Skip to main content
NCBI home page
Ecology and Evolution logoLink to Ecology and Evolution
. 2022 Oct 12;12(10):e9380. doi: 10.1002/ece3.9380

Insights into the phylogeny of the ciliate of class Colpodea based on multigene data

Bailin Li 1, Yumeng Song 1, Tingting Hao 1, Li Wang 1, Weibin Zheng 1, Zhao lyu 2, Ying Chen 3,✉, Xuming Pan 1,✉
PMCID: PMC9595136  PMID: 36304093

Abstract

In the class Colpodea, there are many unresolved evolutionary relationships among taxa. Here, we report 30 new sequences including SSU‐rRNA, ITS1‐5.8S‐ ITS2 rRNA, and the mitochondrial small subunit ribosomal RNA (mtSSU‐rRNA) genes of five colpodeans, and conduct phylogenetic analyses based on each individual gene and a two‐gene concatenated dataset. For the first time, multi‐genes were used to analyze phylogenetic relationships in the class Colpodea. The main findings are: (1) SSU‐rRNA, ITS1‐5.8S‐ ITS2 rRNA, and mtSSU‐rRNA gene sequences of C. reniformis and C. grandis are provided for the first time, and these two species group into the clade including C. inflata, C. lucida, C. cucullus, and C. henneguyi; (2) clustering pattern and morphological similarity indicate that Bresslauides discoideus has a close relation with Colpodidae spp.; (3) Emarginatophrya genus diagnosis is improved to be ‘Hausmanniellidae with sharply shortened and isometric leftmost 1‐4 ciliary rows’ and Colpoda elliotti is transferred to Emarginatophrya; (4) the genus Colpoda is still non‐monophyletic with the addition of 10 populations from five Colpoda species sequences, but there are only two Colpoda groups left based on the present work: Group I comprises C. inflata, C. lucida, C. cucullus, C. henneguyi, C. reniformis, and C. grandis, Group II comprises C. maupasi and C. ecaudata, and the presence of diagonal grooves and the way the vestibular opens might be the two key features that differentiates Colpoda species groups; (5) a close molecular relationship, and highly similar merotelokinetal mode, somatic ciliary pattern, and basic organization of the oral apparatus with P. steinii suggests Bromeliothrix metopoides should be temporarily assigned to Colpodidae.

Keywords: ciliates, Colpoda, ITS1‐5.8S‐ ITS2 rRNA gene, mtSSU‐rRNA gene, phylogeny, SSU‐rRNA gene

In the class Colpodea, there are many unresolved evolutionary relationships among taxa. Here, we report 30 new sequences, including SSU‐rRNA, ITS1‐5.8S‐ ITS2 rRNA, and the mitochondrial small subunit ribosomal RNA (mtSSU‐rRNA) genes of five colpodeans, and conduct phylogenetic analyses based on each individual gene and a two‐gene concatenated dataset. For the first time, multi‐genes were used to analyze phylogenetic relationships in the class Colpodea.

graphic file with name ECE3-12-e9380-g005.jpg

1. INTRODUCTION

Ciliates are a highly differentiated group of microbial eukaryotes that inhabit virtually all environments on the Earth's surface where there is sufficient water for their survival. They play a fundamental role in microbial food web function by mediating the transfer of organic matter and energy between different trophic levels in a wide range of ecosystems (Agatha et al., 2021; Bai et al., 2020; Chi et al., 2021; Dias et al., 2021; Liu et al., 2022; Song et al., 2021; Wang et al., 2021; Weisse & Montagnes, 2021; Wu et al., 2021). Due to their particular nature (e.g., high reproductive capacity, diverse morphology, nuclear dimorphism, chromosomal fragmentation, and sensitivity to environmental changes), ciliates are important model organisms in many disciplines, such as epigenetics, symbiotic relationships, molecular biology, organismal development, evolution, biogeography, and ecology (Berger, 2011; Cheng et al., 2019; Corliss, 1979; Gao et al., 2014; Lynn, 2008; Song et al., 2009; Zhao et al., 2020). Despite this attention, however, there are numerous unresolved issues concerning ciliate phylogeny and systematics (Corliss, 1979; Gao et al., 2016).

Ciliates of the class Colpodea (Small & Lynn, 1981) are an important structural component of soil protozoa. They have been reported from terrestrial habitats world‐wide from the driest deserts to permanently saturated wetlands and bogs (Bourland et al., 2012; Foissner, 1993; Foissner et al., 2014; Vďačný & Foissner, 2019). Characteristically, colpodeans produce resting cysts that allow them to survive desiccation and other adverse environmental conditions and thus often constitute a significant hidden soil biodiversity component (Foissner, 1987; Quintela‐Alonso et al., 2011). Although their oral apparatus is highly diverse, colpodeans share a similar somatic cortex structure which has led to misclassification among species (Foissner, 1993; Lynn, 2008; Quintela‐Alonso et al., 2011; Vďačný & Foissner, 2019). Small and Lynn (1981) recognized colpodeans as a monophyletic taxon and established the class Colpodea on the basis of a set of unique morphological characteristics. The application of molecular techniques supported Colpodea monophyly and its position within the subphylum Intramacronucleata (Dunthorn et al., 2014; Lynn, 2003), but also resulted in a major re‐evaluation of interclass classification and evolution (Foissner et al., 2011; Vďačný & Foissner, 2019).

Four major Colpodea lineages (Bursariomorphida, Platyophryida, Cyrtolophosidida, and Colpodida) have been identified by phylogenetic inference based on nuclear small subunit ribosomal RNA (nSSU‐rRNA) sequences (Dunthorn et al., 2008; Foissner et al., 2011; Vďačný & Foissner, 2019). Among these, Bursariomorphida (Fernández‐Galiano, 1979) was found to include in the taxa previously assigned to the order Bryometopida (Foissner, 1985; Vďačný & Foissner, 2019); the position of Platyophryida is indeterminate (Bourland et al., 2011; Rajter et al., 2020), Cyrtolophosidida is divided into two main clades, and Colpodida is the most complex clade (Foissner et al., 2011). Dunthorn et al. (2014) investigated Colpodea phylogeny based on mtSSU‐rRNA gene sequence data and reported the same four lineages as those from the nSSU‐rRNA analyses mentioned earlier. Nevertheless, there are inconsistencies in the evolutionary relationships among taxa within Colpodea (e.g., the position of Platyophryida), indicating that phylogenetic analysis based on a single gene is problematic (Rajter et al., 2020). Furthermore, species sampling remains unbalanced in Clopodea (e.g., there is only one species in Ilsiellidae and Bardeliellidae) that has led to the formation of paraphyletic groups and uncertainty in the assignment of various taxa (Fan et al., 2013; Foissner & Stoeck, 2009; Quintela‐Alonso et al., 2011; Strüder‐Kypke et al., 2000; Vďačný & Foissner, 2019; Zhao et al., 2016).

Recently, investigations based on multigene trees have been increasingly used for phylogenetic studies and generally produce more robust results than those based on single gene markers (Gao et al., 2013, 2016, 2017; Sun et al., 2020; Wang, Wang, et al., 2017; Zhang et al., 2020). Multigene analyses usually include nSSU‐rRNA, ITS1‐5.8S‐ITS2 and nLSU‐rRNA genes, which are in the same chromosome (Wang, Wang, et al., 2017). However, Colpodea phylogenetic analyses so far have focused on a single gene, (i.e., the nSSU‐rRNA gene or the mtSSU‐rRNA gene), and few studies have used a multigene approach (e.g., Dunthorn et al., 2011). Furthermore, most colpodean sequences in the NCBI database are of SSU‐rRNA, while other gene sequences are very limited. This is one of the main dilemmas of colpodean phylogenetics.

