Diversity of Free-Living Marine Ciliates (Alveolata, Ciliophora): Faunal

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European Journal of Protistology 61 (2017) 424–438

Review

Diversity of free-living marine ciliates (Alveolata, Ciliophora): Faunal

studies in coastal waters of China during the years 2011–2016

a,b,1 a,c,h,1 a,d,1 a,e,1 a,1

Weiwei Liu , Jiamei Jiang , Yuan Xu , Xuming Pan , Zhishuai Qu ,

a,1 f g a,∗ a,c,∗

Xiaotian Luo , Hamed A. El-Serehy , Alan Warren , Honggang Ma , Hongbo Pan

Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China

Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Science,

Guangzhou 510301, China

College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China

State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai 200062, China

College of Life Science and Technology, Harbin Normal University, Harbin 150025, China

Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

Department of Life Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, UK

College of Marine Ecology and Environment, Shanghai Ocean University, Shanghai 201306, China

Available online 21 April 2017

Abstract

In the period 2011–2016, a series of investigations were carried out on the marine and brackish free-living ciliate fauna

of the temperate–tropical coastal waters of China. About 210 morphotypes including over 100 new species within six

groups (cyrtophorians, hypotrichs s.l., karyorelicteans, oligotrichs, pleurostomatids, and scuticociliates) were isolated and

described in detail from observations of live cells and silver-stained specimens. Based on their morphology, morphogene-

sis and molecular phylogeny, three new families (Wilbertomorphidae, Kentrophyllidae, Protolitonotidae) and 22 new genera

(Apotrachelocerca, Wilbertomorpha, Protolitonotus, Paracyrtophoron, Heterohartmannula, Aporthotrochilia, Falcicyclidium,

Paramesanophrys, Pseudodiophrys, Monocoronella, Neourostylopsis, Apobakuella, Parabistichella, Heterokeronopsis, Het-

erotachysoma, Antiokeronopsis, Apoholosticha, Pseudogastrostyla, Antestrombidium, Sinistrostrombidium, Williophrya, and

Varistrombidium) were established. In the present review, we summarize these studies which show there is a large, undiscovered

diversity of ciliates, especially in undersampled habitats, such as subtropical/tropical coastal waters, mangrove wetlands, estuar-

ies and aquaculture ponds. We also highlight the importance of integrative approaches, combining morphology, morphogenesis

and molecular phylogeny, in order to understand ciliate systematics and ecosystem function.

Keywords: Cyrtophoria; Hypotricha; Karyorelictea; Oligotrichea; Pleurostomatida; Scuticociliatia

Introduction

∗ Ciliates have the greatest diversity of cell structure among

Corresponding authors.

all eukaryotic microorganisms and play signiﬁcant roles

E-mail addresses: [email protected] (H. Ma), [email protected] (H.

Pan). in studies of cell biology, genetics, ecology, and evolution

All authors contribute equally.

http://dx.doi.org/10.1016/j.ejop.2017.04.007

W. Liu et al. / European Journal of Protistology 61 (2017) 424–438 425

Fig. 1. Sampling sites and habitats.

(Hausmann and Bradbury 1996). Over 8000 nominal species ing, scanning electron microscopy, and phylogenetic analyses

have been reported to date (Lynn 2008), but most of these mostly of small subunit rRNA (SSU rRNA) gene sequence

are known only from living material or from silver-stained data. Morphogenetic processes have also been investigated

specimens alone. Consequently, the overwhelming majority for selected taxa. This has resulted in the discovery of nearly

of ciliate species are inadequately described by modern stan- 1000 species, mostly from coastal waters of the Bohai Sea,

dards (Song et al. 2009; Warren et al. 2017). Furthermore, it Yellow Sea and South China Sea (Song et al. 2009). Since

is likely that many species await discovery, especially from 2011, much of this work was supported, or at least facili-

undersampled habitats such as the deep ocean and tropical tated, by the International Research Coordination Network

marine environments, and it has been estimated that 83–89% for Biodiversity of Ciliates (IRCN-BC) which is funded by

of free-living ciliate species are undescribed (Foissner et al. the United States National Science Foundation (NSF) and the

