Historical Review of Studies on Cyrtophorian Ciliates (Ciliophora, Cyrtophoria) from China.

The subclass Cyrtophoria are a group of morphologically specialized ciliates which mainly inhabit soil, freshwater, brackish water, and marine environments. In this study, we revise more than 50 publications on the taxonomy, phylogeny, and ecology of cyrtophorian ciliates in China since the first publication in 1925, most of which were carried out in coastal areas. The research history can be divided into three periods: the early stage, the Tibet stage, and the molecular stage. To date, 103 morpho-species (147 isolates) have been formally recorded in China, with ciliature patterns described for 82 of them. A species checklist and an illustrated identification key to the genera are provided. A total of 100 small subunit rDNA sequences have been obtained for 74 taxonomic hits (lowest taxonomic rank to species or genus). These sequences are used for the study of molecular phylogeny. Based on these morphological data and molecular phylogeny analyses, we synthesize the understanding of the phylogeny of cyrtophorian ciliates. We hypothesize that the key evolutionary event of cyrtophorian ciliates lies in the separation of the stomatogenesis zone (postoral kineties) from the left kineties, namely, the formation of an independent "sexual organelle". We, furthermore, briefly summarize the ecological features of cyrtophorian ciliates and provide a comprehensive bibliography of related research from China. Finally, we give an outlook on the future research directions of these taxa.

1. Introduction

As evidenced by an increasing number of publications, China is now recognized as one of the hotspots for the study of the taxonomy and systematics of ciliates [1,2,3,4,5]. Among ciliates, cyrtophorians (subclass Cyrtophoria) are a morphologically specialized group that feature a unique combination of morphological characteristics: (1) The cytostome is reinforced by a special buccal structure called the pharyngeal basket which is made up of fibers that are usually organized in the form of nematodesmal rods [6]. (2) The oral ciliature consists of a few short kinetal segments, with usually one preoral kinety (sometimes in several short fragments) and two circumoral kineties (see oral kineties—OK—in Figure 1) [7]. Some species have a degenerated oral structure or more circumoral kineties. During stomatogenesis, the oral kineties are formed by an anticlockwise rotation of the oral anlagen (see stomatogenetic zone—SZ—in Figure 1) [8]. (3) The cilia are mainly restricted to the ventral side, and only a few are located at the anterior part of the dorsal side [7,9]. (4) Some species have organelles adapted to the adhesion on the substrate, such as podites (e.g., Dysteria and Hartmannula), glandules (e.g., Trichopodiella), and finger-like tentacles (e.g., Lynchella and Chlamydonella) (Figure 1) [7,9,10]. From more “general” cyrtophorians to highly specialized ones, the left kineties tend to degenerate and move to the frontal and right part of the cell. The right kineties are also inclined and mostly restricted to the right margin as a result of the lateral compression of the body in the order Dysteriida (Figure 1).

Figure 1

Schematic ciliary patterns of cyrtophorian ciliates. (A) Order Chlamydodontida. (B) Order Dysteriida. Abbreviations: EF—equatorial fragment; LK—left kineties; OK—oral kineties; P—podite; PK—postoral kineties; RK—right kineties; SZ—stomatogenetic zone; and TF—terminal fragment.

Most cyrtophorian ciliates live as periphyton and feed on bacteria (including cyanobacteria) and/or eukaryotic microalgae [11,12,13,14]. They are widely distributed in a variety of habitats including soil [15,16], freshwater [11,16,17], marine environments [4,6,9,17,18,19,20,21,22], brackish waters [12,13,23,24,25,26], and even glaciers in Antarctica [27,28]. As active bacterivores, some species commonly occur in eutrophic manmade ecosystems, such as Chilodonella uncinata, Gastronauta spp., and Trochilia minuta, which have been used as bio-indicators for monitoring sludge performance in wastewater treatment plants [11,29,30,31,32]. Several free-living species thrive in mariculture [33,34,35] and freshwater fish aquaculture tanks [36]. A few species even live as obligate parasites on the gills and skin of fish [36,37,38,39,40], or on the mucosa of the blowholes of sea mammals [41,42,43], and hence are target organisms in pathogenic studies [44].

To date, more than 180 cyrtophorian ciliate species are known worldwide, divided into 2 orders, 10 families, and 45 genera [3,8,9]. Several milestones can be identified in the research history: (1) Müller [45] reported two cyrtophorian ciliates (Trithigmostoma cucullulus and Chlamydodon triquetrus, according to the current nomination). (2) Klein [46] first applied silver impregnation (dry silver nitrate) on ciliates and revealed the ciliature of the cyrtophorian Chilodonellauncinata. (3) Kahl [17] summarized more than 80 species with fine descriptions based on previous works (e.g., [47,48,49]) and his own. (4) Deroux [6,18,19,20,21,22] conducted a long-term, exclusive study mainly on cyrtophorian ciliates, documenting about 50 marine species with fine ciliary patterns. (5) Snoeyenbos-West et al. [50] analyzed, for the first time, the systematic position of cyrtophorian ciliates using SSU rDNA sequences.

In this paper, we review the research on cyrtophorian ciliates in China, covering aspects of taxonomy, molecular phylogeny, and ecology, and provide an up-to-date literature guide to related research. We, furthermore, present a revised phylogeny of cyrtophorian ciliates based on existing morphological knowledge and the molecular phylogenetic tree inferred from updated SSU rDNA sequences. As a closing remark, we give an outlook on the future research prospects of this special group of ciliates.

2. Morphological Taxonomy

2.1. Brief Research History

For this section, we collected and reviewed 50 publications (Table 1) with morphological descriptions, excluding those where the species identification could not be traced and confirmed. To the best of our knowledge, the very first report of cyrtophorian ciliates from China was given by Wang [51] in the year of 1925. He isolated and described three species, Chilodon caudata Stokes, 1885, C. cucullulus (Müller, 1786) Klein, 1927, and C. vorax Stokes, 1887 (current names are Chilodonella caudata, Trithigmostoma cucullulus and Chilodonella vorax, respectively), from freshwater lakes in Nanjing. Only a couple of years later, he amended the list with a new species, Dysteria amoyensis Wang, 1934, which was discovered in a marine aquarium in Xiamen [52]. Nie and Ho [53] also established a new species, Gastronauta fontzoui Nie and Ho, 1943, extracted from freshwater shrimps; however, they only had a vague morphological description. After an almost three-decade blank, Wang [54] recorded 16 more species, including a new one, during a massive investigation on Mount Everest; unfortunately, only five were described with morphological characteristics. Subsequently, Shen [16] summarized her own novel studies and previous results on ciliate fauna investigation from the Tibet Plateau, and briefly described 22 species (including all the species from Wang [54]). Until then, studies were only conducted using live observation. Since the 2000s, more advanced studies have been based on a combination of live observation and silver staining, mainly focusing on marine and brackish water habitats (e.g., [1,3,4,9,55]). During this period, some scattered studies on soil species and parasites were also published (e.g., [37,38,39,40,56]). Research history in China can, therefore, be divided into three stages (Figure 2): (A) During the early stage from 1925 to 1973, only a handful of studies were published, with six species reported including two new taxa. (B) The Tibet stage (1974–2000) includes the massive faunal investigations summarized by Wang [54] on Mount Everest and the summary by Shen [16] on the Tibet Plateau. In total, they described 22 species, with one new form. This stage also included a few publications by Song and his collaborator [55,57,58]. (C) The molecular stage began after 2000. From then on, most species were described by both live observation and silver staining (mostly protargol staining). During this period, the number of recorded species, new species and species with ciliature patterns increased strongly. The molecular studies also had a rapid growth in this period (see the Molecular Phylogenetic Studies section below).

