Findings on three endocommensal scuticociliates (Protista, Ciliophora) from freshwater mollusks, including their morphology and molecular phylogeny with descriptions of two new species

Species of the ciliate genera Myxophyllum and Conchophthirus are found as endocommensals of terrestrial and freshwater mollusks, respectively. So far, there have been few studies of these genera and morphological data for most members are often incomplete. In the present work, two new species, Myxophyllum weishanense sp. nov. and Conchophthirus paracurtus sp. nov., and a known species, Conchophthirus lamellidens, were isolated from hosts in Lake Weishan Wetland, China. Taxonomic studies indicate that M. weishanense sp. nov. can be recognized mainly by the combination of about 60 somatic kineties on both ventral and dorsal sides and the presence of caudal cilia. Conchophthirus paracurtus sp. nov. differs from congeners in its body shape and size, having a glabrous area on the posterior right side, and having fewer somatic kineties. In addition, differences in their ITS2 (Internally Transcribed Spacer 2) secondary structures support the discrimination of the two new species from their highly similar congeners. An improved diagnosis for the poorly known species, C. lamellidens is also provided. Phylogenetic analyses reveal that members of the genus Myxophyllum belong to a fully supported clade that is sister to a large, poorly supported clade consisting of Hemispeiridae, Ancistridae, and several lineages of the nonmonophyletic Cyclidiidae. The Myxophyllum clade also includes Protophyra ovicola JQ956552, a possible misidentification. Sequences of the two new Conchophthirus species cluster with other congeners in a fully supported clade that is unrelated to either the ‘typical’ thigmotrichs or to pleuronematids, thus conflicting with the traditional classification, and may represent an orphan scuticociliate lineage. Supplementary Information The online version contains supplementary material available at 10.1007/s42995-024-00230-4.


Introduction
The phylum Ciliophora Doflein, 1901 (ciliates) is a highly diverse assemblage of single-celled eukaryotic microbes.Ciliates are heterotrophic or mixotrophic and exhibit complex cortex, nuclear dimorphism (macro-and micronuclei) and sexual reproduction by conjugation as synapomorphies Edited by Jiamei Li.
Two endocommensal scuticociliate genera, Myxophyllum (Stein, 1861) Raabe, 1934 andConchophthirus Stein, 1861, have been known for over a century, but detailed taxonomic features such as the oral and somatic ciliature, remain unclear since most taxa have not been studied by modern methods.To date, all Myxophyllum populations have been identified as the same species, namely M. steenstrupi (Stein, 1861) Raabe, 1934, although they were collected from different hosts and differ from each other in some morphological features (de Puytorac et al. 1992;Kazubski 1964Kazubski , 1973;;Penn 1958;Raabe 1971;Stein 1861).Zhang and Vďačný (2021) carried out a complete redescription of the Slovak population of M. steenstrupi and provided the first sequences of the nuclear ribosomal RNA gene and mitochondrial cytochrome c oxidase I gene of this genus.This allowed us to investigate whether the Myxophyllum populations represent different species, i.e., whether there are cryptic species in M. steenstrupi.
The genus Conchophthirus Stein, 1861 mostly inhabits the mantle cavity of freshwater unionid mollusks (Antipa et al. 2020;Engelmann 1862;Ghosh 1918;Kahl 1931;Raabe 1933Raabe , 1934;;Uyemura 1935;Zhang and Vďačný 2024).According to Raabe (1971), the genus Conchophthirus contains ten valid morphospecies, seven of which have illustrations based on silver nitrate-stained specimens, while all Conchophthirus species lack information on the oral ciliature.Antipa and Small (1971a) revealed the oral ciliature of C. curtus for the first time.More recently, Zhang and Vďačný (2024) documented the oral structure of C. unionis and C. acuminatus.To date, details of the oral ciliature, a crucial taxonomic feature, are lacking in the other seven species.
The role played by endocommensal ciliates in mollusks remains unclear, and collecting them is difficult as compared to free-living species.Consequently, few studies have focused on the diversity of endocommensal ciliates in recent decades, resulting in outdated and limited research data on them.In the present work, three mollusk-dwelling scuticociliates, one Myxophyllum species and two Conchophthirus species collected from the Lake Weishan Wetland in northern China, were thoroughly characterized using a morpho-molecular approach.Based on both morphological features and molecular data, two new species were established, namely Myxophyllum weishanense sp.nov.and Conchophthirus paracurtus sp.nov., while an improved redescription of Conchophthirus lamellidens was provided.Our multifaceted strategy also allows us to better understand the diversity and the evolutionary trajectory of Conchophthirus revealing that this genus is not related either to 'traditional' thigmotrichs or to pleuronematids, but rather represents an orphan lineage.In contrast, Myxophyllum is nested deep within the order Pleuronematida along with other 'traditional' thigmotrichs and some non-thigmotrich taxa.
Myxophyllum weishanense sp.nov.was found in the mantle cavity of the snail Acusta ravida Benson, 1842.Fifteen snails were collected from the humus on the river bank on June 18, 2021 (Fig. 1B).The humus temperature was 24 °C.Samples were observed in 14 of the 15 hosts and each contained about 20-100 ciliates.Twenty-four hours after detaching from their host, about half of the ciliate cells died, the remaining individuals exhibited reduced motility and their bodies became rounded, and all died after about three days.
Three individuals of the freshwater Chinese pond mussel Sinanodonta woodiana (Lea, 1834) were collected from a shallow river channel on March 25, 2021 (Fig. 1C), when the water temperature was 16 °C.Conchophthirus lamellidens was found in extremely high numbers (> 500 cells per host) in the mantle cavity of one of the three individuals, but fewer (about 50 cells per host) in two other individuals.Most C. lamellidens specimens were attached to the mantle cavity and gills.
Six individuals of the cockscomb pearl mussel, Cristaria plicata (Leach, 1815), were collected from a fish-culturing pond on November 24, 2021 (Fig. 1D), the water temperature was 9 °C.Conchophthirus paracurtus sp.nov.was found in moderate abundance (about 150 cells per host) within the mantle cavity and gills of all six mussel individuals.

