#ZooKeys

ZooKeys 1247: 137-149 (2025)
DOI: 10.3897/zookeys.1247.155543

Research Article

Morphology of a novel ciliate, Oxytricha chonggqingica sp. nov.
(Ciliophora, Hypotrichia)

Haifeng Han™, Xiaojin Xue’, Zhisheng Zhang2®, Chen Shao'®

1 Laboratory of Biodiversity and Evolution of Protozoa in Wetland, College of Life Sciences, Shaanxi Normal University, Xi'an 710119, China
2 Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Life Sciences, Southwest University, Chongqing 400715,

China

Corresponding authors: Zhisheng Zhang (zhangzs327@qq.com); Chen Shao (shaochen@snnu.edu.cn)

OPEN Qaccess

Academic editor: Pavel Stoev
Received: 11 April 2025
Accepted: 2 June 2025
Published: 25 July 2025

ZooBank: https://Zoobank.
org/791C10AB-3313-4692-BE65-
39405C11551F

Citation: Han H, Xue X, Zhang Z,
Shao C (2025) Morphology of a
novel ciliate, Oxytricha chonggingica
sp. nov. (Ciliophora, Hypotrichia).

Zookeys 1247: 137-149. https://doi.

org/10.3897/zookeys.1247.155543

Copyright: © Haifeng Han et al.
This is an open access article distributed under
terms of the Creative Commons Attribution

License (Attribution 4.0 International - CC BY 4.0).

Abstract

A novel hypotrichous ciliate, Oxytricha chonggqingica sp. nov., was identified in Chongq-
ing, southwestern China. This species is distinguishable from congeners by various
qualitative and quantitative characteristics, such as body size and shape, pattern of
endoral and paroral, number of macronuclear nodules, presence and arrangement of
cortical granules, as well as ventral and dorsal ciliature. Phylogenetic analyses based on
small subunit ribosomal DNA (SSU rDNA) sequences indicate that Oxytricha chonggin-
gica sp. nov. forms a cluster with O. nauplia, Allotrichides antarcticus, O. paragranulifera,
O. quadricirrata, Paroxytricha longigranulosa, and P. ottowi, albeit with weak or moderate
nodal support (82 ML and 0.87 BI).

Key words: New species, Oxytrichidae, SSU rDNA, taxonomy

Introduction

The genus Oxytricha Bory de Saint-Vincent in Lamouroux et al. 1824 is taxo-
nomically intricate among ciliated protists in comprising about 50 species. Re-
cent discoveries of new species, such as O. acidotolerans Weisse et al., 2013,
O. atypica Fan et al., 2021, O. buxai Bharti & Kumar, 2023, O. granulifera chiapa-
sensis Méndez-Sanchez et al., 2018, O. multilineata Jin et al., 2022, O. paragran-
ulifera Shao et al., 2014, O. seokmoensis Kim & Min, 2019, and O. xianica Wang
et al., 2021, indicate a substantial species diversity within Oxytricha (Weisse et
al. 2013; Shao et al. 2014; Méndez-Sanchez et al. 2018; Kim and Min 2019; Fan
et al. 2021; Wang et al. 2021; Jin et al. 2022; Bharti and Kumar 2023).

In the present study, the living morphology and infraciliature of a novel hy-
potrich, Oxytricha chonggingica sp. nov. is described. Furthermore, sequence
of the small subunit ribosomal DNA (SSU rDNA) and phylogenetic trees were
employed to assess its evolutionary placement.

‘lea

Haifeng Han et al.: Morphology of a novel hypotrichous ciliate, Oxytricha chonggingica sp. nov.

Materials and methods
Sampling and cultivation

Soil (0-5 cm depth, including leaf litter and humus layers) samples containing
Oxytricha chonggingica sp. nov. were collected on 13 August 2024 from Yin-
tiaoling Nature Reserve (31°31'26"N, 109°49'31"E), Chongqing, southwestern
China (Fig. 1A—-D). Ciliates were induced to excyst following Foissner (1987)
non-flooded Petri dish protocol. Cultures were maintained under ambient lab-
oratory conditions (25 + 1 °C) using mineral water (Nongfu Spring) with sterile
rice grains to support bacterial growth as a nutritional source for the ciliates.

Morphology

Live cell observations were conducted using an Olympus BX53 microscope
equipped with differential interference contrast (DIC) optics and bright-field illu-
mination, coupled with a DP74 digital imaging system for photomicrograph cap-
ture. The nuclear apparatus and infraciliature were examined through protargol
staining (Sigma-Aldrich, cat. #448 K2787347) according to Wilbert (1975). Mor-
phometric analyses of silver-impregnated specimens were performed under oil
immersion (100x objective, 12.5x ocular). A depiction of a live specimen was
generated based on photographic records, while stained cell morphologies were
captured using a camera setup. The terminology used follows Berger (1999).

DNA extraction, PCR amplification, and sequencing

Individual ciliates were aseptically isolated from culture media through three-
stage purification with sterile distilled water (0.22 um membrane filtration,
Tianjin Branch Billion Lung Experimental Equipment Co., Ltd, China). Selected
specimens were suspended in 1.5 mL microcentrifuge tubes containing mini-
mal aqueous medium. Genomic DNA was isolated using the DNeasy Blood and
Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
Amplification of SSU rDNA employed KOD OneTM PCR Master Mix-Blue-(TOYO-
BO, Cat# KMM-201) with universal eukaryotic primers (18S-F (5-AAC CTG GTT
GAT CCT GCC AGT-3’) and 18S-R (5'-TGA TCC TTC TGC AGG TTC ACC TAC-3’))
originally described by Medlin et al. (1988). Thermal cycling parameters com-
prised: initial denaturation at 98 °C (30 s); 18 touchdown cycles of 98 °C (10
Ss), 69 °C (30 s, decreasing by 1 °C per cycle), 72 °C (1 min); 18 standard cycles
at 98 °C (10 s), 51°C (30 s), 72 °C (1 min); final extension at 72 °C (2 min). The
product was sequenced bidirectionally by the GeneCreate (Wuhan, China). The
sequence was assembled using SeqMan v7.1.0 (DNA Star).

