New contributions to two ciliate genera (Ciliophora, Heterotrichea) based on morphological and molecular analyses, with description of a new Gruberia species

Background Heterotrichous ciliates are common members of microeukaryote communities which play important roles in both the transfer of material and the flow of energy in aquatic food webs. This group has been known for over two centuries due to their large body size and cosmopolitan distribution. Nevertheless, species identification and phylogenetic relationships of heterotrichs remain challenging due to the lack of accurate morphological information and insufficient molecular data. Results The morphology and phylogeny of two heterotrichous ciliates, namely Gruberia foissneri spec. nov. and Linostomella vorticella (Ehrenberg, 1833) Aescht in Foissner et al., 1999, were studied using rigorous methods (living morphology, stained preparations, and small subunit rDNA sequence data). Gruberia foissneri spec. nov. is morphologically very similar to G. uninucleata Kahl, 1932, however, it can be distinguished from the latter by having more ciliary rows (about 32 vs. about 20) and macronuclear shape (sausage-shaped vs. ellipsoid). Based on a combination of previous and present studies, an improved diagnosis of L. vorticella is supplied and several taxonomic anomalies are clarified. In addition, phylogenetic analyses based on SSU rDNA sequence data support the generic assignment of these two species. Conclusions Modern ciliate taxonomy should be performed by means of detailed living observation, stained preparations and molecular information. For those species that have been reported in previous studies, it is necessary to provide as much useful information as possible using state-of-the-art methods in order to resolve taxonomic anomalies.


Background
Members of the ciliate class Heterotrichea Stein, 1859 are found in a wide range of aquatic biotopes. The heterotrichs are characterized by their typically large body size, somatic kineties composed of dikinetids with postciliodesmata and a prominent oral apparatus composed of a paroral membrane and an adoral zone of membranelles [1,2]. According to the two latest works on the classification of heterotrichs [3,4], the class Heterotrichea contains ten families and about 58 genera, several of which are well-known, e.g., Condylostoma Bory de St. Vincent, 1824, Spirostomum Ehrenberg, 1834, and Stentor Oken, 1815. Gruberia Kahl, 1932 is rarely reported and has only three valid species: G. binucleata Dragesco, 1960, G. lanceolata (Gruber, 1884) Kahl, 1932, and G. uninucleata Kahl, 1932 [5,6]. Of these, only G. lanceolata has been investigated using modern methods while its congeners remain insufficiently described [5,6].
The genus Linostomella Aescht in Foissner et al., 1999 is monotypic and classified within the family Condylostomatidae Kahl in Doflein and Reichenow, 1929. The type species, L. vorticella, was first reported by Ehrenberg [7] as Bursaria vorticella due to the similarity of its body shape with the colpodid B. truncatella. Dujardin [8] doubted Ehrenberg's classification and transferred this species to the heterotrich genus Condylostoma because of it is holotrichous somatic ciliation and the conspicuous, spiraled adoral zone of membranelles. More than a century later, Jankowski [9] established the genus Linostoma for this species because it has no frontal cirrus/cirri, which is a diagnostic characteristics of Condylostoma. Subsequently, Aescht [10] recognized that Linostoma is a homonym and re-named it Linostomella. Recently, Rossi et al. [11] reported the molecular phylogenetic position of this genus.
In the present study, two heterotrich species, namely Gruberia foissneri spec. nov. and Linostomella vorticella, were isolated in Qingdao, China (Fig. 1), giving the opportunity to investigate their taxonomy and phylogeny based on both morphological and molecular data.

Type deposition
One protargol-stained slide containing the holotype specimen marked with an ink circle and one slide with paratype specimens are deposited in the Laboratory of

Gene sequence
The SSU rDNA sequence derived from a single cell isolated from the same population as the holotype is deposited in GenBank (accession number MN783327).
Length of oral area relative to body length highly variable, ranging from 25 to 45% (Fig. 2b, Fig. 3a-e). Adoral zone extends from apical end to main body, oral groove slightly curved to right side, twisted in proximal region making a half-turn as it enters the buccal cavity (Fig. 2h, i, Fig. 3a-g, Fig. 4b, d). About 76-174 adoral membranelles, each composed of one short and two long rows of basal bodies (Fig. 2c, h, Fig. 4b, d). Cilia of membranelles 11-16 μm long in vivo. Paroral membrane fragmented into about 29-75 pieces and arranged along right side of adoral zone of membranelles, almost all fragments composed of two rows of kinetosomes except several anterior ones    and protargol-stained individuals) (present work:  Table 3) [23].
Prior to the current investigation, Linostomella vorticella has been found and reported numerous times, but some details of its morphology remain unknown. Based on both previous and present studies, an improved diagnosis is supplied.
Buccal cavity prominent, length about 35-60% of body length, with numerous oral ribs (Fig. 5h, Fig. 6a-c, Fig. 7c,  d). Oval glabrous protuberance with fiber-like stripes visible in slightly squashed specimens (Fig. 5b, Fig. 6i, m). Adoral zone of membranelles prominent, composed of 36-51 membranelles, most of which consist of two rows of basal bodies of equal length; several adoral membranelles in middle portion consist of three rows of basal bodies, third row with only two or three basal bodies (Fig. 5d, f, Fig. 7e). Cilia of adoral membranelles 20-30 μm long in vivo. Paroral membrane conspicuous, curved and lies along right margin of buccal cavity, anterior portion curves toward the left side of buccal cavity, posterior portion located near distal end of adoral zone (Fig. 6a, e, Fig. 7b-d).

