Phylogeny of two poorly known ciliate genera (Ciliophora, Heterotrichea), with notes on the redefinition of Gruberia uninucleata Kahl, 1932 and Linostomella vorticella (Ehrenberg, 1833) based on populations found in China CURRENT STATUS:

Heterotrichous ciliates are common members of microeukaryote communities which play important roles in the transfer of material and energy flow in aquatic food webs. This group has been known 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. The morphology and phylogeny of two poorly known heterotrichous ciliates, Gruberia uninucleata Kahl, 1932 and Linostomella vorticella (Ehrenberg, 1833) Aescht in Foissner et al. , 1999, were investigated based on their living morphology, infraciliature, and small subunit (SSU) rDNA sequence data. Based on a combination of previous and present studies, detailed morphometric data and the improved diagnoses of both species are supplied here. In addition, molecular data of the two species are reported for the first time. Phylogenetic analyses based on SSU rDNA sequence data support the generic assignment of these two species. ,

Gruberia uninucleata was originally discovered by Kahl [12] from an aquarium in Helgoland, Germany.
According to the original description, this organism is '300-650 μm, slender and spindle-shaped body, pointed caudal region, somatic kineties 16-20, single ellipsoidal macronucleus' ( Table 2). The Qingdao population matches the original description in body shape and size, peristome length, macronucleus, and habitat. The only significant difference is the number of somatic kineties (25-37 in Qingdao population vs. 16-20 in Helgoland population). As the number of somatic kineties was derived from living observation in the original description, it is likely that the author overlooked some shortened and marginal somatic kineties.
Gruberia uninucleata is a rare species. Apart from the original description, it has been reported only four times. Dragesco [20] isolated two populations from fine sands at Roscoff and Green Island, which corresponded closely to the Helgoland population except the Roscoff populations had a longer peristome and the Green Island population had a terminal contractile vacuole (Table2). Based on our observations, we hypothesize that Dragesco [20] mistook a food vacuole for the contractile vacuole ( Figure 3J). Subsequently, Dragesco [21] described a Port-Etienne population which is smaller (200 μm on average) than both the original and the Qingdao populations ( Table 2). Chorik [28] reported a freshwater population of G. uninucleata which corresponds well with Kahl's population (Table 2).
However, we suspect that it is possibly a misidentification because it resembles Blepharisma hyalinum in terms of its body shape, macronucleus shape, a contractile vacuole at posterior end, and habitat [29]. Dragesco [22] Table 2). We believe that this difference is probably a statistical error because only six individuals were counted by Dragesco [22].
In summary, the original population of Kahl [12] and that of Dragesco [22] both have about 20 somatic kineties. In contrast, the Qingdao population and that of Dragesco [21] both have about 40 somatic kineties. Based on the different numbers of somatic kineties, we believe that these populations represent two different species. However, further studies are needed, including molecular data, for the populations from France and Germany in order to test this hypothesis.     Table 3) [6].
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.
With reference to its general infraciliature, body shape and locomotion, Linostomella is also similar to the genus Condylostomides. However, the former can be separated from the latter by the lack of frontal membranelles (present in Condylostomides) [37].
As described above, L. vorticella was originally reported by Ehrenberg [8] under the name Bursaria vorticella. This description, however, was rather superficial and the failure to describe some important features (e.g. body size, macronuclear shape, etc.) 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 and 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).
Gelei [38] reported an organism that resembles L. vorticella in all key characters except the number of somatic kineties (60-70 vs. 26-51 in populations of L. vorticella) ( Table 3). We hypothesize that this population may represent a different species of Linostomella, however the description provided by Gelei [38] was only brief therefore its identity awaits further information. Dragesco [39] described an isolate collected from a freshwater pond in Mokolo, Cameroon, which has fewer adoral membranelles (19-22 vs. 36-51) than other populations of L. vorticella (Table 3). Foissner et al. [7] suspected that this population either represents a different species or was mis-observed. Alekperov et al. [6] reported the only marine population of L. vorticella but provided only a simple description (Table 3).
In general, the habitat is an important character for species circumscription, so further evidence is needed in order to verify the identity of this population.
Phylogenetic analyses based on SSU rDNA sequences 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 uninucleata, 1627bp, 46.22%, MN783327; Linostomella vorticella, 1683bp, 46.88%, MN783328. The ML and BI trees based on SSU rDNA data had nearly identical topologies, therefore only the ML tree is shown with support values from both analyses (Figure 8).
Seven sequences of Gruberia were included in the present analyses, i.e., the newly obtained sequence of G. uninucleata and six sequences obtained from the GenBank database. The seven sequences form a maximally supported clade (100% ML, 1.00 BI) that represents the family Gruberiidae in the SSU rDNA tree (Figure 8). Based on its fragmented paroral membrane, Shazib et al. [3] separated Gruberia from the family Spirostomidae and established for it 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 I' in Figure 8). This is congruent with the findings of previous studies [3][4][5][40][41][42], and supports the scenario proposed by Luo et al. [41], which recognized that only species of 'Clade I' 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 I' [40].
The new sequence of L. vorticella differs from the two unspecified sequences (LN869952, LN870136) by 14 and 9 nucleotides respectively. We suspect that these unspecified sequences may represent different species. The genus Linostomella is most closely related to Condylostomides in the SSU rDNA tree which is consistent with the morphological similarities of these two taxa and their placement in the family Condylostomatidae [1]. The similarities include their habitat (freshwater), body shape (ellipsoidal), oral apparatus (conspicuous buccal cavity with adoral zone membrane on the left and paroral membrane on the right), presence of a contractile vacuole, and moniliform macronucleus [7,37]. The monophyletic family Condylostomatidae comprises two clearly separated sub-clades, namely Linostomella + Condylostomides and Condylostoma + Condylostentor + Chattonidium. This division is probably related to the difference in habitat, members of the former clade inhabiting freshwaters whereas members of the latter clade are marine.