2008). Apart from marine planktonic forms, relatively few Natural Science Foundation of China (NSFC). Here we sum-

ciliates have been investigated in terms of their functional marize the ﬁndings published during the period 2011–2016

ecology. They tend to be lumped together as a group or guild for six major ciliate groups: cyrtophorians, hypotrichs s.l.,

in ecological studies rather than having the functional roles karyorelicteans, oligotrichs, pleurostomatids, and scuticocil-

of individual species described. Therefore, there is a press- iates. In total, about 210 morphospecies have been isolated

ing need for thorough investigations of ciliate biodiversity, and identiﬁed of which over 100 are described for the ﬁrst

especially in marine habitats. time. In addition, three new families and 22 new genera have

Over the past 25 years a series of investigations have been been established. Although there are other groups which

carried out on the ciliate fauna in the temperate and tropical are important and have high diversity, e.g. peritrichs, het-

coastal waters of China (Fig. 1). The vast majority of these cil- erotrichs, nassophoreans, they are excluded in the present

iates were investigated using a combination of morphological review because relatively few reports focusing on these were

and molecular methods, e.g. live observation, silver stain- published during the period 2011–2016.

426 W. Liu et al. / European Journal of Protistology 61 (2017) 424–438

Fig. 2. Selected karyorelicteans from Chinese coastal habitats (original). (A) Apotrachelocerca sp. (B, G) Tracheloraphis spp. (C) Kovalevaia

sp. (D, E) Trachelocerca spp. (F) Geleia sp. (H, M) Kentrophoros spp. (I) Wilbertomorpha sp. (J) Apocryptopharynx sp. (K) Loxodes sp. (L)

Remanella sp. Scale bars = 200 ␮m (A–D, G); 150 ␮m (E, F, H, M); 20 ␮m (I, J); 40 ␮m (K).

Karyorelicteans (Fig. 2A–M) Dragesco 1996). Therefore, knowledge of the infracilia-

ture, especially the structure of the oral ciliature, is lacking

Karyorelictean ciliates are characterized by their elon- for most species (Dragesco and Dragesco-Kernéis 1986;

gated, vermiform, often ﬂattened body shape and numerous Foissner 1995, 1996).

non-dividing paradiploid macronuclei that arise from divi- Since 2011, through integrating taxonomic and genetic

sion of the micronuclei (Lynn, 2008). It is largely on account methods, we studied 27 marine species of karyorelicteans

of this latter character that the class Karyorelictea Corliss, from Chinese coastal waters, including 11 new and nine

1974 is widely considered to represent the ancestral cili- poorly known species, and revised the phylogenetic posi-

ate lineage since the non-dividing macronucleus is thought tions of three families based on small subunit (SSU) rRNA

to be a primitive character (Corliss 1979). With the excep- gene sequences (Table S1 (in the online version at DOI:

tion of the freshwater genus Loxodes Ehrenberg, 1830, all 10.1016/j.ejop.2017.04.007); Xu et al. 2011a,b, 2013, 2015;

karyorelicteans are marine and are commonly found in inter- Yan et al. 2015, 2016). The combination of morphological

stitial environments (Al-Rasheid 1998). Although it has and molecular data provides strong evidence for our taxo-

been nearly two hundred years since the ﬁrst karyorelictean nomic conclusions. These studies are here summarized.

was discovered, few species within this group have been Wilbertomorpha colpoda Xu et al. 2013 was found to rep-

studied in detail both in vivo and after protargol staining. resent a new family, Wilbertomorphidae Xu et al., 2013;

This is largely because the cells are extremely fragile and because of its unique morphological features. Phylogenetic

none have been brought into stable culture (Foissner and analyses based on SSU rRNA gene sequence data support this

W. Liu et al. / European Journal of Protistology 61 (2017) 424–438 427

Fig. 3. Selected pleurostomatid ciliates from China seas (original). (A) Protolitonotus sp. (B, E) Litonotus spp. (C, G, H) Amphileptus spp.

(D, I, K) Loxophyllum spp. (F, J) Kentrophyllum spp. Scale bars = 150 ␮m (A); 100 ␮m (B, C, E, F); 50 ␮m (D, H, I, J, K).