Molecular data and phylogenetic analyses
The two new SSU rDNA sequences obtained in this study were deposited in the GenBank database with lengths, G + C contents, and accession numbers as follows: Gruberia foissneri spec. nov., 1627 bp, 46.22%, MN783327; Linostomella vorticella, 1683 bp, 46.88%, MN783328. The Maximum likelihood (ML) and Bayesian inference (BI) trees based on SSU rDNA data had nearly identical topologies, therefore only the ML tree is shown with support values from both analyses (Fig. 8).
Seven sequences of Gruberia were included in the present analyses, i.e., the newly obtained sequence of G. foissneri spec. nov. and six sequences obtained from the GenBank database. These seven sequences form a maximally supported clade (100% ML, 1.00 BI) that represents the family Gruberiidae in the SSU rDNA tree (Fig. 8).

Discussion
Comments on Gruberia foissneri spec. nov.
The genus Gruberia was established by Kahl [15] with G. uninucleata as the type species. The morphology of Gruberia is similar to that of Spirostomum in having an elongated, slightly contractile body and a well-developed peristomial region, although the body of Gruberia lacks spiraling or torsion [6,27] Lepsi, 1965, and G. uninucleata Kahl, 1932 [15, 20, 28-32]. In their generic review, Campello-Nunes et al. [5] and Chen et al. [6] synonymized G. aculeata, G. beninensis and G. calkinsi with G. lanceolata, and considered G. nematodomorpha as a nomen nudum. We accept these decisions and recognize only four valid species, namely G. uninucleata, G. binucleata, G. lanceolata and G. foissneri spec. nov.
Gruberia foissneri spec. nov. can be easily distinguished from two of its three congeners by its sausageshaped macronucleus (vs. two oval macronuclei in G. binucleata and a moniliform macronucleus in G. lanceolata) ( Table 2) [5,6,29,30]. In contrast, G. foissneri spec. nov. is very similar to G. uninucleata which was originally discovered by Kahl [15] from an aquarium in Helgoland, Germany. Kahl [15] described the organism based on living observations as follows: "Gr. 300-650μ; Schlank spindelförmig, im hinteren Drittel gleichmäßig zu einem dünnen Schwanzstachel ausgezogen, der mit kurzkonischer Spitze endigt; 8-10 Reihen auf einer Seite; Ma, ellipsoid" (translation: size 300-650 μm; slender spindle-shaped, posterior third evenly narrowed to a thin tail ending with short conical tip; 8-10 ciliary rows on one side; macronucleus, ellipsoid) ( Table 2). Dragesco [33] supplied comprehensive data of a Roscoff population based on living morphology and infraciliature ( Table 2). According to these two reports, G. uninucleata can be characterized by: (1)  It is worth noting that Dragesco [34] described a smaller Gruberia uninucleata (200 μm on average) based on living observations of a Port-Etienne population. Like the population described by Kahl [15], this population has an ellispoidal macronucleus but possesses about 40 (vs. 8-10 on one side in the population described by Kahl) somatic kineties. In view of the unavailability of key morphological characters and difference in the number of somatic kineties, we suspect that this population may either be conspecific with Gruberia foissneri spec.
nov. or represent another species. Further studies are needed to test this hypothesis.
Linostomella vorticella was originally reported by Ehrenberg [7] under the name Bursaria vorticella. Ehrenberg's description, however, was rather superficial which made the subsequent re-identification of this organism difficult. According to the original and subsequent investigations, this species should be recognizable by the following characters: (1) body shape spherical to ellipsoidal, posterior end rounded, anterior end always slightly truncated; (2) conspicuous oral cavity that occupies about half the body length; (3) macronucleus moniliform with nodules arranged in a horseshoe-shape or an oblique line; (4) contractile vacuole at the posterior end of the body with a long collecting canal (Table 3). Furthermore, three populations (two from Germany and one from Austria) were investigated using a combination of in vivo observations and histological staining methods and were found to closely resemble the original population [7,19,21]. The Qingdao population corresponds closely with the populations from Europe. We therefore believe that its identification as L. vorticella is correct.