Morphological studies
Living ciliates were observed and photographed using bright field and differential interference contrast microscopy at 100-1000 × magnification.The ciliary pattern and the nuclear apparatus were revealed with the protargol staining method (Wilbert 1975).The silver nitrate staining method (Corliss 1953) was used to reveal the silverline system.Counts and measurements were made under 400-1000 × magnifications.Illustrations of living cells were produced from freehand sketches based on the photographs, while those of protargol-and silver nitrate-stained specimens were made using Adobe Photoshop according to the photomicrographs.Terminology is mainly according to Lynn (2008).

DNA extraction, PCR amplification and sequencing
Three cells of each species were isolated for DNA extraction.All cells were washed five times with filtered in situ water (0.22 µm, Millex-GP filter unit) to exclude contamination (Liu et al. 2022).Genomic DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen, Germany) following the optimized manufacturer's protocol, with 25% of the suggested volume used for each solution.The primers 82-F (5′-GAA ACT GCG AAT GGC TC-3′) and ITS-R (5′-TAC TGA TAT GCT TAA GTT CAG CGG-3′) were used for PCR amplifications of the 18S rRNA gene and ITS1-5.8S-ITS2region (Gao et al. 2012;Jerome et al. 1996).To minimize the possibility of PCR amplification errors, Q5 ® Hot Start High-Fidelity 2 × Master Mix DNA Polymerase (New England BioLabs, USA) was used (Li et al. 2023).The thermal cycler program used was that described by Li et al. (2022).The quality of the amplified DNA was checked by 1% agarose gel electrophoresis.PCR products were purified using the EasyPure R Quick Gel Extraction Kit (TransGen Biotech Co., Ltd., Beijing, China), and then sequenced on an ABI-PRISM 3730 automatic sequencer (Applied Biosystems, Tsingke Biological Technology Company, Qingdao, China).

Phylogenetic analyses
The 18S rRNA gene sequences of Myxophyllum weishanense sp.nov., Conchophthirus lamellidens, and Conchophthirus paracurtus sp.nov.were aligned with 132 other sequences downloaded from GenBank for phylogenetic analyses (for all taxa and accession numbers, see Supplementary Table S1).Four colpodeans, i.e., Colpoda lucida, Colpoda magna, Maryna umbrellata, and Platyophrya bromelicola, were selected as the outgroup.All sequences were aligned with the MAFFT ver.7 server (https:// mafft.cbrc.jp/ align ment/ server/) (Katoh et al. 2019), using the iterative refinement E-INS-i method, the 200PAM/k = 2 scoring matrix, and the gap opening penalty at 1.53.Primer sequences were removed but otherwise the 18S rRNA gene sequence alignment was not masked.The final alignment, comprising 1762 positions, was used for the phylogenetic analyses.The number of unmatched nucleotides, and the identity of the three newly submitted sequences with their most closely sequences, were calculated according to Li et al. (2021).The number of unmatched nucleotides and the pairwise p-distances of five Conchophthirus species were calculated with the program BioEdit ver.7.0 (Hall 1999), using the sequence difference count matrix and sequence identity matrix options.
Maximum likelihood (ML) analysis was performed with 1000 bootstrap replicates to estimate the reliability of the internal branches using the program IQTREE ver.1.6.10 (Nguyen et al. 2015) on the IQ-TREE web server (https:// iqtree.cibiv.univie.ac.at/) (Trifinopoulos et al. 2016).Bayesian inference (BI) was performed using MrBayes v3.2.7 (Ronquist et al. 2012) with the best-fit model GTR + I + G, selected using the IQ-TREE web server.Altogether 5,000,000 generations with a sampling frequency of 100 and a relative burn-in fraction of 25% of sampled trees were generated.All the remaining trees were used to calculate posterior probabilities (PP) using a 50% majority rule consensus.The other parameters were used at default settings.Convergence of the Markov Chain Monte Carlo (MCMC) analyses was assessed as follows: after 5,000,000 generations, the average standard deviation of split frequencies was < 0.01, the potential scale reduction factor approached 1, effective sample sizes were greater than 200, and no obvious trends were recognizable in the plots of generations versus log probability.All trees were computed as unrooted and were rooted in FigTree v.1.2.3 (https:// tree.bio.ed.ac.uk/ softw are/ figtr ee/), using out-group taxa as specified above.
The interpretation of the bootstrap values follows Vďačný and Rajter (2015), that is, values < 70% were considered as low, from 70 to 94% as moderate, and ≥ 95% as high (Hillis and Bull 1993).Following Alfaro et al. (2003), Bayesian posterior probability < 0.95 was considered as low and ≥ 0.95 as high.