Phylogenetic analyses

To determine the phylogenetic position of the new species, the new SSU rDNA
sequences, along with those of 67 other hypotrichs retrieved from GenBank
database (Fig. 4), were utilized for tree construction. Four euplotids, namely
Diophrys scutum JF694040, Uronychia multicirrus EU267929, Apodiophrys ova-
lis GU477634, and Paradiophrys zhangi FJ870076, were employed as outgroup
taxa. Sequence alignment was performed using the MAFFT v. 7.525 module

ZooKeys 1247: 137-149 (2025), DOI: 10.3897/zookeys.1247.155543 138

Haifeng Han et al.: Morphology of a novel hypotrichous ciliate, Oxytricha chonggingica sp. nov.

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Figure 1. Location and surrounding of the sampling site. A. Location of Chongqing city in China (map from the National
Platform for Common Geospatial Information Services (www.tianditu.gov.cn), Revision No. GS (2019) 1654); B. Location
of the sample site; C, D. Surroundings at the sampling site; red arrowhead indicates where the samples were collected.

in PHYLOSUITE v. 1.2.3 (Rozewicki et al. 2019; Zhang et al. 2020; Xiang et al.
2023). Both ends of the alignment were trimmed manually using PHYLOSUITE
v. 1.2.3 (Zhang et al. 2020; Xiang et al. 2023) resulting in a final alignment with
1557 sites. Subsequently, a maximum likelihood (ML) analysis with 1000 stan-
dard bootstrap pseudoreplicates was conducted using IQ-TREE v. 2.4.0, with
the TIM2 + F + R3 model (Minh et al. 2020). Bayesian inference (BI) was carried
out using MRBAYES v. 3.2.7a on MODELFINDER v. 2.2.0 employing the GTR +
F +1 +G4 model (Ronquist et al. 2012; Kalyaanamoorthy et al. 2017). The ITOL
v. 7 server was used to visualize the phylogenetic trees (Ivica and Peer 2024).
Phylogenetic support thresholds were categorized as follows: bootstrap val-
ues = 95 (strong), 71-94 (moderate), 50-70 (weak), and < 50 (unsupported) fol-
lowing Hillis and Bull (1993). Bayesian nodal confidence was evaluated using
posterior probabilities with = 0.95 indicating robust support (Alfaro et al. 2003).
The SSU rDNA sequence of Oxytricha chonggingica sp. nov. was aligned with
phylogenetically relevant homologs following methods described by Rozewic-

ZooKeys 1247: 137-149 (2025), DOI: 10.3897/zookeys.1247.155543 139

Haifeng Han et al.: Morphology of a novel hypotrichous ciliate, Oxytricha chonggingica sp. nov.

ki et al. (2019). The alignment was curated by removing ambiguously aligned
terminal regions and subsequently analyzed in BIOEDIT v. 7.0.5.2 (Hall 1999) to
generate pairwise similarity matrices based on global sequence comparisons.

Results
Taxonomy

Class Spirotrichea Bitschli, 1889

Subclass Hypotrichia Stein, 1859

Order Sporadotrichida Fauré-Fremiet, 1961

Family Oxytrichidae Ehrenberg, 1838

Genus Oxytricha Bory de Saint-Vincent in Lamouroux et al., 1824

Oxytricha chonggqingica sp. nov.
https://Zoobank.org/1638B342-9947-4D0E-BAC2-E5FE5F735AC5
Figs 2A-E, 3A-L; Table 1

Diagnosis. Body size 100-160 x 40-60 um in vivo, elliptical to elongate-elliptical
in outline. Two macronuclear nodules and one to five micronuclei. Contractile
vacuole located at mid-body close to left margin. Cortical granules about 1 um
across, arranged in long rows, colorless. The adoral zone occupying 35-44% of
the body length, consisting of 37-49 adoral membranelles. Paroral and endoral
arranged in a typical Oxytricha-pattern. Typical 18 frontoventral-transverse cirri,
buccal cirrus positioned at the anterior end of paroral, cirrus III/2 close to and
slightly ahead of the level of cirrus VI/3, cirrus V/4 behind cirrus IV/2, the dis-
tances between cirri V/3 and V/4 and between cirri V/2 and VI/2 obvious short-
er than that between cirri V/3 and V/2. Right and left marginal rows with 32-46
and 33-43 cirri, respectively, confluent at the posterior end of cell. Usually, six
dorsal kineties including two dorsomarginal kineties. Dorsal kinety 3 terminat-
ing caudally, dorsal kineties 3 and 4 connected by four or five dikinetids, and
dorsal kinety 5 ending at approximately 80% down length of body. Three caudal
cirri, each one at the posterior end of dorsal kineties 1, 2, and 4.

Type material. The protargol-stained slide (no. XXJ202408130901A) with
the holotype specimen (Figs 2D, E, 3G, H) marked with an ink circle, and two
paratype slides (no. XXJ202408130901B, C) are deposited in the Laboratory of
Protozoological Biodiversity and Evolution in Wetland, Shaanxi Normal Univer-
sity (SNNU), China.

