Ciliophora: Peritrichia) from Coastal Waters of Southern China

Title Morphology and Phylogeny of Four New Vorticella Species (Ciliophora: Peritrichia) from Coastal Waters of Southern China

Authors Liang, Z; Shen, Z; Zhang, Y; Ji, D; Li, J; Warren, A; Lin, X

Date Submitted 2018-07-26 Accepted Article This by is protectedarticle reserved. Allrights copyright. 10.1111/jeu.12668 differences betweenthis version and the Version of Record. Please cite this article doi: as throughpagination copyediting, the proofreadingtypesetting, and process, may which tolead Thishas article been accepted forpublication undergoneand peerfull reviewbut has not been d c b a Liang Ziyao Waters of Southern China New Four of Phylogeny and Morphology LIANG Article type : Original Article LIN ID:0000-0001-7886-7198) (Orcid DR. XIAOFENG 0000-0002-4878-9640) : ID (Orcid JI DR.DAODE SHEN(Orcid: ID0000-0002-6839-0418) MISS ZHUO Four new species of ABSTRACT 0644 +86-20-8521 Telphone/Fax: [email protected]; E-mail: X. Lin, Laboratory Protozoa,of South China Normal University,Guangzhou 510631, China * Correspondence contributed # Theseauthors Zhuhai, Biomonitoring, Schoolof Life Science, South China Normal University, Guangzhou 510631, China 6-11 between aboral trochal band and scopula. scopula. and band trochal aboral between 6-11 band, trochal andaboral peristome between silverlines 21-31 2; row than shorter is 1 row in which 3, polykinetid infundibular two-rowed a vacuole; contractile located dorsally single a macronucleus; staining. silver following and vivo infraciliature, and silverline systemwere investig V. paralima School of Ocean, Yantai University, Yantai 264005, China China 264005, Yantai University, Yantai Ocean, of School Laboratory of Protozoology, Guangzhou Key Laboratory of Subtropical Biodiversity and and Biodiversity of Subtropical Laboratory Key Guangzhou Protozoology, of Laboratory Department of Life Sciences, Natural Histor Natural Sciences, Life of Department Microbial Ecology and Matter CycleGroup, SchoolMarine of Sciences, Sun Yat-sen University, ET AL. 519082, China a,# sp. n., were isolated from coastal brackish waters of southern China. Their morphology, morphology, Their China. southern of waters brackish coastal from isolated were n., sp. – , Zhuo Shen , Zhuo Morphology and Phylogenyof Vorticella a, b, # equally tothis work. , Yong Zhang , Yong , V. parachiangi parachiangi V. Vorticella parachiangi parachiangi Vorticella a Vorticella , Daode Ji , Daode y Museum, Cromwell Rd., London SW7 5BD, UK. 5BD, SW7 London Rd., Cromwell Museum, y Vorticella Vorticella sp. n., Vorticella scapiformis Vorticella ated based on observations of specimens both in in both specimens of observations on based ated Species (Ciliophora: Peritrichia) from Coastal Coastal from Peritrichia) (Ciliophora: Species c , Jiqiu Li , Jiqiu V. scapiformis V. Species sp. n. is distinguished by: a J-shaped J-shaped a by: distinguished is n. sp.

a , Alan Warren Alan ,

sp. n., n., sp. sp. n. is characterized by its its by characterized is n. sp. V. sphaeroidalisV. d , Xiaofeng Lin , Xiaofeng a,* a,* sp. n., and and n., sp.

Accepted Article This by is protectedarticle reserved. Allrights copyright. scopula. band and trochal aboral between two band, trochal aboral and persitome between silverlines 16-18 scopula; the to adjacent band trochal aboral an vacuole; contractile located ventrally a macronucleus; scopula. and band trochal aboral between 8-12 band, trochal aboral and peristome between silverlines infund the of 1 row vacuole; contractile located irregularly and conspicuously thin solitary Vorticellidae with an unbranched, spirally contractile stalk. Subsequently, Subsequently, stalk. contractile spirally unbranched, an with Vorticellidae solitary the to it limited who (1838), Ehrenberg by refined was definition generic The peritrichs. of genus paper. this of scope the outside is family of rank to the rankoforder, i.e., Vorticellida and Operculariida. Discussion of peritrich taxonomy above the molecular phylogeny, al. Utzet suggested (2010) that the two sessilid sub-clades should be assigned Operculariina Jankowski, 1980, as recognised Jankowskiby Based (2007). on analyses of their comprise two major sub-clades, i.e.,the refined sub-orders Vorticellina de Fromentel. 1875and (Foissner etal. 2010; 2015;Lynn Li etal. 2008; et Shen 2017; al. Sun et 2015).al. sessilids The ofciliates thatcommonly are found in a wide varietyof marine, freshwater, and terrestrial habitats THE peritrich order Vorticellida which comprises100 genera, over a large, distinctiveis assemblage Keywords: analyzed. was phylogeny gene rDNA SSU The scopula. to band trochal aboral 3 proximally shortened; silverlines 26-35 from polykinetid infundibular the of and2 1 rows vacuole; contractile positioned dorsally a macronucleus; The exposed part of the oral region is the peristome, a generally flattened surface with a thickened athickened with surface flattened a generally peristome, the is region oral the of part The exposed sub-spherical. or conical cylindroid, maybe but bell inverted an like shaped usually is zooid, or body, additional 113species, varieties, or forms of an to or taxa, ciliate other to genera, other peritrich reduced itsmembership to95species following elimination the many of that obviously belonged to and by1930, genus the to been assigned had that varieties named or species 220 listing genus, the comprehensiverevision thefirst of published Finley(1931) created. and was since Noland it the largest genus of sessile peritrichswithover 300species suspended particles into the oral opening or infundibulum. The shape and size of the zooid, width of of width zooid, the of size and shape The infundibulum. or opening oral the into particles suspended carries that vortex a create to beat and peristome the around counterclockwise run polykinety, border or lip and araised epistomial indisc center. the Two bandscilia, of the haplokinety and og19;Sne l 06 09 07. Song Sun1997; etal. 2006, 2009, 2017). new species of excluded over100dubiousspecies, reducing themembership total of the genus to 86species. Many who (1986) Warren by genus the of revision major next the of publication and paper and Finley's The specialized morphology of The specialized morphology Vorticella sphaeroidalis Vorticella Biodiversity; brackish water; mangrove wetland; Vorticellida; taxonomy. taxonomy. Vorticellida; wetland; mangrove water; brackish Biodiversity; Vorticella Vorticella paralima Vorticella have since been described, raising themore again total to than 100 (e.g., sp. n. can be identified by small, its sub-spherical zooid; aC-shaped Vorticella edged peristomial lip; a J-shaped macronucleus; a single, ventrally ventrally single, a macronucleus; J-shaped a lip; peristomial edged sp. n. can be identified by its ovoidal zooid; a J-shaped J-shaped a zooid; ovoidal byits beidentified can n. sp. reflects its sessile, suspension-feeding lifestyle. The cell Vorticella peristome to aboral trochal band, and 7-13 from from 7-13 and band, trochal aboral to peristome ibular polykinetidproximally 3 shortened; 18-25 Vorticella s of these four species were sequenced and their their and sequenced were species four these of s imal phyla (e.g., rotifers, cnidarians, crinoids). An An crinoids). cnidarians, rotifers, (e.g., phyla imal were described in the period between Noland Noland between period the in described were Linnaeus, was 1767 the first reported