Prediction of ITS2 secondary and tertiary structure
The boundaries of the ITS2 region were determined by constructing the secondary structure of ITS-5.8SrRNA region using R2DT (RNA 2D Templates) web server (https:// rnace ntral.org/ r2dt) (Sweeney et al. 2021) and searching for the highly characteristic 5.8S-28S rRNA proximal stem.Formation of the hybridized 5.8S-28S rRNA proximal stem was forced and all other parameters were left at default settings.Homologies of the secondary structures of the predicted thermodynamically optimal ITS2 region were compared with consensus ITS2 secondary structures of the most closely related oligohymenophorean ciliates (Miao et al. 2008;Zhang and Vďačný 2021).The putative models were then prepared in VARNA v.3.93 (Darty et al. 2009).The consensus structure of the ITS2 region for five Conchophthirus species was also calculated in 4SALE (Seibel et al. 2006).

Etymology
The species-group name weishanense refers to the area (Weishan, China) where the sample was collected.

Deposition of type slide
The protargol-stained slide containing the holotype specimen marked with a red ink circle (Figs.2B, C, 3H, I) and several paratype specimens marked with black ink circles (registration number: LT2021061801-1) is deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China.

Gene sequence
The 18S rRNA gene sequence of Myxophyllum weishanense sp.nov. is deposited in GenBank with the accession number OR042378.The length and G + C content of the sequence are 1670 bp and 45.21%, respectively.The ITS1-5.8S rRNA-ITS2 region sequence is deposited in GenBank with accession number OR148434.The length and G + C content are 477 bp and 35.22%, respectively.
Swimming motion of isolated cells slow, rotating about long axis, or by gliding slowly with dorsal side attached to substrate.

Remarks
This species was established by Ghosh (1918), and then Kahl (1931) redrew it based on Ghosh's figure.Uyemura (1935) rediscovered it in Japan and briefly described the Japanese population.Raabe (1971) collated the historical description of C. lamellidens based on the available in vivo data.Hence, to date, this organism has never been studied in detail regarding its infraciliature and its taxonomic information remains incomplete.Here, we supply an improved diagnosis based on morphologic data from the Chinese population and previously reported populations.Due to the original description of C. lamellidens lacking information about holotype, and no neotype or lectotype designated in previous studies, all the specimens of the type series are automatically syntypes (ICZN 1999).According to the article 74.1 of ICZN (1999), we designate a Chinese specimen as the lectotype for C. lamellidens, and other specimens then become paralectotypes.

Improved diagnosis
Medium-sized Conchophthirus, about 80-110 × 35-65 μm in vivo; anterior end of cell conspicuously narrowed while posterior end is broadly rounded; single macronucleus located in body center with one or two adjacent micronuclei; single contractile vacuole positioned near left cell margin near equator; 62-74 somatic kineties, SK (somatic kinety) 1 to SK7-ninefold into mouth pocket; mouth pocket located near cell equator beneath a lobe-like protrusion of left cell margin; oral apparatus comprises a long, bracket-shaped paroral membrane and a transversely arranged group of three membranelles, each composed of three short rows; paroral membrane extending from caudal end of membranelle 3 to bottom of mouth pocket.

Deposition of type slide
The protargol-stained slide containing the lectotype specimen marked with a red ink circle (Figs.4E, F, 5I, J) and several paralectotype specimens marked with black ink circles (registration number: LT2021032501-1) is deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China.

Gene sequence
The 18S rRNA gene sequence of Conchophthirus lamellidens is deposited in GenBank with accession number OR042379.The length and G + C content of the sequence are 1641 bp and 42.84%, respectively.The ITS1-5.8S rRNA-ITS2 region sequence (accession number OR148435) has a length and G + C content of 542 bp and 37.27%, respectively.