Type locality. Soil from Yintiaoling Nature Reserve in Chongqing, southwest-
ern China (31°31'26"N, 109°49'31"E).

Etymology. The species-group name “chonggingica’” refers to Chongging, the
type locality.

Description. Body size 100-160 x 40-60 um (n = 7) in vivo, 110-170 x 40-
100 um after protargol staining. Ratio of body length to width 2.5-3.0 in vivo. Flex-
ible but not contractile, elliptical to elongate-elliptical in outline with anterior and
posterior ends slightly narrowly rounded. Right margin slightly flat and left margin
distinctly convex (Figs 2A, 3A, B; Table 1). Nuclear apparatus consisting of two
macronuclear nodules and one to five micronuclei. Macronuclear nodules usu-
ally located at 33% and 66% down length of body, respectively, and slightly left of
the cell mid-line, about 25 x 15 um in vivo. Micronuclei attached to macronuclear

ZooKeys 1247: 137-149 (2025), DOI: 10.3897/zookeys.1247.155543 140

Haifeng Han et al.: Morphology of a novel hypotrichous ciliate, Oxytricha chonggingica sp. nov.

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Figure 2. Oxytricha chongqingica sp. nov. from life (A-C) and after protargol staining (D, E). A. Ventral view of a typical
individual; B, C. Cortical granules arranged in long rows on ventral (B) and dorsal (C) side; D, E. Ventral (D) and dorsal (E)
view of the holotype specimen. AZM, adoral zone of membranelles; CC, caudal cirri; DK, dorsal kineties; E, endoral; FC,
frontal cirri; FVC, frontoventral cirri; LMR, left marginal row; Ma, macronuclear nodule; Mi, micronucleus; P, paroral; PTVC,
pretransverse ventral cirri; PVC, postoral ventral cirri; RMR, right marginal row; TC, transverse cirri; 1-6, dorsal kineties
1-6. Scale bars: 50 um (A, D, E); 15 um (B, C).

nodules at variable positions, each about 7 um across in vivo (Figs 2E, 3D, J; Ta-
ble 1). Contractile vacuole about 13 um in diameter when fully expanded, located
at about mid-body close to left margin, contracting at intervals of about 10 s, with
two non-obvious collecting canals (Figs 2A, 3A, B). Cortical granules arranged in
long rows on both sides, colorless, about 1.0 um across (Figs 2B, C, 3C). Cyto-
plasm colorless, filled with numerous food vacuoles (2-10 um in diameter), lipid
droplets (0.5-2 um across), and diatoms (up to 21 um in length). Movement usu-
ally crawling moderately rapidly on the bottom of a petri dish or other substrates,
when in suspension, swimming rapidly while spiraling in a counter-clockwise helix.

Adoral zone occupying 35-44% of body length in protargol preparations and
consisting of 37-49 membranelles, and cilia extending up to 21 um in length
(Figs 2A, D, 3G, |; Table 1). DE-value 0.26-0.37 (Table 1). Buccal cavity flat.
Endoral and paroral bending and optically intersecting with each other. Pharyn-
geal fibres inconspicuous after protargol impregnation (Figs 2D, 3E, 1).

Three slightly enlarged frontal cirri, with cilia approximately 16 um long, ar-
ranged in an oblique pseudorow, with rightmost cirrus located behind distal end
of adoral zone of membranelles. Single buccal cirrus positioned at the anterior
end of the paroral. Four frontoventral cirri, characteristically arranged, namely,

ZooKeys 1247: 137-149 (2025), DOI: 10.3897/zookeys.1247.155543 141

Haifeng Han et al.: Morphology of a novel hypotrichous ciliate, Oxytricha chonggingica sp. nov.

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cates the collecting canal, and arrow marks the contractile vacuole; C. Cortical granules; D, J. Macronuclear nodules and mi-
cronuclei (arrowheads); E. Pharyngeal fibres; F. Dorsal cilia (arrowheads); G, H. Ventral (G) and dorsal (H) view of holotype,
showing cirral pattern; I. Anterior part of ventral surface, showing buccal cirrus (double-arrowhead) and postoral ventral cirri
(in ellipse); K. Individual with five transverse cirri; L. Caudal cirri (in ellipse). AZM, adoral zone of membranelles; FC, frontal
cirri; FVC, frontoventral cirri; LMR, left marginal row; Ma, macronuclear nodule; PTVC, pretransverse ventral cirri; RMR, right
marginal row; TC, transverse cirri; 1—6, dorsal kineties 1-6. Scale bars: 50 um (A, B, G, H); 20 um (C-E, I, J); 10 um (EK, L).

cirrus III/2 close to and slightly ahead of level of cirrus VI/3. Three postoral ven-
tral cirri, cirrus V/4 positioned behind level of cirrus IV/2, the distances between
cirri V/3 and V/4, and between cirri V/2 and VI/2 shorter than that between cirri
V/3 and V/2. Two pretransverse ventral cirri, cirrus VI/2 located ahead of right-
most transverse cirrus. Usually, five (rarely four, only two in 21) slightly enlarged
transverse cirri arranged in hook-shaped pseudorow, with cilia about 20 um long
in vivo. One right and one left marginal row, with cilia about 15 um long, consist-
ing of 32-46 and 33-43 cirri, respectively. Right marginal row commencing at
level of VI/4, while left marginal row starting near posterior end of adoral zone.
Both terminating at posterior end of cell and confluent (Figs 2D, 3G, I, K; Table 1).

