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Morphology and morphogenesis of a new marine ciliate, Apokeronopsis bergeri nov. spec. (Ciliophora, Hypotrichida), from the Yellow Sea, China
ARTICLE IN PRESS European Journal of
PROTISTOLOGY European Journal of Protistology 44 (2008) 208–219 www.elsevier.de/ejop
Morphology and morphogenesis of a new marine ciliate, Apokeronopsis bergeri nov. spec. (Ciliophora, Hypotrichida), from the Yellow Sea, China Liqiong Lia, Weibo Songa,, Alan Warrenb, Khaled A.S. Al-Rasheidc, David Robertsb, Zhenzhen Yia, Saleh A. Al-Farrajc, Xiaozhong Hua a
Laboratory of Protozoology, Ocean University of China, Qingdao 266003, China Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK c Zoology Department, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia b
Received 23 January 2007; received in revised form 21 December 2007; accepted 2 January 2008
Abstract The morphology and morphogenesis of a new marine hypotrich ciliate, Apokeronopsis bergeri nov. spec., collected from mussel-farming waters near Qingdao, China, are described from living and protargol-impregnated specimens. This ciliate has characteristics that place it in the family Pseudokeronopsidae, namely, two long rows of frontal cirri (bicorona), which are continuous with the long midventral rows, and a single row of marginal cirri on each side of the body. It shares with its only congener, Apokeronopsis crassa, the long rows of buccal and transverse cirri and the wide spacing between the midventral rows of cirri. These characters separate the genus Apokeronopsis from Pseudokeronopsis, which has a single buccal cirrus, fewer transverse cirri and midventral rows of cirri arranged in a typical zig-zag pattern. A. bergeri differs from A. crassa in its shape, colour and in the numbers of membranelles and transverse cirri. Although morphogenesis in A. bergeri is similar to that of A. crassa in most respects, the mode of formation of the buccal cirri is slightly different. The close relationship of A. bergeri with A. crassa, and the more distant relationship with three Pseudokeronopsis species, is supported by a comparison of the sequences of their ITS15.8S-ITS2 rDNA regions. r 2008 Elsevier GmbH. All rights reserved. Keywords: Apokeronopsis bergeri nov. spec.; ITS and 5.8S rDNA sequence; Morphogenesis; Morphology; Stichotrichia; Taxonomy
Introduction The well-known hypotrich genus Pseudokeronopsis is distinguished by having frontal cirri arranged as a bicorona, whose rows continue posteriorly to form two midventral rows and one row of marginal cirri on each side of the body (Berger 2006; Borror and Wicklow 1983; Song et al. 2006). The genus Apokeronopsis, which Corresponding author.
E-mail address: [email protected] (W. Song). 0932-4739/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2008.01.001
was recently created by Shao et al. (2007), also possesses these characters but differs from Pseudokeronopsis as they have (1) a long row of buccal cirri (vs. a single buccal cirrus in Pseudokeronopsis); (2) widely separated midventral rows (vs. midventral rows closely juxtaposed with cirri in pairs forming the midventral complex in Pseudokeronopsis); and (3) at least 13 transverse cirri in a long row (vs. generally o10 transverse cirri in Pseudokeronopsis) (Berger 2006). Furthermore, the marginal rows and dorsal kineties, as observed in the type species A. crassa, originate de novo in both dividers
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(vs. within the old structure in Pseudokeronopsis) (Berger 2006; Hu and Song 2000; Hu et al. 2004; Shao et al. 2007). In the summer of 2005, we isolated an unknown urostylid ciliate from coastal waters off Qingdao. Pure cultures were established enabling detailed descriptions of its morphology and morphogenesis to be made and sequencing of the ITS1-5.8S-ITS2 rDNA region to be performed. The results of our investigations demonstrate that this is a new species within the genus Apokeronopsis.
Material and methods Apokeronopsis bergeri nov. spec. was collected from open mussel-farming waters near Qingdao (361 080 N; 1201 430 E) on August 1, 2005, where the salinity was 29–31%, the water temperature 20 1C, and pH 8.0. Long-term cultures were established in Petri dishes containing boiled seawater to which squeezed rice grains were added to enrich the bacterial food. Observations on live specimens were undertaken using differential interference microscopy. Protargol staining (Wilbert 1975) was used to reveal the infraciliature and nuclear apparatus. Counting and measurements were made at a magnification of 1250 . Drawings were performed with the help of a camera lucida. To illustrate the changes occurring during morphogenesis parental cirri are depicted by contour whereas new ones are shaded black. Terminology is mainly according to Hemberger (1982), Wirnsberger (1987) and Berger (2004, 2006). Genomic DNA extraction, PCR amplification, and ITS1-5.8S-ITS2 rDNA region cloning and sequencing of A. bergeri were performed according to Shang et al. (2003). Two primers were used: 5.8S-F: 50 -GTAGGTGAACCTGCGGAAGGATCATTA-30 ; 5.8S-R: 50 -TACTGATATGCTTAAGTTCAGCGG-30 . The nucleotide sequences used in this paper are available from the GenBank database under the following accession numbers: Pseudokeronopsis flava (DQ503579), Pseudokeronopsis rubra (DQ640313), Pseudokeronopsis carnea (DQ503580) and Apokeronopsis crassa (DQ537483). PHYLIP package, version 3.75c (Felsenstein 1995), was used to calculate the evolutionary similarities among three Pseudokeronopsis and two Apokeronopsis species.
Results Order Hypotrichida Stein, 1859 Family Pseudokeronopsidae Borror and Wicklow, 1983 Genus Apokeronopsis Shao et al., 2007
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Apokeronopsis bergeri nov. spec. Diagnosis Large marine Apokeronopsis 150–400 70–90 mm in vivo, body flexible, elongate to fusiform with both ends broadly rounded; yellow-brown to dark brown in colour; bicorona comprising about 38 frontal cirri; 9–13 buccal and 1–3 frontoterminal cirri; 13–22 transverse cirri densely arranged in a J-shaped row; midventral complex consisting of two conspicuously separated rows with ca. 35 pairs of cirri; adoral zone composed of about 80 membranelles; invariably 3 dorsal kineties. Two kinds of cortical granules: one tiny and colourless, densely arranged in short lines; the other large (about 2 mm across), biconcave and distributed sparsely, yellowbrown to yellow-green in newly sampled specimens, becoming dark-red in cultured cells. Numerous (i.e. 150–200) macronuclear nodules. Single contractile vacuole positioned in anterior 1/3 of the body. Type locality Mariculture waters of Yellow Sea coast at Qingdao, China (361 080 N; 1201 430 E). Type specimens One permanent holotype of protargol-impregnated specimens is deposited in the Natural History Museum, London, UK, with registration no. 2007:5:14:1. One paratype slide is deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China. Dedication We dedicate this new species to our Austrian colleague, Dr. Helmut Berger, in recognition of his contribution to ciliate morphogenesis and taxonomy, especially in the field of hypotrichs.
