Integrative studies on the taxonomy and molecular phylogeny of four new Pleuronema species (Protozoa, Ciliophora, Scuticociliatia)

The ciliate genus Pleuronema comprises approximately 30 nominal species and has been reported in freshwater, brackish water, and marine habitats. Nevertheless, recent studies have indicated that there might be a large undiscovered species diversity. In the present work, four new Pleuronema species, namely P. foissneri sp. nov., P. parasmalli sp. nov., P. parasalmastra sp. nov., and P. paraorientale sp. nov., collected from Shenzhen, southern China, was investigated using taxonomic methods. The diagnosis, description, comparisons with morphologically related species and detailed morphometric data are supplied for each. The small subunit ribosomal RNA (SSU rRNA) gene of the four new species is sequenced and their molecular phylogeny is analyzed. The SSU rRNA gene tree shows that Pleuronema is polyphyletic comprising several separate clades. All four new species cluster consistently with P. orientale KF206429, P. puytoraci KF840520 and P. setigerum FJ848874 within the core Pleuronematidae + Peniculistomatidae clade. Phylogenies of Pleuronematidae-related taxa are also discussed. Supplementary Information The online version contains supplementary material available at 10.1007/s42995-022-00130-5.


Introduction
The ciliate genus Pleuronema was established by Dujardin (1841), based on its ovoidal body with a depression, long oral opening, and prominent oral cilia, in a redescription of "Paramecium chrysalis" by Ehrenberg (1838), which is very distinct from other reports on Paramecium. Dujardin (1841) also described another two Pleuronema species: P. crassa and P. marina. Today, Pleuronema is a speciose and cosmopolitan genus comprising approximately 30 nominal species which can be found in various aquatic environments (Fan and Pan 2020;Hu et al. 2019;Lynn 2008;Pan et al. 2015aPan et al. , 2016Song et al. 2009). In recent years, several new species have been discovered, suggesting there is a large undescribed diversity and a need to conduct further studies on this genus (Pan et al. 2015a(Pan et al. , b, 2016Wang et al. 2008aWang et al. , b, 2009. Molecular phylogenetic analyses have increasingly been applied in modern taxonomic studies on ciliated protists, and the polyphyly of Pleuronema has been widely reported (Antipa et al. 2016(Antipa et al. , 2020Gao et al. 2012Gao et al. , 2013Gao et al. , 2017Pan et al. 2010Pan et al. , 2015aPan et al. , b, 2016Pan et al. , 2020Yi et al. 2009).
Although Pleuronema is a common genus with a long research history, there are still problems with its taxonomy. These include: (1) Incomplete morphological information: Edited by Jiamei Li.
Mingjian Liu, Yujie Liu have contributed equally to this work.
* Xiaozhong Hu xiaozhonghu@ouc.edu.cn * Weibo Song wsong@ouc.edu.cn some species, especially those described in the eighteenth and nineteenth centuries, lack detailed descriptions or graphic illustrations. For example, eight species lack illustrations or photomicrographs of cells in vivo and the whole ciliature pattern remains unknown for P. grassei, P. prunulum, and P. simplex. Consequently, these poorly known taxa have not been reported since they were first described (Agatha et al. 1993;Corliss and Snyder 1986;Dragesco 1960Dragesco , 1968Fernandez-Leborans and Novillo 1994;Kahl 1926).
(2) Confusion in taxonomic status: some characters from live observation (e.g., right ventrolateral side straight or convex) and silver-stained specimens (e.g., position and length of the buccal field relative to the body length, anterior position of membranelle 2b) were overlooked until the middle of the twentieth century causing unreliable identifications and synonyms/homonyms (Agamaliev 1968;Borror 1963Borror , 1972Dragesco 1960Dragesco , 1968Noland 1937).
