The Diverse Morphogenetic Patterns in Spirotrichs and Philasterids

Available online at www.sciencedirect.com

ScienceDirect

European Journal of Protistology 61 (2017) 439–452

REVIEW

The diverse morphogenetic patterns in spirotrichs and philasterids:

Researches based on ﬁve-year-projects supported by IRCN-BC and NSFC

a,b,1 a,c,1 a,1 a,d,1 e,∗

Xumiao Chen , Xiaoteng Lu , Xiaotian Luo , Jiamei Jiang , Chen Shao ,

f g a,h

Khaled A.S. Al-Rasheid , Alan Warren , Weibo Song

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

Department of Marine Organism Taxonomy and Phylogeny, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China

University of Innstruck, Research Institute for Limnology, Mondseestrasse 9, 5310 Mondsee, Austria

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

The Key Laboratory of Biomedical Information Engineering, Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong

University, Xi’an 710049, China

Zoology Department, King Saud University, Riyadh 11451, Saudi Arabia

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

The Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237,

China

Available online 17 May 2017

Abstract

In the ﬁve years 2012–2016, the ciliate research group at Ocean University of China and their collaborators have performed

several investigations on the morphogenesis of ciliated protists during binary division. Multiple samples were collected from

17 cities and cortical development studied in 42 species belonging to 32 genera and 13 families (Amphisiellidae, Euploti-

dae, Kahliellidae, Oxytrichidae, Philasteridae, Pseudokeronopsidae, Pseudourostylidae, Schmidingerotrichidae, Spiroﬁlidae,

Strobilidiidae, Uroleptidae, Uronychiidae and Urostylidae). Among these, 12 genera were investigated morphogenetically for

the ﬁrst time, revealing some unusual pattern formations and allowing four new genera to be established: Heterokeronopsis,

Apobakuella, Parabistichella and Apoholosticha. The objective of this review is to: 1) summarize the morphogenetic studies

supported by the IRCN-BC and NSFC projects during these ﬁve years; 2) summarize the patterns of development and document

deviations from normal morphogenetic events within a group; 3) discuss how studies on morphogenesis have helped to advance

understanding in the three dimensions of biodiversity, i.e. taxonomy, genetics and function; and 4) suggest potential future

directions for the morphogenetic study of ciliated protists.

creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Ciliates; Cortical patterns; Diversity; Morphogenesis; Ontogenesis

Abbreviations: FVC, frontoventral cirri; FVT-anlage, anlage for fronto-

ventral transverse cirri (sing.); FVTA, anlagen for frontoventral transverse Introduction

cirri (pl.); FVTC, frontoventral transverse cirri; UM-anlage, anlage for undu-

lating membranes.

∗ Among the protists, ciliates have some of the most complex

Corresponding author at: The Key Laboratory of Biomedical Information

and highly differentiated surface organelles. They exhibit

Engineering, Ministry of Education, School of Life Science and Technology,

high species diversity with over 4000 free-living ciliates hav-

Xi’an Jiaotong University, Xi’an 710049, China.

E-mail address: [email protected] (C. Shao). ing been described. The majority of these, however, were

All authors contribute equally. described only from living or ﬁxed cells without details of

http://dx.doi.org/10.1016/j.ejop.2017.05.003

440 X. Chen et al. / European Journal of Protistology 61 (2017) 439–452

the infraciliature or silver-line system. With improvements in Family Amphisiellidae Jankowski, 1979 is characterized

optical microscopy, the wider application of silver staining by the presence of the amphisiellid median cirral row derived

and the development of molecular methods, guidelines for from the two or three rightmost anlagen (Berger 2008; Huang

acceptable species descriptions have recently been revised et al. 2016; Li et al. 2007). In the work performed by our

(Warren et al. 2017). While not a requirement for inclusion group, four species of the genus Amphisiella, i.e. A. candida,

in species descriptions, it was recommended that ontogenesis A. milnei, A. pulchra, and A. sinica were described in terms

(i.e. cell division including stomatogenesis, morphogene- of their living features, infraciliature and morphogenetic pro-

sis, and the regeneration of parental structures) should be cesses. The main morphogenetic features can be summarized

included when it provides taxonomically relevant features, as follows: (1) in the proter, the parental adoral zone of mem-

e.g. the position of the oral primordium in oligotrichid ciliates branelles is completely retained; and (2) the FVTC originate

(Agatha 2004). from six cirral anlagen (Chen et al. 2013c; Li et al. 2016;

Ontogenetic information has long provided a basis for Fig. 1A, B).

inferring phylogenetic relationships among ciliates (Foissner In the family Kahliellidae Tuffrau, 1979, morphogene-

1996). Morphogenesis, which concerns the pattern of forma- sis in Perisincirra paucicirrata, Deviata parabacilliformis,

tion of cortical structures during binary division, is of interest D. brasiliensis and D. rositae was described. The main

for researchers engaged in cell differentiation, systematics characteristic features are: (1) the parental adoral zone of

and evolution because these disciplines play an important role membranelles is retained unchanged by the proter; and (2) the

both in deducing phylogenetic relationships and in outlining FVC originate from four cirral anlagen in Perisincirra pauci-

or separating closely related taxa. In addition, the patterning cirrata and six cirral anlagen in Deviata parabacilliformis (Li

of cortical structure development during the division process, et al. 2013, 2014; Luo et al. 2016; Fig. 1D–F). Berger (2011)

such as the origin of the oral primordium and of various cirri, speculated that Perisincirra may have three FVTA and could

indicate essential mechanisms of gene expression and cell not determine whether the inner right row is a marginal row

differentiation (Kumar et al. 2014; Pan et al. 2011; Hu and or a frontoventral row (Berger 2011). Li et al. (2013) showed

Song 2000; Song et al. 2011). that P. paucicirrata has four frontoventral anlagen, and that

In the ﬁve years from 2012 to 2016, the International the inner right row is a marginal row.