Morphological description (Figs 1–3; Table 1) Specimens mostly about 300 80 mm in vivo. Body shape elongate to fusiform with both ends broadly rounded, conspicuously flexible and slightly contractile. Specimens in newly collected samples generally much slimmer than those in culture with plentiful food (Figs 1A and 2A, F). Cells dorsoventrally flattened about 2:1. Buccal field narrow, about 1/3 of the body length. Cells appear brown to dark brown in colour under low magnification and yellow-brown when observed under high magnification (Fig. 2A–D). Two types of cortical granules: type I very small, about 0.2 mm across and colourless, arranged in longitudinally oriented lines that are evenly distributed over the body (Figs 1C, arrowheads; 2J, arrowheads); type II, biconcave (i.e. similar to erythrocytes of mammals),
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Fig. 1. Morphology and infraciliature of Apokeronopsis bergeri nov. spec. from life (A, C, D) and after protargol impregnation (B, E, F). (A) Ventral view of a typical individual; arrow marks the contractile vacuole. (B) Oral field, showing the buccal apparatus and the strong fibres, arrowheads mark the buccal cirri. (C) Details of the cortex, to show the very small type I granules (arrowheads) and the large biconcave type II granules (arrows). (D) Dorsal view; note the sparsely distributed type II granules. (E) Ventral view, arrow indicates the distal end of the adoral zone, and double-arrowheads depict the frontoterminal cirri. (F) Dorsal view of the same specimen as (E), to demonstrate the macronuclear nodules and the dorsal kineties. AZM—adoral zone of membranelles, BC—buccal cirri, DK—dorsal kineties, EM—endoral membrane, FC—frontal cirri, LMR—left marginal row, Ma—macronuclei, MVR—midventral rows (midventral complex), PM—paroral membrane, RMR—right marginal row, TC— transverse cirri. Bars ¼ 120 mm (in A, D, E, F); 20 mm (in C).
flattened, ca. 2 mm across, yellow-brown to yellow-green in colour, densely distributed, but not grouped, within the cortex (Figs 1C, arrows; 2C, J, arrows). Significantly, after several weeks’ culture, some type II granules changed colour from yellow-green to darkreddish; all type II granules turned dark-red during encystment (Fig. 2E, H, I, arrows). Cytoplasm opaque and colourless, containing numerous light-reflecting globules. Contractile vacuole pulsating infrequently, positioned near the left margin at about anterior 1/3–1/4 of the cell length (Figs 1A, arrow; 2A, arrow). About 150–200 macronuclear nodules, oval to elongate in shape, scattered within the cytoplasm (Fig. 1F). Micronuclei (Mi) not observed in protargolimpregnated specimens. Locomotion by crawling slowly without pausing on the debris or on bottom of the Petri dish. Adoral zone (AZM) curves across the anterior cell margin and extends conspicuously down the right side of the body (Fig. 1A); the distal end of AZM extends somewhat obliquely towards the buccal cavity (Figs 1E, arrow; 3C, double-arrowheads). Bases of the longest membranelles are about 20 mm wide, and those at the
distal end of AZM are distinctly shorter than those in the central portion. Paroral and endoral membranes (PM, EM) almost equal in length and spatially crossed; PM is thicker than EM (Fig. 1B, E). Most somatic cirri relatively fine, about 15 mm long. Bicorona consisting of about 40 slightly enlarged frontal cirri that are not separated from the midventral complex although in some specimens an inconspicuous gap was observed. Usually there are two frontoterminal cirri on the right side of the cell near the distal end of the adoral zone which are easy to recognize due to the distinct gap between them (Figs 1E, double-arrowheads; 3C, arrows). One long row of 9–13 buccal cirri (BC) is close to the paroral membrane (Figs 1B, arrowheads; 3B, arrows). Two rows of midventral cirri (MVR) are parallel to and considerably separated from each other, usually terminating just above the transverse cirri (Figs 1A, E; 3A, E). Thirteen to twenty-two transverse cirri (TC) about 20 mm long, are densely arranged in a J-shaped row near the posterior margin of the cell (Figs 1E; 3E, arrows). Cirri in marginal rows narrowly spaced; right marginal row (RMR) commencing near the distal end of the adoral zone. Marginal rows almost
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Fig. 2. Photomicrographs of Apokeronopsis bergeri nov. spec. (A–F, H–J) and A. crassa (G, K) from life. (A) Ventral view of a typical specimen, arrow indicates the contractile vacuole. (B) Ventral view of anterior portion, to show a light-coloured specimen. (C) A dark-coloured specimen, arrows show the distribution of type II granules. (D, F) Ventral view of two specimens, to show the different body shapes. (E) Cyst, arrows mark the dark-reddish type II granules. (G) Ventral view of a typical specimen of A. crassa. (H, I) Type II granules, arrows indicate the dark-reddish granules; note that most of the others are yellow-greenish. (J) Dorsal view, arrowheads depict the colourless type I granules, arrows indicate the type II granules. (K) Dorsal view of A. crassa, to show the brown-reddish granules. Bars ¼ 100 mm (in A, C, D, F, G); 30 mm (in E).
confluent posteriorly (Figs 1E; 3E). Invariably 3 complete dorsal kineties, each comprising about 50 pairs of basal bodies, cilia about 3 mm long (Figs 1F; 3F, arrows).
Divisional morphogenesis (Figs 4–7) Stomatogenesis and development of the somatic ciliature in the opisthe Stomatogenesis commences with the formation of small groups of basal bodies close to several of the intact left midventral cirri (Fig. 4A, arrows; 4B, arrowheads). With the proliferation of basal bodies, these groups join to make a longish field which is the anarchic primordium (AP) of the opisthe (Figs 4C, small doublearrowheads; 6A, arrow). During this process the left midventral cirri remain intact. Subsequently, a long streak of basal bodies that extends to the posterior end of the buccal field appears as a result of the splitting of the anterior end of the AP (Fig. 4D, small doublearrowheads). The middle part of the AP will become the anlage of the undulating membrane. By this stage, the development of membranelles in the right anterior part of this area has already begun (Fig. 4D). Soon after, when the thread-like anlagen arrange as a ladder-like structure, the UM-anlage (UMA) starts aggregating
into the undulating membranes (Figs 4E and 6G, double-arrowheads). The UM-anlage remains as a single unit until the late stage and then splits longitudinally to form the paroral and endoral membranes (Fig. 5D, F). Division continues with the complete formation of the FVT-cirral anlagen at the right of the AP, within which each streak seems to break into several segments. Meanwhile, a single frontal cirrus derives from the UMA (Fig. 5D, arrows). Along with the formation of the new cirri, several segments from the posterior portion of the FVT-cirral anlagen streaks begin migrating towards the newly formed UM which seem to give rise to the row of buccal cirri of the opisthe (Fig. 5C, double-arrowheads). Each of the remaining streaks divides into 2 cirri except for about 15 posterior-most streaks that form 3 cirri (Fig. 5D, F). The two anteriormost cirri from the last streak will migrate anteriad to become the frontoterminal cirri (Figs 5F, double-arrowheads; 7F, arrowhead). Each of the long posterior streaks contributes one transverse cirrus to the row of transverse cirri (TC), formed by the shortest streaks, the remaining new cirri becoming the frontal and midventral cirri (Figs 5F and 7F, arrow). The new anlagen of the marginal rows (MA) originate independently from the old structures on each side of the cell at almost the same time as the AP appears
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Fig. 3. Photomicrographs of Apokeronopsis bergeri nov. spec. after protargol impregnation. (A) Ventral view, to show the general ciliature. (B) Ventral view of the anterior-right portion, arrows mark the buccal cirri. (C) Ventral view of the anterior portion, arrows indicate the frontoterminal cirri, double-arrowhead marks the distal end of the adoral zone. (D) Ventral view of the buccal field, arrowhead indicates the paroral membrane, and the arrow marks the endoral membrane. (E) Ventral view of the posterior portion, to show the transverse cirri (arrows). (F). Detail of dorsal side, arrows indicate the cilia of the dorsal kineties and arrowheads mark the macronuclear nodules. Bar ¼ 100 mm (in A); 20 mm (in F). Table 1.
