The Morphology, Taxonomy, and Phylogenetic Analyses of Five Freshwater Colonial Peritrich Ciliates (Alveolata, Ciliophora), Including the Descriptions of Two New Species

The morphology and phylogeny of two new sessilid species, Zoothamnium weishanicum n. sp. and Epicarchesium sinense n. sp., two insufficiently known species, Zoothamnium arbuscula Ehrenberg, 1831 and Zoothamnium hentscheli Kahl, 1935, and a well-known species, Carchesium polypinum (Linnaeus, 1767) Ehrenberg, 1838, collected from freshwater habitats of China, were investigated. Zoothamnium weishanicum n. sp. is characterized by its inverted bell-shaped zooids, double-layered peristomial lip, alternately branched stalk, and two different-length rows in infundibular polykinety 3 (P3). Epicarchesium sinense n. sp. is recognized by its asymmetric-pyriform zooids, single-layered peristomial lip, conspicuous cortical blisters on the pellicle, dichotomously branched stalk, and P3 containing one short inner row and two long outer rows. Based on previous and newly obtained data of the three known species, improved diagnoses and redescriptions are provided including, for the first time, data on the infraciliature of Z. arbuscula and Z. hentscheli. In addition, we analyzed the phylogeny of each species based on SSU rDNA sequence data.

In the classification of Lynn (2008), Peritrichia is composed of two orders: Sessilida Kahl, 1933 andMobilida Kahl, 1933. The species of order Sessilida are either solitary or colonial and are commonly attached to a substrate via a stalk, a scopula, or a lorica (Lynn, 2008). Although investigations of sessilids have been carried out for more than 300 years, many species are poorly described since they are known only from in vivo observations and information on their infraciliature, silverline system, and molecular phylogeny is lacking (Kahl, 1935;Precht, 1935;Nenninger, 1948;Sommer, 1951;Stiller, 1971;Bernerth, 1982;Foissner et al., 1992). These insufficient descriptions make the species identification of many sessilids extremely difficult, thus highlighting the need for their reinvestigation based on modern methods (Warren et al., 2018). Furthermore, new species are continuously being reported, suggesting that there is a large undiscovered diversity of sessilids (Canals and Salvadó, 2016;Kühner et al., 2016;Wang et al., 2017;Zhou et al., 2019a,b;Lu et al., 2020;Wu et al., 2020Wu et al., , 2021. In the present study, five species representing three genera (Zoothamnium Bory de St. Vincent, 1824, Epicarchesium Jankowski, 1985, and Carchesium Ehrenberg, 1831 and two families (Zoothamniidae Sommer, 1951 andVorticellidae Ehrenberg, 1838) are investigated. Zoothamnium is characterized by its colonial habit, transverse silverline system, continuous spasmoneme, and the contraction of the stalk in a "zig-zag" fashion (Bory de St. Vincent, 1824;Corliss, 1979). It contains more than 140 nominal species, about two-thirds of which lack data on their silverline system and/or infraciliature (Ji et al., 2015;Schuster and Bright, 2016;Shen et al., 2017;Lu et al., 2020;Mayen-Estrada and Dias, 2021). Epicarchesium is characterized by its colonial habit, discontinuous spasmoneme, tuberculate pellicle, reticulate silverline system, and the contraction of the stalk in a spiral fashion (Jankowski, 1985;Leitner and Foissner, 1997). Carchesium is similar to Epicarchesium but has a transverse silverline system, and its pellicle is not tuberculate (Ehrenberg, 1831;Kahl, 1935;Shen and Gu, 2016). Compared with Zoothamnium, Epicarchesium and Carchesium are poorly studied in terms of their morphology and phylogenetics and, with the exception of one or two species, morphological information based on modern standards and accurately identified SSU rDNA sequences are lacking for both genera.
During faunal surveys of freshwater ciliates in two widely separated locations in Shandong Province, China, five colonial sessilid peritrichs representing these three genera were isolated, giving the opportunity to investigate them using modern methods. Here we provide detailed morphological information based on the observations of specimens in vivo and after silver staining. We also sequenced their small subunit ribosomal DNA (SSU rDNA) and analyzed their phylogenetic relationships.

Sample Collection
All the species were isolated in 2019 from freshwater habitats in either Weishan or Qingdao, Shandong Province, China ( Figure 1A), using glass microscope slides as artificial substrates. Briefly, the slides were fixed onto a frame that was immersed in water at a depth of 1-2 m for 7-10 days to allow colonization by ciliates (Small, 1973).

