Exploring the biogeography, morphology, and phylogeny of the condylostomatid ciliates (Alveolata, Ciliophora, Heterotrichea), with establishment of four new Condylostoma species and a revision including redescriptions of five species found in China

Species of the ciliate class Heterotrichea Stein, 1859 are a cosmopolitan group of unicellular eukaryotic microorganisms, many of which have been widely used as models in various fields of research such as regenerative biology, functional ecology, environmental toxicology, and symbiotic behavior. However, species identification in the heterotrich family Condylostomatidae, especially the most species-rich and type genus Condylostoma Bory de Saint-Vincent, 1824, remains challenging due to incomplete original descriptions, few reliable distinguishing characters, and overlapping features between different species. This study presents an updated revision of Condylostoma and its related genus Condylostomides da Silva Neto, 1994 based on descriptions of five species, including nine populations collected from China, using both morphological and molecular methods. The main findings are as follows: (1) 43 nominal species and about 130 populations are reviewed, resulting in the recognition of 30 valid species of Condylostoma and eight valid species of Condylostomides; (2) keys, synonyms, biogeographic distributions and amended/improved diagnoses of all valid species are provided; (3) based on the available data, four new Condylostoma species (C. marinum sp. nov., C. petzi sp. nov., C. villeneuvei sp. nov., and C. microstomum sp. nov.), one new combination (Condylostomides minimus (Dragesco, 1954) comb. nov. & nom. corr.), and two corrected names (Condylostoma ancestrale Villeneuve-Brachon, 1940 nom. corr. and Condylostomides nigrus (Dragesco, 1960) nom. corr.) are suggested; (4) cryptic species are detected and proposed for the first time to form the Condylostoma curvum species complex; (5) three highly confusing Condylostoma species, C. kris, C. spatiosum, and C. minutum, are redefined for the first time based on modern taxonomic methods; (6) a ‘flagship’ species, Condylostomides coeruleus, is recorded for the first time from the continent of Asia, substantially expanding its biogeography; (7) ciliature adjacent to the distal end of the paroral membrane within the family Condylostomatidae is uniformly defined as frontal membranelles and is classified into three patterns according to the arrangement of kinetosomes, which serve as important key features. Supplementary Information The online version contains supplementary material available at 10.1007/s42995-024-00223-3.


Introduction
Ciliated protists, a diverse group of single-celled organisms, are vital components of various aquatic and terrestrial environments where they are important contributors to nutrient cycling and energy transfer (Hu et al. 2019;Lu et al. 2023;Lynn 2008;Ma et al. 2022a;Wang et al. 2022;Ye et al. 2021a;Zhang et al. 2022a).Members of the class Heterotrichea Stein 1859, a primitive group of ciliates characterized by their large cell size, somatic dikinetids associated with postciliodesmata, and a welldeveloped oral apparatus composed of an adoral zone of membranelles and a paroral membrane, have been widely used as model organisms in numerous biological disciplines including regeneration, ecology, nuclear function, and symbiotic relationships (Ahsan et al. 2022;Chi et al. 2021;Fernandes et al. 2016;Hao et al. 2022;Shazib et al. 2014;Yan et al. 2019;Ye et al. 2022).
The family Condylostomatidae is a relatively well-studied family within the Heterotrichea, and several studies on the diversity of its five genera, Chattonidium Villeneuve-Brachon, 1937, Condylostentor Jankowski, 1978, Condylostoma Bory de Saint-Vincent, 1824, Condylostomides da Silva Neto, 1994, and Linostomella Aescht in Foissner et al., 1999, have been reported in recent years (Chen et al. 2007;Chi et al. 2020b;Fernandes et al. 2015;Jin et al. 2021;Kim et al. 2012;Modeo et al. 2006;Shao et al. 2006;Yan et al. 2015).The type genus, Condylostoma, is the most speciose of these genera and is commonly found in brackish or marine habitats.It is easily recognized by its large cell size and prominent buccal region (Chen et al. 2007;Fernandes et al. 2015;Kim et al. 2012;Shao et al. 2006;Yan et al. 2015).Although the genus Condylostoma has a research history spanning over two centuries, with species recognizable in texts from as early as the 1780's (Müller 1786), it remains one of the most confusing taxa among ciliates in terms of species identification and separation.Song et al. (2003) summarized the reasons for this as follows: (1) many species exhibit variable morphological characters (e.g., cell shape, size, pattern of ciliature); 2) only a few characters can be reliably used for species separation; (3) the morphology of most species has not been fully described; (4) many species have overlapping key features such as the number of somatic kineties, cell shape, and cell size; and (5) a mass of misinterpretations or inadequate descriptions have accumulated in the literature as a result of these problems.So far, only four Condylostoma species, namely C. arenarium, C. curvum, C. elongatum, and C. tropicum, have been defined using an integrative taxonomic approach that includes living morphology, stained preparations, and molecular data (Fernandes et al. 2015;Yan et al. 2015).Nevertheless, with the rapid development of molecular methodologies in ciliate research, large amounts of molecular data on Condylostoma have been submitted to the GenBank database in recent years (Chen et al. 2019;Gao et al. 2016;Guo et al. 2008;Shazib et al. 2014;Zhou et al. 2010).However, due to the lack of reliable data for taxonomic identification, many of these sequences are likely assigned to the wrong species, which further exacerbates the understanding of the systematics of Condylostoma.The last revision to the genus Condylostoma, over 90 years ago, predates modern methods of morphological investigation including several commonly used fixatives and histological staining techniques and, of course, the molecular era, and was based only on 10 nominal species (Kahl 1932).Therefore, a comprehensive revision of Condylostoma is needed to better understand the evolutionary relationships, morphology, and biogeography of species within this group.
Unlike Condylostoma, which mainly comprise marine species, the genus Condylostomides da Silva Neto, 1994 is generally encountered in freshwater or terrestrial soil habitats (da Silva Neto 1994;Foissner 2016;Foissner et al. 2002;Hines et al. 2020).The overall morphology of these two genera is very similar and is characterized by a prominent buccal area, which has been a contributing factor to potential misidentifications (Foissner 2016;Foissner et al. 2002;Kahl 1932;Penard 1922).Interestingly, Condylostomides coeruleus was perceived as an endemically distributed species in the original report based on soil samples from Venezuela (Foissner 2016).Subsequently, however, Hines et al. (2020) discovered this 'flagship' ciliate species on the North American continent, indicating that additional sampling is needed to fully reveal its true geographical distribution.
Here we conducted a comprehensive reassessment of 43 nominal species and about 130 populations belonging to Condylostoma and Condylostomides.Ultimately, we recognized 30 valid Condylostoma species and eight valid Condylostomides species, and provided the synonyms, diagnoses, and biogeography for each.Drawing on the present review and the redescriptions of four highly confusable Condylostoma species (C.kris, C. curvum, C. spatiosum, and C. minutum) and one Condylostomides species (C.coeruleus), we proceeded to refine the diagnostic features of both genera.Finally, based on current phylogenetic analyses inferred from SSU rDNA sequences, we re-evaluated the relationships between species within the family Condylostomatidae and revealed for the first time the presence of cryptic species in Condylostoma curvum.

Sample collection and observation
Five species, i.e., Condylostoma kris (two populations), C. curvum (three populations), C. spatiosum, C. minutum (two populations), and Condylostomides coeruleus, were collected from Qingdao, Ningbo, and Lake Weishan Wetland in China.The collection sites and dates are shown in Table 1, and the geographical information and physicochemical parameters are shown in Fig. 4B-J.All samples were taken from the surface layer of sediment using a pipette and transferred into Petri dishes for processing in the laboratory.
Living cells were haphazardly selected from the original samples and observed at 100-1000× magnification using both bright field and differential interference contrast microscopy (Olympus BX53; Zeiss AXIO Imager.D2) and photographed with Olympus DP74 and Axiocam 506 color cameras.The protargol staining method (Wilbert 1975) was used to determine the ciliary pattern.The protargol powder was synthesized according to Pan et al. (2013).Hoechst 33,342 solution was used to reveal the nuclear apparatus (Jiang et al. 2019).Counts and measurements of stained specimens were conducted at 100× and 1000× magnifications.Drawings of living and stained cells were accomplished with the help of Adobe Photoshop.Terminology and systematics are mainly according to Chi et al. (2021), Lynn (2008), and Shazib et al. (2014).

