Study on Analysis of Several Molecular Identification Methods for Ciliates of Colpodea (Protista, Ciliophora)

The application of molecular techniques to accurately identify protozoan species can correct previous misidenti ﬁ cations based on traditional morphological identi ﬁ cation. Colpodea ciliates have many toxicological and cytological applications, but their subtle morphological di ﬀ erences and small body size hinder species delineation. Herein, we used Cox I and β - tubulin genes, alongside ﬂ uorescence in situ hybridization (FISH), to evaluate each method in delineating Colpodea species. For this analysis, Colpoda harbinensis n. sp., C. reniformis , two populations of C. in ﬂ ata , Colpoda compare grandis , and ﬁ ve populations of Paracolpoda steinii , from the soil in northeastern China, were used. We determined that (1) the Cox I gene was more suitable than the β - tubulin gene as a molecular marker for de ﬁ ning intra- and interspeci ﬁ c level relationships of Colpoda . (2) FISH probes designed for Colpoda sp., C. in ﬂ ata , Colpoda compare grandis , and Paracolpoda steinii , provided rapid interspeci ﬁ c di ﬀ erentiation of Colpodea species. (3) Colpoda harbinensis n. sp. was established and mainly characterized by its size in vivo (approximately 80 × 60 μ m ), a reniform body in outline, one macronucleus, its spherical shape, a sometimes nonexistent micronucleus, 11 – 15 somatic kineties, and ﬁ ve or six postoral kineties. In conclusion, combining oligonucleotide probes, DNA barcoding, and morphology for the ﬁ rst time, we have greatly improved the delineation of Colpodea and con ﬁ rmed that Cox I gene was a promising DNA barcoding marker for species of Colpodea, and FISH could provide useful morphological information as complementing traditional techniques such as silver carbonate.

Several surveys of DNA barcoding in Ciliophora have shown a high prevalence [18][19][20][21][22]. The Cox I gene is a suit-able marker for resolving the interspecific and intraspecific relationships of Paramecium spp. [22]. The internal transcribed spacer 2 region (ITS2) is also a strong barcoding candidate for identifying the closely related Tintinnids [23]. Molecular phylogenies and genetic measurements based on variable regions of nuclear genes demonstrated that the ITS2 and LSU-D1/D2 regions are more suitable for delineating Euplotes [24]. Fluorescent probes targeting small subunit ribosomal RNA (SSU-r RNA) have been designed and optimized for fluorescence in situ hybridization (FISH), resulting in the accurate and rapid identification of pathogenic ciliates (e.g., Pseudocohnilembus persalinus, Boveria labialis, and B. subcylindrica) [25][26][27][28]. FISH allows for molecular identification of targeted organisms in mixed populations, overcoming the negatives of morphological methods and producing timely detection results. However, there are currently no available fluorochrome-labeled oligonucleotide probes for the genus Colpoda.
The class Colpodea (Small and Lynn [29]) comprises approximately 60 genera and 200 species, with most living in terrestrial and semiterrestrial habitats, such as mosses, leaf litter, soil, and tree holes [30][31][32][33][34]. However, this is likely only a subset of the total diversity, with a high number of species likely undiscovered [35]. Colpodea is typically characterized by high technical requirements for staining, environmental sensitivity, susceptibility to dormant cysts, and few multigene sequences, resulting in long-standing problems with species identification and taxon attribution [13,30,[36][37][38][39][40][41]. To date, the identification of ciliates of Colpodea has relied solely upon morphological features and SSU rDNA sequence analysis. However, with the conservative evolution of SSU rDNA alongside various issues such as asynchronous evolution with morphology, delineation remains problematic. Therefore, other methods, including DNA barcoding and oligonucleotide probes, should be developed to accurately and rapidly identify Colpodea. The uses of DNA barcoding and FISH are universally applicable tools that can identify ciliates and confirm taxonomic relationships previously based on ultrastructural and other morphological features [22,[26][27][28]42].
Nonetheless, there is still no universal gene marker for species discrimination of ciliates. In the present investigation, we assessed the suitability of DNA barcoding and oligonucleotide probe techniques to delineate ten newly isolated Chinese populations of five Colpodea species. Specifically, we investigated the barcoding utility of β-tubulin and the mitochondrial cox1 genes, both at the congeneric and conspecific levels, in order to analyze the reliabilities of molecular identification methods for ciliates of Colpodea.