Description
Body size about 80-100 × 45-65 μm in vivo (Figs.4A, 5A,  B).Body variable in shape, with upper half triangular and lower half broad to narrow semi-elliptical in right ventrolateral view, caudal end with a prominent ventral bulge posteriorly (Figs.4A, B, G, 5A, B, D).Length to width ratio about 1.5-2:1.Dorsoventrally compressed about 2:1.In lateral view, anterior end pointed and curved towards dorsal side, posterior end slightly broader, and mid-region of ventral surface concave (Figs.4B, 5D).Oral apparatus located in deep vestibulum on left ventral cell margin, its opening located beneath a lobe-like protrusion in mid-region of left margin (Figs.4A, E, 5A, B, F, I).No cortical granules or extrusomes observed.Cytoplasm colorless, grey particles about 2 μm in diameter usually concentrated in upper part of body giving cells grey-black color (Fig. 5B, C).Single contractile vacuole located to left of midline at cell equator, about 10-18 µm in diameter in diastole, contracts at about 50 s intervals (Figs.4A, B, G, 5A).Single spherical macronucleus about 20 μm in diameter, located right of midline at cell equator; single micronucleus about 2-3 μm in diameter, adjacent to macronucleus (Figs.4C, F, 5B, H).
Locomotion by gliding at moderate speed with dorsal side attached to substrate or by swimming at moderate speed while rotating about long body axis.

Etymology
Composite of the Greek prefix para-(beside) and the species-group name curtus, referring the similarity between the new species and Conchophthirus curtus Engelmann, 1862.

Deposition of type slide
The protargol-stained slide containing the holotype specimen marked with a red ink circle (Figs.6H, I, 7H, I) and several paratype specimens marked with black ink circles (registration number: LT2021112401-1) is deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China.

Gene sequence
The 18S rRNA gene sequence of Conchophthirus paracurtus sp.nov. is deposited in GenBank (accession number OR042380).The length and G + C content of the sequence are 1629 bp and 43.16%, respectively.The ITS1-5.8S rRNA-ITS2 region sequence (accession number OR148436) has a length and G + C content of 563 bp and 37.83%, respectively.
Locomotion usually by gliding at moderate speed with dorsal side attached to substrate, occasionally swims at moderate speed while rotating about long axis.

Phylogenetic analyses (Fig. 8)
ML and BI trees based on 18S rRNA gene sequences were constructed to determine the phylogenetic positions of Myxophyllum weishanense sp.nov., Conchophthirus lamellidens, and C. paracurtus sp.nov.Topologies of the ML and BI trees were nearly congruent, hence only the ML tree is shown with branch support from both methods (Fig. 8).Myxophyllum weishanense sp.nov.clusters with its congener M. steenstrupi (Stein, 1861) Raabe, 1934 and another (possibly misidentified) "thigmotrich" ciliate, namely Protophyra ovicola Kofoid, 1903, with full support within the scuticociliate order Pleuronematida.The sequence of "Protophyra ovicola" is not associated with published morphological information or a vouchered specimen, therefore its identification is questionable.In addition, the order Pleuronematida is a fully supported monophyletic group that is sister to the scuticociliate order Philasterida with high to moderate support (92% ML and 0.94 BI).The clade consisting of M. weishanense sp.nov.+ M. steenstrupi + so-called "Protophyra ovicola" is sister to a highly heterogenous clade encompassing "thigmotrich" ciliates isolated from mollusks and belonging to the genera Ancistrum Maupas, 1883 and Boveria Stevens, 1901, as well as the free-living cyclidiid species Protocyclidium citrullus (Cohn, 1866) Foissner et al., 2002, Cyclidium glaucoma Müller, 1773, C. marinum Borror, 1963, C. varibonneti Song, 2000, and C. vorax Pan et al., 2020, with

Comparisons of the 18S rRNA gene and ITS1-5.8S-ITS2 region sequences (Fig. 9; Table 2)
A BLAST (Basic Local Alignment Search Tool) search of the three newly obtained 18S rRNA gene sequences against the nucleotide NCBI database was carried out to find the most closely related species.The BLASTn algorithm revealed that Myxophyllum weishanense sp.nov.(OR042378) is most closely related to its congener M. steenstrupi (MT649635-MT649640) and so-called "Protophyra ovicola" (JQ956552).The Weishan population of Conchophthirus lamellidens is most closely related to a Qingdao population of C. lamellidens (JQ956542) for which no published morphologic information exists.The newly obtained sequence of Conchophthirus paracurtus sp.nov. is most closely related to two of its congeners, namely C. cucumis (JQ956543) and C. curtus (OR127229).The sequence of M. weishanense sp.nov.(OR042378) differs from M. steenstrupi at seven nucleotide positions and from so-called "Protophyra ovicola" at five nucleotide positions (Fig. 9C).The newly obtained sequence of C. lamellidens (OR042379) differs from the Qingdao population C. lamellidens (JQ956542) at only one nucleotide position and from seven other closely related species at four to 11 nucleotide positions (Fig. 9D).2).