Investigation of Morphology
Colonies were removed from the slides using acupuncture needles and transferred with glass micropipettes. Live specimens were observed using differential interference contrast microscopy at magnifications of ×40 to ×1,000. The infraciliature was revealed by the protargol staining method (Wilbert, 1975;Ji and Wang, 2018). The silverline system was demonstrated using the "dry" silver nitrate method (Song and Wilbert, 1995;Foissner, 2014). Counts and measurements were performed at ×400-1,000 magnifications. Drawings of live organisms were performed based on actual observations and photomicrographs, while those of stained specimens were made with the help of a drawing device. The terminology is according to Warren (1986) and Foissner et al. (1992).

DNA Extraction, PCR Amplification, and Sequencing
For each species, five zooids were isolated and washed five times with distilled water to remove potential contamination. Genomic DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the instruction of the manufacturer. The SSU rDNA was amplified using the primers 82F (5 -GAA ACT GCG AAT GGC TC -3 ) (Jerome et al., 1996) and 18SR (5 -TGA TCC TTC TGC AGG TTC ACC TAC-3 ) (Medlin et al., 1988). Q5 R Hot Start High-Fidelity DNA Polymerase (NEB, Ipswich, MA) was used to minimize the possibility of PCR amplification errors. The PCR programs were designed according to Bai et al. (2020). The PCR products were sequenced bidirectionally by Tsingke Biological Technology Company (Qingdao, China).

Phylogenetic Analyses
The five newly obtained SSU rDNA sequences and 54 sequences of other peritrichs downloaded from GenBank (accession numbers are shown in Figure 12) were used for phylogenetic analyses. Four hymenostomatians (Glaucoma chattoni X56533, Ichthyophthirius multifiliis U17354, Tetrahymena corlissi U17356, and Tetrahymena pyriformis EF070254) were selected as outgroup taxa. All the SSU rDNA sequences were aligned using the GUIDANCE2 algorithm 1 with default parameters (Landan and Graur, 2008;Sela et al., 2015). The two ends of the resulting alignment were trimmed manually in BioEdit v.7.0 (Hall, 1999). The final length of the alignment was 2,287 bp.

Improved Diagnosis
The colony was up to 3,500 µm high. Accessory branches radiate from the main stalk, forming an inverted dome-like outline, with micro-and macrozooids. The microzooids were inverted bell-shaped, 40-80 × 30-65 µm in vivo. The macrozooids were nearly globular, up to 150 µm in diameter. The peristomial lip was single-layered and strongly everted. One contractile vacuole was dorsally located, at the same level as the peristomial lip. The macronucleus is typically C-shaped and transversely oriented. The infundibular polykinety 3 (P3) consists of three equal-length rows, terminating adstomally above infundibular polykinety 1 (P1). Transverse silverlines numbered about 75 from the peristome to the trochal band and about 50 from trochal band to scopula. Freshwater is the habitat.
The cytoplasm was colorless, usually containing numerous vacuoles with yellow and/or green contents, possibly the remains of ingested algae. A single contractile vacuole was located at the dorsal wall of the infundibulum, about the same level as the peristomial lip (Figures 2A,B, 3F,G). The macronucleus of most microzooids was typically C-shaped and transversely oriented (Figures 2A, 3M,N), the macronucleus of microzooids at the end of the branches varied in shape (Figures 2B,F, 3G), and the macronucleus of macrozooids was usually C-shaped ( Figure 3J). A micronucleus was not observed.
The colony was up to 3,500 µm tall, usually containing more than 100 zooids and with accessory branches that radiate from the apical end of the main stalk forming an inverted dome-like outline (Figures 2C,G, 3A,B,H). The main stalk consists of three parts: a basal part without spasmoneme, about 25 µm across; a middle part with a central bundle of transparent fibrils, about 40 µm across; and an upper part with sturdy spasmoneme, about 60 µm in diameter ( Figure 2G). The spasmoneme was covered by a sheath with a rough surface, comprising bundles of fibrils (stalk myonemes) within a transparent membrane, which was about 40 µm across its widest point (Figures 2F, 3K).
The oral ciliature was of the usual type for sessilid peritrichs. Haplokinety and polykinety make approximately 1.25 circuits around the peristome before entering the infundibulum where they make a further circuit (Figures 2E, 3M-O). Three infundibular polykineties (P1-P3) were each composed of three rows of kinetosomes ( Figures 2E, 3M-Q). P1 is continuous with polykinety and terminates adstomally below P2 and P3, with P2 about twice the length of P3 and terminating adstomally at the convergence of P1 and P3 (Figures 2E, 3M-Q). The rows of P1 were nearly equal in length (Figures 2E, 3O,P). The inner two rows of P2 converge abstomally with P1, and the outer row of P2 separated abstomally from the inner two rows (Figures 2E, 3P,S). P3 consists of three almost-equallength rows of kinetosomes, terminating adstomally above P1 CV, coefficient of variation in %; Max, maximum; Min, minimum; Mean, arithmetic mean; n, number of specimens investigated; SD, standard deviation; -, data not available. a Rough data. b Data based on "dry" silver nitrate-stained specimens. All data are based on the populations investigated in the present study. ( Figures 2E, 3N-Q). There were two epistomial membranes (EM1 and EM2): EM1, long, was located at the entrance of the infundibulum (Figures 2E, 3O,R), while EM2 was located in front of the distal ends of haplokinety and polykinety ( Figures 2E, 3M). The germinal kinety lies parallel to haplokinety in the upper half of the infundibulum (Figures 2E, 3O). The trochal band consists of dikinetids, located about two-thirds down the length of zooid (Figures 2D, 3M,N).
The silverline system consists of closely spaced transverse silverlines, numbering about 75 (N = 3) from the peristome to  (1884). b Inferred from "the length being nearly twice the breadth" in Grenfell (1884). c 1/11 inch in Grenfell (1884). Zoothamnium hentscheli was first described by Hentschel (1916) without a species name (Zoothamnium spec. a). Kahl (1935) named it Zoothamnium hentscheli. To date, this species has been reported several times; however, the details of its infraciliature remain unknown, which necessitates a reinvestigation (Hentschel, 1916;Kahl, 1935;Hammann, 1952;Xu, 1988). We collected a population of this species from Weishan and made a detailed redescription. An improved diagnosis based on previous and present data is also supplied.