With the conservative evolution of SSU rDNA alongside various issues such as asynchronous evolution with morphology, delineation of Colpodea species remains problematic. And, the usefulness of combination of nuclear and mitochondrial gene is rarely tested in Colpodea. The mtSSU‐rRNA gene is more variable and has a higher evolutionary rate compared to the nSSU‐rRNA gene (Boore & Brown, 1998; Moore, 1995; Wang, Zhang, et al., 2017), and it is reasonable to assume that it can effectively uncover deep nodes within Colpodea (Dunthorn et al., 2011, 2014; Rand, 2003; Zhang et al., 2019). In this study, 30 new sequences of 10 populations from five colopdeans (Colpoda cf. inflata, C. reniformis, C. inflata pop1‐2, C. grandis, and Paracolpoda steinii pop1‐5) isolated from soil and water samples in Harbin, Shuangyashan and Mudanjiang, China, are presented. For the first time, Colpodea phylogenetic analyses based on multiple genes (SSU‐rRNA gene, mtSSU‐rRNA gene), along with analyses of morphological data are carried out.

2. MATERIALS AND METHODS

2.1. Taxon sampling, observation, and terminology

Soil and water samples of nine populations of four colopodeans were obtained from several sites in Harbin, Shuangyashan and Mudanjiang, Heilongjiang province, northern China. Colpoda cf. inflata, C. inflata pop1 (population 1), and Colpoda grandis were collected from a farmland at the Hulan Beet Research Institute of Heilongjiang University (45° 59′ 47″ N, 126° 38′ 18″ E), Harbin. Colpoda inflata pop2 was collected from an experimental plot in Harbin Normal University (45° 86′ 46″ N, 126° 56′ 15″ E), Harbin. Colpoda reniformis was collected from Mudanjiang Xuanwu Lake in National Agricultural Park (44° 9′ 10″ N, 129° 9′ 19″ E), Mudanjiang. Paracolpoda steinii pop1‐3 were collected from several waters in Qixing River wetland (46° 41′ 20″ N, 132° 2′ 30″ E; 46° 42′ 48″ N, 132° 11′ 36″ E; 46° 43′ 41″ N, 132° 18′ 12″ E), Shuangyashan, and Paracolpoda steinii pop4‐5 were collected from soil at the same location (46° 42′ 59″ N, 132° 8′ 21″ E; 46° 42 13″ N, 132° 7′ 29″ E). Ciliates were isolated from soil using the non‐flooded Petri dish method at room temperature (ca. 25°C) with rice or wheat grains added to enrich bacteria growth as a food source. Clonal cultures were established for each of the 10 populations from five colpodean species isolated. Silver carbonate staining (Foissner, 1992) was used to reveal the infraciliature. Five slides containing silver carbonate‐stained voucher specimens encircled in black ink are deposited in the Laboratory of Protozoology, Harbin Normal University of China with registration numbers HTT‐20211006‐01; −20,211,006‐02; −20,211,006‐03; −20,211,006‐04 and −2,021,100,605. Terminology and classification was mainly according to Foissner et al. (2011) and Lynn (2008).

2.2. DNA extraction, amplification, and sequencing

For each species, 1–10 cells from clonal culture were isolated under a stereomicroscope using a micropipette, washed with distilled water at least thrice to remove potential contaminants, and then incubated in non‐nutrient distilled water for 24 h. Cells were then transferred to an Eppendorf tube in a volume of no more than 5 μl distilled water. Total genomic DNA was extracted using the DNeasy & Tissue Kit (Hilden, QIAGEN) following the manufacturer's instructions.

SSU‐rRNA, ITS1‐5.8S‐ITS2 rRNA, and mtSSU‐rRNA genes were amplified by the polymerase chain reaction (primers see Appendix Table A1). High‐fidelity Taq polymerase (Takara Ex Taq; Takara Biomedicals) was used to reduce amplification errors. PCR condition for the SSU‐rRNA gene amplification was denaturation for 5 min at 94°C, followed by 5 cycles of denaturation for 30 s at 94°C, annealing for 1 min 45 s at 56°C, extension for 2 min at 72°C and the other 25 cycles of denaturation for 45 s at 94°C, annealing for 1 min 45 s at 60°C, extension for 2 min at 72°C and a final extension at 72°C for 8 min. The ITS1‐5.8S‐ITS2 rRNA gene was amplified as follows: 5 min initial denaturation 94°C, followed by 35 cycles of denaturation for 30 s at 94°C, annealing for 45 s at 58°C, extension for 1 min at 72°C and a final extension at 72°C for 10 min. The mtSSU‐rRNA gene was amplified as follows: 5 min initial denaturation 94°C, 5 cycles of 45 s at 94°C, 1 min 45 s at 58°C and 2 min at 72°C, and the other 25 cycles of denaturation for 45 s at 94°C, annealing for 1 min 45 s at 60°C, and extension for 2 min at 72°C with a final extension of 10 min at 72°C. Cloning and sequencing were both routine operations (Zhang et al., 2019). A total of 30 new SSU‐rRNA, ITS1‐5.8S‐ ITS2 rRNA, and mtSSU‐rRNA gene sequences from five Colpodea species were determined (GenBank accession numbers see Appendix Table A2).

2.3. Data sets and alignments

Other colpodeans and outgroup sequences were obtained from GenBank, and their accession numbers are shown in Figures 1, 2, 3, 4. Sequences downloaded with questionable morphological information, unknown source, or of too short length were not included. SSU‐rRNA gene was aligned using Clustal W implemented in BioEdit 7.0.1 (Hall, 1999). The ITS1‐5.8S‐ITS2 rRNA gene and mtSSU‐rRNA gene sequences were aligned using the default parameters implemented in Guidance server (http://guidance.tau.ac.il/) (Penn et al., 2010). To remove ambiguously aligned positions in the multiple sequence alignments all the sequence datasets were edited by eye in BioEdit 7.0.1 (Hall, 1999). Sequences used for phylogenetic analyses were compiled into four data sets: (i) 1833 characters of SSU‐rRNA (83 taxa); (ii) 722 characters of ITS1‐5.8S‐ rRNA –ITS2 (19 taxa); (iii) 1138 characters of mtSSU‐rRNA (31 taxa); and (iv) 2895 characters of concatenated sequence data of the above two genes (29 taxa in total). The two alignments were concatenated to be contiguous in SeaView V4 for gene analysis. All new sequences were deposited in the NCBI database (https://www.ncbi.nlm.nih.gov/), accession numbers, lengths, and G&C contents are shown in Appendix Table A2. For all trees, the outgroup was composed of three species of tetrahymenid taxa (Tetrahymena pyriformis, T. tropicalis and T. americanis).

FIGURE 1.

FIGURE 1

Open in a new tab

The maximum‐likelihood (ML) tree based on the concatenated genes (SSU‐rRNA and mtSSU‐rRNA genes) of major members of the class Colpodea. Newly added sequences in this study are bolded in red. Node support is shown as: ML bootstraps/BI posterior probability. “‐” indicate mismatch in topology between Bayesian and ML tree. Fully supported (100%/1.00) branches are marked with solid circles. The scale bar corresponds to five substitutions per 100 nucleotide sites.

FIGURE 2.

FIGURE 2

Open in a new tab

The maximum‐likelihood (ML) tree based on the SSU‐rRNA gene of major members of the class Colpodea. Newly added sequences in this study are bolded in red type. Node support is shown as: ML bootstraps/BI posterior probability. “‐” indicate mismatch in topology between Bayesian and ML tree. Fully supported (100%/1.00) branches are marked with solid circles. Two long branches has been shortened, as shown by “//”, and the other branches are drawn to scale. The scale bar corresponds to five substitutions per 100 nucleotide sites. (a–j) The five newly sequenced species after silver carbonate staining. (a, b) Colpoda inflata pop1‐2; (c) Colpoda grandis; (d) Colpoda reniformis; (e) Colpoda cf. inflata; (f–j) Paracolpoda steinii pop1‐5.

