Pseudourostyla cristatoides, Jung, Jae-Ho, Park, Kyung-Min & Min, Gi-Sik, 2012

Jung, Jae-Ho, Park, Kyung-Min & Min, Gi-Sik, 2012, Morphology, morphogenesis, and molecular phylogeny of a new brackish water ciliate, Pseudourostyla cristatoides n. sp., from Songjiho lagoon on the coast of East Sea, South Korea, Zootaxa 3334, pp. 42-54 : 44-50

Description of Pseudourostyla cristatoides n. sp. ( Tables 1–2 and Figs. 1–18 , 24–42 ).

Size 220–265 × 85–125 μm in vivo ( Figs. 1, 2 , 24–28 ), on average 243.8 × 89 μm in protargol preparations ( Table 1 and Figs. 3–5 ; 32, 33). Body flexible and slightly contractile; cell colour grayish to colourless. Extrusomes trichocyst type, densely arranged all over cell surface ( Figs. 28, 29 , arrows) and observed in some protargolimpregnated specimens ( Fig. 44 , arrowhead); extruded extrusomes are observed from methyl green-pyronin and occasionally from protargol impregnated specimens and the extruded ones are ca. 5–10 μm in length, with oval shape of distal end ( Figs. 30, 31 ). Two contractile vacuoles on left side of cell at 25% and 75% of body length, about 14 μm in diameter when fully extended; contractile vacuoles with anterior and posterior collecting canals about 180–200 μm in length. The two contractile vacuoles observed in either anterior or posterior or both positions ( Figs. 1, 2 ; 24–26, arrows). On average 78 ellipsoidal macronuclear nodules; ca. four micronuclei of oval shape ( Figs. 4 , 32–34 ). Crawling moderately fast on bottom of Petri dish.

*Numbered from left to right, ** Numbered from inside to outside, DK - dorsal kineties, TC - transverse cirri, CV - coefficient of variation in

%, Max - maximum, Min - minimum, n - number of specimens investigated, SD - standard deviation, SE - standard error of arithmetic mean.

Measurements in μm.

* Number from Fig. 110 9 in Kahl (1932), ** Number from Fig. 150h in Berger (2006), *** Number of cirral pairs including frontal and midventral cirri from Kumar et al. (2010), **** Number of cirri including frontal and midventral cirri from Fig. 2 a in Wiackowski (1988), ***** The positions of the two contractile vacuoles are estimated from Fig. 1 b in Paiva and Silva-Neto (2006), ****** According to Berger (2006), all species in the genus Pseudourostyla likely have trichocyst type of extrusomes, FVT anlagen – fronto-ventral-transverse cirral anlagen, n/a – unavailable.

All cirri relatively fine, mostly 10–15 μm long; frontal cirri in a bicorona 13 μm long; midventral, marginal and frontoterminal cirri 10 μm in length; transverse cirri 15 μm in length ( Figs. 1 , 27 ). 20–30 frontal cirri in a bicorona and 17–25 midventral pairs; distinct gap between frontal and midventral cirri near posterior frontoterminal cirrus; one buccal cirrus. Invariably two frontoterminal cirri at distal end of adoral zone. Two pretransverse ventral and 6–12 transverse cirri. 5–7 left, 4–5 right marginal rows, number of cirri in each row decreasing gradually from inside to outside row. 10–13 complete dorsal kineties ( Figs. 5 , 33 , arrows). Number of dorsal bristles gradually decreases from outside to inside row and leftmost row of dorsal bristles have distinctly higher number of bristles, about 51 ( Table 1 ). Dorsal cilia about 3 μm long ( Fig. 29 ).

Adoral zone of membranelles about 33% of body length in fixed specimens, base of largest membranelles about 12 μm long, cilia of membranelles about 15 μm long. Distal end of adoral zone extends far posteriorly ( Figs. 3 , 32 ). Paroral and endoral membranes are rather short, slightly curved and almost parallel ( Fig. 3 ).

Morphogenesis.

Stomatogenesis and development of fronto-ventral-transverse cirral anlagen. The oral primordium of the opisthe close to the left of midventral pairs ( Figs. 6, 7 , arrows) and the proter’s oral primordium is formed on the dorsal wall of the buccal cavity ( Fig. 7 , asterisk). The buccal cirrus begins to dedifferentiate ( Figs. 6, 7 , arrowheads) and the patches of the opisthe’s oral primordium form an ordinary longitudinal primordium ( Fig. 8 ). The posterior cirri of the rear corona dedifferentiate and participate in fronto-ventral-transverse cirral anlagen (FVT anlagen) development ( Fig. 8 ).