Improved Diagnosis
The colony was up to 1,500 µm high. The stalk was alternately branched. The zooids were inverted bell-shaped, about 50-85 × 30-45 µm in vivo, and often densely covered with detritus. The peristomial lip was single-layered and moderately everted. The peristomial disc was slightly elevated. A contractile vacuole was dorsally located at the same level as the peristomial lip. The macronucleus was C-shaped and transversely oriented. The infundibular polykinety 3 (P3) consists of three approximately equal-length rows and terminates adstomally above infundibular polykinety 1 (P1). Transverse silverlines numbered about 65 from the peristome to the trochal band and about 30 from the trochal band to the scopula. Freshwater is the habitat.
The cytoplasm was colorless and contained several gray or colorless granules. A single contractile vacuole was located at the dorsal wall of the infundibulum at the same level as the peristomial lip (Figures 4A,B, 5F-H). The macronucleus was C-shaped and transversely oriented (Figures 4A,B, 5L,M). The micronucleus not observed.
The colony was up to 2,500 µm tall. The stalk alternately branched. The branches progressively narrowed and shortened from the main stalk to the terminal branches (Figures 4D,E, 5A,B). The spasmoneme was with numerous mitochondria ( Figure 5J).
The oral ciliature was of the usual type for sessilid peritrichs. Haplokinety and polykinety make approximately 1.25 circuits around the peristome before entering the infundibulum (Figures 4G, 5L,M). The infundibular polykineties (P1-P3) were of three rows (Figures 4G, 5L-O,S). The rows of P1 were nearly equal in length (Figures 4G, 5L,N,O,S). The adstomal end of P2 terminates at the convergence of P1 and P3 (Figures 4G, 5L-O,S). The abstomal end of the inner row of P2 converges with P1; the abstomal end of the outer row of P2 was detached from the inner two rows (Figures 4G, 5P). P3 terminates adstomally above P1, with the rows equal in length (Figures 4G, 5L-O,S). There were two epistomial membranes (EM1 and EM2): EM1, located at the entrance of the infundibulum (Figures 4G, 5Q), and EM2, located near the distal ends of haplokinety and polykinety (Figures 4G, 5R). Germinal kinety lies parallel to haplokinety in the upper half of the infundibulum (Figures 4G, 5L,M). The trochal band consists of dikinetids, located about two-thirds down the length of zooid (Figures 5L,M).
The silverline system consists of closely spaced transverse silverlines, numbering about 65 (N = 1) from the peristome to the trochal band and about 30 (N = 1) from the trochal band to the scopula (Figures 4F, 5K).