Material and Methods
2.1. Ciliate Isolation, Observation, and Identification. Five species were collected from soil in northeastern China and treated with nonflooded Petri dish cultures as described in Foissner et al. [43]. After isolation, specimens were maintained in Petri dishes in the laboratory for three days. Clonal cultures were then established and maintained at room temperature in boiled water amended with a grain of wheat to enrich natural bacteria as food for the ciliates. Isolated cells were observed and photographed in vivo using differential interference contrast microscopy. The silver carbonate [44] was used to reveal the infraciliature in different morphogenetic stages. Stained specimens were counted and measured at magnifications of ×100-1250, and mapping was performed with the help of a drawing device. Classification and terminology are mainly according to Foissner [30] and Lynn [45].
2.2. DNA Extraction, PCR Amplification, and Sequencing. Five cells from each monoclone were isolated under the stereomicroscope using micropipettes and washed with double distilled water to remove contaminants. Cells were then transferred to an Eppendorf tube with a small amount of water. Total genomic DNA of the cells was extracted with the DNeasy & Tissue Kit (Shanghai, QIAGEN, Germany) according to the manufacturer's instructions.
The β-tubulin and the Cox I genes were amplified using the polymerase chain reaction (PCR). PCR primers are listed in Table 1, and conditions of the respective PCR reactions  are summarized in Table 2. Sequencing was performed Shanghai Sangon Biological Engineering and Technical Service Company (Shanghai, China). 36 new molecular sequences of β-tubulin and Cox I genes were generated from five species of Colpodea. All the sequences were aligned using Clustal W implemented in BioEdit 7.0.1 [46]. (Table 3) were designed using the probe design tool as implemented in the ARB software package for the SSU-rDNA sequences of the present Colpoda harbinensis n. sp, C. inflata, Colpoda compare grandis, and Paracolpoda steinii. Generated probes were checked against the GenBank sequence collection by a standard nucleotide-nucleotide BLAST search [47]. FISH was used to visualize Colpodea spp. above both in field samples and a mixture of species as well as Coleps hirtus that frequently occurred in the same habitats as the negative control. Cells were fixed with 50% Bouin's solution and filtered onto a 2 μm-pore-size cellulose nitrate membrane (25 mm in diameter) using low under pressure. The membrane was then washed five times with 2 ml of filtered sterile water. The basic hybridization follows the protocol of Stoeck et al. [48] and Zhan et al. [26].

Haplotype Networks.
A β-tubulin haplotype network was constructed for Paracolpoda steinii and Colpoda inflata, using the TCS method [53] as implemented in PopART ver. 1.7 [54]. Mutations in β-tubulin sequences were displayed as line segments on the haplotype network.

Phylogenetic Analyses Based on 18S rRNA Gene Sequence
Data. Phylogenetic trees were constructed using ML and BI and produced similar topologies; therefore, only the ML trees and their support values from both methods are shown. According to the 18S-rRNA gene tree, all four orders within Colpodea were monophyletic (Figure 3). Colpodida and Cyrtolophosidida clustered together to form a clade, with Bursariomorphida as a sister clade, while the order Platyophryida occupied the basal position within Colpodea.
The newly sequenced species Paracolpoda steinii was sister to the clade clustered by P. steinii (KJ607914) and

Cellular Microbiology
Bromeliothrix metopoides (100% ML, 0.9 BI). All nine newly sequenced species were clustered within the core of the Colpodea clade. The two newly sequenced species, Colpoda compare grandis and C. reniformis, formed a sister group, which then grouped with C. henneguyi and Bresslauides discoideus. The newly sequenced Colpoda harbinensis n. sp., C. inflata pop1, and C. inflata pop2 clustered together. The seven Paracolpoda steinii sequences, including the five newly sequenced populations, clustered together as a sister group to Bromeliothrix metopoides with full support (100% ML, 1.00 BI).  (Table 1) yielded a single DNA band of the predicted length (~945 bp) from Colpoda compare grandis, C. inflata pop. 2, Paracolpoda steinii pop. 2, and Paracolpoda steinii pop. 3 isolates. Therefore, each PCR product was cloned, and the partial Cox I sequences were deposited in GenBank under the respective accession numbers OM752200, OM752201, OM752202, and OM752203.

Cellular Microbiology
Their GC contents were 28.56%, 26.42%, 27.87%, and 27.75%, respectively, with sequence differences shown in Figure 4. Base variations between populations of Colpoda compare grandis, C. inflata, and Paracolpoda steinii were large, ranging from 12.01% to 14.88%, while the base variation between individuals within the Paracolpoda steinii population was small, at 0.35%.