Putative internal transcribed spacer 2 secondary structure (Figs. 10, 11)
The boundaries of the ITS2 region were determined by searching for the 5.8S-28S rRNA hybridization stem with R2DT (Sweeney et al. 2021).There were 15 nucleotide pairs in the hybridized 5.8S-28S rRNA stem.However,   Zhang and Vďačný (2021) utilized the putative ITS2 secondary structures of most closely related oligohymenophorean ciliates (Gao et al. 2013;Miao et al. 2008) and used only five pairs to constrain the 5.8S-28S rRNA imperfect helix, which led to an incorrect determination of the 3′-end of the ITS2 region.Here, we considered 15 nucleotide pairs in the 5.8S-28S rRNA hybridization stem as described in Zhang and Vďačný (2024).The length of the ITS2 region is 155 nucleotides in M. weishanense sp. to helices I-III (Fig. 10A, B).The common loop of the Myxophyllum ITS2 region consists of 24 nucleotides.Helix I is highly conserved and exhibits a 5′-GUU versus AAC-3′ motif at its stem and a terminal loop of seven nucleotides.Helix II is 20 nucleotides long and exhibits a 5′-GUGG versus CCAU-3′ motif at its stem, a single pyrimidine-pyrimidine bulge consisting of 5′-UU versus UC-3′ (brown blocks in Fig. 10A, B), and a terminal loop of four nucleotides.Helix III is the longest, having as many as 98 nucleotides in M. weishanense sp.nov.and 102 nucleotides in M. steenstrupi.Helix III of M. weishanense sp.nov. is almost identical to that of M. steenstrupi, except for four unpaired nucleotides in M. weishanense sp.nov.
(arrowheads in Fig. 10A).10C-G).Helix I is the shortest and most highly conserved, and exhibits a 5′-UUA versus UAA-3′ motif at its stem and a terminal loop of nine nucleotides, in five Conchophthirus species (Fig. 10C-G

Comparison of Myxophyllum weishanense sp. nov. with congeners (Table 3)
Myxophyllum, which was established as a monotypic genus (type species M. steenstrupi), lives mostly in the mantle cavity of terrestrial pulmonate gastropods and has been reported from a variety of hosts from Europe and the USA (de Puytorac et al. 1992;Kazubski 1964Kazubski , 1973;;Penn 1958;Raabe 1971).According to a recent redescription (Zhang and Vďačný 2021), M. steenstrupi has the following features: (1) globular to ovoid body; (2) multiple macronuclear nodules grouped in the center of the cell; (3) a contractile vacuole in the mid-region of the body near the group of macronuclei; (4) densely-spaced somatic cilia; (5) oral cavity situated in the left-posterior quarter of the cell; (6) oral apparatus comprising one paroral membrane and one adoral membranelle, both sunken in the buccal cavity.
In previous studies, all Myxophyllum isolates have a similar morphology but lack molecular data, and have been identified as M. steenstrupi, although some differences are recognizable among such "populations" (e.g., numbers of somatic kineties and macronuclear nodules).In their redescription and review of M. steenstrupi, based on populations from Slovakian hosts, Zhang and Vďačný (2021) provided the first 18S rRNA gene sequence for this species.This enabled our new species to be compared with M. steenstrupi, mainly represented by the Slovak population.
The new species was isolated from a terrestrial snail, Acusta ravida, collected from the Weishan Wetland, China.It differs from the Slovak population of M. steenstrupi in two features: (1) fewer somatic kineties (55-61 vs. 87-97 on the ventral side and 56-62 vs. 76-89 on the dorsal side); (2) caudal cilia (conspicuous vs. absent/not mentioned in M. steenstrupi).In terms of molecular data, comparison of the 18S rRNA gene sequences reveals seven unmatched nucleotide positions, which supports the distinctness of the two taxa and supports the validity of Myxophyllum weishanense sp.nov.as a new species.

Remarks on the genus Conchophthirus
Although the 18S rRNA gene sequence of C. paracurtus sp.nov.(OR042380) differs from that of C. curtus (OR127229) at only one nucleotide position, its ITS1-5.8S-ITS2region sequence differs substantially from that of C. curtus, i.e., the genetic divergence is 15.6% (85 unmatched nucleotide positions), and the secondary structure of the ITS2 region for C. paracurtus sp.nov. is distinctly different from that of C. curtus (e.g., having the extra subhelix III-3 in Helix III as shown in Fig. 11C).Taken together, the morphologic and genetic differences support the validity of C. paracurtus as a new species.