Diagnosis
The colony was up to 1,400 µm high. The stalk was alternately branched. The zooids were inverted bell-shaped, usually 55-90 × 30-45 µm in vivo. The peristomial lip was double-layered and strongly everted. The peristomial disc was moderately elevated. A single contractile vacuole was dorsally located, at the same level as the peristomial lip. The macronucleus was C-shaped and transversely oriented. The infundibular polykinety 3 (P3) consists of two different-length rows of kinetosomes and terminates adstomally above infundibular polykinety 1 (P1). There were transverse silverlines numbering about 55 from the peristome to the trochal band and about 33 from the trochal band to the scopula.

Deposition of Slides
One protargol slide (registration number: WT2019061401-01) with the holotype specimen circled in ink, a second protargol slide with paratype specimens (registration number: WT2019061401-02) and one "dry" silver nitrate slide with paratype specimens (registration number: WT2019061401-03), were deposited in the Laboratory of Protozoology, Ocean University of China (OUC), Qingdao, China.

Etymology
The species-group name "weishanicum" refers to the area (Weishan) where the sample was collected.

Description
The zooids were inverted bell-shaped, about 55-90 × 30-45 µm in vivo (Figures 6A,B, 7C-H). The peristomial lip was about 30-50 µm in diameter, double-layered, and strongly everted (Figures 6A,B, 7C-E). The peristomial disc convex was clearly elevated above the peristomial lip in fully extended zooids (Figures 6A,B, 7C-H). The pellicular striations were extremely fine ( Figure 7J).   The cytoplasm was colorless, usually containing numerous vacuoles with yellow and/or green contents, possibly the remains of ingested algae. A contractile vacuole was situated at the dorsal wall of the infundibulum, at the same level as the peristomial lip (Figures 6A,B, 7G,H). The macronucleus was C-shaped and transversely oriented (Figures 6A,B, 7L,M). The micronucleus was located within the curvature of the macronucleus (Figures 6B, 7O).
The colony was up to 1,400 µm tall, usually with fewer than 50 zooids. The stalk was alternately branched; the branches progressively narrowed and shortened from the main stalk to the terminal branches (Figures 6D, 7A). The stalk sheath was colorless, with inconspicuous longitudinal striations ( Figure 7I).
The oral ciliature was typical for sessilid peritrichs. Haplokinety and polykinety make approximately 1.5 circuits around the peristome and a further turn within the infundibulum (Figures 6E, 7L,M). P1 and P2 had three rows each; P3 had two rows (Figures 6E, 7L,M,P,Q). The three rows of P1 were nearly equal in length. P2 terminates adstomally at the convergence of P1 and P3 (Figures 6E, 7L,M,P,Q). The abstomal ends of the inner two rows in P2 converge with P1 and diverge from the outer row (Figures 6E, 7K). The inner row of P3 was longer than the outer row which terminates adstomally above the inner row (Figures 6E, 7P,Q). There were two epistomial membranes (EM1 and EM2): EM1 was located at the entrance of the infundibulum (Figures 6E, 7K); EM2 was located close to the distal ends of haplokinety and polykinety (Figures 6E, 7N). The germinal kinety runs parallel to haplokinety in the upper half of the infundibulum (Figures 6E, 7L,M). The trochal band consists of dikinetids, located about two-thirds down the length of zooid (Figures 7L,M).
The silverline system consists of closely spaced transverse silverlines, numbering about 55 (N = 3) from the peristome to the trochal band and about 33 (N = 3) from the trochal band to the scopula (Figures 6C, 7J).

Diagnosis
The colony was up to 750 µm tall. The stalk was dichotomously branched. The zooids were asymmetric pyriform, about 45-60 × 30-40 µm in vivo. The peristomial lip was singlelayered and everted. The peristomial disc was slightly elevated. A contractile vacuole was ventrally located below the level of the peristomial lip. The macronucleus was J-shaped. The infundibular polykinety 3 (P3) has three rows and terminates adstomally at the same level as infundibular polykinety 1 (P1). The inner row of P3 was about half the length of the other two rows. The reticulate silverlines system, with about 37 transverse silverlines from the peristome to the trochal band and about 23 from the trochal band to the scopula. Freshwater is the habitat.