ﬁnding with W. colpoda occupying a deep-branching position Pleurostomatids (Fig. 3A–K)

between the families Geleiidae Kahl, 1933 and Loxodidae

(Xu et al. 2013). Pleurostomatids belong to the class Litostomatea Small

Following a detailed reinvestigation of its oral ciliature, and Lynn, 1981. Unlike other litostomateans, members of

Trachelocerca arenicola Kahl, 1933 was revealed to rep- the order Pleurostomatida Schewiakoff, 1896 have a lat-

resent a new genus, Apotrachelocerca Xu Y. et al., 2011. erally compressed body, conspicuously fewer and shorter

After integrating recent morphological and molecular phylo- left somatic kineties, and a ventrally positioned slit-like

genetic studies on the family Trachelocercidae Kent, 1881, cytostome (Lynn 2008).

two new hypotheses about the evolution of its seven genera Most pleurostomatids are periphytic and can be found in a

were posited (Yan et al. 2016). wide range of habitats such as lakes, streams, marine water,

Following studies on six Remanella spp., we summa- and wastewater-treatment plants and have been reported from

rized the morphological and ecological differences between (sub)tropical, temperate and polar regions. According to

Remanella Foissner, 1996 and Loxodes Ehrenberg, 1830 (Xu Lynn (2008), Pleurostomatida includes 15 genera that are

et al. 2015). Phylogenetic analyses based on SSU rRNA gene assigned to two families, Amphileptidae Bütschli, 1889 and

sequence data suggested that the monophyletic freshwater Litonotidae Kent, 1882. Previous studies revealed that cer-

genus Loxodes evolved from its marine relatives (Xu et al. tain characters are reliable for species identiﬁcation, i.e. body

2015). Based on morphological and morphometric charac- shape, number and position of the contractile vacuoles, shape

teristics, an illustrated key to the identiﬁcation of all valid and the distribution of extrusomes, number of macronuclei

species of Remanella was provided by Xu et al. (2013). In and number of somatic kineties (Foissner 1984; Song and

addition, we improved the diagnosis of the genus Geleia Kahl Wilbert 1989; Song et al. 2009). To date, nearly two hun-

in Foissner, 1998, following a study of the oral structure of dred nominal species of pleurostomatids have been reported

Geleia sinica Xu et al. 2011 (Xu et al. 2011b). although few have been described with details both of their

428 W. Liu et al. / European Journal of Protistology 61 (2017) 424–438

characters in vivo and of their infraciliature (Pan et al. 2014, Cyrtophorians (Fig. 4A–O)

2015c).

During our investigations, 24 species, including 20 new The cyrtophorians are a highly specialized and divergent

species and four poorly known species, were described (Table group of ciliates with more than 160 species reported globally

S1 in the online version at DOI: 10.1016/j.ejop.2017.04.007). to date. Most can be found in the periphyton in aquatic habi-

Among these, three isolates showed a special ciliary pattern tats, i.e., marine, brackish and freshwater, or in soil, and few

with the right somatic kineties being progressively shortened are parasitic (Deroux 1970; Foissner et al. 1991; Kahl 1931;

along the rightmost full kineties (Fig. 3A). Furthermore, phy- Song et al. 2009). Cyrtophorians are characterized as follows:

logenetic analyses based on SSU rRNA gene sequence data (i) the cilia are mainly conﬁned to the ventral side with only a

showed that these isolates formed a clade that occupied a few somatic kineties and terminal fragments on the anterior

basal position within the Pleurostomatida (Wu et al. 2016). dorsal side; (ii) there is usually an equatorial fragment on

Based on these ﬁndings, a new family, Protolitonotidae Wu the right margin; (iii) the funnel-shaped cyrtos is encircled

et al., 2016, and a new genus, Protolitonotus Wu et al., 2016; by toothed nematodesmal rods; and (iv) the macronucleus is

were proposed. The classiﬁcation of Pleurostomatida was heteromerous (Song et al. 2009). The subclass Cyrtophoria

revised in the light of these ﬁndings (Wu et al. 2016). Fauré-Fremiet in Corliss, 1956 is thought to be monophyletic

Morphologically, Kentrophyllum Petz, Song and Wilbert, (Lynn 2008).

1995 can be distinguished from Epiphyllum Lin, Song and In the period 2011–2016, 43 marine or brackish water

Warren, 2005 by having spines along the cell margin (vs. species, belonging to six families and 20 genera, were isolated

absent in the latter). However, phylogenetic analyses based from coastal waters of China and studied in detail using both

on SSU rRNA gene sequence data showed that they clus- morphological and molecular methods (Chen et al. 2011c,

tered together in a strongly supported clade that is highly 2012; Pan et al. 2012, 2013, 2016a; Qu et al. 2015a,b,c, 2016).

divergent from other members of the family Amphiletidae As well as revealing further the true diversity of this group,

Bütschli, 1889 (Wu et al. 2015). Therefore, Epiphyllum with these studies have enriched the SSU rDNA database by the

Kentrophyllum were synonymized and a new family, Ken- deposition of sequences from carefully identiﬁed, vouchered

trophyllidae Wu et al., 2015; was established for this group. specimens.