Identification of Conchophthirus lamellidens Ghosh, 1918 and comparison with related species (Table 5)
In the original description of C. lamellidens (Gosh 1918), the mouth pocket is located on the right margin of the ventral surface, opposite to not only other Conchophthirus taxa but to almost all 'typical' thigmotrichs in which the mouth pocket is on the left margin of the ventral surface (Kahl 1931).Thus it is likely that Gosh (1918) drew a cell oriented "ventral side down" (i.e., with the dorsal surface toward the viewer).On the other hand, Uyemura (1935) rediscovered C. lamellidens in Japan and briefly redescribed the population based on some in vivo data, i.e., body size and shape, the position of the nuclear apparatus, contractile vacuoles, the entrance to the buccal cavity and host locality (Raabe 1971).The following characteristics of the Lake Weishan population of C. lamellidens overlap with that described by Uyemura (1935): body size (80-100 × 45-65 μm vs. 90-108 × 35-54 μm), number of vestibular kineties (7-9 vs. about 7), body shape, position of the nuclear apparatus, position of the contractile vacuole, and location and morphology of the buccal cavity.In addition, these two populations share similar hosts (Sinanodonta species) and host localities (China vs. Japan).Additionally, one published 18S rRNA gene sequence (JQ956542) marked with the name C. lamellidens was found in GenBank.It was collected in Qingdao, China, many years ago, but was not associated with morphological information.The 18S rRNA gene sequence of the Lake Weishan population of C. lamellidens differs at only one nucleotide position from the Qingdao population, so we consider these to be conspecific.For elucidating the relationships among Conchophthirus species, taxon sampling must be increased and a broader range of marker genes should be analysed.

Phylogenetic positions of Myxophyllum and Conchophthirus (Figs. 8, 9A, B)
The genus Myxophyllum was first described by Stein (1861).The only species of Myxophyllum, M. steenstrupi, originally belonged to Conchophthirus.Owing to significant differences in the oral ciliature, however, Raabe (1934) established the genus Myxophyllum with Conchophthirus steenstrupi as type species.Raabe (1971) assigned Myxophyllum to the family Thigmophryidae, order Thigmotrichida.The family was placed in the order Philasterida in the subclass Scuticociliatia, because of its short paroral membrane (Lynn and Small 2002;Small and Lynn 1985).Lynn (2008) went on to classify Myxophyllum in the family Thigmophryidae, order Philasterida, subclass Scuticociliatia.Zhang and Vďačný (2021) first analysed the phylogenetic position of Myxophyllum based on the molecular data of the 18S, 5.8S, 16S, and 28S regions of the ribosomal RNA gene and the mitochondrial cytochrome c oxidase I gene.Phylogenetic analysis strongly rejected the inclusion of Myxophyllum in the order Philasterida, and fully supported Myxophyllum as belonging to the order Pleuronematida.Furthermore, Zhang andVďačný (2021, 2023b) proposed to transfer all members of the order Thigmotrichida into the order Pleuronematida, and proposed possible reasons for the divergence of Myxophyllum from typical pleuronematids, i.e., that the symbiotic lifestyle of Myxophyllum is likely responsible for the dramatic remodelling of its oral apparatus and ciliature (e.g., the transfer of the mouth pocket to the posterior body region, the strong reduction of the paroral membrane and adoral organelles, and the formation of vestibular kineties).The general topology of the 18S rRNA gene phylogenetic tree in the present study is consistent with previously published phylogenetic studies (Antipa et al. 2016(Antipa et al. , 2020;;Gao et al. 2013;Poláková et al. 2021;Rataj andVďačný 2018, 2022;Zhang and Vďačný 2024), and the phylogenetic position of Myxophyllum is consistent with Zhang and Vďačný (2021), that is Myxophyllum belongs to the family Thigmophryidae, in the order Pleuronematida.
The present 18S rRNA gene phylogenetic analysis shows that all Conchophthirus sequences cluster together as a monophyletic group with full statistical support and are nested deep within the genus Dexiotricha with very strong support (99% ML, 1.00 BI), which is consistent with the previous studies (Antipa et al. 2020;Poláková et al. 2021;Zhang and Vďačný 2024).Within this group, the Conchophthirus clade clusters with the D. colpidiopsis (MG819725) + Dexiotricha sp.1 (MN704273) clade.Dexiotricha colpidiopsis (MG819725) was reported by Qu et al. (2019) and its ciliature has a typical Dexiotricha-pattern, which is distinctly different from that of Conchophthirus.However, the longitudinal cortical ridge microtubule of C. curtus is in the identical location as that of D. colpidiopsis (Antipa 1971(Antipa , 2014;;Peck 1977), which might be regarded as an ultrastructural feature that is common to both clades, thus explaining their close relationship.Other Dexiotricha species that are not clustered with D. colpidiopsis might have a different ultrastructure, therefore further studies are needed to provide insights into the polyphyly of Dexiotricha.
Examination of the ten valid Conchophthirus species reveals different correlations between individual species and their host organisms.Some species possibly have "universal occurrence" in unionids and these species also show changes in host preferences according to the geographical region.For example, the most commonly studied species, C. curtus, is cosmopolitan and inhabits Anodonta cygnea and Unio species in Europe, Sinanodonta lauta in Japan, and various host species belonging to 13 genera in the USA (Supplementary Table S2).All of these host species belong to the family Unionidae Rafinesque, 1820.Moreover, C. anodontae and C. unionis share the same host species as C. curtus and may coexist in a single host, although their abundance may differ in various locations in the mantle cavity (Antipa et al. 2020;Fenchel 1965;Kidder 1934).Conchophthirus magna has only been reported once in the USA and was found to coexist with C. anodontae in a single host (about 10-20 individuals in one host specimen) (Kidder 1934).On the other hand, C. acuminatus, C. discophorus, and C. klimentinus appear to show a higher degree of host specificity.Conchophthirus acuminatus and C. klimentinus have so far been reported only from Dreissena polymorpha of the family Dreissenidae, and C. discophorus only from members of the family Sphaeriidae.Three Conchophthirus species, namely, C. cucumis, C. elongatus, and C. lamellidens from India and Japan, were incompletely described due to the lack of silver stained specimens, hence taxon sampling must be increased and detailed morphological data based on the modern methods, together with molecular data from additional marker genes, are required to verify the reliability of identifications.
The findings of the present study reveals a broader host range for Conchophthirus than had previously been documented, including one new species C. paracurtus sp.nov.from the Chinese pond mussel Sinanodonta woodiana (Lea, 1834) and C. lamellidens from the pearl mussel Cristaria plicata (Leach, 1815).It is generally believed that the majority of endocommensal ciliates are not restricted to a single host species, which also appears to be the case for most Conchophthirus species.Therefore, a broader range of potential host organisms needs to be investigated.