Usually six (rarely seven, only four in 21) dorsal kineties including two (rarely
three, only four in 21) dorsomarginal kineties, cilia about 4 um in vivo. Dorsal kine-
ty 1 commencing subapically, while kineties 2 and 3 starting apically, all ending at

ZooKeys 1247: 137-149 (2025), DOI: 10.3897/zookeys.1247.155543 142

Haifeng Han et al.: Morphology of a novel hypotrichous ciliate, Oxytricha chonggingica sp. nov.

Table 1. Morphometric characterization of Oxytricha chonggqingica sp. nov.

Character* H Min Max Mean Med SD CV n
Body, length 128.6 | 109.2 | 169.1 140.4 145.3 19.1 13:6 21
Body, width 53.7 43.6 100.9 F2n #V,.2 18.1 25.1 21
Body length: width, ratio 2.39 L595 2.54 2.02 1.92 0.34 16.78 21
Macronuclear nodules, number 2 2 2 2.0 Ea 0 0 21
Macronuclear nodules, length 25.6 22.0 64.9 38.8 40.1 10.7 27.6 21
Macronuclear nodules, width 11.6 6.1 22.8 12.8 12.3 4.3 34.0 21
Micronuclei, number 2 1 5 2.8 3 t A1.1 21
Micronuclei, length 4.3 oe 6.3 4.9 4.9 0.9 18.6 21
Micronuclei, width 4.3 37 6.0 4.0 3.9 0.8 20.2 21
Adoral zone of membranelles, length 48.9 43.3 70.8 56.2 58.1 8.5 15-1 21
Adoral zone of membranelles length, % of body length | 38.0 34.9 44.4 39.8 39.5 3.1 77 21
Adoral membranelles, number 36 37 49 43.6 43 3.6 8.2 21
DE-value 0.32 0.26 0.37 0.31 0.30 0.03 10.28 21
Cell apex to buccal cirrus, distance 15:5 12 22.5 Ne 173 2.8 16.1 21
Buccal cirrus, number 1 1 1 1.0 1 0 0 21
Frontal cirri, number 3 3 3 3.0 3 0 0 21
Frontoventral cirri, number 4 4 4 4.0 4 0 0 21
Postoral ventral cirri, number 3 3 3 3.0 3 0 0 21
Pretransverse ventral cirri, number 2 2 2 2.0 2 0 0 21
Transverse cirri, number 5 4 ) 4.9 5 0.3 6.1 21
Cirri IV/2 and V/4, distance 4.5 2,8 8.0 4.8 4.5 13 97.2 21
Cirri V/3 and V/4, distance 11.2 9.0 15.5 11.6 11.4 1.6 T3.7 zal
Cirri V/3 and V/2, distance 31.5 24.7 50.2 33.6 31.8 #3 24.7 21
Cirri in right marginal row, number 32 OZ 46 39.8 40 3.8 9.5 21
Cirri in left marginal row, number oF 33 43 38.1 39 2.9 7.7 21
Dorsal kineties, number 6 6 P 6.1 6 0.4 5.8 21
Dikinetids in dorsal kinety 1, number 34 33 52 43.1 43 52 12.0 21
Dikinetids in dorsal kinety 2, number 29 25 39 33:1 33 3.6 10.8 21
Dikinetids in dorsal kinety 3, number 20 19 29 230] 23 3.4 14.8 21
Dikinetids in dorsal kinety 4, number 13 9 1 11.0 11 LZ 15.4 2}
Dikinetids in dorsal kinety 5, number 17 15 28 21.4 21 3.6 16.8 21
Dikinetids in dorsal kinety 6, number 14 9 19 12.9 13 2.4 18.6 21
Caudal cirri, number 3 3 3 3.0 3 0 0 21

*All data are based on protargol-stained specimens and measurements in um. Abbreviations: CV, coefficient of variation in %; H, holo-
type; Max, maximum; Mean, arithmetic mean; Med, median; Min, minimum; n, sample size; SD, standard deviation.

posterior end of cell, comprising 33-52, 25-39, and 19-29 dikinetids, respective-
ly. Dorsal kinety 4 commencing at about 55% down length of body, terminating at
posterior end of cell, and consisting of 9-15 dikinetids. Dorsal kinety 4 not clearly
separated from kinety 3, four to six dikinetids existing between them. Dorsal kine-
ty 5 (dorsomarginal kinety) stretching to approximately 80% down length of body
and composed of 15-28 dikinetids, while dorsal kinety 6 (dorsomarginal kinety)
terminating near midbody and consisting of 9-19 dikinetids. Dorsal kinety 7 (dor-
somarginal kinety), if presenting, positioned at about 25% down length of body
near right body margin, consisting of one or two dikinetids. Three caudal cirri,
each one at the posterior end of dorsal kineties 1, 2, and 4 (Figs 2E, 3H, L; Table 1).

ZooKeys 1247: 137-149 (2025), DOI: 10.3897/zookeys.1247.155543 143

Haifeng Han et al.: Morphology of a novel hypotrichous ciliate, Oxytricha chonggingica sp. nov.

SSU rDNA
ML/BI
0.02

@:100/1.00

99/0.99}. |

30/0.40)

39/0.44-
90/0.74
65/0.42-1-

82/0.87-

50/-

O7/-

7410.61

92/0.96

79/0.63;
89/0.87

91/0.99)

Phylogenetic analyses (Fig. 4)

The SSU rDNA sequence of Oxytricha chonggingica sp. nov. has been deposited
in GenBank with the accession number PV476686. The length and GC content
of the novel species are 1672 bp and 45.78%, respectively.