Table 1

Checklist of cyrtophorian ciliate species/isolates with morphological descriptions from China. New species are highlighted in bold. Species are presented with current names. * Ref. [4] by Song et al. summarizes 32 species found by Song’s group (Ocean University of China) from 1991 to 2008. Here, only seven species/populations not published before 2009 are listed. † This species was isolated from marine water, which is different from those populations of Chilodonella uncinata isolated from soil or freshwater habitats; thus, we count it as a different species. § Previous name was Chilodonella parauncinata [59].

Publication	Species Name (Current Name)	Ciliature	Site	Habitat
Wang (1925) [51]	Chilodonella caudata Stokes, 1885	-	Nanjing	Freshwater
	Chilodonella vorax Stokes, 1887	-	Nanjing	Freshwater
	Trithigmostoma cucullulus (Müller, 1786) Jankowski, 1967	-	Nanjing	Freshwater
Wang (1934) [52]	Dysteria amoyensis Wang, 1934	-	Xiamen	Marine
	Hartmannula entzi Kahl, 1931	-	Xiamen	Marine
Nie and Ho (1943) [53]	Gastronauta fontzoui Nie & Ho, 1943	-	-	Freshwater shrimps
Wang (1974) [54]	Chilodonella aplanata Kahl, 1931	-	Mount Everest	Soil
	Chilodonella parauncinata Wang, 1974	-	Mount Everest	Soil
	Chilodonella uncinata (Ehrenberg, 1838) Strand, 1928	-	Mount Everest	Soil
	Odontochlamys convexa (Kahl, 1931) Blatterer & Foissner, 1992	-	Mount Everest	Soil
	Pseudochilodonopsis algivora (Kahl, 1931) Foissner, 1979	-	Mount Everest	Soil
Shen (1983) [16]	Chilodonella aplanata Kahl, 1931	-	Tibetan plateau	Freshwater
	Trithigmostoma bavariensis (Kahl, 1931) Foissner,1987	-	Tibetan plateau	Sodium sulfate lake
	Chilodonella capucina (Penard, 1922) Kahl, 1931	-	Tibetan plateau	Freshwater
	Chilodonella dentata Fauguè, 1876	-	Tibetan plateau	Freshwater
	Chilodonella fluviatilis Stokes, 1885	-	Tibetan plateau	Freshwater
	Chilodonella granulate Penard, 1922	-	Tibetan plateau	Freshwater
	Chilodonella nana Kahl, 1928	-	Tibetan plateau	Freshwater
	Chilodonella parauncinata Wang, 1974	-	Tibetan plateau	Freshwater
	Chilodonella piscicola Zacharias, 1894	-	Tibetan plateau	Freshwater
	Chilodonella turgidula Penard, 1922	-	Tibetan plateau	Freshwater
	Chilodonella uncinata (Ehrenberg, 1838) Strand, 1928	-	Tibetan plateau	-
	Chlamydonellopsis calkinsi (Kahl, 1928) Blatterer & Foissner, 1990	-	Tibetan plateau	Freshwater
	Lophophorina capronata Penard, 1922	-	Tibetan plateau	Freshwater
	Odontochlamys convexa (Kahl, 1931) Blatterer & Foissner, 1992	-	Tibetan plateau	Freshwater
	Odontochlamys gourandi Certes, 1891	-	Tibetan plateau	-
	Phascolodon vorticella Stein, 1859	-	Tibetan plateau	Freshwater
	Pseudochilodonopsis algivora (Kahl, 1931) Foissner, 1979	-	Tibetan plateau	Freshwater
	Pseudochilodonopsis labiata (Stokes, 1891) Packroff, 1988	-	Tibetan plateau	Freshwater
	Trithigmostoma cucullulus (Müller, 1786) Jankowski, 1967	-	Tibetan plateau	Freshwater
	Trochilia minuta (Roux, 1901) Kahl, 1931	-	Tibetan plateau	Freshwater
	Trochilia palustris Stein, 1859	-	Tibetan plateau	Freshwater
	Trochilia sulcata (Claparède & Lachmann, 1858)	-	Tibetan plateau	Freshwater
Song (1991) [55]	Pseudochilodonopsis marina Song, 1991	√	Qingdao	Marine
Song (1997) [57]	Chilodonella uncinata (Ehrenberg, 1838) Strand, 1928	√	Qingdao	Freshwater
Song and Packroff (1997) [58]	Dysteria brasiliensis Faria et al., 1922	√	Qingdao	Marine
Gong et al. (2002) [60]	Dysteria cristata (Gourret & Roeser, 1888) Kahl, 1931	√	Zhanjiang	Marine
	Dysteria monostyla (Ehrenberg, 1838) Kahl, 1931	√	Qingdao	Marine
Song (2003) [61]	Chlamydonella derouxi Song, 2003	√	Qingdao	Marine
	Orthotrochilia piluta (Deroux, 1976) Song, 2003	√	Qingdao	Marine
Gong and Song (2003) [33]	Dysteria magna Gong & Song, 2003	√	Qingdao	Mariculture water
	Dysteria procera Kahl, 1931	√	Qingdao	Marine
Gong et al. (2003) [35]	Dysteria pusilla (Claparède & Lachmann, 1859) Kahl, 1931	√	Qingdao	Mariculture water
Gong and Song (2004) [34]	Coeloperix sleighi Gong & Song, 2004	√	Qingdao	Mariculture water
Gong and Song (2004) [62]	Hartmannula angustipilosa Deroux & Dragesco, 1968	√	Qingdao	Mariculture water
	Hartmannula derouxi Gong & Song, 2004	√	Qingdao	Mariculture water
Gong and Song (2004) [63]	Dysteria derouxi Gong & Song, 2004	√	Qingdao	Mariculture water
Gong et al. (2005) [64]	Chlamydodon mnemosyne Ehrenberg, 1835	√	Qingdao	Mariculture water
	Chlamydodon obliquus Kahl, 1931	√	Qingdao	Marine
	Chlamydodon triquetrus (Müller, 1786) Kahl, 1931	√	Qingdao	Mariculture water
Gong and Song (2006) [65]	Brooklynella sinensis Gong & Song, 2006	√	Qingdao	Mariculture water
Gong and Song (2006) [66]	Chlamydonella derouxi Song, 2003	√	Qingdao	Mariculture water
	Chlamydonella pseudochilodon (Deroux, 1970) Petz et al., 1995	√	Qingdao	Mariculture water
	Chlamydonellopsis calkinsi (Kahl, 1928) Blatterer & Foissner, 1990	√	Qingdao	Mariculture water
Gong et al. (2007) [67]	Dysteria brasiliensis Faria et al., 1922	√	Qingdao	Marine
	Dysteria crassipes Claparède & Lachmann, 1859	√	Qingdao	Mariculture water
	Dysteria pectinata (Nowlin, 1913) Kahl, 1931	√	Qingdao	Marine
	Dysteria semilunaris (Gourret & Roeser, 1886) Kahl, 1931	√	Qingdao	Mariculture water
Liu et al. (2008) [68]	Dysteria subtropica Qu et al., 2015	√	Qingdao	Marine
	Pseudochilodonopsis marina Song, 1991	√	Huizhou	Marine
Shao et al. (2008) [69]	Hartmannula sinica Shao et al., 2008	√	Qingdao	Mariculture water
Gong et al. (2008) [70]	Trichopodiella faurei Gong et al., 2008	√	Qingdao	Marine
Fan et al. (2009) [71]	Chlamydodon obliquus Kahl, 1931	√	Huizhou	Marine
	Dysteria derouxi Gong & Song, 2004	√	Huizhou	Marine
* Song et al. (2009) [4]	Aegyria oliva Claparede & Lachmann, 1859	√	Qingdao	Marine and Mariculture
	Agnathodysteria littoralis Deroux, 1976	√	Qingdao	Mariculture water
	† Chilodonella cf. uncinata (Ehrenberg, 1838) Strand, 1928	√	Qingdao	Marine
	Microxysma acutum Deroux, 1976	√	Qingdao	Marine
	Orthotrochilia agamalievi (Deroux, 1976) Song, 2003	√	Qingdao	Marine
