Two new colonial peritrich ciliates (Ciliophora, Peritrichia, Sessilida) from China: With a note on taxonomic distinction between Epistylididae and Operculariidae

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ScienceDirect European Journal of Protistology 70 (2019) 17–31

Two new colonial peritrich ciliates (Ciliophora, Peritrichia, Sessilida) from China: With a note on taxonomic distinction between Epistylididae and Operculariidae Tong Zhoua,b , Zhe Wanga,b , Hao Yanga,b , Zemao Gua,b,∗ a b

Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan, 430070, PR China Hubei Engineering Technology Research Center for Aquatic Animal Diseases Control and Prevention, Wuhan, 430070, PR China

Received 1 February 2019; received in revised form 24 May 2019; accepted 24 May 2019 Available online 30 May 2019

Abstract Two new colonial sessilid species, Opercularia miaoxinensis spec. nov. and Epistylis conica spec. nov., were isolated from the pereopods of freshwater crayfish, Procambarus clarkii in Hubei Province, China from 2016 to 2017. Both species were investigated by living observation, protargol impregnation, and molecular methods. Opercularia miaoxinensis spec. nov. is morphologically characterized by the following characteristics: spindle-shaped zooid, zooid size of 48–74 × 20–35 ␮m in vivo, contractile vacuole ventrally located above macronucleus, and dichotomously branched stalk. Epistylis conica spec. nov. is characterized by conical zooid shape, zooid size of 56–70 × 22–39 ␮m in vivo, C-shaped macronucleus with transverse orientation, dorsally located contractile vacuole, and dichotomously branched stalk. To further identify both species, phylogenetic trees were constructed based on small subunit ribosomal DNA (SSU rDNA) and ITS1-5.8S-ITS2 sequences. Both results showed that E. conica spec. nov. was part of a clade consisting of the majority of Epistylis species. Surprisingly, O. miaoxinensis spec. nov. also clustered within this large clade of Epistylis species and had a distant relationship with the other Opercularia species. These findings challenged the distinguishing morphological characteristics between Epistylididae and Operculariidae. © 2019 Elsevier GmbH. All rights reserved.

Keywords: ITS1-5.8S-ITS2 sequence; Phylogenetic analyses; Sessilida; SSU rDNA sequences; Taxonomy

Introduction Sessilida Kahl, 1933 is assigned to the subclass Peritrichia Stein, 1859 (Jiang et al. 2016; Lynn 2008; Sun et al. 2017; Wang et al. 2017). Traditionally, sessilids have been widely recognized as a well-defined group of 14 families distinguished by morphological characteristics, such as stalk, spasmoneme, lorica, and peristome (Lynn 2008). During the last decade, some distinguishing characteristics have ∗ Corresponding author at: Huazhong Agricultural University, No.1, Shizishan Street, Hongshan District, Wuhan, 430070, PR China. E-mail address: [email protected] (Z. Gu).

https://doi.org/10.1016/j.ejop.2019.05.003 0932-4739/© 2019 Elsevier GmbH. All rights reserved.

been challenged by the results of molecular phylogenetic investigations (Sun et al. 2013; Utz et al. 2010; Zhuang et al. 2018), indicating that the entire sessilids might require revision. Epistylididae Kahl, 1933 and Operculariidae FauréFremiet in Corliss, 1979 are typical problematic sessilids. Morphologically, species of both families generally have similar features but are distinct with respect to their peristome structure: Epistylididae has a well-defined epistomial lip, while Operculariidae lacks such a structure (Lynn 2008). In combination with the utilization of phylogenetic analyses based on molecular data, the validity of family-level systematics has been questioned. Species in Epistylididae,

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namely Epistylis galea Ehrenberg, 1831 and Campanella umbellaria (Linnaeus, 1758) Goldfuss, 1820, usually clustered with Opercularia species rather than with the other species assigned to Epistylididae in phylogenetic trees (Miao et al. 2004; Sun et al. 2016; Utz et al. 2010; Wang et al. 2017). Although these findings implied that a taxonomic revision is needed, the morphological and molecular data of discovered species are insufficient. Thus, further sample collections are urgently needed. We isolated two new colonial sessilid species from the pereopods of Procambarus clarkii Girard, 1852 in Qianjiang city, Hubei Province, China. They were identified as new members of the genera Opecularia Goldfuss, 1820 and Epistylis Ehrenberg, 1830 via live observation and silver staining. The names Opercularia miaoxinensis spec. nov. and Epistylis conica spec. nov. were assigned, respectively. Detailed morphological descriptions of both species as well as the results of the phylogenetic analyses based on SSU rDNA and ITS1-5.8S-ITS2 sequence are provided. The validity of the taxonomic distinction between the families Epistylididae and Operculariidae is briefly discussed.

Material and Methods Sample collection and identification The two sessilids were found adhering to the pereopods of different Procambarus clarkii specimens from a crayfish pond (31,302 m2 ) of a crayfish aquaculture base (6.7 × 105 m2 ) in Miaoxin Village, Longwan Town, Qianjiang City, Hubei Province, China (30◦ 5 35 N; 112◦ 47 56 E). Opercularia miaoxinensis spec. nov. was collected on the 28th May, 2016. Epistylis conica spec. nov. was collected on the 7th July, 2017. Both were treated using the same methods as follows. The colonies were washed three to four times with distilled water to remove impurities. Living specimens were observed at magnifications from 200× to 1,000× using a bright field and differential interference contrast microscope (Olympus BX53 F, Japan). Images were captured by a digital camera (Olympus DP73, Japan) mounted to the microscope. The morphometric data were measured with the microscope. The oral ciliature was obtained by protargol impregnation according to the protargol procedure B described in Foissner (2014). The protargol used in the present study was produced according to Pan et al. (2013). Illustrations of live and stained specimens were drawn using Photoshop CS6 (Adobe Systems Inc). Terminology (e.g., epistomial lip) and systematics mainly follow Lynn (2008). Colonies of Epistylis conica spec. nov. were prepared for scanning electron microscopy according to Wang et al. (2017). The colonies were fixed with 3% glutaraldehyde and 0.2 M phosphate buffer saline (PBS, pH 7.2) at 4 ◦ C. The fixed colonies were then placed on a slide by 0.1% PolyL-lysine (PLL) and thoroughly rinsed in PBS. Thereafter,

the colonies were dehydrated using a graded ethanol series and propylene oxide. Colonies were further dehydrated with CO2 and sputter coated with gold. Observations were carried out with a scanning electron microscope (Hitachi SU8010, Japan) at 20 kV with a working distance of 18 mm.