DNA extraction and gene sequencing
One cell of each population was isolated under a dissecting microscope and washed five times in filtered habitat water to remove potential contaminants.Genomic DNA was extracted from the single cleaned cell using a DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions but modified by using 1/4 of the suggested volume for each solution (Chi et al. 2022a).SSU rDNA was amplified using Q5 Hot Start high-fidelity DNA polymerase (NEB, Ipswich, MA, USA) with the universal eukaryotic primers, 18 s-F (5'-AAC CTG GTT GAT CCT GCC AGT-3') and 18 s-R (5'-TGA TCC TTC TGC AGG TTC ACC TAC -3') (Medlin et al. 1988;Ye et al. 2021b).PCR amplifications were according to the following procedure: 1 cycle of initial denaturation at 98 ℃ for 30 s, followed by 18 cycles of amplification (98 ℃, 10 s; 69-51 ℃ touchdown, 30 s; 72 ℃, 1 min), and another 18 cycles (98 ℃, 10 s; 51 ℃, 30 s; 72 ℃, 1 min), with a final extension of 72 ℃ for 5 min (Ma et al. 2022b;Zhang et al. 2022b).PCR products were checked using agarose gel and then sequenced bidirectionally in TSINGKE (Qingdao, China).Sequence fragments were assembled into contigs using Seqman (DNAStar).

Molecular analyses
Apart from the eight new sequences produced during this study, all sequence data used in the phylogenetic inference analyses were obtained from the GenBank database (accession numbers are provided in Fig. 5A).Five karyorelictean species were used as the outgroup.Sequence alignments were performed using MAFFT with the L-INS-I strategy (Katoh and Standley 2013).Ambiguously aligned regions were manually edited using BioEdit (Hall 1999).The final alignment, including 1586 sites, was used to construct phylogenetic trees.
A maximum likelihood (ML) tree was constructed by IQ-TREE v2.0 with 10,000 ultrafast bootstrap, using SYM + I + G4 as the best-fit model (Minh et al. 2020).Bayesian inference (BI) analysis was carried out using MrBayes version 3.2.7a on XSEDE (Ronquist et al. 2012) on the CIPRES Science Gateway (Miller et al. 2010).GTR + I + G was selected as the best model by MrModeltest version 2.2 according to the Akaike Information Criterion (Nylander 2004).Markov chain Monte Carlo (MCMC) simulations were run for ten million generations at a sample frequency of 100 generations, with the first 25% discarded as burn-in.Convergence of the Bayesian analysis was determined by the average standard deviation of split frequencies (< 0.01) (Ronquist et al. 2011).MEGA 5.2 (Tamura et al. 2011) was used to visualize the tree topology.Nucleotide differences were analyzed by BioEdit (Hall 1999).

Improved diagnosis
Medium-sized (about 100 µm) to very large (up to 2.5 mm) heterotrich ciliates, freely motile, usually crawling on substrates or between sediment particles, occasionally swimming.Cell moderately contractile; when swimming or crawling slender ribbon-shaped, elongate-ellipsoidal or fusiform; when fixed and contracted, usually ellipsoidal or ovoidal.Somatic ciliature holotrichous, somatic cilia arranged in evenly spaced longitudinal rows, several shortened kineties form a suture on ventral side near posterior end of cell.

Etymology
The generic name is a composite of the Greek words "condylo = kondylo" (knuckle, knuckle-like knob) and "stoma" (mouth), meaning a ciliate with distinct mouth.Neuter gender.

Identification characteristics
Recent studies of Condylostoma species (Chen et al. 2007;Fernandes et al. 2015;Kim et al. 2012;Shao et al. 2006;Song et al. 2003;Yan et al. 2015), including the present investigation, have allowed for a more precise understanding of the taxonomic significance of various features.Among the main living characters, the cell shape, nuclear apparatus, and cortical granules play crucial roles in distinguishing among different species.Additionally, specific characteristics that are only recognizable in silver-stained specimens, such as the number of somatic kineties and the pattern of frontal membranelles, are also important for accurate species identification.
Cell shape: The cell shape is highly variable, both between and within species.In fully extended or freeswimming forms, at least four typical cell shapes can be recognized (Fig. 1C): (1) ribbon-shaped or slender with a sharp posterior end or needle-like tail; (2) flattened slender with a rounded posterior end; (3) flattened ellipsoidal with a rounded posterior end; and (4) elongated cylindrical with a rounded posterior end.Some other peculiarities can also occur, such as in Condylostoma subterraneum the cell of which is triangular with a long tail.Song and Wilbert (1997), as a synonym of C. spatiosum in the present study.K, L C. spatiosum reported by Shao et al. (2006).M, N C. minutum reported by Chen et al. (2007).O, P C. tropicum reported by Yan et al. (2015).Q, R C. elongatum reported by Yan et al. (2015).Arrows mark the paroral membrane; arrowheads mark the frontal membranelles Nuclear apparatus: The shape of the macronucleus is a reliable character for identifying different Condylostoma species.In the majority of species, the macronucleus is moniliform, and is formed by a chain of beads or nodules connected by thread-like extensions of the nuclear membrane.However, the macronucleus can also take other forms, such as many scattered spherical nodules in C. fjeldi, a single ribbon-shaped macronucleus in C. enigmaticum, or a multi-nodular macronucleus that can be considered a special ribbon-shaped type in C. acutum.In some cases the macronucleus may be composed of only a few isolated, spherical or sausage-like nodules (Fig. 1D).
Number of somatic kineties and adoral membranelles: Some available data suggest that there is slight variability in the number of somatic kineties and adoral membranelles both between and within populations.Specifically, smaller cells tend to have fewer than larger ones.Most species have a relatively stable range of these characters, although there is also some variability in certain species, possibly due to some data being based on living cells and the variable number of shortened somatic kineties in some specimens.Nonetheless, the numbers of somatic kineties and adoral membranelles are generally considered reliable characters for distinguishing Condylostoma species, based on the current state of knowledge.
Frontal membranelles: The frontal membranelles are located at the distal end of paroral membrane and have thus far been referred to as "frontal cirri" until the present study.However, the term "frontal cirri" is commonly used in descriptions of hypotrichs and euplotids and is considered a unique characteristic of these groups.Our results indicate that there are two patterns of kinetosome arrangement in this structure in Condylostoma, with one being block-like (Pattern I) (Fig. 2A-D) and the other being strip-like (Pattern II) (Fig. 2E-R).The latter pattern is obviously not suitable to be referred to as frontal cirri.Additionally, in the closely related genus Condylostomides, which has a similar morphology to Condylostoma, this structure is also present in the same position and is referred to as "frontal membranelles".Hence, to avoid confusion and to maintain consistency, we propose  C. kasymovi; 5, C. caudatum; 6, C. subterraneum; 7, C. tenue; 8, C. pauculum; 9, C. longicaudatum; 10, C. elongatum; 11, C. reichi; 12, C. tropicum; 13, C. remanei; 14, C. psammophium; 15, C. kahli; 16, C. granulosum; 17, C. ancestrale nom. corr.; 18, C. vorax; 19, C. marinum sp. nov.; 20, C. patens; 21, C. petzi sp. nov.; 22, C. rugosum; 23, C. villeneuvei sp. nov.; 24, C. magnum; 25, C. microstomum sp. nov.; 26, C. curvum; 27, C. kris; 28, C. spatiosum; 29, C. minu-tum; 30, C. arenarium using the term "frontal membranelles" to refer to the ciliary organelle located at the distal end of paroral membrane in both Condylostoma and Condylostomides.However, it should be noted that the frontal membranelles in Condylostomides differ from Patterns I and II described above.In Condylostomides, they are closely connected to the paroral membrane and do not seem to represent a completely separate structure.Hence, it can be considered as another unique pattern of frontal membranelles (Pattern III) within the family Condylostomatidae.
The frontal membranelles are a valuable taxonomic feature in Condylostoma.Unfortunately, it is not easy to detect in vivo and was usually overlooked until the widespread use of silver staining methods.Four patterns have been reported within the genus Condylostoma: Pattern I, Pattern II, and two misinterpreted patterns (Fig. 1B).The pattern of two fragmented frontal membranelles was derived from a possibly extruded specimen of C. magnum (Song and Wilbert 1997), which is regarded as a synonym of C. spatiosum in the present work (Fig. 2I, J).As for the pattern of two parallel frontal membranelles (Shao et al. 2006), it has been confirmed that the second frontal membranelle, located near the distal end of paroral membrane, is homologous to the paroral membrane (Chen et al. 2007) (Fig. 2K, L).Additionally, the Based on the aforementioned analyses and criteria, we conducted a reassessment of 35 nominal Condylostoma species (including approximately 120 populations), utilizing available morphological data such as published illustrations and key diagnostic characters.Through this comprehensive examination, the results are summarized as follow: (1) a total of 30 valid species were recognized, four of which are new (Fig. 3 (Chi et al. 2020b;Jin et al. 2021).