Research Coordination Network for Biodiversity of Ciliates The family Schmidingerotrichidae Foissner, 2012 is proba-

(IRCN-BC) and the Natural Science Foundation of China bly related to the family Cladotrichidae Small and Lynn, 1985

(NSFC) supported projects to study the formation of corti- (Foissner 2012). The ontogeny of Paracladotricha salina,

cal patterns during the morphogenetic process in 42 ciliate isolated from hypersaline waters, was described. The main

species. The three main aims of these studies were: 1) to features of the morphogenetic process are as follows: (1) the

investigate morphogenetic patterns during cell division, par- parental adoral zone is inherited unchanged by the proter; (2)

ticularly in unknown and poorly-known species; 2) to outline the FVC form independently in the proter and opisthe; and

the modes of morphogenesis of new taxa and to compare the (3) no paroral, dorsal ciliature, and buccal, transverse and

diversity of their pattern formation; and 3) to re-evaluate, caudal cirri formed during binary ﬁssion (Shao et al. 2014b;

using morphological and molecular biological methods in Fig. 1H). Shao et al. (2014b) assigned Paracladotricha to the

combination with ontogenetic data, the systematic position previously monotypic family Schmidingerotrichidae because

of groups whose phylogenetic relationships are unclear. both Schmidingerothrix and Paracladotricha lack a paroral

In the data given below, ‘our group’ designates all and a buccal cirrus, have three-rowed adoral membranelles,

researchers led by W. Song and his collaborators. The term a very strongly reduced dorsal infraciliature, and are only

‘research projects’ refers to those supported by the IRCN-BC known from highly saline habitats (Shao et al. 2017).

and NSFC. In the family Spiroﬁlidae von Gelei, 1929, the ontoge-

netic processes of three species have been described, namely

Strongylidium orientale, Hypotrichidium paraconicum and

Morphogenesis in stichotrichids

Pseudouroleptus caudatus caudatus (Chen et al. 2013a,b,

2015a). The main features of the morphogenetic process in

In the subclass Stichotrichida Faure-Fremiet, 1961, eight

Strongylidium orientale are as follows: (1) the posterior part

new species/subspecies representing four families and eight

of the parental adoral zone of membranelles is renewed by

genera were established during the course of these research

dedifferentiation; (2) the left and right ventral cirral rows

projects (Chen et al. 2013a,b,c, 2015a; Fan et al. 2014a; Li

originate intrakinetally and the ﬁnal left ventral cirral row

et al. 2013, 2014, 2016; Shao et al. 2014b).

is composed of an anterior portion formed by anteriorly

migrating cirri of FVTA VI, a middle portion formed by the

The names of the new taxa established in the online-only works of Chen

anteriormost cirrus/cirri of FVTA IV, and a posterior por-

et al. (2014), Jung et al. (2015), Kim et al. (2014), Kumar et al. (2014),

tion formed by FVTA V; and (3) the dorsal kineties replicate

Shao et al. (2014b) and mentioned in the present review are disclaimed

de novo and do not fragment (Chen et al. 2013b; Fig. 1G).

for nomenclatural purposes (ICZN, 1999, Article 8.3) because they are not

available due to lack of ZooBank registration in the work itself (ICZN 2012, Chen et al. (2013b) speculated a close relationship between

Article 8.5.3). Pseudouroleptus and Strongylidium based on a combination

X. Chen et al. / European Journal of Protistology 61 (2017) 439–452 441

Fig. 1. A–H. Diagram of the ventral (A, C, D, F-H) and dorsal (B, E) ciliature showing formation patterns of frontoventral transverse

cirri (with dotted lines connecting cirri that develop from the same cirral streaks), marginal cirri and dorsal ciliature of species in the order

Stichotrichida. (A) Amphisiella spp. Note that the amphisiellid median cirral row derived from the two rightmost anlagen (arrowheads).

(B) Amphisiella spp. and Bistichella cystiformans. Note that dorsal ciliature is formed in Gonostomum-pattern (arrows). (C) Bistichella

cystiformans. Note that the cirral rows generated by the two rightmost FVTA will not migrate to form one single cirral row (arrows). (D)

Perisincirra paucicirrata. Note the FVTC originate from four cirral anlagen (arrowheads). (E) Perisincirra paucicirrata. Note that dorsal

ciliature is formed in Gonostomum-pattern (arrowheads). (F) Deviata parabacilliformis. Note the FVTC originate from six cirral anlagen

(arrowheads). (G) Pseudouroleptus caudatus and Strongylidium orientale. Note that the ﬁnal left ventral cirral row is composed of an anterior

portion formed by anteriorly migrating cirri of FVT-anlage VI, a middle portion formed by the anteriormost cirrus/cirri of FVT-anlage IV, and

a posterior portion formed by FVT-anlage V (arrowheads). (H) Paracladotricha salina. Note that no paroral (arrow), and buccal (arrowhead)

and transverse cirri (double-arrowhead) formed.

of molecular (SSU rDNA sequence similarity), morphologi- row 4 and cirral row 2 develop from anlage V, and the other

cal (similar infraciliature patterns) and morphogenetic (same four anlagen for cirral rows 3–6 arise de novo (Chen et al.

formation pattern of the left ventral row) data. 2013a).

