Morphology and Morphogenesis of A New Soil Ciliate, Holostichides Songi Nov. Spec. with Notes on the Phylogeny of Holostichides (Ciliophora, Hypotrichia)

Background: Hypotrichia are a group with the most complex morphology and morphogenesis within the ciliated protists. The classication of the genus Holostichides is poorly understood particularly due to the lack of molecular data. Hence, the systematic relationship between this genus and other taxa in the subclass Hypotrichia remains unresolved. In this paper, the morphology and morphogenesis of Holostichides songi nov. spec. were studied, in addition, genomic DNA was extracted in order to sequence the small subunit rDNA. The main aims of this study were to document morphogenesis in H. songi nov. spec. and investigate the systematics of Holostichides based on morphology, morphogenetic, and molecular data. Results: A new bakuellid ciliate, Holostichides songi nov. spec. isolated from China, is investigated in terms of its morphology, ontogenesis, and molecular phylogenesis. It is characterized by size in vivo 150–180 µm × 45–60 µm; 27–46 macronuclear nodules; cortical granules dark, spherical, about 0.5 µm across, clustered in groups and then arranged longitudinally; three frontal cirri with one to four smaller cirri below the middle one, one buccal cirrus, 9–15 frontoterminal cirri, and midventral complex composed of ve to eight cirral pairs and two long midventral rows. The main events during binary ssion are as follows: (1) in the proter, the undulating membrane anlage is formed from both the dedifferentiation of old undulating membranes and the basal bodies developing de novo, and the posterior part of the parental adoral zone of membranelles is renewed by new structure formed from the oral primordium; (2) in the opisthe, the oral primordium is formed intrakinetally; (3) Besides the second frontal cirrus and buccal cirrus, the frontoventral-transverse cirral anlage II produces the extra one to four cirri. Phylogenetic analyses based on SSU rDNA sequence data suggest a close relationship between Holostichides songi nov. spec.


Background
In recent years there have been signi cant advances in the knowledge and understanding of the taxonomy and systematics of hypotrichous ciliates. This is largely due to the application of silver impregnation methods to reveal the infraciliature, silverline system and other characters of taxonomic importance [1][2][3][4][5][6]. Moreover, a great diversity of species and basic modes of formation of ciliature and developmental events have been reported [7][8][9][10][11][12][13].
The bakuellid genus Holostichides was established by Foissner [14] with H. chardezi Foissner, 1987 as the type species, and was de ned as follows: adoral zone of membranelles continuous; three frontal cirri; buccal cirrus(i) present; and three or more frontoterminal cirri; midventral complex composed of midventral pairs and one or more midventral rows; transverse cirri lacking; one left and one right marginal row; four or ve dorsal kineties; caudal cirri present. Up to date, ve species have been assigned to this genus: H. chardezi Foissner, 1987, H. dumonti Foissner, 2000, H. typicus (Song & Wilbert, 1988)  In May 23, 2018, an unknown hypotrichous ciliate was isolated from China. Observations of its morphology both in vivo and after protargol staining demonstrate that it represents a novel species within the genus Holostichides. In the present study, its morphology and morphogenesis are described. The small subunit ribosomal DNA (SSU rDNA) of the new isolate was sequenced and analysed in order to estimate its phylogenetic position.  . Cytoplasm hyaline colorless to grayish, containing some lipid droplets (0.5-4 µm around) and food vacuoles (6-10 µm around) in posterior portion that render cell opaque and dark in posterior portion at low magni cation, usually many irregular crystals located in posterior portion of cell ( Fig. 2A). Locomotion by crawling slowly on plant debris or on the bottom of the Petri dish, occasionally swimming with slight rotation around long body axis.

Results
Most cirri relatively ne, cilia about 10-12 µm long in vivo except for frontal cirri, cilia about 15 µm. Three slightly enlarged frontal cirri arranged in a transverse "pseudo" row with one to four smaller cirri longitudinally arranged below the middle frontal cirrus. Buccal cirrus located slightly anterior to Subsequently, the undulating membranes anlagen appear on the right both in opisthe and proter, interestingly, another spherical eld of basal bodies appears at the proximal end of old adoral zone of membranelles, which will form the new membranelles for the proter (Figs. 3F, 5H). In the middle stage, the undulating membranes anlage splits longitudinally to form two streaks from which the endoral and paroral derive, and gives rise to the leftmost frontal cirrus in each the proter and opisthe. The proximal membranelles in the proter are replaced by newly formed structures (Figs. 3H, 5L). In the late stages, the differentiation of membranelles is almost complete, forming the new structures for both the daughter cells, anterior end of the new adoral in opisthe bends to the right. Initially, the newly formed endoral and paroral initially lie close together in parallel, but later they separate, arch, and intersect in each lia product (Fig. 4A, C, E, G).

Development Of The Frontoventral-transverse Cirri
In the early stage, some cirri in the midventral complex dedifferentiate to join in the construction of the frontoventral-transverse cirral anlagen (FVT-anlagen) (Figs. 3D, E, 5F). Subsequently, the FVT-anlagen form and lengthen by increasing the number of basal bodies, to the right of the undulating membranes anlagen in both the opisthe and proter (Figs. 3E, F, 5G, J). In the middle stage, these anlagen broaden and break apart (Figs. 3H, 5L). In late stages, the FVT-anlagen commence to fragmentize (Figs. 4A, C, 5N). Later, the segregation of cirri from the FVT-anlagen is almost complete and, the old structures are almost resorbed. New cirri migrate to their nal positions as distinct cirri (Fig. 4E, G). Consequently, anlage I provides the left frontal cirrus; anlage II forms the middle frontal cirrus, the cirri below the middle frontal cirrus and the buccal cirrus; anlage III produces the rightmost frontal cirrus and the cirrus III/2; midventral pairs develop from anlagen IV to n-4 (in some individuals, n-3) and midventral rows are formed from anlagen n-3 (in some individuals, n-2) to n-1; anlage n forms the frontoterminal cirri (Fig. 4E, G).