Key to valid Condylostoma species
Note: The numbers within parentheses correspond to the order of species in synonym lists, diagnoses, and distributions.
1 Gradually tapered at posterior portion or forming a conspicuous tail ……… …………..…………..............      Note: In the list of synonyms for each species, we have directly cited the unaltered species names (as well as author names) from the original sources.This is to demonstrate the transition of species names from historical references to the contemporary era, despite several identifiable errors that are subsequently present throughout.
Comments: Fjeld (1955) described two types of C. remanei: type A with a moniliform macronucleus and type B with numerous scattered macronuclei as shown in C. fjeldi.We agree with Hartwig (1973) who listed type B of C. remanei as a synonym of C. fjeldi.
Comments: It is noteworthy that the cell size and the ratio of peristome length to cell length exhibit significant variation among different populations.Additionally, the two populations reported by Dragesco (1963) and Kattar (1970) do not demonstrate an obvious tapered posterior end.Considering that only one species has been found to have symbiotic algae thus far, we suggest that these are populations of Condylostoma tenue.However, further research is needed to confirm this.

Condylostoma tropicum
Comments: Before Yan et al. (2015) established this species based on comprehensive morphological data of a Chinese population, the SSU rDNA sequence of this population had been released for exploring the phylogenetic position of Condylostoma (Zhou et al. 2010).In the present work, we noticed that a population of C. longicaudatata from Saudi Arabia (Al-Rasheid 1999) differed significantly from the original description (Dragesco 1996), especially in size (400-600 μm vs. 1500 μm), but closely resembled C. tropicum in terms of various morphological characters.Therefore, we suggest that the Saudi Arabian population is conspecific with C. tropicum.caudatum Lauterborn, 1908).[2] 1928 Condylostoma longissimum Grimm?- Kahl, (two size types of specimens, the smaller of which is identified as C. remanei).
Comments: When he established this species, Spiegel (1926) was unaware that the name had been occupied by Condylostoma caudatum Lauterborn, 1908. Subsequently, Kahl (1932) renamed it C. remanei with the agreement of Dr. Spiegel.Condylostoma remanei was the first marine species in this genus reported to have a pointed tail, which led to the identification of many morphologically similar populations as being conspecific with it, making this one of the most confusing species.
There are two size types of individuals described in Kahl (1928Kahl ( , 1932)).We agree with Yan et al. (2015) and Dragesco (1996) to identify the larger one as Condylostoma longicaudatum and the small one as C. remanei.In addition, Kahl (1932) also observed two individuals from Kiel with a length of 350 μm, however, due to the lack of key morphological data and illustrations, we are unable to determine their identity.A Brazilian population reported by Kattar (1970) is similar in appearance to C. pauculum, however it has a cell length of more than 500 μm (vs.400 μm) and lives in marine habitat (vs.brackish with a salinity of 4‰), thus we retain this as a population of C. remanei pending further investigations.It should also be noted that Villeneuve-Brachon (1940) reported a population collected from freshwater in France.Considering that most of freshwater populations of Condylostoma have been proven to be misidentified (Foissner 2016;Foissner et al. 2002), its identity should be verified by future research.Finally, there are still many reports related to C. remanei that are not included in present work, e.g., see Al-Rasheid (1996) and the list in Hartwig and Parker (1977), which cannot be reasonably identified due to the lack of sufficiently detailed descriptions and illustrations as well as unreliable reported data.
Amended diagnosis: Cell size about 360 μm; inverted pyriform with short spines at posterior end; peristome closed apically, occupying about 2/5 (from original drawing) of cell length; about five nearly ellipsoidal macronuclei scattered throughout the cytoplasm; small cortical granules; marine habitat.
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Comments: According to the original report, Condylostoma ancestrale nom.corr.has two parallel elongate rows formed by the coalescing kinetosomes at the base of the peristome.This structure appears simpler than cirri, suggesting that it may represent the primitive species of this genus (Villeneuve-Brachon 1940).Jankowski (1979) established the genus Procondylostoma for this species based on the absence of cirri and classified it into the order Hypotrichia.
As we discussed earlier, the distal end of the paroral membrane broadens in some Condylostoma species, giving it the appearance of the stripy frontal membranelles.Therefore, it is possible that the author misidentified the distal end of the paroral membrane as the second frontal membranelle.Therefore, we propose that this species should be transferred back to Condylostoma, and Procondylostoma should be considered invalid.[5] 1932 Condylostoma arenarium Spiegel, 1926-Kahl, Tierwelt Dtl. 25: 455 [Fig. S. 454, 25] (containing three different morphotypes; the forms about 500 μm in size and with an enlarged posterior end should be classified as C. marinum sp.nov.).
Type material: Information unavailable.Etymology: "marinum" was given by Claparède and Lachmann (1858) who first discovered it.See the Comments section of Condylostoma patens.
Z o o B a n k r e g i s t r a t i o n : u r n : l s i d : z o o b a n k .org:act:FC1714B3-E2B9-4AA0-8EF8-4F41AF00A713.
Comments: See the Comments section of Condylostoma patens for details on the establishment of this species.In addition to the inclusion of C. patens morphotypes with an enlarged posterior end, we note that there are three different morphotypes of the species named C. arenarium with 4 or 5 "frontal cirri" and 20-32 somatic kineties (Kahl 1932): Type 1, 400-700 μm in size, typical C. arenariumlike individuals, listed here as a synonym of C. kris (see the Comments section of C. arenarium); Type 2, 200 μm in size, has been listed as a synonym of C. curvum (Song et al. 2003); Type 3, 500 μm in size and with an enlarged posterior end.Kahl (1932) mentioned that the "frontal cirri" of Type 3 are weakly visible compared to the other two types, leading us to exclude this feature in the species diagnosis.Similarly, it is inadvisable to include the number of somatic kineties (20-32) in the species diagnosis due to the inclusion of multiple morphotypes.Therefore, we believe it is reasonable to list the Type 3 as a synonym of C. marinum sp.nov.[6] 1999 Condylostoma patens Dujardin 1841-Al-Rasheid, Arab Gulf J. Scient.Res.17: 133 [Fig.17] (brief redescription without illustration of living cell).
Comments: The type species, Condylostoma patens, was originally reported by (Müller 1786) under the name Trichoda patens.Although its description is brief, an easily recognizable illustration was provided, which leads us to recognize its identifying features as a cylindrical cell slightly expanding in the middle and the peristome occupying 1/6-1/5 of cell length (Type I).However, numerous subsequent researchers appear to have overlooked this illustration and mistakenly identified a type of organism whose posterior portion expanded into a saccular shape (Type II) as C. patens (Calkins 1902;Claparède and Lachmann 1858;Kahl 1932;Stein 1867).Claparède and Lachmann (1858) misidentified Type II as C. patens and regarded a population with a relatively smaller peristome as a new species, C. patulum.Furthermore, descriptions and illustrations of C. patulum provided by the original and subsequent reports (Calkins 1902;Claparède and Lachmann 1858;Kahl 1932;Stein 1867) are consistent with of C. patens Type I in terms of cell shape and oral region.Thus, we propose that C. patulum is a synonym of C. patens and that C. patens Type II represents a separate species.In addition, we noticed that in Claparède and Lachmann's (1858) report on C. patens Type II, it was mentioned that "if one day it can be proved that his species is different from the C. patens reported by Dujardin (1841), then his species can be named C. marinum".Thus, we here classify individuals with a sac-like posterior end as novel species, C. marinum sp.nov.
We need to mention that a Saudi Arabian population with an elongate cell shape, peristome occupying one-fifth of cell length, and 60-73 somatic kineties resembles both Condylostoma patens and C. spatiosum (Al-Rasheid 1999).This population, however, can be distinguished from C. spatiosum by the number of adoral membranelles (50-64 vs. 80-200 in C. spatiosum).In addition, the number of adoral membranelles in the type species (C.patens) has not been reported, thus we classify this Saudi Arabian population as C. patens pending further information.Maupas (1883) reported an Algerian population under the name of Condylostoma patens, however this is inconsistent with C. patens in terms of cell size (305-495 μm) and shape (flattened and slender), ratio of peristome to cell length (almost 30%), and number of somatic kineties (about 25), but matches C. kris very well, especially in the patterns of frontal membranelles (Pattern I) and cortical granules (Pattern 1).We therefore propose to classify this Algerian population as a synonym of C. kris.Several other populations have been reported under the name C. patens (Al-Rasheid 1996, 1997;Dragesco 1960;Fauré-Fremiet 1912), however, we could not confidently verify their identity due to the lack of information on key diagnostic characters in these works.Wilbert and Kahan (1981) found a population collected from Solar Lake (Egypt) and identified it as Condylostoma patulum.This population is, however, easily distinguishable from other congeners with a rounded posterior end by its small peristome (1/15-1/10 of cell length), number of adoral membranelles (60-70), number of somatic kineties (36-42), and frontal membranelles composed of six blocks of kinetosomes (Pattern I).Thus, we suggest that this population be classified as a new species, C. microstomum sp.nov.Petz et al. (1995) reported an unidentified Condylostoma species from Antarctica, which resembles C. patens only by having a vermiform cell that is more than 1 mm long and with a rounded posterior end.However, it can be separated from C. patens by the cell shape (anterior right and left margins parallel, gradually narrowed to posterior end vs. anterior slightly narrowed, widest in the middle), arrangement of cortical granules (loose vs. dense), ratio of peristome length to cell length (< 1/6 vs. 1/6-1/5), numbers of adoral membranelles (ca.180 vs. 50-64) and somatic kineties (39 vs. 48-80), and habitat (hypersaline vs. brackish to marine).We therefore propose that this Antarctic population represents a novel species C. petzi sp.nov.