The Utility of β-Tubulin Gene Tested for Accurate
Identification. The β-tubulin amplification primers 349A and 349B (Table 1) generated a total of 33 DNA sequences of predicted length (~980 bp) from C. inflata (populations 1-2), Paracolpoda steinii (populations 1-5), and C. harbinensis n. sp. isolates. The interspecific genetic distances of β-tubulin of Colpoda ranged from 0.59% to 8.80%, and intraspecific genetic distances ranged from 0.89% to 5.81%. The TCS network of β-tubulin genes revealed the C. inflata, the largest difference between pop. 1 and pop. 2 was 60 genetic steps (5a and 2a), while the smallest difference was nine genetic steps (1b and 4). There were large genetic step differences among individuals within the same pop, e.g., 60 genetic step differences between 5a and 4 in pop. 2

Gene Sequence Data.
The SSU rDNA sequence of Colpoda harbinensis sp. nov. has been deposited in the GenBank database with the accession number, length, and G + C content as follows: MZ557804, 1716 bp, and 44.23%. reniformis is similar to previous populations, as they share a distinctly nephrogenic body shape in vivo and an ellipsoid macronucleus between their vestibulum and dorsal side but is distinct in their large body size (123 -130 × 85 -95 μm in the present study vs. 90-100 μm) and absence of micronucleus (vs. presence in the previous populations [30,55].

Colpoda
Compare grandis Smith, 1899. Colpoda compare grandis has many features that are similar to those of C. grandis: body reniform in vivo (about 2 : 1) with a distinct indentation at its vestibular entrance sometimes absent, laterally flattened, no postoral sack, contractile vacuole, cytopyge near its posterior end, extrusomes conspicuous and numerous, left oral polykinetid on the vestibular bottom, and elongate square [30,56]. However, Colpoda compare grandis differs from C. grandis by the shape of the macronuclei (round vs. distinctly oval in C. grandis; Smith [56]). However, the morphology of macronucleus alone is not sufficient to distinguish Colpoda species. Considering the slightly variable shape of the macronucleus in Colpoda, the insufficient number of specimens investigated in this study, and the close phylogenic relationship with C. grandis based on the SSU-rRNA gene sequences, we temporarily identify our isolate as Colpoda compare grandis. Stokes, 1884. Both the two Chinese populations of C. inflata have typical "L"-shaped body with a marked preoral narrowing and a hemispherical postoral portion, similar numbers of somatic kineties, and postoral kineties with those of previous studies [57][58][59]. The body size of pop. 1 did not differ much from previous studies; although, the body size of pop. 2 was much larger (40 -60 μm × 30 -50 μm in the previous populations compared to 85 -88 μm × 65 μm in the present study) [59]. Maupas, 1883. Compared with the previous studies, the four Chinese populations of P. steinii are similar in the following characteristics: dikinetid, two longer caudal cilia, a distinctly ellipsoidal macronucleus

Phylogeny of Genus Colpoda.
Among the polygenes with small subunit ribosomal RNA genes (SSU-rRNA), the genus Colpoda was nonmonophyletic, consistent with previous studies [13,36]. Typical Colpoda species are unlikely to unite into a single clade because they are spread throughout the order Colpodida, and some species (e.g., Colpoda maupasi and C. ecaudata) often form unexpected clades with two or more genera that have little in common morphologically [13]. This is also observed in previously constructed phylogenies (e.g., [10,35,36,56]) by Foissner et al. [61]. Dunthorn et al., [19] even proposed that there exists a strongly radiating Colpoda, in which several species subsequently evolved independently to form new genera and families. We augmented the taxon sampling within the genus Colpoda with seven newly sequenced taxa, and our results support these earlier analyses, indicating a nonmonophyletic topology of Colpoda. In the 18S-rRNA gene phylogenetical analysis, five Colpoda species (C. reniformis, Colpoda compare grandis, C. inflata, Paracolpoda steinii, and C. harbinensis n. sp.) appeared in the core of Colpodidae with medium to high support. Paracolpoda steinii pops. 1-4 were sister to the clade clustered by P. steinii and Bromeliothrix metopoides. This discrepancy may be due to the fact that the SSU rRNA gene is too conservative in Colpoda to differentiate species. Extensive barcode analyses of the animal kingdom indicate that sequence divergences in mitochondrial genes encoding Cox I can distinguish closely related animal species [62][63][64]. In the model protist genus Tetrahymena, intraspecific Cox I divergence is typically >4% [65][66][67]. Interestingly, Colpoda compare grandis, C. inflata, and Paracolpoda steinii differed by 12.01%-14.88% in the Cox I gene, strongly suggesting that the three species were distinct. In contrast, the intraspecific genetic variation of Paracolpoda steinii was only 0.35%, indicating that the Cox I gene could represent an applicable DNA barcoding region for accurate and rapid identification of Colpoda. However, based on our experience, we conclude that it is difficult to design primers to amplify the Cox I gene in Colpoda.