having beendescribed inthe 250years

Vorticella became Accepted Article This by is protectedarticle reserved. Allrights copyright. Vorticella parachiangi collection Sample AND METHODS MATERIALS genus the of members benew to them revealed sequences rDNA gene (SSU) subunit small their of analysis and nitrate), silver and (protargol staining silver and following diversitycoastal in waters of southern China. Detailed investigationsof specimens, the both vivoin species have shown that se gene on based studies Phylogenetic 2012). al. information has also been included in speciesdescriptions and redescriptions etal. (Ji 2015; Sun et sequence gene recently, More 2017). 2006, al. et (Sun species of determination in the used routinely arenow characters These protargol. with bystaining revealed be can which infundibulum, the within polykineties infundibular the of structure the and microscopy, electron scanning the zooid andcan be visualiz species circumscription and identification. includeThese transversethe pellicular ridges that encircle Inthe last 50years, silver staining methods have usedbeen toreveal additional characters for Noland and Finley 1931; Warren 1986). creationof numerous species andvarieties of doubtful taxonomic validity (Itabashi al. 2002;et the in resulting nature contractile highly its and size and shape zooid variable of its because difficult of characters. Species been usedas taxonomic identification have lip peristomial the of thickness and disc epistomial the of contour and height peristome, the salinity of 5‰, and apH of about 7. a °C, 23 of temperature water a at China, Province, Guangdong ofShantou, 116°68É) (23°38Ń; Vorticella scapiformis °C, a salinity of ‰,25 a and pH of about 7. Island 110°44É) (21°16Ń; in Zhanjiang,Guangdong Province, China, at awater temperature of 25 Wilbert (1995), respectively. Counts and measurem and Counts respectively. (1995), Wilbert following and Wilbert (1975) Chatton-Lwoff the nitrate-stain silver method following Song and (Nikon 80i,Japan). The infraciliature and silverline were system revealed byprotargol the method Ciliates were observed in usingvivo bright field studies Morphological salinity of 23‰, and of apH about 7.8. a °C, 23 of temperature water a at China, Province, Guangdong Zhanjiang, in 110°28É) (21°53Ń; Vorticella sphaeroidalis temperature of 25°C, a salinity of 12‰,andof a pH about 9. Donghai Island 110°34É)(21°02Ń; in Zhanjiang,Guangdong Province, China,a atwater Vorticella paralima In the present study, four new solitary vorticellids were discovered during a survey of ciliate of survey discovereda during were new solitary four vorticellids In thepresent study, sp. n.sp. collected was on 11 sp. n.collected was on 14 sp. n. was collected sp. was n. on 21 Vorticella sp. n. wascollected sp. n. on 5 ed either as as silverlines by staining with silver nitrate or by directly withby silverlines ed eitheras nitrate as silver staining i aahltcgop(a ta.21;Sne l 02 03. a paraphyletic is group (Gao al.et 2016; Sun etal. 2012, 2013). and differential interference contrast microscopy contrast interference differential and th th quence data derived from carefully identified identified carefully from derived data quence October 2009 from Gaoqiao mangrove wetland wetland mangrove Gaoqiao from 2009 October March from 2010 a mangrovewetland on Techeng st th March 2010from asewagedischarge outlet on ents of stained specimens performedwere at May 2011from a small ditch near the coast coast the near ditch asmall 2011from May Vorticella

is, however, frequently very frequently however, is, Vorticella . Accepted Article n 9rgos h egho h ia lgmn a ,1 p bp. 1,616 was alignment final of the Thelength regions. and V9 V4 variable highly the in and alignment of the termini both at gaps ambiguous removing by refined Sequences were aligned with the ClustalW Multiple alignment in BioEdit 7.2.5(Hall 1999) and then and lengths. The hymenostomes Thehymenostomes lengths. rDNA three of obtained sequenced previously 52 and sequenced newly This by is protectedarticle reserved. Allrights copyright. phyl usedfor were taxa Fifty-six likelihood (ML). min; 72 °C for 2min) and one cycle for the final extension (72 °C 10min).for cycle for initial denaturation °C (94 for 35cycles 5min); for amplification °C(94 for 56°C 1min; for 2 (5’-TGATCCTTCTGCAGGTTCACCTAC-3’) Cycling etal. 1988). (Medlin parameters wasamplified PCR,following using by the prim rDNA sequence SSU The Kit. PCR Tissue Extract-N-Amp RED the using extracted, was DNA Genomic analysis phylogenetic and sequencing gene rDNA SSU (2008). Lynn to according are terminology and Classification magnification. 1,000× at lucida camera 1,000× magnification.Drawings of stained specimens wereconducted by handwith thehelp of a faoa rca ad irncesntosre. observed. not Micronucleus band. trochal of aboral level to longitudinally almost extends portion posterior hook-shaped infundibulum, around oriented horizontally portion apical J-shaped, Macronucleus 2D). 1A, (Fig. expanded fully when in diameter µm 10 approximately infundibulum, of wall dorsal near vacuole Contractile C). 1A, (Fig. in diameter visible at1000×magnification (Fig. 2E,arrows). Telotroch not observed. occasionally slightly foldedonventral and dorsal(Fig. sides 1C, 2D). Transverse pellicular striations cilia 12 about oral of slightly Length C, C). elevated (Fig. 2A, above1A, peristome Perist peristome. at width maximum 1:1, about width to length of ratio vivo, µm in µm ×55-70 50-65 size expanded, when campanulate inverted Zooid of Description RESULTS of ICZN urn:lsid:zoobank.org:pub: 2012): C1F52FCF-46AB-4444-900E-1CD9B58FA00A. ZooBank registration: 1,000. of frequency base were constructedwithPhyML the 3.0 program (Gui sequences the from inferred trees likelihood TheMaximum 2015). al. et (Li burn-in as discarded 1,000,000 generations with trees sampled 100generations;every the were firstof 25%trees with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). chainThe length for the analysis was constructed was tree inference Bayesian majority-rule % A 50 2004). (Nylander 2.2 MrModeltest imtr preydsrbtdaogislnt Fg F. 2F). (Fig. length its along distributed sparsely diameter) spasmoneme about 2µm diameter,in with astring of The tree topologies were inferred, using the best-fit model GTR model best-fit the using inferred, were topologies tree The Phylogenetic trees were constructed by two methods: Bayesian inference (BI) and maximum maximum and (BI) inference Bayesian methods: two by constructed were trees Phylogenetic Stalk up to 350 µm long and 4 µm in diameter, surface smooth without attached bacteria. Stalk Stalk bacteria. attached without smooth surface diameter, in µm 4 and long µm 350 upto Stalk µm 10-15 about vacuoles food few a containing usually gray, slightly semi-transparent, Cytoplasm V. natans, V. natans, Vorticella were removed from the alignment after pre after alignment removed the were from Vorticella parachiangi parachiangi Vorticella ZooBank registration number of the present work (see Recommendation 8A 8A Recommendation (see work present the of number registration ZooBank species sequenced by Sun et al. (2013), namely, namely, (2013), al. et by Sun sequenced species Tetrahymena bergeri p . n. sp. ers: EukA (5’-AACCTGGTTGATCCTGCCAGT-3’), EukB EukB EukA (5’-AACCTGGTTGATCCTGCCAGT-3’), ers: ogenetic analyses, includ and ndon 2005) etal. with a parametric bootstrap omial lip thin, ca. 4 µm thick. Epistomial disc disc Epistomial thick. µm ca. 4 thin, lip omial light gray thecoplasmic granules (ca. 0.5 µm in T. corlissi T.