Although only two late dividers of Hypotrichidium para- The morphogenetic process of Pseudouroleptus caudatus

conicum were observed, some morphogenetic features were caudatus is characterized as follows: (1) the parental adoral

identiﬁed as follows: (1) the parental adoral zone of mem- zone of membranelles is retained unchanged by the proter; (2)

branelles remains completely unchanged; and (2) meridional the FVC originate from six anlagen whereas the right ventral

row 3 and cirral row 1 originate from anlage IV, meridional row originates de novo; and (3) dorsal kineties anlagen 1

442 X. Chen et al. / European Journal of Protistology 61 (2017) 439–452

and 2 develop intrakinetally whereas dorsal kinety anlage 3 Morphogenesis in Trichototaxis marina and T. songi show

develops de novo in the proter and intrakinetally in the opisthe several similarities including: (1) the parental oral apparatus

(Chen et al. 2015a; Fig. 1G). is completely renewed by the independently formed oral pri-

Bistichella cystiformans was described in terms of its liv- mordium; and (2) the macronuclear nodules fuse into many

ing features, infraciliature and morphogenetic processes. The masses. However, Trichototaxis marina differs from T. songi

main morphogenetic features can be summarized as follows: in the following features: (1) parental basal bodies are not

(1) in the proter, the parental adoral zone of membranelles is incorporated into the oral primordium in the opisthe (vs.

completely retained; (2) the FVTC originate from six cirral incorporated in T. songi); (2) FVTA I forms only one coronal

anlagen; and (3) the cirral rows generated by the rightmost cirrus (vs. two cirri in T. songi); (3) the marginal row anla-

two FVTA will not migrate to form one single cirral row, that gen are formed by the disaggregation of the parental cirri

is, no amphisiellid median cirral row is formed (Fan et al. within the parental right marginal row and the innermost left

2014a; Fig. 1B, C). marginal row only (vs. within each parental structure in T.

songi); and (4) the formation of multiple left marginal rows

is due to the retention of the parental structures (vs. the de

Morphogenesis in urostylids novo development of small anlagen to the left of the intrakine-

tally formed left marginal anlagen, or the fragmentation of

In the research performed by our group, ontogenesis in the left marginal anlage(n) with no parental marginal rows

17 urostylid species belonging to 13 genera and four families being retained after morphogenesis, in T. songi) (Lu et al.

have been investigated (Chen et al., 2013d,e, 2014, 2016; Fan 2014; Shao et al. 2014a; Fig. 2D, E). Shao et al. (2014a) and

et al. 2014b; Jiang et al. 2013; Lu et al. 2014; Luo et al. 2015; Lu et al. (2014) concluded that Trichototaxis, with its variable

Pan et al. 2013; Shao et al. 2013b, 2014a). number of marginal rows and the fusion of its macronuclear

Family Pseudokeronopsidae Borror & Wicklow, 1983 is nodules into many masses during morphogenesis, represents

characterized by having numerous macronuclear nodules that an intermediate form between the families Pseudourostylidae

fuse into a strongly branched mass or many masses during cell and Pseudokeronopsidae, and is more closely related to the

division and the parental adoral zone of membranelles being latter than the former (Chen et al. 2014; Pan et al. 2016).

totally replaced during cell division (Berger 2006). Mor- Two species in Pseudourostylidae Jankowski, 1979 were

phogenesis was described for ﬁve species belonging to this investigated, namely Pseudourostyla subtropica and P. nova.

family, i.e. Apoholosticha sinica, Thigmokeronopsis stoecki, The main morphogenetic events during binary ﬁssion of both

Heterokeronopsis pulchra, Trichototaxis marina and T. songi species can be characterized as follows: (1) the parental

(Chen et al. 2013e; Fan et al. 2014b; Lu et al. 2014; Pan et al. adoral membranelles are partially renewed with the ante-

2013; Shao et al. 2014a). The key morphogenetic features of rior membranelles being retained; (2) the parental midventral

Apoholosticha sinica are similar to those of the type genus complex dedifferentiates to form the FVTA; and (3) the

Pseudokeronopsis except no buccal cirrus is formed in A. marginal rows of each side originate from a common anlage

sinica and its macronuclear nodules fuse into a single mass in both the proter and opisthe (Chen et al. 2014; Fig. 2F). The

during cell division (Fan et al. 2014b; Hu and Song 2001b; pattern of morphogenesis supports the monophyly of Pseu-

Fig. 2A). Based partly on these morphogenetic features, the dourostyla Borror, 1972, which is consistent with previous

genus Apoholosticha was newly erected and assigned to Pseu- ﬁndings (Berger 2006).

dokeronopsidae (Fan et al. 2014b). In the family Urostylidae Bütschli, 1889, the ontogenetic

The main ontogenetic events in Thigmokeronopsis stoecki events for nine species were investigated, viz., Bakuella sub-

are as follows: (1) the cirral anlagen form a large ﬁeld of tropica, Uncinata gigantea, Holosticha heterofoissneri, H.