FIGURE 3.

FIGURE 3

Open in a new tab

The Bayesian inference (BI) tree based on the ITS1‐5.8S‐ITS2 gene of major members of the class Colpodea. Newly added sequences in this study are bolded in red type. Node support is shown as: ML bootstraps/BI posterior probability. “‐” indicate mismatch in topology between Bayesian and ML tree. Fully supported (100%/1.00) branches are marked with solid circles. The scale bar corresponds to 0.2 expected substitutions per site.

FIGURE 4.

FIGURE 4

Open in a new tab

The Bayesian inference (BI) tree based on the mtSSU‐rRNA gene of major members of the class Colpodea. Newly added sequences in this study are bolded in red type. Node support is shown as: ML bootstraps/BI posterior probability. “‐” indicate mismatch in topology between Bayesian and ML tree. Fully supported (100%/1.00) branches are marked with solid circles. The scale bar corresponds to 0.1 expected substitutions per site.

2.4. Phylogenetic analyses

Phylogenetic trees were inferred using maximum likelihood (ML) and Bayesian inference (BI) methods. ML analyses were constructed by RAxML‐HPC2 v8.2.12 (Stamatakis, 2014), and BI analyses by MrBayes v3.2.7a (Ronquist et al., 2012), both on the CIPRES Science Gateway (URL: http://www.phylo.org/sub_sections/portal). The most appropriate model for phylogenetic analyses of nSSU‐rRNA gene (SSU‐rRNA, ITS1‐5.8S‐ITS2 rRNA gene) was GTR + I + G as selected by Modeltest v3.4 (Posada & Crandall, 1998). The ML and BI trees based on mtSSU‐rRNA gene and concatenated dataset were constructed according to the GTR + I + G model chosen by MrModeltest v.2.2 program (Nylander, 2004). ML analysis was conducted using rapid bootstrap with 1000 non‐parametric bootstrap replicates. Bayesian posterior probabilities were calculated by running four chains for 10,000,000 generations, with cold chain sampling every 10,000 generations. The first 25% of sampled trees were discarded as burn‐in. Support value <70%/0.94 (ML/BI) was considered as low, 70%–94% (ML) as moderate, and >95%/0.95 (ML/BI) as high. MEGA 7.0 (Kumar et al., 2016) was utilized to visualize tree topologies.

2.5. Sequence analyses and putative secondary structure modeling

The secondary structures of ITS2 sequences of Paracolpoda steinii pop1‐5, Colpoda cf. inflata and C. inflata pop2 were predicted based on the models of Cardiostomatella vermiformis (EU262621) (http://www.rna.ccbb.utexas.edu) using the Mfold website (http://unafold.rna.albany.edu/?q=mfold/RNA‐Folding‐Form) with default settings (Miao et al., 2008; Zuker, 2003). The 5′ and 3′ ends of the ITS2 sequences were determined via Rfam (available on the web http://www. sanger.ac.uk/Software/Rfam/; Griffiths‐Jones et al., 2003).

3. RESULTS

3.1. Phylogeny based on concatenated dataset

The phylogenetic trees constructed using ML and BI had similar topologies, therefore, only the ML trees and their support values from both methods are shown in Figure 1. The order Colpodida contains six families (Colpodidae, Tillinidae, Hausmanniellidae, Ilsiellidae, Marynidae, and Bardeliellidae; Figure 1). However, due to sequence paucity, there is only one available sequence from single species in the Tillinidae, Hausmanniellidae, Ilsiellidae, Marynidae, and Bardeliellidae. Hausmanniella discoidea and Ilsiella palustris form a sister clade (85% ML, 0.99 BI). One Marynidae species (Maryna umbrellata), H. discoidea and I. palustris cluster together to form a clade (80% ML, 0.86 BI). The family Tillinidae is represented by one species (Tillina magna) and Emarginatophrya aspera cluster together to form a sister clade. The family Bardeliellidae (Bardeliella pulchra) is the sister of the order Colpodida. The order Cyrtolophosidida and Colpodida cluster together with high support (96% ML, 0.95 BI). The class Colpodea is monophyletic with four main lineages (Platyophryida, Bursariomorphida, Cyrtolophosidida, and Colpodida), each of which is monophyletic. Platyophryida is the deepest branching lineage within the Colpodea. Bursariomorphida is a sister group to the clade formed by the orders Colpodida and Cyrtolophosidida.

The newly sequenced species C. reniformis and C. henneguyi cluster as a sister group, and the resulting clade then groups with the other newly sequenced species C. grandis (100% ML, 1.00 BI) and Bresslauides discoideus (100% ML, 1.00 BI). The two newly sequenced Colpoda cf. inflata and C. inflata pop2 cluster together with full support (100% ML, 1.00 BI), and the resulting clade then groups with the clade formed by newly sequenced C. inflata pop1 (100% ML, 1.00 BI). The genus Colpoda is monophyletic.

The five newly sequenced populations of Paracolpoda steinii (1–5) cluster together. The five new populations group cluster in the Colpodida clade ‘core position’. Noticeably, Paracolpoda steinii pop1 is a sister to P. steinii pop2 (98% ML, 0.80 BI) and forms a sister group with P. steinii pop3 (100% ML, 1.00 BI) and P. steinii pop4 (100% ML, 1.00 BI). P. steinii pop5 is a sister to this clade (100% ML, 1.00 BI).

3.2. Phylogenetic analyses based on SSU‐rRNA gene sequence data

Each of the Colpodea four orders are monophyletic and the genus Colpoda is non‐monophyletic. The orders Colpodida and Cyrtolophosidida cluster together to form a clade that is a sister to Bursariomorphida. The order Platyophryida is the deepest branching lineage within the Colpodea.

The order Colpodida contains eight families (Colpodidae, Tillinidae, Grossglockneriidae, Bryophryidae, Hausmanniellidae, Ilsiellidae, Marynidae, and Bardeliellidae; Figure 2). Hausmanniella discoidea clusters with Kreyellidae‐like taxa and Emarginatophrya aspera (71% ML, 0.99 BI). The family Grossglockneriidae is represented by two species (Mykophagophrys terricola and Pseudoplatyophrya nana) that cluster together with high support (98% ML, 1.00 BI). Family Bryophryidae is monophyletic. Jaroschia sumptuosa, Bryophrya gemmea, and Bryophryoides ocellatus cluster together as a clade that then forms a sister clade with Notoxoma parabryophryides. Bardeliella pulchra and Ilsiella palustris cluster together to form a clade. Family Tillinidae is monophyletic and contains three species. Marynidae is paraphyletic because Maryna meridiana and Pseudomaryna sp. cluster together with full support and then group with M. umbrellata (87% ML, 1.00 BI). Maryna ovata clusters with the M. meridiana + Pseudomaryna sp. + M. umbrellata clade with a full support. Marynidae has a sister relationship with Sandmanniella terricola.

Two Colpodidae species (C. ecaudata and C. maupasi JF747215) and Repoma cavicola cluster together to form a clade that is a sister group to Exocolpoda augustini. Colpoda ecaudata and C. maupasi (JF747215) cluster together with full support but far away from most congeners. Colpoda elliotti is a sister to the clade formed by Hausmanniella discoidea, Kreyellidae‐like taxa and an Emarginatophrya aspera cluster. The five newly sequenced species cluster at the Colpodida clade core position. Colpoda reniformis and C. grandis are sisters with moderate support (76% ML, 0.95 BI) as a sister group to C. henneguyi with full support. The clade formed by C. inflata pop2 and Colpoda cf. inflata (98% ML, 1.00 BI) is a sister to another C. inflata population (M97908), while C. inflata pop1 clusters with the other two C. inflata populations (KM222106 and KJ607918). The 12 Paracolpoda steinii sequences including four newly sequenced species (P. steinii pop1‐4) cluster together with Bromeliothrix metopoides. In the SSU‐rRNA gene tree (Figure 2), Bardeliella pulchra clustered with Ilsiella palustris.