from the NCBI GenBank database. The SSU TheSSU database. GenBank NCBI the from liminary analysis because of short their

were selected as outgroup taxa. outgroup as selected were ＋ I ＋ G selected by AIC criterionin AIC G by selected V. chlorostigma, striata V. ingfour the thatwere

were as follows: one one follows: as were μ m. Pellicle Accepted Article urudn pnn fifniuu Fg D D ) irncesntosre. observed. not Micronucleus H). 6D, 5D, (Fig. of infundibulum opening surrounding oriented, transversely C-shaped, Macronucleus 6C). B, 5A, (Fig. expanded when fully diameter µm in 6 approximately infundibulum, of wall near ventral located vacuole Contractile 6D). (Fig. diameter granules visibleon zooid surface × magnification at 400 (Fig. 5E, 6E, G). Telotroch observed.not persitome (Fig. 5A, 6A, Length B). B, of ciliaoral about7 moderate Peristomial across. approximatelylip 15µm Zooid almost globular,20-30 µmsize µm ×15-25 vivo, in maximumwidth at mid-body. Peristome periphery (Fig. 3D, 4F). Haplokinety and polykinety make approximately 1¼ turns around peristome peristome around turns 1¼ approximately make polykinety and Haplokinety 4F). 3D, (Fig. periphery µm diameter in withdark gray thecoplasmicgranules (ca. 0.5µm indiameter) sparsely distributed at Pelliclular striationsclosely spaced, visible atmagnification 1,000× (Fig. 4E). almost longitudinally tolevel of aboral trochal band (Fig. Micronucleus3E, 4G). not observed. extends portion posterior hook-shaped infundibulum, around oriented horizontally portion apical J-shaped, Macronucleus B,4C). 3A, (Fig. expanded whenfully diameter in µm 12 approximately length, body % of 25 about at infundibulum of wall ventral near located vacuole Contractile (Fig. D). 3A,vacuoles 4A, µm (8-10 food in diameter) sphericalin diameter) and µm (3-6 granules Length of oral cilia about 10 rim.Epis Peristome on with protuberances irregular peristome,withconstriction posterior peristomialto lip, which approximately is thick. 2µm at width maximum vivo, in µm ×40-55 µm 40-50 size state, expanded in obconical elongate Zooid of Description trochal band and scopula;with sparsely dist aboral between 6-11 and band trochal aboral and peristome between silverlines of 21-31 consists pe from %distance 80 at zooid encircles dikinetidal, band trochal Aboral 2H). 1F, (Fig. infundibulum of entrance at membranelocated Epistomial haplokinety. to parallel infundibulum, of half distal in lies kinety Germinal 2H). 1F, (Fig. P1 end of proximal the to extends P3 of 2 row while P2, with together terminates and 2 row of length half only is P2, to nearest one the i.e. P3, of 1 Row F). 1E, (Fig. two-rowed is P3 kinetosomes, of rows distinct three of composed are P2 and P1 infundibulum. of half lower at (P1-3) polykineties infundibular three into transforms Polykinety turn. afurther andmake separate they where infundibulum Description of of Description 1). Table 4I; (Fig. scopula and band trochal aboral between 8-12 and band trochal aboral peristome scopulato Silverline (Fig.3E). system consists of silverlines18-25 between peristome and from distance % 85 at zooid encircles dikinetidal, band trochal Aboral infundibulum. of entrance near located membrane Epistomial 4H). F, 3E, (Fig. haplokinety to parallel infundibulum, of portion distal in lies kinety Germinal P3. 1 of row above just P1, of curvature the near terminates P2 of P1. terminates at same level latter.ofas P3 Rowthe shortened 1 proximally and terminates at curvature of kinetosome three each.converges rows P1 wi composed P1-3 infundibulum. of half lower at (P1-3) polykineties infundibular three into transforms Polykinety half-turn. further a make and separate they where infundibulum entering before This by is protectedarticle reserved. Allrights copyright. Cytoplasm semi-transparent, slightly grayish and containing a few food vacuoles, 3-5 µm in in µm 3-5 vacuoles, food few a containing and grayish slightly semi-transparent, Cytoplasm Haplokinety and polykinety describe about 1¼ turns around peristome and continue into into continue and peristome around turns 1¼ about describe polykinety and Haplokinety Stalk 500-600 µm in length and 6 µm in diameter with smooth surface. Stalk spasmoneme about 2 2 about spasmoneme Stalk surface. smooth with diameter in µm and 6 length µm in 500-600 Stalk Cytoplasm semi-transparentslightly andgrayish, us Vorticella sphaeroidalis sphaeroidalis Vorticella scapiformis Vorticella μ m. Telotroch not observed. Telotroch not m. p . n. sp. p . n. sp. ributed pellicular pores (Fig. 1D, Table 1). 2I; th P3 in proximal portion of infundibulum and and infundibulum of portion proximal in P3 th tomial disc slightly convex (Fig. 4A, 3A, B). C, ristome to scopula (Fig. 1E, 2G). Silverline ristomesystem 2G). 1E, toscopula(Fig. ually containing afew toseveral ellipsoidal or ly thick. Epistomial disc elevated high above ly elevated high above disc thick. Epistomial μ m. Pellicular striations several and grayish

Accepted Article bacteria and algae. Spasmoneme about 2 µm in diameter; thecoplasmic granules not observed (Fig. (Fig. observed not granules thecoplasmic diameter; in µm 2 about Spasmoneme algae. and bacteria observed. not Micronucleus 8F). (Fig. band trochal aboral of level below slightly terminates portion posterior hook-shaped infundibulum, around oriented horizontally portion apical J-shaped, Macronucleus C). 8A, (Fig. expanded whenfully diameter in µm 7 about zooid, of fourth anterior in infundibulum of wall dorsal at located vacuole Contractile diameter. µm in 3-5 striations observable at x 1,000 magnification (Fig. 8D, E). elevated about 4µm above peristome.cilia Oral about µm long12 C). (Fig. Pellicular 8A, B, 7A, lip (Fig. 8A, 7A,B, PeristomialB). lip everted and thin,approximately µm thick.Epistomial 3 disc 20-30 µm in vivo. Cell usually proper widest mid-body, at withconstriction posterior peristomialto Zooid obovoid, with truncated oral end and narrowedaboral end inexpanded state, size 35-50µm × of Description Table 1). between aboral trochal band and scopula; with sparsely distributed pellicular pores (Fig. 5E, 6J, K; distance % 88 at zooid encircles band dikinetidal, improper orientationspecimen observation.for P3very likely two-rowed (Fig. D).Aboral5C, trochal P3 of Arrangement kinetostomes. of rows distinct three of composed are P2 and P1 infundibulum. of half lower at (P1-3) polykineties infundibular three into transforms Polykinety turn. afurther andmake separate they where infundibulum granules (ca. 0.3µm indiameter) distributed in periphery. bacteria and algae. Spasmoneme about µm 1 in diameter, with several slightlygrayish thecoplasmic GCcontent as follows: and length with database GenBank in the beendeposited have sequences rDNA SSU new The four sequence rDNA SSU of Analyses Table 1). I; 8H, 7F, (Fig. pores pellicular distributed irregularly numerous with scopula; to band trochal aboral 75 % distance from peristome to scopula (Fig. 8F).7D, at zooid encircles dikinetid, band trochal Aboral 7E). (Fig. infundibulum of entrance near membrane 8G).Germinal kinety distal liesin portion of infundibulum parallel to haplokinety.Epistomial the end Rows of P1. 2 ofP3 1and halfare of the length row3,terminating together with P2 (Fig. 7D, near terminates it where infundibulum of end the to extends P3 of 3 Row latter. the of curvature at converge P3 and P1 three-rowed. P1-3 infundibulum. of half lower at (P1-3) polykineties infundibular three into transforms turn. Polykinety another andmake separate they where infundibulum 7B). This by is protectedarticle reserved. Allrights copyright. only here tree is shown the (Fig. ML 9). In all phylogenetic analyses, new three species the clustered Phylogenetic trees constructed with analyses had almost and BI ML identical topologies, therefore 43.66%; Silverline system consists of 16-18 silverlines between peristome and aboral trochal band and two two and band trochal aboral and peristome between silverlines 16-18 of consists system Silverline into extend and peristome around turns 1¼ approximately make polykinety and Haplokinety attached some with occasionally smooth surface µmacross, 2 µm long, 40-50 approximately Stalk from 7-13 and band trochal aboral to peristome from silverlines 26-35 of consists system Silverline into extend and peristome around turns 1½ approximately make polykinety and Haplokinety attached a few with smooth surface diameter, in µm 4 and µmlong 80-120 approximately Stalk vacuoles food several to few a containing usually grayish, slightly and semi-transparent Cytoplasm V. sphaeroidalis V. Vorticella paralima paralima Vorticella Vorticellaparachiangi sp. n., 1,742 bp, 43.51%; sp. n.

sp. n.,sp. 1,729 bp, 44.07%; fo eitm osoua(i.5,6) 6G). 5D, (Fig. scopula to peristome from unclear due to limited numberof specimens and V. paralima sp. 1,651 n. bp, 43.50%.