“thigmotactic” cirri between the midventral complex and the cf. heterofoissneri, Anteholosticha randani, A. paramanca,

left marginal row; (2) the anlagen of marginal rows and dorsal Apobakuella fusca, Parabistichella variabilis and Metau-

kineties develop de novo; and (3) macronuclear nodules fuse rostylopsis salina (Chen et al. 2013d; Fan et al. 2016; Jiang

into numerous masses and then divide (Chen et al. 2013e; et al. 2013; Luo et al. 2015; Shao et al. 2013b). In B. sub-

Fig. 2B). Chen et al. (2013e) showed that morphogenesis tropica, the main features are as follows: (1) the parental

during the binary ﬁssion in T. stoecki is almost the same as its adoral zone of membranelles is completely renewed by new

two congeners except for the behavior of the macronucleus structures and the old midventral pairs join the formation of

(Berger 2006). the FVTA; (2) the FVTA are formed with some kinetosomal

In Heterokeronopsis pulchra, the main morphogenetic fea- contribution from the parental midventral cirri; and (3) the

tures are as follows: (1) the posterior FVTA generates a posterior FVTA generate the midventral rows (Chen et al.

midventral row; (2) no frontoterminal, transverse or cau- 2013d; Fig. 3A).

dal cirri are formed; and (3) macronuclear nodules fuse into Uncinata gigantea shows rather unusual features in its dor-

single mass (Fig. 2C). Based partly on this combination sal morphogenesis; dorsal kineties develop in a two-group

of morphogenetic features, the genus Heterokeronopsis was mode, that is, two groups of dorsal anlagen form de novo near

newly established and assigned to the family Pseudokeronop- the leftmost and rightmost dorsal kineties respectively, and as

sidae (Pan et al. 2013). these develop they replace the parental structures (Fig. 3C,

X. Chen et al. / European Journal of Protistology 61 (2017) 439–452 443

Fig. 2. A–H. Diagram of the ventral ciliature showing formation patterns of frontoventral transverse cirri (with dotted lines connecting cirri

that develop from the same cirral streaks), marginal cirri and dorsal ciliature in species of the order Urostylida. (A) Apoholosticha sinica. Note

that no buccal cirrus is formed (arrowhead). (B) Thigmokeronopsis stoecki. Note that the cirral anlagen form a large ﬁeld of “thigmotactic”

cirri between the midventral complex and the left marginal row (arrowheads). (C) Heterokeronopsis pulchra. Note that the posterior FVT-

anlage generates a midventral row (arrow), as well as no frontoterminal (arrowhead) and transverse (double-arrowhead) cirri are formed. (D)

Trichototaxis marina. Note that FVT-anlage I forms only one coronal cirrus (arrowhead), as well as the formation of multiple left marginal rows

is due to the retention of the parental structures (arrow). (E) Trichototaxis songi. Note that FVT-anlage I forms two coronal cirri (arrowhead),

as well as the de novo development of small anlagen to the left of the intrakinetally formed left marginal anlagen (arrows). (F) Pseudourostyla

subtropica. Note that the marginal rows of each side originate from a common anlage in both the proter and opisthe (arrows). (G) Uroleptus

longicaudatus. Note that the ventral ciliature is formed in a typical urostylid mode. (H) Holosticha heterofoissneri.

D). Based on the morphological and morphogenetic data a Morphogenesis in Holosticha heterofoissneri and H. cf.

new combination, Uncinata bradburyae (Gong et al., 2001) heterofoissneri has the following features: (1) the parental

Luo et al., 2015 (basionym Holosticha bradburyae Gong adoral membranelles are retained unchanged and are inher-

et al., 2001), was made (Luo et al. 2015). ited by the proter; (2) the old undulating membranes are

444 X. Chen et al. / European Journal of Protistology 61 (2017) 439–452

Fig. 3. A–H. Diagram of the ventral (A–C, E, G, H) and dorsal (D, F) ciliature showing formation patterns of frontoventral transverse

cirri (with dotted lines connecting cirri that develop from the same cirral streaks), marginal cirri and dorsal ciliature in species of the order

Urostylida. (A) Bakuella subtropica. Note that the posterior FVTA generate the midventral rows (arrows). (B) Parabistichella variabilis.

Note the intrakinetal development of the midventral row (arrows). (C, D) Uncinata gigantea. Note that two groups of dorsal anlagen form de

novo near the leftmost (arrows) and rightmost dorsal kineties respectively (arrowheads). (E, F) Metaurostylopsis salina. Note that multiple

left and right marginal rows are generated from individual anlage within each parental row (arrowheads). (G) Anteholosticha randani. (H)

Apobakuella fusca. Note that the anteriormost cirri develop from the rightmost FVT-anlage do not migrate anteriorly (arrow).

completely renewed; (3) the FVTA are formed as primary The morphogenetic process of Anteholosticha randani is

primordia; and (4) four dorsal kineties anlagen are formed similar to that of its congener, A. petzi, and is characterized by:

next to the parental 1st, 4th and 5th kineties in the proter and (1) the parental adoral membranelles are completely replaced

next to the parental 1st, 3rd, 4th and 5th kineties in the opis- by new structures; and (2) the FVTA in both dividers develop

the. The ﬁrst anlage fragments into two anlagen posteriorly, independently (Fan et al. 2016; Shao et al. 2011; Fig. 3G).