3.3. Phylogenetic analyses based on ITS1‐5.8S‐ITS2 region sequence data and the secondary structures of ITS2

In the ITS1‐5.8S‐ITS2 rRNA gene tree, bootstrap values for the maximum likelihood (ML) and posterior probabilities for the Bayesian inference (BI) were mapped onto the best BI tree (Figure 3). Due to sequence shortages, only the order Colpodida (family Colpodidae, genus Colpoda and Paracolpoda) and outgroup were included in this tree. Colpodida, Colpoda, and Paracolpoda form a monophyletic clade. Colpoda reniformis is still separated from the other newly sequenced Colpoda species (C. inflata pop1‐2 and Colpoda cf. inflata), and forms a sister clade with the newly sequenced C. grandis (96% ML, 1.00 BI). Two populations of C. inflata (KM222071 and MZ562700), C. inflata pop1, and Colpoda cf. inflata cluster together, and the clade then forms a sister clade with C. inflata. Colpoda inflata pop2 cluster with the C. inflata + C. inflata pop1 + Colpoda cf. inflata clade. Seven Paracolpoda species cluster in one clade, and the clade formed by the five newly sequenced Paracolpoda steinii cluster as a sister group to P. steinii (AB684403) with moderate support (77% ML, 1.00 BI). Paracolpoda steinii (AB684403) groups with the P. steinii pop1‐5 clade, which then clusters with Paracolpoda steinii MK912139 with full support.

Secondary structures of the ITS2 transcript of the seven species are shown in Figure 5. The same taxa shared a very similar pattern of secondary structure. Only three helice spins were found in the genus Paracolpoda while six in Colpoda. There are differences in the helix 1 among different populations of the genus Paracolpoda, while mainly in helix 3 in the genus Colpoda.

FIGURE 5.

FIGURE 5

Open in a new tab

The putative secondary structures of ITS2 in the present study.

3.4. Phylogenetic analyses based on mtSSU‐rRNA gene sequence data

The BI tree topology is similar to the ML tree, so only the BI tree with branch‐support values is shown in Figure 4. The mtSSU‐rRNA and SSU‐rRNA gene phylogenies show similar relationships within Colpodea. Noticeably, both the order Colpodida and the family Colpodidae are monophyletic. Bardeliella pulchra groups within the Colpodida clade.

Within Colpodea, the order Bursariomorphida is a sister to the clade formed by Platyophryida and Cyrtolophosidida. The newly sequenced species C. reniformis and C. henneguyi HM246408 cluster with another population of C. henneguyi HM246407, and the clade then clusters with C. grandis (ML 100%, BI 1.00). The results shown above are similar with those of concatenated and SSU‐rRNA gene phylogenies. The five new Paracolpoda steinii populations cluster together and form a sister clade with Emarginatophrya aspera (ML 99%, BI 1.00). Three Colpoda species (C. inflata pop1‐2 and Colpoda cf. inflata) cluster in one group, C. inflata pop2 and Colpoda cf. inflata form a sister clade with a full support, and the resulting clade then forms a sister relationship with C. inflata pop1 (ML 98%, BI 1.00). The clustering pattern of the three Colpoda species is relatively stable in all phylogenetic trees. Due to sequence shortages, there are only single species sequences available for Tillinidae, Ilsiellidae, Marynidae, and Bardeliellidae. Tillina magna clustered with Colpodida, Maryna umbrellata is a sister to the clade formed by five new Paracolpoda steinii populations and Emarginatophrya aspera.

4. DISCUSSION

4.1. Phylogeny of genus Colpoda

In previously reported SSU rRNA gene trees, typical Colpoda species are distributed over the whole Colpodea tree, and mainly form four groups: C. inflata + C. cucullus + C. lucida, C. henneguyi, C. maupasi + C. ecaudata, and C. elliotti clades (Foissner et al., 2011, 2014; Foissner & Stoeck, 2009; Lynn et al., 1999; Vďačný & Foissner, 2019). This motivated Foissner et al. (2011) and Dunthorn et al. (2012) to suggest rapid Colpoda radiation that produced several new genera and families. We added 30 new multiple gene sequences of 10 populations from five colopdeans. These additions change the Colpoda species into three main groups: Group I is composed of C. inflata, two Chinese C. inflata populations, Colpoda cf. inflata, C. lucida, C. cucullus, C. henneguyi, C. reniformis, and C. grandis; Group II comprises C. maupasi and C. ecaudata; and Group III only contains C. elliotti in the SSU rRNA gene trees.

Notably, for the first time, we provide C. reniformis and C. grandis SSU‐rRNA, ITS1‐5.8S‐ ITS2 rRNA, and mtSSU‐rRNA gene sequences. Colpoda reniformis and C. grandis clustered with C. henneguyi, and the resulting clade then clustered with Bresslauides discoideus. In this case, B. discoideus groups into the Colpoda clade including C. inflata, C. lucida, C. cucullus, C. henneguyi, C. reniformis, and C. grandis in the SSU rRNA, mtSSU‐rRNA gene, and concatenated trees. Bresslauides species differ mainly by the unique right semicircular oral polykinetid that is longer than the left, as opposed to being of equal length in the Colpodidae (Foissner, 1987, 1993), but similar with the features of Colpodidae sp. in vivo, which indicates that Bresslauides species have a close relation with Colpodidae sp. Broader sampling and molecular sequencing of Bresslauides species would be necessary to clarify this relationship, although we also believe that the character of the right semicircular curved oral polykinetid may have evolved more than once (Bourland et al., 2011; Dunthorn et al., 2008).

Colpoda elliotti KJ873047 clusters in the clade formed by Kreyellidae‐like species, Emarginatophrya aspera, and it unites with E. aspera in trees comprised colpodids only (Foissner et al., 2011). Emarginatophrya, morphologically distinguished by a distally emarginated left oral polykinetid, was established by Foissner (2016). However, establishing this new colpodid genus has not helped to completely erase the Colpoda paraphyly problem. Colpoda aspera and C. elliotti are highly similar morphologically and according to Foissner (1993), ‘the left oral polykinetid of C. elliotti is slightly variform’. Then, we found in fig. 53b in Foissner's book that the left oral polykinetid was slightly emarginated (as a result of its leftmost kinety always consisting of only two basal bodies) in some representative individuals. The only two Emarginatophrya species, E. aspera and E. terricola, also have three to four sharply shortened leftmost ciliary rows (almost of equal length), which were described as ‘emarginated left oral polykinetid’. Therefore, we decided to change the diagnosis of Emarginatophrya to ‘Hausmanniellidae with sharply shortened and isometric leftmost 1–4 ciliary rows’. So, we transferred Colpoda elliotti to Emarginatophrya, and establish a new combination Emarginatophrya elliotti (Bradbury & Outka, 1967) nov. comb., which partially solves the Colpoda problem.