Phylogenetic trees resulting from maximum likelihood (ML) and Bayesian
inference (BI) analyses demonstrate analogous topologies; consequently, sole-
ly the ML tree is presented, accompanied by both bootstrap values (ML) and
posterior probabilities (Bl) (Fig. 4). In the phylogenetic tree, the new species
Oxytricha chonggingica sp. nov. clusters within the clade containing O. nauplia
Berger & Foissner, 1987, Allotrichides antarcticus (Berger, 1999) Foissner 2016,
O. paragranulifera Shao et al., 2014, O. quadricirrata Blatterer & Foissner, 1988,

Urosomoida sejongensis KT723011
Uroleptus piscis AF164131
Uroleptus longicaudatus KF734979
27 Urosoma caudata MK385610
4 Urosoma emarginata MF289777

[ Uroleptidae

Hemiamphisiella granulifera KP266623
98/1.00) Oxytricha granulifera KJO81199
81/] | Oxytricha granulifera granulifera AM412768
82/- 85/4 Architricha indica KJO000536
92/0.2 Oxytricha granulifera JX899421
Oxytricha granulifera granulifera AM412770
Oxytricha granulifera chiapasensis KX889988
Oxytricha granulifera americanum M1622649
Oxytricha multilineata OK299176
Hemiurosoma clampi MT448251
710.49 Hemiurosoma terricola AY498651
Oxytricha elegans AM412767
47/037 Urosoma salmastra KF951419
s9/.— Urosoma salmastra MF289778
Oxytricha lithofera MT364897
Oxytricha seokmoensis MT622651
Sea Heterourosomoida lanceolata AM412773
_p Paroxytricha longigranulosa JX899420
Paroxytricha ottowi JQ723977
| Oxytricha quadricirrata OQ231489
y Allotrichides antarcticus AY498652

40/0.27
66/0.69

—_—

Oxytricha paragranulifera KJO81200

Oxytricha nauplia MHO35977
Oxytricha chongqingica nov. spec. PV476686

96/0.517- Paraurostyla weissei AJ310485
Apoamphisiella vernalis KU522216
Notohymena apoaustralis KC430934
Paraurosomoida indiensis JX139117
Oxytricha auripunctata MH844496
Cyrtohymena citrina AF164135

85/0.85

23/-

Neokeronopsis asiatica KM061386
Ponturostyla enigmatica KC896649
Oxytricha ferruginea AF370027
Sterkiella cavicola GU942565

__ |, Sterkiella histriomuscorum KC193240
96/0.99 ) Oxytricha trifallax AF164121
Gastrostyla steinii AF164133
Pleurotricha lanceolota AF 164128
Pleurotricha curdsi KP262051
Onychodromus grandis AJ310486
Stylonychia bifaria FM209296
Oxytricha nova X03948
Rigidohymena candens KC414885
Stylonychia mytilus EF535730
Stylonychia lemnae AM233909
Hemiurosomoida longa AF164125

Anteholosticha antecirrata MHO00682
Bakuella granulifera KY873998
9911.00 Neobakuella flava GU967698
Hemicycliostyla franzi KY874008

| Urostylida

VIHOIYLOdAH SSV 1IO€8Ns

Gonostomum namibiense AY498655
Gonostomum strenuum AJ310493
Trachelostyla pediculiformis KU594642
Spirotrachelostyla tani FJ870093
Hemigastrostyla enigmatica EF194085
Oxytricha saltans AF370028

| Gonostomatidae
| Trachelostylidae
_ Oxytrichidae

Amphisiella annulata KX138644
Holosticha bradburyae FJ775716

Apodiophrys ovalis GU477634 | Euplotida

(outgroup)

Paradiophrys zhangiFJ870076
Uronychia multicirrus EU267929
Diophrys scutum JF694040

Figure 4. Maximum-likelihood (ML) tree based on 18S rRNA gene sequences, showing the phylogenetic position of
Oxytricha chongqingica sp. nov. The new sequence is indicated in bold red. Support values for nodes are for ML and

BI, respectively (ML/BI). Disagreements in ML and BI tree topologies are indicated by

au

. Fully supported branches are

marked with solid circles at the nodes. All branches are drawn to scale. The scale bar corresponds to 0.02 expected

substitutions per site.

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144

Haifeng Han et al.: Morphology of a novel hypotrichous ciliate, Oxytricha chonggingica sp. nov.

Paroxytricha longigranulosa (Berger & Foissner, 1989) Foissner 2016, and P ot-
towi (Foissner, 1997) Foissner 2016, with support (82 ML and 0.87 BI). These
six species were selected as molecularly related taxa of O. chonggingica sp.
nov. The sequence similarities among O. chonggingica sp. nov. and these six
species vary from 96.9% (0. paragranulifera KJO81200) to 98.7% (P. longigranu-
losa JX899420), with unmatched sites from 19 (P. longigranulosa JX899420) to
48 (0. paragranulifera KJO81200) (Table 3).

Discussion
Comparison Oxytricha chonggqingica sp. nov. with congeners

Based on the body size, shape, ventral and dorsal ciliature, pattern of endoral
and paroral and number of macronuclear nodules, Oxytricha chonggingica sp.
nov. should be compared with the following congeners, namely, O. aeruginosa
Wrzesniowskiego, 1866, O. bimembranata Shibuya, 1929, O. granulifera Foiss-
ner & Adam, 1983, O. longicirrata Kahl, 1932, O. matritensis Ramirez-Montesi-
nos & Perez-Silva, 1966, O. multiseta Dragesco, 1966, O. paragranulifera Shao
et al., 2014, O. procera Kahl, 1932, O. proximata Shibuya, 1930, O. quadricirrata
Blatterer & Foissner, 1988, O. seokmoensis Kim & Min, 2019, and O. variabilis
Groliére, 1975 (Berger 1999; Shao et al. 2014; Foissner 2016; Méndez-Sanchez
et al. 2018; Kim and Min 2019; Zhu et al. 2021; Bharti and Kumar 2023).