(3) Insufficient molecular data: SSU rRNA gene sequence data are available for only half of nominal Pleuronema species and a large proportion of these sequences lack corresponding morphological information, thus their identity needs to be confirmed (Gao et al. 2013). In addition, the large disparity in gene sequences among species within the genus suggests there is a large undiscovered species diversity of Pleuronema.
In the present study, four Pleuronema species collected from brackish and freshwater habitats in Shenzhen, southern China ( Fig. 1), were investigated using modern taxonomic methods. The morphological and molecular data indicate that each of these is a new species.
Dedication We dedicate this new species to Dr. Wilhelm Foissner (Salzburg University) in recognition of his significant contributions to the taxonomy of ciliates.
Deposition of type slides The protargol slide containing the holotype specimen (Fig. 2B, C) and several paratype specimens (registration number: LMJ2016040401-1), and a second protargol slide containing paratype specimens (registration number: LMJ2016040401-2), were deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China.
Small subunit ribosomal RNA (SSU rRNA) gene sequence The sequence of Pleuronema foissneri sp. nov. was deposited in GenBank with accession number OL654416. The length and G + C content of the sequence are 1637 bp and 43.01%, respectively.  Description Cell size in vivo approximately 60-75 μm × 30-40 μm. Elliptical or oval in outline ( Fig. 2A, D, F). Anterior end slightly narrowed, posterior end rounded, right ventrolateral and dorsal sides convex ( Fig. 2A, D, F). Buccal field occupying 70-85% of cell length ( Fig. 2A, D; Table 1). Oral cilia approximately 30 μm long. Pellicle slightly notched with shallow longitudinal grooves (Fig. 2H). Extrusomes bar-shaped, located beneath pellicle, approximately 5 μm in length (Fig. 2E). Cytoplasm colorless to greyish, containing several food vacuoles, refractile globules and crystals that usually posteriorly distributed ( Fig. 2A, D, F). Single contractile vacuole dorsally located about 85% down length of cell, approximately 8-10 μm in diameter when fully expanded, pulsating at intervals of approximately 20-40 s ( Fig. 2A, D, F). Somatic cilia densely packed and approximately 8-10 μm long, perpendicular to surface when cell is at rest ( Fig. 2A, D Thirty-two to 40 somatic kineties (SK) extending almost entire length of body. Each SK consisting of dikinetids in anterior three-quarters of SK and monokinetids in posterior quarter ( Fig. 2B, C, I-L; Table 1). Four to eight preoral kineties located to left of buccal field, commencing at anterior end of cell and terminating posteriorly approximately twothirds down length of body (Fig. 2B, I; Table 1). Postoral kinety usually absent, one present in five out 15 individuals examined. Single macronucleus located one-third down length of body, approximately 25-35 μm × 15-30 μm after protargol staining, generally ellipsoidal to spherical in shape; in 14 out of 24 individuals examined, macronucleus notched in mid-portion giving it a heart-or pear-shaped appearance ( Fig. 2C, I-K, N; Table 1). Micronucleus not detected.
Anterior quarter of membranelle 1 (M1) three-rowed while rest of M1 two-rowed (  Table 1). Posterior portion of M2a hook-like (Fig. 2B, E, I, K). Membranelle 2b (M2b) basically V-shaped, with basal bodies arranged in several single-rowed groups in zig-zag pattern (Fig. 2B, E, I). Each group with two to six basal bodies. Length of M2b approximately 13-22% of body length, commencing at same level as posterior end of M2a (Fig. 2B, E, I, K; Table 1). Membranelle 3 composed of three densely arranged rows, rightmost row shortened, length approximately 20% of two left rows (Fig. 2B, E, I, L, M). Paroral membrane double-rowed in zig-zag pattern, occupying about 70-80% of cell length (Fig. 2B, E, I, K). Pleuronema parasmalli sp. nov. (Fig. 3 Etymology Composite of the Greek word para (beside) and the species-group name smalli, indicating the similarity between the new species and Pleuronema smalli in terms of their small body size and ciliature pattern.