As a result, there are now two remaining Colpoda groups: Group I comprises C. inflata, C. lucida, C. cucullus, C. henneguyi, C. reniformis, and C. grandis, and Group II comprises C. maupasi and C. ecaudata. Colpoda maupasi and C. ecaudata group with Exocolpoda and Ropoma species, which have distinctly different characteristics from the former species, including different life cycles, boomerang‐shaped left oral ciliary fields, and a very thick resting cyst wall. Colpoda maupasi and C. ecaudata are two common Colpoda species, but they group far away from other congeners. A probable explanation for this grouping pattern is that both C. maupasi and C. ecaudata lack the diagonal groove and their vestibular opening is marked on the left body margin by shallow indentation, but other Colpoda species having molecular information possess diagonal grooves (except C. inflata, but its left body margin with right‐angular notch, in the center where the vestibulum opens; Foissner, 1993). Morphologically, Tillina magna also possess diagonal grooves (Foissner, 1993), which might explain why Tillina magna and the Group I of the genus Colpoda cluster together in the SSU‐rRNA and mtSSU‐rRNA gene trees. Collectively, the presence of diagonal grooves and the way the vestibular opens might be the two key features that differentiate Colpoda species groups. Due to sequence shortages, only one species of Tillinidae used in the concatenated tree, which leads to the clustering of Tillina magna with Emarginatophrya aspera. Therefore, we suggest that sampling should be expanded to further address this issue.

4.2. Paracolpoda and Bromeliothrix phylogeny

Totally twelve populations (including five from the present work) of Paracolpoda steinii firmly cluster with Bromeliothrix metopoides in the SSU rRNA gene trees. Bromeliothrix metopoides has the same merotelokinetal mode with Paracolpoda steinii, and its somatic ciliary pattern is similar to that of Colpoda species (Foissner, 1993, 2010; Weisse et al., 2013). Bromeliothrix has a ciliary and silverline pattern typical for members of the family Colpodidae, and its basic organization of the oral apparatus is also the same as that known from genera of Colpodidae families (Foissner, 2010; Foissner et al., 2002). Foissner pointed out that Bromeliothrix neither belong to the Exocolpodidae nor the Hausmanniellidae due to its complex morphological characteristics, and several phylogenetic analyses based on SSU rRNA gene sequence also indicated that B. discoideus was more closely related to a clade formed by large Colpoda species than with Hausmanniella discoidea, a type of the hausmanniellids (Bourland et al., 2011; Dunthorn et al., 2008). The Paracolpoda genus was assigned to the Colpodidae and established by Lynn (1978), who noted that Colpoda species have a somatic groove while Paracolpoda species do not. Foissner (1985) subsumed C. steini into Paracolpoda. A close molecular relationship, highly similar merotelokinetal mode, somatic ciliary pattern and basic organization of the oral apparatus with P. steinii suggests Bromeliothrix metopoides could be temporarily assigned to Colpodidae (Foissner, 1993, 2010, Foissner et al., 2002, 2011). Additionally, the rather long, semicircular curved right oral polykinetid character, among other features of the Hausmanniellidae (Foissner, 1993) might be interpreted as convergence. Until now, the molecular data available in the NCBI database for Bromeliothrix and Paracolpoda are very sparse, with sequences from only one species for each. Broader sampling and molecular sequencing of Paracolpoda species is necessary to further clarify these relationships. Paracolpoda steinii pop5 separates from P. steinii pop1‐4 in concatenated tree, the reason may be due to morphological differences, e.g., a larger body size compared with the other four populations.

4.3. Bardeliella pulchra evolutionary position

In previous work, and in our study, B. pulchra was always the earliest divergent branch of Colpodida in the SSU‐rRNA gene tree, which means it has been stable between Colpodida and Cyrtolophosidida (Bourland et al., 2011; Dunthorn et al., 2012; Rajter et al., 2020; Vďačný & Foissner, 2019). But, we established multiple gene trees and they all confirmed the instability of the position of B. pulchra in Colpodida.

Morphologically, three traits indicated that Bardeliella pulchra belongs to the Colpodida: (1) like most of colpodids, B. pulchra can divide in cysts; (2) B. pulchra's silverline pattern consists of highly ordered, comparatively large meshes extending between two ciliary rows each, and this silverline pattern is widely found in Colpodida species (Foissner, 1993; Foissner et al., 2011); (3) the oral ciliary fields of B. pulchra. Bardeliella and Colpoda are similar, i.e., the left oral ciliary field consisting of a shorter proximal portion with equidistantly spaced, monokinetidal rows (Foissner et al., 2011).

AUTHOR CONTRIBUTIONS

Bailin Li: Formal analysis (lead); software (lead); writing – original draft (lead); writing – review and editing (equal). Yumeng Song: Formal analysis (equal); investigation (equal); software (equal). Tingting Hao: Formal analysis (equal); investigation (equal); software (equal). Li Wang: Formal analysis (equal); investigation (equal); software (equal). Weibin Zheng: Formal analysis (equal); investigation (equal); software (equal). Zhao Lyu: Methodology (equal); software (equal); writing – review and editing (equal). Ying Chen: Conceptualization (equal); funding acquisition (supporting). Xuming Pan: Conceptualization (lead); formal analysis (lead); funding acquisition (lead); methodology (lead); supervision (lead); writing – review and editing (equal).

CONFLICT OF INTEREST

All authors declare that they have no conflicts of interest.

ACKNOWLEDGMENTS

This work was supported by the Natural Science Foundation of China (project numbers: 31970498, 32000302), and Shenzhen Science and Technology Program (Grant No. KQTD20190929172630447). Many thanks are given to Xinyuan Zhang, Rong Gao and Lei Zhu, for their help on sampling.

APPENDIX A.

TABLE A1.

Primers used in the polymerase chain reactions for ciliate SSU‐rRNA, ITS1‐5.8S‐ ITS2 rRNA, and mtSSUrRNA gene in the present study.

Primer name Primer sequence
SSU‐rRNA Euk‐A 5′‐AAC CTG GTT GAT CCT GCC AGT‐3′ (Medlin et al., 1988)
Euk‐B 5′‐TGA TCC TTC TGC AGG TTC ACC TAC‐3′ (Medlin et al., 1988)
ITS1‐5.8S‐ ITS2 rRNA 5.8S‐F 5′‐GTA GGT GAA CCT GCG GAA GGA TCA TTA‐3′ (Goggin, 1994)
5.8S‐R 5′‐TAC TGA TAT GCT TAA GTT CAG CGG‐3′ (Goggin, 1994)
mtSSU‐rRNA mt‐F 5′‐TGT GCC AGC AGC CGC GGT AA‐3′ (Hoek et al., 2000)
mt‐R 5′‐CCC A(C)T ACC A(G)G TAC CTT GTG T‐3′ (Hoek et al., 2000)
Open in a new tab

TABLE A2.

Newly sequenced genes in the present work

Taxon Accession numbers Lengths (bp) G&C % Sampling site
Class Colpodea
Colpoda inflata pop1 MZ557833 1725 44.52% Harbin
MZ562697 1041 33.14%
MZ562700 721 38.00%
Colpoda inflata pop2 MZ557838 1717 44.09% Harbin
MZ562692 1027 32.13%
MZ562693 656 38.87%
Colpoda cf. inflata MZ557804 1715 44.26% Harbin
MZ562691 1036 31.66%
MZ562698 722 37.67%
Colpoda grandis MZ562690 1711 45.30% Harbin
MZ562696 1066 32.55%
MZ562694 647 37.25%
Colpoda reniformis OL872369 1717 44.55% Mudanjiang
OL873584 1065 31.92%
OL873585 664 38.10%
Paracolpoda steinii pop1 OL894854 1716 44.55% Shuangyashan
OL894851 1013 27.54%
OL894847 542 36.16%
Paracolpoda steinii pop2 OL961825 1715 44.55% Shuangyashan
OL894855 1019 28.07%
OL894849 543 36.10%
Paracolpoda steinii pop3 OL894848 1714 44.69% Shuangyashan
OL894858 1010 27.62%
OL894856 538 36.06%
Paracolpoda steinii pop4 OL894852 1726 44.38% Shuangyashan
OL894853 1025 28.00%
OL894850 543 36.10%
Paracolpoda steinii pop5 MZ562701 1714 44.34% Shuangyashan
MZ562699 1014 28.01%
MZ562695 533 36.40%
Open in a new tab

Li, B. , Song, Y. , Hao, T. , Wang, L. , Zheng, W. , lyu, Z. , Chen, Y. , & Pan, X. (2022). Insights into the phylogeny of the ciliate of class Colpodea based on multigene data. Ecology and Evolution, 12, e9380. 10.1002/ece3.9380

Contributor Information

Ying Chen, Email: chenying@hit.edu.cn.