V. scapiformis V. sp. n., 1,727 bp, Accepted Article vacuoles (Song and Warren 2000). 2000). Warren and (Song vacuoles parachiangi transverse) silverline system. low magnifications, although can separated all three be from the by reticulatelatter having a (vs. 1870) Ji et al., 2009and 1991). 24-32 has a similar zooid shape, but these species differ thezooid in size invivo (50-65 closely resembles that congener Another (two-rowed three-rowed) vs. (Sun al. 2017).et ciliarywreath; 6-11 vs.10-14 between aboralciliary wreath and scopula),structure and the of P3 35-50 (50-65 vivo in size zooid the by latter fromthe separated be can it Nevertheless, Vorticella parachiangi of Comparison of thesecharacters. combinations on based taxa similar other and congeners their from separated be each could species structure of P3), features of silverlinethe system, and habitat etal. (Sun 2006, four The 2017). new morphology (appearance of peristomial lip and epistomial disc),oral infraciliature (especially oral macronucleus, the of position and shape vacuoles, contractile of location and number in circumscription species for characters accepted Commonly DISCUSSION of grouping the to sister is that clade form a with differential support (99% ML, 0.53 BI). together with high support ML, (95% sister 1.00BI)and to are the This by is protectedarticle reserved. Allrights copyright. maximal support (100% ML, 1.00BI), Vorticella withof species sp. n. can be separated from from separated be can n. sp. n. sp. of Comparison (6-11 vs. 11-14), and structure the (two-rowed of P3 vs. three-rowed) al. 2006). et (Ji zhengae zooid vivo (50-65 sizein transverse silverlines both between the peristome and the aboral trochal band and between the the between and band trochal aboral the and peristome the between both silverlines transverse Vorticella similis (two-rowed three-rowed) vs. (Sun al. 2009). et contractile vacuole (in the anterior 1/4 of zooid vs. in mid-zooid) in vs. zooid of 1/4 anterior the (in vacuole contractile and 8- (18-25 scopula the and band trochal aboral Living cellsLiving three of

in terms of zooid shape, macronucleus shape, and habitat (Song 1997). 1997). (Song habitat and shape, macronucleus shape, zooid of terms in μ μ m ×24-37 m ×35-50 by the number of transverse silverlines between the aboral trochal band and the scopula scopula the and band trochal aboral the between silverlines transverse of number the by - Pseudovorticella Pseudovorticella sp. n.(55-110 Vorticella scapiformis scapiformis Vorticella Vorticella Vorticella parachiangi parachiangi Vorticella Stokes, 1887 and μ μ m) and the shape of the macronucleus (J-shaped vs. horseshoe-shaped) (Song (Song horseshoe-shaped) vs. (J-shaped macronucleus the of shape m) andthe m), number the of silverlines (21-31 vs. between 33-37 peristome and aboral sp. n. resembles Pseudovorticella P. zhengae P. μ and and m × 50-65 50-65 m × μ subclade, subclade, m × 50-100 50-100 m ×

V. similis In addition, addition, In Pseudovorticella V. spuripicta Vorticella parachiangi Vorticella Ji et al., 2006, bear some resemblance to to resemblance some bear 2006, al., et Ji μ V. parachiangi V. V. sphaeroidalis sphaeroidalis V. m vs. 40-50 by by zooidthe shorter (40-50 sp. n. with congeners. congeners. n.with sp. sp. n. with related species. species. with related sp. n. species,namely, V. chiangi μ m vs. 50-65 P. patellina V. parachiangi V. V. parachiangi parachiangi V. Vorticella paralima Vorticella withinthe family Vorticellidae. Within the Song, 1997bear some resemblance to Vorticella parachiangi Sun et al., 2017in termsof itszooid shape. μ 12 vs. 75-80and 28-34) and in the position of the m × 35-45 ×35-45 m sp. issister groupn. to hasmuch a zooid larger vivoin than μ sp. n. and m 50-65 × P. patellina P. sp. n. can be separated from separated be can n. sp. sp. is n. sp. and n.

μ Vorticella m) and the structure of m)P3 of thestructure and Vorticellides astyliformis

sp. and n. Sn 97. (Song 1997). μ μ Vorticellides aquadulcis- Vorticellides m) and two (vs. one)contractile V. fornicata Müller, 1776, m vs.52-58 sp. n. can be separated from from separated be can n. sp. V. scapiformis scapiformis V. include zooid shape and size, size, and shape zooid include V. scapiformis Pseudovorticella V. parachiangi V. Vorticella scapiformis (Dons, which 1915), μ μ μ m), fewer fewer m), P. marina m 50-65 × m × 50-65 50-65 m × p . n. sp. P.marina V. scapiformis scapiformis V. cluster cluster V. sp.with n. complex complex sp. sp. atn. (Greeff, (Greeff,

species μ μ m vs. m vs. m vs. m vs. by by P.

Accepted Article from distinguished and indistinct (vs. distinct)pellicular striations (Song 1991; Stokes 1885). utriculus Comparison of Comparison 1997). (Song vacuoles contractile one) (vs. two having in and respectively), and 8-12, 18-25 vs. 11-15 and (35-39 scopula the and peristome the both and band trochal aboral the between This by is protectedarticle reserved. Allrights copyright. congeners, close scopula. Three the band to of trochal theaboral location the and zooid, globular and small its of combination by the congeners its from distinguished observed, clearly be not could P3 of structure the Although Vorticella sphaeroidalis pulchella V. paralima Like 2006). al. et (Sun habitat marine) positionof aboralits trochal band, the globular (vs. barrel-shaped) zooid, and its brackish water (vs. Nevertheless, from latter the conspicuously formerin the tothe more respect differs posterior Living Living cells of number of silverlines between aboralthe trochal band and the scopula vs. 7-13) (Song (5-7 1991). (22-40 vivo in zooid shorter vivo (35-50 Vorticella transferred to the genus closely The congener most that resembles of Comparison 2009). al. et (Sun band trochal aboral its of position posterior more conspicuously the peristomial lip, double-layered) single-layered (vs. spaced pellicular silverlines. However, positionof aboral the trochal band (Sommer 1951). (20-30 size zooid shorter much its by separated be can species appearance in vivo. However, the latter species can be clearly separated from from separated clearly be can species latter the However, vivo. in appearance 1991). (Song J-shaped) vs. (C-shaped macronucleus the of shape the by be separated can two the Nevertheless, of silverlines. number and habitat and size, position contractileof the vacuole (Kahl 1933). ventral) (vs. dorsal the (4) marine); vs. (brackish. habitat (3) posteriorly); recurved not and anteriorly curved slightly only elongate, vs. portion posterior recurved a and portion anterior horizontal Vorticella spuripicta The presentform issimilar to The Vorticella hamata Vorticella sinica V. sphaeroidalis V. can be distinguished from from bedistinguished can Sommer, 1951, and byWarren (1986). μ sp. n. sp. m vs. 60-70 m vs. 60-70 Vorticella sphaeroidalis sphaeroidalis Vorticella Vorticella paralima paralima Vorticella V. utriculus V.

in terms of their zooid size and shap and size zooid of their terms in resembles V. paralima paralima V. Ehrenberg, also 1831 resembles differsfrom sp. n., n., sp. sp. n.resembles sp. Pseudovorticella μ m); (2) the shape of the macronucle the of shape m); (2)the Stokes, 1885 and μ Vorticella m vs. 35-50 35-50 vs. m V. sphaeroidalis sphaeroidalis V. V. sinica V. V. pulchella V. sp. n. its by C-shaped (vs. J-shaped) macronucleus (Kahl 1935). V. striata V. scapiformis V. V. paralima paralima V. sp. n. with congeners.