Diagnosis: Cell size more than 1200 μm; elongate and vermiform, right and left margins parallel in anterior cell half, posteriorly tapering; peristome length about 15% of cell length; macronucleus moniliform with about 15 nodules; cortical granules large, circular to ellipsoid, colorless, loosely arranged in two rows between adjacent somatic kineties (Pattern 3); adoral zone with about 180 adoral membranelles; about 39 somatic kineties; frontal membranelles composed of a single stripe of kinetosomes (Pattern II); hypersaline habitat.
Type material: A protargol slide has been deposited in the Oberösterreichische Landesmuseum (LI), A-4040 Linz, Austria.
Etymology: We dedicate this species to Dr. Wolfgang Petz, the discoverer.
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Type material: Information unavailable.Etymology: We dedicate this species to Dr. Simone Villeneuve-Brachon, the discoverer.
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Comments: Song and Wilbert (1997) reported a Chinese population with the following characters: cell size 450-800 μm; peristome about 1/4-1/3 of cell length; 47-56 somatic kineties; about 150-200 adoral membranelles; frontal membranelles composed of a single stripe of kinetosomes (Pattern II).However, this population is distinctly different from the original report by having significantly fewer somatic kineties (47-56 vs. 80) (Spiegel 1926), and can also be clearly distinguished from the Benin population which has seven kinetosomes blocks (Pattern I) (Dragesco and Dragesco-Kernéis 1986).In contrast, the Chinese population matches well with Condylostoma spatiosum in various characters except the number of adoral membranelles (150-200 vs. 80-153).Considering that this character is highly variable among different populations of this species, we believe it is reasonable to classify the Chinese population as Condylostoma spatiosum.Similarly, an Antarctic population with a small cell size, about 30 somatic kineties, frontal membranelles composed of a single stripe of kinetosomes (Pattern II), and about 120 adoral membranelles was identified by Wilbert and Song (2005) as Condylostoma cf.magnum.It bears a closer resemblance to Condylostoma minutum than C. magnum, so we here classify it as C. minutum.
Comments: Condylostoma curvum was originally reported by Burkovsky (1970), and has been redescribed several times (Chen et al. 2011;Kim et al. 2012;Song et al. 2003;Yan et al. 2015).Considering the high variability in the morphological characters of Condylostoma, it is reasonable to summarize the recognizable features of C. curvum by combining the characters of all populations with reliable descriptions: (1) small-sized (< 400 μm); (2) cell shape flattened and elongated, posterior end rounded, anterior end slightly truncated; (3) oral region about 25-45% of cell length; (4) macronucleus moniliform; 5) 20-42 somatic kineties; (6) 60-112 adoral membranelles; (7) frontal membranelles with Pattern I arrangement; (8) cortical granules with Pattern 4 arrangement; (9) brackish or marine habitat.The three Chinses populations described in the present study closely resemble the other populations, except for the cell size of population 1 (maximum length 505 μm) (Fig. 9A, D).As cell size is thought to be population-dependent and the average size of population 1 is less than 400 μm, we believe that our identification of the three Chinese populations is correct.
Comments: Like the remarks on Condylostoma spatiosum in Shao et al. (2006), C. kris was first mentioned at an annual meeting of Japanese zoologists in 1942 and was cited as C. kris Ozaki & Yagiu, 1942 in the subsequent detailed description (Yagiu 1944).According to the regulations of ICZN (1999), the original report ( 1942) is a nomen nudum, and hence the valid name should be cited as C. kris Ozaki & Yagiu in Yagiu, 1944.The original description of Condylostoma kris, which was based on fixed material with a certain degree of shrinkage, gives the diagnostic characters as follows: (1) medium size in vivo (325-410 μm in fixed cells); ( 2) slender ellipsoidal and slightly flattened cell shape; (3) peristome length approximately a quarter of cell length; (4) moniliform macronucleus; ( 5) 36-48 somatic kineties; ( 6) about 80 adoral membranelles; (7) kinetosomes of frontal membranelles arranged in Pattern I; (8) pale yellowish-green (depending on microscopy method used) cortical granules, about 1 μm in diameter (0.6-0.8 μm after fixation); (9) marine habitat (Yagiu 1944).Given that the number of adoral membranelles varies among different populations and that the description of the Japanese population was based on both live and fixed cells (without using silver staining methods), we believe the number of adoral membranelles (84-114) falls within a reasonable range for the two Chinese populations.In addition, Yagiu (1944) observed only one type of cortical granules with a loosely-spaced arrangement, which is consistent with Type I of cortical granules in Chinese populations.However, considering the Type II cortical granules are extremely fine and light-colored, these may have been overlooked due to the limitations of the microscopes available.Therefore, we consider the identification of the two Chinese populations as C. kris is correct.
Comments: Condylostoma spatiosum was originally described by Yagiu (1944) with the following characteristics: (1) medium to large size (364-1320 μm in fixed material); ( 2) cell with a truncated anterior end and a rounded posterior end; (3) peristome length about a quarter of cell length; (4) moniliform macronucleus; (5) 47-64 somatic kineties; (6) about 80 adoral membranelles; (7) three cirri-like projections in front of the paroral membrane; (8) one type of pale yellowishgreen (depending on method of microscopy used) cortical granules, about 1 μm in diameter (0.4-0.6 μm after fixation); (9) marine habitat.Our present population closely matches the original description in terms of cell size and shape, general morphology, and habitat, except for the number of adoral membranelles (115-177 vs. ca. 80) and the arrangement of kinetosomes of the frontal membranelles (Pattern II vs. three cirri-like projection, i.e., Pattern I).These differences have also been observed in subsequent redescriptions (Kim et al. 2012;Shao et al. 2006;Song and Wilbert 1997;Wilbert and Song 2008), where a higher number of adoral membranelles (ca. 110-200) and frontal membranelles with Pattern II were reported.It is important to note that the original description was based on both live and fixed cells (without using silver staining methods), which could have led to the overlooking or misinterpretation of these two characters.Considering the consistent redescription of these characters in subsequent reports, we believe that our identification is correct.
Comments: Condylostoma minutum was first described by Bullington (1940) based on observations of cells in vivo, but without details of several diagnostic features.Later, Chen et al. (2007) redescribed it based on three Chinese populations using observations of both live and protargolstained specimens and provided an improved diagnosis.Combining the original and subsequent investigations (Bullington 1940;Chen et al. 2007;Kim et al. 2012), this species can be recognized by the following characters: (1) small size, 200-390 μm in vivo; (2) cell flattened and slender; (3) peristome length 25-40% of cell length; (4) moniliform macronucleus; (5) 26-44 somatic kineties; ( 6) 67-107 adoral membranelles; (7) frontal membranelles with Pattern II arrangement; (8) cortical granules with Pattern 4; (9) brackish or marine water habitat.Our present populations closely resemble the previously described populations in terms of all morphological characters with the exception of the color of the cortical granules (gray green vs. dark-gray in Chen et al. 2007 vs. greenish yellow in Kim et al. 2012).As mentioned earlier, this feature usually varies among different observers based on their equipment, such as the magnification combined with the lighting and microscopy technique used, resulting in a rather subjective interpretation.Nevertheless, it still deserves attention in future research as it has been found to be a useful distinguishing character in numerous taxa (Hao et al. 2022;Ma et al. 2022b;Shao et al. 2023).
Comments: Condylostoma arenarium was originally reported based on living cells but lacked a description of the ciliature on the right side of the paroral membrane (Spiegel 1926).Subsequently, it was identified as having several frontal cirri-like structures (Pattern I) (Borror 1961(Borror , 1972;;Dragesco and Dragesco-Kernéis 1986;Kahl 1932;Villeneuve-Brachon 1940).However, a recent detailed study, including living observation, protargol staining, scanning electron microscopy, and phylogenetic analyses, redefined C. arenarium as having one strip-like "frontal cirri" structure (Pattern II) (Fernandes et al. 2015).Therefore, all populations under the name C. arenarium, except for the original and Brazilian populations, need to be reassessed, particularly those with several frontal cirri-like structures (Pattern I).
As discussed in the Comments section of Condylostoma marinum sp.nov., Kahl (1932)'s report included three morphotypes, with Types 2 and 3 being synonymous with C. curvum and C. marinum sp.nov., respectively.Considering the frontal membranelles (Pattern I) and cell size (400-700 μm), C. arenarium-like Type 1 corresponds well with C. kris, thus we suggest classifying Type1 as a synonym of C. kris.We only included cell size in the diagnosis and did not consider other morphological data due to the existence of multiple morphotypes.Additionally, we agree with the proposal by Song et al. (2003) to classify the populations reported by Borror (1961) and Dragesco and Dragesco-Kernéis (1986) as synonyms of C. curvum.
There are four other populations under the name of Condylostoma arenarium in which the ciliature at right side of paroral membrane was undescribed (Agamaliev 1972;Al-Rasheid 1999;Dragesco 1960;Hartwig 1973).There are large overlaps between C. minutum and C. curvum, as well as between C. arenarium and C. kris, in terms of cell size and shape, ratio of peristome to cell length, and number of somatic kineties.However, we can only attempt to classify these populations (marked with asterisks in the synonym list) based on weakly supported reasons and have not included the associated morphological data in the diagnoses and geographical distributions.The individuals reported by Dragesco (1960) have two sizes.We propose that the larger one (300-600 μm) be retained in C. arenarium because it has only one type of cortical granules (vs.two types in C. kris), and that the smaller one (ca.200 μm) should be classified as C. curvum due to its slightly stubby cell shape.Similarly, the North Sea population (Hartwig 1973) which has only one type of cortical granules, should be retained as C. arenarium.In contrast, two types of cortical granules appear to present in the drawing of the Azerbaijani population (Agamaliev 1972), which can serve as evidence for provisionally listing it as a synonym of C. kris.In his description of C. arenarium, Al-Rasheid (1999) noted that it resembles the Cotonou population (Dragesco and Dragesco-Kernéis 1986).Since the latter is here classified as C. curvum, thus we suggest that Saudi Arabian population should also be recognized as C. curvum.