DNA extraction, polymerase chain reaction (PCR), and sequencing For DNA extraction, colonies (approximately 20–40 zooids) were washed six to seven times with distilled water, and observed under a microscope with 1,000× magnification to ensure that the zooids were identical in morphology. Genomic DNA was extracted using the TransDirect Animal Tissue PCR Kit (Trans, Beijing, China). Small subunit (SSU) rDNA sequences and large subunit (LSU) rDNA sequences were amplified with the primers Euk A & Euk B (Medlin et al. 1988) and 28S-1 F & 28S-3R (Moreira et al. 2007), respectively. The following cycling parameters were applied: 5 min initial denaturation at 94 ◦ C, followed by 30 cycles (30 s at 94 ◦ C for denaturation, 30 s at 55 ◦ C for annealing, and 1 min at 72 ◦ C for extension) and a final extension step at 72 ◦ C for 10 min. The entire ITS1-5.8S-ITS2 sequence was amplified with the primers ITSF and ITSR (Yi et al. 2009). The PCR program started with an initial denaturation step of 5 min at 94 ◦ C, followed by 30 cycles (30 s at 95 ◦ C for denaturation, 30 s at 55 ◦ C for annealing, and 45 s at 72 ◦ C for extension) and a final extension step at 72 ◦ C for 5 min. The PCR products were purified by using the HighPure PCR Product Purification Kit (Cwbio, Beijing, China). High-pure fragments were linked with the pMD-19Tvector (TaKaRa, Dalian, China), and target fragments were subsequently transformed into Escherichia Trans5␣ competent cells (Trans, Beijing, China). Finally, these ® samples were sequenced by an ABI PRISM 3730 DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA).

Phylogenetic analyses To determine the phylogenetic positions of both species, the sequences were blasted (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Highly matching sequences were acquired from GenBank and aligned by using MAFFT v7.245 (Katoh and Standley 2013). The alignment of SSU rDNA sequences and ITS15.8S-ITS2 sequence was refined by GBLOCKS (http://www.phylogeny.fr/one task.cgi?task type=gblocks). Phylogenetic trees were constructed by two different methods: bayesian inference (BI) and maximum likelihood (ML). Maximum likelihood (ML) trees were constructed with IQTREE 1.6.2 (Nguyen et al. 2015) under the best selected evolutionary model (SSU rDNA sequences, GTR + F + R4;

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Fig. 1. a–i. Opercularia miaoxinensis spec. nov. in vivo. a. Contracted and extended zooids. b. Anterior portion of extended zooid, arrow indicates hemispherical bulge on epistomial disk. c. Extended zooid, arrow indicates infundibulum. d. Macronucleus and contractile vacuole. e. Trochal band at primary stage of telotroch morphogenesis. f. Trochal band of extended zooid. g. Pellicular striations. h. Dichotomously branched stalk. i. Stalk, arrow indicates transverse striations on surface. CV, contractile vacuole; ED, epistomial disk; Ma, macronucleus; TB, trochal band. Scale bars = 10 ␮m.

ITS1-5.8S-ITS2 sequences, TPM2u + F + I + G4) with 10,000 UF-Boot replicates. Bayesian analyses were performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) under the model of GTR + I + G. Markov chain Monte Carlo chains of SSU rDNA sequences and of ITS1-5.8S-ITS2 sequences were determined for 1,000,000 generations using the default heating parameter. Sampling was conducted once every 100 generations. The first 25% of the trees were discarded as burn-in. The trees were edited and annotated with MEGA 6 (Tamura et al. 2013).

Results Genus Opercularia Goldfuss, 1820 Opercularia miaoxinensis spec. nov. (Figs. 1, 2, 6a, c, Table 1) Diagnosis. Extended zooids spindle-shaped, 48–74 × 20–32 ␮m in vivo. Contractile vacuole on ventral wall of infundibulum. C-shaped macronucleus underneath

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Fig. 2. a–f. Photomicrographs of protargol-stained Opercularia miaoxinensis spec. nov. a. Macronucleus. b. Polykinety and haplokinety. c. Adoral region of peniculi 1 and 2. d. Aboral ends of peniculi 1 and 2. e. Peniculi 1–3. f. Pellicular striations and trochal band. GK, germinal kinety; HK, haplokinety; Ma, macronucleus; P1–3, peniculi 1–3; PK, polykinety; TB, trochal band. Scale bars = 10 ␮m.

contractile vacuole. Number of pellicular striations from peristome to trochal band 63–88, from trochal band to scopula 11–20. Row 1 of peniculus 1 longer than other two ciliary rows. Three ciliary rows of peniculus 2 equal in length. Peniculus 3 composed of two ciliary rows. Stalk dichotomously branched with longitudinal and transverse striations. Freshwater habitat. Type locality. Freshwater crayfish pond in Miaoxin Village, Longwan Town, Qianjiang City, Hubei Province, China (30◦ 15 35 N; 112◦ 47 56 E); water temperature 19 ◦ C. Colonies attached to the pereopods of Procambarus clarkii. Type material. The protargol-stained slide containing the holotype (Fig. 2a, b) circled in ink has been deposited in the National Zoological Museum of China, Institute of Zoology, Chinese Academy of Sciences, Beijing, China, with registration number QJ201605283. The protargol-stained slide containing the paratype specimen circled in ink (Fig. 2d, registration number QJ201605282) has been deposited in the Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan, China. Etymology. The species-group name miaoxinensis refers to the village (Miaoxin) where the species was first detected.

ZooBank registration. Registration number of Opercularia miaoxinensis spec. nov.: urn:lsid:zoobank.org:act:681D7147 -CB40-42E0-B79C-BBA5613EA65E. Description. Extended zooids spindle-shaped, 48–74 × 20–32 ␮m in vivo, 60 × 24 ␮m (n = 12) on average (Figs. 1a, 6 a). Length to width ratio 2.5:1 (n = 12) on average (Fig. 1b). Peristome margin smooth, lacking epistomial lip, 10.9–17.3 ␮m (n = 12) in diameter. Epistomial disk 5.6–8.4 ␮m (n = 12) in diameter, 1.9–3.2 ␮m (n = 12) in height (Table 1). A hemispherical bulge visible on top of epistomial disk (Fig. 1b). Infundibulum extends to mid-zooid (Fig. 1c). Cytoplasm gray, with numerous granules (Fig. 1c). Single contractile vacuole on ventral wall of infundibulum (Figs. 1d, 2a). C-shaped macronucleus transversely located below contractile vacuole (Figs. 1d, 2a). Trochal band clearly detectable at primary stage of telotroch morphogenesis (Fig. 1e). Contracted zooids of ovoid shape (Fig. 1a). Colonies composed of 2–6 (n = 10) zooids (Fig. 1a). Stalk not hollow, dichotomously branched (Fig. 1h) with longitudinal and transverse striations on surface (Fig. 1i). Secondary stalk morphometric characterization: width 3.0–4.5 ␮m (n = 14), length 39.8–121.8 ␮m (n = 16). After protargol impregnation, haplokinety and polykinety make one turn together around epistomial disk before plunging down into infundibulum (Figs. 2b, 6c). Germinal

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Table 1. Morphometrical characteristics of Opercularia miaoxinensis spec. nov. and Epistylis conica spec. nov. Characteristic

Species

Mean

M

SD

SE

CV

Min

Max

n

Zooid, lengtha

O. miaoxinensis E. conica O. miaoxinensis E. conica O. miaoxinensis E. conica O. miaoxinensis E. conica E. conica O. miaoxinensis E. conica O. miaoxinensis E. conica O. miaoxinensis E. conica O. miaoxinensis E. conica O. miaoxinensis E. conica O. miaoxinensisa/b E. conicac O. miaoxinensisa/b E. conicac

