Ontogeny and Phylogeny of a New Hypotrichous Ciliate (Protista, Ciliophora), Metaurostylopsis alrasheidi n. sp., With Establishment of a New Genus Monourostylopsis n. gen.

In the present study, based on both morphologic and phylogenetic analyses, a new genus, Monourostylopsis n. gen., and new species, Metaurostylopsis alrasheidi n. sp. as well as a new combination, Monourostylopsis antarctica (Jung et al., 2011) n. comb. (original combination: Metaurostylopsis antarctica Jung et al., 2011), are suggested. The new genus is diagnosed mainly by having three or more frontoterminal cirri, a midventral complex with midventral pairs and a single midventral row, one right marginal row and two or more left marginal rows. The new genus can be easily separated from the morphologically similar genera mainly by having single right marginal row (vs. two or more right marginal rows). Based on live observation and protargol staining, the morphology and morphogenesis of a new species, M. alrasheidi n. sp. isolated from China, were investigated. The new species can be characterized by: two types of cortical granules; about 22 adoral membranelles; three or four frontoterminal, four or five transverse cirri; about eight midventral pairs and a midventral row of three or four unpaired midventral cirri; three or four left and right marginal rows. The main morphogenetic features of Metaurostylopsis alrasheidi n. sp. can be summarized as: (1) the entire parental ciliature, including the oral apparatus, is renewed; (2) the oral primordium of the proter probably originates within a pouch; (3) the oral primordium of the opisthe forms de novo on the cell surface; (4) the anlagen of marginal rows and dorsal kineties are formed intrakinetally, and (5) the fusion of macronuclear nodules results in an irregular branched mass prior to karyokinesis. In the phylogenetic trees, all the available Metaurostylopsis sequences cluster together in a clade with full support (ML/BI: 100/1.00) revealing that the genus is monophyletic within the large group of core urostylids.

Urostylids, with a diversity of more than 200 species, are one of the largest groups in hypotrichs, and are commonly found in marine, soil and freshwater habitats. Morphologically, they are characterized by a midventral complex composed of zigzag-patterned midventral cirral pairs (Berger, 2006;Kumar et al., 2010;Chen et al., 2011;Pan et al., 2016;Kim et al., 2017;Hu et al., 2019;Jung and Berger, 2019). Based mainly on the presence of frontoterminal cirri, the clearly differentiated frontal cirri and several morphogenetic features, Song et al. (2001) transferred Urostyla marina Kahl, 1932 to a newly established urostylid genus, Metaurostylopsis, with M. marina (Kahl, 1932) Song et al., 2001 as the type species. In the following decades, more species of this genus have been identified and described (Song and Wilbert, 2002;Lei et al., 2005;Shao et al., 2008a,b;Chen et al., 2011Chen et al., , 2013Jung et al., 2011;Lu et al., 2016). Nevertheless, detailed ontogenetic and molecular information has consistently questioned the assignments of some Metaurostylopsis species and further revisions of the genus have been made. Based on comprehensive analyses, Song et al. (2011) recognized five species in the genus Metaurostylopsis (M. marina, M. rubra Song and Wilbert, 2002, M. salina Lei et al., 2005, M. struederkypkeae Shao et al., 2008, and M. cheni Chen et al., 2011 and reassigned M. sinica Shao et al., 2008 into a new genus Apourostylopsis. While M. flavicans Wang et al., 2011 andM. songi Lei et al., 2005 were treated as incertae sedis. In the same year, a new Metaurostylopsis species, M. antarctica Jung et al., 2011was reported. Subsequently, Chen et al. (2013 transferred the two incertae sedis, M. flavicans and M. songi, into a new genus Neourostylopsis and provided improved diagnoses for both the genus Metaurostylopsis and Apourostylopsis. Most recently, Lu et al. (2016) added a new species, Metaurostylopsis parastruederkypkeae to the genus. As of this writing, seven species are included in Metaurostylopsis.
In the present work, morphological, ontogenetic, and molecular data for a new urostylid species, Metaurostylopsis alrasheidi n. sp., are provided. In addition, morphological, morphogenetic, and phylogenetic analyses comparing Metaurostylopsis antarctica and other Metaurostylopsis species are carried out, leading to the establishment of a new genus for Metaurostylopsis antarctica.