SSU rRNA gene sequence The sequence of Pleuronema parasmalli sp. nov. was deposited in GenBank with accession number OL654417. The length and G + C content of the sequence are 1632 bp and 43.26%, respectively.
Twenty-six to 32 somatic kineties extending almost entire body length, each with densely spaced dikinetids in anterior three-fifths, and monokinetids in posterior two-fifths ( Fig. 3B, C, I, J; Table 1). Four to six preoral kineties located to left of buccal field, commencing near anterior end of cell and terminating posteriorly 65% down length of cell (Fig. 3B, I; Table 1). Thirteen out of 25 cells examined with one postoral kinety (PoK), others without PoK. Usually (in 19 out of 25 cells examined) with a single spherical macronucleus positioned at 33 to 40% down length of body, diameter approximately 15-22 μm after protargol staining (Fig. 3C, I-K); six out of 25 cells examined with two to 14 spherical macronuclei, diameter of each varying from 5-15 μm. Single spherical micronucleus, 5-6 μm in diameter, adjacent to macronucleus (Fig. 3C, H).
First quarter of membranelle 1 (M1) three-rowed while rest two-rowed (Fig. 3B, D, I, K). Length of M1 10-15% of cell length (Fig. 3B, I; Table 1). Anterior end of M1 located approximately 10% down length of cell (Fig. 3B, I). Anterior one-eighth and posterior one-third of membranelle 2a (M2a) two-rowed, mid-portion single-rowed  Table 1).  Etymology Composite of the Greek word para (beside) and the species-group name salmastra, indicating that the new species resembles the large individuals of Pleuronema salmastra in having an entirely posterior-positioned buccal field.
SSU rRNA gene sequence The sequence of Pleuronema parasalmastra sp. nov. was deposited in GenBank with accession number OL654418. The length and G + C content of the sequence are 1630 bp and 44.36%, respectively.
Thirty-seven to 43 somatic kineties extending entire body length, each with closely arranged dikinetids in anterior 33-75% portion, and monokinetids in remaining portion ( Fig. 4B, C, K, L; Table 2). Four to six preoral kineties located to left of buccal field, commencing near anterior end of cell and terminating posteriorly 75% down length of cell (Fig. 4B, K; Table 2). One postoral kinety in seven out of ten cells examined, postoral kinety lacking in remaining three cells (Fig. 4B). Usually (in 12 out of 16 cells examined) with a single ellipsoidal macronucleus, centrally positioned, approximately 23-36 μm in length after protargol staining (Fig. 4C); four out of 16 cells examined with three to six spherical macronuclei, each approximately 15-20 μm across (Fig. 4K, L). Micronucleus not detected.
Deposition of type slides The protargol slide containing the holotype specimen (Fig. 5B, C) and several paratype specimens (registration number: LMJ2017010601-1), and one protargol slide containing paratype specimens 1 3 (registration number: LMJ2017010601-2), were deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China.
SSU rRNA gene sequence The sequence of Pleuronema paraorientale sp. nov. was deposited in GenBank with accession number OL654419. The length and G + C content of the sequence are 1639 bp and 43.50%, respectively.