The Utility of the β-Tubulin Gene Inaccurate
Identification. The β-tubulin gene is another strong candidate gene for the delineation of Colpoda, given that it displays a diverse array of microtubules composed of tubulin with highly similar sequences [68,69]. Specific regions of the β-tubulin gene are highly conserved, making it possible to design universal primers, while regions containing hypervariable sequences can be used to generate species-specific primers for accurate identification. In this study, there were no clear boundaries between intra-and interspecific genetic distances for each of the Colpoda. The intraspecies variation in the β-tubulin gene in the Colpoda was considerable, as indicated by the haplotype network, with a difference of 60 genetic steps between pop. 1 and pop. 2 in C. inflata genetic steps (5a and 2a) (Figure 5(a)) and 69 genetic steps between pop. 3 (7) and pop. 5 (11b) in Paracolpoda steinii ( Figure 5(b)). Therefore, the β-tubulin gene may be less suitable for Colpoda DNA barcoding than Cox I.

Species Identification by FISH.
In this study, five probes were developed to accurately identify Colpoda (Table 3). Using Coleps hirtus instead of Colpoda species as a negative control is more effective to test the probe's specificity. Following Fried and Foissner [25], we evaluated our probes with the ARB software package and the GenBank BLAST tool to analyze the probe's specificity. Previous studies have already demonstrated the power of this method for specific delineation. Nevertheless, the probes still require consolidation with the support of isolation and/or sequencing of Colpoda. Our study reveals that FISH can be used for rapid and interspecific identification of Colpoda and can also provide some morphological information such as body shape, (y) Figure 8: (a, b) C. inflata (Liu [77]). (c, d) C. ecaudata (Small and Lynn [78]). (e, f) C. cucullus (Small and Lynn [70]). (g, h) C. magna (Small and Lynn [70]). (i, j) C. lucida (Kim and Min [55]). (k, l) C. aspera (Foissner and Schubert [79]). (m, n) C. maupasi. (o, p) C. henneguyi (Kim et al. [55]). (q, r) C. elliotti (Foissner and Schubert [79]). (s, t) C. spiralis (Novotny et al. [80]). (u, v) Colpoda steinii (Liu [69]). (w)-(y) C. minima (Díaz Silvia et al. [81]). 28 Cellular Microbiology macronucleus shape, and macronucleus number, which will help verify morphotypes in mixed taxa samples. However, while Colpoda species are geographically dispersed (e.g., Korea, U.S.A, and China), limited molecular data from disparate isolates are available [12,13,34,36,70]. The FISH probes designed here can potentially be used to investigate the geographic distribution of Colpoda and potentially even their dispersal.

Conclusion
In conclusion, our analysis is consistent with previous study showing that no single marker can delineate microbial species [73]. Combining morphological and molecular biology techniques can greatly improve the delineation of Colpodea. We suggest that Cox I is a promising DNA barcoding marker for species of Colpodea, as shown in this and previous studies [22,65,74,75]. However, difficulties with amplification may challenge its utility in identifying this group. The FISH can provide some morphological information, thus complementing traditional techniques such as silver carbonate. Furthermore, the establishment of a character-based database may be a useful tool for resolving conflicts between morphological or molecular approaches to the differentiation of not only Colpodea but also ciliate species in general.
In conclusion, we investigated and compared the morphological features of Colpoda reniformis, Colpoda compare grandis, Colpoda inflata, and Paracolpoda steinii, revealed the phylogeny of Colpoda, explored the feasibilities of Cox I and β-tubulin as DNA barcoding, and supplied the identification of Colpoda species using oligonucleotide probes. In addition, we have established a new species of Colpoda. The novelty of this study mainly displays in following several aspects: (1) molecular techniques are used for the identification of Colpoda for the first time; (2) oligonucleotide probes and haplotype network analysis are firstly conducted for the identification of Colpoda species; and (3) the comparative exploration is made for the feasibility of Cox I and β-tubulin genes as DNA barcoding.

Data Availability
The data presented in the study are deposited in the NCBI database repository, accession numbers: OM752200, OM752201, OM752202, and OM752203.