Development Of Marginal Rows And Dorsal Kineties
The

Division Of Nuclear Apparatus
The nuclear apparatus divides in the usual way for urostylids. Brie y, the two macronuclear nodules fuse to form a single mass during the mid-divisional stage and then divide twice prior to cytokinesis. Micronuclei were observed to divide mitotically (Figs. 3G, I, 4B, D, F, H, 5I, M).
Phylogenetic analyses based on SSU rDNA gene sequences (Fig. 6) The SSU rDNA sequence of Holostichides songi nov. spec. was deposited in GenBank with the accession number MT557692. The length and GC content of the new sequence are 1651 bp and 45%, respectively.
Phylogenetic trees using two different methods (ML and BI) generated nearly congruent relationships. Therefore, only the ML tree is presented with support values from both algorithms at the nodes (Fig. 6)

Phylogenetic Analyses
The SSU rDNA sequence of Holostichides songi nov. spec. and those of 66 other hypotrichs were downloaded from GenBank database for the phylogenetic analyses (for accession numbers, see Fig. 6).
Four euplotid species, namely Apodiophrys ovalis, Diophrys scutum, Paradiophrys zhangi and Uronychia multicirrus, were used as outgroup taxa. Sequences were aligned using the GUIDANCE web server (http://guidance.tau.ac.il/, [29]). Both ends of the alignments were trimmed and ambiguous columns were removed based on con dence scores calculated by GUIDANCE. Maximum likelihood (ML) analyses were performed using RAxML-HPC2 on XSEDE v8.2.9 [30,31] on the online server CIPRES Science Gateway (http://www.phylo.org/portal2/login!input.action, [32]). The reliability of internal branches was assessed using a nonparametric bootstrap method with 1000 replicates. Bayesian inference (BI) analyses were carried out using MrBayes on XSEDE v3.2.6 [33] on CIPRES Science Gateway with the GTR + I + G model selected by Akaike Information Criterion (AIC) in MrModeltest v2 [34]. Markov chain Monte Carlo simulations were run with two sets of four chains for 2 000 000 generations with a sample frequency of 100 generations and discarding the rst 5000 trees as a burn-in (25%). All remaining trees were used to calculate posterior probabilities using a 50% majority rule consensus. MEGA v5 was used to visualize the tree topologies [35].

Conclusions
In this manuscript, the new soil species, Holostichides songi nov. spec. collected from the wetland, expand the knowledge of biodiversity of ciliates. Moreover, the monophyly of Holostichides is con rmed by the molecular phylogeny as the Holostichides species clustered in a well-supported clade.

Sample collection, observation, and identi cation
Samples (about 250 g) were collected from the surface (top 10 cm) of soil in Egret Wetland Park, Suzhou, China (31°16'18''N; 120°43'18''E) on May 23, 2018. Ciliates were made to excyst by employing the nonooded Petri dish method as described by Foissner [14]. A non-clonal culture was established at room temperature (about 24 °C) in Petri dishes containing mineral water (Nongfu Spring) with squeezed rice grains to enrich the bacterial food. The species was accurately identi ed based on its in vivo morphologic characteristics. Moreover, no other Holostichides-morphotypes were present in the protargol preparation. The probability is therefore extremely high that the present morphological, morphogenetic and molecular studies deal with the same species.
Living cells were observed in vivo using bright eld and differential interference contrast microscopy (Olympus BX53), and photographed using a digital camera. Protargol staining was used to reveal the ciliary pattern and nuclear apparatus [26]. Counts and measurements of stained specimens were performed with an ocular micrometer. Drawings were made with the help of a camera lucida. To illustrate the changes occurring during morphogenetic processes, old (parental) ciliary structures are depicted by contour whereas new structures are shaded black. Terminology is mainly according to Berger [1].

Dna Extraction, Pcr Ampli cation, And Gene Sequencing
One cell of Holostichides songi nov. spec. was isolated and repeatedly washed using sterile distilled water. It was then transferred to a 1.5-ml microfuge tube with a minimum volume of water. Genomic DNA was extracted from cells using DNeasy Blood & Tissue Kit (Qiagen, CA) following the manufacturer's instructions. The PCR was ampli ed according to Gao et al. [27], using the eukaryotic universal SSU rDNA The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. One permanent slide containing the protargol-impregnated holotype specimen of Holostichides songi nov. spec. (registry no. WJY2018052301B) and one paratype slide (registry no. WJY2018052301C) were deposited in the Laboratory of Protozoological Biodiversity and Evolution in Wetland, Shaanxi Normal University, China.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was supported by the Natural Science Foundation of China (Project number: 31872190). The funding body had no role in study design, data collection, analysis and interpretation, and in writing of the manuscript.
Authors' contributions JW collected the samples, carried out the live observation, protargol preparations and morphometrics and drafted the manuscript; JL was responsible for DNA ampli cation and sequencing, and drafted the manuscript; YW carried out the molecular phylogenetic analyses. CS did the identi cation of the species, taxonomic analysis and revision of the manuscript. All authors prepared the manuscript and approved the nal version.