thus ﬁve dorsal kinety anlagen are formed in total (Hu and In Apobakuella fusca, the main morphogenetic events are

Song 2001a; Luo et al. 2015; Fig. 2H). as follows: (1) the parental oral apparatus is completely

X. Chen et al. / European Journal of Protistology 61 (2017) 439–452 445

Fig. 4. A–C. Diagram of the ventral (A, C) and dorsal (B) ciliature showing formation patterns of frontoventral transverse cirri (with dotted

lines connecting cirri that develop from the same cirral streaks), marginal cirri and dorsal ciliature in species of the families Euplotidae (A, B)

and Uronychiidae (C). (A, B) Euplotes balteatus and Euplotoides amieti. Note that the marginal anlage is formed de novo (arrow), as well as

the caudal cirri are formed at the posterior ends of the two rightmost dorsal kineties (arrowheads). (C) Pseudodiophrys nigricans. Note that

the leftmost frontal cirrus develops from the UM-anlagen (arrow) and the marginal cirral anlage is formed de novo (arrowhead).

replaced by new structures derived from the oral primordium anlagen streaks in two groups for differentiation of the ventral

of the proter which originates de novo just posterior to the somatic ciliature.

old undulating membranes; (2) the parabuccal cirri originate Oxytrichidae Ehrenberg, 1838 is one of the largest fam-

from the posterior part of FVTA III-V; (3) the midventral rows ilies of the class Spirotrichea Bütschli, 1889, with almost

originate from the last few FVTA; and (4) some segment is 50 genera and over 200 species, and exhibits a high diver-

missing her origin of anteriormost cirri that will migrate ante- sity of morphogenetic features (Berger 1999; Fan et al. 2015;

riad, i.e. frontoterminal cirri are absent (Fig. 3H). Both the Foissner 2016; Jung et al. 2015; Kim et al. 2014; Kumar et al.

morphological and the morphogenetic data for this species 2014, 2015; Luo et al. 2017; Shao et al. 2012, 2015; Singh

support the validity of the genus Apobakuella (Jiang et al. and Kamra 2015a,b; Song 2001; Song and Hu 1999). Mor-

2013). phogenesis of seven species, including those that were newly

Morphogenesis in Parabistichella variabilis is charac- described, from six genera, has been reported by our group

terized by the formation of more than six FVTA and the (Chen et al., 2013f, 2015b,c; Hu and Kusuoka 2015; Lu et al.

intrakinetal development of the midventral row (Jiang et al. 2015; Lv et al. 2013; Shao et al. 2013a).

2013; Fig. 3B). Both the morphological and the morpho- Morphogenesis of ﬁve typical oxytrichids with 18 FVTC

genetic data for this species support the validity of the genus was investigated: Notohymena australis, N. apoaustralis,

Parabistichella. Rigidohymena candens, Rubrioxytricha haematoplasma, and

The main characteristic morphogenetic events in Metau- Sterkiella subtropica (Chen et al., 2013f, 2015b,c; Hu and

rostylopsis salina are as follows: (1) the parental adoral zone Kusuoka 2015; Lv et al. 2013). The ventral morphogenetic

and undulating membranes are completely replaced by the events of the ﬁve species have a high similarity whereas

proter’s oral primordium; (2) FVT-anlage n-1 generates the the dorsal morphogenesis varies as follows: (1) new dorsal

midventral row; and (3) multiple left and right marginal rows kineties of Rigidohymena candens and Sterkiella subtrop-

are generated from individual anlage within each parental ica are formed in a typical Oxytricha-pattern, with three

row (Shao et al. 2013b; Fig. 3E, F). caudal cirri being formed, one each at the posterior end of

dorsal kinety 1, 2 and 4; (2) in Notohymena australis and

N. apoaustralis, the dorsal kineties are also formed in an

Oxytricha-pattern, but two to four caudal cirri are formed at

Morphogenesis in sporadotrichids the posterior end of each of dorsal kinety 1, 2 and 4; and (3) in

Rubrioxytricha haematoplasma, morphogenesis of the dor-

Sporadotrichida Faure-Fremiet, 1961 is characterized by: sal kineties is simpler than the Oxytricha-pattern, i.e. without

1) frontoventral cirri that are typically strong and conspicuous fragmentation of dorsal kinety 3 anlage and with only a single

and arranged in speciﬁc, localized frontal and ventral groups; caudal cirrus originating from dorsal kinety 3 anlage (Chen

and 2) apokinetal stomatogenesis, usually with ﬁve or six

446 X. Chen et al. / European Journal of Protistology 61 (2017) 439–452

Fig. 5. A–H. Diagram of the ventral (A, E, G, H) and dorsal (B–D, F) ciliature showing formation patterns of frontoventral transverse cirri

(with dotted lines connecting cirri that develop from the same cirral streaks), marginal cirri and dorsal ciliature of species in the family