Oxytricha aeruginosa contrasts with Oxytricha chonggingica sp. nov. in hav-
ing black and orange-yellow (vs colorless) cortical granules (Berger 1999).

Compared to Oxytricha chonggingica sp. nov., O. bimembranata is very differ-
ent in cortical granules absent (vs. present), and contractile vacuole at level of
buccal vertex (vs at about mid-body) (Berger 1999).

Although O. procera is a poorly known species, it can still be distinguished
from Oxytricha chonggingica sp. nov. in cell shape (slender spindle-shaped vs
usually elliptical), positions of contractile vacuole (distinctly in front of mid-
body vs at about mid-body) and transverse cirri (caudally vs subcaudally), as
well as length of cilia of caudal cirri (distinctly longer than cilia of transverse
cirri vs. inconspicuous) (Berger 1999).

Oxytricha granulifera is mainly distinguishable from Oxytricha chonggingica
sp. nov. in dorsal kineties 3 and 4 clearly separated (vs. connected), and dorsal
kinety 5 terminates at about mid-body (vs 80% of cell length) of cell. In addi-
tion, differences exist in sequence similarities (97.6%—97.8%) and nucleotides
32-38 (Table 2) (Berger 1999; Shao et al. 2014; Méndez-Sanchez et al. 2018;
Zhu et al. 2021).

Oxytricha multiseta is different from Oxytricha chonggingica sp. nov. in num-
bers of transverse cirri (six or seven vs four or five), and adoral membranelles
(26-29 vs 37-49), as well as ratio of body length: width (about 3.6 vs 2.5-3.0)
(Berger 1999).

In comparison with Oxytricha chonggingica sp. nov., Oxytricha matritensis
manifests distinguishing characteristics in the absence (vs presence) of cau-
dal cirri, and numbers of cirri in right (about 17, data from drawing vs 32-46)
and left (about 15, data from drawing vs 33-43) marginal rows (Berger 1999).

Oxytricha proximata can be separated from Oxytricha chonggingica sp. nov.
by cortical granules absent (vs present), ratio of body length to width about 2.2

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Haifeng Han et al.: Morphology of a novel

hypotrichous ciliate, Oxytricha chonggingica sp. nov.

Table 2. Sequence similarities (below diagonal) and nucleotide differences (above diagonal) among Oxytricha chonggin-

gica sp. nov. and O. granulifera.

Sequences 1 2 3 4 5 6 7 8
1. Oxytricha chonggingica sp. nov. oF, 34 35 a0 38 32 36
2. O. granulifera AF164122 0.976 3 18 4 20 12
3. O. granulifera AF508762, AM412768, AM412769, 0.978 0.998 TS 1 FF 9 2
AM412771, AM412772, MG836525, MG836526, MG836527,
MG836528, MG836529, MG836530
4. O. granulifera AM412770 0.978 0.988 | 0.99 16 26 18 17
5. O. granulifera KJ081199, MG836531, MG836532, 0.978 | 0.997 | 0.999 | 0.989 16
MG836533, MG836534, MG836535, MG836536
6. O. granulifera ssp. MT622649 0.976 | 0.987 | 0.989 | 0.983 | 0.989 16 19
7. O. granulifera chiapasensis KX889988 0.979 0.992 | 0.994 0.988 | 0.994 0.989 11
8. O. granulifera granulifera MW143561, MW143562 0.977 | 0.996 | 0.998 | 0.989 | 0.998 | 0.988 | 0.993

Table 3. Sequence similarities (below diagonal) and nucleotide differences (above diagonal) among Oxytricha chonggin-
gica sp. nov. and six molecularly related species.

Sequences 1 2 3 4 5 6 7
1. Oxytricha chongqingica sp. nov. PV476686 19 22 25 24 48 22
2. Paroxytricha longigranulosa JX899420 0.987 7 19 13 35 9
3. Paroxytricha ottowi JQ723977 0.986 | 0.995 22 18 40 14
4. Allotrichides antarcticus AY498652 0.984 0.991 0.988 18 46 20
5. Oxytricha quadricirrata 0Q231489 0.984 | 0.987 | 0.986 | 0.988 36 10
6. Oxytricha paragranulifera KJO81200 0.969 0.977 | 0.974 | 0.977 0.97 26