Fig. 1
Fig. 1 Sampling site and host mollusks.A The landscape of the sampling site, and partial map of China.Red dot marks the location of the Lake Weishan Wetland.B Snail Acusta ravida, host of Myxophyllum

Fig. 2
Fig. 2 Myxophyllum weishanense sp.nov. in vivo (A, D, E), after protargol (B, C, F, G) and silver nitrate (H) staining.A Ventral view of a representative cell, arrow marks the caudal cilia.B, C Ventral (B) and dorsal (C) views, to show the ciliature and location of the nuclear apparatus.Blue arrows denote the anterior suture, green arrows mark the right lateral suture, red arrows show the left lateral suture, orange arrows mark the postoral (posterior) suture, red arrowhead denotes the micronucleus.D Lateral view, arrow shows the contractile vacuole.E Individuals with different body shapes.F

Fig. 3
Fig. 3 Myxophyllum weishanense sp.nov. in vivo (A-C, E, G), after protargol (D, H-L) and silver nitrate (F) staining.A, B Ventral view of two representative individuals, arrow in A shows the contractile vacuole, arrowheads show the opening of the mouth pocket; arrow in B shows caudal cilia.C Left-lateral view.D Oral apparatus.E Ventral-dorsal border of the anterior body region, arrows show the thigmotactic cilia, arrowheads show the somatic cilia.F Reticulate silverline system.G Arrows show the caudal cilia.H, I Ventral

Fig. 4
Fig. 4 Conchophthirus lamellidens in vivo (A, B, G), after protargol (C-F) and silver nitrate (H, I) staining.A Right ventrolateral view of a representative cell.B Right-lateral view, arrow shows the bulge at posterior body end, arrowhead shows the contractile vacuole.C Nuclear apparatus, arrow shows the spherical micronucleus.D Oral apparatus, the green lines represent the SK wrapped within the mouth pocket, and the purple lines represent the exposed somatic kineties.E, F Ventral (E) and dorsal (F) views, to show the ciliature and location of the nuclear apparatus.Blue arrows denote the anterior suture,

Fig. 5
Fig. 5 Conchophthirus lamellidens in vivo (A-F), after protargol (G-J, L) and silver nitrate (K, M, N) staining.A Right ventrolateral view of a slightly compressed cell, arrow shows the contractile vacuole, arrowhead shows the mouth pocket.B Right ventrolateral view of a representative cell.C Particles concentrated in the upper part of the body.D Lateral view of a representative cell, arrow shows the mouth pocket, arrowhead shows the bulge at posterior body end.E Cell surface, arrows show the ribs distributed along the somatic kineties.F Opening of the mouth pocket.G Thigmotactic field at the anterior

Fig. 6
Fig. 6 Conchophthirus paracurtus sp.nov. in vivo (A, C, G), after protargol (B, D, H, I) and silver nitrate (E, F) staining.A Right ventrolateral view of a representative cell, arrow shows the contractile vacuole.B Nuclear apparatus, arrow shows the micronucleus.C Lateral view, arrow marks the contractile vacuole.D Oral apparatus, the blue lines represent the somatic kineties wrapped within the mouth pocket, and the orange lines represent the exposed somatic kineties.E, F Silverline system for the posterior (E) and anterior (F) parts of the body.G Variation of body shapes.H, I Right ventrolateral (H)

Fig. 7
Fig. 7 Conchophthirus paracurtus sp.nov. in vivo (A-G), after protargol (H, I, L-N) and silver nitrate (J, K) staining.A, B Right ventrolateral view of representative cells, arrow in A shows contractile vacuole, arrowhead in A shows the opening of the mouth pocket.C Particles concentrated in the body.D Lateral view of a representative cell.E Anterior dorsal side of the cell, arrows show the thigmotactic cilia, arrowheads show the somatic cilia.F Arrow shows the opening of the mouth pocket.G Arrows show the transversely arranged caudal cilia, arrowhead shows the nonciliated area.H, I Right ventro-