Oxytrichidae s. l. (A) Rigidohymena candens, Sterkiella subtropica, Notohymena australis, Notohymena apoaustralis and Rubrioxytricha

haematoplasma. (B) Rigidohymena candens, Sterkiella subtropica and Pleurotricha curdsi. Note that the dorsal ciliature is formed in typical

Oxytricha-pattern, with three caudal cirri being formed, one each at the posterior end of dorsal kinety 1, 2 and 4 (arrowheads). (C) Notohymena

australis and Notohymena apoaustralis. Note that the dorsal ciliature is formed in typical Oxytricha-pattern, but two to four caudal cirri are

formed at the posterior end of each of dorsal kinety 1, 2 and 4 (arrowheads). (D) Rubrioxytricha haematoplasma. Note that the morphogenesis

of the dorsal kineties is simpler than the Oxytricha-pattern, i.e. without fragmentation of dorsal kinety 3 anlage and with only a single caudal

cirrus originating from dorsal kinety 3 anlage (arrow). (E) Hemigastrostyla enigmatica. (F) Hemigastrostyla enigmatica. Note that three

caudal cirri are formed one each at the posterior ends of dorsal kineties 2, 4 and 5 (arrowheads). (G, H) Pleurotricha curdsi. Note that, in

some dividers, several small anlagen are formed to the left of the right marginal anlage (arrows).

et al., 2013f, 2015b,c; Hu and Kusuoka 2015; Lv et al. 2013; dedifferentiate into the new oral primordium for the proter,

Fig. 5A–D). and hence the adoral zone in the proter comprises a mixture

The morphogenetic features of Hemigastrostyla enigmat- of unchanged old membranelles and new membranelles gen-

ica, a species with two “extra cirri”, can be summarized erated from the oral primordium; (2) the “extra cirri” in the

as follows: (1) the parental undulating membranes and the opisthe are retained until the end of the division process; (3)

posterior end of the parental adoral zone of membranelles the dorsal kinety anlagen are formed de novo as three anlagen

X. Chen et al. / European Journal of Protistology 61 (2017) 439–452 447

each in the proter and opisthe although the left and middle like ontogenetic features which are as follows: (1) the oral

dorsal kinety anlagen fragment thus generating ﬁve dorsal primordium in the opisthe forms hypoapokinetally; (2) the

kineties altogether; and (4) three caudal cirri are formed one frontal, ventral and transverse cirri develop from ﬁve FVTA

each at the posterior ends of dorsal kineties 2, 4 and 5 (Shao which are formed in a secondary mode; (3) the leftmost

et al. 2013a; Fig. 5E, F). These ﬁndings represent a new frontal cirrus develops from the UM-anlagen; and (4) the

formation pattern of dorsal ciliature in sporadotrichids and marginal cirral anlagen are formed de novo. The origins of the

suggest that morphogenesis is not uniform among members UM-anlagen, the dorsal kineties and the caudal cirri remain

of the genus Hemigastrostyla. unclear. Compared to the closely related genera Diophrys

Pleurotricha curdsi is an oxytrichid species with more than and Diophryopsis, Pseudodiophrys shows a distinct morpho-

18 FVTC and more than one right marginal row (Shi et al. genetic characteristic: the FVTA develop in the secondary (vs.

2002; Gupta et al. 2003). Morphogenesis in an Indian popula- primary) mode (Fig. 4C) (Fan et al. 2013; Shao et al. 2010).

tion of P. curdsi was described by Gupta et al. (2003) in which

an anlage of the right marginal rows develops within the out-

ermost right marginal row, splits into two, and these two parts Morphogenesis in strombidiids

eventually form two new rows 1 and 2. The population from

China investigated by Lu et al. (2015) revealed the following Ciliates of the family Strobilidiidae Kahl in Doﬂein &

additional ontogenetic feature: in some dividers, several small Reichenow, 1929 are mostly small to medium-sized, spheroid

anlagen are formed to the left of the right marginal anlagen, to conoid in shape, and are characterized by the posses-

which elongate and develop into an additional right marginal sion of somatic kineties arranged in spiral or longitudinal

row. It was concluded that the instability of the number of rows and cortical ﬂaps covering the bases of the somatic

right marginal anlagen leads to the intraspeciﬁc variation of cilia. Morphogenesis has been documented for fewer than ten

the number of right marginal rows in P. curdsi (Lu et al. 2015; choreotrich ciliates. In the research performed by our group,

Fig. 5B, G, H). three species of the family Strobilidiidae have been reported,

namely Rimostrombidium veniliae (Montagnes and Taylor,

1994) Petz et al., 1995, Pelagostrobilidium paraepacrum Liu

Morphogenesis in other dorsomarginalian

et al., 2012, and P. minutum Liu et al., 2012. These share

hypotrichs

the following ontogenetic features: (1) the oral primordium

of the opisthe forms de novo on the left-dorsal side; (2) the

In the family Uroleptidae Foissner and Stoeck, 2008,

endoral originates de novo; (3) the somatic kineties lengthen

ontogenesis in one species, Uroleptus longicaudatus, was

by intrakinetal proliferation of basal bodies and each somatic

investigated (Chen et al. 2016). The main characteristic mor-

kinety splits into two, one each for the opisthe and proter; and

phogenetic events are as follows: (1) the parental adoral

(4) one replication band forms at each end of the macronu-

membranelles are completely retained; (2) FVTA appear to

cleus and migrates towards the middle, following which the

be primary primordia; and (3) dorsal kineties develop in

macronucleus condenses into an ellipsoidal or globular mass

the Urosomoida-pattern. These ﬁndings are consistent with

and then divides into two. These three species exhibit differ-

previous studies of congeners of U. longicaudatus suggest-

ent locations for the oral primordium, i.e. anterior to kineties

ing that morphogenesis is uniform among members of the

4 and 5 in Rimostrombidium veniliae and between kineties

genus Uroleptus (Berger 2006; Chen et al. 2016; Foissner

2 and 3 in Pelagostrobilidium paraepacrum and P. minutum

and Stoeck 2006; He et al. 2011; Fig. 2G).