Sampling, Observation, and Identification
Metaurostylopsis alrasheidi n. sp. was discovered on June 3, 2019 in a freshwater sample from Lake Weishan (34 • 46 14 N, 117 • 12 56 E) (Figures 1A-D), China. A raw culture was established in Petri dishes with rice grains added to facilitate the growth of bacteria as food source for the ciliates. A single cell was transferred with a micropipette to a mini Petri dish containing mineral water with squeezed rice grains to establish a clonal culture which was maintained for about 1 month at room temperature about 25 • C, and from which DNA extraction was done.
Live observation, protargol preparation, and drawings of protargol-impregnated specimens, live cells, and dividers are according to Shao et al. (2019). Classification is mainly according to Jankowski (2007) and terminology is according to Berger (2006).

Diagnosis
Body 60-115 × 20-60 µm in vivo; two types of cortical granules: larger yellow-greenish ones and smaller colorless ones; about 22 adoral membranelles; three frontal, three or four frontoterminal, one buccal, four or five transverse cirri; 5-13 midventral pairs and a midventral row of three or four unpaired ventral cirri; three or four left and right marginal rows respectively; invariably three dorsal kineties; over 40 macronuclear nodules; freshwater habitat.

Material Deposited
The protargol slide (accession no. SWY2019060301-1) containing the holotype and several paratype slides (accession

Dedication
We dedicate this new species to our eminent colleague, Prof. Khaled A.S. Al-Rasheid, King Saud University, Saudi Arabia, in recognition of his contributions to ciliatology.

Description
Cell size about 60-115 × 20-60 µm in vivo, with ratio of length to width about 2-3:1, body outline broad to elongated elliptical (Figures 2A,B,I,J). Cortex rather flexible, but not contractile. Two types of cortical granules on both ventral and dorsal sides (Figures 2E-H): larger ones about 1.0 µm in size, yellow-greenish, wheatgrain shaped along the cirral rows, dorsal kineties and between dorsal dikinetids, giving cells a yellow-greenish appearance; smaller ones colorless, about 0.4-0.5 µm in diameter, relatively densely arranged. Cytoplasm colorless, usually with many tiny lipid droplets (about 0.6-1.0 µm across) and food vacuoles containing algae. Contractile vacuole about 15-18 µm across, located at two-fifth of body length, behind buccal vertex, near left body margin, collecting canals not observed (Figures 2A,B Adoral zone extending about 38% of body length, with cilia up to 10 µm long in vivo, consisting of 19-30 (on average 22) membranelles (Table 1), distal end of adoral zone of membranelles (AZM) bending only slightly to the right of the midline in protargol preparations ( Figures 2C,K). Paroral and endoral of nearly equal length, slightly curved and optically intersect at about posterior quarter of the paroral ( Figures 2C,K