Morphometrical data of Apokeronopsis bergeri nov. spec. (all data are based on protargol-impregnated specimens)
Character
Min
Max
Mean
SD
CV
n
Length of the body in mm Width of the body in mm Length of the adoral zone in mm No. of membranelles No. of anterior frontal cirri No. of posterior frontal cirri No. of buccal cirri No. of frontoterminal cirri No. of transverse cirri Cirral pairs in midventral rows, No. of left cirri Cirral pairs in midventral rows, No. of right cirri No. of cirri in the left marginal row No. of cirri in the right marginal row No. of dorsal kineties
184 104 108 72 14 15 9 1 13 26 28 44 54 3
404 208 148 93 23 25 13 3 22 43 48 70 88 3
310.3 145.3 127.8 80.2 16.9 20.6 10.9 2.0 15.6 33.0 35.9 55.3 63.2 3
50.1 25.9 11.9 5.3 2.2 2.5 1.0 0.3 2.3 4.6 4.8 6.2 7.6 0
16.1 17.8 9.3 6.6 13.0 12.1 9.2 15.0 14.7 13.9 13.4 11.2 12.0 0
25 25 25 25 25 25 25 25 25 25 25 25 25 25
Abbreviations: CV—coefficient of variation in %; Max—maximum; Mean—arithmetic mean; Min—minimum; n—number of cells measured; SD— standard deviation; SE—standard error of the mean.
(Fig. 4C, arrows). All these anlagen then stretch both anteriorly and posteriorly while remaining separate from the old structures (Figs 4G and 6E, double-
arrowheads). The old marginal cirri do not participate in the formation and development of the cirral anlagen and are eventually resorbed.
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Fig. 4. Morphogenesis of Apokeronopsis bergeri nov. spec. after protargol impregnation. (A) Ventral view of an early divider, arrows indicate the newly formed basal bodies close to the left midventral cirri. (B) Detailed structure from the same specimen as (A), to show the groups of basal bodies (arrowheads). (C) Early stage of morphogenesis, small double-arrowheads mark the OP of the opisthe and arrows indicate the marginal row anlagen; large double-arrowhead marks the OP of the proter. (D) Ventral view, double-arrowhead and small arrow indicate fronto-ventral-transverse cirral anlagen in both dividers; the large arrow shows the disorganization of the parental adoral zone; arrowhead depicts the OP of the proter; inset—macronuclear nodules with replication bands. (E, F) Ventral and dorsal views of the same specimen, arrows indicate the dorsal kinety anlagen. (G) Ventral view, arrow indicates the newly formed membranelles of the proter, arrowheads mark the FVT-cirral anlagen of the proter, double-arrowheads depict the marginal row anlagen. (H) Dorsal view of the same cell as (G), to show the partially fused macronuclear nodules. DA— dorsal kinety anlagen, MA—marginal row anlagen, Ma—macronuclei, MVR—midventral rows (midventral complex). Bar ¼ 120 mm.
Dorsal kineties are formed in an unusual way. Just after the formation of the marginal row anlagen, three streaks of basal bodies appear de novo (DA) in both dividers (Figs 4D–H and 6E, arrows). With the proliferation of basal bodies, these anlagen elongate and ultimately replace the old structures (Fig. 5B, E, G). Stomatogenesis and development of the somatic ciliature in the proter At about the same time as the appearance of the AP in the opisthe, a scoop-like oral primordium forms deep
beneath the parental buccal cavity (Figs 4C, large double-arrowheads; 6C, arrows). Later the thread-like FVT-cirral anlagen of the proter emerges de novo close to the right of the endoral membrane near the cell surface (Fig. 4D, small arrow). Meanwhile, the oral primordium starts to split to form the UM-anlage (UMA). The parental UMs appear not to join in the formation of the new anlagen and are soon resorbed (Fig. 5A). As a result of the joining of new basal bodies in the anarchic field, the new adoral membranelles are gradually organized in a posteriad direction (Fig. 4D,
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Fig. 5. Morphogenesis of Apokeronopsis bergeri nov. spec. after protargol impregnation. (A, B) Ventral and dorsal views of the same specimen at the middle stage; double-arrowheads mark the dorsal kinety anlagen and arrows indicate the marginal row anlagen. (C) Ventral view, double-arrowheads indicate that some segments have migrated from the fronto-ventral-transverse cirral anlagen; arrows mark the marginal row anlagen; inset—macronuclear nodules in division. (D) Ventral view of a late divider, arrows mark the first frontal cirrus generated from the undulating membrane anlage in each divider; double-arrowhead depicts the right marginal row anlage. (E) Dorsal view of the same specimen as (D), arrows indicate dorsal kinety anlagen, double-arrowheads mark the marginal row anlagen. (F, G) Ventral and dorsal view of the same specimen; arrows show the newly formed transverse cirri and double-arrowheads mark the frontoterminal cirri. Bars ¼ 100 mm.
E). Simultaneously, the UMA detaches and generates a single frontal cirrus at its anterior end. As in the opisthe, the FVT-anlagen give rise to the FC, BC, MVC and TC. Marginal cirri and dorsal kineties are also formed in the same way as those of the opisthe. Macronuclear division The division process of the macronuclear apparatus appears to be unique. The replication bands of macronuclear nodules appear at an early stage (Figs 4D, inset; 6B). In the middle stage there is an obvious merging process of the macronuclear nodules although there is no fusion into a single mass prior to division (Figs 4H, 5B and 6F, arrows).
Information derived from ITS1-5.8S-ITS2 rDNA sequence analysis (Fig. 8; Table 2) The ITS1-5.8S-ITS2 region sequence of Apokeronopsis bergeri has been deposited in GenBank with the accession number DQ503583. The ITS1-5.8S-ITS2 region sequences within the genera Pseudokeronopsis and Apokeronopsis share evolutionary similarities of 88.85%–89.95% and 82.92%, respectively, while the evolutionary similarities of Pseudokeronopsis to Apokeronopsis range from 65.15% to 69.73% (Table 2). These data, along with the comparisons of the nucleotide sequences of the ITS1-5.8S-ITS2 rDNA regions (Fig. 8), indicate that
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Fig. 6. Photomicrographs of morphogenesis of Apokeronopsis bergeri nov. spec. after protargol impregnation. (A) Ventral view of an early divider, arrow indicates the oral primordium (OP) of the opisthe. (B, D) Ventral view of the middle portion, arrows show the anarchic primordium of the opisthe. (C) Ventral view of the parental buccal field, arrows mark the OP of the proter. (E) Ventral view, double-arrowheads indicate the marginal row anlagen, arrows indicate the dorsal kinety anlagen. (F) Ventral view, arrows show the macronuclear nodules. (G) Ventral view of the opisthe, arrowheads indicate the fronto-ventral-transverse cirral anlagen, arrows mark the oral primordium, double-arrowhead depicts the undulating membrane anlage. Bars ¼ 100 mm (in A, E); 50 mm (in D).
A. bergeri is more closely related to A. crassa than to Pseudokeronopsis spp. In addition, the small subunit rRNA gene of A. bergeri has also been sequenced (Yi et al., pers. comm.) and has been submitted to GenBank with the accession number DQ777742.
Discussion Morphology comparison (Table 3) Prior to the present investigation only one species of Apokeronopsis, A. crassa (Clapare`de and Lachmann,
1858) Shao et al., 2007, was known and this had been reported under at least four different names: Oxytricha crassa, Trichotaxis (Oxytricha) crassa, Pseudokeronopsis qingdaoensis and Thigmokeronopsis crassa (Berger 2006; Hu and Song 2000; Song et al. 2004b; Shao et al. 2007). Compared with A. crassa, the new species described here can be distinguished mainly in body shape (with round posterior cell end vs. with conspicuously narrowed taillike caudal portion in A. crassa), yellow-brownish cell colour (vs. colourless), the number of membranelles in the adoral zone (72–93 vs. 46–65 in A. crassa), the number and arrangement of the transverse cirri (ca. 16 in a short row vs. ca. 34 in an extremely long row that is parallel to the midventral complex and extends to about
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Fig. 7. Photomicrographs of morphogenesis of Apokeronopsis bergeri nov. spec. after protargol impregnation. (A) Ventral view to show the development of the fronto-ventral-transverse cirral anlagen and the disorganization of the old AZM. (B) Ventral view of the posterior portion of the same cell as (A), arrow indicates the undulating membrane anlage of the opisthe. (C–E) Late stages of morphogenesis, indicating the migration of the newly generated cirri and division of cell, arrowheads and arrows in (C) mark the marginal row anlagen at the right and left side, respectively, arrows in (D) indicate the newly built buccal cirri. (F) Ventral view, arrowhead indicates the frontoterminal cirri moving anteriad, arrow marks the newly generated transverse cirri. Bars ¼ 100 mm (in A); 60 mm (in B); 150 mm (in E).