Phylogenetic analyses based on SSU rDNA sequences
Based on its fragmented paroral membrane, Shazib et al. [4] separated Gruberia from the family Spirostomidae and established the new family Gruberiidae. This assignment is supported by the present phylogenetic analyses, in which Gruberia is clearly divergent from the family Spirostomidae. All sequences of Gruberia form a clade that is the sister-group of the Stentoridae + Blepharismidae + Folliculinidae + Maristentoridae + Fabreidae clade ('Clade SBFMF' in Fig. 8). This is consistent with the findings of previous studies [3][4][5][6][39][40][41], and supports the scenario proposed by Luo et al. [39], which recognized that only species of 'Clade SBFMF' possess hypericin-like pigment granules. It is suggested that these pigment granules probably play important roles in the evolution of the class Heterotrichea, including the separation of Gruberia from 'Clade SBFMF' [3].
The genus Linostomella is most closely related to Condylostomides in the SSU rDNA tree which is consistent with the phylogenetic analyses in Rossi et al. [11]. The similarities of these two taxa in terms of habitat (freshwater), body shape (ellipsoidal), oral apparatus (conspicuous buccal cavity with adoral zone membrane on the left and paroral membrane on the right), contractile vacuole (present), and macronuclear shape (moniliform) [22,42] support their close evolutionary relationship. The monophyletic family Condylostomatidae comprises two clearly separated sub-clades, namely Linostomella + Condylostomides and Condylostoma + Condylostentor + Chattonidium, which is broadly consistent with the findings of Rossi et al. [11]. We suspect that the separation of these sub-clades is probably related to differences in habitat, members of the former clade inhabiting freshwaters whereas members of the latter clade are marine.
The new sequence of Linostomella vorticella differs from the two unspecified Linostomella sequences (LN869952, LN870136) by 14 and 9 nucleotides respectively. This finding, combined with descriptions of populations that differ significantly in their morphology, suggests that the genus Linostomella may be not be monotypic.

Conclusions
In the present paper we describe two heterotrich ciliates, Gruberia foissneri spec. nov. and Linostomella vorticella, collected from Qingdao, China, using an integrative approach as suggested by Warren et al. [43]. Although G. foissneri spec. nov. closely resembles G. uninucleata, we provide evidence that these are separate species. In addition, an improved diagnosis of L. vorticella is supplied based on present and previous descriptions. Based on analyses of its morphology and molecular phylogeny, we posit that the genus Linostomella is not monotypic.
Living cells were randomly selected from the original samples and observed at 100-1000× magnification using both bright field and differential interference contrast The protargol staining method of Wilbert [44] was used to reveal the infraciliature. The protargol powder was made according to Pan et al. [45]. The invertible function in Photoshop was used to adjust the photomicrographs of the infraciliature to show the structure more clearly. Hoechst 33342 solution was used to reveal the nuclear apparatus [46]. Counts, measurements, and drawings of stained specimens were made from photomicrographs (Nikon Y-IDT). Terminology and systematics are mainly according to Foissner et al. [22], Lynn [2] and Shazib et al. [4].

DNA extraction, PCR amplification, and sequencing
A single cell of each species was isolated from the original sample and washed five times with filtered habitat water to remove potential contaminants. Extraction of genomic DNA was performed using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. Q5® Hot Start high-fidelity DNA polymerase (NEB, Ipswich, MA) was used to amplify the SSU rDNA using universal eukaryotic primers 82F (5′-GAAACTGCGAATGGCTC-3′) and 18 s-R (5′-TGATCCTTCTGCAGGTTCACCTAC-3′) [47,48]. Cycling parameters of touchdown PCR were as follows: 1 cycle of initial denaturation at 98°C for 30 s, followed by 18 cycles of amplification (98°C, 10 s; 69-51°C touchdown, 30 s; 72°C, 1 min), and another 18 cycles (98°C, 10 s; 51°C, 30 s; 72°C, 1 min), with a final extension of 72°C for 5 min. PCR products were checked using agarose gel and were sequenced in TSINGKE (Qingdao, China). Sequence fragments were assembled into contigs using Seqman (DNAStar).

Phylogenetic analyses
A total of 96 taxa were used for phylogenetic analyses, including the two newly sequenced species and 94 sequences obtained from the GenBank database (see Fig. 8 for accession numbers). Five karyorelictean species were used as the outgroup. Sequences were aligned using MUSCLE on the web server GUID-ANCE (http://guidance.tau.ac.il/ver2/) with default parameters [49]. Ambiguously aligned regions were excluded before phylogenetic analyses using G-blocks version 0.91b [50,51]. The final alignment with 1431 characters was used to construct phylogenetic trees. Maximum likelihood (ML) analysis was carried out on the CIPRES Science Gateway [52] using RAxML-HPC2 on XSEDE v8.2.12 [53]. Bayesian inference (BI) analysis was performed with MrBayes version 3.2.6 on XSEDE [54,55] of the CIPRES Science Gateway. GTR+ I+ G was selected as the best fitting evolutionary model by MrModeltest version 2.2 according to the Akaike Information Criterion (AIC) [56]. Markov chain Monte Carlo simulations were then run with two sets of four chains using the default settings. The chain length for the analysis was 10,000,000 generations with trees sampled every 100 generations. The first 10% of trees were discarded as burn-in. MEGA 5.2 [57] was used to visualize tree topology.