Morphology of Two Novel Species of Chaenea (Ciliophora, Litostomatea): Chaenea paucistriata spec. nov. and C. sinica spec. nov.

Abstract

During faunistic studies of ciliates in coastal waters of Daya Bay and Bohai Bay, China, two previously unknown ciliates were discovered and investigated using standard taxonomic methods. Morphological comparative analyses revealed that they represent two novel species in the genus Chaenea. Chaenea paucistriata spec. nov. can be distinguished from its congeners by the following traits: body length in vivo about 180-250 µm; eight somatic kineties; dorsal brush rows 1-4 consisting of three, five, seven, and two dikinetids, respectively; rod-like extrusomes, 8 µm long; 63-94 macronuclei; cortical granules minute and colourless. Chaenea sinica spec. nov. differs from its congeners in having: body length in vivo about 140-240 µm; 17-21 somatic kineties; dorsal brush rows 1-4 consisting of 3-7, 10 or 11, 11-13, and 3-6 dikinetids, respectively; rod-like extrusomes about 6-8 µm long; 71-164 macronuclei. A key is presented to assist the identification of all Chaenea species.

Headnote

Abstract. During faunistic studies of ciliates in coastal waters of Daya Bay and Bohai Bay, China, two previously unknown ciliates were discovered and investigated using standard taxonomic methods. Morphological comparative analyses revealed that they represent two novel species in the genus Chaenea. Chaenea paucistriata spec. nov. can be distinguished from its congeners by the following traits: body length in vivo about 180-250 µm; eight somatic kineties; dorsal brush rows 1-4 consisting of three, five, seven, and two dikinetids, respectively; rod-like extrusomes, 8 µm long; 63-94 macronuclei; cortical granules minute and colourless. Chaenea sinica spec. nov. differs from its congeners in having: body length in vivo about 140-240 µm; 17-21 somatic kineties; dorsal brush rows 1-4 consisting of 3-7, 10 or 11, 11-13, and 3-6 dikinetids, respectively; rod-like extrusomes about 6-8 µm long; 71-164 macronuclei. A key is presented to assist the identification of all Chaenea species.

Key words: Chaenea, ciliary pattern, identification key, marine ciliates, new species, taxonomy.

INTRODUCTION

The widespread haptorid genus Chaenea Quennerstedt, 1867 has been found in marine sand, freshwater, brackish water and moist soil (Borror 1963; Carey 1992; Dragesco 1960, 1966; Dragesco and Dragesco-Keméis 1986; Fauré-Fremiet and Ganier 1969; Foissner 1984; Fryd-Versavel et al. 1975; Gao et al. 2008; Kahl 1926, 1927, 1928, 1933; Kwon et al. 2014; Lipscomb and Riordan 1990; Song et al. 2009; Wang 1934). Its members are characterized by the following features: cell elongate and contractile; cytostome apically located and surrounded by dikinetid circumoral kinety; somatic kineties which are slightly spiralled when contracted and mainly composed of monokinetids; dorsal brush consisting of four dikinetidal rows; one permanent contractile vacuole located at the posterior end of the body; extrusomes rod-like or thom-like, attached to the oral tbulge and scattered in the cell (Foissner et al. 1995, Petz et al. 1995, Song et al. 2009). Since being established, 14 nominal species have been assigned to this genus, namely, C. crassa Masked, 1887, C. gigas Kahl, 1933, C. limicola Lauterbom, 1901, C. minor Kahl, 1926, C. mirabilis Kwon et al., 2014, C. psammophila Dragesco, 1960, C. robusta Kahl, 1930, C. sapropelica Kahl, 1930, C. simulons Kahl, 1930, C. stricta (Dujardin, 1841) Foissner et al., 1995, C. teres (Dujardin, 1841) Kent, 1881, C. tesselata (Kahl, 1935) Dragesco and Dragesco-Kemeis, 1986, C. torrentícola Foissner, 1984, and C. vorax Quennerstedt, 1867. In 1995, Foissner et al. synonymized C. torrentícola with C. stricta. Consequently, 13 species and an unidentified species from Petz et al. (1995) remain in the genus. Among these species, most have only been reported once, mainly based on living observation. Data on the ciliary pattern, especially detailed information regarding the dorsal brush, is only available for C. teres and C. mirabilis (Kwon et al. 2014, Petz et al. 1995). For C. vorax and C. stricta, although no statistical data is available, the number of dikinetids in the dorsal brush is shown in illustrations (Foissner et al. 1995, Song and Packroff 1997). Since this genus shares a similar pattern of general ciliature and body shape, some morphological characters, such as the number of dikinetids in the dorsal brush as well as the length and shape of extrusomes, have been used to distinguish closely related species (Petz et al. 1995). When describing novel species of this genus, therefore, these characters should be paid close attention to.