Sun et al., 2009, have compared tobe with the new species. V. sphaeroidalis sphaeroidalis V. paralima sp. n. with congeners. congeners. with n. sp. V. parapulchella V. by Jankowski (1976), and transferred back to the genus genus the to back transferred and (1976), Jankowski by has widely spaced pellicular spaced widely has μ Dujardin, 1841 in termsof body shapeand general Vorticellaparalima m), C-shaped J-shaped) (vs. macronucleus, andthe V. aequilata V. sp. in n. terms of zooid size and shape and widely sp. n. by C-shaped sp. n. its J-shaped) (vs. macronucleus sp. n. differs from from differs n. sp. sp. n. sp. inrespectnumber tothe of silverlines very likelytwo-rowed (vs. three-rowed) P3, and V. paralima e and numbers of silverlines. e andnumbers However, sp. n. can be separated from the former by its its by former the from separated be can n. sp. in zooid size and number of silverlines. Kahl, also 1935 bear some resemblance to V. sphaeroidalis sphaeroidalis V.

sp. n.sp. is us (very long, with a distinctly distinctly a with long, (very us μ m vs. 40-46 sp. n. in terms of its zooid shape shape zooid its of terms in n. sp. V. parapulchella V. V. lima striations. However, the new new the However, striations. V. lima V. Vorticella aequilata by: (1) shorter by: (1) its zooid in sp. n. can be easily easily can be n. sp. μ Kahl, 1933which was m) and m) and more posterior V.

paralima Sun et al., 2006, sp. nby its canbe V. V. V. Accepted Article Vorticella chiangi Vorticella Etymology: Etymology: NHMUK2018:3:1.1. registration with History UK, number the Natural London, Museum, SZ-2010-0314-01-3. paratypeOne slidewith silver nitrate-stained specimens is deposited in the Laboratory of Protozoology, Ocean of Qingdao, China, University China with the registration number slides: of Deposition Guangdong Province, China. Water temperature 25 °C, salinity pH 25 ‰, about7. Type habitat: locality and length together of and row terminates with2, 2of P3extends P2; toproximalrow of endP1. the half only 1 row two-rowed; P3 scopula. and band trochal aboral between 6-11 band, trochal transv 21-31 located. dorsally vacuole contractile Diagnosis. Vorticella parachiangi This by is protectedarticle reserved. Allrights copyright. Genus Family Vorticellidae Ehrenberg, 1838 Order Sessilida Kahl, 1933 SUMMARY TAXONOMIC P3. of structure shape, andthe globular µm), × 15-25 µm (20-30 sphaeroidalis Opisthonecta species, such as barrel-shaped between small, relationship aclosephylogenetic supported data also Molecular P3. <60 (usually barrel-shaped zooid asmall membranes,epistomial genus the newly established transferred afew (2010) Foissneral. the Astylozoidae.et to sister is subcladethat Pseudovorticella of subclade the to is sister that group a toform support maximal with cluster together 2006). 2004, 2003, al. et (Ji system silverline Pseudovorticella a indicates veryof close phylogenetic relationship with two cluster andboth maximalsupport . 2013). al. 2012, et Sun 2015; al. Li et 2015; al. et (Ji studies previous with tree Thetopology Vorticellidae. our isconsistent of of group the sister the Astylozoidae, infusionum V. Pseudovorticella of Vorticellidae, genera assemblage of morphologically groups diverse (M In the decade,past studies several have suggested that the family Vorticellidae a is nonmonophyletic four of positions Phylogenetic and In this In this study, Vorticella sphaeroidalis Vorticella new Among the Epicarchesium Vorticella Zooid inverted bell-shaped, µm 50-65 50-65 × The Greek word spp. and spp. sp. n. clusters with members of members of with clusters n. sp. , Linnaeus, 1767 1767 Linnaeus, species. However, they can be clearly differentiated by the zooid the by and shape zooid differentiated they beHowever, can clearly species. V. mayeri V.

species and and species species in the family Vorticellidae whereas the remainder, including including remainder, the whereas Vorticellidae family the in species Sun et al., 2016. Vorticella Vorticella Jankowski, were 1985 reported nonmonophyletic be to et al. (Sun 2016). V. astyliformis V. astyliformis

species, species, A protargol slide with the holotype specimen circled by ink is deposited in the the in deposited is ink by circled specimen holotype the with slide A protargol Astylozoon enriquesi Astylozoon sp. n. n. sp. Vorticella Mangrove wetland on Techeng Island in Zhanjiang (21°16´N; 110°44´E), sp. n. clusters sp. n. with , para para species are distributed among two clades: the majority cluster with cluster majority clades: among the two are distributed species V. paralima V. paralima V. paralima V. paralima V. sphaeroidalis V. Vorticellides Vorticella (similar, near) indicates that the type population is similar to to similar is population type the that indicates near) (similar, Linnaeus, 1767, and species within the Vorticellidae Vorticellidae the within species V. microstoma V. sp. n. n. sp. sp. n. groups with with groups n. sp. Foissner et al., 2010 based on the possession of of two et 2010basedon al., possession the Foissner (Foissner et al. 2010). Thus, it is not surprising that that surprising not is it Thus, 2010). etal. (Foissner Pseudovorticella Vorticellides Vorticella parachiangi sp. n. sp.n. and Vorticellides erse silverlines between peristome and aboral erse silverlines aboral peristome and between iaoet al. Sun 2004; etal. 2012, 2013). Among the Pseudovorticella Pseudovorticella , and certain stalkless peritrichs such as as such peritrichs stalkless certain and , µm in vivo. Macronucleus J-shaped. Single Single J-shaped. Macronucleus vivo. µm in and Astylozoidae based on its small zooid zooid small its on based Astylozoidae and V. paralima Vorticellides species to form a strongly supported supported astrongly form to species

species with a strongly support. This speciesa strongly with support. Pseudovorticella μ Foissner and Schiffmann, 1974, m long), m and along), two-rowed minute sp. n. and sp. n.and species, group with the with group species, sp. n. with these three these three with sp. n. V. scapiformis

Vorticella punctata

V. microstoma V.

first with first species to to species

sp. n. n. sp. V. V. , Accepted Article dorsally located. 26-35 silverlines between perist between silverlines 26-35 located. dorsally vacuole contractile Single vermiform. or J-shaped Macronucleus width. body maximum to equal Diagnosis paralima Vorticella of ICZN urn:lsid:zoobank.org: 2012): act:F91AC656-E89C-4309-9FB8-5F9BE331F530 ZooBank registration: KY594766. sequence. Gene Etymology: Natural History Museum, London, UK, with registrationthe number NHMUK 2018.3.1.3. SZ-2011-0505-01-2. paratypeOne slidewith silver nitrate-stained specimens is deposited in the Laboratory of Protozoology, Oceanof Qingdao,University China, China with the registration number Type specimens: Province, China. Water temperature °C, 23 salinity‰, pH 5 about 7. Type habitat: locality and scopula. and band trochal aboral between two band, trochal aboral and peristome between Macronucleus C-shaped. Single contractilevacuole ventrally located. transverse 16-18 silverlines Diagnosis: sphaeroidalis Vorticella of ICZN urn:lsid:zoobank.org: 2012): act:06390D5C-670F-4899-87FC-A9D6E05D23A8 ZooBank registration: KY594768. sequence. Gene body shape. This by is protectedarticle reserved. Allrights copyright. Etymology: Natural History Museum, London, UK, with registrationthe number NHMUK 2018.3.1.2. SZ-2010-0321-01-3. paratypeOne slidewith silvernitrate-stained specimens is deposited in the Laboratory of Protozoology, Ocean with the registration ofChina University number Type specimens: Guangdong Province, China. Water temperature 25 °C, salinity pH 12‰, about9. Type habitat: locality and terminating with curvature P2of P1. at three-rowed;terminate rows 3together withand 2 is proximally 1ofP1; row P3 shortened P3 scopula. and band trochal aboral between 8-12 band, trochal aboral and peristome between peristomial lip. Macronucleus J-shaped. Single contractile vacuole ventrally located. silverlines 18-25 Diagnosis: scapiformis Vorticella of ICZNurn:lsid:zoobank.org: 2012): act:87851AFC-EB3D-4F02-A909-D78ECF79CAEB ZooBank registration: KY594767. sequence. Gene : Zooid inverted bell-shaped, 35-50 µm × 20-30 µm in vivo. Width of peristomial lip about about lip peristomial of Width vivo. µm in 20-30 µm × 35-50 bell-shaped, inverted : Zooid Zooid globular, 20-30 µm ×15-25 µm vivo, in constricted not below peristome. Zooid elongate obconical, µm 40-5540-50 µm × vivo, inwith conspicuously everted The Latin adjective The Latinadjective The Latin adjective The Latinadjective The SSU rDNA sequence has been deposited in GenBank with the accession number number accession the with GenBank in beendeposited has sequence rDNA SSU The The SSU rDNA sequence has been deposited in GenBank with the accession number number accession the with GenBank in beendeposited has sequence rDNA SSU The The SSU rDNA sequence has been deposited in GenBank with the accession number number accession the with GenBank in beendeposited has sequence rDNA SSU The A protargol slide with the holotype specimen circled by ink is deposited in the the in deposited is by ink circled specimen holotype the with slide A protargol A protargol slide with the holotype specimen circled by ink is deposited in the the in deposited is by ink circled specimen holotype the with slide A protargol p . n. sp. sp. n. ZooBank registration number of the present work (see Recommendation 8A 8A Recommendation (see work present the of number registration ZooBank 8A Recommendation (see work present the of number registration ZooBank ZooBank registration number of the present work (see Recommendation 8A 8A Recommendation (see work present the of number registration ZooBank sp. n. n. sp. A small ditch near the coast of Shantou (23°38Ń; 116°68É),Guangdong Sewage outlet onDonghai Island Zhanjiang (21°02Ń; 110°34É), sphaeroidalis scapiform·is, -is, -e refers to the globular zooid shape. shape. zooid globular the to refers ome and aboral trochal band, 7-13 between aboral aboral between 7-13 band, trochal ome and aboral ([m, bacilliform)([m, f, n]; the to bacilliform refers