Table 2. Gene sequence similarities between the ITS1-5.8S-ITS2 rDNA regions of 5 closely related morphotypes: three Pseudokeronopsis and two Apokeronopsis species using the PHYLIP package based on the Kimura (1980) two-parameter model
P. carnea P. rubra P. flava A. crassa A. bergeri
P. carnea (%)
P. rubra (%)
P. flava (%)
A. crassa (%)
88.85 89.95 67.15 69.73
89.32 66.63 65.15
69.04 68.02
82.92
A. bergeri
Table 3.
Comparison of marine species of Pseudokeronopsis and Apokeronopsis investigated using silver impregnation techniques P. carnea
140–250 50–70
200–300 40–55
140–200 28–40
150–400 70–90 Slender, posteriorly tapered Yellow-brownish
150–300 40–70 Belt-like, caudally narrowed Yellow
Yellow-brownish
Yellowish
1 43–51 ca. 9 24–36
P. rubra
P. pararubrab
A. crassaa
P. sepetibensis
A. bergeri nov. spec.
150–250 30–55
180–350 50–90
Elongated with round ends
Elongate to fusiform with round ends Yellow-green to yellowish Brown-yellowish
Colourless
Elongated with tapering posterior end Brown-reddish
Belt-like, caudally slightly or not narrowed Orange-red
Slender, both cell ends narrowed Brick-red
Plump, frontal area wide, seldom twisted Orange-red
Brick-reddish
Brown-reddish
1 46–66 ca. 14 25–40
Brick-reddish to brown-reddish 1 39–80 10–14 22–44
Light yellowgreenish Yellow-greenish
1 40–53 10–12 23–35
1 64–92 15–26 ca. 30–45
1 38–55 8–11 35–57
9–13 72–93 29–48 ca. 26–48
6–10 46–60 27–32 ca. 40–60
3–4 3 Conspicuous
3–6 4–5 Small to inconspicuous
6–7 5–6 Small to conspicuous
3–4 3–6 Absent
7–11 5–8 Absent
3 4–6 Small to inconspicuous
13–22 3 Absent
25–36 3 Absent
Song et al. (2004a)
Song et al. (2002)
Wirnsberger et al. (1987)
Hu and Song (2001)
Hu et al. (2004b)
Wanick and Silva-Neto (2004)
Present work
Song et al. (2004b)
Measurements are in m m. AM—adoral membranelles, BC—buccal cirri, DK—dorsal kineties, FC—frontal cirri, MVC—midventral complex, TC—transverse cirri. a Misidentified as Pseudokeronopsis qingdaoensis, which is a junior synonym of Apokeronopsis crassa. b A marine form recently reported by Hu et al. (2004b), which is regarded as a junior synonym of P. carnea and has been synonymized with the latter (Song et al. 2006).
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Colour of large cortical granules Colour of cell at low magnification Number of BC Number of AM Number of FC Number of pairs of MVC Number of TC Number of DK Gap between posterior end of MVC and TC Data source
P. flavicans
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Cell size in vivo mm 110–140 20–26 Body shape
P. flava
217
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Fig. 8. Comparative nucleotide sequence data from ITS1-5.8S-ITS2 rDNA regions of three Pseudokeronopsis spp. and two Apokeronopsis spp. Matched sites are marked with black, while sites marked in grey are incomplete matches between the two genera.
cytostome level in A. crassa) and the arrangement of the type I cortical granules (densely aligned in longitudinal rows vs. sparsely distributed in A. crassa) (Table 3). Hence, the two forms are clearly separated. The separation of these two taxa is also firmly supported by the molecular data, which demonstrate that there is a considerable discrepancy (i.e. an evolutionary distance value 17.09%) between Apokeronopsis bergeri and A. crassa (Table 2; Fig. 8). In terms of its general morphology, infraciliature and marine habitat, Apokeronopsis bergeri should also be compared with five Pseudokeronopsis species: P. flava, P. flavicans, P. carnea, P. rubra and P. sepetibensis (Hu and Song 2001; Hu et al. 2004; Song et al. 2002, 2004a, b; Wanick and Silva-Neto 2004; Wirnsberger et al. 1987). Compared with these forms A. bergeri can be distinguished by a combination of the following features: (1) 9–13 buccal cirri (vs. one in the all the Pseudokeronopsis spp.); (2) 13–22 transverse cirri (vs. fewer than 11 in the Pseudokeronopsis spp.); (3) conspicuously separated midventral rows (vs. midventral rows closely aligned with cirri in pairs in the Pseudokeronopsis spp.) (Table 3). Similarly the molecular data also reveal a clear separation between A. bergeri and the Pseudokeronopsis spp. (Table 3). Apokeronopsis can also be readily distinguished from another closely related genus, Thigmokeronopsis, by the absence of thigmotactic cirri (vs. present in Thigmokeronopsis) and 9–13 buccal cirri (vs. only one or two in Thigmokeronopsis) (Berger 2006; Shao et al. 2007; Wicklow 1981).
et al. 2004; Ruthmann 1972; Sun and Song 2005; Wirnsberger 1987). These share very similar characteristics during the division process, namely: (1) complete replacement of the old AZM in the proter by the newly built structure; (2) marginal rows and dorsal kinety anlagen develop within the old structures; (3) macronuclear nodules partially fuse but do not form a single mass before division; and (4) the FVT-cirral anlagen in both daughter cells give rise to the buccal, transverse, frontoterminal, bicorona and the midventral complex, the cirri of the latter being typically arranged in a zig-zag pattern. Compared with the morphogenetical mode revealed in the genus Pseudokeronopsis, Apokeronopsis bergeri exhibits some particular characters which are similar to those found in its congener, A. crassa (Shao et al. 2007), e.g. marginal row and dorsal kinety anlagen develop independently alongside the old structures (vs. within parental structures in Pseudokeronopsis). One noteworthy difference between Apokeronopsis bergeri and A. crassa is the method by which the buccal cirral row is formed from the FVT-cirral anlagen. In A. bergeri several anterior streaks from the FVT-cirral anlagen appear to contribute their last segments to form the row of buccal cirri, which is an unusual feature, while in A. crassa the buccal cirri are generated from the first FVT-cirral anlage, as seen in most other related taxa (Berger 2006). It is, however, too early to evaluate the significance of this difference. Overall, the similarities in morphology, morphogenesis and ITS1-5.8SITS2 region gene sequences exhibited by A. crassa and our new species support the conclusion that they belong to the same clearly defined genus within the urostylids.
Divisional morphogenesis
Acknowledgments Morphogenesis has been described in four species of Pseudokeronopsis: P. carnea, P. pulchra, P. rubra and P. flava (Berger 2006; Borror 1972; Hu and Song 2001; Hu
This work was supported by the Natural Science Foundation of China (Project nos.: 40676076,
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30430090), the Darwin Initiative Programme (Project no.: 14-015), which is funded by the UK Department of Environment, Food and Rural Affairs, and from Center of Excellence in Biodiversity, King Saud University. We also thank Mr. Xiangri Chen and Mr. Yangang Wang, postgraduates in our laboratory, for help with collecting samples.