Xuming Pan, Email: pppppp206@126.com.

DATA AVAILABILITY STATEMENT

The data presented in the study are deposited in the NCBI database (https://www.ncbi.nlm.nih.gov/) repository, accession numbers, lengths, and G&C contents are shown in Appendix Table A2.

REFERENCES

  1. Agatha, S. , Ganser, M. H. , & Santoferrara, L. F. (2021). The importance of type species and their correct identification: A key example from tintinnid ciliates (Alveolata, Ciliophora, Spirotricha). The Journal of Eukaryotic Microbiology, 68, e12865. 10.1111/jeu.12865 [DOI] [PubMed] [Google Scholar]
  2. Bai, Y. , Wang, R. , Song, W. , Suzuki, T. , & Hu, X. Z. (2020). Redescription of five tintinnine ciliates (Alveolata: Ciliophora: Oligotrichea) from coastal waters of Qingdao, China. Marine Life Science & Technology, 2, 209–221. 10.1007/s42995-020-00034-2 [DOI] [Google Scholar]
  3. Berger, H. (2011). Monograph of the gonostomatidae and kahliellidae (Ciliophora, Hypotricha). Monographiae Biologicae., 90, 1–741. 10.1007/978-94-007-0455-8 [DOI] [Google Scholar]
  4. Boore, J. L. , & Brown, W. M. (1998). Big trees from little genomes: Mitochondrial gene order as a phylogenetic tool. Current Opinion in Genetics & Development, 8, 668–674. 10.1016/s0959-437x(98)80035-x [DOI] [PubMed] [Google Scholar]
  5. Bourland, W. , Hampikian, G. , & Vďačný, P. (2012). Morphology and phylogeny of a new woodruffifiid ciliate, Etoschophrya inornata sp. n. (Ciliophora, Colpodea, Platyophryida), with an account on evolution of platyophryids. Zoologica Scripta, 41, 400–416. 10.1111/j.1463-6409.2012.00539.x [DOI] [Google Scholar]
  6. Bourland, W. , Vďačný, P. , Davis, M. C. , & Hampikian, G. (2011). Morphology, morphometrics, and molecular characterization of Bryophrya gemmea n. sp. (Ciliophora, Colpodea): Implications for the phylogeny and evolutionary scenario for the formation of oral ciliature in the order Colpodida. The Journal of Eukaryotic Microbiology, 58, 22–36. 10.1111/j.1550-7408.2010.00522.x [DOI] [PubMed] [Google Scholar]
  7. Bradbury, P. C. , & Outka, D. E. (1967). The structure of Colpoda elliotti n. sp. The Journal of Protozoology, 14(2), 344–348. 10.1111/j.1550-7408.1967.tb02006.x [DOI] [PubMed] [Google Scholar]
  8. Cheng, T. , Wang, Y. Y. , Huang, J. , Chen, X. , Zhao, X. L. , Gao, S. , & Song, W. B. (2019). Our recent progress in epigenetic research using the model ciliate, Tetrahymena thermophila . Marine Life Science & Technology, 1, 4–14. 10.1007/s42995-019-00015-0 [DOI] [Google Scholar]
  9. Chi, Y. , Chen, X. R. , Li, Y. Q. , Wang, C. D. , Zhang, T. T. , Ayoub, A. , Warren, A. , Song, W. B. , & Wang, Y. Y. (2021). New contributions to the phylogeny of the ciliate class Heterotrichea (Protista, Ciliophora): Analyses at family‐genus level and new evolutionary hypotheses. Science China Life Sciences, 64, 606–620. 10.1007/s11427-020-1817-5 [DOI] [PubMed] [Google Scholar]
  10. Corliss, J. O. (1979). The ciliated protozoa: Characterization, classification and guide to the literature. Pergamon Press. 10.1016/B978-0-08-018752-5.50010-0 [DOI] [Google Scholar]
  11. Dias, R. J. P. , de Souza, P. M. , Rossi, M. F. , Wieloch, A. H. , Silva‐Neto, I. D. , & D'Agosto, M. (2021). Ciliates as bioindicators of water quality: A case study in the neotropical region and evidence of phylogenetic signals (18S‐rDNA). Environmental Pollution, 268, 115760. 10.1016/j.envpol.2020.115760115760 [DOI] [PubMed] [Google Scholar]
  12. Dunthorn, M. , Foissner, W. , & Katz, L. A. (2008). Molecular phylogenetic analysis of class Colpodea (phylum Ciliophora) using broad taxon sampling. Molecular Phylogenetics and Evolution, 46, 316–327. 10.1016/j.ympev.2007.08.006 [DOI] [PubMed] [Google Scholar]
  13. Dunthorn, M. , Foissner, W. , & Katz, L. A. (2011). Expanding character sampling in ciliate phylogenetic inference using mitochondrial SSU‐rDNA as a molecular marker. Protist, 162, 85–99. 10.1016/j.protis.2010.06.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dunthorn, M. , Hall, M. , Foissner, W. , & Katz, L. A. (2014). Broad taxon sampling of ciliates using mitochondrial small subunit ribosomal DNA. Acta Protozoologica, 53, 207–213. 10.4467/16890027AP.14.017.1598 [DOI] [Google Scholar]
  15. Dunthorn, M. , Katz, L. A. , Stoeck, T. , & Foissner, W. (2012). Congruence and indifference between two molecular markers for understanding oral evolution in the Marynidae sensu lato (Ciliophora, Colpodea). European Journal of Protistology, 48, 297–304. 10.1016/j.ejop.2012.01.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fan, X. P. , Lin, X. F. , Liu, W. W. , Xu, Y. , Al‐Farraj, S. A. , Al‐Rasheid, K. A. S. , & Warren, A. (2013). Morphology of three new marine Frontonia species (Ciliophora; Peniculida) with note on the phylogeny of this genus. European Journal of Protistology, 49, 312–323. 10.1016/j.ejop.2012.06.003 [DOI] [PubMed] [Google Scholar]
  17. Fernández‐Galiano, D. (1979). Transfer of the widely known “spirotrich” ciliate Bursaria truncatella O. F. M. to the Vestibulifera as a separate order there, the Bursariomorphida. Transactions of the American Microscopical Society, 98, 447–454. [Google Scholar]
  18. Foissner, W. (1985). Klassifikation und Phylogenie der Colpodea (Protozoa: Ciliophora). Archiv für Protistenkunde, 129, 239–290. 10.1016/S0003-9365(85)80025-7 [DOI] [Google Scholar]
  19. Foissner, W. (1987). Soil protozoa: Fundamental problems, ecological significance, adaptations in ciliates and testaceans, bioindicators, and guide to the literature. Progress in Protistology, 2, 69–212. [Google Scholar]
  20. Foissner, W. (1992). The silver carbonate methods. In Lee J. J. & Soldo A. T. (Eds.), Protocols in protozoology (pp. C7.1–C7.3). Society of Protozoologists, Allen Press Inc. [Google Scholar]
  21. Foissner, W. (2010). Life cycle, morphology, ontogenesis, and phylogeny of Bromeliothrix metopoides nov. gen., nov. spec., a Peculiar Ciliate (Protista, Colpodea) from Tank Bromeliads (Bromeliaceae). Acta Protozoologica, 49(3), 159–193. [PMC free article] [PubMed] [Google Scholar]
  22. Foissner, W. (2016). Terrestrial and semterrestrial ciliates (Protozoa, Ciliophora) from Venezuela and Galápagos. Denisia, 35, 1–912. [Google Scholar]
