Triangulopteris lacunata gen. et sp. nov. (Centroplasthelida), a New Centrohelid Heliozoan from Soil †

: A new genus and species of centrohelid heliozoans, Triangulopteris lacunata gen. et sp. nov. (Pterocystidae Cavalier-Smith and Heyden, 2007), from four geographically remote locations (the Crimean Peninsula, the Dnieper Lowland (the East European Plain), Franz Josef Land, and the Kolyma Lowland (North–Eastern Siberia) was examined using light and electron microscopy. The novel centrohelid is characterized by round shape, 4.3–16.3 µ m in diameter, covered with two types of scales: 1.06–4.54 µ m long triangular spine scales and 1.22–2.05 µ m oval plate scales. Studied centrohelid heliozoan possesses a unique spine scale morphology. The base of scales is represented by a horse hoof-shaped basal plate. The inner surface and lateral wings of spine scales have numerous radial ribs with two ‘pockets’ that are located on both sides of the spine shaft. These pockets are formed by the lateral wings and ends of the basal plate. The cyst formation and transition to a spicules-bearing stage were noted. Additionally, phylogenetic tree was constructed based on SSU rRNA sequences including the strain HF-25 from the permafrost of Kolyma Lowland. The resulting phylogeny recovered it within the clade Pterista, while forming a separate sister lineage to H2 clade, which only had included freshwater environmental sequences.


Introduction
Centrohelid heliozoans (Centroplasthelida Febvre-Chevalier et Febvre, 1984) are a group of free-living heterotrophic protists, found throughout global marine and freshwaters bodies, and soils [1]. Based on multigene phylogeny, centrohelids form a eukaryotic supergroup, Haptista, along with haptophytes [2]. The characteristic features of centrohelids are a peculiar organization of the microtubule-organizing center (MTOC), extrusive organelles (kinetocysts), ribbon-like cristae of mitochondria, narrow pseudopodia (axopodia) with a complex arrangement of microtubule inside, and external skeletal elements [1,[3][4][5][6]. Most centrohelids are covered with various types of silica scales and/or organic needle-like spicules and the morphology of these coverings have been used to distinguish different genera and species. However, several centrohelid genera can lose silica scales and produce radial needle-shaped organic spicules in clonal cultures [7,8] making morphology-based species identification more challenging.
However, most studies on centrohelid heliozoans are focused on ones found in freshwater. Specifically, reports on diversity and abundance of centrohelids from soil biotopes are handful [12,13]. Rather, soil biotopes were discarded as unsuitable habitats for centrohelids due to frequent changes in moisture level [1]. There are only a few works that mention centrohelids in soil samples [12,13]. On the contrary, modern metabarcoding investigations show that soil centrohelids are numerous and even more diverse than marine and freshwater ones [14,15].
Despite the ubiquity of centrohelids across various ecological niches, very little is known about their biogeography [27,30]. In this study, to better understand the diversity, ecology and phylogeny of this group, in depth morphological investigation was carried out using light and electron microscopy and a molecular phylogenetic analysis. Furthermore, centrohelids from different types of soil biotopes were obtained and clone-cultured for a detailed investigation of the life cycles.
Here, we describe the novel Triangulopteris lacunata gen. et sp. nov., a representative of the Pterista lineage.