Membranelle 1 (M1) three-rowed in anterior 15 to 20% portion, two-rowed in remaining portion (Fig. 5B, D, I, K). Length of M1 8-13% of cell length (Fig. 5B, I; Table 2). M1 commencing about 12% down body length (Fig. 5B, I). Anterior 15% and posterior 33% of membranelle 2a (M2a) two-rowed, basal bodies in remaining portion arranged in a zig-zag pattern (Fig. 5B, D, I, K). M2a occupying approximately 40-60% of body length, commencing near level of anterior end of M1 (Fig. 5B, I; Table 2). Posterior portion of M2a hook-like, located approximately 60% down length of cell (Fig. 5B, D, I, K). Membranelle 2b (M2b) V-shaped, (marked with red triangle) deviates from the other three P. coronatum sequences, unfortunately, available information is not sufficient to determine its identity. Main morphological features of Pleuronema species and the number of unmatched nucleotides within each Pleuronema clade are both provided. "Exceeding" in anterior end of M2b means that M2b commences above level of posterior end of M2a, while "not exceeding" means it commences below level of posterior end of M2a. BW brackish water, FW freshwater, M2a membranelle 2a, M2b membranelle 2b, MW marine water basal bodies in right anterior 20% arranged in a continuous single row, those in remaining portion arranged in several single-rowed groups, each group composed of two to six basal bodies and linked with other groups (Fig. 5B, D, I, K). M2b occupying approximately 15 to 28% of body length, commencing significantly above level of posterior end of M2a (Fig. 5B, D, I, K). Membranelle 3 three-rowed and closely packed, posterior end of rightmost row slightly separated from other rows by diverging rightwards (Fig. 5B, D, I, K). Paroral membrane (PM) double-rowed, basal bodies arranged in zig-zag pattern, occupying approximately 60-80% of cell length (Fig. 5B, D, I, K).

Molecular data and phylogenetic analyses (Figs. 6, 7; Supplementary Table S1)
The topologies of the maximum likelihood (ML) and Bayesian inference (BI) trees were similar, therefore, only the ML tree, with support values from both algorithms, is shown (Fig. 6). The order Pleuronematida is monophyletic with high support (97% ML/1.00 BI). Within the Pleuronematida, the families Ctedoctematidae and Eurystomatidae form a fully supported clade that branches first, followed by the family Histiobalantiidae. The family Peniculistomatidae is nested within the family Pleuronematidae forming a single clade (59% ML/0.88 BI). Within this clade, members of the genera Schizocalyptra, Mytilophilus, and Peniculistoma are scattered among members of Pleuronema, making the genus Pleuronema polyphyletic.

Comments on Pleuronema coronatum Kent, 1881
Pleuronema coronatum is one of the most common and well-studied species of Pleuronema. Since its establishment based on a freshwater population (Kent 1881), several populations isolated from freshwater, brackish water or marine water, mainly collected from Europe, North America, Africa, and East Asia, have been reported under the name "P. coronatum" (Agamaliev 1968;Borror 1963Borror , 1972Chorik 1968;Dragesco 1960Dragesco , 1968Dragesco and Dragesco-Kernéis 1986;Foissner et al. 1994;Kahl 1926Kahl , 1931Noland 1937;Small and Lynn 1985;Song 2000;Wang et al. 2008a). This species, which was originally called "Pleuronema coronata", is shorter and thicker than the morphologically related congener "Pleuronema chrysalis", and has extrusomes and caudal cilia, both of which are absent in the latter (Ehrenberg 1838; Kent 1881). In addition, the former has a straight right ventrolateral side and a spherical macronucleus according to the original drawing of a cell in vivo (Kent 1881), which we consider to be diagnostic features of P. coronatum.
The body length in vivo of two populations of P. coronatum described by Wang et al. (2008a) range from 55 to 170 μm, the extremes of which deviate significantly from the original description (approximately 90 μm in length), suggesting that these populations may have included multiple species. It is noteworthy that Wang et al. (2008a) regarded Pleuronema balli, P. borrori, and P. smalli as synonyms of P. coronatum since they share some similar characteristics. After reinvestigating those descriptions, we agree that P. balli is a synonym of P. coronatum, since the former matches well with the original and other reliable populations of the latter in terms of body size, ciliature pattern, and macronucleus shape (Borror 1963;Chorik 1968;Dragesco 1968;Foissner et al. 1994;Kent 1881;Small and Lynn 1985;Song 2000). As a result, both P. balli populations (Dragesco 1968;Small and Lynn 1985) should be considered as P. coronatum populations. However, we disagree that Pleuronema borrori and P. smalli are synonyms of P. coronatum (Wang et al. 2008a). Pleuronema borrori has a much wider body (70-77 μm after silver staining vs. usually 25-65 μm in P. coronatum) and a smaller ratio of buccal field/body length (approximately 0.55 in P. borrori vs. 0.64-0.75, data measured from the drawings of silver-stained cells) than P. coronatum (Borror 1963;Dragesco 1968;Foissner et al. 1994;Small and Lynn 1985;Song 2000). Pleuronema smalli has fewer somatic kineties than P. coronatum (28-36 vs. 35-48) and an ellipsoid macronucleus (vs. spherical in P. coronatum) (Borror 1963(Borror , 1972Chorik 1968;Dragesco 1968;Foissner et al. 1994;Kent 1881;Small and Lynn 1985;Song 2000). Hence, we recognize P. borrori and P. smalli as valid species.