Deposition of Slides
One protargol slide (registration number: WT2019102301-01) with the holotype specimen circled in ink, a second protargol slide with paratype specimens (registration number: WT2019102301-02), and one "dry" silver nitrate slide with paratype specimens (registration number: WT2019102301-03) were deposited in the Laboratory of Protozoology, Ocean University of China (OUC), Qingdao, China.

Etymology
The species-group name "sinense" refers to the country (China) where it was first isolated.
The cytoplasm was colorless, usually containing numerous vacuoles with yellow and/or green contents, possibly the remains of ingested algae. A single contractile vacuole was located at the ventral wall of the infundibulum below the level of the peristomial lip (Figures 8A,C, 9B,C). The macronucleus was J-shaped ( Figures 8A,C, 9K,L). A micronucleus was not observed.  The colony was up to 750 µm tall, usually with fewer than 20 zooids. The stalk was dichotomously branched. The spasmoneme is discontinuous and extends throughout the colony (Figures 8D, 9A).
The oral ciliature was genus-typical. Haplokinety and polykinety make approximately 1.25 circuits around the peristome before entering the infundibulum (Figures 8E, 9K,L). All three infundibular polykineties (P1-P3) were of three rows (Figures 8E, 9M,O). P2 terminates adstomally at the convergence of P1 and P3 (Figures 8E, 9K-M,O). P2 converges abstomally with P1 (Figures 8E, 9N). P3 terminates adstomally at the same level as P1; the inner row of P3 was about half the length of the other two rows, and the inner row terminates abstomally ahead of the other two rows (Figures 8E, 9K,M,O). Only one epistomial membrane was observed, located at the entrance of the infundibulum (Figures 8E, 9K). The germinal kinety lies parallel to haplokinety in the upper half of the infundibulum (Figures 8E, 9K,L). The trochal band consists of dikinetids, located about two-thirds of the way down the length of zooid (Figures 9K,L).
The silverline system consists of reticulate silverlines, with about 37 (N = 1) transverse silverlines between the peristome and the trochal band and 23 (N = 1) between the trochal band and the scopula ( Figure 9P).