Additionally, a taxonomic revision and a key to the identi- Three new genera were established, namely Heterohart-

ﬁcation of the species of Kentrophyllum were provided (Wu mannula Pan et al. 2012; Aporthotrochilia Pan et al. 2012;

et al. 2015). and Paracyrtophoron Chen et al. 2012. The genus Trochil-

In contrast to other genera in the family Litonotidae Kent, ioides (Kahl, 1931) was re-established with T. recta (Kahl,

1882, the extrusomes of the genus Loxophyllum Dujardin, 1928) Chen et al. 2011 as the type species (Chen et al. 2011c).

1841 are distributed along both the ventral and the dorsal mar- Another two species, Trochilioides tenuis (Deroux, 1976)

gins, usually forming warts along the latter. We collected nine Chen et al. 2011 and Trochilioides recta (Kahl, 1923) Chen

Loxophyllum spp., eight of which were previously unknown et al. 2011; were redescribed.

(Wu et al. 2014). In addition, marine and brackish water The genus Chlamydodon Ehrenberg, 1835 was taxonom-

species of the genus were reviewed. As a result, Loxophyllum ically revised, a key to the identiﬁcation of species was

fasciolatus Dragesco, 1966 and Loxophyllum multiplicatum provided and three new and one poorly known species

Kahl, 1931 were transferred to the genus Litonotus Wres- were described (Pan et al. 2013, 2016a). The species-rich

niowski, 1870 based on the distribution of their extrusomes. genus Dysteria Huxley, 1857 was reviewed with 11 species

For Amphileptus Ehrenberg, 1830 and Litonotus, we reported, indicating its high diversity (Pan et al. 2016a; Qu

reported ﬁve new species of the former and two new species et al. 2015c). In addition, three species of Pseudochilodonop-

of the latter, provided a detailed redescription of Litonotus sis Foissner, 1979 were reported from brackish waters and a

duplostriatus (Maupas, 1883) Kahl, 1931 and emended the key to the identiﬁcation of the species was provided (Qu et al.

diagnosis of A. marinus (Kahl, 1931) Song et al., 2004 (Pan 2015c).

et al. 2014, 2015c). By adopting an integrative approach, i.e. combining mor-

Compared to other litostomateans, pleuostomatids have phological and molecular data, we have elucidated a number

relatively few diagnostic characters and most of these of taxonomic problems within the Cyrtophoria. Nevertheless

are numerical parameters. Integrating morphology with much work remains to be done, in particular with respect to

molecular information has therefore greatly improved our under-studied groups such as those within the family Ple-

understanding of the taxonomy and systematics of the Pleu- siotrichopidae. With sample collections extended, there are

rostomatida, The molecular data not only provide another also cryptic species in cyrtophorians (Katz et al. 2011). In

kind of character for species delineation, but also indicate if a order to resolve the status of cryptic species, more effort

group likely represents a new taxon, such as Protolitonotidae with combination of morphological, molecular and ecologi-

and Kentrophyllidae. cal characters is required.

W. Liu et al. / European Journal of Protistology 61 (2017) 424–438 429

Fig. 4. Selected cyrtophorid ciliates found in China Seas (original). (A, B) Heterohartmannula sp. (C, D) Orthotrochilia sp. (E, F) Coeloperix

sp. (G, H) Chlamydodon sp. (I) Paracyrtophoron sp. (J) Dysteria sp. (K) Trochilia sp. Scale bars = 30 ␮m (A–D, G–I, L–N); 20 ␮m (E, F, J,

K).