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PROTISTOLOGY European Journal of Protistology 44 (2008) 208–219 www.elsevier.de/ejop
Morphology and morphogenesis of a new marine ciliate, Apokeronopsis bergeri nov. spec. (Ciliophora, Hypotrichida), from the Yellow Sea, China Liqiong Lia, Weibo Songa,, Alan Warrenb, Khaled A.S. Al-Rasheidc, David Robertsb, Zhenzhen Yia, Saleh A. Al-Farrajc, Xiaozhong Hua a
Laboratory of Protozoology, Ocean University of China, Qingdao 266003, China Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK c Zoology Department, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia b
Received 23 January 2007; received in revised form 21 December 2007; accepted 2 January 2008
Abstract The morphology and morphogenesis of a new marine hypotrich ciliate, Apokeronopsis bergeri nov. spec., collected from mussel-farming waters near Qingdao, China, are described from living and protargol-impregnated specimens. This ciliate has characteristics that place it in the family Pseudokeronopsidae, namely, two long rows of frontal cirri (bicorona), which are continuous with the long midventral rows, and a single row of marginal cirri on each side of the body. It shares with its only congener, Apokeronopsis crassa, the long rows of buccal and transverse cirri and the wide spacing between the midventral rows of cirri. These characters separate the genus Apokeronopsis from Pseudokeronopsis, which has a single buccal cirrus, fewer transverse cirri and midventral rows of cirri arranged in a typical zig-zag pattern. A. bergeri differs from A. crassa in its shape, colour and in the numbers of membranelles and transverse cirri. Although morphogenesis in A. bergeri is similar to that of A. crassa in most respects, the mode of formation of the buccal cirri is slightly different. The close relationship of A. bergeri with A. crassa, and the more distant relationship with three Pseudokeronopsis species, is supported by a comparison of the sequences of their ITS15.8S-ITS2 rDNA regions. r 2008 Elsevier GmbH. All rights reserved. Keywords: Apokeronopsis bergeri nov. spec.; ITS and 5.8S rDNA sequence; Morphogenesis; Morphology; Stichotrichia; Taxonomy
Introduction The well-known hypotrich genus Pseudokeronopsis is distinguished by having frontal cirri arranged as a bicorona, whose rows continue posteriorly to form two midventral rows and one row of marginal cirri on each side of the body (Berger 2006; Borror and Wicklow 1983; Song et al. 2006). The genus Apokeronopsis, which Corresponding author.
E-mail address: [email protected] (W. Song). 0932-4739/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2008.01.001
was recently created by Shao et al. (2007), also possesses these characters but differs from Pseudokeronopsis as they have (1) a long row of buccal cirri (vs. a single buccal cirrus in Pseudokeronopsis); (2) widely separated midventral rows (vs. midventral rows closely juxtaposed with cirri in pairs forming the midventral complex in Pseudokeronopsis); and (3) at least 13 transverse cirri in a long row (vs. generally o10 transverse cirri in Pseudokeronopsis) (Berger 2006). Furthermore, the marginal rows and dorsal kineties, as observed in the type species A. crassa, originate de novo in both dividers
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(vs. within the old structure in Pseudokeronopsis) (Berger 2006; Hu and Song 2000; Hu et al. 2004; Shao et al. 2007). In the summer of 2005, we isolated an unknown urostylid ciliate from coastal waters off Qingdao. Pure cultures were established enabling detailed descriptions of its morphology and morphogenesis to be made and sequencing of the ITS1-5.8S-ITS2 rDNA region to be performed. The results of our investigations demonstrate that this is a new species within the genus Apokeronopsis.
Material and methods Apokeronopsis bergeri nov. spec. was collected from open mussel-farming waters near Qingdao (361 080 N; 1201 430 E) on August 1, 2005, where the salinity was 29–31%, the water temperature 20 1C, and pH 8.0. Long-term cultures were established in Petri dishes containing boiled seawater to which squeezed rice grains were added to enrich the bacterial food. Observations on live specimens were undertaken using differential interference microscopy. Protargol staining (Wilbert 1975) was used to reveal the infraciliature and nuclear apparatus. Counting and measurements were made at a magnification of 1250 . Drawings were performed with the help of a camera lucida. To illustrate the changes occurring during morphogenesis parental cirri are depicted by contour whereas new ones are shaded black. Terminology is mainly according to Hemberger (1982), Wirnsberger (1987) and Berger (2004, 2006). Genomic DNA extraction, PCR amplification, and ITS1-5.8S-ITS2 rDNA region cloning and sequencing of A. bergeri were performed according to Shang et al. (2003). Two primers were used: 5.8S-F: 50 -GTAGGTGAACCTGCGGAAGGATCATTA-30 ; 5.8S-R: 50 -TACTGATATGCTTAAGTTCAGCGG-30 . The nucleotide sequences used in this paper are available from the GenBank database under the following accession numbers: Pseudokeronopsis flava (DQ503579), Pseudokeronopsis rubra (DQ640313), Pseudokeronopsis carnea (DQ503580) and Apokeronopsis crassa (DQ537483). PHYLIP package, version 3.75c (Felsenstein 1995), was used to calculate the evolutionary similarities among three Pseudokeronopsis and two Apokeronopsis species.
Results Order Hypotrichida Stein, 1859 Family Pseudokeronopsidae Borror and Wicklow, 1983 Genus Apokeronopsis Shao et al., 2007
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Apokeronopsis bergeri nov. spec. Diagnosis Large marine Apokeronopsis 150–400 70–90 mm in vivo, body flexible, elongate to fusiform with both ends broadly rounded; yellow-brown to dark brown in colour; bicorona comprising about 38 frontal cirri; 9–13 buccal and 1–3 frontoterminal cirri; 13–22 transverse cirri densely arranged in a J-shaped row; midventral complex consisting of two conspicuously separated rows with ca. 35 pairs of cirri; adoral zone composed of about 80 membranelles; invariably 3 dorsal kineties. Two kinds of cortical granules: one tiny and colourless, densely arranged in short lines; the other large (about 2 mm across), biconcave and distributed sparsely, yellowbrown to yellow-green in newly sampled specimens, becoming dark-red in cultured cells. Numerous (i.e. 150–200) macronuclear nodules. Single contractile vacuole positioned in anterior 1/3 of the body. Type locality Mariculture waters of Yellow Sea coast at Qingdao, China (361 080 N; 1201 430 E). Type specimens One permanent holotype of protargol-impregnated specimens is deposited in the Natural History Museum, London, UK, with registration no. 2007:5:14:1. One paratype slide is deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China. Dedication We dedicate this new species to our Austrian colleague, Dr. Helmut Berger, in recognition of his contribution to ciliate morphogenesis and taxonomy, especially in the field of hypotrichs.