	Trochilia petrani Dragesco, 1966	√	Qingdao	Marine
	Trochilia sigmoides Dujardin, 1841	√	Qingdao	Mariculture water
Ning et al. (2009) [56]	Pseudochilodonopsis quadrivacuolata Qu et al., 2015	√	Gansu	Soil
Chen et al. (2011) [72]	Chlamydonyx paucidentatus Deroux, 1976	√	Qingdao	Mariculture water
	Dysteria lanceolata Claparède & Lachmann, 1859	√	Qingdao	Mariculture water
	Lynchella nordica Jankowski, 1968	√	Qingdao	Mariculture water
Pan et al. (2011) [73]	Dysteria derouxi Gong & Song, 2004	√	Qingdao	Marine
	Dysteria legumen (Dujardin, 1841) Kahl, 1931	√	Qingdao	Marine
	Dysteria proraefrons Clark, 1865	√	Qingdao	Marine
	Mirodysteria decora Deroux, 1976	√	Qingdao	Marine
Chen et al. (2012) [23]	Aegyria rostellum Chen et al., 2012	√	Shenzhen	Brackish water
	Paracyrtophoron tropicum Chen et al., 2012	√	Shenzhen	Brackish water
Hu (2012) [38]	Chilodonella hexasticha Kiernik, 1909	√	Luzhou	Ectoparasite of fish
Pan et al. (2012) [74]	Aporthotrochilia pulex (Deroux, 1976) Pan et al., 2012	√	Zhanjiang	Marine
	Heterohartmannula fangi Pan et al., 2012	√	Zhanjiang	Marine
	Trochilia alveolata Pan et al., 2012	√	Hong Kong	Brackish water
	Trochochilodon flavus Deroux, 1976	√	Qingdao	Marine
Pan et al. (2013) [12]	Chlamydodon caudatus Pan et al., 2013	√	Guangzhou	Brackish water
	Chlamydodon paramnemosyne Pan et al., 2013	√	Zhanjiang	Mariculture water
	Chlamydodon salinus Pan et al., 2013	√	Changyi	Mariculture water
Pan et al. (2013) [75]	Orthotrochilia sinica Pan et al., 2013	√	Qingdao	Marine
	Trochilioides recta (Kahl, 1923) Chen et al. 2011	√	Qingdao	Marine
	Trochilioides tenuis (Deroux, 1976) Chen et al., 2011	√	Qingdao	Marine
Zhao et al. (2014) [76]	Coeloperix sleighi Gong & Song, 2004	√	Zhanjiang	Marine
Deng et al. (2015) [37]	Chilodonella piscicola Zacharias, 1894	√	Tibet	Ectoparasite of fish
Qu et al. (2015) [77]	Pseudochilodonopsis fluviatilis Foissner, 1988	√	Yantai	Mariculture water
	Pseudochilodonopsis mutabilis Foissner, 1981	√	Zhanjiang	Brackish water
	Pseudochilodonopsis quadrivacuolata Qu et al., 2015	√	Zhuhai	Brackish water
Qu et al. (2015) [59]	§ Chilodonella apouncinata nom. nov.	√	Qingdao	Freshwater
	Chlamydonella derouxi Song, 2003	√	Qingdao	Marine
	Chlamydonella derouxi Song, 2003	√	Zhanjiang	Brackish water
	Chlamydonella irregularis Qu et al., 2015	√	Qingdao	Mariculture water
Qu et al. (2015) [78]	Dysteria brasiliensis Faria et al., 1922	√	Yantai	Mariculture water
	Dysteria brasiliensis Faria et al., 1922	√	Zhanjiang	Brackish water
	Dysteria crassipes Claparède & Lachmann, 1859	√	Dongying	Mariculture water
	Dysteria cristata (Gourret & Roeser, 1888) Kahl, 1931	√	Zhuhai	Brackish water
	Dysteria derouxi Gong & Song, 2004	√	Qingdao	Marine
	Dysteria nabia Park & Min, 2014	√	Zhanjiang	Brackish water
	Dysteria paraprocera Qu et al., 2015	√	Zhanjiang	Brackish water
	Dysteria proraefrons Clark, 1865	√	Yantai	Mariculture water
	Dysteria subtropica Qu et al., 2015	√	Huizhou	Marine
Pan et al. (2016) [24]	Chlamydonellopsis calkinsi (Kahl, 1928) Blatterer & Foissner, 1990	√	Shanghai	Brackish water
	Dysteria ovalis (Gourret & Roeser, 1886) Kahl, 1931	√	Zhejiang	Marine
	Dysteria semilunaris (Gourret & Roeser, 1888) Kahl, 1931	√	Shanghai	Brackish water
	Phascolodon vorticella Stein, 1859	√	Changzhou	Freshwater
Pan et al. (2017) [79]	Atopochilodon distichum Deroux, 1976	√	Hong Kong	Brackish water
	Chlamydodon rectus Ozaki & Yagiu, 1941	√	Hangzhou	Brackish water
	Coeloperix sinica Pan et al., 2017	√	Qingdao	Marine
	Dysteria compressa (Gourret & Roeser, 1886) Kahl, 1931	√	Zhanjiang	Marine/brackish water
	Odontochlamys alpestris biciliata Foissner et al., 2002	√	Guangzhou	Brackish water
Qu et al. (2017) [25]	Aegyria foissneri Qu et al., 2017	√	Zhanjiang	Brackish water
	Lynchella minuta Qu et al., 2017	√	Zhuhai	Brackish water
Chen et al. (2018) [80]	Aegyria apoliva Chen et al., 2018	√	Qingdao	Marine
	Trithigmostoma cucullulus (Müller, 1786) Jankowski, 1967	√	Guangzhou	Brackish water
Li et al. (2018) [39]	Chilodonella hexasticha Kiernik, 1909	√	Wuhan; Dali; Jiangsu; Hanchuan; Liangshan	Ectoparasite of fish
	Chilodonella piscicola Zacharias, 1894	√	Wuhan; Dali; Jiangsu; Hanchuan; Liangshan	Ectoparasite of fish
Qu et al. (2018) [13]	Chlamydodon crassidens Qu et al., 2018	√	Qingdao	Marine
	Chlamydodon oligochaetus Qu et al., 2018	√	Qingdao	Marine
	Chlamydodon similis Qu et al., 2018	√	Shenzhen	Brackish water
Qu et al. (2018) [81]	Chlamydodon bourlandi Qu et al., 2018	√	Zhanjiang	Brackish water
	Chlamydodon triquetrus (Müller, 1786) Kahl, 1931	√	Yantai	Mariculture water
	Chlamydodon wilberti Qu et al., 2018	√	Zhanjiang	Brackish water
Wang et al. (2019) [26]	Chlamydodon bourlandi Qu et al., 2018	√	Qingdao	Marine
	Chlamydodon pararoseus Wang et al., 2019	√	Qingdao	Marine
	Dysteria crassipes Claparède & Lachmann, 1859	√	Zhanjiang	Brackish water
	Dysteria monostyla (Ehrenberg, 1838) Kahl, 1931	√	Haikou	Marine/brackish water
Wang et al. (2019) [40]	Chilodonella uncinata (Ehrenberg, 1838) Strand, 1928	√	Hubei	Ectoparasite of fish
	Chilodonella hexasticha Kiernik, 1909	√	Hubei	Ectoparasite of fish
Jin et al. (2021) [41]	Kyaroikeus paracetarius Jin et al., 2021	√	Ningbo	Parasite of beluga whale
	Planilamina ovata Ma et al., 2006	√	Ningbo	Parasite of beluga whale
Qu et al. (2021) [14]	Gastronauta paraloisi Qu et al., 2021	√	Shenzhen	Freshwater
	Trithigmostoma cucullulus (Müller, 1786) Jankowski, 1967	√	Shenzhen, Qingdao,Zhanjiang	Freshwater
Wang et al. (2021) [82]	Dysteria brasiliensis Faria et al., 1922	√	Haikou	Brackish water
	Dysteria compressa (Gourret & Roeser, 1886) Kahl, 1931	√	Haikou	Brackish water
	Dysteria ozakii Wang et al., 2021	√	Qingdao	Marine
Zhao et al. (2022) [83]	Dysteria brasiliensis Faria et al., 1922	√	Ningbo	Brackish water
	Dysteria crassipes Claparède & Lachmann, 1859	√	Ningbo	Brackish water
	Dysteria paracrassipes Zhao et al., 2022	√	Ningbo	Brackish water
Sum	103 species, 147 populations, 39 new species	82	-	-