Improved Diagnosis
The colony was up to 2,500 µm high. The stalk was dichotomously branched. The zooids were inverted bell-shaped, 35-140 × 35-70 µm in vivo. The peristomial lip was singlelayered and moderately everted. One contractile vacuole was located at the ventral wall of the infundibulum below the level of the peristomial lip. The macronucleus was J-shaped. Infundibular polykinety 3 (P3) terminates adstomally at the same level as P1; the inner row of P3 was about half the length of the other two rows. There were transverse silverlines numbering 70-80 from the peristome to the trochal band and about 30-50 from the trochal band to the scopula. Freshwater is the habitat.
The cytoplasm was colorless, usually containing several gray or colorless granules. A single contractile vacuole was ventrally located below the level of the peristomial lip (Figures 10A,B, 11C). The macronucleus was J-shaped ( Figures 10A,B, 11H,I). A micronucleus was not observed. The colony was up to 2,500 µm tall. The stalk was dichotomously branched, and the spasmoneme was discontinuous, extending throughout colony (Figures 10E, 11A).
The oral ciliature was genus-typical. Haplokinety and polykinety make approximately 1.25 circuits around the peristome before entering the infundibulum (Figures 10D, 11J,H,I). The infundibular polykineties (P1-P3) had three rows (Figures 10D, 11J-K). The three rows of P1 were nearly equal in length (Figures 10D, 11J-K). P2 terminates adstomally at the convergence of P1 and P3 (Figures 10D, 11K). P2 converges adstomally with P1 (Figures 10D, 11K). The inner row of P3 was about half the length of the other two rows. P3 terminates adstomally at the same level as P1 (Figures 10D,K). Only one epistomial membrane was observed, located at the entrance of the infundibulum (Figures 10D, 11J). Germinal kinety lies parallel to haplokinety in the upper half of the infundibulum (Figures 10D, 11H). The trochal band consists of dikinetids, located about three-quarters of the way down the zooid length (Figures 11H,I).
The silverline system consists of closely spaced transverse silverlines, numbering about 75 (N = 3) between the peristome and the trochal band and about 40 (N = 3) between the trochal band and the scopula (Figures 10C, 11E,M).
The phylogenetic trees based on SSU rDNA sequences using BI and ML methods have similar topologies; therefore, only the ML tree is shown here with support values from both algorithms (Figure 12). In the phylogenetic tree, the members of Zoothamniidae were grouped into three clades (clades I-III), resulting in the polyphyly of Zoothamniidae. Clade I clusters with one group of Epistylididae (ML 94% and BI 1.00), forming a clade that is sister to clade II (ML 97% and BI 1.00). Clade III is located outside the assemblage formed by clade I, clade II, and Epistylididae (ML 94% and BI 1.00). Zoothamnium arbuscula and Z. hentscheli nest within clade I. The new sequence of Z. arbuscula is sister to the previously reported sequence (KU363261), while the Weishan population of Z. hentscheli groups with Z. arbuscula rather than with Z. hentscheli (KM222118). Zoothamnium weishanicum n. sp. is located in clade II and is sister to Z. arcuatum with full support. Epicarchesium sinense n. sp. and Carchesium polypinum fall within the Vorticellidae assemblage. Epicarchesium sinense n. sp. clusters with E. pectinatum with moderate to high support (ML 81% and BI 1.00), forming a clade that is sister to the crown group comprising Vorticella, Carchesium, Pseudovorticella, and three species of Epicarchesium (E. corlissi, E. variable, and E. abrae). The Qingdao population of C. polypinum groups with two previously sequenced populations (GU187053 and HM852990) to form a clade that is sister to Vorticella. The molecular data based on the alignments of SSU rDNA sequences supports the validity of each of the five species investigated here and their separation from morphologically similar species (Figures 13, 14). Table 2 Zoothamnium arbuscula is a very common species that has been recorded many times (Ehrenberg, 1831(Ehrenberg, , 1838Ayrton, 1903;Wesenberg-Lund, 1925;Furssenko, 1929;Kahl, 1935;Biernacka, 1962;Müller, 1980;Xu, 1988;Foissner et al., 1992;Foissner and Berger, 1996;Shen and Gu, 2016). Ehrenberg (1831) gave the first description of this species and redescribed it 7 years later (Ehrenberg, 1838). Kahl (1935) made a revision including historical reports, a list of synonyms, an improved diagnosis, and notes on its distribution. He synonymized Zoothamnium geniculatum Ayrton, 1903 andZ. geniculatum sensu Wesenberg-Lund (1925) with Z. arbuscula and deemed that the marine population of Z. arbuscula sensu Kent (1880Kent ( -1882 needed to be re-examined. Foissner et al. (1992) also made a revision of this species and questioned the identity of marine populations reported under the name Z. arbuscula. Ji et al. (2005a) and Wu et al. (2020) reported two marine species (Z. pararbuscula Ji et al., 2005 andZ. apoarbuscula, Wu et al., 2020, respectively) that are morphologically similar to Z. arbuscula. Thus, we speculate that Z. arbuscula is a freshwater species and the marine populations reported under the name Z. arbuscula are populations of Z. pararbuscula, Z. apoarbuscula, or other species. Foissner et al. (1992) proposed the following diagnostic characteristics for the identification of Z. arbuscula: (i) differentiated zooids, microzooids that were bell-shaped, and macrozooids that were mostly ellipsoidal and rarely spherical; (ii) macronucleus that was usually C-shaped and located in the center of the zooid; (iii) a contractile vacuole was located at the dorsal wall of the infundibulum; (iv) the primary stalk was divided into three parts, including a basal part where the spasmoneme is absent and an upper part that is very thick; and (v) colony umbellate, each main branch feather-like. The Weishan population fits sufficiently well with all the above-mentioned characters and the original description of Z. arbuscula for us to conclude that they are conspecific.

Comments on Zoothamnium arbuscula
Zoothamnium arbuscula is characterized by its umbellate colony shape and differentiated zooids, which distinguished it from most other congeners except Z. pararbuscula and Z. apoarbuscula.