Accepted Article ICZN (The International Commission on Zoological Nomenclature), 2012. Amendment of of Amendment 2012. Nomenclature), Zoological on Commission International (The ICZN Hall, T. A.1999. BioEdit:user-friendly a biological sequence alignment editor and analysis program Guindon,Lethiec, S., Duroux, F., &Gascuel, P. 2005.O. PHYML Online-aweb for server fast Z.W. & Yi, Song, J., D., Huang, Xu, Sun, M., P., Miao, J., Gong, Q., Zhang, A., F., Warren, Gao, Itabashi, T., Mikami, K., Fang, J. & Asai, H. 2002. Phylogenetic relationships between between Phylogenetic relationships Fang, 2002. H. K., & Mikami, J. T., Itabashi, Asai, molecular and Morphological H.W. 2010. Breiner, T. K., & Stoeck, Wolf, N., W., Blake, Foissner, das tiefere in Blick Ein Organismen. vollkommene als DieInfusionsthierchen 1838. C. G. Ehrenberg, LITERATURE CITED for the improving of this manuscript. Song (OUC, China), the late John C. Clamp (NCCU JohnSong C. late the (OUC, China), 41576148, 41476128, 31772413, 31430077). We acknowledgehelpful the suggestions of Weibo numbers: (project China” of Foundation Science Natural “National the by supported was study This ACKNOWLEDGMENTS of ICZNurn:lsid:zoobank.org: 2012): act:6E42E1AE-8F12-4E53-9920-0C7ACD25BE0D ZooBank registration: KY594765. sequence. Gene This by is protectedarticle reserved. Allrights copyright. that the typesimilar population is to Etymology: Natural History Museum, London, UK, with registrationthe number NHMUK 2018.3.1.4. the in deposited is specimens nitrate-stained silver with slide paratype One SZ-2009-1011-01-2; Laboratory of Protozoology, Ocean Qingdao, ofChina, University China with the registration number Type specimens: Guangdong Province, China. Water temperature 23 °C, salinity pH 23‰, 7.8. Type habitat: locality and curvature of P1. at terminate P3 and P2 3; row of length the half are P3 of 2 and 1 Rows scopula. and band trochal refine methods of publication. publication. of methods refine Articles 8,9, of 10, 78 the and 21 International Code of Zoological Nomenclatureto expand and for Windows95/98/NT. Server):W557–W559. inference. phylogenetic likelihood-based maximum Alveolata). (Eukaryota, the phylumCiliophora of a classification revised with protists ciliated hypothesis of The evolutionary 2016. all-data-based Orborhabdostyla Orborhabdostyla twonew bromeliads genera including some from tank of peritrichs characterization 1-612. L. Leipzig: i-xviii+(4), Leben Voss, Natur. der organische 19:931–937. 19:931–937. convallaria

Composite Greekof the word and other species inferred from small subunit rRNA gene sequences. sequences. gene rRNA subunit small from speciesinferred other and The SSU rDNA sequence has been deposited in GenBank with the accession number number accession the with GenBank in beendeposited has sequence rDNA SSU The A protargol slide with the holotype specimen circled by ink is deposited in the the in deposited is by ink circled specimen holotype the with slide A protargol and and ZooBank registration number of the present work (see Recommendation 8A 8A Recommendation (see work present the of number registration ZooBank Vorticellides. Acta Vorticellides. Acta Protozool Mangrove wetlandGaoqiao at town, Zhanjiang (21°53´N; 110°28´E), Nucl. Acids Symp Acids Nucl. Bull. Zool. Nom Zool. Bull. Vorticella lima para· . Ser (similar, near) and the specific epithet ., 41:95–98. . 69:161–169. , USA), the associate edit associate the , USA), Sci. Rep. Sci. Kahl, 1933. Kahl, ., 48:291–319. 48:291–319. ., Nucl. Acids Res., , 6, 24874. 6,24874. ,

33 (Web 33 or, anonymous reviewers Zool. Scr. Zool.

lima Vorticella Vorticella

indicating indicating , Accepted Article Li, L., Al-Rasheid, Ma, H. & K. A. Monophyly2015. S. or polyphyly? Possibleconflict between Kahl, 1935.Urtiere oderA. Protozoa I: oder CiliataWimpertiere (Infusoria). 4.Peritricha und Kahl, 1933.CiliataA. liberaet ectocommensalia. Song,W.A. 2004. D., &Warren, Ji, 2006. Song,W.J. D., & Ji, Clamp, ciliate, of a Description marine peritrichous K.A. Song,W.S. 2003. D., & Ji, Al-Rasheid, This by is protectedarticle reserved. Allrights copyright. Miao, Fen,W., W. H., Yu,Y.S., Zhang, &Shen, X. Y. Y.F.2004.Phylogenetic relationships of the Medlin,L., Elwood, H. J., Stickel, S.&Sogin, L. 1988. M. characterization The of enzymatically Lynn, D. CiliatedThe 2008. H. Protozoa: Characterization, Classification, andGuide toLiterature the Ji, D., Kim, J. H., Shazib, S. U., Sun, P., Li,L. & Shin, M. K. 2015. Two new species of of Materials (Ed.), N. Dumka, In: (Peritricha). Sessilida order the of revision A W.1976. Jankowski, Noland, Finley,L. E.& H. Studies E.1931. on taxonomy the of the genus Sommer, G. 1951. Die peritrichen Ciliaten des Gro des Ciliaten peritrichen Die 1951. G. Sommer, Shen, Z., Ji,D.D., Yi, Z., Z. Al-Rasheid, F. K.A. &Lin,S. 2017. Morphology X. and phylogenetic MrBayes 2003. P. J. Huelsenbeck, & F. Ronquist, Nylander, 2004.MrModeltest J.A. A. version Distributed 2. by the author. Uppsala, Sweden: Song, 1997.A W. new genus and two new species marine of peritrichousciliates (Protozoa, Song, 1991.Contribution W. commensal tothe ciliates on ciliate (Ciliophora: Peritrichida) from north China. from north Peritrichida) (Ciliophora: ciliate morphological and molecular interpretations of well-known the genus Chonotricha. Akademische Ostsee Nord- Leipzig., und Verlag. China. north from and 1976, Jankowski, 360-365. Pseudovorticella sinensis 2009. et al., Ciliophora, Peritrichida) from Qingdao, China. China. Qingdao, from Peritrichida) Ciliophora, amplified eukaryotic 16S-like rRNA-coding regions. p. 605 Dordrecht, Springer, edn. 3rd Peritrichia). Zoothamnium Russian). (in pp. 168-170 Part Protozoology, General I. Protozoologists. of theConference IIUnion All sequences. sequences. gene rRNA subunit fromsmall inferred Ciliophora) (Oligohymenophorea, Peritrichia subclass Microsoc. Soc. Microsoc. Southern China. China. Southern new placementthree of models. Evolut Zoology, ofSystematic Department Peritrichida). Peritrichida). Bioinformatics J. Eukaryot.J. Microbiol. Chin. J. Oceanol. Limnol J. Eukaryot. Microbiol. Eukaryot. J. Tierwelt. Dtl., J. Ocean Univ. Qingdao, Qingdao, Univ. Ocean J. (Ciliophora, Peritrichia) from Korea, with new observations of of from Korea, with new observations Peritrichia) (Ciliophora, , 50:80–123. J. Eukaryot.J. Microbiol. Eur. J. Protistol. P. punctata , 19:1572–1574. Zoothamnium n. sp. Peritrichia) (Ciliophora, n. fromChina. 30: 651–886. Pseudovorticella zhengae Pseudovorticella (Dons, 1918) Warren, 1987, three marine peritrichous ciliates ciliates peritrichous 1987, three Warren, marine 1918) (Dons, , 51:180–186. Pseudovorticella paracratera Pseudovorticella , 42:269–279. 42:269–279. , , 62:505–518. 62:505–518. , ., 33:490–499. 21:45–55 (in21:45–55 Chinese with English summary). species (Ciliophora: Peritrichia)coastal from waters of , DOI: 10.1111/jeu.12358 ionary Biology Centre, Uppsala University. University. Uppsala Centre, Biology ionary 3: Bayesian phylogeneti Bayesian 3: Ophelia In β