Figure 2

Timeline and accumulated numbers of studied cyrtophorian ciliates (species) in China. The four lines represent the numbers of recorded species, new species, species with ciliature information, and taxonomic hits (lowest taxonomic rank to species or genus) with SSU rDNA sequences. A, B, and C represent three study periods: the early stage (A) 1925–1973)), the Tibet stage (B) 1974–2000)), and the molecular stage (C) since the 2000s)).

2.2. Species List and Classification

As of May 2022, a total of 103 morpho-species (147 isolates) have been reported in China which can be assigned to 8 families and 31 genera (Table 1). Among them, 82 species with ciliature (mainly from protargol staining) have been reported. Three new genera, Aporthotrochilia Pan et al., 2012, Heterohartmannula Pan et al., 2012, and Paracyrtophoron Chen et al., 2012, and thirty-nine new species have been erected. Here, we list the genus classifications regarding the species (with reported morphology) found in China. The class, subclass, order and family assignments are mainly based on Lynn [8] and subsequent modifications (see references in Table 1). Accordingly, Figure 3 and Figure 4 provide an illustrated key to identify the genera existing in China on the basis of morphological characters (mainly ciliary patterns).

Figure 3

Illustrated key for the identification to cyrtophorian genera found in China. All the illustrations are original. The arrows and colored circles indicate dichotomic characteristics. Abbreviations: CSB—cross-striated band; TF—terminal fragment.

Figure 4

Illustrated key for the identification to cyrtophorian genera found in China (Figure 3 continued). All the illustrations are original. The arrows and colored structures indicate dichotomic characteristics. Abbreviations: NR—nematodesmal rods; PK—postoral kineties; and Pr—preoral kinety.

A list of cyrtophorian genera recorded in China and their systematic assignments.