Pleuronema foissneri sp. nov. resembles P. salmastra in the slightly narrowed anterior end and in the body size after silver staining. The former, however, differs from the latter by having fewer somatic kineties (32-40 vs. 43-63 in P. salmastra), fewer preoral kineties (four to eight, five on average vs. six to ten, eight on average in P. salmastra), and a longer M1 relative to the body length (0.14-0.21 vs. 0.09-0.10 in P. salmastra) (Dragesco and Dragesco-Kernéis 1986).
Pleuronema parasmalli sp. nov. closely resembles P. smalli in its small body size after silver staining and similar ciliature pattern. The former, however, can be distinguished from the latter by having a single micronucleus (vs. two in P. smalli) and its freshwater habitat (vs. brackish and marine water in P. smalli). Morphological information of P. smalli in vivo is not available, therefore, a comparison with P. parasmalli sp. nov. in vivo cannot be performed (Borror 1972;Dragesco 1968).
Pleuronema parasmalli sp. nov. has a similar body size, shape, and oral structure to P. paucisaetosum, but the former differs from the latter in having more somatic kineties (26-32 vs. 21-23), a single micronucleus (vs. one to five, two on average), and a freshwater (vs. brackish water) habitat (Pan et al. 2015a).
When compared with Pleuronema coronatum, P. parasalmastra sp. nov. has a larger body size in silver-stained specimens (95-160 μm in length vs. 50-115 μm in P. coronatum) and a more posteriorly positioned buccal field (distance between cell apex to anterior end of M1 in P. parasalmastra sp. nov. is 0.16-0.25 of body length vs. 0.09-0.21 in P. coronatum). In addition, the mid-portion of M2a is clearly single-rowed in P. parasalmastra sp.

Phylogenetic relationships between Pleuronema and Schizocalyptra
Studies on the molecular phylogeny of Pleuronema began with the sequencing of the large subunit rRNA gene of P. marinum (Baroin-Tourancheau et al. 1992). Then the SSU rRNA gene and internal transcribed spacer 2 region data of P. coronatum were added and phylogenetic analyses showed that Pleuronema was closely related to Schizocalyptra and Cyclidium (Lynn and Strüder-Kypke 2005;Miao et al. 2008).
According to the phylogenetic analysis in Yi et al. (2009), the genus Pleuronema is polyphyletic since two Schizocalyptra sequences nest within it forming a fully supported clade. The polyphyly of Pleuronema caused by Schizocalyptra was subsequently confirmed in most phylogenetic studies based on the SSU rRNA gene (Antipa et al. 2016(Antipa et al. , 2020Gao et al. 2012Gao et al. , 2013Gao et al. , 2017Pan et al. 2010Pan et al. , 2015aPan et al. , b, 2016Pan et al. , 2020. However, in phylogenetic analyses based on nuclear or mitochondrial data, the genus Schizocalyptra falls outside of Pleuronema (Gao et al. 2014;Lu et al. 2021;Zhang et al. 2019). Consistent with previous studies based on SSU rRNA gene sequence data, our ML tree shows that Schizocalyptra sequences nest within Pleuronema (Fig. 6). However, the support values are very low and the phylogenetic position of Schizocalyptra is unstable, which may be due to the inclusion of insufficient taxa in the analyses. Considering the low support values and inconsistency of the phylogenetic position of Schizocalyptra based on previous and present studies, the relationship between Pleuronema and Schizocalyptra is still uncertain, and more morphological and molecular data are needed to further clarify their positions.