Comments on Zoothamnium hentscheli
We identified the Weishan population as Z. hentscheli after comparing it with the original descriptions and redescriptions of all morphologically similar species of Zoothamnium. It closely resembles Z. hentscheli in the characteristic detritus coat on the zooids and stalk, the elongated inverted bellshaped zooids, the single-layered and everted peristomial lip, the irregular alternately branched stalk, the shape and position of the macronucleus, the position of the contractile vacuole, and freshwater habitat. Thus, we identified our population as Z. hentscheli.
Comments on Epicarchesium sinense n. sp. Table 5 Considering that the pellicular striations of Epicarchesium sinense n. sp. in vivo are fine, it could easily be misidentified as a Carchesium species. To confirm that our new species is not a misidentified known Carchesium species, it should also be compared with two morphologically similar Carchesium species, i.e., C. epistylis Claparède andLachmann, 1858, andC. cyclopidarum Nenninger, 1948. Carchesium epistylis can be distinguished from E. sinense n. sp. by its C-shaped (vs. J-shaped) macronucleus and stalk with (vs. without) septa (Claparède and Lachmann, 1858;Kahl, 1935). Carchesium cyclopidarum can be easily separated from E. sinense n. sp. by its C-shaped (vs. J-shaped) macronucleus (Nenninger, 1948;Stloukal and Matis, 1997). Table 6 Carchesium polypinum is a well-known peritrich with a global distribution and is widely used in studies of ecology, cytology, and genetics (Foissner et al., 1992;Miao et al., 2004;Lynn, 2009, 2012;Boas et al., 2018;Vlaičević et al., 2021). However, many populations have been reported without morphological information or voucher specimens. Furthermore, the zooid shape and the size of C. polypinum collected from different environments were variable during our study. The Qingdao population matches closely the main characters of C. polypinum as described by Ehrenberg (1831Ehrenberg ( , 1838 and as summarized in the revision by Foissner et al. (1992), including the following: (i) the single-layered and everted peristomial lip, (ii) the dichotomously branched stalk, (iii) the J-shaped macronucleus, (iv) the contractile vacuole located at the ventral wall of the infundibulum below the level of the peristomial lip, (v) the height and the shape of the colony, (vi) the pattern of the oral ciliature, and (vii) the freshwater habitat (Ehrenberg, 1830(Ehrenberg, , 1838Foissner et al., 1992). Because the zooids of the Qingdao population are smaller than those of other populations, we suggest that the size range of C. polypinum zooids should be extended.

Comments on Carchesium polypinum
One other freshwater species with a similar zooid shape should be compared with the present population, namely, C. epistylis Claparede and Lachmann, 1850. Carchesium epistylis differs from C. polypinum by its C-shaped (vs. J-shaped) macronucleus and stalk with (vs. without) septa (Claparède and Lachmann, 1858;Kahl, 1935). Therefore, the identity of the Qingdao population as C. polypinum is not in doubt.

Phylogenetic Analyses
The phylogenetic tree inferred from SSU rDNA sequence data shows that the genus Zoothamnium is non-monophyletic and the species are grouped into three clades, which is consistent with previous studies (Li et al., 2008;Zhuang et al., 2018;Lu et al., 2020;Wu et al., 2020). The Weishan population of Zoothamnium hentscheli and Z. arbuscula are nested within clade I along with Z. arbuscula (KU363261), Z. pararbuscula, and Z. apoarbuscula. Within this clade, however, Z. hentscheli does not cluster with Z. hentscheli (KM222118), although no morphological information is available for the latter, so its identity could not be confirmed. Zoothamnium weishanicum n. sp. clusters with Z. arcuatum with maximal support (100% ML and 1.00 BI) in clade II. These two sequences differ by only two base pairs and share several morphological similarities including zooid shape, the double-layered peristomial lip, the shape and the position of the macronucleus, and the position of the contractile vacuole. However, the oral ciliature differs significantly in that P3 is two-rowed in Z. weishanicum n. sp. but is three-rowed in Z. arcuatum (Ji et al., 2015).
As expected, Epicarchesium and Carchesium group within the family Vorticellidae. Epicarchesium is non-monophyletic, which is consistent with previous studies Zhuang et al., 2018;Lu et al., 2020). Epicarchesium sinense n. sp. is most closely related to E. pectinatum which is supported by morphological and ecological data such as their pellicle with conspicuous cortical blisters and their freshwater habitat (Jankowski, 1985;Leitner and Foissner, 1997). The Qingdao population of C. polypinum groups with the other two populations of C. polypinum (GU187053 and HM852990). It is noteworthy that several of the sequences identified as C. polypinum differ significantly from each other. However, since most of these lack morphological information or voucher specimens, it is difficult to verify the species identity of these sequences.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ supplementary material.

AUTHOR CONTRIBUTIONS
TW performed the experiments and drafted the manuscript. LD performed the phylogenetic section. ZW, HE-S, SA-F, YL, and AW checked all the data related and helped to improve the draft. BL and CW supervised and organized to complete the work. All authors read and approved the final manuscript.