research (sampling and partial morphological investigation of P. aparasomata and P. karteshi) and 17-74-10103 from the Russian Science Foundation (the part of mitochondrial genome of P. aparasomata containing the COI gene).

Appendix A. Supplementary data

Type material: Type culture (accession No RC CCMAm0454) and purified DNA sample (accession No A516) are deposited with the Core Facility Centre “Culture Collection of Microorganisms” of the Saint-Petersburg State University Research Park.

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10. 1016/j.ejop.2019.125630.

Molecular sequence data: GenBank accession numbers MK168790-MK168793 (SSU rRNA gene), MK168803 (COI gene).

References

ZooBank LSID: urn:lsid:zoobank.org:act:D76E074F86FB-4488-A42E-1EA3093A0440. Etymology: aparasomata refers to the absence of a parasome (an older term for PLO).

Author contributions Ekaterina Volkova designed the study, performed isolation of P. karteshi n. sp., light and electron microscopic investigation, primer design, sequencing and phylogenetic analysis, wrote the manuscript; Eckhard Völcker performed SEM sample preparation and scanning electron microscopic investigation; Steffen Clauß performed SEM sample preparation and scanning electron microscopic investigation; Natalya Bondarenko obtained and provided partial sequence of the mitochondrial genome of P. aparasomata n. sp. containing COI gene; Alexander Kudryavtsev designed the study, performed marine sampling, isolation of P. aparasomata, wrote the manuscript. All authors participated in editing of the manuscript, discussed and agreed on the final version.

Acknowledgements We are grateful to a diver and photographer Mr. Mikhail Fedyuk (St. Petersburg, Russia) for collecting the sample WS13.21T containing P. aparasomata, and Mr. Ilya Borisenko (Faculty of Biology, St. Petersburg State University) for the Halisarca dujardini sample. The study utilized equipment of the Resource Centers for Culture Collection of Microorganisms and Development of Molecular and Cell Technologies of the Saint-Petersburg State University Research Park and White Sea Biological Station “Kartesh”

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