Clone Isolation
Samples were obtained from geographically distant biotopes of the Crimean Peninsula, the Dnieper Lowland (East European Plain), Franz Josef Archipelago and the Kolyma Lowland (North-Eastern Siberia) (see Table 1 and Figure 1). Soil samples were placed into 15 or 50 mL plastic tubes and kept at 4 • C until processed in a laboratory. Each sample was processed by diluting with autoclaved mineral water (Aqua Minerale, PepsiCo, Inc., Moscow oblast, Russia). The diluted samples were then plated onto plastic or glass Petri dish (60 mm in diameter), enriched with a suspension of Pseudomonas fluorescens Migula, 1895 bacteria or an autoclaved grain of rice.
The processed samples were kept at 22 • C in the dark and observed for 10 days to reveal the hidden species diversity [31]. To obtain clonal cultures, the individual heliozoan cells were transferred using a glass micropipette onto Petri dish containing a clonal culture of eukaryotic prey. The prey cultures of flagellates Parabodo caudatus (Dujardin 1841) Moreira et al., 2004 (strain BAS-1, IBIW RAS) or Bodo saltans Ehrenberg 1832 (strain Pl-1, IBIW RAS) were grown in mineral water (Aqua Minerale, PepsiCo, Inc., Moscow oblast, Russia).  Table 1 for the description of the sampling sites (1-4).
The processed samples were kept at 22°C in the dark and observed for 10 days to reveal the hidden species diversity [31]. To obtain clonal cultures, the individual heliozoan cells were transferred using a glass micropipette onto Petri dish containing a clonal culture of eukaryotic prey. The prey cultures of flagellates Parabodo caudatus (Dujardin 1841) Moreira et al., 2004 (strain BAS-1, IBIW RAS) or Bodo saltans Ehrenberg 1832 (strain Pl-1, IBIW RAS) were grown in mineral water (Aqua Minerale, PepsiCo, Inc., Moscow oblast, Russia).
Preparations for studying skeletal elements were carried out as follows. For scanning electron microscopy (SEM), live cells of heliozoans were transferred using a glass micropipette into a small drop of autoclaved mineral water on a coverslip. The drop was air dried after which glass was washed three times with distilled water, air-dried, and attached to the aluminum SEM specimen stub by a conductive carbon adhesive tab (Nisshin EM Co., Ltd., Tokyo, Japan). The preparation was coated with 15 nm gold layer using Auto Fine Coater JFC-1600 (JEOL, Tokyo, Japan) and observed with a JSM-6510LV (JEOL, Tokyo, Japan) electron microscope at an acceleration voltage of 15 kV. For transmission electron microscopy, suspension of live heliozoan cells was transferred onto a formvar-coated copper grids, air-dried, and then washed three times with distilled water. After drying, the preparation was shadowed with tungsten oxide (WO2) using a JEE-4X vacuum evaporator (JEOL, Tokyo, Japan) and observed with a JEM-1011 (JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV.
Morphological parameters of centrohelids such as cell diameter, spine scale length, basal plate width, marginal rim of basal plate, shaft diameter, height, width and depth of basal plate pockets, plate scales' length and width, length and width of medial thickening of plate scales, and cysts diameter were measured using ImageJ 1.52a [32].  Table 1 for the description of the sampling sites (1-4).
Preparations for studying skeletal elements were carried out as follows. For scanning electron microscopy (SEM), live cells of heliozoans were transferred using a glass micropipette into a small drop of autoclaved mineral water on a coverslip. The drop was air dried after which glass was washed three times with distilled water, air-dried, and attached to the aluminum SEM specimen stub by a conductive carbon adhesive tab (Nisshin EM Co., Ltd., Tokyo, Japan). The preparation was coated with 15 nm gold layer using Auto Fine Coater JFC-1600 (JEOL, Tokyo, Japan) and observed with a JSM-6510LV (JEOL, Tokyo, Japan) electron microscope at an acceleration voltage of 15 kV. For transmission electron microscopy, suspension of live heliozoan cells was transferred onto a formvar-coated copper grids, air-dried, and then washed three times with distilled water. After drying, the preparation was shadowed with tungsten oxide (WO 2 ) using a JEE-4X vacuum evaporator (JEOL, Tokyo, Japan) and observed with a JEM-1011 (JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV.
Morphological parameters of centrohelids such as cell diameter, spine scale length, basal plate width, marginal rim of basal plate, shaft diameter, height, width and depth of basal plate pockets, plate scales' length and width, length and width of medial thickening of plate scales, and cysts diameter were measured using ImageJ 1.52a [32].
To infer Bayesian phylogenetic tree, MrBayes version 3.2.6 [37] was used with four categories of Gamma-distributed among-site rate variation under GTR+I+GAMMA4 substitution model, calculating proportion of invariable sites. To calculate posterior probability, four independent Metropolis-coupled Markov chains were run for 20 million generations, and summarized with a 50% burn-in.
Additionally, maximum likelihood phylogeny was inferred using IQ-TREE v1.6.12 [38] under the best fit model determined by the in-built ModelFinder with and 1000 nonparametric bootstraps. The cells are covered with plate scales ( Figure 3A,E,F) and spine scales ( Figure 3B-D). Spine scales are 1.06-3.33 µm long, have a triangular appearance, consist of a shaft, a basal plate and lateral wings. The shaft is 0.08-0.12 µm wide ( Figure 3C), tapering to a sharp apex. The horse hoof-shaped basal plate is 0.60-0.92 µm in diameter ( Figure 3C) and surrounded by a small 0.06-0.09 µm wide marginal rim. Two so-called 'pockets' (Figure 3B,D) are located on both sides of the shaft. They are formed by the lateral wings and ends of the hoof-shaped basal plate. Each pocket size is by 0.14-0.36 × 0.07-0.13 µm with a depth of 0.14-0.36 µm. The lateral wings taper from the basal plate to the apex of the shaft ( Figure 3B). Radial ribs are located on the inner side of the basal plate and on the lateral wings ( Figure 3B). Spine scales are slightly curved distally from the inner part of the basal plate. Plate scales are oval with a size dimension of 1.25-2.05 × 0.86-1.93 µm and its medial thickening with the size 0.47-1.05 × 0.060-0.116 µm ( Figure 3E,F).  In addition to the type strain HF-25 isolated from the permafrost of the Kolyma Lowland, cells with similar morphology were found in the soil samples from Crimea, an island on the Dnieper River, and in the wet moss with sand from Franz Josef Land archipelago (Figure 4). Comparison of the morphological characteristics of the skeletal elements can be found in Table 2. In addition to the type strain HF-25 isolated from the permafrost of the Kolyma Lowland, cells with similar morphology were found in the soil samples from Crimea, an island on the Dnieper River, and in the wet moss with sand from Franz Josef Land archipelago (Figure 4). Comparison of the morphological characteristics of the skeletal elements can be found in Table 2.