(Liu et al. 2012).

Morphogenesis in euplotids

Morphogenesis in philasterids

In the family Euplotidae Ehrenberg, 1838, morphogenesis

in two species, i.e., Euplotes amieti and E. balteatus were The scuticociliate order Philasterida Small, 1967 com-

investigated (Liu et al. 2015; Pan et al. 2012b). The main prises 16 families and over 70 genera (Lynn 2008). The

morphogenetic features suggest that morphogenesis is con- main morphogenetic events of Pseudocohnilembus persal-

servative among the members of the genus Euplotes: (1) in inus Evans and Thompson, 1964, a species of the family

the opisthe, the UM-anlage develops independently from the Philasteridae Kahl, 1931, are as follows: (1) the parental

oral primordium; (2) the leftmost frontal cirrus develops de scutica proliferates and becomes the primordial ﬁeld, which

novo; (3) the marginal anlage is formed de novo; and (4) eventually forms membranelle 3 of the opisthe; (2) the sec-

the caudal cirri are formed at the posterior ends of the two ondary proliferated ﬁeld, which derives from the zigzag

rightmost dorsal kineties (Liu et al. 2015; Pan et al. 2012b; conﬁguration of the parental paroral membrane, develops into

Fig. 4A, B). two parts, with the anterior part becoming the paroral mem-

In the family Uronychiidae Jankowski, 1979, morphogen- brane as well as the scutica of the opisthe and the posterior

esis in Pseudodiophrys nigricans was described based on part developing into membranelles 1 and 2 of the opisthe;

three dividing cells only (Fan et al. 2013). Several Diophrys- and (3) the remnants of the parental paroral membrane give

448 X. Chen et al. / European Journal of Protistology 61 (2017) 439–452

rise to the paroral membrane and scutica of the proter (Pan analyses of SSU rDNA sequence data, Gong et al. (2007)

et al. 2012a). erected the genus Protogastrostyla with Protogastrostyla

pulchra (formerly Gastrostyla pulchra) the type species by monotypy.

Conclusion

(2) Evolutionary relationships. Morphogenetics can also

improve our understanding of molecular phylogeny. The

Summary of morphogenetic studies carried out

genus Kiitricha has a unique and probably ancestral pat-

by our group in the period 2012–2016

tern of ciliature (Song and Wilbert 1997). It was long

assumed to be the most primordial taxon in the subclass

In the ﬁve years from 2012 to 2016, investigations

Hypotrichia. Both molecular and ontogenetic data sup-

supported by the IRCN-BC and NSFC were carried

port the suggestion that: (i) Kiitricha, which may be an

out on the morphogenesis of 42 species representing

intermediate group between the heterotrichs (s. l.) and

32 genera and 13 families (Amphisiellidae, Euplotidae,

hypotrichs (s. l.), is probably an ancestor-like form of the

Kahliellidae, Oxytrichidae, Philasteridae, Pseudokeronopsi-

latter and (ii) Kiitricha is separated from the hypotrichs

dae, Pseudourostylidae, Schmidingerotrichidae, Spiroﬁlidae,

(s. l.) and is instead placed in the class Spirotrichea at

Strobilidiidae, Uroleptidae, Uronychiidae and Urostylidae).

about subclass level, supporting the establishment of the

Based on these investigations, new data on the cortical devel-

subclass Protohypotrichia (Li et al. 2009).

opment process and unusual patterns of formation have

been documented including the ﬁrst morphogenetic inves- Sometimes, molecular trees show discrepancies with mor-

tigations of 12 genera and 40 species. Based on these results, phogenetic data. For example, based on morphogenetic data,

closely related groups were outlined and separated leading some Oxytrichidae genera, e.g. Cyrtohymena, Sterkiella and

to the establishment of four new genera (Heterokeronopsis, Tetmemena, are monophyletic. However, in the molecular

Apobakuella, Parabistichella and Apoholosticha) and 16 new trees, congeners in these three genera do not cluster with each

species. The systematic relationships of several taxa were other (Berger 1999; Chen et al. 2015c, 2017; Song 2004). We

evaluated using a combination of ontogenetic and molecular believe that the ambiguous relationship of species in these

data (Gao et al. 2016; Lv et al. 2015). genera might be due to the limited sampling of representative

taxa in the SSU rDNA database and that the systematics of

these taxa is more accurately reﬂected by the morphogenetic

Morphogenesis and three dimensions of

biodiversity data.