59.2 60.1 23.8 27.1 2.5 13.1 13.1 27.6 6.3 6.8 16.0 2.7 5.4 4 75 3.7 6.3 85.2 596.9 72/71 97 15/13 27

48.4 59.3 22.1 26.7 2.4 2.2 12.0 27.1 6.2 6.3 15.7 2.7 5.7 3 76 3.67 5.5 81.24 600.3 70/72 89 15/13 26

9.5 3.8 3.9 4.3 0.3 0.3 2.2 2.9 0.82 1.0 1.6 0.4 0.8 1.3 33.5 0.46 2.7 29.1 93.0 6.9/3.9 16.8 2.7/1.7 5.1

2.7 1.0 1.3 1.1 0.1 0.1 0.6 0.4 0.2 0.3 0.4 0.1 0.2 0.4 10.6 0.1 0.7 7.2 26.8 2/0.1 4.3 0.7/0.6 1.4

16 6.3 16.4 15.9 12.0 13.0 16.8 10.5 13.0 14.7 10.0 10.0 14.8 32.5 44.7 12.4 42.9 34.1 15.6 9.6/1.2 17.3 18/13.1 18.9

47.7 55.9 19.8 21.8 2.2 1.8 10.9 24.1 5.8 5.6 13.9 13.9 4.2 2 26 3 3.6 39.8 441.8 63/65 84 11/11 21

73.6 69.7 31.8 38.8 2.9 2.7 17.3 36.6 7.9 8.4 19.1 19.1 6.6 6 151 4.5 13.2 121.8 732.7 88/76 128 20/15 38

12 16 12 16 12 16 12 16 16 12 16 12 16 10 10 14 15 16 12 12/11 15 12/7 13

Zooid, widtha Zooid length: width, ratioa Peristome / epistomial lip, diametera Epistomial lip, thicknessa Epistomial disk, diametera Epistomial disk, heighta Zooids in colony, numbera Stalk, widtha Stalk, lengtha Pellicular striations above trochal band, number Pellicular striations below trochal band, number

Abbreviations: CV, coefficient of variation (%); M, median; Max, maximum; Mean, arithmetic mean; Min, minmum; n, number of specimens investigated; SD, standard deviation; SE, standard error of arithmetic mean. a Data based on live observation, measurements in ␮m. b Data based on protargol-stained specimens. c Data based on scanning electron microscopy.

kinety located above haplokinety (Fig. 2c). Both peniculi 1 and 2 (P1 and P2) composed of three ciliary rows. Aboral end of row 1 of P1 slightly longer than other two ciliary rows (Figs. 2d, 6c). Three ciliary rows of P2 terminate at same level and shorter than P1 (Fig. 2d). Two ciliary rows of P3 emerge at aboral end of P2 and curve toward aboral end of P1 (Fig. 2e). Pellicular striations (= silverlines) clearly observed in vivo (Fig. 1e–g) and in protargol preparations (Fig. 2f), numbering 63–88 (n = 23) from peristome to trochal band, 11–20 (n = 19) from trochal band to scopula (Table 1).

Genus Epistylis Ehrenberg, 1830 Epistylis conica spec. nov. (Figs. 3–5, 6b, d, Table 1) Diagnosis. Extended zooids conical-shaped, 56–70 × 22–39 ␮m in vivo. Macronucleus C-shaped with transverse orientation. One contractile vacuole dorsally located at level of epistomial lip. Number of pellicular striations 84–128 from peristome to trochal band, 21–38 from trochal band to scopula. Row 2 of peniculus 1 shorter than other two ciliary rows. Three ciliary rows of peniculus 2 equal in length. Aboral end of row 1 of peniculus 3 curves toward row 3 of peniculus 2. Aboral end of row 3 of peniculus 3 next to

aboral end of peniculus 1. Stalk dichotomously branched with longitudinal and transverse striations on surface. Freshwater habitat. Type locality. Freshwater crayfish pond in Miaoxin Village, Longwan Town, Qianjiang City, Hubei Province, China (30◦ 15 35 N; 112◦ 47 56 E); water temperature 31 ◦ C. Colonies attached to the pereopods of Procambarus clarkii. Type material. The protargol-stained slide containing the holotype (Fig. 4a–c) circled in ink has been deposited in the National Zoological Museum of China, Institute of Zoology, Chinese Academy of Sciences, Beijing, China, with registration number QJ201707071. The protargol-stained slide containing paratype specimens (Fig. 4d–f, registration number QJ201707072) circled in ink has been deposited in the Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan, China. Etymology. The species-group name conica refers to the conical zooid shape of the species in vivo. ZooBank registration. Registration number of Epistylis conica spec. nov.: urn:lsid:zoobank.org:act:C79BDF69E993-4BD8-9144-D98F684FEDEF. Description. Zooids conical, 56–70 × 22–39 ␮m in vivo, 60 × 27 ␮m (n = 16) on average (Figs. 3b, 6b). Length-to-

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Fig. 3. a–f. Epistylis conica spec. nov. in vivo. a. Colony of zooids. b. Extended zooids, arrow indicates infundibulum. c. Anterior portion of extended zooid. d. Contracted zooid, arrow indicates folds of posterior portion of zooid. e. Dichotomously branched stalk, arrows indicate transverse striations on surface. f. Longitudinal striations on stalk surface. CV, contractile vacuole; ED, epistomial disk; EL, epistomial lip; Ma, macronucleus. Scale bars: a = 100 ␮m; b–f = 10 ␮m.

width ratio 2.3:1 (n = 16) on average (Fig. 3b). Maximum zooid width approximately equal to diameter of epistomial lip of fully extended zooid (Fig. 3b). Epistomial lip distinctly everted, parallel to transverse axis of zooid, 24–37 ␮m (n = 16) in diameter, 5.8–7.9 ␮m (n = 16) thick. Epistomial disk sloped, 13.9–19.1 ␮m (n = 16) in diameter, 4.2–6.6 ␮m (n = 16) high (Table 1). Single contractile vacuole dorsally located at level of epistomial lip (Fig. 3b). Cshaped macronucleus transversely located at anterior 25% of zooid length (Fig. 3c). Contracted zooid ovoid-shaped, with clear folds on posterior portion of zooid (Fig. 3d). Infundibulum extends to mid-zooid (Fig. 3b). Colonies composed of 26–151 (n = 10) zooids (Fig. 3a). Stalk not hollow, dichotomously branched with longitudinal and transverse striations on surface (Fig. 3e, f). Secondary stalk morphometric characterization: width 3.6–13.2 ␮m (n = 12), length 442–733 ␮m (n = 15). After protargol impregnation, epistomial membrane near opening of infundibulum (Fig. 4a). Trochal band appeared at posterior 25% of zooid length (Fig. 4b). Polykinety and haplokinety circle one and one-quarter turns around epistomial disk (Figs. 4a, 6d). Germinal kinety located above haplokinety (Fig. 4c). Polykinety divided into two peniculi (P1 and P2) after plunging into infundibulum (Fig. 4c). Both P1 and P2 composed of three ciliary rows. Row 2 of P1

slightly shorter than other two ciliary rows (Figs. 4e, 6d). Three ciliary rows of P2 end at same level and shorter than P1 (Fig. 4f). Row 1 of P3 shorter than three ciliary rows of P2, row 2 of P3 extends to aboral end of P2, and row 3 of P3 extends to aboral end of P1 (Fig. 4f). Pellicular striations clearly recognizable in scanning electron micrographs (Fig. 5a–d), numbering 84–128 (n = 15) from peristome to trochal band and 21–38 (n = 13) from trochal band to scopula (Table 1).