Class Phyllopharyngea de Puytorac et al., 1974

Subclass Cyrtophoria Fauré-Fremiet in Corliss, 1956

Order Chlamydodontida Deroux, 1976

Family Chilodonellidae Deroux, 1970

Chilodonella Strand, 1928

Odontochlamys Certes, 1891

Pseudochilodonopsis Foissner, 1979

Phascolodon Stein, 1859

Trithigmostoma Jankowski, 1967

Family Chlamydodontidae Stein, 1859

Chlamydodon Ehrenberg, 1835

Paracyrtophoron Chen et al., 2012

Family Gastronautidae Deroux, 1994

Gastronauta Engelmann in Bütschli, 1889

Family Lynchellidae Jankowski, 1968

Atopochilodon Kahl, 1933

Chlamydonella Deroux in Petz et al., 1995

Chlamydonellopsis Blatterer & Foissner, 1990

Coeloperix Deroux in Gong & Song, 2004

Lynchella Jankowski, 1968

Family Plesiotrichopidae Deroux, 1976

Trochochilodon Deroux, 1976

Incertae sedis

Lophophorina Penard, 1922

Order Dysteriida Deroux, 1976

Family Dysteriidae Claparède & Lachmann, 1859

Agnathodysteria Deroux, 1976

Dysteria Huxley, 1857

Microxysma Deroux, 1977

Mirodysteria Kahl, 1933

Trochilia Dujardin, 1841

Family Hartmannulidae Poche, 1913

Aegyria Chen et al., 2012

Aporthotrochilia Pan et al., 2012

Brooklynella Lom & Nigrelli, 1970

Chlamydonyx Deroux, 1976

Hartmannula Poche, 1913

Heterohartmannula Pan et al., 2012

Orthotrochilia Deroux in Song, 2003

Trichopodiella Corliss, 1960

Trochilioides Chen et al., 2011

Family Kyaroikeidae Sniezek & Coats, 1996

Kyaroikeus Sniezek, Coats & Small, 1995

Planilamina Ma et al., 2006

2.3. Comments on New Taxa Described from China

Three new genera and thirty-seven new species have been established in China. We carefully examined all these new taxa using the descriptions in the original publications and by checking the deposited specimens, and found that the establishment of one genus was not necessary, and the name of one species was already pre-occupied.

Paracyrtophoron Chen et al., 2012, was established mainly because it differed from its closest congener Cyrtophoron by “the lack of a fragment near anterior ends of right kineties and transpodial fragments in the posterior portion of the ventral surface” [23]. Here, the “fragment near anterior ends of right kineties” seems to be a structure only described in Cyrtophoron isagogicum [6], but not in other Cyrtophoron species; thus, it cannot be used as a promising generic difference. Additionally, it is not convincing that the transpodial fragments can be considered as a genus-level discrepancy, because these fragments are not necessarily present among the species of the related genus Chlamydodon (e.g., present in C. pararoseus, but absent in C. wilberti) [26,81]. Therefore, we doubt the necessity and the validation of the establishment of Paracyrtophoron from Cyrtophoron.

Furthermore, we have some comments on another genus, Aporthotrochilia Pan et al., 2012. It has been stated that the difference between Aporthotrochilia and Orthochilia is that the former has fragments on the right, posterior part of the frontoventral kineties and a higher number of terminal fragments [74]. This description is, however, somewhat unclear. In our opinion, the outer right kineties in Aporthotrochilia are interrupted in the middle, which leaves anterior parts (called “several terminal fragments” by the original authors) and posterior parts (corresponding to the posterior fragments). This is supported by L, S and T in the original publication [74], in which the anterior fragments are clearly a part of the outer right kineties. Thus, Aporthotrochilia has only one terminal fragment. Nevertheless, this interruption of the outer right kineties could be considered as a sufficient generic difference, and the establishment of the new genus is hence reasonable.

A new species of Chilodonella, C. parauncinata, was suggested by Qu et al. [59], and the separation from its congeners is undoubtable. However, the authors neglected the preoccupation of the species name, which was also a new species established by Wang [54] from Mount Everest (no deposited type material). This highlights the importance of an extensive literature search, especially for taxonomic study. C. parauncinata Wang, 1974, was originally published in Chinese; we provide herein a translation of the vague diagnosis of the poorly known species (no staining information): “Cell length 45–48 µm in vivo; body shape irregularly oval, beak-shaped protrusion to left in anterior end; posterior end more or less rounded, concave on right margin; four right and four or five left kineties; isolated from soil on the Mount Everest”. These two isolates can be clearly distinguished by the numbers of somatic kineties: the form in Qu et al. [59] had five right (vs. four) and six or seven left kineties (vs. four or five). Therefore, Chilodonella parauncinata sensu Qu et al., 2015 is a junior primary homonym, and it is permanently invalid according to article 57.2 of the ICZN [84]. Thus, we replace Chilodonella parauncinata sensu Qu et al., 2015 with a new name, Chilodonella apouncinata nom. nov.

Chilodonella parauncinata—Qu et al., J. Eukaryot. Microbiol. 2015, 62, 267–279 (primary homonym, non Chilodonella parauncinata Wang, 1974).

ZooBank registration number of present paper. urn:lsid:zoobank.org:pub:CEB2291F-08EC-4840-8979-01F28FF6DB83.

ZooBank registration number of urn:lsid:zoobank.org:act:5E887B0E-B6DC-462A-9C20-9A1C6721CE44.

Type specimen. See Qu et al. [59] (p. 269, Figure 3H).

Type locality. See Qu et al. [59] (p. 275).

Deposition of type materials. See Qu et al. [59] (p. 275).

Etymology. The species-group name apouncinata is a composite of the Greek adjective apo- (from) and the species-group name uncinata, indicating that the species is similar to Chilodonella uncinata.

Morphological description and morphogenesis. See Qu et al. [59] (pp. 269–271).

Comparison with congeners. See Qu et al. [59] (pp. 272–273).

3. Molecular Phylogenetic Studies

A new perspective on the systematics of cyrtophorid ciliates emerged at the turn of the millennium with the advent of molecular techniques. Molecular research corresponds to the molecular stage of morphological research (stage C in Figure 2). The first two SSU rDNA (small subunit ribosome DNA) sequences from China of the species Dysteria derouxi (AY378112) and Hartmannula derouxi (AY378113) were released in 2003. Shortly after that, the number of species studied with molecular sequences increased rapidly. By May 2022, 100 SSU rDNA sequences of Chinese origin had been deposited in the GenBank database with to 74 taxonomic hits (lowest taxonomic rank to species or genus), of which 66 had verifiable morphological records (Table 2). The molecular signatures (mainly the SSU rDNA) were then used to study the phylogeny of cyrtophorian ciliates. By this, Li and Song [85] revealed the phylogenetic positions of Dysteria derouxi and Hartmannula derouxi, the monophyly of families Chilodonellidae, Chlamydodontidae and Dysteriidae, and the clustering of Hartmannulidae with Isochona species (subclass Chonotrichia). Gong et al. [70] constructed phylogenetic trees using SSU rDNA and group I introns. Although only a few representatives were involved, the possible close relationship of Hartmannulidae and the subclass Chonotrichia was also indicated.