Genus Condylostomides da Silva Neto, 1994
A clear definition is lacking, therefore an improved diagnosis is provided here based on the present and previous studies.

Improved diagnosis
Medium-sized condylostomatid heterotrich, usually 100-300 µm; freely motile, crawling on substrates or between sediment particles.Cell non-contractile, ellipsoidal, obliquely truncated at anterior end.Somatic ciliature holotrichous, including some shortened kineties; suture absent.Buccal area prominent, adoral zone of membranelles at left margin of peristome.Frontal membranelles connected to distal end of paroral membrane.Macronucleus moniliform or composed of two or three ellipsoidal nodules.Contractile vacuole present at posterior end, with two collecting canals extending along margin of cell to anterior end.Cortical granules usually pigmented, mostly yellow or blue-green.Mainly soil or freshwater habitat.

Identification characteristics
Condylostomides species had been discovered before the genus was established in 1994, however, they were classified as Condylostoma due to their close resemblance in general morphology, particularly the prominent buccal area (da Silva Neto 1994;Dragesco 1954Dragesco , 1960;;Foissner 1995;Kahl 1932;Penard 1922).While Condylostoma species are commonly found in marine or brackish water, members of Condylostomides typically inhabit freshwater or soil habitat.A notable distinguishing feature is the presence of a contractile vacuole at the posterior end in Condylostomides, which is absent in Condylostoma.Additionally, protargol-stained specimens reveal further distinctions between the two genera, such as the absence of suture in Condylostomides (present in Condylostoma), the ciliation of dikinetids (only one basal body ciliated in Condylostomides vs. both basal bodies ciliated in Condylostoma), and the arrangement of kinetosomes of the frontal membranelles (Pattern III in Condylostomides vs. Patterns I and II in Condylostoma).
Currently, the macronucleus and cortical granules, i.e., their shape, arrangement, and color, serve as important identifying features within the genus Condylostomides (Foissner et al. 2002).Based on the review of Condylostoma and Condylostomides in the present work, we propose the transfer of Condylostoma minima to Condylostomides, resulting in a total of eight valid Condylostomides species (Fig. 3).