Phylogenetic analyses SSU rDNA sequences of Opercularia miaoxinensis spec. nov. (GenBank accession number: MK949363; length: 1713) and Epistylis conica spec. nov. (GenBank accession number: MK949362; length: 1732) were blasted with other available sequences in GenBank. The results showed that E. conica spec. nov. was most similar to E. riograndensis (KM594566, identity 99%, 13 bp substitutions), while O. miaoxinensis spec. nov. was more closely related to Telotrochidium matiense (AY611065, identity 98%, 35 bp substitutions) and E. wuhanensis (KU869709, identity 97%, 46 bp substitutions) than to Opercularia species, such as O. microdiscum (AF401525, identity 85%, 262

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Fig. 4. a.f. Photomicrographs of protargol-strained Epistylis conica spec. nov. a. Frontal view of polykinety and haplokinety. b. Trochal band. c. Adoral region of peniculi 1 and 2. d. Macronucleus and aboral ends of peniculi 1–3. e. Aboral end of peniculus 1. f. Aboral ends of peniculi 2 and 3. EM, epistomial membrane; GK, germinal kinety; HK, haplokinety; Ma, macronucleus; P1–3, peniculi 1–3; PK, polykinety; TB, trochal band. Scale bars: a, c-f = 10 ␮m; b = 20 ␮m.

bp substitutions). The topologies of the ML and BI trees based on SSU rDNA sequences were almost congruent, and a consensus tree (Fig. 7) was constructed based on the topology of BI trees. Seven Opercularia species (Opercularia sp. 2 LRPU-2010 (HM627240), Opercularia sp. 3 LRPU2010 (HM627241), O. allensi (HM627240), Opercularia sp. 1 LRPU-2010 (HM627239), O. microdiscum (AF401525), Opercularia sp. JP clone 121operCul (KU363267), and Opercularia sp. 102 clone 121operCul (KU363257)) were nested with clade II of Epistylididae in the basal clade of Sessilida. Opercularia miaoxinensis spec. nov. clustered with T. matiense (AY611065) in a well-supported clade (100 ML, 100 BI). This nested inside clade I of Epistylididae comprising E. conica spec. nov., E. wuhanensis (KU869709), E. riograndensis (KM594566), E. chrysemydis (AF335514), E. chlorelligerum (KM096375), E. wenrichi (AF335515), and E. urceolata (AF335516). ITS1-5.8S-ITS2 sequences of Opercularia miaoxinensis spec. nov. (GenBank accession number: MK949365; length: 488) and Epistylis conica spec. nov. (GenBank accession number: MK949364; length: 494) were deposited in GenBank. According to the BLAST results, the most similar species of E. conica spec. nov. was E. chrysemydis (GU586187, identity 93%, 36 bp substitutions), while O. miaoxinensis spec. nov. was most similar to E. wuhanensis

(KU869710, identity 87%, 64 bp substitutions). The topologies of the ML and BI trees based on ITS1-5.8SITS2 sequences were almost congruent, and a consensus tree (Fig. 8) was constructed based on the topology of BI trees. Epistylis conica spec. nov. nested into a well-supported clade with other Epistylis species, such as E. plicatilis (AF429890), E. portoalegrensis (KT358503, KT358504), and E. chrysemydis (GU586187). Opercularia miaoxinensis spec. nov. was sister to E. wuhanensis (KU869710), which together formed a clade that was sister to the clade of above Epistylis species. The basal clade of Sessilida included E. galea (AF429888), Campanella umbellaria (GU586188), and O. microdiscum (AF429893). The following LSU rDNA sequences were deposited in GenBank: Opercularia miaoxinensis spec. n. (GenBank accession number: MK949361; length: 1843) and Epistylis conica spec. nov. (GenBank accession number: MK949360; length: 1843). The BLAST results showed that E. chrysemydis (KY675197) was the most similar species to E. conica spec. nov. (identity 97%, 56 bp substitutions) and O. miaoxinensis spec. nov. (identity 96%, 73 bp substitutions). No phylogenetic tree was constructed based on LSU rDNA sequences, because few LSU rDNA sequences (no sequence of Opercularia species, 1 sequence of Epistylis species) are available in GenBank.

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Fig. 5. a–d. Scanning electron micrographs of Epistylis conica spec. nov. a. Contracted zooid. b. Anterior portion of contracted zooid. c. Pellicular striations. d. Trochal band. TB, trochal band. Scale bars: a, b = 10 ␮m; c = 5 ␮m; d = 2 ␮m.

Discussion Identification of Opercularia miaoxinensis spec. nov. Opercularia miaoxinensis spec. nov. shows the typical morphological characteristics of the genus Opercularia and can be distinguished from other sessilid genera by the presence of a rigid stalk and a lacking epistomial lip. Opercularia microdiscum Fauré-Fremiet, 1905 resembles O. miaoxinensis spec. nov. in zooid shape, contractile vacuole location, and epistomial disk shape. However, the following characteristics are different between O. miaoxinensis spec. nov. and O. microdiscum, respectively: (1) infundibulum (regular shape vs. irregular shape); (2) epistomial disk diameter (50% of peristome diameter vs. onesixth of peristome diameter); (3) striations on stalk surface (longitudinal and transverse striations vs. only longitudinal striations) (Shen and Gu 2016). In addition, the identity of SSU rDNA sequences between O. miaoxinensis spec. nov. and O. microdiscum is 85% (262 substitutions in 1763 bp), which is beyond the intraspecific variation (Irwin et al. 2017).