Scuticociliates (Fig. 5A–S) 2016b; Pan et al. 2015a,b, 2016b). Morphometrical com-

parison of known “ophrys”-species populations, i.e. species

Ciliates of the subclass Scuticociliatia Small, 1967 are of Mesanophrys, Paramesanophrys and Metanophrys, was

common inhabitants of freshwater, brackish water, and also provided in Pan et al. (2016b). The genus Uronemita

marine environments (Wiackowski et al. 1999; Mallo et al. Jankowski, 1980 (junior synonym: Uronemella Song and

2014). Many are free-living whereas others are symbionts or Wilbert, 2002) was re-activated and re-deﬁned with Urone-

facultative pathogens of fishes and invertebrates (de Puytorac mita filificum (Kahl, 1931) Jankowski 1980 as the type

et al. 1974). Owing to their small body size and lack of species. Five species formerly assigned to Uronemella were

distinguishing morphological characters, the taxonomy of transferred to Uronemita (Liu et al. 2016b).

this group remains difﬁcult and confused. Scuticociliates Following studies of ﬁve species of Pleuronema Dujardin,

are characterized by a paroral that is divided into three 1841, it was concluded that the most important morphological

segments: anterior, middle, and posterior (or scutica). The characters for species delimitation in this genus are body

scutica, named for its hook- or whip-like conﬁguration dur- shape, the location of the contractile vacuole pore and the

ing stomatogenesis, is the major synapomorphy for the group number of somatic kineties (Pan et al. 2015a).

(Lynn 2008). The subclass Scuticociliatia comprises three With the aid of the combination of the morphological and

orders: Pleuronematida Kent, 1881, Philasterida Small, 1967 molecular methods, our studies revealed the high diversity of

and Thigmotrichida Chatton and Lwoff, 1922. scuticociliates in China and accurately circumscribed several

During the period 2011–2016, 31 species, including 10 closely related species. In contrast to the high diversity, only

new species and one new combination, were described from few molecular data of scuticociliates are available in database

coastal waters of China. Most of these studies involved a (e.g. GenBank), which greatly impedes on its diversity, tax-

combination of morphology and molecular methods. Based onomy and phylogeny research. Further investigation with

on its unique oral apparatus, the genus Paramesanophrys Pan extending the sampling region and the DNA collection are

et al., 2016; was proposed and assigned to the family Orchi- required.

tophryidae Cépède, 1910 (Table S1 (in the online version at

DOI: 10.1016/j.ejop.2017.04.007; Fan et al. 2011; Liu et al.

430 W. Liu et al. / European Journal of Protistology 61 (2017) 424–438

Fig. 5. Selected scuticociliates from China seas (original). (A–C, E, P) Pleuronema spp. (D, M, O) Uronema spp. (F) Pseudocohnilembus

sp. (G, I) Metanophrys spp. (H, K) Histiobalantium spp. (J) Parauronema sp. (L, N, S) Philaster spp. (Q, R) Pseudoplatynematum spp. Scale

bars = 50 ␮m (A–C, E, K, L, N, P–S); 20 ␮m (D, F–J, M, O).

Oligotrichs sensu lato (Fig. 6A–Z) functional ecology of this group has been extensively stud-

ied worldwide, accurate species-level identiﬁcations were

Oligotrich ciliates are common marine microplankters rarely included in these works because of the lack of olig-

that usually make a signiﬁcant contribution to the energy otrich diversity data (Agatha 2011). Owing to their small

ﬂow of the microbial loop (Azam et al. 1983). There are size and fragile body, many oligotrichs s.l. were tradition-

about 133 species of oligotrichians, over 1550 species of ally rather superﬁcially investigated with descriptions usually

choreotrichians comprising 53 aloricate species and over based on observations either of living cells or of Lugol’s-ﬁxed

1500 loricate (tintinnid) species, and with a single Lynnel- specimens (Kahl 1932). Quantitative Protargol Stain (QPS)

lid species (Agatha 2011; Liu et al. 2015a). Although the gives helps to make accurate identiﬁcation for oligotrichs by

W. Liu et al. / European Journal of Protistology 61 (2017) 424–438 431

Fig. 6. Selected oligotrich ciliates found in coastal water of China since 2011–2016 (original). (A, B) Sinistrostrombidium sp. (C, D) Ante-

strombidium sp. (E, F, K, L) Spirostrombidium spp. (G–J, M, N) Parallelostrombidium spp. (O, P) Apostrombidium sp. (Q, R) Strombidium

sp. (S–V) Pelagostrobilidium spp. (W, X) Strombidinopsis sp. Scale bars = 20 ␮m (A, M, O, Q, S, W), 30 ␮m (B–L, N, P, R, T–V, X–Z).

revealing their infraciliature, which remarkably increase the (Song et al. 2015). Following the comparison of girdle kinety

discovery of new oligotrich taxa (Lynn and Gilron 1993). patterns of species of Strombidium, it was concluded that the