Table 2

NCBI-deposited SSU rDNA sequences of cyrtophorian ciliates from China. One hundred sequences in total belonging to seventy-four cyrtophorian taxonomic hits.

Taxonomic Hit	Accession(s)	Taxonomic Hit	Accession(s)
Aegyria apoliva	FJ998028	Dysteria ovalis	KX258193
Aegyria foissneri	KX364493	Dysteria ozakii	MW046154
Aegyria oliva	FJ998029	Dysteria paracrassipes	OL527698
Agnathodysteria littoralis	KC753482	Dysteria paraprocera	KM103263
Aporthotrochilia pulex	HQ605947	Dysteria pectinata	FJ870068
Atopochilodon distichum	KT461933	Dysteria procera	DQ057347
Brooklynella sinensis	KC753483	Dysteria proraefrons	KM103261
§ Chilodonella apouncinata	KJ509197	* Dysteria sp.	FJ868205
Chilodonella hexasticha	MH342041, MH342042, MH342045, MH341591	Dysteria semilunaris	KX258194
Chilodonella piscicola	MH341624, MH342043	Dysteria subtropica	KC753494
Chlamydodon bourlandi	MG566059, MK882887	Gastronauta paraloisi	MW072507
Chlamydodon caudatus	JQ904058	Hartmannula derouxi	AY378113
Chlamydodon mnemosyne	FJ998031	Hartmannula sinica	EF623827
Chlamydodon obliquus	FJ998030	Heterohartmannula fangi	HQ605946, FJ868204
Chlamydodon oligochaetus	KY496620	Kyaroikeus paracetarius	MN830168
Chlamydodon paramnemosyne	JQ904059	Lynchella minuta	KX364494
Chlamydodon pararoseus	MK882886	Lynchella nordica	FJ998036
Chlamydodon rectus	KT461932	* Lynchella_sp.	FJ998036
Chlamydodon salinus	JQ904057	Microxysma acutum	FJ870069
Chlamydodon similis	KY496621	Mirodysteria decora	JN867020
Chlamydodon triquetrus	KX302700, MG566058	Odontochlamys alpestris biciliata	KC753484
Chlamydodon wilberti	MG566060	Paracyrtophoron tropicum	FJ998035
Chlamydonella derouxi	KJ509198	Phascolodon vorticella	KX258192
Chlamydonella irregularis	KC753486	* Pithites vorax	FJ870070
Chlamydonella pseudochilodon	FJ998032	Planilamina ovata	MN830169
Chlamydonellopsis calkinsi	FJ998033, KC753487	Pseudochilodonopsis fluviatilis	JN867021, KR611083
Coeloperix sinica	FJ998034	Pseudochilodonopsis mutabilis	KR611084, KC753498
Coeloperix sleighi	KC753489	Pseudochilodonopsis quadrivacuolata	KR611082
* Coeloperix sp.	FJ998034	* Pseudochilodonopsis sp.1	KC753495
Dysteria brasiliensis	EU242512 MW046155, OL527700-OL5277004	* Pseudochilodonopsis sp.2	KC753496
Dysteria compressa	KC753491, MW046156	* Pseudochilodonopsis sp.3	KC753497
Dysteria crassipes	FJ868206, KC753492, KC753493, MK882889, OL527699	* Spirodysteria kahli	KC753499
Dysteria cristata	KC753488	Trichopodiella faurei	EU515792, KC753500, FJ870071
Dysteria derouxi	AY378112, KX302697	Trithigmostoma cucullulus	FJ998037, MW116158, MW116159
Dysteria lanceolata	KC753490	Trochilia petrani	JN867016
Dysteria monostyla	MK882888	Trochilioides recta	JN867017
Dysteria nabia	KM103262	Trochochilodon flavus	JN867018

* No corresponding morphological data. § The deposited species name in NCBI is Chilodonella parauncinata.

All subsequent work followed the systematic scheme proposed by Lynn [8] that cyrtophorian ciliates belong to the subclass Cyrtophoria, with two orders assigned: Chlamydodontida Deroux, 1976 and Dysteriida Deroux, 1976. Gao et al. [86] comprehensively analyzed the phylogeny of cyrtophorian ciliates using SSU rDNA sequences from 7 families and 17 genera, and portraited the overall phylogenetic structure for the first time, which then became the template for the following studies: The non-monophyly of the order Chlamydodontida and the monophyly of Dysteriida were confirmed; a new family, Pithitidae Gao et al., 2012, was established based on its special phylogenetic position and unique morphological feature; and the position of the family Plesiotrichopidae was found to be uncertain. However, we agree with Lynn [87] that the sequence of Plesiotrichopus, the type genus of Plesiotrichopidae, is still missing; thus, the certainty of the family transfer needs further testing in the future. Following that, Chen et al. [88] conducted complementary analyses with additional SSU rDNA sequences. The phylogenetic topology was consistent with that in Gao et al. [86], but with further discussion on some newly sequenced taxa such as Brooklynella. Based on these results, the possible evolutionary pattern of cyrtophorian ciliates was proposed. However, this evolutionary routine was without sufficient references from either morphological data or molecular phylogeny. The authors also showed the prediction of the secondary structures of the hypervariable region 4 (V4) of the SSU rDNA of the representative cyrtophorian genera. Later, Wang et al. [89] analyzed the phylogeny of class Phyllopharyngea—including Cyrtophoria—using two genes: the mitochondrial SSU rDNA (mtSSU rDNA) and the nuclear SSU rDNA. The phylogenetic results were generally consistent with previous works. Recently, Pan et al. [90] employed high-throughput sequencing to obtain the genomic and transcriptomic data of seven species, including five cyrtophorians. The emphasis of this paper, however, was to solve the uncertain phylogenetic position of Synhymenia, and was not focused on cyrtophorian ciliates.

Other works involving molecular phylogeny (SSU rDNA) were combined with morphological descriptions, serving as a complementary method for species identification [12,13,14,24,25,26,37,39,41,59,74,77,78,79,80,81].

In the present work, we reconstruct a comprehensive phylogenetic tree inferred from updated SSU rDNA sequences (Figure 5). Sequence selection and alignment, and phylogenetic tree construction methods are described in the Supplementary Materials. The topology of the present phylogenetic tree is consistent with previous works (e.g., [14,41,59,77,78,79,80,81,85,88,89,90]), and similar conclusions can be drawn: (1) The order Clamydodontida are monophyletic, while the order Dysteriida are non-monophyletic. (2) The subclass Chonotrichia are nested within Dysteriida, on the level of the family Hartmannulidae. (3) Kyaroikeidae display as a subfamily of Dysteriidae. (4) Gastronautidae represent a transitional family among Chilodonellidae, Chlamydodontidae, and Lynchellidae.