Morphological description (Figs 1–3; Table 1) Specimens mostly about 300 80 mm in vivo. Body shape elongate to fusiform with both ends broadly rounded, conspicuously flexible and slightly contractile. Specimens in newly collected samples generally much slimmer than those in culture with plentiful food (Figs 1A and 2A, F). Cells dorsoventrally flattened about 2:1. Buccal field narrow, about 1/3 of the body length. Cells appear brown to dark brown in colour under low magnification and yellow-brown when observed under high magnification (Fig. 2A–D). Two types of cortical granules: type I very small, about 0.2 mm across and colourless, arranged in longitudinally oriented lines that are evenly distributed over the body (Figs 1C, arrowheads; 2J, arrowheads); type II, biconcave (i.e. similar to erythrocytes of mammals),
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Fig. 1. Morphology and infraciliature of Apokeronopsis bergeri nov. spec. from life (A, C, D) and after protargol impregnation (B, E, F). (A) Ventral view of a typical individual; arrow marks the contractile vacuole. (B) Oral field, showing the buccal apparatus and the strong fibres, arrowheads mark the buccal cirri. (C) Details of the cortex, to show the very small type I granules (arrowheads) and the large biconcave type II granules (arrows). (D) Dorsal view; note the sparsely distributed type II granules. (E) Ventral view, arrow indicates the distal end of the adoral zone, and double-arrowheads depict the frontoterminal cirri. (F) Dorsal view of the same specimen as (E), to demonstrate the macronuclear nodules and the dorsal kineties. AZM—adoral zone of membranelles, BC—buccal cirri, DK—dorsal kineties, EM—endoral membrane, FC—frontal cirri, LMR—left marginal row, Ma—macronuclei, MVR—midventral rows (midventral complex), PM—paroral membrane, RMR—right marginal row, TC— transverse cirri. Bars ¼ 120 mm (in A, D, E, F); 20 mm (in C).
flattened, ca. 2 mm across, yellow-brown to yellow-green in colour, densely distributed, but not grouped, within the cortex (Figs 1C, arrows; 2C, J, arrows). Significantly, after several weeks’ culture, some type II granules changed colour from yellow-green to darkreddish; all type II granules turned dark-red during encystment (Fig. 2E, H, I, arrows). Cytoplasm opaque and colourless, containing numerous light-reflecting globules. Contractile vacuole pulsating infrequently, positioned near the left margin at about anterior 1/3–1/4 of the cell length (Figs 1A, arrow; 2A, arrow). About 150–200 macronuclear nodules, oval to elongate in shape, scattered within the cytoplasm (Fig. 1F). Micronuclei (Mi) not observed in protargolimpregnated specimens. Locomotion by crawling slowly without pausing on the debris or on bottom of the Petri dish. Adoral zone (AZM) curves across the anterior cell margin and extends conspicuously down the right side of the body (Fig. 1A); the distal end of AZM extends somewhat obliquely towards the buccal cavity (Figs 1E, arrow; 3C, double-arrowheads). Bases of the longest membranelles are about 20 mm wide, and those at the
distal end of AZM are distinctly shorter than those in the central portion. Paroral and endoral membranes (PM, EM) almost equal in length and spatially crossed; PM is thicker than EM (Fig. 1B, E). Most somatic cirri relatively fine, about 15 mm long. Bicorona consisting of about 40 slightly enlarged frontal cirri that are not separated from the midventral complex although in some specimens an inconspicuous gap was observed. Usually there are two frontoterminal cirri on the right side of the cell near the distal end of the adoral zone which are easy to recognize due to the distinct gap between them (Figs 1E, double-arrowheads; 3C, arrows). One long row of 9–13 buccal cirri (BC) is close to the paroral membrane (Figs 1B, arrowheads; 3B, arrows). Two rows of midventral cirri (MVR) are parallel to and considerably separated from each other, usually terminating just above the transverse cirri (Figs 1A, E; 3A, E). Thirteen to twenty-two transverse cirri (TC) about 20 mm long, are densely arranged in a J-shaped row near the posterior margin of the cell (Figs 1E; 3E, arrows). Cirri in marginal rows narrowly spaced; right marginal row (RMR) commencing near the distal end of the adoral zone. Marginal rows almost
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Fig. 2. Photomicrographs of Apokeronopsis bergeri nov. spec. (A–F, H–J) and A. crassa (G, K) from life. (A) Ventral view of a typical specimen, arrow indicates the contractile vacuole. (B) Ventral view of anterior portion, to show a light-coloured specimen. (C) A dark-coloured specimen, arrows show the distribution of type II granules. (D, F) Ventral view of two specimens, to show the different body shapes. (E) Cyst, arrows mark the dark-reddish type II granules. (G) Ventral view of a typical specimen of A. crassa. (H, I) Type II granules, arrows indicate the dark-reddish granules; note that most of the others are yellow-greenish. (J) Dorsal view, arrowheads depict the colourless type I granules, arrows indicate the type II granules. (K) Dorsal view of A. crassa, to show the brown-reddish granules. Bars ¼ 100 mm (in A, C, D, F, G); 30 mm (in E).
confluent posteriorly (Figs 1E; 3E). Invariably 3 complete dorsal kineties, each comprising about 50 pairs of basal bodies, cilia about 3 mm long (Figs 1F; 3F, arrows).
Divisional morphogenesis (Figs 4–7) Stomatogenesis and development of the somatic ciliature in the opisthe Stomatogenesis commences with the formation of small groups of basal bodies close to several of the intact left midventral cirri (Fig. 4A, arrows; 4B, arrowheads). With the proliferation of basal bodies, these groups join to make a longish field which is the anarchic primordium (AP) of the opisthe (Figs 4C, small doublearrowheads; 6A, arrow). During this process the left midventral cirri remain intact. Subsequently, a long streak of basal bodies that extends to the posterior end of the buccal field appears as a result of the splitting of the anterior end of the AP (Fig. 4D, small doublearrowheads). The middle part of the AP will become the anlage of the undulating membrane. By this stage, the development of membranelles in the right anterior part of this area has already begun (Fig. 4D). Soon after, when the thread-like anlagen arrange as a ladder-like structure, the UM-anlage (UMA) starts aggregating
into the undulating membranes (Figs 4E and 6G, double-arrowheads). The UM-anlage remains as a single unit until the late stage and then splits longitudinally to form the paroral and endoral membranes (Fig. 5D, F). Division continues with the complete formation of the FVT-cirral anlagen at the right of the AP, within which each streak seems to break into several segments. Meanwhile, a single frontal cirrus derives from the UMA (Fig. 5D, arrows). Along with the formation of the new cirri, several segments from the posterior portion of the FVT-cirral anlagen streaks begin migrating towards the newly formed UM which seem to give rise to the row of buccal cirri of the opisthe (Fig. 5C, double-arrowheads). Each of the remaining streaks divides into 2 cirri except for about 15 posterior-most streaks that form 3 cirri (Fig. 5D, F). The two anteriormost cirri from the last streak will migrate anteriad to become the frontoterminal cirri (Figs 5F, double-arrowheads; 7F, arrowhead). Each of the long posterior streaks contributes one transverse cirrus to the row of transverse cirri (TC), formed by the shortest streaks, the remaining new cirri becoming the frontal and midventral cirri (Figs 5F and 7F, arrow). The new anlagen of the marginal rows (MA) originate independently from the old structures on each side of the cell at almost the same time as the AP appears
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Fig. 3. Photomicrographs of Apokeronopsis bergeri nov. spec. after protargol impregnation. (A) Ventral view, to show the general ciliature. (B) Ventral view of the anterior-right portion, arrows mark the buccal cirri. (C) Ventral view of the anterior portion, arrows indicate the frontoterminal cirri, double-arrowhead marks the distal end of the adoral zone. (D) Ventral view of the buccal field, arrowhead indicates the paroral membrane, and the arrow marks the endoral membrane. (E) Ventral view of the posterior portion, to show the transverse cirri (arrows). (F). Detail of dorsal side, arrows indicate the cilia of the dorsal kineties and arrowheads mark the macronuclear nodules. Bar ¼ 100 mm (in A); 20 mm (in F). Table 1.