During a faunistic survey of ciliates in the coastal waters of Daya Bay and Bohai Bay, China, two species of Chaenea were isolated. Investigation of the morphology of living cells and their ciliature revealed that they represent two new forms.

MATERIALSAND METHODS

Chaenea paucistriata spec. nov. was collected from coastal waters of Daya Bay, China (22°42'N, 114°32'E), on December 12, 2007 when the water temperature was 21°C and the salinity was 30%o. Chaenea sínica spec. nov. was collected from coastal waters of Bohai Bay, China (37°37'N, 121°22'E), on March 27, 2006 when the water temperature was 8°C and the salinity was 34%o. Water samples were taken directly with a plastic jar, and then transported to the laboratory and maintained in Petri dishes. Rice grains were added to increase the amount of food for the bacteria (Chen et al. 2013). Large numbers of both species were present after about a week, when the rice granules had decomposed (Li et al. 2013). Living cells were observed using bright field and differential interference contrast microscopy (100 × to 1,000 × magnifications). Protargol staining was used to reveal the ciliature (Wilbert 1975). Counts and measurements of stained specimens were performed at a magnification of 1,000 ×. Drawings were made with the help of a camera lucida. Terminology mainly follows Kwon et al. (2014).

RESULTS AND DISCUSSION

Chaenea paucistriata spec. nov. (Figs 1, 2; Table 1)

Diagnosis: Extended cell size in vivo usually about 220 × 18 pm. Eight somatic kineties. Dorsal brush rows 1-4 consisting of three, five, seven and two dikinetids, respectively. About 63-94 macronuclei. Cortical granules minute and colourless.

Type locality: Coastal water of Daya Bay (22°42'N, 114°32'E), China.

Type material: A protargol slide containing the holotype specimen marked with an ink circle is deposited in the Laboratory of Protozoology, Ocean University of China (Registiy no. FXP2007122006).

Etymology: The species group name is a composite of the Latin prefix pauci ("few"), and the Latin adjective striatus, -a, -um [m, f, n] ("striated"), which reflects the fact that this species possesses fewer somatic kineties than its congeners.

Gene sequence data: The small subunit rRNA gene sequence of Chaenea paucistriata spec. nov. was deposited in GenBank with accession number FJ876970 (Zhang et al. 2012).

Description: Extended cells measuring about 180250 x 15-25 pm in vivo, with a length: width ratio of about 12-14:1; narrowly flask-shaped; cell very flexible and contractile; when contracted, cell measuring about 100-180 × 20-30 pm, with ratio of length to width about 4-7:1 (Figs IA, D, 2A-C, H, I). Anterior portion of body distinctly narrowed, with an inconspicuous head; posterior part tapering to rounded (Figs IA, D, 2A-C, H, I). Oral bulge, ca. 2 × 6 pm, on top of anterior body end, forming a short snout and usually bent (Figs 1 A, B, D, 2A, D, H, I). 63-94 ellipsoid macronuclei, with size about 2-5 × 1-2 pm, scattered in the whole cell except for the anterior and posterior portion (Figs 1G, 20). Single contractile vacuole located at the posterior end (Figs IA, D, 2A-C, H, I). Extrusomes rod-like, about 8 pm long, usually in batches attached to oral bulge and scattered in cell (Figs IA, B, G, 2E, F, K). Cortex flexible, and furrowed by somatic kineties (Fig. 2G). Cell colour brownish in middle of body due to packed food vacuoles and cytoplasmic granules, while anterior portion and posterior end transparent (Figs 2A-C). Fine cortical granules colourless, with diameter less than 0.5 pm, distributed between somatic kineties (Fig. 2J). Cytoplasmic granules ellipsoid or round, with diameter about 2-5 pm (Figs 2E, F, K). Movement by slowly crawling on bottom of Petri dish. Typically, whole of somatic kineties consist of monokinetids (Figs IE, F, 2L-N, P). Cilia about 7-8 pm long and arranged in longitudinal rows, although these become spiral in form in contracted specimens (Figs IE, F, 2P). Consistently, eight somatic kineties, each of which consists of six or seven narrowly spaced oralized somatic monokinetids and 60-89 ordinarily spaced somatic monokinetids (Figs IE, F, 2L, P). Four dorsal brush rows consistently comprising three, five, seven and two dikinetids respectively (number of specimens = 15) (Figs 1C, F, 2M, N). Cilia of dorsal brush about 3-4 µm long.