Opercularia asymmetrica Aescht and Foissner, 1992 resembles O. miaoxinensis spec. nov. in zooid shape. The following characteristics are different between O. miaoxinensis spec. nov. and O. asymmetrica, respectively: (1) contractile vacuole location (ventral vs. dorsal); (2) stalk length (3.0–4.5 ␮m vs. 1.0–2.0 ␮m); (3) peniculus 1 (row 1 is slightly longer than the other two ciliary rows vs. three ciliary rows terminate at the same level) (4) peniculus 2 (emerges at the beginning of P1 vs. does not emerge at the beginning of P1) (Aescht and Foissner 1992). Opercularia miaoxinensis spec. nov. can be distinguished from other similar Opercularia species by the following characteristics: (1) macronuclei are S-shaped or J-shaped in several species, such as O. articulata Goldfuss, 1820 and O. fabrei Fauré-Fremiet, 1905 (vs. macronucleus is C-shaped in O. miaoxinensis spec. nov.) (Guhl 1979; Fernández-Galiano et al. 1988; Foissner et al. 1992; Shen and Gu 2016); (2) peristome margins are undulating in several species, such as O. gracilis Fauré-Fremiet, 1905 and O. corethrae Keiser, 1921 (vs. peristome margin is smooth in O. miaoxinensis spec. nov.) (Kahl 1935); (3) contractile vacuoles are dorsally located in several species, such as Opercularia nutans

Table 2. Morphological comparison of Opercularia miaoxinensis spec. nov. with similar species. Characteristic

O. miaoxinensis

Zooid, size in vivo (␮m) Zooid, shape

48–74 × 20–32 65–88 × 23–37 28–75 × 13–40 75–78, length SpindleSpindleSpindleSpindleshaped shaped shaped shaped Smooth Smooth Smooth Undulating

Ma, orientation Pellicular striations above and below TB, number Peniculi, feature

Stalk, branching pattern Stalk, striations on surface Data source

O. asymmetrica

O. corethrae

O. articulata

O. fabrei

O. gracilis

O. allensi

O. nutans

O. coarctata

80–105 × 37–47 –

80, length

58–65 × 31–38 25–45 × 75–90 45–56 × 19–23

Spindleshaped Undulating

Spindleshaped Smooth

Spindleshaped Undulating

Spindleshaped Smooth

Spindleshaped Smooth

Spindleshaped Smooth

Flask-shaped

Irregular

Flask-shaped

Flask-shaped

Flask-shaped

Flask-shaped

Flask-shaped

Flask-shaped

Flask-shaped

Flask-shaped

Ventral C-shaped

Ventral C-shaped

– C-shaped

Dorsal S-shaped,

Ventral J-shaped

Ventral C-shaped

– C-shaped

Dorsal C-shaped

– C-shaped

Transverse

Transverse

Transverse

Transverse

Longitudinal

Transverse

–

Transverse

Transverse

63–88, 11–20

–

Dorsal Horseshoeshaped, reniform, or elipsoid Longitudinal or transverse 52–97, in total

–

90–120, 20–30

–

–

–

105–122, 25–35

–

P1, row 1 longest; P3, consisted of 2 ciliary rows Dichotomous

–

–

–

–

–

–

Dichotomous

Dichotomous

–

Dichotomous

Extensive

P1, three rows equal; P3, consisted of 3 ciliary rows Dichotomous

P1, three rows equal; P3, consisted of 2 ciliary rows Dichotomous

Longitudinal and transverse Shen and Gu (2016)

Longitudinal

Longitudinal

Transverse

No striation

Guhl (1979)

Kahl (1935)

Viljoen and Van As (1983)

Longitudinal and transverse Jiang and Miao (2017)

Longitudinal and transverse Present study

Dichotomous

P1, three rows equal; P3, consisted of 2 ciliary rows Usually unbranched

Longitudinal

No striation

Transverse

Shen and Gu (2016)

Aescht and Foissner (1992)

Kahl (1935)

T. Zhou et al. / European Journal of Protistology 70 (2019) 17–31

Peristome margin, shape Infundibulum, shape CV, location Ma, shape

O. microdiscum

Foissner et al. (1992); FernándezGaliano et al. (1988)

Abbreviations: CV, contractile vacuole; Ma, macronucleus; TB, trochal band. –, data are unavailable. 25

26

T. Zhou et al. / European Journal of Protistology 70 (2019) 17–31

Fig. 6. a–d. Drawing from in vivo and protargol-stained specimens. a. Opercularia miaoxinensis spec. nov. b. Epistylis conica spec. nov. c. Oral ciliature of Opercularia miaoxinensis spec. nov. d. Oral ciliature of Epistylis conica spec. nov. 1–3, rows 1–3; EM, epistomial membrane; GK, germinal kinety; HK, haplokinety; P1–3, peniculi 1–3; PK, polykinety. Scale bars = 10 ␮m.

(Müller, 1773) Stein, 1854 and O. articulata (vs. ventrally located in O. miaoxinensis spec. nov.) (Jiang and Miao 2017; Shen and Gu 2016); (4) stalks are extensively branched in several species, such as O. allensi Stokes, 1887 (vs. stalk is dichotomously branched in O. miaoxinensis spec. nov.) (Viljoen and Van As 1983); (5) three ciliary rows of peniculus 1 were of equal length in several species, such as O. nutans and O. coarctata (Claparède and Lachmann, 1858) Roux, 1901 (vs. row 1 of P1 is longer than the other two ciliary rows in O. miaoxinensis spec. nov.) (Fernández-Galiano et al. 1988; Jiang and Miao 2017); (6) pellicular striations above trochal band of several species are more than 90, such as in O. articulata and O. nutans (pellicular striations above trochal band of O. miaoxinensis spec. nov. are less than 88) (Foissner et al. 1992; Jiang and Miao 2017). Detailed comparisons are presented in Table 2. Considering the typical morphological characteristics of the genus Opercularia and the discrepancies with congeners,

O. miaoxinensis spec. nov. was identified as a new species of Opercularia.

Identification of Epistylis conica spec. nov. Epistylis conica spec. nov. was assigned to the genus Epistylis according to the following typical morphological characteristics: noncontractile stalk, well-defined epistomial lip, and oral ciliary rows circling for less than two turns around epistomial disk. The majority of Epistylis species have cylindroid or vase-shaped zooids, such as E. chlorelligerum Shen, 1980, E. chrysemydis Bishop and Jahn, 1941, E. plicatilis Ehrenberg, 1831, E. riograndensis Utz et al., 2014, and E. entzii Stiller, 1935 (Foissner et al. 1992; Shi et al. 2014; Utz et al. 2014; Jiang et al. 2016), which are obviously different from E. conica spec. nov. Considering zooid shape, several species should still be compared to E. conica spec. nov.

T. Zhou et al. / European Journal of Protistology 70 (2019) 17–31

27

Fig. 7. Consensus tree constructed from BI and ML trees generated via phylogenetic analyses of nuclear SSU rDNA sequences. The sequences investigated in the present study are in bold. Numbers at nodes of branches indicate the posterior probability (BI) and bootstrap (ML) values, respectively. The scale bar corresponds to 5 substitutions per 100 nucleotide positions.

Epistylis galea Ehrenberg, 1831 has a strong resemblance to E. conica spec. nov. in terms of contractile vacuole location and zooid shape. However, the following characteristics are different between E. conica spec. nov. and E. galea, respectively: (1) macronucleus (C-shaped with transverse orientation vs. J-shaped with longitudinal orientation); (2) zooid size (56–70 × 22–39 ␮m vs. 160–225 × 50–70 ␮m); (3) epistomial lip (thick vs. thin) (Foissner et al. 1992). When blasting based on SSU rDNA sequences, the identity of both species is 86% (244 substitutions in 1761 bp). Therefore, E. conica spec. nov. differs from E. galea in both morphological characteristics and molecular data. Epistylis conica spec. nov. is similar to E. hentscheli Kahl, 1935 in zooid shape and macronucleus orientation. The following characteristics are different between E. conica spec. nov. and E. hentscheli, respectively: (1) contractile vacuole (at the same level of epistomial lip vs. below epistomial lip); (2) stalk (not hollow vs. hollow); (3) zooid size (56–70 × 22–39 ␮m vs. 110–130 × 60–65 ␮m); (4) peniculus 1 (row 2 is shorter than the other two ciliary rows vs. three ciliary row were of equal length (Wang et al.