Condylostomides species
Note: In the list of synonyms for each species, we have directly cited the unaltered species names (as well as author names) from the original sources.This is to demonstrate the transition of species names from historical references to the contemporary era, despite several identifiable errors that are subsequently present throughout.
Diagnosis summarized from original population: Cell size 100-130 μm; narrowly sac-shaped, anterior portion slightly widened, posterior end bluntly rounded; peristome about 1/3 of cell length; two macronuclear nodules; one type of yellowish cortical granules arranged in approximately four closely spaced rows between adjacent kineties; freshwater habitat.
Comments: The genus Condylostomides was established by da Silva Neto (1994) with C. grolieri as the type species, however, the author did not make any comparisons with other similar taxa.We agree with the analyses of Foissner et al. (2002) that: (1) C. grolieri should be listed as a junior synonym of C. tardus; (2) C. tardus possesses colorless cortical granules.As a result, we agree with the change of the type species of this genus to C. tardus.
Z o o B a n k r e g i s t r a t i o n : u r n : l s i d : z o o b a n k .org:act:1326A00C-9CB0-4E3B-92DC-26C1EE63ADEE.
Comments: In the original report (Dragesco 1960), it was mentioned that there were numerous "vacuoles pulsatiles" on the edge of the cell, which Foissner (2016) later considered to be small contractile vacuoles.We speculate this species has only one contractile vacuole at the posterior end, similar to other congeners, and that other small contractile vacuoles could be either food vacuoles or the expansion of collecting canals as shown in the illustrations for Condylostomides tardus in Dragesco (1960) and Condylostomides coeruleus in the present study (Fig. 15C).Al-Rasheid (1996) reported a Saudi Arabian population as Condylostoma nigra (marked with asterisks in the synonym list), but with limited morphological data to support this identification.Therefore, we did not include this population's morphological data in the diagnosis as its identity cannot be verified.Foissner (2016) transferred Condylostoma nigra to Condylostomides when comparing with C. coeruleus.As the genus Condylostomides is masculine gender, we propose a compulsory change to the name, i.e., Condylostomides nigrus (Dragesco, 1960) nom.corr.
Comments: Condylostomides coeruleus was originally discovered in slightly saline mud and soil and was recognized by its large size and conspicuous color (Foissner 2016).Hines et al. (2020) rediscovered it in Florida, USA, with diagnostic features matching the original description, except they reported a smaller cell width of 40-64 μm (mean 55 μm).This can be explained by measurements taken in vivo while swimming from soil cultures rather than from fixed cells, and overall slight variation in size within a population.The Weishan population is the first record of this species from Asia and matches closely with the original description in all morphological characters, leaving little doubt as to its identity.
An additional Condylostomides species with blue cortical granules, C. nigrus nom.corr., should be compared with C. coeruleus.The former has a second type of cortical granules that are densely arranged and colorless (Dragesco 1960) and so can easily be distinguished from the latter, which has only one type of cortical granules.Foissner et al., 2002 [1] 2002 Condylostomides etoschensis nov.spec.-Foissneret al., 406,407] (detailed morphological description based on living observations, protargol preparations, and scanning electron microscopy).
Comments: It is noteworthy that this distinct and colorful soil species was, like C. coeruleus, thought to be an endemic ciliate with a restricted distribution upon its first discovery in Africa.Despite its apparent absent from South American soil surveys, and indeed the present study in China, it was discovered in Florida on the North American continent (Hines et al. 2020).
Z o o B a n k r e g i s t r a t i o n : u r n : l s i d : z o o b a n k .org:act:570CBC5A-C958-4E21-82E5-4404F321EE98.
Comments: This species is characterized by a terminal contractile vacuole, which is the most distinguishing character between Condylostoma and Condylostomides.In addition, its small cell size, ellipsoidal cell shape, large oral region, and comparatively few macronuclear nodules make it consistent with the diagnosis of Condylostomides.Thus, we suggest that this species should be transferred to Condylostomides as Condylostomides minimus (Dragesco, 1954) comb. nov. & nom. corr.to match the masculine genus.It should be noted that both reported populations of this species were collected from sand, although the exact environmental parameters of these sands were not precisely reported (Dragesco 1954(Dragesco , 1960)).Also, as Dragesco (1960) mentioned, the salinity of sandy habitats is highly variable due to the influence of freshwater flow, rainfall, and evaporation, therefore further sampling of potential habitats of this species is needed, despite the original descriptions being collected within a marine beach area.(Dragesco 1960;Foissner 2016;Foissner et al. 2002).Condylostomides terricola most closely resembles C. minimus comb.nov.& nom.corr.Since the color of cortical granules for C. minimus comb.nov.& nom.corr. is not listed, it is assumed to be inconspicuous like other ciliates without mention of pigments (vs.bright yellowish cortical granules in C. terricola).In addition, C. terricola can still be separated from C. minimus comb.nov.& nom.corr.by its smaller size (90-140 μm vs. 130-220 μm), smaller proportion of peristome (ca.33% vs. 40-50%), and in having fewer somatic kineties (15-18 vs. 20-22) (Dragesco 1954(Dragesco , 1960;;Foissner 1995).Therefore, we consider that C. minimus comb.nov.& nom.corr. is a valid species.

Geographic distribution
The global distribution patterns of Condylostoma and Condylostomides are derived from the data generated from the review of all valid species recognized in the present studies (Fig. 4A).Both genera have been recorded on multiple continents, with the exceptions of Condylostoma in Oceania and Condylostomides in Antarctica.Europe is the predominant region for records of Condylostoma, with more than 70% of the species recorded from this continent.Additionally, approximately 40% of Condylostoma species have been found thus far in Asia.In contrast, some members of the genus Condylostomides were at first considered representative biogeographical flagship species (Foissner 2016;Foissner et al. 2002).For example, C. etoschensis was recorded from Africa and was thought to be restricted there, especially after similar sampling from South America did not recover this species.However, like other species that benefit from intensive sampling, it was later recorded from another geographical region, in this case Florida, USA (Hines et al. 2020).Similarity, C. coeruleus was at first found in South America, but later was also found in Florida, USA.Notably, in the present study, we report the first record of C. coeruleus on the Asian continent, providing further evidence for the global distribution of Condylostomides and indeed ciliates in general when sampling efforts are increased (Finlay 2002).
Condylostoma species are characterized as large heterotrichous ciliates with a prominent buccal region (Chen et al. 2007;Chi et al. 2021).This distinctive buccal region allows them to be macrophagous omnivores capable of feeding on a variety of organisms, including diatoms, flagellates, green algae, other ciliates, and small metazoans (Fenchel 1968).Their broad dietary range indicates their adaptability to diverse environments, enabling them to inhabit brackish and marine waters, and even extreme habitats such as those with high salinity and in extreme temperatures found in polar regions.In contrast, the habitats of Condylostomides differ completely from those of Condylostoma.They are primarily found in terrestrial soils and freshwater environments, occasionally exhibiting some degree of salinity tolerance.It is noteworthy that only two Condylostoma species (C.kasymovi and C. caudatum) have been recorded from freshwater habitats, and no further redescriptions have been reported since their original descriptions.Additionally, the full extent of geographical distribution for Condylostomides is not fully understood due to undersampling.Therefore, broader sampling efforts are needed to further explore the occurrence, distribution, and population-specific morphology of these two genera.