  23. Foissner, W. , & Stoeck, T. (2009). Morphological and molecular characterization of a new protist family, Sandmanniellidae n. fam. (Ciliophora, Colpodea), with description of Sandmanniella terricola n. g., n. sp. from the Chobe floodplain in Botswana. The Journal of Eukaryotic Microbiology, 56, 472–483. 10.1111/j.1550-7408.2009.00429.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Foissner, W. (1993). Colpodea (Ciliophora). Protozoenfauna, 4/1(i–x), 1–798. [Google Scholar]
  25. Foissner, W. , Agatha, S. , & Berger, H. (2002). Soil ciliates (Protozoa, Ciliophora) from Namibia (Southwest Africa) with emphasis on two contrasting environments, the Etosha region and the Namib Desert. Denisia, 5, 1–1459. [Google Scholar]
  26. Foissner, W. , Bourland, W. A. , Wolf, K. W. , Stoeck, T. , & Dunthorn, M. (2014). New SSU‐rDNA sequences for eleven colpodeans (Ciliophora, Colpodea) and description of Apocyrtolophosis nov. gen. European Journal of Protistology, 50, 40–46. 10.1016/j.ejop.2013.09.003 [DOI] [PubMed] [Google Scholar]
  27. Foissner, W. , Stoeck, T. , Agatha, S. , & Dunthorn, M. (2011). Intraclass evolution and classification of the Colpodea (Ciliophora). The Journal of Eukaryotic Microbiology, 58, 397–415. 10.1111/j.1550-7408.2011.00566.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Gao, F. , Gao, S. , Wang, P. , Katz, L. A. , & Song, W. B. (2014). Phylogenetic analyses of cyclidiids (Protista, Ciliophora, Scuticociliatia) based on multiple genes suggest their close relationship with thigmotrichids . Molecular Phylogenetics and Evolution, 75, 219–226. 10.1016/j.ympev.2014.01.032 [DOI] [PubMed] [Google Scholar]
  29. Gao, F. , Huang, J. , Zhao, Y. , Li, L. F. , Liu, W. W. , Miao, M. , Zhang, Q. Q. , Li, J. M. , Yi, Z. Z. , El‐Serehy, H. A. , Warren, A. , & Song, W. B. (2017). Systematic studies on ciliates (Alveolata, Ciliophora): Progress and achievements based on molecular information. European Journal of Protistology, 61, 409–423. 10.1099/ijs.0.028464-0 [DOI] [PubMed] [Google Scholar]
  30. Gao, F. , Katz, L. A. , & Song, W. B. (2013). Multigene‐based analyses on evolutionary phylogeny of two controversial ciliate orders: Pleuronematida and Loxocephalida (Protista, Ciliophora, Oligohymenophorea). Molecular Phylogenetics and Evolution, 68, 55–63. 10.1016/j.ympev.2013.03.018 [DOI] [PubMed] [Google Scholar]
  31. Gao, F. , Warren, A. , Zhang, Q. Q. , Gong, J. , Miao, M. , Sun, P. , Xu, D. P. , Huang, J. , Yi, Z. Z. , & Song, W. B. (2016). The all‐data‐based evolutionary hypothesis of ciliated protists with a revised classification of the phylum Ciliophora (Eukaryota, Alveolata). Scientific Reports, 6, 24874. 10.1038/srep24874 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Goggin, C. L. (1994). Variation in the two internal transcribed spacers and 5.8S ribosomal RNA from five isolates of the marine parasite Perkinsus (Protista, Apicomplexa). Molecular and Biochemical Parasitology, 65, 179–182. 10.1016/0166-6851(94)90126-0 [DOI] [PubMed] [Google Scholar]
  33. Griffiths‐Jones, S. , Bateman, A. , Marshall, M. , Khanna, A. , & Eddy, S. R. (2003). Rfam: An RNA family database. Nucleic Acids Research, 31(1), 439–441. 10.1093/nar/gkg006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Hall, T. A. (1999). BioEdit: A user‐friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41(41), 95–98. [Google Scholar]
  35. Hoek, A. H. , Alen, T. A. , Sprakel, V. S. , Leunissen, J. A. , Brigge, T. , Vogels, G. D. , & Hackstein, J. H. (2000). Multiple acquisition of methanogenic archaeal symbionts by anaerobic ciliates. Molecular Biology and Evolution, 17, 251–258. 10.1093/oxfordjournals.molbev.a026304 [DOI] [PubMed] [Google Scholar]
  36. Kumar, S. , Stecher, G. , & Tamura, K. (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870–1874. 10.1093/molbev/msw054 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Liu, M. J. , Liu, Y. J. , Zhang, T. T. , Lu, B. R. , Gao, F. , Gu, J. , Al‐Farraj, S. A. , Hu, X. Z. , & Song, W. B. (2022). Integrative studies on the taxonomy and molecular phylogeny of four new Pleuronema species (Protozoa, Ciliophora, Scuticociliatia). Marine Life Science & Technology, 4, 179–200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lynn, D. H. (1978). Comparative ultrastructure and systematics of the Colpodida. Dissertation Abstracts International, 38, 1–2. [Google Scholar]
  39. Lynn, D. H. (2003). Morphology or molecules: How do we identify the major lineages of ciliates (Phylum Ciliophora)? European Journal of Protistology, 39, 356–364. [Google Scholar]
  40. Lynn, D. H. (2008). The ciliated protozoa: Characterization, classification and guide to the literature (3rd ed.). Springer Verlag. 10.1016/B978-0-08-018752-5.50010-0 [DOI] [Google Scholar]
  41. Lynn, D. H. , Wright, A. D. G. , Schlegel, M. , & Foissner, W. (1999). Phylogenetic relationships of orders within the class Colpodea (phylum Ciliophora) inferred from small subunit rRNA gene sequences. Journal of Molecular Evolution, 48, 605–614. 10.1007/pl00006503 [DOI] [PubMed] [Google Scholar]
  42. Medlin, L. , Elwood, H. J. , Stickel, S. , & Sogin, M. L. (1988). The characterization of enzymatically amplified eukaryotic 16S‐like rDNA‐coding regions. Gene, 71, 491–499. 10.1016/0378-1119(88)90066-2 [DOI] [PubMed] [Google Scholar]
  43. Miao, M. , Warren, A. , Song, W. B. , Wang, S. , Shang, H. , & Chen, Z. (2008). Analysis of the internal transcribed spacer 2 (ITS2) region of scuticociliates and related taxa (Ciliophora, Oligohymenophorea) to infer their evolution and phylogeny. Protist, 159(4), 519–533. 10.1016/j.protis.2008.05.002 [DOI] [PubMed] [Google Scholar]
  44. Moore, W. S. (1995). Inferring phylogenies from mtDNA variation: Mitochondrialgene trees versus nuclear‐gene trees. Evolution, 49, 718–726. 10.1111/j.1558-5646.1995.tb02308.x [DOI] [PubMed] [Google Scholar]
  45. Nylander, J. A. (2004). MrModeltest v2. Evolutinary Biology Centre, Uppsala University. [Google Scholar]
  46. Penn, O. , Privman, E. , Ashkenazy, H. , Landan, G. , Graur, D. , & Pupko, T. (2010). GUIDANCE: A web server for assessing alignment confidence scores. Nucleic Acids Research, 38, W23–W28. 10.1093/nar/gkq443 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Posada, D. , & Crandall, K. A. (1998). Modeltest: Testing the model of DNA substitution. Bioinformatics, 14, 817–818. [DOI] [PubMed] [Google Scholar]
  48. Quintela‐Alonso, P. , Nitsche, F. , & Arndt, H. (2011). Molecular characterization and revised systematics of Microdiaphanosoma arcuatum (Ciliophora, Colpodea). The Journal of Eukaryotic Microbiology, 58, 114–119. 10.1111/j.1550-7408.2010.00527.x [DOI] [PubMed] [Google Scholar]