7. Oxytricha nauplia MH035977

ZooKeys 1247: 137-149 (2025), DOI: 10.3

0.986 0.994 0.991 0.993 0.987 0.983

(vs 2.5-3.0), and distance between cirri V/2 and V/3 almost equal to (vs largely
greater than) those between V/4 and V/3, and V/2 and VI/2 (Berger 1999).
Oxytricha paragranulifera differs from O. chonggingica sp. nov. in having Sty-
lonychia-patterned (vs Oxytricha-patterned) endoral and paroral, as well as low-
er numbers of adoral membranelles (25-29 vs 37-49), cirri in right (18-25 vs
32-46) and left (18-25 vs 33-43) marginal rows, and dikinetids in kineties 1
(15-23 vs 33-52), 2 (16-23 vs 25-39), 3 (13-17 vs 19-29), 4 (six or seven vs
9-15), 5 (six or seven vs 15-28), and 6 (two vs 9-19) (Shao et al. 2014).
Oxytricha quadricirrata differs from Oxytricha chonggingica sp. nov. ina small-
er size in life (70-100 x 20-30 um vs 100-160 x 40-60 um), lower numbers
of adoral membranelles (19-21 vs 37-49), cirri in right (14-17 vs 32-46) and
left (13-18 vs 33-43) marginal rows, dikinetids in kineties 1 (9-14 vs 33-52), 2
(7-11 vs 25-39), 3 (5-14 vs 19-29), 4 (three to seven vs 9-15), 5 (three or four
vs 15-28) and 6 (one or two vs 9-19), as well as dorsal kinety 5 terminates at
40% (vs 80%) down length of cell (Berger 1999, Bharti and Kumar 2023).
Oxytricha seokmoensis is distinct from Oxytricha chonggingica sp. nov. in
lower numbers of dikinetids in dorsal kineties 1 (20-26 vs 33-52), 3 (12-17 vs
19-29), and 6 (four to six vs 9-19), as well as ends of right and left marginal
rows separate (vs confluent) (Kim and Min 2019).
Oxytricha variabilis can be distinguished from Oxytricha chonggingica sp.
nov. by numbers of postoral ventral cirri (five vs three) and dorsomarginal kine-
ties (one vs two or three) (Berger 1999).

897/zookeys.1247.155543 146

Haifeng Han et al.: Morphology of a novel hypotrichous ciliate, Oxytricha chonggingica sp. nov.

Phylogenetic analyses

The SSU rDNA phylogenetic construction revealed a non-monophyletic group
of Oxytricha species, consistent with previous molecular phylogenetic studies
(Kwon and Shin 2008; Fan et al. 2021; Wang et al. 2021; Jin et al. 2022; Bharti and
Kumar 2023). Notably, Oxytricha chongqgingica sp. nov. formed a weak to mod-
erately supported clade with O. nauplia, Allotrichides antarcticus, O. paragranulif-
era, O. quadricirrata, Paroxytricha longigranulosa, and P. ottowi. This phylogenetic
association is supported by sharing morphological characteristics (e.g. 18 fron-
toventral-transverse cirri, dorsal kinety 3 produces kinety 4 during ontogenesis
and 3 caudal cirri produced by dorsal kineties 1, 2, 4 in total) between the novel
species and the known species. Interestingly, despite their striking morphologi-
cal similarities, the SSU rDNA sequence of O. chonggingica sp. nov. was found to
cluster phylogenetically distant from O. granulifera. This result possibly indicates
the necessity of incorporating multiple genetic loci, in addition to SSU rDNA, to
clarify phylogenetic relationships. Moreover, sequencing molecular data for mor-
phologically defined species is critical for robust evolutionary reconstructions.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Use of Al

This study utilized DeepSeek solely for language polishing and grammatical refinement.

Funding

This work was supported by the Fund for survey of Invertebrates from Yintiaoling Na-
tional Nature Reserve (CQS21C00739, CQS24C00333).

Author contributions

Haifeng Han: Writing — original draft, Data curation. Xiaojin Xue: Methodology. Zhisheng
Zhang, Chen Shao: Writing — review and editing, Validation, Supervision.

Author ORCIDs

Haifeng Han © https://orcid.org/0009-0009-8151-572X
Zhisheng Zhang ® https://orcid.org/0000-0002-9304-1789
Chen Shao © https://orcid.org/0000-0001-8474-3204

Data availability

All of the data that support the findings of this study are available in the main text.

References

Alfaro ME, Zoller S, Lutzoni F (2003) Bayes or bootstrap? A simulation study comparing
the performance of Bayesian Markov chain Monte Carlo sampling and bootstrap-

ZooKeys 1247: 137-149 (2025), DOI: 10.3897/zookeys.1247.155543 147

Haifeng Han et al.: Morphology of a novel hypotrichous ciliate, Oxytricha chonggingica sp. nov.

ping in assessing phylogenetic confidence. Molecular Biology and Evolution 20(2):
255-266. https://doi.org/10.1093/molbev/msg028

Berger H (1999) Monograph of the Oxytrichidae (Ciliophora, Hypotrichia). Monographi-
ae Biologicae 78: 1-1080. https://doi.org/10.1007/978-94-011-4637-1_1

Bharti D, Kumar S (2023) Description of a new oxytrichid ciliate, Oxytricha buxai n. sp.
and redescription of O. guadricirrata Blatterer and Foissner, 1988 based on morphol-
ogy and 18S rDNA analyses. European Journal of Protistology 88: e125959. https://
doi.org/10.1016/j.ejop.2023.125959

Fan X, Yao S, Luo X, Dong T, Xu Y, Chen L, Bourland W, Zhao Y, Huang J (2021) Some
morphologically distinguishable hypotrich ciliates share identical 18S rRNA gene
sequences-taxonomic insights from a case study on Oxytricha species (Protista,
Ciliophora). Zoological Journal of the Linnean Society 193(1): 356-379. https://doi.
org/10.1093/zoolinnean/zlaa145

Foissner W (1987) Soil protozoa: Fundamental problems, ecological significance, adap-
tations in ciliates and testaceans, bioindicators, and guide to the literature. Progress
in Protistology 2: 69-212.