Spicules-Bearing Stage
After several years of cultivating the strain HF-25 using B. saltans flagellate as a prey with the addition of rice grains, a loss of silica scales by the HF-25 cells was observed. No silica scales could be observed both in live cell images (Figure 2A-E) and in SEM and TEM images ( Figure 5). Instead, irregularly placed needle-shaped spicules (1.85-3.64 µm long and 0.035-0.056 µm wide) were observed in electron microscope over the entire surface of the cells ( Figure 5).

Spicules-Bearing Stage
After several years of cultivating the strain HF-25 using B. saltans flagellate as a prey with the addition of rice grains, a loss of silica scales by the HF-25 cells was observed. No silica scales could be observed both in live cell images (Figure 2A-E) and in SEM and TEM images ( Figure 5). Instead, irregularly placed needle-shaped spicules (1.85-3.64 μm long and 0.035-0.056 μm wide) were observed in electron microscope over the entire surface of the cells ( Figure 5).

Phylogenetic Analysis
The SSU rRNA based phylogenetic tree recovered the strain HF-25 within the clade Pterista ( Figure 6). Strain HF-25 forms a separate lineage in an unresolved trichotomy with the environmental sequence Env H7.1 (AY749492) and the clade H2, consisted of Env H1.10 (AY749488), Env H19.3 (AY749502), Env H5.6 (AY749489), and Env H19.7 (AY749486) from freshwater biotopes of Botswana, New Zealand, and Singapore. The sequence of HF-25 strain has a rather low similarity with the closest NCBI GenBank se-   We propose that the strain HF-25 represents a new genus and species of centrohelid heliozoans due to its isolated placement on the phylogenetic tree and the unique morphology of silica scales.