Figure 5

Phylogenetic tree inferred from SSU rDNA sequences. The trees were reconstructed by two algorithms: IQTREE and MrBayes. Support values from the two methods are provided at the branching points (IQTREE/MrBayes). Bold dots at the branching points indicate full support from both analyses. “-” indicates discrepancy between the topologies of IQTREE and the MrBayes trees. The scale bar represents five substitutions per hundred nucleotide positions.

4. Proposed Phylogeny

With some key members not yet uncovered, as well as missing molecular representatives, a detailed evolutionary relationship (phylogeny) of the subclass Cyrtophoria cannot be drawn satisfactorily. As mentioned above, Chen et al. [88] have attempted to illustrate the evolutionary relationships among the recognized cyrtophorian genera. However, these relationships were not strictly based on morphological and molecular data. For example, the solid separation of the two orders, Chlamydodontida and Dysteriida, were not supported by molecular phylogenetic analyses (Figure 5), and the positions of Chlamydodon and Cyrtophoron at the end of the Lychellidae branch were confusing. Nevertheless, what we can infer is that there is an obvious discrepancy between the considerable knowledge on the morphological diversity of cyrtophorian ciliates and their phylogeny and evolution. It has been emphasized that the morphogenetic data have to be taken into account in order to reconstruct evolutionary and phylogenetic relationships in ciliates. However, investigations show that the morphogenetic process of cyrtophorians seems quite identical among all groups [6,13,14,19,57,59,91,92], thus providing only limited information. Based on the review of the morphological (including feeding- and motility-associated morphological traits) and phylogenetic works mentioned above, and the one constructed ourselves (Figure 5), we propose the phylogeny of 29 genera from 9 families for which molecular data are available (Figure 6).

Figure 6

Evolutionary pattern of the subclass Cyrtophoria inferred from species with both morphological and molecular data. All illustrations are original.

The separation of the two orders, Chlamydodontida and Dysteriida, is mainly based on the form of the cell (dorso-ventrally compressed in the former and laterally compressed in the latter) and the posterior adhere organelles (podite and/or glandule absent in the former and present in the latter). However, nothing has been mentioned about the trend of the morphogenesis-related postoral kineties (see stomatogenesis zone—SZ—in Figure 1). In the order Chlamydodontida, the postoral kineties are not well separated from either the left or right kineties (Lynchellidae, Chlamydodontidae, and Trithigmostoma), or are merged as the inner part of the left kineties (Chilodonellidae and Gastronauta). Despite the highly degenerated left kineties, the postoral kineties are still connected with the left kineties in Hartmannulidae. Then, the postoral kineties tend to separate from the left kineties and become independent in Dysteriidae and Kyaroikeidae. We here hypothesize that the key evolutionary event of cyrtophorian ciliates lies in the separation of the stomatogenesis zone (postoral kineties) from the left kineties, namely, the formation of an independent “sexual organelle”.

As shown in Figure 5, the phylogenetic positions of the families Lynchellidae, Chilodonellidae, Chlamydodontidae, Hartmannulidae, and Dysteriidae are clear, with strong support from abundant morphological and molecular data [86,88,89]. In contrast, the positions of the families Pithitidae, Gastronautidae, Plesiotrichopidae, and Kyaroikeidae appear vague, mainly because of the lack of morphological and/or molecular data. It should be kept in mind that one should not over interpret the topology of phylogenetic trees derived from limited molecular data. Pithitidae and Plesiotrichopidae comprise only a few morpho-species and only one molecular representative each, which largely obscures phylogenetic study. Nevertheless, we agree with Gao et al. [86] that these two families are intermediate groups between the orders Chlamydodontida and Dysteriida, mainly from the aspect of morphological comparison. Similarly, although Gastronautidae has only one sequence positioned within the family Chilodonellidae, this family is valid and represents an intermediate group within the order Chlamydodontida based on its unique combination of morphological characteristics [14]. The separation of Kyaroikeidae (Kyaroikeus and Planilamina) from Dysteriidae is not well supported by either morphological or molecular data, and Kyaroikeidae species could be seen as highly specialized Dysteriidae adapted to parasitism [41]. Therefore, we place it at the basal position of Dysteriidae.

Several intermediate genera are also recognized: Trithigmostoma belongs to the family Chilodonellidae, but shares some characteristics with the Chlamydodontidae family, which is why it is placed at the basal position within the Chilodonellidae [14]. Brooklynella, Trochilioides and Microxysma represent transitional genera between Hartmannulidae and Dysteriidae [86,88] with intellectual morphological characteristics [65,72,75,76].

5. Ecology

5.1. Sites, Habitats, and Distribution

China is characterized by a vast diversity of habitats, including plateau regions, deserts, rain forests, wetlands, rivers, lakes, coastal areas, and deep sea. Accordingly, it is reasonable to assume that there should also be a high degree of ciliate biodiversity. However, apart from in the very early stage, respective investigations have mostly been carried out along coastal areas (Figure 7), while other habitats are only sporadically studied. The sampled sites can, therefore, be categorized into coastal areas and inland areas. Coastal areas can further be classified into three parts: (1) The northern part (the coasts of the Bohai Sea and the Yellow Sea) is located in a temperate zone. Sampling sites include Qingdao, Yantai, Changyi, and Dongying. (2) The eastern part (the estuary of Yangtze River) includes Shanghai, Ningbo, and Hangzhou. (3) The southern part (the coasts of the Southern China Sea and the estuary of the Pearl River) includes Xiamen, Huizhou, Hong Kong, Shenzhen, Guangzhou, Zhuhai, Zhanjiang, and Haikou. The inland areas cover the Tibet Plateau (including Mount Everest), Gansu province, and cities along the Yangtze River and its tributary (Dali, Liangshan, Luzhou, Hanchuan, Wuhan, Nanjing, and Changzhou).

Figure 7

Sampling sites and habitat categories (or lifestyles) of studied cyrtophorian ciliate populations from China. The dots represent study sites, and the sizes of the dots indicate the number of populations studied. Colors represent different habitat types or lifestyles.

The investigated areas covered five habitat types or lifestyles (Figure 7), namely, marine, brackish water, freshwater, soil, and parasitism. Studies on the northern coastal areas focused mainly on marine habitats (coastal waters, indoor or open sea mariculture waters), while studies on the eastern and southern coasts were mainly conducted from brackish waters. Freshwater habitats were mainly investigated along the Yangtze River and its tributary, and on the Tibetan Plateau. Most evidence of cyrtophorian parasitic lifestyles also comes from these areas. Soil cyrtophorians were mainly isolated from Gansu and Mount Everest.