However, many critical morphological characters from live sinistrally spiraled girdle kinety in S. tropicum Liu et al., 2015

cells were lacked in these descriptions, such as extrusomes probably represents a derived stage during the evolution of

and hemitheca which usually disappear after QPS staining. this structure (Liu et al. 2015b). In addition, comparison of the

A combination of living observation and silver staining is ciliary patterns of the genera Spirostrombidium Jankowski,

now regarded as essential for adequate species description 1978 and Novistrombidium Song and Bradbury, 1998 indi-

(Warren et al. 2017). cated that those species in which the posterior regions of the

In the past six years, 25 species belonging to seven gen- girdle overlap ventral kineties should be assigned to the genus

era of oligotrichs s.l. from the coastal waters of China Spirostrombidium. (Liu et al. 2015b).

have been investigated, including 15 new species and Four new oligotrich genera have been established: Willio-

four new genera (Table S1 in the online version at DOI: phrya Liu et al., 2011; Sinistrostrombidium Liu et al., 2015,

10.1016/j.ejop.2017.04.007). The distinctive hemitheca of Antestrombidium Liu et al., 2015 and Varistrombidium Xu

Strombidium caudispina Song et al., 2015; composed of et al., 2011. The bipartite horizontal girdle kinety in Willio-

square-shaped cortical platelets, was reported for the ﬁrst phrya suggests a close relationship between Strombidium

time in the genus Strombidium Claparède and Lachman, 1859 and Williophrya, which is supported by molecular phylo-

432 W. Liu et al. / European Journal of Protistology 61 (2017) 424–438

Fig. 7. Selected hypotrichid ciliates from China (original). (A) Polystichothrix sp. (B) Bakuella sp. (C, E, K) Pseudokeronopsis spp. (D)

Trichototaxis sp. (F, H, O) Holosticha spp. (G) Rubrioxytricha sp. (I) Pseudogastrostyla sp. (J) Uncinata sp. (L) Anteholosticha sp. (M)

Hypotrichidium sp. (N) Pseudourostyla sp. (P, R) Pseudouroleptus spp. (Q) Monocoronella sp. Scale bars = 50 ␮m (A–I, K–M, O–R); 300 ␮m

(J); 100 ␮m (N).

genetic analyses (Liu et al. 2011). The sinistrally spiraled et al., 1988 respectively, and the taxonomic signiﬁcance of

girdle kinety (GK) in Sinistrostrombidium was assumed to these characters was investigated (Liu et al. 2011a).

originate from the Strombidium-pattern by elongation and Among the choreotrichians, four aloricate species were

sinistral spiraling of the right portion of GK (Liu et al. reported including two new species of Pelagostrobilidium

2015b). The three parts of somatic ciliary pattern in Ante- Petz et al., 1995 and one new species of Strombidinop-

strombidium probably originated in parallel with that of sis Kent, 1881 (Liu et al. 2012, 2016a). By comparing the

Omegastrombidium from the Propecingulum pattern con- ciliary patterns in all known species of Rimostrombidium

cerning the orientation and location of the kineties (Liu et al. Jankowski, 1978 and Pelagostrobilidium, it was found that the

2015b). The unique ﬁve somatic kineties in Varistrombidium spiralling of the somatic kinety around the left side of the body

suggest its close relationship to Omegastrombidium pattern represents the main distinguishing character of Pelagostro-

(Xu et al. 2011c). bilidium. Based on these ﬁndings, improved diagnoses of

In addition, ten poorly known species have been both genera were supplied (Liu et al. 2012). The morphology

redescribed based on populations isolated from coastal waters of three tintinnid ciliates, namely Tintinnopsis tocantinensis

of China with supplementary data and improved species diag- Kofoid and Campbell, 1929, Tintinnopsis radix (Imhof, 1886)

noses (Liu et al. 2011a, 2016; Xu et al. 2011c). For example, Brandt, 1907, and Tintinnopsis cylindrica Daday, 1887, was

the presence of an endoral membrane in a Chinese popula- also investigated. This resulted in the ﬁrst detailed descrip-

tion of Cyrtostrombidium paralongisomum Tsai et al., 2015 tions of the ciliary patterns and improved diagnoses for T.

was reported for the ﬁrst time in the family Cyrtostrom- tocantinensis and T. radix (Jiang et al. 2012).