Oral bulge inconspicuous after protargol staining (Fig. 2L, M, P). Circumoral kinety inconspicuous and composed of dikinetids which are at the anterior end of each somatic kinety (Figs 1C, E, F, 2L, M, P).

Comparison: Considering the general morphology in terms of body length and the number of macronuclei, five species should be compared with Chaenea paucis- triata spec, nov., namely C. teres, C. vorax, C. simulans, C. stricta and an unidentified Chaenea species from Petz et al. (1995) (Figs 3A-E, G-M; Table 2).

Chaenea teres is similar to the new species in terms of body size, length of the extrusome and the presence of fine cortical granules; it can be distinguished, however, in having more somatic kineties (12-14 vs. 8), and more dikinetids in dorsal brush row 3 (14-17 vs. 7) and 4 (5-7 vs. 2) (Figs 3A-D; Table 2; Petz et al. 1995).

Chaenea vorax differs from C. paucistriata in having a smaller body length (100-180 pm vs. 180-250 pm), more somatic kineties (11 or 12 vs. constantly 8) and shorter extrusomes (5-6 µm vs. 8 µm) (Figs 3L, M; Table 2; Song and Packroff 1997).

Chaenea simulons can be separated from the new species by having a longer body length (250-350 pm vs. 180-250 µm), more somatic kineties (12-14 vs. constantly 8) and a different habitat (brackish water with salinity l%o vs. marine water with salinity about 30%o) (Fig. 3E; Table 2; Kahl 1930).

Chaenea stricta (Dujardin, 1841) Foissner et al., 1995 can be distinguished from the new organism through its smaller body length (90-130 pm vs. 180250 µm), greater number of somatic kineties (11 or 12 vs. constantly 8), and different habitat (freshwater vs. marine water) (Figs 3I-K; Table 2; Foissner et al. 1995).

Although in vivo characteristics of Chaenea sp. sensu Petz et al., 1995 are not available, it differs from the new species in having more somatic kineties (16-20 vs. constantly 8), and longer extrusomes (12-15 µm vs. ca. 8 µm) (Figs 3G, H; Table 2; Petz et al. 1995).

Chaenea sínica spec. nov. (Figs 4, 5; Table 1)

Diagnosis: Extended cell size in vivo usually about 200 × 20 pm. On average 19 somatic kineties. Dorsal brush rows 1-4 consisting of 3-7, 10 or 11, 11-13, and 3-6 dikinetids, respectively. About 71-164 macronuclei.

Type locality: Coastal waters of Bohai Bay (37°37'N, 121°22'E), China.

Type material: A protargol slide containing the holotype specimen marked with an ink circle has been deposited in the Laboratory of Protozoology, Ocean University of China (Registiy no. WYG2006032701).

Etymology: The species-group name sinicus, -a, -um [m, f, n] reflects the fact that this organism was discovered in China.

Description: Extended cell size about 140-240 * 14-25 µm in vivo, with a length:width ratio of about 10-13:1; cell flexible and contractile; anterior body portion slightly narrowed and posterior part rounded (Figs 4A, C, 5A-E). Oral bulge inconspicuous (Fig. 5A-C). 71-164 ellipsoid macronuclei, with size about 2-4 × 1-2 µm, scattered in cytoplasm (Figs 4F, 5N). Single contractile vacuole located at the posterior body end (Figs 4A, C, 5B). Extrusomes rod-like, about 6-8 µm long, attached in batches to oral bulge, and scattered in cell (Figs 4A, C, 5F, G); extruded ones can be observed outside oral bulge, about 12-16 µm long (Figs 4F, 5L). Cortex flexible with distinct furrows present in some contracted specimens (Fig. 5E). Cell colour greyish due to packed food vacuoles, ca. 10 µm in diameter, and cytoplasmic granules, ellipsoid or round, ca. 1-4 µm in diameter (Figs 4A, C, 5C). Movement by slowly crawling on bottom of Petri dish, with anterior body portion continually contracting. 17-21 somatic kineties mainly consisting of monokinetids and extending the entire body length, each of which consists of 6-9 narrowly spaced oralized somatic monokinetids and 89-188 ordinarily spaced somatic monokinetids (Figs 4D, E, 5H-K). Somatic cilia about 5-6 µm long (Fig. 5D). Four dorsal brush rows consisting of 3-7, 10 or 11, 11-13, and 3-6 dikinetids respectively (Figs 4B, 5H-J). Cilia of dorsal brush undetectable in living cells, but observable in protargol stained specimens and about 2.5 µm long (Figs 4B, 5H-J, M). Circumoral kinety inconspicuous, composed of dikinetids which are at anterior end of each somatic kinety (Figs 4D, E, 51). Nematodesmata, which can be observed after protargol staining, arising from circumoral kinety (Figs 4F, 51).