Conclusions
In the present paper we redescribe two poorly known heterotrich ciliates, Gruberia uninucleata and Linostomella vorticella, based on populations collected from Qingdao, China, using the integrative approach suggested by Warren et al. [43]. Moreover, the molecular data of these two species are provided for the first time. Improved diagnoses of these two species are supplied based on present and previous descriptions, and the phylogenetic relationships among related genera and species are discussed.
Living cells were randomly selected from the original samples and observed at 100-1000× magnification using both bright field and differential interference contrast microscopy (Olympus BX53; Zeiss AXIO Imager. D2). The protargol staining method of Wilbert [44] was used to reveal the infraciliature. The protargol powder was made according to Pan et al. [45]. 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 Lynn [1] and Shazib et al. [3]. 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 18s-R (5'-TGATCCTTCTGCAGGTTCACCTAC-3') [47,48]. Cycling parameters of touchdown PCR were as follows: 1 cycle of initial denaturation at 98 °C for

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 Figure 8 for accession numbers). Five karyorelictean species were used as the outgroup. Sequences were aligned using MUSCLE on the web server GUIDANCE ((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 1,431 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.

Declarations
Ethics approval and consent to participate Not applicable.

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Availability of data and materials
All data generated or analysed during this study are included in the published article.

Competing interests
The authors declare that they have no competing interests.  Tables   Table 1. Morphometric data for Qingdao populations Gruberia uninucleata and Linostomella vorticella. arithmetic mean; Min, minimum; n, number of specimens; SD, standard deviation. * Data based on protargol-stained specimens, † Macronuclear nodules were selected randomly in each individual, -Data not available.  Ehrenberg [8] almost spherical body, large and oblique oral cavity in front -Wrześniowski [10] ovoid body with a broadly rounded rim 210 Penard [11] ovoid body with broad back and truncated forward 200 Fauré-Fremiet [31] globular, hemispherical or ovoid body with obliquely truncated anterior 100-125 Kahl [12] bag-shaped, truncated in front 100-200 Wang & Nie [19] ovoid body, large and evenly rounded toward the posterior extremity, truncated at the anterior end  [17] bag-shaped, truncated in front, rounded at the back 170-200 Foissner et al. [7] saccular to ellipsoidal, both ends broadly rounded, ventral anterior half obliquely truncated