biidae (Liu et al. 2016a). Furthermore, in vivo characters The systematic position of Lynnella Liu et al., 2011 is

such as the distribution of extrusomes and the presence of ambiguous based on analyses of its molecular phylogeny (Liu

the hemitheca were described for Strombidium basimorphum et al. 2012). Our morphological analysis suggests that Lyn-

Martin & Montagnes, 1993 and S. chlorophilum Montagnes nella may represent an evolutionary lineage separate from

W. Liu et al. / European Journal of Protistology 61 (2017) 424–438 433

both the subclasses Oligotrichia and Choreotrichia. There- were newly established. The family Oxytrichidae was revised

fore, a new order Lynnellida was established for Lynnella, based on analyses of the living morphology, ciliary patterns

characterized by an open adoral zone of membranelles with- and dorsal morphogenetic patterns of representative mem-

out differentiation of anterior and ventral membranelles (Liu bers, including schematic illustrations and a key to typical

et al. 2015). ‘18 frontal-ventral-transverse-cirri genera’ of Oxytrichidae

Partly due to advancements in our knowledge and under- (Shao et al. 2014b).

standing of oligotrich diversity and systematics, increasing

attention is being paid to the functional ecology of olig-

Family Urostylidae Bütschli, 1889

otrichs s.l. (Agatha, 2011). The availability of morphological,

molecular and ecological data opens the door to integrative

The Urostylidae, which comprises specialized hypotrichs

approaches for the study of oligotrichs s.l. and their role in

with ventral cirri arranged in a zig-zag pattern, is also a

ecosystems worldwide.

highly diverse family. Eighteen species were collected, 12

of which were previously unknown. Three new genera were

established: Apobakuella Jiang et al., 2012; Parabistichella

Hypotrichs sensu lato (Figs. 7A–Q and

Jiang et al., 2011, and Neourostylopsis Chen et al., 2013. The

8A–R) establishment of the genus Neourostylopsis, which is well

supported by both its morphology and its molecular phy-

Hypotrichs s.l. are dorsoventrally ﬂattened spirotrichs with logeny based on SSU rDNA sequence analyses, is a good

prominent ventral cirri and inconspicuous dorsal bristle cilia. example of the advantage of data integration for improving

Members of two subclasses, Hypotrichia sensu Lynn 2008 taxonomy. Subsequently, a new combination Neourostylopsis

and Stichotrichia sensu Lynn 2008; were investigated in these ﬂavicana (Wang et al., 2011) was suggested. The place-

studies. ment of the little known genus Uncinata Bullington, 1940

Free-living hypotrichs show a considerable diversity in within the family Urostylidae was suggested based on an

terms of their morphology, morphogenesis, and molecu- investigation of the morphology and phylogenetic analy-

lar phylogeny (Hu 2014). To date, over 200 genera have ses of the type species, U. gigantea Bullington, 1940 (Luo

been described (Berger 1999, 2006, 2008, 2011) which are et al. 2015). In addition, Holosticha bradburyae Gong et al.,

assigned to 21 families (Lynn 2008). With a history of 2001 was transferred to Uncinata as U. bradburyae (Luo

over 150 years of investigation, hypotrichs are one of the et al. 2015). Three Metaurostylopsis spp., one of which was

best-known but most systematically ambiguous groups of new, were investigated and the genus Metaurostylopsis Song,

ciliates. Numerous nominal species need to be redescribed, Petz and Warren, 2001 was revised (Chen et al. 2011b;

misidentiﬁed taxa need to be reviewed, and many new taxa Song et al. 2011). Gene sequence data for the well-known

probably await discovery (Chen et al. 2015; Hu and Kusuoka genus Bakuella was made available for the ﬁrst time as a

2015; Luo et al. 2014). consequence of the investigation of the novel species B. sub-

Since 2011, 43 papers have been published that focus tropica Chen et al., 2013 (Chen et al. 2013). In addition, 13

mostly or entirely on hypotrichs from the coastal waters new or poorly-known species belonging to Anteholosticha

of China. Fifty-ﬁve species from 36 genera have been iso- Berger, 2003, Holosticha Wrzesniowski, 1877, Metaurosty-

lated, including 35 new species and 10 new genera (Table S1 lopsis Song, Petz and Warren, 2001, Uncinata or Tunicothrix

in the online version at DOI: 10.1016/j.ejop.2017.04.007). wilberti (Lin & Song, 2004) Xu et al., 2006, were described

Compared to the studies of other groups, many reports on in detail (Fan et al. 2014b, Luo et al. 2015).