Foissner W (2016) Terrestrial and semiterrestrial ciliates (Protozoa, Ciliophora) from
Venezuela and Galapagos. Denisia 35: 1-912.
Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and anal-
ysis program for windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98.
Hillis DM, Bull JJ (1993) An empirical test of bootstrapping as a method for assessing
confidence in phylogenetic analysis. Systematic Biology 42(2): 182-192. https://doi.
org/10.1093/sysbio0/42.2.182

Ivica L, Peer B (2024) Interactive tree of life iTOL v6: Recent updates to the phylogenetic
tree display and annotation tool. Nucleic Acids Research 52(W1): W78-W82. https://
doi.org/10.1093/nar/gkae268

Jin D, Li L, Lyu J, Warren A, Shao C (2022) Morphogenesis and molecular phylogeny of
a freshwater ciliate, Oxytricha multilineata n. sp. (Ciliophora, Hypotrichia). European
Journal of Protistology 82: e125864. https://doi.org/10.1016/j.ejop.2022.125864

Kalyaanamoorthy S, Minh BQ, Wong TKF, Von Haeseler A, Jermiin LS (2017) ModelFind-
er: Fast model selection for accurate phylogenetic estimates. Nature Methods 14(6):
587-589. https://doi.org/10.1038/nmeth.4285

Kim K-S, Min G-S (2019) Morphology and molecular phylogeny of Oxytricha seokmoen-
sis sp. nov. (Hypotrichia: Oxytrichidae), with notes on its morphogenesis. European
Journal of Protistology 71: e125641. https://doi.org/10.1016/j.ejop.2019.125641

Kwon CB, Shin HC (2008) Two newly recorded ciliates, Oxytricha longigranulosa and O.
marina (Ciliophora: Spirotrichea: Sporadotrichida) from Korea. Animal Systematics,
Evolution and Diversity 24(1): 81-88. https://doi.org/10.5635/KJSZ.2008.24.1.081

Medlin LK, Elwood Hu, Stickel SK, Sogin ML (1988) The characterization of enzymatical-
ly amplified eukaryotic 16S-like rRNA-coding regions. Gene 71(2): 491-499. https://
doi.org/10.1016/0378-1119(88)90066-2

Méndez-Sanchez D, Mayén-Estrada R, Luo X, Hu X (2018) A new subspecies of Oxytricha
granulifera (Hypotrichia: Oxytrichidae) from Mexico, with notes on its morphogenesis
and phylogenetic position. The Journal of Eukaryotic Microbiology 65(3): 357-371.
https://doi.org/10.1111/jeu.12479

Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, Lan-
fear R (2020) IQ-TREE 2: New models and efficient methods for phylogenetic infer-
ence in the genomic era. Molecular Biology and Evolution 37(5): 1530-1534. https://
doi.org/10.1093/molbev/msaa015

ZooKeys 1247: 137-149 (2025), DOI: 10.3897/zookeys.1247.155543 148

Haifeng Han et al.: Morphology of a novel hypotrichous ciliate, Oxytricha chonggingica sp. nov.

Ronquist F, Teslenko M, van der Mark P Ayres DL, Darling A, Hohna S, Larget B, Liu L,
Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic
inference and model choice across a large model space. Systematic Biology 61(3):
539-542. https://doi.org/10.1093/sysbio/sys029

Rozewicki J, Li S, Amada KM, Standley DM, Katoh K (2019) MAFFT-DASH: Integrated
protein sequence and structural alignment. Nucleic Acids Research 47: W5-W10.
https://doi.org/10.1093/nar/gkz342

Shao C, Lv Z, Pan Y, Al-Rasheid KAS, Yi Z (2014) Morphology and phylogenetic analysis
of two oxytrichid soil ciliates from China, Oxytricha paragranulifera n. sp. and Oxytri-
cha granulifera Foissner and Adam, 1983 (Protista, Ciliophora, Hypotrichia). Interna-
tional Journal of Systematic and Evolutionary Microbiology 64(Pt_9): 3016-3027.
https://doi.org/10.1099/ijs.0.062281-0

Wang J, Zhang T, Li F, Warren A, Li Y, Shao C (2021) A new hypotrich ciliate, Oxytricha
xianica sp. nov., with notes on the morphology and phylogeny of a Chinese popula-
tion of Oxytricha auripunctata Blatterer & Foissner, 1988 (Ciliophora, Oxytrichidae).
Marine Life Science & Technology 3(3): 303-312. https://doi.org/10.1007/s42995-
020-00089-1

Weisse T, Moser M, Scheffel U, Stadler P Berendonk T, Weithoff G, Berger H (2013) Sys-
tematics and species-specific response to pH of Oxytricha acidotolerans sp. nov. and
Urosomoida sp. (Ciliophora, Hypotricha) from acid mining lakes. European Journal of
Protistology 49(2): 255-271. https://doi.org/10.1016/j.ejop.2012.08.001

Wilbert N (1975) Eine verbesserte Technik der Protargolimpragnation fiir Ciliaten.
Mikrokosmos 64: 171-179.

Xiang C, Gao F, Jakovlié |, Lei H, Hu Y, Zhang H, Zou H, Wang G, Zhang D (2023) Using
PhyloSuite for molecular phylogeny and tree-based analyses. iMeta 2(1): e87. https://
doi.org/10.1002/imt2.87

Zhang D, Gao F, Jakovlié |, Zou H, Zhang J, Li WX, Wang GT (2020) PhyloSuite: An in-
tegrated and scalable desktop platform for streamlined molecular sequence data
management and evolutionary phylogenetics studies. Molecular Ecology Resources
20(1): 348-355. https://doi.org/10.1111/1755-0998.13096

Zhu R, Qu Z, Zhang Q, Filker S, Stoeck T, Li F, Hu X (2021) A new record of Oxytricha gran-
ulifera granulifera Foissner and Adam, 1983 (Protozoa, Ciliophora, Oxytrichidae) from
a hot spring in Iceland, with notes on its abnormal form during cultivation. Frontiers in
Marine Science 8: e621349. https://doi.org/10.3389/fmars.2021.621349

ZooKeys 1247: 137-149 (2025), DOI: 10.3897/zookeys.1247.155543 149