Stomatogenesis and Development of Frontal-Ventral-Transverse Cirral Anlagen
Stomatogenesis commences with the proliferation of densely arranged basal bodies forming a slender anarchic field, that is, the opisthe's oral primordium, which occurs de novo to the left of posterior midventral pairs (Figures 3A, 5A).
Simultaneously, a small field of basal bodies, the proter's oral primordium, appears de novo in a subsurface pouch beneath the buccal field and near the parental undulating membranes (UM). Subsequently, the opisthe's oral primordium widens and lengthens between the midventral complex and the inner left marginal row during which time the parental structures remain unchanged. At the same time, the proter's oral primordium continues to develop within the pouch, which is clearly outlined (Figures 3B, 5B,C). The oral primordium then differentiates into new adoral membranelles, and the UM anlage appears to the right of the oral primordium in both proter and opisthe (Figures 3D,E, 5D-G). During this time, the parental oral structures remain unchanged, while the frontal-ventral-transverse cirral anlagen (FVTA) appear as several streak-like collections of basal bodies to the right of the UM anlage for both proter and opisthe (Figures 3D,E, 5D,E,G).
In the next stage, the differentiation of adoral membranelles is almost complete, forming new structures for both proter and opisthe. The UM anlagen split and give rise to the paroral and endoral membranes and the leftmost frontal cirrus is generated from the anterior end of the UM anlage (=FVTA I) (Figures 3G, 5G). Simultaneously the FVTA begin to break apart and differentiate into new cirri. In the following stages, the new cirri formed from the FVTA and migrate to their final positions as follows: FVTA II produces the middle frontal cirrus and the buccal cirrus; FVTA III provides the rightmost frontal cirrus and the parabuccal cirrus (III/2); FVTA IV to n-2 each develops a midventral pair, with the last two or three streaks forming one transverse cirrus; FVTA n-1 provides a short midventral row composed of three or four cirri and one transverse cirrus; FVTA n, the last anlage, develops three or four frontoterminal cirri and probably the rightmost transverse cirrus (Figures 4A,C,D, 5K,M,N).

Development of Marginal Rows and Dorsal Kineties
At the early-to-middle stages, the marginal anlagen are formed intrakinetally both in proter and opisthe. Subsequently, these anlagen elongate toward both ends and generate new cirri and gradually replace the parental structures (Figures 3D,E,G, 4A,C,D, 5F,K,M,N). In the same way, the dorsal kineties develop by intrakinetal basal body proliferation. During cell division, no caudal cirri are formed. As some key stages were not observed, the origins of the extra dorsal bristles (usually only two) are not clear (Figures 3C,F,H, 4B,E, 5H,L).

Division of Nuclear Apparatus
Although macronuclear replication bands were not definitely identified in M. alrasheidi n. sp., they have been identified (but seen with some difficulty) in M. rubra and M. marina which, otherwise, have the same macronuclear morphology as M. alrasheidi n. sp. during division and replication bands were very possibly overlooked in the new species (Song et al., 2001;Song and Wilbert, 2002). During the middle stage, all macronuclear nodules fuse into a single somewhat branched complex which then divides into numerous nodules (Figures 3C,F,H, 4B,E, 5I,J,N). Division of the micronuclei was not observed.

Phylogeny Analyses Based on 18S rRNA Gene Sequence
The partial 18S rRNA gene sequence of Metaurostylopsis alrasheidi n. sp. has been submitted into GenBank database (acc. no. MT911525), with 1,643 bp long and G + C content of 44.92%. According to sequence comparisons of the 18S rRNA gene, the sequence of Metaurostylopsis alrasheidi n. sp. differs by 8 bp from an unidentified Metaurostylopsis sequence (FJ870098) (from a taxonomic research lab), 13-14 bp from five populations of M. cheni, 16 bp from M. salina, 34-44 bp from four M. struederkypkeae sequences, and 73 bp from Monourostylopsis antarctica (Jung et al., 2011) n. comb (Figure 6).
The topologies of the ML and BI trees are mostly congruent. As a result, only the ML tree with nodal supports inferred from both algorithms is shown (Figure 7). According to our 18S rRNA gene tree, M. alrasheidi n. sp. clusters with

Geographic Distribution
Metaurostylopsis alrasheidi n. sp. was isolated from freshwater habitat. All the other Metaurostylopsis species have been reported in marine or brackish water (Song et al., 2001;Lei et al., 2005;Berger, 2006;Shao et al., 2008a,b;Chen et al., 2011;Lu et al., 2016). These studies have shown that Metaurostylopsis species can be found in a variety of habitats (Figure 8). Metaurostylopsis marina, has been reported in Mauritania, Poland, China, Korea, and United States, and is probably a cosmopolitan species. While most of the other Metaurostylopsis species have been found in one place only, this likely reflects undersampling.

