Morphology, Ontogeny and Molecular Phylogeny of a New Urostylid Ciliate, Bakuella (Pseudobakuella) Guangdongica n. sp. (Protista, Ciliophora, Hypotrichia) from Southern China


 Background: Spirotrich ciliates are one of the most diverse groupsin the phylum Ciliophora and are widely distributed in marine, freshwater, and terrestrial biotopes. Many nominal species are, however, poorly known and their systematic positions remain uncertain due to the lack of information concerning their infraciliature, morphogenesis, and gene sequences. In this paper, the morphology and morphogenesis of Bakuella (Pseudobakuella) guangdongica n. sp. were studied, in addition, genomic DNA was extracted in order to sequence the small subunit rDNA. Results: Bakuella (Pseudobakuella) guangdongica n. sp. is characterized by 150–225 µm in vivo; 35–42 adoral membranelles; three to five buccal, two frontoterminal and eight to 13 transverse + pretransverse cirri; midventral complex comprised of 10–20 midventral pairs and two midventral rows extending to transverse cirri; posterior part of marginal rows slightly overlapped; colorless cortical granules about 1 μm across, arranged in small groups; soil habitat. Its main ontogenetic features are: (1) in the proter, the parental adoral zone of membranelles is completely renewed by new structures; (2) in the opisthe, the oral primordium originates apokinetally, some old midventral cirri join the formation of frontoventral-transverse cirral anlagen; (3) the anlagen for marginal rows and dorsal kineties develop intrakinetally; and (4) the numerous macronuclear nodules fuse into a single mass before dividing. Phylogenetic analyses based on the SSU rDNA sequence suggests the non-monophyly of the genus Bakuella. Conclusions: The morphology, morphogenesis and molecular phylogeny of the new hypotrichous ciliate, Bakuella (Pseudobakuella) guangdongica n. sp. were studied, the phylogenetic analyses show that the Bakuella is polyphyletic in the SSU rDNA.

The genus Bakuella was erected by Agamaliev and Alekperov [12] with Bakuella marina Agamaliev & Alekperov, 1976 as the type species. This genus is characterized by adoral zone of membranelles continuous, three enlarged frontal cirri, one or more buccal cirri, two or more frontoterminal cirri, midventral complex composed of obliquely arranged midventral pairs and midventral rows, one left and one right marginal cirral row, transverse cirri present and caudal cirri absent. So far, there are 13 species/subspecies that belong to this genus [1,[13][14][15]. Berger [1] divided Bakuella into two subgenera, Bakuella (Bakueklla) and B. (Pseudobakuella), mainly according to the number of frontoterminal cirri. The former has more than two frontoterminal cirri, while the latter has exactly two.
In March 23, 2016, a new soil urostylid ciliate was isolated from Guangdong, China. Observations of its morphology both in vivo and after protargol staining demonstrate that it represents a novel species within the genus Bakuella. In the present study, its morphology and morphogenesis were described. The small subunit ribosomal DNA (SSU rDNA) of the new isolate was sequenced and analysed in order to estimate its phylogenetic position.

Division of nuclear apparatus
The nuclear apparatus divides in the usual way for urostylids, i.e. all macronuclear nodules fuse to form a single mass during the ontogenetic process and then divide into many nodules. Micronuclei were observed to divide mitotically during the morphogenesis (Figs. 3E, G, I, 4B, D Consistent with previous studies [16][17][18][19], the urostyloids are non-monophyletic in our phylogenetic tree. Bakuella (Pseudobakuella) guangdongica n. sp. is placed within the core urostylids and clusters together with Bakuella (Bakuella) granulifera, Neobakuella aenigmatica, Anteholosticha antecirrata and Urostyla grandis with full support. This group is sister to a clade formed by three Bakuella spp., two Diaxonella spp., Neobakuella ava and Apobakuella fusca (ML/BI, 97/1.00).