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50: 50: Sun Accepted Article infundibular polykinety1-3; Po, polykinety. Scale bars: 40 40 bars: Scale polykinety. Po, polykinety1-3; infundibular P1-3, macronucleus; Ma, kinety; germinal G, haplokinety; H, membrane. epistomial marks the pore; pellicular indicates arrowhead Arows folds indicate on the surface; This by is protectedarticle reserved. Allrights copyright. Sun, P., Al-Farraj, A.S., Warren, Ma, H.G. A. & Morphology 2017. of four solitarynew sessile Song, &Wilbert, W. 1995. N. Benthische Ciliaten des Sü Song, &Warren, W. A.2000. A redescriptionof staining. u,P,Cap .C,X,D,Hag . hn .K unr .21.A T-ae Sun, P., Clamp, J. C., Xu, D., Huang, B., Shin, M. K. & Turner, F. 2013. An ITS-based Sun, P., Clamp, J., Huang,Xu D., B.&Shin, M.K.2016. An integrative approach phylogeny to reveals Warren, Arevision A.1986. of thegenus of representation phylogenetic Expanded 2010. E. Eizirik, & L. S. L. Safi, L., L. T. Simão, P., R. L. Utz, sessile three colonial of A.& Morphology S. new Song, W. Al-Farraj, Warren, A., 2015. Sun, P., of Taxonomic K.A.S. characterization 2006. & C. Song,W., J. Al-Rasheid, Sun, P., Clamp, peritrichs. sessilid Solitary W.2009. Song, & A. Warren, D., Ji, P., Sun, W. Y. & Miao, Xu, C., 2012. Kusuoka, D., J. Clamp, P., Sun, Figure 1 Figure legends Ciliaten. für Protargolimprägnation der Technik verbesserte Eine 1975. N. Wilbert, Paravorticella peritrichciliates from the Yellow China, Sea, with description of an unidentified species of Protozoologie. Gustav Fischer, New York. 156–168. P. in north China. comb., aperitrichous ciliate (Protozoa: Ciliophora: Peritrichida) isolated from mariculture biotopes phylogenetic framework for the genus for framework phylogenetic ciliates. of major group a in relationships evolutionary novel and distribution ofenvironmental patterns peritrich ciliates, ciliates, peritrich from China. Oligohymenophorea) Vorticella fusca 217–256. Hu, X. (ed.), Free-living ciliates in the Bohai and Yellow Seas, China. Science Press, Beijing. p. RNA. ribosomal subunit small for gene coding the derived from characters molecular using Astylozoidae and Vorticellidae and thefamilies of clade: redefinition a not grade a is Peritrichia) Oligohymenophora, (Ciliophora, in a taxonomicallygroup. difficult (Zool.). Peritrichia). (Ciliophora: Ciliates peritrich genera notes on with sp., 64:171–179.

Vorticella parachiangi Vorticella A. 50:1-57. Opercularia Sci. Rep. Sci. General view of typical zooid; zooid; typical of view General (Ciliophora,Peritrichia). Acta Protozool. Acta Precht, 1935 and 1935 Precht, and , 6:21695. Pseudepistylis paramphora paramphora Pseudepistylis Epicarchesium variabile Epicarchesium and and Epistylis sp. in n. vivo 39:43–50. 39:43–50. sheds light on the evolution evolution the on light sheds Vorticella parapulchella Vorticella J. Microbiol Eukaryot. D. E. Proc. Soc.B. R. Infraciliature and macronucleus; and Infraciliature Silverline system. Arrowmarks the aboraltrochal band; Vorticella Vorticella Eur. J. Eur. Protistol B.

J. Eukaryot. Microbiol Eukaryot. J. Showing proportion of stalk and zooid; zooid; and stalk of proportion Showing ( (Ciliophora, Peritrichia). Peritrichia). (Ciliophora, A-C n. sp., sp., n. Pseudovorticella patellina ) and after silver nitrate ( nitrate silver after and ) Protist (Ciliophora: Peritrichida). Peritrichida). (Ciliophora: : finding the molecular and morphological gaps morphological and molecular finding the : 280, 20131177. 280, Zoothamnium paranii Zoothamnium β , 163:129–142. , 163:129–142. ., 57:73–84. wassers. μ m 200 (A); ., 53:348–357. 53:348–357. ., n. sp., two marine peritrichs (Ciliophora, (Ciliophora, peritrichs marine two n.sp., Vorticella

and higher-level taxonomy of higher-level and ., 57:415-420. In : Röttger, Praktikum: R. (ed.), der μ Eur. J. Protistol J. Eur. m (B); 25 25 m (B); Lnau,16 1767 Linnaeus, F. In F.Müller,(O. 1776) nov. D Oral ciliature. Oral Arowhead :

) and protargol) and ( Song, W., Warren, A. & n. and n. sp. Bull. Br. Mus. nat. Hist. Hist. nat. Mus. Br. Bull. μ m (E). Mikrokosmos C. Typical zooid. zooid. Typical Z. hartwigi ,5:8–9. 51:186–195. ., Vorticella E, F E,

) , n.