2016). The BLAST result showed that both species share 93% identity (116 substitutions in 1739 bp). Other conical-shaped Epistylis species can be distinguished from E. conica spec. nov. by the following morphological characteristics: (1) contractile vacuoles of several species are below epistomial lip, such as in E. anastatica (Linnaeus, 1767) Kent, 1881, E. vaginula Stokes, 1884, E. digitalis (Linnaeus, 1758) Ehrenberg, 1838, and E. callinectes Ma and Overstreet, 2006, while contractile vacuole is at the level of epistomial lip in E. conica spec. nov. (Kahl 1935; Viljoen and Van As 1987; Ma and Overstreet 2006; Shen and Gu 2016); (2) contractile vacuole of several species is on the ventral wall of infundibulum, such as in E. articulata Fromentel, 1874 and E. hospes Fromentel, 1874, while contractile vacuole of E. conica spec. nov. is on the dorsal wall of infundibulum (Kahl 1935); (3) macronuclei of several species are J-shaped or band-shaped, such as in E. digitalis, E. hospes, E. lacustris Imhoff, 1884, and E. articulata, while macronucleus is C-shaped in E. conica spec. nov. (Kahl 1935; Jankowski 2007; Shen and Gu 2016); (4) macronuclei of several species are longitudinally

28

Table 3. Morphological comparison of Epistylis conica spec. nov. with similar species. Characteristic E. conica

Ma, shape Ma, orientation Pellicular striations above and below TB, number Peniculi, feature Stalk, branching pattern Stalk, striations on surface Data source

E. hentscheli

E. lacustris

E. E. helicostylum callinectes

E. digitalis

E. hospes

E. coronata E. anastatica

45–75 × 15–35

160–225 × 50–70

100–130 × 60–65

50–70, length

50, length

Parallel

Declining

Declining

Parallel

Dorsal, not below EL C-shaped

Dorsal, not below EL J-shaped,

Ventral, below EL C-shaped

Transverse

40–57 × 18–33

60–120, length

125, length

70–120, length

Parallel

Parallel

Parallel

Parallel

Parallel

–, Below EL Bandshaped Longitudinal

–, Below EL C-shaped

Dorsal, below EL J-shaped

Longitudinal Transverse

Dorsal, below EL Bandshaped Longitudinal

84–128, 21–38

150, 100

–

–

48–70, 19–26

P1, row 2 shortest

–

E. vaginula

48–94 × 51–84

120, length

66–85 × 23–41

Parallel

Parallel

Parallel

Ventral, not Dorsal, – below EL BandC-shaped shaped Longitudinal Longitudinal Transverse

–, Below PL C-shaped

Ventral, below EL Bandshaped Longitudinal

–, Below EL C-shaped

–

Transverse

Transverse

–

–

54–64, 90–94

–

–

P1, three – P1, three – P1, three – ciliary ciliary ciliary rows equal rows equal rows equal Dichotomous Dichotomous Dichotomous Dichotomous Dichotomous Dichotomous Dichotomous –

–

–

–

–

Longitudinal and transverse Present study

71–74, 35–47

Transverse

E. articulata

Longitudinal transverse and transverse Foissner Wang et al. et al. (2016) (1992)

Dichotomous Dichotomous Dichotomous Dichotomous

–

Transverse

Longitudinal Transverse

–

Longitudinal Transverse

Jankowski (2007)

Vavra (1962)

Ma and Overstreet (2006)

Kahl (1935)

Foissner et al. (1992)

Shen and Gu (2016)

Abbreviations: CV, contractile vacuole; EL, epistomial lip; Ma, macronucleus; TB, trochal band. –, data are unavailable.

Longitudinal Longitudinal and transverse Viljoen and Kahl Shen and Van As Gu (2016) (1935) (1987)

T. Zhou et al. / European Journal of Protistology 70 (2019) 17–31

Zooid, size in vivo (␮m) EL, orientation to zooid transverse axis CV, location

E. galea

T. Zhou et al. / European Journal of Protistology 70 (2019) 17–31

29

Fig. 8. Consensus tree constructed from BI and ML trees generated by phylogenetic analyses of nuclear ITS1-5.8S-ITS2 sequence. The sequences investigated in the present study are in bold. Numbers at the nodes of branches indicate the posterior probability (BI) and bootstrap (ML) values, respectively. The scale bar corresponds to 2 substitutions per 100 nucleotide positions.

oriented, such as in E. helicostylum Vavra, 1962, while macronucleus of E. conica spec. nov. is transversely oriented (Vavra 1962); (5) zooid length is longer than 70 ␮m in several species, such as E. hospes and E. coronata Nusch, 1970, while zooid length of E. conica spec. nov. is shorter than 75 ␮m (Kahl 1935; Foissner et al. 1992); (6) pellicular striations above trochal band of several species are less than 80, such as in E. callinectes and E. anastatica, while pellicular striations above trochal band of E. conica spec. nov. are more than 88 (Ma and Overstreet 2006; Viljoen and Van As 1987). Detailed comparisons are presented in Table 3. The BLAST results showed that Epistylis conica spec. nov. is most similar to E. riograndensis (KM594566, SSU rDNA sequence) and E. chrysemydis (GU586187, ITS1-5.8S-ITS2 sequence; KY675197, LSU rDNA sequence), which can be morphologically distinguished from E. conica spec. nov. (see above). In addition, phylogenetic analyses based on SSU rDNA and ITS1-5.8S-ITS2 sequences both indicated that E. conica spec. nov. is clearly separated from other Epistylis species. Thus, E. conica spec. nov. was identified as a new member of the genus Epistylis based on morphological and molecular comparisons.

Relationship between Operculariidae and Epistylididae Epistylididae has long been considered to be distinct from Operculariidae by the presence of everted epistomial

lip (Lynn 2008). However, recent molecular phylogenetic analyses queried the taxonomic distinction between Operculariidae and Epistylididae (Utz et al. 2010; Wang et al. 2017). Previous phylogenetic studies based on rDNA sequences reported that two species in Epistylididae (Epistylis galea and Campanella umbellaria) always clustered with Opercularia microdiscum in a basal clade, which is distinct from the other species in Epistylididae (Utz et al. 2010; Zhuang et al. 2018; Jiang et al. 2019). The present study described two species, Opercularia miaoxinensis spec. nov. and Epistylis conica spec. nov., which were morphologically assigned to family Operculariidae and family Epistylididae, respectively. Phylogenetic analyses based on SSU rDNA sequences indicated that both species fall into clade I of Epistylididae. As a result, species in both distinct clades (clade I of Epistylididae and basal clade), such as E. galea (in basal clade) vs. E. conica spec. nov. (in clade I of Epistylididae), O. microdiscum (in basal clade) vs. O. miaoxinensis spec. nov. (in clade I of Epistylididae), and Telotrochidium cylindricum (in basal clade) vs. T. matiense (in clade I of Epistylididae), cannot be morphologically delimited at the genus level. This finding implied that both Operculariidae and Epistylididae may be non-monophyletic. Considering the widely accepted suggestion that the species in the basal clade is the ancestral morphotype of sessilids (Zhuang et al. 2018), this study inferred that the morphological consistency in both clades may be a consequence of atavism.