Discussion
Morphological characteristics and the measurements of the newly described centrohelids in this study (Table 2) do not overlap with those of other known centrohelids. Horse hoof-shaped basal plates and triangular lateral wings narrowing toward a sharp apex of clone HF-25 distinguish it from all other centrohelid genera and species except Raineriophrys scaposa (Dürrschmidt, 1987) Mikrjukov, 2002. The size of the scales, the shape of lateral wings and the basal plates between R. scaposa and HF-25 are quite similar. However, the descriptions of R. scaposa do not report any structures resembling 'pockets' described above. Additionally, no information about the ribs on the surface of the lateral and basal wings are recounted. Such distinctive features are not visualized on the microphotographs in the original description either [39]. Another R. scaposa described in the work of Wujek [40], also lacks characteristic features described in spine scales of our strain. It is noteworthy that Raineriophrys scaposa was first described as Acanthocystis scaposa, from samples obtained from freshwater in Chile and Sri Lanka [39]. Later it was transferred to the genus Pterocystis [6], and then to Raineriophrys [1].
To this day, NCBI GenBank contains merely one sequence of Raineriophrys which was morphologically identified. Raineriophrys erinaceoides (type species of the genus Raineriophrys) belongs to the clade Pterocystida C together with Pterocystis canadensis (AY749633). The problems associated with SSU rRNA sequences belonging to Pterocystida C clade were discussed previously [27], one of which is that the two similar sequences (98.86%), P. canadensis (AY749633) and R. erinaceoides (AY268043) are assigned to different genera. Considering the morphology of the skeletal elements of T. lacunata and R. scaposa is similar to some extent, we assume that they are representatives of the same genus. It would be necessary to obtain molecular data on R. scaposa to resolve this ambiguity. The presence of horse hoof-shaped basal plates, triangular lateral wings narrowing to the sharp apex, and isolated phylogenetic position of clone HF-25 distant from the type species of the genus Raineriophrys, allow us to consider it a member of a new genus.
The morphological characteristics of four T. lacunata strains from geographically distant biotopes are very similar. Interestingly, T. lacunata was found in several soil samples at once and has never been found in freshwater bodies. Apparently, this organism is a representative of specific soil microbial ecosystems. Soils and temporary shallow freshwater biotopes remain poorly explored [41][42][43][44]. The information about centrohelid heliozoans from soils is especially scanty. No special studies have been conducted. There are only a few works that mention centrohelids in soil samples [12,13]. However, growing metabarcoding studies have just started to unveil new and intriguing information about the soil-associated protist communities including centrohelids. For example, Singer et al. [15], have shown that species diversity of soil protists is more numerous than that of both marine and freshwater: the number of operating taxonomic units (OTUs) assigned to centrohelids in soils is twice as much as the ones in freshwater biotopes and 10 times higher than that of marine waters.
A metatranscriptomic study of protists of soil systems in Europe [14] revealed the presence of centrohelids with relative abundance of SSU rRNA sequences up to 1%. Thus, centrohelid heliozoans are diverse and ubiquitous component of soil microbial communities.
Two strains of centrohelid studied here were isolated from frozen soil and peat. It has been known that protists can remain viable at permafrost conditions for hundreds to hundreds of thousands of years [13,[45][46][47][48][49][50]. The discovery of a centrohelid heliozoan in the permafrost of the Kolyma River basin was reported earlier [13]: viable cysts of a heliozoan, identified as Choanocystis perpusilla (Petersen et Hansen, 1960) Siemensma, 1991, were isolated from sediments of the Late Pleistocene fossil burrows of ground squirrels [13]. In our study, centrohelid heliozoans were found in two sites near Cape Maliy Chukochiy: (1) buried peat from a depth of 35-40 cm; (2) mineral soil from a depth of 60-65 cm, both of which are layers belonging to Holocene deposits. The average thickness of active layer of the permafrost in Cape Maliy Chukochiy is 42 cm, but in some years, it can go up to 64 cm [51]. The isolated strains of T. lacunata gen. et sp. nov. and their locations complement our knowledge of paleoprotists. It should be noted that at present there is an increase in the thickness of active permafrost layer and thawing of permafrost leading to a change in the ecological balance and composition of microbial communities [52][53][54].
We observed that T. lacunata gen. et sp. nov. cells lose silica scales in the studied old cultures and produce radial needle-shaped spicules. A similar change in the type of scales is shown for Raphidocystis glabra Dürrschmidt 1985 [7]. The stages of a complex life cycle are described in detail for Raphidiophrys heterophryoidea Zlatogursky, 2012, and at a certain stage, it has been described to possesses organic spicules [8,20]. A similar change of cells covers is therefore, a likely typical character for other genera.
Here we present the third confirmed case of silica scales being replaced to spicules. It is also highly unlikely a contamination as the two monoclonal cultures were isolated from the samples from the adjacent collection points. Morphology of cells from both cultures were visualized using electron microscopy and silica scales were noted. SSU rRNA genes of both cultures were also sequenced (the sequence identity was 99.9%). A few years later, a change to spicule-bearing stage was found in both cultures.
Strains of T. lacunata studied here were isolated from geographically distant biotopes ( Figure 1). Global distribution of centrohelids and some other protists have already been reported [1,44]. The five most closely related environmental sequences ( Figure 5) are also obtained from remote freshwater locations in Botswana, New Zealand, and Singapore. However, the factors influencing centrohelid diversity and distribution remain unclear. More research is needed to understand the relationships within the different phylogenetic clades of centrohelids and boundaries of their main taxa. It is also necessary to sequence more SSU rRNA genes and thoroughly investigate the morphology of skeletal elements of closely related strains of centrohelids, including those from poorly studied soil biotopes. Diagnosis: Centrohelid heliozoans with two types of silica covering elements. Spine scales have a triangular appearance, they consist of a hoof-shaped basal plate, a hollow pointed shaft, and lateral wings tapering to sharp apex. Plate scales are oval, smooth, with a poorly developed medial thickening.

Taxonomic Summary
Type species: Triangulopteris lacunata sp. nov. Etymology: From Latin "triangulus"-triangular and from Greek "πτερóν"-wing; due to the shape of spine scales, which has a triangular appearance.