Molecular data and phylogenetic analyses
In the present study, eight new SSU rDNA sequences were obtained and deposited in the GenBank database.The lengths, GC-contents, and accession numbers of these sequences are provided in Table 1.The maximum likelihood (ML) and Bayesian inference (BI) trees based on SSU rDNA sequences are generally congruent, and therefore, only the ML tree with support values from both analyses is presented (Fig. 5A).
Within the class Heterotrichea, eight families, i.e., Stentoridae, Fabreidae, Folliculinidae, Gruberiidae, Condylostomatidae, Peritromidae, Climacostomidae, and Spirostomidae, are revealed to be monophyletic (Fig. 5A).The exceptions are Maristentoridae, which are monotypic, and Blepharismidae, which are paraphyletic with Blepharisma halophilum grouping in the family Fabreidae.The five genera with available molecular data within the family Condylostomatidae are divided into two lineages.One lineage consists of Condylostentor, Chattonidium, and Condylostoma, which inhabit brackish or marine habitats.The other lineage comprises Condylostomides and Linostomella, which are commonly found in soil or freshwater habitats (Fig. 5B).(Chen et al. 2007;Shao et al. 2006;Spiegel 1926), making it challenging to distinguish them in vivo.In addition, none of the existing sequences of these species are supported by morphological information or vouchered specimens, raising the potential for misidentification.Based on these analyses, we suggest that: Clade I, exhibiting 0-4 nucleotide differences, represents C. spatiosum; Clade II represents C. minutum; and Clade III represents a C. spatiosum-like species.However, future studies combing both morphological and molecular data are needed to further explore the identifications and classifications of these species within their respective clades.
Although the three newly sequenced Chinese populations of Condylostoma curvum (OR553809, OR553810, and OR553811) cluster with other populations of C. curvum, they are divided into two distinct clades (Clades VI and VII).The presence of 21-25 stable nucleotide differences between Clade VI and Clade VII supports their significant divergence.Despite this differentiation, the current knowledge and morphometric data obtained from in vivo observations and protargol staining do not allow us to classify them as separate species.This suggests the existence of cryptic species within C. curvum, similar to the findings reported for Spirostomum minus and S. teres (Boscaro et al. 2014;Chi et al. 2020aChi et al. , 2022b)).To accurately determine and define the boundaries of the C. curvum species complex, a multidisciplinary integrative approach combining traditional taxonomy and modern molecular technologies is required (Serra et al. 2020).
All populations of Condylostoma kris are clustered in Clade IX, which also includes Condylostoma sp., Condylostoma cf.arenarium, and C. arenarium.Condylostoma arenarium was redefined based on a Brazilian population using an integrative approach including in vivo observation, protargol staining, scanning electron microscopy, and phylogenetic analysis (Fernandes et al. 2015).This Brazilian population (JQ28289) is placed within Clade VIII in our phylogenetic tree.In the literature, many descriptions of populations under the name C. arenarium have been recognized as having a "blocky" arrangement of frontal membranelles (Pattern II), which is same as the frontal membranelles of C. kris, leading to potential misidentifications of these two species.However, it is noteworthy that the sequence divergence within Clade IX, ranges from 0 to 10 nucleotide differences.Therefore, broader sampling with more detailed morphological information and reliable molecular data is needed to determine whether Clade IX exclusively comprises C. kris.
The genus Condylostomides is divided into two fully supported clades.One clade consists of three populations of Condylostomides coeruleus, which includes the newly sequenced Chinese population (OR553815), as well as an unidentified species (KP970236).The other clade solely comprises the three sequences from a single population of Condylostomides etoschensis.However, the Chinese population differs from the American (MK543445) and Venezuelan (AM713188) populations by 10 and 3 nucleotide sites, respectively.These differences may contribute to the inconsistency of the topologies between the ML and BI trees with additional samples being necessary to fully investigate any potential differences between different populations.
Finally, mapping the two patterns of frontal membranelles onto the phylogenetic tree reveals that Condylostoma species are roughly divided into two major groups according to different patterns (Fig. 5B).One group (G1) contains Clades I-V and Clade VIII (C.arenarium), all of which are Pattern II.The other group (G2) consists of Clade VI, Clade VII, and Clade IX, all of which are Pattern I. Interestingly, Chattonidium setense possesses a distinct bundle of cilia in the same location as the frontal membranelles of Condylostoma species (Modeo et al. 2006).Based on scanning electron micrographs, this structure appears to be arranged in a striped pattern, suggesting a potential homology with the Pattern II of frontal membranelles in Condylostoma (G2).These homologous structures may play important roles in establishing the sister relationship between Chattonidium setense and G2 of Condylostoma.However, the frontal membranelles of Condylostomides do not correspond well with the two patterns observed in Condylostoma.It is nearly connected to the paroral membrane and does not seem to be a fully independent structure.As a result, the frontal membranelles serve not only as a crucial taxonomic feature, but also likely play a significant role in the evolution of the family Condylostomatidae.To further investigate and verify this hypothesis, broader sampling from different genera, including detailed morphological characters and robust molecular data, are required.

D e s c r i p t i o n o f p o p u l a t i o n 1 :
C e l l s i z e 385-560 × 85-110 μm in vivo, on average about 450 × 95 μm with length to width ratio about 5:1, slightly contractile.Cell narrowed ellipsoidal and flattened, truncated at anterior end, widest in mid-region and gradually tapered to rounded posterior end (Figs.6A, 7A-C).Macronucleus moniliform, composed of 11-15 nodules, located along right side of cell; numerous micronuclei, closely associated with macronuclear nodules (Figs.6B, D, 7G, H; Table 2).Pellicle flexible and thin, with two types of cortical granules: Type I, ellipsoidal, grayish-green, loosely arranged, about 1.5 μm in length; Type II, spherical, colorless, densely arranged, about 0.5 μm in diameter (Figs.6F, H, 7I, J).Unknown structures (possibly mitochondria) ellipsoidal, dark gray, about 2.0 μm in length, located underneath cortex (Figs.6F, 7I).Cytoplasm colorless to slight yellowish at low magnification, with numerous small granules and food vacuoles filled with algae (Figs.6A, 7A-C).Locomotion by gliding over substratum, occasionally swimming while rotating about the main cell axis.

D e s c r i p t i o n o f p o p u l a t i o n 1 :
C e l l s i z e 315-505 × 55-95 μm in vivo, on average about 395 × 70 μm, with length to width ratio about 5:1.Cell elongate-ellipsoidal and dorsoventrally flattened, with a truncated anterior end and a narrowly rounded posterior end (Figs.8A, B, 9A, D).Macronucleus moniliform, composed of 7-13 nodules, aligned along the right margin of cell; micronuclei difficult to recognize either in vivo or in protargol preparations (Figs.8A, B, 9J, N; Table 2).Pellicle rough with ellipsoidal, brownish-green cortical granules (about 1 μm in length), distributed with medium density between somatic kineties (Figs.8G, 9H, I).Cytoplasm brownish at low magnification, with numerous small granules and a few food vacuoles containing algae (Figs.8A, 9A, K).Movement relatively slow, usually gliding over substrate, occasionally swimming.
Buccal field about 25-40% of cell length, with a conspicuous cytopharynx (Figs.10A, B, 11A-E).Adoral zone comprises 115-177 membranelles, each composed of one short and two long rows of basal bodies, twisted in proximal region (Figs.10C, E, F, 11H, M-P).Transversely arranged fiber-like stripes visible on inner wall of oral cavity (Fig. 11H).Paroral membrane highly developed and visible in vivo, occasionally slightly broaden at distal end (Figs.10E, 11H, N, P).Frontal membranelles located to right of distal end of paroral membrane, consist of a single stripe formed by aggregations of kinetosomes (Pattern II) (Figs. 10E, F, 11I, N, P).