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T. Zhou et al. / European Journal of Protistology 70 (2019) 17–31

The present study supplemented morphological and molecular data of Epistylis and Opercularia species. For the first time, these results revealed that family Operculariidae may be non-monophyletic based on phylogenetic analyses, further highlighting the conflict between morphological taxonomy and molecular analyses. Thus, we suggested that enlarging taxon sampling with morphological and molecular characters is urgently needed to get an improved taxonomy.

Conclusion The present study provided detailed morphological and molecular descriptions of two new colonial sessilids isolated from Procambarus clarkii, namely Opercularia miaoxinensis spec. nov. and Epistylis conica spec. nov. In addition, molecular analyses challenged the validity of the shape of the peristome (epistomial lip present vs. absent) as a taxonomic character for separating Epistylididae from Operculariidae. These results will promote the revision of a more reliable taxonomy within sessilid.

Author contributions Tong Zhou collected samples; Tong Zhou and Zhe Wang carried out all laboratory works (in vivo observation, protargol impregnation, DNA extraction, analyses, etc.); Hao Yang assisted with part experiments (in vivo observation and protargol impregnation); Zemao Gu provided the synopsis of the manuscript; Tong Zhou wrote the manuscript; all authors revised the manuscript and agreed to the final version.

Acknowledgements This study was supported by the Technical Innovation Project of Hubei Province (No. 2018ABA103) and the Hubei Agricultural Science and Technology Innovation Center (2016620000001046).

References Aescht, E., Foissner, W., 1992. Biology of a high-rate activated sludge plant of a pharmaceutical company. Arch. Hydrobiol. 90, 207–251. Bishop, E.L., Jahn, T.L., 1941. Observation on colonial peritrichs (Ciliata: Protozoa) of the Okoboji region. Proc. Iowa Acad. Sci. 48, 417–421. Claparède, E., Lachmann, J., 1858. Etudes sur less infusoires et les rhizopodes. Mem. Inst. Nat. Genevois. 5, 1–260. Corliss, J.O., 1979. The Ciliated Protozoa: Characterization, Classification and Guide to the Literature, 2nd ed. Pergamon Press, London. Ehrenberg, C.G., 1830. Beiträge zur Kenntniss der Organisation der Infusorien und ihrer geographischen Verbreitung, besonders in Sibirien. Abh. dt. Akad. Wiss. Berl., pp. 1–88.

Ehrenberg, C.G., 1831. Über die Entwickelung und Lebensdauer der Infusionsthiere; nebst ferneren Beiträgen zu einer Vergleichung ihrer organischen Systeme. Abh. dt. Akad. Wiss. Berl., pp. 1–154. Ehrenberg, C.G., 1838. Die Infusionsthierchen als vollkommene Organismen. Ein Blick in das tiefere organische Leben der Natur. Voss, Leipzig. Fauré-Fremiet, E., 1905. Note sur un groupe nouveau d’Operclaria. Arch. Anat. Micr. 7, 181–197. Fernández-Galiano, D., Esteban, G., Mu˜noz, A., 1988. The stomatogenic process in Opercularia coarctata (Ciliophora, Peritrichida). J. Eukaryot. Microbiol. 35, 1–4. Foissner, W., 2014. An update of ‘basic light and scanning electron microscopic methods for taxonomic studies of ciliated protozoa’. Int. J. Syst. Evol. Microbiol. 64, 271–292. Foissner, W., Berger, H., Kohmann, F., 1992. Taxonomische und ökologische Revision der Ciliaten des SaprobiensystemsBand II: Peritrichida, Heterotrichida, Odontostomatida. Informationsberichte des Bayer. Landesamtes für Wasserwirtschaft, 5/92: 1–502. Fromentel, E., 1876. Études sur les Microzoaires du Infusoires Prosprement Dits, Comprenant de Nouvelles Recherches sur Leur Organization, Leur Classification, et la Description des Espèces Nouvelles ou Peu Cornus. G. Masson, Paris. Girard, C., 1852. A revision of the North American Astaci, with observations on their habits and geographic distribution. Proc. Acad. Sci. Phila. 6, 87–91. Goldfuss, G.A., 1820. Handbuch der Zoologie. In: Schubert, G.H. (Ed.), Handbuch der Naturgeschichte, zum Gebrauch bei Vorlesungen. J. L. Schrag, Nürnberg. Guhl, W., 1979. Opercularia coarctata, ein variables Peritrich. Arch. Protistenk. 121, 308–346. Imhoff, O.E., 1884. Resultate meiner Studien über die pelagische Fauna kleinerer und größerer Süßwasserbecken der Schweiz. Z. Wiss. Zool. 40, 154–178. Irwin, N.A.T., Sabetrasekh, M., Lynn, D.H., 2017. Diversification and phylogenetics of mobilid peritrichs (Ciliophora) with description of Urceolaria parakorschelti sp. nov. Protist 168, 481–493. Jankowski, A.V., 2007. Phylum Ciliophora Doflein, 1901. Review of taxa. In: Alimov, A.F. (Ed.), Protista: Handbook on Zoology, vol. 2. Nauka, St Petersburg. Jiang, C., Shi, X., Liu, G., Jiang, Y., Warren, A., 2016. Morphology and molecular phylogeny of two freshwater peritrich ciliates, Epistylis chlorelligerum Shen 1980 and Epistylis chrysemydis Bishop and Jahn 1941 (Ciliophora, Peritrichia). J. Eukaryot. Microbiol. 63, 16–26. Jiang, C.Q., Miao, W., 2017. Morphology of four freshwater peritrichous ciliates. Acta. Hydrobiologia Sin. 41, 652–660. Jiang, C.Q., Wang, G.Y., Xiong, J., Yang, W.T., Sun, Z.Y., Feng, J.M., Warren, A., Miao, W., 2019. Insights into the origin and evolution of Peritrichia (Oligohymenophorea, Ciliophora) based on analyses of morphology and phylogenomics. Mol. Phylogenet. Evol. 132, 25–35. Kahl, A., 1933. Ciliata libera et ectocommensalia. In: Grimpe, G., Wagler, E. (Eds.), Die Tierwelt der Nord- und Ostsee, Lief. 23 (Teil II, c3), pp. 29–146. Kahl, A., 1935. Urtiere oder Protozoa I: Wimpertiere oder Ciliata (Infusoria) 4. Peritricha und Chonotricha. Tierwelt Dtl. 30, 651–886.