Comparison: Considering the cell size, the number of macronuclei and somatic kineties, Chaenea sínica spec. nov. can be distinguished from most congeners. But, Chaenea sp. sensu Petz et al., (1995) and Chaenea robusta Kahl, 1930 need to be compared with our new species.

Although no information regarding its living cell is available, Chaenea sp. sensu Petz et al. (1995) still differs from the new form in possessing longer extrusomes (12-15 µm vs. 6-8 µm) and more dikinetids, in particular in the dorsal brush rows (22-28, 25-30, 25-32, 24-29 vs. 3-7, 10 or 11, 11-13, 3-6). These two species can therefore be separated (Figs 3G, H; Table 2; Petz et al. 1995).

Chaenea robusta can be distinguished from the new species by having: (1) a longer body (300-400 µm vs. 140-240 µm), (2) longer dorsal brush cilia (8 µm vs. ca. 2.5 µm) and extrusomes (12-15 µm vs. 6-8 µm), and (3) fewer somatic kineties (about 15 vs. 19 on average) (Fig. 3F; Table 2; Kahl 1930).

Key to the identification of fifteen Chaenea species:

1 Posterior body end distinctly pointed.....................................................................................2

Posterior body end rounded....................................................................................................3

2 Body length in vivo 625-833 µm...........................................................................................C. crassa

Body length in vivo 130-150 µm...........................................................................................C. limicola

3 Body length in vivo ca. 1000 µm............................................................................................C. gigas

Body length in vivo smaller than 650 µm...............................................................................4

4 Macronuclear nodules doughnut-shaped or horseshoe-shaped..............................................5

Macronuclear nodules ellipsoid..............................................................................................6

5 Five or six macronuclear nodules...........................................................................................C. minor

11-21 macronuclear nodules..................................................................................................C. mirabilis

6 Two or three macronuclear nodules........................................................................................7

More than 20 macronuclear nodules.......................................................................................8

7 Anterior body part distinctly swollen.....................................................................................C. tesselata

Anterior body part not distinctly swollen...............................................................................C. sapropelica

8 Fewer than 11 ormorethan21 somatic kineties....................................................................9

11-21 somatic kineties............................................................................................................10

9 Eight somatic kineties.............................................................................................................C. paucistriata

34 somatic kineties.................................................................................................................C. psammophila

10 Freshwater or brackish water (196o) habitat............................................................................11

Marine habitat.........................................................................................................................12

11 Body length in vivo 250-350 pm, more than 100 macronuclear nodules..............................C. simulans

Body length in vivo 90-130 pm, 20-30 macronuclear nodules.............................................C. stricta

12 Extrusome 12-15 pm long.....................................................................................................C. robusta

Extrusome 5-9 pm long.........................................................................................................13

13 17-21 somatic kineties...........................................................................................................C. sinica

11-14 somatic kineties............................................................................................................14

14 Extrusome wedge-shaped, 5-6 pm long; cilia of dorsal brush 2 pm long.............................C. vorax

Extrusome rod-shaped, 9 pm long; cilia of dorsal brush 10 pm long....................................C. teres

Acknowledgements. This work was supported by the National Nature Science Foundation of China (project numbers: 31201708, 41376141, 31111120437) and the International Research Group Program (IRG14-22), and by the Deanship of Scientific Research, King Saud University. Thanks are due to Dr Yangang Wang for his help with finishing expenments.

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Received on 13th March, 2014; revised on 11th May, 2014; accepted on 2nd July, 2014