Morphometrical data of Apokeronopsis bergeri nov. spec. (all data are based on protargol-impregnated specimens)
Character
Min
Max
Mean
SD
CV
n
Length of the body in mm Width of the body in mm Length of the adoral zone in mm No. of membranelles No. of anterior frontal cirri No. of posterior frontal cirri No. of buccal cirri No. of frontoterminal cirri No. of transverse cirri Cirral pairs in midventral rows, No. of left cirri Cirral pairs in midventral rows, No. of right cirri No. of cirri in the left marginal row No. of cirri in the right marginal row No. of dorsal kineties
184 104 108 72 14 15 9 1 13 26 28 44 54 3
404 208 148 93 23 25 13 3 22 43 48 70 88 3
310.3 145.3 127.8 80.2 16.9 20.6 10.9 2.0 15.6 33.0 35.9 55.3 63.2 3
50.1 25.9 11.9 5.3 2.2 2.5 1.0 0.3 2.3 4.6 4.8 6.2 7.6 0
16.1 17.8 9.3 6.6 13.0 12.1 9.2 15.0 14.7 13.9 13.4 11.2 12.0 0
25 25 25 25 25 25 25 25 25 25 25 25 25 25
Abbreviations: CV—coefficient of variation in %; Max—maximum; Mean—arithmetic mean; Min—minimum; n—number of cells measured; SD— standard deviation; SE—standard error of the mean.
(Fig. 4C, arrows). All these anlagen then stretch both anteriorly and posteriorly while remaining separate from the old structures (Figs 4G and 6E, double-
arrowheads). The old marginal cirri do not participate in the formation and development of the cirral anlagen and are eventually resorbed.
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Fig. 4. Morphogenesis of Apokeronopsis bergeri nov. spec. after protargol impregnation. (A) Ventral view of an early divider, arrows indicate the newly formed basal bodies close to the left midventral cirri. (B) Detailed structure from the same specimen as (A), to show the groups of basal bodies (arrowheads). (C) Early stage of morphogenesis, small double-arrowheads mark the OP of the opisthe and arrows indicate the marginal row anlagen; large double-arrowhead marks the OP of the proter. (D) Ventral view, double-arrowhead and small arrow indicate fronto-ventral-transverse cirral anlagen in both dividers; the large arrow shows the disorganization of the parental adoral zone; arrowhead depicts the OP of the proter; inset—macronuclear nodules with replication bands. (E, F) Ventral and dorsal views of the same specimen, arrows indicate the dorsal kinety anlagen. (G) Ventral view, arrow indicates the newly formed membranelles of the proter, arrowheads mark the FVT-cirral anlagen of the proter, double-arrowheads depict the marginal row anlagen. (H) Dorsal view of the same cell as (G), to show the partially fused macronuclear nodules. DA— dorsal kinety anlagen, MA—marginal row anlagen, Ma—macronuclei, MVR—midventral rows (midventral complex). Bar ¼ 120 mm.
Dorsal kineties are formed in an unusual way. Just after the formation of the marginal row anlagen, three streaks of basal bodies appear de novo (DA) in both dividers (Figs 4D–H and 6E, arrows). With the proliferation of basal bodies, these anlagen elongate and ultimately replace the old structures (Fig. 5B, E, G). Stomatogenesis and development of the somatic ciliature in the proter At about the same time as the appearance of the AP in the opisthe, a scoop-like oral primordium forms deep
beneath the parental buccal cavity (Figs 4C, large double-arrowheads; 6C, arrows). Later the thread-like FVT-cirral anlagen of the proter emerges de novo close to the right of the endoral membrane near the cell surface (Fig. 4D, small arrow). Meanwhile, the oral primordium starts to split to form the UM-anlage (UMA). The parental UMs appear not to join in the formation of the new anlagen and are soon resorbed (Fig. 5A). As a result of the joining of new basal bodies in the anarchic field, the new adoral membranelles are gradually organized in a posteriad direction (Fig. 4D,
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Fig. 5. Morphogenesis of Apokeronopsis bergeri nov. spec. after protargol impregnation. (A, B) Ventral and dorsal views of the same specimen at the middle stage; double-arrowheads mark the dorsal kinety anlagen and arrows indicate the marginal row anlagen. (C) Ventral view, double-arrowheads indicate that some segments have migrated from the fronto-ventral-transverse cirral anlagen; arrows mark the marginal row anlagen; inset—macronuclear nodules in division. (D) Ventral view of a late divider, arrows mark the first frontal cirrus generated from the undulating membrane anlage in each divider; double-arrowhead depicts the right marginal row anlage. (E) Dorsal view of the same specimen as (D), arrows indicate dorsal kinety anlagen, double-arrowheads mark the marginal row anlagen. (F, G) Ventral and dorsal view of the same specimen; arrows show the newly formed transverse cirri and double-arrowheads mark the frontoterminal cirri. Bars ¼ 100 mm.
E). Simultaneously, the UMA detaches and generates a single frontal cirrus at its anterior end. As in the opisthe, the FVT-anlagen give rise to the FC, BC, MVC and TC. Marginal cirri and dorsal kineties are also formed in the same way as those of the opisthe. Macronuclear division The division process of the macronuclear apparatus appears to be unique. The replication bands of macronuclear nodules appear at an early stage (Figs 4D, inset; 6B). In the middle stage there is an obvious merging process of the macronuclear nodules although there is no fusion into a single mass prior to division (Figs 4H, 5B and 6F, arrows).
Information derived from ITS1-5.8S-ITS2 rDNA sequence analysis (Fig. 8; Table 2) The ITS1-5.8S-ITS2 region sequence of Apokeronopsis bergeri has been deposited in GenBank with the accession number DQ503583. The ITS1-5.8S-ITS2 region sequences within the genera Pseudokeronopsis and Apokeronopsis share evolutionary similarities of 88.85%–89.95% and 82.92%, respectively, while the evolutionary similarities of Pseudokeronopsis to Apokeronopsis range from 65.15% to 69.73% (Table 2). These data, along with the comparisons of the nucleotide sequences of the ITS1-5.8S-ITS2 rDNA regions (Fig. 8), indicate that
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Fig. 6. Photomicrographs of morphogenesis of Apokeronopsis bergeri nov. spec. after protargol impregnation. (A) Ventral view of an early divider, arrow indicates the oral primordium (OP) of the opisthe. (B, D) Ventral view of the middle portion, arrows show the anarchic primordium of the opisthe. (C) Ventral view of the parental buccal field, arrows mark the OP of the proter. (E) Ventral view, double-arrowheads indicate the marginal row anlagen, arrows indicate the dorsal kinety anlagen. (F) Ventral view, arrows show the macronuclear nodules. (G) Ventral view of the opisthe, arrowheads indicate the fronto-ventral-transverse cirral anlagen, arrows mark the oral primordium, double-arrowhead depicts the undulating membrane anlage. Bars ¼ 100 mm (in A, E); 50 mm (in D).
A. bergeri is more closely related to A. crassa than to Pseudokeronopsis spp. In addition, the small subunit rRNA gene of A. bergeri has also been sequenced (Yi et al., pers. comm.) and has been submitted to GenBank with the accession number DQ777742.
Discussion Morphology comparison (Table 3) Prior to the present investigation only one species of Apokeronopsis, A. crassa (Clapare`de and Lachmann,
1858) Shao et al., 2007, was known and this had been reported under at least four different names: Oxytricha crassa, Trichotaxis (Oxytricha) crassa, Pseudokeronopsis qingdaoensis and Thigmokeronopsis crassa (Berger 2006; Hu and Song 2000; Song et al. 2004b; Shao et al. 2007). Compared with A. crassa, the new species described here can be distinguished mainly in body shape (with round posterior cell end vs. with conspicuously narrowed taillike caudal portion in A. crassa), yellow-brownish cell colour (vs. colourless), the number of membranelles in the adoral zone (72–93 vs. 46–65 in A. crassa), the number and arrangement of the transverse cirri (ca. 16 in a short row vs. ca. 34 in an extremely long row that is parallel to the midventral complex and extends to about
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Fig. 7. Photomicrographs of morphogenesis of Apokeronopsis bergeri nov. spec. after protargol impregnation. (A) Ventral view to show the development of the fronto-ventral-transverse cirral anlagen and the disorganization of the old AZM. (B) Ventral view of the posterior portion of the same cell as (A), arrow indicates the undulating membrane anlage of the opisthe. (C–E) Late stages of morphogenesis, indicating the migration of the newly generated cirri and division of cell, arrowheads and arrows in (C) mark the marginal row anlagen at the right and left side, respectively, arrows in (D) indicate the newly built buccal cirri. (F) Ventral view, arrowhead indicates the frontoterminal cirri moving anteriad, arrow marks the newly generated transverse cirri. Bars ¼ 100 mm (in A); 60 mm (in B); 150 mm (in E).