Accepted Article ( (arrow); Arrows margin mark of peristomial lip; silver nitrate ( This by is protectedarticle reserved. Allrights copyright. Figure 15 bars: Scale (arrowheads). pores pellicular band (double-arrowheads); system; silver nitrate ( 6 Figure indeterminate. is rows kinetosomal of number * In ( 200 3; polykinety infundibular indicates Arrow ciliature. Oral H. macronucleus; (arrows); lip ofedge peristomial on irregular protuberances silver nitrate ( stalk; striations; pellicular indicate Arrows optics. contrast interference differential and with Figure 2 stalk and zooid; zooid; and stalk Figure 5 5 Figure Details of stalk. indicates Arrow thecoplasmic granules in the spasmoneme;G. Arrows show D. Figure 4 (arrows); zooid; and stalk of proportion ( staining protargol 3 Figure 3; polykinety infundibular (arrow); (arrowhead); macronucleus and (arrowhead) vacuole food Showing (A); 40 15 bars: Scale polykinety. Po, 1-3; polykinety infundibular P1-3, kinety; G, germinal haplokinety; infraciliature and myoneme system; zooid; and stalk of proportion infundibular polykinety 1-3; Po, polykinety. Scale bars: 40 40 bars: Scale polykinety. Po, 1-3; polykinety infundibular P1-3, kinety; germinal G, haplokinety; H, membrane. epistomial indicates Arrowhead ciliature. (B); 15 C-D C, D Showing food vacuole (arrow); (arrow); vacuole food Showing C μ ) and silver nitrate ( ) and ( silver nitrate and ) , initial the (i.e. proximal) position is of P3 shown, but the terminal distal) (i.e. position and the m (A); 20 m (A); G. 7 μ μ E. Photomicrographs of of Photomicrographs

Photomicrographs of of Photomicrographs G. m (F); 15 15 m (F); m (B); 6 (B); m Macronucleus (arrows) and aboral trochal band (arrowhead); (arrowhead); band trochal aboral and (arrows) Macronucleus D. Morphology and infraciliature of of Photomicrographs Morphology and infraciliature of Morphology and infraciliature of D. Showing macronucleus (arrow), infundibular polykinety 3 (arrowhead) and aboral trochal trochal andaboral (arrowhead) 3 polykinety infundibular (arrow), macronucleus Showing Zooid high at magnification. Arrowspellicular show striations; Contractile vacuole (arrow) and food vacuole (arrowhead); vacuole food and (arrow) vacuole Contractile Details of stalk; I H-I I ) staining. ) staining. ) staining. ) staining. μ C. m (B, D, G). G). D, (B, m ) staining. ) staining. μ μ Showing myoneme macronucleus; system and D. m (D). E, F E, m (G). m (G). ). E E A. A. A. ) staining. ) staining. staining. ) I. General lateral view of typicalzooid; Lateral view Lateral showing stalk the relative to zooid; length the Lateral view, showing the proportion of stalk and zooid; zooid; and stalk of proportion the showing view, Lateral A. H. Lateral viewshowing silverline system. Scale bars: 135 E. Typical zooidvivo; in Showing general Showing Showing silverline system. Arrowindicates silverline; C. C. Vorticella parachiangi Vorticella Vorticella Vorticella scapiformis Vorticella Oral ciliature. Oral Arrowhead marks epistomial membrane; lip peristomial of edge on protuberances irregular Showing E. A. A. Zooid high at magnification. Arrows show pellicular striations; E. General view of typical zooid; zooid; typical of view General General view of typical zooid; zooid; typical of view General Silverline system. Arrow themarks aboral trochal band. H, C.

sphaeroidalis Lateral view of zooid showing thickness of peristomial lip Vorticella Vorticella scapiformis Vorticella sphaeroidalis sphaeroidalis Vorticella μ m (A); 10 m (A);

infraciliature and macronucleus (arrow); (arrow); and macronucleus infraciliature B.

sp. n. in vivo ( in vivo sp. n. paralima sp. n. in vivo ( in vivo sp. n. Showing contractile vacuole (arrow); vacuole Showing contractile sp. n. in vivo ( sp. vivo in n. μ m (H, J). J). m (H, μ m (A); 200 (A); 200 m C. sp. n.sp.

Showing contractile vacuole (arrow); contractile (arrow); vacuole Showing sp. n. in in vivo ( n. sp. B. D. sp. n. in vivo ( vivo in n. sp. A-F Lateral view showing the A-F

in vivo ( General infraciliature. Arrow A-E D. B. B. ) and after protargol ( protargol after and ) ) and after protargol ( protargol after and ) E. μ Showing the proportion of proportion the Showing Granules on the surface surface the on Granules H. Lateral view, showing the ), after protargol (F-G) and (F-G) protargol after ), m (B); 15 15 m (B); Zooid highat magnification Oral ciliature. Arrow marks marks Arrow ciliature. Oral F. I. A-B Arrow shows myoneme Arrowshows Silverlines. Scale bars: bars: Scale Silverlines. A-D ), after protargol protargol after ), A-B μ ) and after m (E). m (E). μ ), after protargol B. I. m (A);30 m Showing Showing F. B. B. Detail of of Detail D. F. Apical view. view. Apical Oral General General C. G-H G-H

) and m ) and μ m F.

Accepted Article Showing oral infraciliature. Arrow indicates infundibular polykinety infundibular indicates Arrow infraciliature. oral Showing striations (arrow); on surface; depression indicates Arrow vacuole. contractile marks Arrowhead ( nitrate 30 bars: Scale Po, polykinety. 1-3; polykinety infundibular P1-3, kinety; G, germinal haplokinety; H, membrane. marks epistomial This by is protectedarticle reserved. Allrights copyright. and of positions phylogenetic Figure 9 Figure 8 band; trochal aboral marks Arrowhead macronucleus. indicates Showing silverlines . Scale bars: Showing 60 . Scale silverlines bar indicates two substitutions 100nucleotides. per scale The value. support a of instead a hyphen by indicated one thetwophylogenetic of are analyses posterior probabilities. GenBank accession number values inthe followingorder: MaximumLikelihood (ML) bootstrapvalues and Bayesian inference (BI) Vorticella paralima F-G) Maximum likelihood tree inferred from small subunit rDNA gene sequences indicating indicating sequences gene rDNA subunit small from inferred tree likelihood Maximum of Photomicrographs staining. staining. D. Showing macronucleus (arrow) and aboral trochal band (arrowhead); (arrowhead); band trochal aboral and (arrow) macronucleus Showing A. sp. n. (bold typeface and arrows). Numbers at the nodes represent support support represent nodes the at Numbers arrows). and typeface (bold n. sp. Lateral view of zooid at low magnification; magnification; low at zooid of view Lateral Vorticella parachiangi μ Vorticella m (A); 90 m (A); μ m (A); 20 (A); m

paralima μ m (B); 20 m (B); μ m (B, E); 15 m (B, sp. n., sp. n. in vivo ( in vivo n. sp. s each follow speciesname. Nodes absent from μ m (E). m (E). V. scapiformis V. μ m (F, m (F, H).

A-C E. Showing oral ciliature. Arrowhead ciliature. oral Showing

), after protargol ( protargol ), after B. 3; sp. n., n., sp.

Typical zooid in vivo. vivo. in zooid Typical F. F. Showing pellicular pores; pores; pellicular Showing V.

C. sphaeroidalis Showing pellicular D-E ) and silver silver ) and E. sp. n.

Accepted Article This by is protectedarticle reserved. Allrights copyright. d c b number. n, sample deviation; standard SD, mean; arithmetic Mean, maximum; Max, Min, minimum; ªData of silverlines basedon Chatton-Lwoff silver nitrate-stained specimens. scapiformis Table 1.

Number of silverlines between peristome and aboral trochal band. band. trochal aboral and peristome between silverlines of Number Number of silverlines between aboral trochal band and scopula. scopula. and band trochal aboral between ofsilverlines Number Measurements in Number of silverlines Zooid widthvivo in hrces pce i a enS n SD Mean Max Charactersª Species Min Zooid length invivo

Number of silverlines Characterization of zooid size and silverline system of silverline sizeand system zooid of Characterization sp. n., V. sphaeroidalis sphaeroidalis V. μ m. b b

d c V V V V. V. sphaeroidalis scapiformis V. V V. V. sphaeroidalis scapiformis V. V. V. sphaeroidalis scapiformis V. V. V. sphaeroidalis scapiformis V. . . . . p p p p p p p p aralima arachiangi aralima arachiangi aralima arachiangi aralima arachiangi sp. and n. 8 3.54 23.75 30 8 20 4.63 42.50 50 35 V. paralima 03 33 .86 7 4.08 4.50 23.33 44.31 30 50 20 40 6 11 7.50 1.41 22 11 6 26 3530.50 6 2.70 14 8 17.50 18 16 7 2 4.08 10 18 2522.57 2.70 6.07 7 18.33 21 3125.72 3.50 44.30 2.66 22 25 53.00 55 15 65 10 40 50 4.76 55.70 65 50 7 13 10.36 1.7 2 14 2 9.86 1.35 7 12 2 08 8 s.n sp. n.

Vorticella parachiangi parachiangi Vorticella sp. n., n., sp. V. Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.