  49. Rajter, Ľ. , Ewers, I. , Graupner, N. , Vďačný, P. , & Dunthorn, M. (2020). Colpodean ciliate phylogeny and reference alignments for phylogenetic placements. European Journal of Protistology, 77, 125747. 10.1016/j.ejop.2020.125747 [DOI] [PubMed] [Google Scholar]
  50. Rand, D. M. (2003). The units of selection on mitochondrial DNA. Annual Review of Ecology and Systematics, 32, 415–448. [Google Scholar]
  51. Ronquist, F. , Teslenko, M. , Mark, P. , Ayres, D. L. , Darling, A. , Höhna, S. , Larget, B. , Liu, L. , Suchard, M. A. , & Huelsenbeck, J. P. (2012). MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61, 539–542. 10.1093/sysbio/sys029 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Small, E. B. , & Lynn, D. H. (1981). A new macrosystem for the phylum Ciliophora Doflein, 1901. Bio Systems, 14, 387–401. 10.1016/0303-2647(81)90045-9 [DOI] [PubMed] [Google Scholar]
  53. Song, W. B. , Warren, A. , & Hu, X. Z. (2009). Free‐living ciliates in the Bohai and Yellow Seas, China. Science Press. [Google Scholar]
  54. Song, W. Y. , Zhang, T. Y. , Zhang, X. , Warren, A. , Song, W. B. , Zhao, Y. , & Luo, X. T. (2021). Taxonomy, ontogenesis and evolutionary phylogeny of the algae‐bearing ciliate Limnoholosticha viridis (Kahl, 1932), with establishment of a new genus and new family (Protista, Ciliophora, Hypotrichia). Frontiers in Microbiology, 11, 560915. 10.3389/fmicb.2020.560915 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Stamatakis, A. (2014). RAxML version 8: A tool for phylogenetic analysis and post‐analysis of large phylogenies. Bioinformatics, 30, 1312–1313. 10.1093/bioinformatics/btu033 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Strüder‐Kypke, M. , Wright, A. D. G. , & Fokin, S. I. (2000). Phylogenetic relationships of the subclass Peniculia (Oligohymenophorea, Ciliophora) inferred from small subunit rDNA gene sequence. The Journal of Eukaryotic Microbiology, 47, 419–429. 10.1111/j.1550-7408.2000.tb00069.x [DOI] [PubMed] [Google Scholar]
  57. Sun, M. , Li, Y. , Cai, X. L. , Liu, Y. Q. , Chen, Y. , & Pan, X. M. (2020). Further insights into the phylogeny of peniculid ciliates (Ciliophora, Oligohymenophorea) based on multigene data. Molecular Phylogenetics and Evolution, 154, 107003. 10.1016/j.ympev.2020.107003 [DOI] [PubMed] [Google Scholar]
  58. Vďačný, P. , & Foissner, W. (2019). Re‐analysis of the 18S rRNA gene phylogeny of the ciliate class Colpodea. European Journal of Protistology, 67, 89–105. 10.1016/j.ejop.2018.11.003 [DOI] [PubMed] [Google Scholar]
  59. Wang, C. D. , Zhang, T. T. , Wang, Y. R. , Katz, L. A. , Gao, F. , & Song, W. B. (2017). Disentangling sources of variation in SSU rDNA sequences from single cell analyses of ciliates: Impact of copy number variation and experimental error. Proceedings of the Royal Society B: Biological Science, 284, 20170425. 10.1098/rspb.2017.0425 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wang, J. Y. , Zhang, T. T. , Li, F. C. , Warren, A. , Li, Y. B. , & Shao, C. (2021). A new hypotrich ciliate, Oxytricha xianica sp. nov., with notes on the morphology and phylogeny of a Chinese population of Oxytricha auripunctata Blatterer & Foissner, 1988 (Ciliophora, Oxytrichidae). Marine Life Science & Technology, 3, 303–312. 10.1007/s42995-020-00089-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wang, P. , Wang, Y. R. , Wang, C. D. , Zhang, T. T. , Al‐Farraj, S. A. , & Gao, F. (2017). Further consideration on the phylogeny of the Ciliophora: Analyses using both mitochondrial and nuclear data with focus on the extremely confused class Phyllopharyngea. Molecular Phylogenetics and Evolution, 112, 96–106. 10.1016/j.ympev.2017.04.018 [DOI] [PubMed] [Google Scholar]
  62. Weisse, T. , & Montagnes, D. J. S. (2021). Ecology of planktonic ciliates in a changing world: Concepts, methods, and challenges. The Journal of Eukaryotic Microbiology, 69(5), e12879. 10.1111/jeu.12879 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Weisse, T. , Scheffel, U. , Stadler, P. , & Foissner, W. (2013). Bromeliothrix metopoides, a boom and bust ciliate (Ciliophora, Colpodea) from tank bromeliads. European Journal of Protistology, 49, 406–419. 10.1016/j.ejop.2013.02.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wu, L. , Li, J. Q. , Warren, A. , & Lin, X. F. (2021). Taxonomy and systematics of a new pleurostomatid ciliate, Pseudolitonotus spirelis gen. et sp. n. (Protozoa, Ciliophora, Haptoria). Marine Life Science & Technology, 4, 31–41. 10.1007/s42995-021-00123-w [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zhang, T. T. , Fan, X. P. , Gao, F. , Al‐Farraj, S. A. , El‐Serehy, H. A. , & Song, W. B. (2019). Further analyses on the phylogeny of the subclass Scuticociliatia (Protozoa, Ciliophora) based on both nuclear and mitochondrial data. Molecular Phylogenetics and Evolution, 139, 106565. 10.1016/j.ympev.2019.106565 [DOI] [PubMed] [Google Scholar]
  66. Zhang, T. Y. , Wang, Y. R. , Cheng, T. , Ma, J. Y. , Vďačný, P. , Song, W. B. , & Shao, C. (2020). Systematics and multi‐gene phylogeny of the subfamily Nothoholostichinae (Ciliophora, Hypotrichia), with integrative description of a new marine species Nothoholosticha luporinii n. sp. Frontiers in Marine Science, 7, 610886. [Google Scholar]
  67. Zhao, X. L. , Li, Y. , Duan, L. L. , Chen, X. , Mao, F. B. , Juma, M. , Liu, Y. F. , Song, W. B. , & Gao, S. (2020). Functional analysis of the methyltransferase SMYD in the single‐cell model organism Tetrahymena thermophila . Marine Life Science & Technology, 2, 109–122. 10.1007/s42995-019-00025-y [DOI] [Google Scholar]
  68. Zhao, Y. , Yi, Z. Z. , Gentekaki, E. , Zhan, A. B. , Al‐Farraj, S. A. , & Song, W. B. (2016). Utility of combining morphological characters, nuclear and mitochondrial genes: An attempt to resolve the conflicts of species identification for ciliated protists. Molecular Phylogenetics and Evolution, 94, 718–729. 10.1016/j.ympev.2015.10.0 [DOI] [PubMed] [Google Scholar]
  69. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research, 31(13), 3406–3415. 10.1093/nar/gkg595 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data presented in the study are deposited in the NCBI database (https://www.ncbi.nlm.nih.gov/) repository, accession numbers, lengths, and G&C contents are shown in Appendix Table A2.

Articles from Ecology and Evolution are provided here courtesy of Wiley

RESOURCES