Table 2. Gene sequence similarities between the ITS1-5.8S-ITS2 rDNA regions of 5 closely related morphotypes: three Pseudokeronopsis and two Apokeronopsis species using the PHYLIP package based on the Kimura (1980) two-parameter model
P. carnea P. rubra P. flava A. crassa A. bergeri
P. carnea (%)
P. rubra (%)
P. flava (%)
A. crassa (%)
88.85 89.95 67.15 69.73
89.32 66.63 65.15
69.04 68.02
82.92
A. bergeri
Table 3.
Comparison of marine species of Pseudokeronopsis and Apokeronopsis investigated using silver impregnation techniques P. carnea
140–250 50–70
200–300 40–55
140–200 28–40
150–400 70–90 Slender, posteriorly tapered Yellow-brownish
150–300 40–70 Belt-like, caudally narrowed Yellow
Yellow-brownish
Yellowish
1 43–51 ca. 9 24–36
P. rubra
P. pararubrab
A. crassaa
P. sepetibensis
A. bergeri nov. spec.
150–250 30–55
180–350 50–90
Elongated with round ends
Elongate to fusiform with round ends Yellow-green to yellowish Brown-yellowish
Colourless
Elongated with tapering posterior end Brown-reddish
Belt-like, caudally slightly or not narrowed Orange-red
Slender, both cell ends narrowed Brick-red
Plump, frontal area wide, seldom twisted Orange-red
Brick-reddish
Brown-reddish
1 46–66 ca. 14 25–40
Brick-reddish to brown-reddish 1 39–80 10–14 22–44
Light yellowgreenish Yellow-greenish
1 40–53 10–12 23–35
1 64–92 15–26 ca. 30–45
1 38–55 8–11 35–57
9–13 72–93 29–48 ca. 26–48
6–10 46–60 27–32 ca. 40–60
3–4 3 Conspicuous
3–6 4–5 Small to inconspicuous
6–7 5–6 Small to conspicuous
3–4 3–6 Absent
7–11 5–8 Absent
3 4–6 Small to inconspicuous
13–22 3 Absent
25–36 3 Absent
Song et al. (2004a)
Song et al. (2002)
Wirnsberger et al. (1987)
Hu and Song (2001)
Hu et al. (2004b)
Wanick and Silva-Neto (2004)
Present work
Song et al. (2004b)
Measurements are in m m. AM—adoral membranelles, BC—buccal cirri, DK—dorsal kineties, FC—frontal cirri, MVC—midventral complex, TC—transverse cirri. a Misidentified as Pseudokeronopsis qingdaoensis, which is a junior synonym of Apokeronopsis crassa. b A marine form recently reported by Hu et al. (2004b), which is regarded as a junior synonym of P. carnea and has been synonymized with the latter (Song et al. 2006).
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Colour of large cortical granules Colour of cell at low magnification Number of BC Number of AM Number of FC Number of pairs of MVC Number of TC Number of DK Gap between posterior end of MVC and TC Data source
P. flavicans
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Cell size in vivo mm 110–140 20–26 Body shape
P. flava
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Fig. 8. Comparative nucleotide sequence data from ITS1-5.8S-ITS2 rDNA regions of three Pseudokeronopsis spp. and two Apokeronopsis spp. Matched sites are marked with black, while sites marked in grey are incomplete matches between the two genera.
cytostome level in A. crassa) and the arrangement of the type I cortical granules (densely aligned in longitudinal rows vs. sparsely distributed in A. crassa) (Table 3). Hence, the two forms are clearly separated. The separation of these two taxa is also firmly supported by the molecular data, which demonstrate that there is a considerable discrepancy (i.e. an evolutionary distance value 17.09%) between Apokeronopsis bergeri and A. crassa (Table 2; Fig. 8). In terms of its general morphology, infraciliature and marine habitat, Apokeronopsis bergeri should also be compared with five Pseudokeronopsis species: P. flava, P. flavicans, P. carnea, P. rubra and P. sepetibensis (Hu and Song 2001; Hu et al. 2004; Song et al. 2002, 2004a, b; Wanick and Silva-Neto 2004; Wirnsberger et al. 1987). Compared with these forms A. bergeri can be distinguished by a combination of the following features: (1) 9–13 buccal cirri (vs. one in the all the Pseudokeronopsis spp.); (2) 13–22 transverse cirri (vs. fewer than 11 in the Pseudokeronopsis spp.); (3) conspicuously separated midventral rows (vs. midventral rows closely aligned with cirri in pairs in the Pseudokeronopsis spp.) (Table 3). Similarly the molecular data also reveal a clear separation between A. bergeri and the Pseudokeronopsis spp. (Table 3). Apokeronopsis can also be readily distinguished from another closely related genus, Thigmokeronopsis, by the absence of thigmotactic cirri (vs. present in Thigmokeronopsis) and 9–13 buccal cirri (vs. only one or two in Thigmokeronopsis) (Berger 2006; Shao et al. 2007; Wicklow 1981).
et al. 2004; Ruthmann 1972; Sun and Song 2005; Wirnsberger 1987). These share very similar characteristics during the division process, namely: (1) complete replacement of the old AZM in the proter by the newly built structure; (2) marginal rows and dorsal kinety anlagen develop within the old structures; (3) macronuclear nodules partially fuse but do not form a single mass before division; and (4) the FVT-cirral anlagen in both daughter cells give rise to the buccal, transverse, frontoterminal, bicorona and the midventral complex, the cirri of the latter being typically arranged in a zig-zag pattern. Compared with the morphogenetical mode revealed in the genus Pseudokeronopsis, Apokeronopsis bergeri exhibits some particular characters which are similar to those found in its congener, A. crassa (Shao et al. 2007), e.g. marginal row and dorsal kinety anlagen develop independently alongside the old structures (vs. within parental structures in Pseudokeronopsis). One noteworthy difference between Apokeronopsis bergeri and A. crassa is the method by which the buccal cirral row is formed from the FVT-cirral anlagen. In A. bergeri several anterior streaks from the FVT-cirral anlagen appear to contribute their last segments to form the row of buccal cirri, which is an unusual feature, while in A. crassa the buccal cirri are generated from the first FVT-cirral anlage, as seen in most other related taxa (Berger 2006). It is, however, too early to evaluate the significance of this difference. Overall, the similarities in morphology, morphogenesis and ITS1-5.8SITS2 region gene sequences exhibited by A. crassa and our new species support the conclusion that they belong to the same clearly defined genus within the urostylids.
Divisional morphogenesis
Acknowledgments Morphogenesis has been described in four species of Pseudokeronopsis: P. carnea, P. pulchra, P. rubra and P. flava (Berger 2006; Borror 1972; Hu and Song 2001; Hu
This work was supported by the Natural Science Foundation of China (Project nos.: 40676076,
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30430090), the Darwin Initiative Programme (Project no.: 14-015), which is funded by the UK Department of Environment, Food and Rural Affairs, and from Center of Excellence in Biodiversity, King Saud University. We also thank Mr. Xiangri Chen and Mr. Yangang Wang, postgraduates in our laboratory, for help with collecting samples.
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