(3) Ecology and Occurrence. Morphogenesis can also

Studies of morphogenesis have helped to advance stud-

help improve our understanding of function (ecology).

ies in the three dimensions of biodiversity, i.e. taxonomy,

Species of the family Uronychidae, which are almost

genetics and function.

invariably found in periphytic habitats, are character-

ized by their distinctive mode of locomotion which is

(1) Taxonomy. Morphogenetic data has helped to inform

by rapidly jumping sideways or backwards. This behav-

morphology-based classiﬁcations and improve our

ior can be traced back to the development of the dorsal

understanding of taxonomy. Apokeronopsis crassa, for

kinety anlagen, the rightmost one (Diophrys-complex) or

example, was long considered a member of the genus

two (Uronychia) appearing earlier than the others and the

Pseudokeronopsis (Berger 2006). Based on a study

posterior ends of these anlagen being larger during the

of its morphogenesis, Shao et al. (2007) erected the

early stages enabling the generation of the three conspic-

genus Apokeronopsis with Apokeronopsis crassa the type

uously large, strong caudal cirri that are characteristic of

species having noted that, in contrast to Pseudokeronop-

this group (Song et al. 2004, 2009).

sis, it generates distinctly separated midventral rows,

and the marginal and dorsal kineties anlagen are formed

Schmidingerothrix is extraordinary in that the paroral, the

de novo. The separation of Apokeronopsis from Pseu-

dorsal bristle rows, and the buccal, transverse and caudal

dokeronopsis was also supported by molecular data, with

cirri are not generated during morphogenesis. Foissner (2012)

congeners of the two genera clustering into separate

considered that its simple ciliature is an adaptation to highly

clades (Lu et al. 2014).

saline habitats where competition is low and bacterial food

abundant.

Gastrostyla was long considered to be monophyletic

(Eigner 1997; Walker and Grim 1973). Hu and Song

(2000) proposed that Gastrostyla pulchra occupies a lower Future tasks

systematic position within the hypotrichs s.l. because its mor-

phogenesis differs from that of the type species G. steinii Ontogenetic data, together with features of the phys-

and other typical oxytrichids by possessing many “primitive” iological reorganization, play important roles in deﬁning

features. Following further studies including phylogenetic relationships among ciliates. Nevertheless, there are still

X. Chen et al. / European Journal of Protistology 61 (2017) 439–452 449

many difﬁculties in determining the systematic relationships sis stoecki Shao et al., 2008 (Ciliophora, Hypotricha) and a

of many groups using morphogenetic features. A major difﬁ- comparison with members of the family Pseudokeronopsidae.

Acta Protozool. 52, 65–72.

culty is deciding which characteristics should have the greater

Chen, X., Yan, Y., Hu, X., Zhu, M., Ma, H., Warren, A., 2013f.

weight at different taxonomic levels. Future directions in

Morphology and morphogenesis of a soil ciliate, Rigidohymena

morphogenetic studies should include: obtaining complete

candens (Kahl, 1932) Berger, 2011 (Ciliophora, Hypotricha,

ontogenetic information for as many taxa as possible, par-

Oxytrichidae), with notes on its molecular phylogeny based on

ticularly those for which such data are lacking; giving the

small-subunit rDNA sequence data. Int. J. Syst. Evol. Microbiol.

appropriate weight to relevant morphogenetic features at dif-

63, 1912–1921.

ferent taxonomic levels; critically analyzing morphogenetic

Chen, X., Miao, M., Ma, H., Al-Rasheid, K.A.S., Xu, K., Lin, X.,

data in order to discriminate between convergent and diver- 2014. Morphology, ontogeny, and phylogeny of two brackish

gent evolutionary events. urostylid ciliates (Protist, Ciliophora, Hypotricha). J. Eukaryot.

Microbiol. 61, 594–610.

Chen, L., Zhao, X., Ma, H., Warren, A., Shao, C., Huang, J., 2015a.

Acknowledgements Morphology, morphogenesis and molecular phylogeny of a soil

ciliate, Pseudouroleptus caudatus caudatus Hemberger, 1985

(Ciliophora, Hypotricha), from Lhalu Wetland, Tibet. Eur. J.

This work was supported by the International Research

Protistol. 51, 1–14.

Coordination Network for Biodiversity of Ciliates (IRCN-

Chen, W., Chen, X., Li, L., Warren, A., Lin, X., 2015b. Morphol-

BC, project no. 31111120437), Natural Science Foundation

ogy, morphogenesis and molecular phylogeny of an oxytrichid

of China (projects no. 31110293, 31401954), the Applied ciliate, Rubrioxytricha haematoplasma (Blatterer & Foissner,

Basic Research Plan of Qingdao (15-12-1-1-jch), Dean- 1990) Berger, 1999 (Ciliophora, Hypotricha). Int. J. Syst. Evol.

ship of Scientiﬁc Research, King Saud University, Proliﬁc Microbiol. 65, 309–320.

research group (PRG-1436-01), the China Postdoctoral Sci- Chen, X., Gao, F., Al-Farraj, S.A., Al-Rasheid, K.A.S., Xu, K.,

Song, W., 2015c. Morphology and morphogenesis of a novel

ence Foundation (no. 2014M550378 and 2015T80753) and

mangrove ciliate, Sterkiella subtropica sp. nov. (Protozoa, Cil-

a BBSRC China Partnering Award.

iophora, Hypotrichia), with phylogenetic analyses based on

small-subunit rDNA sequence data. Int. J. Syst. Evol. Microbiol.

65, 2292–2303.

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