D e s c r i p t i o n o f p o p u l a t i o n
Thirty-eight to 44 longitudinal somatic kineties, including 6-12 shortened rows both on ventral and dorsal sides (Figs.12C, D, 13P).All somatic kineties composed of dikinetids; each basal body of dikinetids bears a cilium (Figs. 12C,D,G,13M).Suture on ventral side of posterior portion of cell formed by shortened somatic kineties (Fig. 13N).
Frontal membranelles located at distal end of paroral membrane, composed of a single stripe formed by aggregations of kinetosomes (Pattern II) (Figs. 12E, 13E, L, O).
Somatic cilia about 10 μm long in vivo.Forty-two to 52 somatic kineties composed of densely spaced dikinetids, only one basal body of each dikinetid bears a cilium (Figs.14E, G

Conclusions
Limited by a variety of factors, the correct species identification of individuals from the most species-rich heterotrich genus, Condylostoma, has long been a challenging task.This difficulty has, in turn, raised concerns regarding potential misidentifications of numerous gene sequences in publicly accessible databases, thus impeding our understanding of their evolutionary relationships.To address this issue, we conducted a comprehensive review of two genera, based on morphological redescriptions and phylogenetic analyses of four highly confusable Condylostoma species and one Condylostomides species isolated for the first time from the Asian continent.In this review, a total of 43 nominal species and about 130 populations were reassessed from the perspective of modern taxonomy, ultimately identifying 30 valid Condylostoma species and eight Condylostomides species.Furthermore, we updated the identification criteria for members of these two genera, examined their current documented biogeographical distributions, explored their phylogenetic relationships, and, interestingly, revealed the presence of cryptic species within Condylostoma for the first time.The present study provides a solid foundation upon which further future investigations into these and other related genera can be built.

Fig. 1
Fig. 1 Introduction to morphological characters of Condylostoma.A A typical idealized individual to show the key morphological structures.B Kinetosome arrangement patterns of frontal membranelles (arrows).C Outlines of different cell shapes.D Various macronuclear

Fig. 5 A
Fig. 5 A Maximum likelihood (ML) tree based on SSU rDNA sequences of heterotrichous ciliates.Numbers near branches show the bootstrap values from ML and posterior probabilities from Bayesian inference (BI).Disagreements between ML and BI are shown by asterisks.Fully supported clades are marked with solid circles.The nine populations investigated in the present study are marked

Fig. 6 6 4. 1 5 5 6 . 1
Fig. 6 Schematic drawings of Chinese populations of Condylostoma kris from life (A, B, F, H) and after protargol staining (C-E, G).A General view of a representative individual.B Different cell shapes and presence of moniliform macronuclei.C, D Ventral (C) and dorsal (D) views of the same specimen, showing the ciliature and macronucleus.E Detailed view of anterior portion of cell, arrows mark the paroral membrane, arrowheads mark the frontal membranelles.F

Fig. 7
Fig. 7 Photomicrographs of Condylostoma kris pop.1 from life (A-G, I, J), after protargol staining (K-O), and after Hoechst 33,342 staining (H).A-C Various individuals to show different cell shapes.D Details of cilia (arrows).E Buccal region, arrows mark the adoral zone of membranelles, arrowheads show the paroral membrane.F Anterior portion of cell, arrows mark the frontal membranelles.G Moniliform macronucleus.H Hoechst 33,342-stained individual to show the nuclear apparatus, arrows mark the micronuclei.I, J Details of cortex, arrows mark the ellipsoidal unknown structures (possibly mitochondria), arrowheads show the Type II of cortical granules,

Fig. 8
Fig. 8 Schematic drawings of Chinese populations of Condylostoma curvum from life (A, B, G) and after protargol staining (C-F).A General view of a typical individual.B Different cell shapes and moniliform macronuclei.C, D Ventral (C) and dorsal (D) views of the same specimen, showing the suture (arrows), paroral membrane (arrowhead), frontal membranelles (double-arrowhead), moniliform

Fig. 9
Fig. 9 Photomicrographs of Condylostoma curvum from life (A, D, G-K from pop.1; E, F from pop.2; B, C from pop.3) and after protargol staining (L-O from pop.1).A-F Various individuals to show different cell shapes, arrows mark the moniliform macronucleus.G Anterior portion of cell, arrows mark the paroral membrane, arrowheads show the frontal membranelles, double-arrowhead indicates the adoral membranelles.H Cortical granules (arrows) between adjacent somatic kineties.I Tangential section of the cell, arrows mark

Fig. 11
Fig. 11 Photomicrographs of Chinese population of Condylostoma spatiosum from life (A-L) and after protargol staining (M-R).A-G Various individuals showing different cell shapes, arrow marks the cytopharynx.H Anterior portion of cell, arrows mark the adoral zone of membranelles, arrowheads show the paroral membrane, doublearrowheads indicate the fiber-like stripes.I Details of frontal membranelles (arrows).J Cortical granules (arrows) between adjacent somatic kineties.K Tangential section of the cell, arrows mark the ellipsoidal cortical granules.L Moniliform macronucleus.M Ventral

Fig. 12
Fig. 12 Schematic drawings of Chinese populations of Condylostoma minutum from life (A, B, G) and after protargol staining (C-F).A General view of a typical individual.B Different cell shapes and moniliform macronuclei.C, D Ventral (C) and dorsal (D) views of same specimen, showing the ciliature and macronucleus.E Detailed

Fig. 13
Fig. 13 Photomicrographs of Condylostoma minutum pop.1 from life (A-K) and after protargol staining (L-P).A-D Various individuals showing different cell shapes.E Anterior portion of cell, arrows mark the paroral membrane, arrowheads show the frontal membranelles.F Buccal region, arrows mark the paroral membrane, arrowheads show the fiber-like stripes, double-arrowheads indicate the adoral zone of membranelles.G Cortical granules (arrows).H Tangential section of the cell, arrows mark the ellipsoidal cortical granules.I, J Cytoplasmic granules and food vacuoles containing algae.K Moniliform

Fig. 14
Fig. 14 Schematic drawings of Chinese population of Condylostomides coeruleus from life (A, B, E, F) and after protargol staining (C, D, G, H).A General view of a typical individual, arrows mark the contractile vacuole.B Different cell shapes and moniliform macronuclei (arrowheads).C Most adoral membranelles are composed of three rows (two long and one short) of basal bodies.D Details of buccal region, arrows mark the paroral membrane, arrowheads show the frontal membranelles, green double-arrowheads indicate the adoral membranelles composed of one short and two long rows of basal

number of specimens, NA not available, SD standard deviation, SK somatic kineties a Macronuclear nodules were selected haphazardly in each specimen 18 Transverse furrows absent on the cortex ………… 19 18.1 Transverse furrows present on the
Three other congeners are brightly colored and have a moniliform macronucleus, namely C. etoschensis, C. coeruleus, and C. nigrus nom.corr.Condylostomides minimus comb.nov.& nom.corr.can be distinguished from all of these by having loosely arranged spindle-shaped cortical granules (vs.closely arranged minute cortical granules in C. etoschensis vs. closely arranged ellipsoidal cortical granules in C. coeruleus vs. two types of cortical granules in C. nigrus nom.corr.) and about 20 somatic kineties (vs.30-42 in C. etoschensis vs. 34-52 in C. coeruleus vs. ca.40 in C. nigrus nom.corr.) Three genera, namely Condylostentor, Condylostomides, and Linostomella, are monophyletic, while Condylostoma are paraphyletic due to the monotypic genus Chattonidium nesting within it.the existing C. minutum sequences in the GenBank database.The three species, i.e., C. spatiosum, C. minutum, and C. magnum, are very similar in overall appearance