DISCUSSION
Comparison of Metaurostylopsis alrasheidi n. sp. With Closely Related Species (Table 2) With respect to the ciliature, that is, frontal and transverse cirri clearly differentiated, buccal cirri present, more than two frontoterminal cirri, midventral complex with midventral pairs and a single short midventral row, more than two marginal rows on each body side; pretransverse cirri and caudal cirri absent, the new isolate corresponds well with the diagnosed characteristics of the genus Metaurostylopsis (Chen et al., 2013).
Metaurostylopsis alrasheidi n. sp. is most similar to M. cheni, especially in body shape, yellow-greenish cell color, two types of cortical granules, and most aspects of the ciliature, but differs from the latter by having a relatively smaller body size in vivo 60-115 × 20-60 µm vs. 90-140 × 40-60 µm, a dense distribution pattern of the smaller type of cortical granules on dorsal side (vs. sparse), contractile vacuole located anteriorly (2/5 of body length vs. equatorial region), relatively more midventral pairs (5-13 vs. 5-9) and less transverse cirri (4-5 vs. 5-8) (Chen et al., 2011). The 18S rRNA gene sequence of the newly isolated organism differs from that of M. cheni (GU170204) by 13 nucleotides (Figure 6), which also reveals these two species are not conspecific.
The character of cortical granules, that is, two types of cortical granules, of the new species is distinguishable from some of the congeners with only one type of cortical granules present ( Table 2). However, it is possible that earlier researchers overlooked the tiny type of cortical granules. Considering the habitat, Metaurostylopsis alrasheidi n. sp. can be separated from all the other congeners (freshwater vs. marine or brackish, Table 2).

Emended Diagnosis of Genus
Metaurostylopsis and Reassignment of the Species, M. antarctica Jung et al., 2011 In the most recent revision of the genus Metaurostylopsis, Chen et al. (2013) provided an improved diagnosis for the genus Metaurostylopsis, which were defined as marine urostylids with characteristics mentioned in the above section. However, the new species in the present study, Metaurostylopsis alrasheidi n. sp., which was isolated from a freshwater lake, fits the morphological characteristics of the genus Metaurostylopsis very well. Moreover, our new species clusters in a clade together with all the other Metaurostylopsis sequences, indicating the close relationship among them and supporting the classification of the new species in the genus Metaurostylopsis. Thus, an emended diagnosis including marine, brackish and freshwater habitats is supplied.
As discussed above, the genus Metaurostylopsis is characterized by having more than one marginal row on each body side. However, M. antarctica was characterized by having only one right marginal row, which does not fit the diagnosis of Metaurostylopsis. Also, in the phylogenetic trees, M. antarctica is placed as sister to the clade including all the other Metaurostylopsis sequences with strong to full support. Therefore, we propose that M. antarctica should be excluded from the genus Metaurostylopsis. As M. antarctica also can be separated from other morphologically similar genera, i.e., Neourostylopsis and Apourostylopsis, by having only one right marginal row (vs. two or more right marginal rows) ( Table 2), this suggests that M. antarctica represents a new genus. Hence, an emended diagnosis of the genus Metaurostylopsis and diagnosis of a new genus, Monourostylopsis n. gen., are supplied here.
Metaurostylopsis Song et al., 2001 Improved Diagnosis Marine, brackish or freshwater urostylids with frontal and transverse cirri clearly differentiated; three or more frontoterminal cirri; midventral complex with midventral pairs extending to the level of the buccal vertex and a single midventral row; buccal and transverse cirri present; pretransverse ventral and caudal cirri absent; more than two marginal rows on each body side.

Diagnosis
Urostylids with frontal and transverse cirri clearly differentiated; three or more frontoterminal cirri; midventral complex with midventral pairs extending to the level of the buccal vertex and a single midventral row; buccal and transverse cirri present; pretransverse ventral and caudal cirri absent; one right marginal row and two or more left marginal rows.