Phylogenetic relationships between the families Peniculistomatidae and Pleuronematidae
Based on SSU rRNA gene data, Antipa et al. (2016) was the first to reveal that the monophyletic family Peniculistomatidae falls within the Pleuronema spp., resulting in the polyphyly of Pleuronematidae. In subsequent studies, the polyphyly of Pleuronematidae caused by Peniculistomatidae was verified (Antipa et al. 2020;Lu et al. 2021;Zhang et al. 2019). In the present study, the family Peniculistomatidae nests within the Pleuronematidae in both the ML and BI trees, albeit with low support, supporting previous finding.
Peniculistoma and Mytilophilus are two endocommensal genera of Peniculistomatidae. Both are characterized by an irregular oval-shaped outline when viewed from the lateral aspect, the cytostome lying at the bottom of a deeply concaved depression, and having numerous somatic kineties, which clearly differentiates these genera from Pleuronema Antipa et al. 2016;Dolan and Antipa 1985;Fenchel 1965). The oral structures (a long and prominent paroral membrane and three membranelles, with M2 bipartite) and stomatogenesis of Peniculistoma and Mytilophilus, however, are generally similar with those in Pleuronema Dolan and Antipa 1985;Fenchel 1965). Furthermore, their phylogenetic positions in the SSU rRNA gene tree reflect the morphological similarities between the families Peniculistomatidae and Pleuronematidae (Fig. 6).

Phylogeny of the family Histiobalantiidae
In the phylogenetic analyses by Foissner et al. (2009) (when Histiobalantiidae sequences were first used in molecular phylogeny) and by Antipa et al. (2016Antipa et al. ( , 2020, Histiobalantiidae clustered with Schizocalyptra, which was nested within the Pleuronematidae, whereas in other phylogenetic analyses, including the present study (Fig. 6), Histiobalantiidae is invariably placed outside the Pleuronematidae with full support (Antipa et al. 2016;Gao et al. 2012Gao et al. , 2013Gao et al. , 2014Gao et al. , 2017Lu et al. 2021;Pan et al. 2010Pan et al. , 2015aPan et al. , b, 2016Pan et al. , 2020Zhang et al. 2019). In consideration of the insufficient sampling of Pleuronema sequences in Foissner et al. (2009) and the relatively low support for the position of Schizocalyptra in Antipa et al. (2016Antipa et al. ( , 2020, it is more credible that the phylogenetic position of Histiobalantiidae is outside the Pleuronematidae + Peniculistomatidae clade.  Habitat Brackish water (salinity 12) Brackish water (salinity 6.8) Marine water salinity of approximately 10. Pleuronema paraorientale sp. nov. was collected from a mangrove wetland on the west coast of Shenzhen Bay (22°30′8.2′′ N; 113°57′10.7′′ E) on 6th January, 2017, where the water temperature was approximately 24 °C and the salinity was approximately 12. For Pleuronema foissneri sp. nov. and P. paraorientale sp. nov., water samples were collected from naturally formed small puddles during ebb tide. An approximately 200 ml water sample with wilted leaves and sediment was placed into a 400 ml sampling bottle using bottle caps, and stirring was avoided. For P. parasmalli sp. nov., an approximately 200 ml volume of well-stirred freshwater sample with humus was placed into a 400 ml bottle. For P. parasalmastra sp. nov., several holes were excavated in the sand to a depth of approximately 10 cm. After water gradually seeped into the holes, water and sand at the bottom of the holes were mixed and collected. An approximately 200 ml water sample was placed into a 400 ml bottle. In all cases, samples were transferred to the laboratory and stored at room temperature (~ 25 °C).