hypotrichs integrate aspects of their morphology, morpho-

genesis and molecular systematics, thus offering a more

Family Pseudokeronopsidae Borror and

comprehensive understanding of their taxonomy. Due to

Wicklow, 1983

limitations of space, only a selection of these studies is

summarized here. For a list of all groups investigated

The pseudokeronopsids are characterized by the frontal

please refer to Table S1 (in the online version at DOI:

10.1016/j.ejop.2017.04.007). cirri arranged as a bicorona. A greater diversity of ciliary

patterns has been revealed for this group and four new

genera, Monocoronella Chen et al., 2011; Heterokeronop-

Family Oxytrichidae Ehrenberg, 1830 sis Pan et al., 2013; Antiokeronopsis Fan et al., 2014 and

Apoholosticha Fan et al., 2014, were established (Fan et al.

The family Oxytrichidae, which typically possesses 2014a,b). In addition, a population of Thigmokeronopsis

18-frontal-ventral-transverse cirri, is extremely species-rich stoecki Shao et al., 2008 was redescribed and a new species

and morphologically diverse. In these studies, 13 species Pseudokeronopsis erythrina Chen et al., 2011 was described

belonging to ten genera were investigated (Fan et al. 2015; (Chen et al. 2011a). Phylogenetic analyses based on molec-

Shao et al. 2014a). Two of these genera, Pseudogastrostyla ular, morphological and morphogenetic data demonstrated

Fan et al., 2015 and Heterotachysoma Shao et al., 2013, that the genera Thigmokeronopsis and Apokeronopsis are not

434 W. Liu et al. / European Journal of Protistology 61 (2017) 424–438

Fig. 8. Selected euplotid ciliates from China seas (original). (A-F) Euplotes spp. (G, H) Paradiophrys sp. (I, J) Pseudodiophrys sp. (K–N)

Diophrys spp. (O, P) Discocephalus sp. (Q, R) Aspidisca sp. Scale bars = 50 ␮m (A–N); 20 ␮m (O–R).

confamilial with Pseudokeronopsis and Nothoholosticha but, Diversity of Free-living Marine and

instead, must be transferred to the family Urostylidae (Chen

Brackish Water Ciliates in China

et al. 2011a).

During the period 2011–2016, faunistic studies of

free-living marine and brackish water ciliates in coastal

waters of China have resulted in the isolation of 210 mor-

Order Euplotida Small and Lynn, 1985

phospecies, 100 of which are new. This indicates that there

is a large undiscovered ciliate diversity, which is consistent

Four species of Euplotes Ehrenberg, 1831 (Euplotidae

with the conclusions of Agatha (2011) and Foissner et al.

Ehrenberg, 1838), four species of the Diophrys-complex

(2008). These studies also highlight the value of an integrative

Dujardin, 1841 (Uronychiidae Jankowski, 1975) and two

approach for improved understanding of ciliate systematics

species of Aspidisca Ehrenberg, 1830 (Aspidiscidae Ehren-

and biodiversity.

berg, 1830) were investigated, ﬁve of which were novel (Liu

Environmental factors (e.g. temperature, salinity, pH,

et al. 2015c). A new genus Pseudodiophrys Jiang et al., 2011

etc.) shape ciliate communities and determine species dis-

was established based on its reduced undulating membrane.

tributions (Finlay et al. 1997). In our investigation, most

Discocephalus pararotatorius Jiang et al., 2013 (Disco-

pleurostomatids and oligotrichs were collected from trop-

cephalidae Jankowski, 1979) was described (Jiang et al.

ical or subtropical coastal waters of China. Although these 2013).

W. Liu et al. / European Journal of Protistology 61 (2017) 424–438 435

two groups had previously been well investigated in the north to Prof. W. Song, OUC, the China-based leading scientist

China seas (Song et al. 2009), the proportion of new species of the IRCN-BC program for his helpful suggestions during

discovered since 2011 is still high (83% in pleurostomatids, drafting of the manuscript. We are also grateful to Prof. Denis

53% in oligotrichs). This is likely a result of few studies hav- H. Lynn and two anonymous reviewers for their constructive

ing previously been made of ciliates in tropical/subtropical comments that greatly improved the manuscript.

seas, especially of certain speciﬁc habitats such as mangrove

wetlands, estuaries and aquaculture ponds. Therefore further

faunistic investigations on such habitats are likely to reveal

signiﬁcant numbers of new taxa awaiting discovery.

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