Many studies on pelagic and soil habitats indicate that the dispersal and distribution of ciliates follow the moderate endemicity model (e.g., [93]). Apparently, this also holds true for cyrtophorian ciliates. Currently, more than one third of cyrtophorians discovered in China are new and possibly endemic, while the rest can be found in other countries or on other continents as well. The only attempt to analyze the biogeographic distribution patterns of cyrtophorian ciliates was briefly conducted on a well-studied genus: Chlamydodon [13]. This work summarized the historical studies on the morphology of this genus worldwide, showed the global distribution of Chlamydodon species, and indicated possible cosmopolitan and endemic species. However, as stated by the authors, the analyses were very limited, mainly because of the scattered studies on this genus. This limit also applies to the studies on cyrtophorian ciliates in China. For instance, a cosmopolitan species, Chlamydodon mnemosyne, has only been formally reported once in a publication from China [64], but sampled and recorded in master’s and PhD theses several times (by personal communication). This issue prevents a thorough diversity study and also hinders intra- and interspecific comparisons for both morphological and molecular aspects. Therefore, in order to obtain more detailed data (at population level) with a higher geographical resolution, extensive sampling must be carried out in different habitats at a larger scale.

5.2. Lifestyles

Most cyrtophorian species display a free-living periphyton life, and can thus be easily sampled by gently scratching the substrate or enrichment with artificial substrates such as glass slides [35,67]. Some species are found mainly in aquaculture water bodies, especially mariculture waters [33,34,35] such as Dysteria crassipes [67,79]. A few species exhibit a parasitic lifestyle. Chilodonella uncinata [40], C. piscicola [37,39], and C. hexasticha [38,39,40] have been reported to be opportunistic or obligate parasites on gills and the skin of freshwater fish. Furthermore, two mammal parasites, Kyaroikeus paracetarius and Planilamina ovata, were isolated from the mucus of an unhealthy captive beluga [41].

5.3. Food Source and Feeding Types

Cyrtophorian ciliates mainly feed on bacteria (including cyanobacteria) and/or eukaryotic microalgae, and they are obligate bacterivores, algivores, or omnivores [12,13,14]. This could be inferred by a direct check of food vacuoles or cultivation attempts using grain-enriched bacteria. Some of this information can be used for species identification. For instance, Pseudochilodonopsis algivora is filled with algae inclusions, which is a promising character to quickly identify this species [16]. Some species such as Aegyria oliva, A. paroliva, Chlamydodon bourlandi, C. obliquus, and Paracyrtophoron tropicum have unusual violet digested inclusions that can be used to aid species identification [4,23,26,82]. We have tried to summarize food inclusions by checking descriptions, photomicrographs, and cultivation attempts in the literature. A large part of cyrtophorian ciliates feed on microalgae (e.g., Chlamydodon, Hartmannula, Pseudochilonopsis, and Trithigmostoma). This can be best explained by their strong pharyngeal basket which is adapted to undertake microalgae food. Small, spherical algae are mostly observed as food inclusions (e.g., [12,16,55,59]). Large diatoms are also commonly found, and most of them belong to Naviculaceae (e.g., [14,24,80,81]), while Cyclotella is casually identified [24]. Thus far, filamentous cyanobacteria have only been reported in Chlamydodon mnemosyne [64] and a Chilodonella species [59]. Bacterivorous species are also common, especially for those species found in the aquaculture waters (Chilodonella spp. and Dysteria spp.). These species can be cultivated by the enrichment of bacteria by adding rice or wheat grains. A few species (Pseudochilodonopsis marina, Dysteria brasiliensis, and Gastronauta paraloisi) have been reported to feed on both bacteria and small, spherical algae, and thus are possibly omnivorous [14,55,67,68,77]. No predation has been found in cyrtophorian ciliates. Although Aegyria oliva has been reported once to contain small scuticociliates and Aspidisca in its inclusions [4], this was likely the result of non-selective feeding, and no predatory behavior was confirmed.

5.4. Abundance

Cyrtophorian ciliates have relatively low abundance in natural habitats compared to other ciliates, but some species (bacterivores or omnivores) are found to be quite abundant in aquaculture or saprobic environments, such as Dysteria spp. [26,67,77] and Gastronauta paraloisi [14]. Although some diatom consumers, such as Trithigmostoma, may be dominant when food diatoms are enriched [14,81], the population density of obligate algivores is usually low.

5.5. Others

A few studies have reported that dysteriid species (mainly Dysteria) have possible ectosymbiotic bacteria (most likely bacterial epibionts) on cell surface, e.g., Aegyria foissneri [25], Dysteria brasiliensis [67], D. compressa [80], D. crassipes [26,67,79], D. lanceolata [72], D. monostyla [26], D. paraprocera [79], and D. subtropica [68,79]. The occurrence and the type of these bacteria seem to be environment-induced [67,77]. However, the exact function of these bacteria is yet unknown.

6. Prospects

With almost a century of research history, studies on the taxonomy, phylogeny, and ecology of cyrtophorian ciliates in China have accumulated substantial results. In addition to this, we offer an outlook on the future research of this group of ciliates.

Despite the considerable number of studies published in China, as described above, it is clear that there are still many unknown species that prevent a more detailed and cohesive systematic scheme of Cyrtophoria. Thus, more investigations into fauna need to be conducted on a large regional scale with different habitats. In contrast to massive investigations from marine and brackish water habitats, sampling from freshwater and soil as well as extreme environments such as hypersaline lakes (mostly on the Tibetan Plateau, western China) and cold regions (northern China) are urgently needed;

The de facto standard for the taxonomic study of ciliates combining morphology (live observation and silver staining) and molecular phylogeny [94] has been well practiced for Cyrtophoria over the last decade. As high-throughput sequencing becomes cost-efficient and more effective bioinformatic tools are developed, it is possible to use these techniques to perform phylogenomic reconstruction, as indicated by [91]. Thus, in the near future, genomic/transcriptomic data should also be included in taxonomy or phylogenetic study routines to achieve higher phylogenetic resolution;

Similarly to other ciliate groups, species separation/circumscription is still a major problem for the taxonomy of cyrtophorian ciliates. Different geographical populations of the same species should be recorded, described and compared on the aspects of morphology and different marker genes;

Attention should be paid to the ectosymbiotic bacteria (or bacterial epibionts) on the cell surface of Dysteria, with emphasis on trophic function as well as possible phylogenetic signals to their hosts;

Detailed ecological roles (niches) of cyrtophorian ciliates should be elucidated. This could be performed by annual or seasonal sampling, cultivation experiments, and checking food vacuoles or fluorescence in situ hybridization (FISH) to detect species occurrence, food inclusion, and trophic relationships (autecology).