T. Zhou et al. / European Journal of Protistology 70 (2019) 17–31

Katoh, K., Standley, D.M., 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780. Keiser, A., 1921. Die sessilen peritrichen Infusorien und Suctorien von Basel und Umgebung. Rev. Suisse Zool. 28, 221–241. Kent, W.S., 1881. A Manual of the Infusoria. Including a Description of All Known Flagellate, Ciliate, and Tentaculiferous Protozoa British and Foreign, and an Account of the Organization and Affinities of the Sponges, Vol. II. David Bogue, London. Linnaeus, C., 1758. Systemae Naturae, Vol. 1., 10th ed., pp. 823. Linnaeus, C., 1767. Systemae Naturae, 12th ed., pp. 1327. Lynn, D.H., 2008. The Ciliated Protozoa: Characterization, Classification, and Guide to the Literature, 3rd ed. Springer, Dordrecht. Ma, H., Overstreet, R.M., 2006. Two new species of Epistylis (Ciliophora: Peritrichida) on the blue crab (Callinectes sapidus) in the gulf of Mexico. J. Eukaryot. Microbiol. 53, 85–95. Medlin, L., Elwood, H.J., Stickel, S., Sogin, M.L., 1988. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71, 491–499. Miao, W., Fen, W.S., Yu, Y.H., Zhang, X.Y., Shen, Y.F., 2004. Phylogenetic relationships of the subclass Peritrichia (Oligohymenophorea, Ciliophora) inferred from small subunit rRNA gene sequences. J. Eukaryot. Microbiol. 51, 180–186. Moreira, D., Heyden, S.V.D., Bass, D., López-García, P., Chao, E., Cavalier-Smith, T., 2007. Global eukaryote phylogeny: combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata. Mol. Phylogenet. Evol. 44, 255–266. Müller, O.F., 1773. Vermium Terrestrium et Fluviatilium, seu Animalium Infusoriorum, Helminthicorum et Testaceorum, non Marinorum, Succincta Historia. Heineck et Faber, Havniae et Lipsiae. Nguyen, L.T., Schmidt, H.A., Von Haeseler, A., Minh, B.Q., 2015. Iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274. Nusch, E.A., 1970. Ökologische and systematische Untersuchungen der Peritricha (Protozoa, Ciliata) im Aufwuchs von Talsperren und Flussstauen mit verschiedenem Saprobitätsgrad (mit Modellversuchen). Arch. Hydrobiol. 37, 243–383. Pan, X., Bourland, W.A., Song, W., 2013. Protargol synthesis: an in-house protocol. J. Eukaryot. Microbiol. 60, 609–614. Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Roux, J., 1901. Faune infusorienne des eaux stagnates des environs de Genève. Mém. Inst. Natn. Génev. 19, 1–148. Shen, Y., Gu, M., 2016. Fauna sinica, Invertebrata vol. 45, Ciliophora, Oligohymenophorea, Peritrichida. Science Press, Beijing. Shen, Y.F., 1980. Description of six new species of periphytic protozoa in lake Dong Hu of Wuhan. Acta. Hydrobiologia. Sin. 7, 245–251. Shi, X., Meng, Q., Liu, G., Qi, G., Jiang, C., Meng, X., Warren, A., 2014. Morphology and morphogenesis of Epistylis plicatilis Ehrenberg, 1831 (Ciliophora, Peritrichia) from Wuhan, China. J. Morphol. 275, 882–893.

31

Stein, F., 1854. Die Infusionsthiere auf ihre Entwickelungsgeschichte untersucht. Wilhelm Engelmann, Leipzig. Stiller, J., 1935. Drei neue Peritrichen-Arten aus dem Balaton-See. Acta Litt. Scient. R. Univ. hung 3, 149–157. Stokes, A.C., 1884. Notices of new freshwater infusoria. Amer. Month. Microsc. J. 5–6, 226–230. Stokes, A.C., 1887. Notice of new freshwater infusoria. Amer. Month. Microsc. J. 8, 141–147. Sun, P., Al-Farraj, S.A., Warren, A., Ma, H., 2017. Morphology of four new solitary sessile peritrich ciliates from the Yellow Sea, China, with description of an unidentified species of Paravorticella (Ciliophora, Peritrichia). Eur. J. Protistol. 57, 73–84. Sun, P., Clamp, J.C., Xu, D., Huang, B., Shin, M.K., Turner, F., 2013. An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group. Proc. Biol. Sci. 280, 20131117. Sun, P., Clamp, J., Xu, D., Huang, B., Shin, M.K., 2016. An integrative approach to phylogeny reveals patterns of environmental distribution and novel evolutionary relationships in a major group of ciliates. Sci. Rep. 6, 21695. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA 6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Utz, L.R., Farias, A.C., Freitas, E.C., Araújo, G.O., 2014. Description of Epistylis riograndensis n. sp. (Ciliophora: Peritrichia) found in an artificial lake in Southern Brazil. Zootaxa 3869, 557–564. Utz, L.R., Simao, T.L., Safi, L.S., Eizirik, E., 2010. Expanded phylogenetic representation of genera Opercularia and Epistylis sheds light on the evolution and higher-level taxonomy of peritrich ciliates (Ciliophora: Peritrichia). J. Eukaryot. Microbiol. 57, 415–420. Vavra, J., 1962. Epistylis helicostylum n. sp. A new peritrichous ciliate with an allometric stalk formation. J. Eukaryot. Microbiol. 9, 469–473. Viljoen, S., Van As, J.G., 1983. A taxonomic study of sessile peritrichians of a small impoundment with notes on their substrate preferences. J. Limnologica. Soc. South. Afr. 9, 33–42. Viljoen, S., Van As, J.G., 1987. Notes on the morphology and asexual reproductive processes of sessile peritrichs. Hydrobiologia 154, 75–86. Wang, L.Q., Hao, R.J., Li, X.Q., Pan, H.B., 2016. Morphology of six freshwater ciliates. Acta Hydrobiol. Sin. 40, 343–349. Wang, Z., Zhou, T., Guo, Q., Gu, Z., 2017. Description of a new freshwater ciliate Epistylis wuhanensis n. sp. (Ciliophora, Peritrichia) from China, with a focus on phylogenetic relationships within family Epistylididae. J. Eukaryot. Microbiol. 64, 394–406. Yi, Z., Song, W., Clamp, J.C., Chen, Z., Gao, S., Zhang, Q., 2009. Reconsideration of systematic relationships within the order Euplotida (Protista, Ciliophora) using new sequences of the gene coding for small-subunit rRNA and testing the use of combined data sets to construct phylogenies of the Diophrys-complex. Mol. Phylogenet. Evol. 50, 599–607. Zhuang, Y., Clamp, J.C., Yi, Z., Ji, D., 2018. Phylogeny of the families Zoothamniidae and Epistylididae (Protozoa: Ciliophora: Peritrichia) based on analyses of three rRNA-coding regions. Mol. Phylogenet. Evol. 57, 155–161.