Etymology
The generic name is a composite of the Greek mono-(single), referring to the single right marginal row, and the posterior part (-urostylopsis) of the genus-group name Metaurostylopsis.

Morphogenetic Comparison
As of this writing, the morphogenetic process of four Metaurostylopsis species, i.e., M. alrasheidi n. sp., M. cheni, M. marina, and M. rubra has been investigated (Wilbert and Song, 2005;Chen et al., 2011;Song et al., 2011). The morphogenetic pattern of the genus Metaurostylopsis shows high stability, and shares similar morphogenetic features with Apourostylopsis, Monourostylopsis, and Neourostylopsis as follows: (1) in the proter, the oral primordium (OP) is formed de novo in a subcortical pouch beneath (i.e., dorsal to) the buccal field, the new undulating membranes (UM) anlage is generated near the OP, and the parental oral structures are completely replaced; (2) in the opisthe, both the OP and the UM anlage develop apokinetally on the cell surface; (3) the FVT cirral anlagen are formed apokinetally, and none of the old ciliature seems to be involved in the development of the new primordia; (4) marginal anlagen and dorsal anlagen develop intrakinetally; (5) all macronuclear nodules fuse to a single branching mass during the middle divisional stage (Wilbert and Song, 2005;Shao et al., 2008a;Chen et al., 2011;Jung et al., 2011;Song et al., 2011;Zhang et al., 2018). Ontogenetic features of Metaurostylopsis show striking differences when compared with genera Apourostylopsis and Neourostylopsis, namely: (1) FVTA n-1 forms a row of unpaired ventral cirri which migrates along with the midventral pairs, whereas in Apourostylopsis and Neourostylopsis, no unpaired ventral cirri are generated; (2) FVTA n formed a transverse cirrus and more than two frontoterminal cirri in Metaurostylopsis (i.e., typical Metaurostylopsis mode), whereas the FVTA n forms two frontoterminal cirri, a pretransverse and a transverse cirrus in Apourostylopsis (i.e., typical urostylid mode), and whether FVTA n forms pretransverse cirrus in Neourostylopsis is variable (Wilbert and Song, 2005;Shao et al., 2008a;Chen et al., 2011;Jung et al., 2011;Song et al., 2011;Song and Shao, 2017;Zhang et al., 2018).

Phylogenetic Analyses
Consistent with previous studies (Shao et al., 2008a;Chen et al., 2011;Song et al., 2011;Lu et al., 2016), we confirm the monophyly of genus Metaurostylopsis based on the phylogenetic analyses in present work.
In the 18S rRNA gene tree, our new taxon, Metaurostylopsis alrasheidi n. sp., clusters with Metaurostylopsis species and falls into the monophyletic clade of Metaurostylopsis, which can also be supported by the shared morphological features, such as, similar body shape and size, as well as semblable ciliature. Additionally, 8-44 different nucleotides of 18S rRNA gene are discovered between M. alrasheidi n. sp. and its congeners, further indicating the validity of our isolate as a distinct species. It is worth noting that the genus Metaurostylopsis forms a sister clade to Monourostylopsis n. gen., with which it shares some similar characteristics: (1) three or more frontoterminal cirri; (2) midventral complex with midventral pairs and a single midventral row; (3) buccal and transverse cirri present; (4) pretransverse ventral and caudal cirri absent. However, in addition to its molecular phylogenetic position, Metaurostylopsis can be clearly separated from Monourostylopsis n. gen. morphologically by having more than two marginal rows on each body side (vs. one left marginal row and two right marginal rows) (Jung et al., 2011).

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI GenBank (accession: MT911525).

AUTHOR CONTRIBUTIONS
XL and TZ conceived the study. WS carried out the live observation, protargol staining, DNA extraction, and data analyses. All authors contributed to the manuscript and approved the final version.

ACKNOWLEDGMENTS
We would like to express our gratitude to Dr. Xumiao Chen, Institute of Oceanology, Chinese Academy of Sciences, for her kind help and advice on the identification of this species.