Morphological comparison with congeners
According to Berger [1] and recent studies [13,15], there are 13 species included in the genus Bakuella, which consists of two subgenera discriminated by the number of frontoterminal cirri (more than two in Bakuella (Bakuella) vs. exactly two in Bakuella (Pseudobakuella). According to this de nition, our form should be assigned to Bakuella (Pseudobakuella) and compared with three congeners, i. slightly overlapped), shorter length of midventral complex (extending to 70% of body length vs. near transverse cirri), and two types, yellowish or yellowish to colorless (vs. one type, colorless) cortical granules [14].
The ambiguous relationship of these ve Bakuella species might be due to the limited sampling of representative taxa in the SSU rDNA tree. Therefore, SSU rDNA sequences from more taxa, especially the type species Bakuella marina, and sequence data for additional gene markers, are needed in order to enhance our understanding of the molecular phylogeny of Bakuella.

Conclusions
In this manuscript, the new soil species, Bakuella (Pseudobakuella) guangdongica n. sp. collected from the Seaside Garden, expand the knowledge of biodiversity of ciliates. Moreover, the molecular data of the species is provided for the rst time and the phylogenetic relationships among related genera and species are discussed. Ciliates were made to excyst by employing the non-ooded Petri dish method as described by Foissner [23]. A non-clonal culture was established at room temperature (about 24 °C) in Petri dishes containing mineral water (Nongfu Spring) with rice grains to enrich the bacterial food. The probability is therefore extremely high that our morphological, morphogenetic and molecular studies deal with the same species although we were unable to establish clonal cultures, because we were able to identify the species accurately based on its in vivo morphologic characteristics.

Morphology and morphogenesis
Cells were observed in vivo using bright eld and differential interference contrast microscopy. The protargol staining [24] was used to reveal the ciliature and nuclear apparatus, protargol was made according to Pan et al. [25]. Counts and measurements of stained specimens were performed at a magni cation of 1,000×. Drawings of protargol-prepared cells 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 and Systematics are mainly according to Berger [1].
DNA extraction, PCR ampli cation, and sequencing One cell of Bakuella (Pseudobakuella) guangdongica n. sp. was isolated and repeatedly washed using sterile distilled water. Then it was 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. PCR ampli cation and sequencing of the SSU rDNA were performed according to Wang et al., [26] using the eukaryotic universal SSU rDNA primers 18S-F (5'-AAC CTG GTT GAT CCT GCC AGT-3') and 18S-R (5'-TGA TCC TTC TGC AGG TTC ACC TAC-3') [27]. High-delity Taq polymerase (Takara Ex Taq; Takara Biomedicals) was used to minimize the possibility of ampli cation errors. Polymerase chain reaction conditions for the SSU rDNA ampli cation were as follows: 2 min initial denaturation (98 °C) followed by 30 cycles of denaturation for 10 s at 98 °C, annealing for 15 s at 56 °C, and extension for 1 min 50 s at 72 °C, with a nal extension of 7 min at 72 °C. Sequencing of the PCR products was performed bidirectionally on an ABI 3700 sequencer (Invitrogen sequencing facility, Shanghai, China).

Phylogenetic analyses
In order to perform phylogenetic analyses, the SSU rDNA of Bakuella (Pseudobakuella) guangdongica n. sp. was aligned with sequences of 63 other hypotrichs downloaded from GenBank database (for accession numbers, see Fig. 6). Four euplotid species, namely Apodiophrys ovalis, Diophrys scutum, Paradiophrys zhangi, and Uronychia multicirrus, were used as outgroup taxa. All sequences were aligned using the GUIDANCE web server (http://guidance.tau.ac.il/, [28]). 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 v8.1.11 [29,30] on the online server CIPRES Science Gateway [31]. The reliability of internal branches was assessed using a nonparametric bootstrap method with 1,000 replicates. Bayesian inference (BI) analyses were carried out using MrBayes on XSEDE v3.2.6 [32] on CIPRES Science Gateway with the GTR + I + G model selected by Akaike Information Criterion (AIC) in MrModeltest v2 [33]. 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 5,000 trees as a burn-in (25%). All remaining trees were used to calculate posterior probabilities using a 50% majority rule consensus. TreeView v1.6.6 and MEGA v5 were used to visualize the tree topologies [34,35].

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
Sequence data are available in GenBank (Accession Number: MT435536).
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 Bakuella (Pseudobakuella) guangdongica n. sp. (registry no. WJY2016032301B) and one paratype slide (registry no. WJY2016032301C) 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.