Sampling and cultivation
After gently mixing the samples in the bottles, water with sediment of each sample was poured into five 90 mm Petri dishes to establish the initial cultures at ~ 25 °C. No food source was added to these Petri dishes. Three to five cells from the initial cultures were then isolated with micropipettes and transferred into a 35 mm disposable Petri dish with 0.22 µm-filtered water in situ for pure cultivation. Rice grains were added to promote the growth of bacterial food for the ciliates.

Morphological studies
Living cells were isolated with micropipettes and observed using bright-field and differential interference contrast microscopy at × 100-1000 magnification. The protargol staining method was used to reveal the ciliature and nuclear apparatus (Wilbert 1975). Counts and measurements were performed according to Bai et al. (2020). Drawings of living cells were produced according to Wu et al. (2020). Drawings of silver-stained cells were made using Adobe Photoshop based on photomicrographs of the holotype specimen of each species. Terminology and systematics follow Pan et al. (2016) and Gao et al. (2016), respectively.

DNA extraction, PCR amplification, and sequencing
Six to ten cells of each species were selected from pure cultures and washed five times with filtered in situ water (0.22 µm, Millex-GP filter unit) to exclude contamination. For each species, cells were then distributed in three Eppendorf tubes (Axygen, USA) with one, three, and multiple individuals. Genomic DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen, Germany) following the optimized manufacturer's protocol, modified such that 1/4 of the suggested volume was used for each solution. The primers 18S-F, 18S-R (Medlin et al. 1988) and 82F (Jerome et al. 1996) were used for PCR amplifications of the SSU rRNA gene. To minimize the errors caused by PCR, Q5 Hot Start High-Fidelity 2 × Master Mix (New England BioLabs, USA) was used as DNA polymerase. The PCR parameters were utilized according to Jiang et al. (2021). After amplification, PCR products were sequenced bidirectionally by the Tsingke Biological Technology Company (Qingdao, China).

Phylogenetic analyses
The SSU rRNA gene sequences of the four Pleuronema species in the present work were combined with 64 sequences of related taxa downloaded from GenBank, forming the dataset for phylogenetic analyses (for accession numbers, see Fig. 6). Ten sequences from Philasterida were selected as the outgroup. All sequences were aligned using the MAFFT algorithm on GUIDANCE2 Server (http:// guida nce. tau. ac. il) with default parameters (Penn et al. 2010;Sela et al. 2015). The resulting alignment was manually edited using the program BioEdit version 7.0.5.2 (Hall 1999), and both ends of the alignment were trimmed. The final alignment, including 1802 positions, was used to construct the phylogenetic trees.
Maximum likelihood (ML) analysis with 1000 bootstrap replicates was performed using RAxML-HPC2 on XSEDE 8.2.10 (Stamatakis 2014) under the GTRGAMMA model at CIPRES Science Gateway (http:// www. phylo. org/ sub_ secti ons/ portal) (Miller et al. 2010). Bayesian inference (BI) was performed with MrBayes 3.2.6 on XSEDE 3.2.6 (Ronquist and Huelsenbeck 2003) at the CIPRES Science Gateway with the best-fit model GTR + I + G, selected under the Akaike Information Criterion using MrModeltest 2 (Nylander 2004). Markov chain Monte Carlo (MCMC) simulations were run with two sets of four chains for 10,000,000 generations at a sampling frequency of 100 and a burn-in of 10,000 trees (10%). Convergence of the MCMC analyses was confirmed in that the average standard deviation of split frequencies was well below 0.01. All remaining trees were used to calculate posterior probabilities (PP) using a 50% majority rule consensus. Tree topologies were visualized using SeaView version 4 (Gouy et al. 2010). The SSU rRNA gene comparison of all Pleuronema sequences in the present work was performed by BioEdit version 7.0.5.2 (Hall 1999). The alignment length had 1623 positions after trimming both ends.