Fig. 8
Fig. 8 Maximum likelihood (ML) tree inferred from 18S rRNA gene sequences, showing the positions of the newly submitted sequences (in red color).Numbers near branches denote bootstrap values (ML)/ Bayesian posterior probabilities (BI).Fully supported (100%/1.00)branches are marked with red solid circles.Black dashes indicate the disagreement between ML and BI trees.Sequences with doubtful

Fig. 9
Fig. 9 A, B Relationships among families within Pleuronematida (A) and Loxocephalida (B), respectively.C Myxophyllum weishanense sp.nov.OR042378 with related sequences, triangular marker indicates that this sequence may be a misidentification.D Conchophthirus paracurtus sp.nov.OR042380 and Conchophthirus lamellidens nov., 224 nucleotides in C. paracurtus sp.nov., and 195 nucleotides in C. lamellidens.The predicted secondary structures of the ITS2 region are shown in Figs. 10, 11.In the genus Myxophyllum, ITS2 has one central loop model and three helices of unequal length, corresponding

Fig. 11
Fig. 11 Putative secondary structure models of ITS2 region for the genus Conchophthirus.A Consensus secondary structure showing a central loop with four helices corresponding to helices I, II, III and IV.Note that helix II has a pyrimidine-pyrimidine mismatch (yel-

Table 1
Morphometrical characterization ofMyxophyllum weishanense sp.nov.(Mwe, the 1st lines), Conchophthirus lamellidens (Cla, the 2nd lines) and Conchophthirus paracurtus sp.nov.(Cpa, the 3rd lines)CV coefficient of variation in %, Max maximum, Mean arithmetic mean, Min minimum, n number of specimens investigated, SD standard deviation, SE standard errorCharacterSpecies Min Max Mean Median SD SE CV n ventral kineties at anterior suture on left cell margin (Figs.2B, C, 3H).Dorsal kineties similar to that on ventral side, anterior half of each kinety obliquely oriented, posterior half vertical; left half of dorsal somatic kineties conspicuously curved at posterior ends, extending onto ventral side and into buccal cavity, terminating along rightmost end of buccal cavity (Fig.2C, G).All ventral kineties and posterior dorsal kineties consist of monokinetids.Anterodorsal dikinetids forming an irregular triangular thigmotactic field, recessed upwards at base, about two-fifths of body length, thigmotactic cilia about 3 µm long in vivo (Figs.2B, C, G, 3E, H-J).Conspicuous suture running around body along ventral-dorsal border, commencing at left end of mouth pocket and terminating at right end of mouth pocket (Figs.2B, C, G, 3H, I).Upper left suture broad, forming a nonciliated area (Figs.2B, C, 3H, I).About 20-30 caudal cilia directed slightly to right, each about 15-20 µm long (Figs. 2A, 3B, G).Silverline system reticulate, silverlines extending to somatic kineties, with short, loose and irregular transverse silverlines connecting adjacent somatic kineties, forming closed rectangular grids(Figs.2H full statistical support.The sequences obtained from Conchophthirus lamellidens and C. paracurtus sp.nov.cluster with those from other Conchophthirus species and together form a fully supported clade confirming the monophyly of the genus Conchophthirus.The Conchophthirus clade is placed outside the "core" scuticociliate clade (which consists of the orders Pleuronematida and Philasterida) and nests within the heterogenous cluster of loxocephalid Dexiotricha spp.and the mouthless Haptophrya spp., with full support.Within the Conchophthirus + Dexiotricha + Haptophrya clade, Conchophthirus first group with Dexiotricha sp. 1 and D. colpidiopsis with full statistical support.The Conchophthirus + Dexiotricha clade is sister to the genus Haptophrya with weak support (82% ML, 0.79 BI).Finally, the Conchophthirus + Dexiotricha + Haptophrya clade together groups with Dexiotricha sp. 2, D. elliptica, and Dexiotricha cf.granulosa with full statistical support.The separation of Dexiotricha taxa among different clades indicates that the genus Dexiotricha is paraphyletic.

Table 2
Numbers of unmatched nucleotides (above diagonal) and pairwise p-distances (below diagonal) of ITS1-5.8S-ITS2sequences among members of the genus Conchophthirus

Comparison of Conchophthirus paracurtus sp. nov. with its most morphologically similar congeners (Table 4 )
Based on its body shape and general morphology, Conchophthirus paracurtus sp.nov.should be compared with five congeners: C. curtus, C. unionis, C. klimentinus, C. discophorus, and C. magna.Morphologically, four of these, i.e., Conchophthirus unionis, C. klimentinus, C. discophorus, and C. magna can be easily distinguished from C. paracurtus sp.nov.by the absence of a glabrous right posterolateral area (vs.present in C. paracurtus), and body size (C.paracurtus is larger than all four)

Table 5
Comparison of Conchophthirus lamellidens with closely related congenersNo.number, NA not available, SK somatic kineties