In the next stage, the FVT anlagen have grown into long oblique streaks and each FVT anlagen of proter and opisthe form about 33 streaks ( Fig. 8 , asterisks; Fig. 9 ). The leftmost frontal cirrus originates from the anterior end of undulating membranes anlagen (FVT I anlagen; Fig. 9 , asterisks) and both anlagen for undulating membranes begin to differentiate ( Fig. 9 ). The proximal portion of the old adoral membranelles will be gradually replaced by a new one and the opisthe’s adoral membranelles begin to differentiate posteriorly ( Figs. 8, 9 ). The anterior end of opisthe’s adoral membranelles arches to the right. Later, the anterior part of the FVT anlagen begins to fragment and form new cirri. All FVT anlagen develop into two cirri, except posterior anlagen, which form three or four cirri comprising of midventral pairs, pretransverse, transverse, and frontoterminal cirri. The rearmost anlage (streak n) forms two frontoterminal cirri along with one pretransverse, and one transverse cirrus. The other (streak n-1) forms one midventral pair as well as one pretransverse and one transverse cirrus. The posterior cirrus of FVT anlagen II forms the buccal cirrus and migrates posteriorly ( Fig. 11 , asterisks).

In late dividers, the new adoral membranelles will be completely developed in both daughter cells and the anterior parts of the membranelles arch to the right ( Figs. 13 , 15, 17 ). The new frontoterminal cirri migrate anteriad ( Figs. 13 , 15, 17 , arrows). The parental cirri will be resorbed and newly developed ones migrate towards their final positions. The undulating membranes anlagen split into endoral and paroral membranes in both dividers.

Marginal and dorsal anlagen. In the early stage, a few cirri of the anterior and middle part of the innermost left and outermost right marginal row likely begin to dedifferentiate and old cirri are incorporated into the primordium ( Fig. 8 , arrows, arrowheads). Later, each primordium forms 4–7 anlagen and elongates posteriorly. The posterior part of the proter’s left marginal anlagen is distinctly curved to the left.

The dorsal kinety anlagen intrakinetally develop within the parental dorsal kineties. Within each parental row, two anlagen form at the anterior and posterior quarters of the body ( Figs. 10 , arrows, 12, 14, 16, 18). The anlagen are arranged parallel and obliquely, that is, the anterior positions of each parallel dorsal kinety anlage are gradually arranged posteriorly from left to right. Each anlage stretches in both directions and the parental dorsal kineties are incorporated or resorbed.

Division of nuclear apparatus. Nuclear division proceeds in the usual way of urostylid. Briefly, during middle division all macronuclear nodules fuse to form a single and rod-shaped mass ( Fig. 10 ). This mass is likely formed during first division after complete fusion. The single mass of macronuclei is continuously separated during the entire process of morphogenesis. The micronuclei are inflated and located at the left side of the cell ( Figs. 10, 12 ). In late dividers, the micronuclei are finished division ( Fig. 16 ).

Physiological reorganisation ( Figs. 19–23 , 43–47 ). Middle late reorganizers are observed and the main process of reorganisation is similar to that of the dividers. Briefly, the new adoral membranelles arise via dedifferentiation of the old ones. Posterior cirri of the rear corona are incorporated into FVT anlagen. Each marginal row anlage develops from the anterior parts of innermost left and outermost right marginal row ( Figs. 19 , arrow; 20, arrowhead) and extend posteriorly. The differentiation of all FVT anlagen is completely identical to that of the dividers, that is, anlagen I–n develop into undulating membranes and frontal, buccal, midventral, pretransverse, transverse, and frontoterminal cirri ( Figs. 21, 22 ). The frontoterminal cirri migrate anteriorly ( Fig. 22 , arrow). Each dorsal kinety anlage originates on the center of the parental row and extends in both directions ( Figs. 20, 23 ). The posterior cirrus of FVT anlage II becomes the buccal cirrus and migrates posteriorly ( Fig. 21 , asterisk).

Molecular phylogeny ( Table 3 and Fig. 48 ). The 18S rRNA gene sequence of the new species was 1,674 bp in length ( JN887467 ) and the range of pairwise distances using the Kimura (1980) two-parameter model was 0.93–1.00% between P. cristatoides n. sp. and the three Asian populations of P. cristata . Intra-specific distances among three P. cristata populations were ranged between 0.07–0.12% ( Table 3 ). The pairwise distances between Pseudourostyla and H. franzi , recently transferred from Pseudourostyla , were 4.41–4.67%. In the phylogenetic analysis, H. franzi represented a distinct clade from Pseudourostyla . Three Asian populations of P. cristata and Korean population of P. cristatoides were clustered monophyly with a high bootstrap value and posterior probability ( Fig. 48 ).

* A gap, 108 bp in length, in the conserved middle portion of the SSU rRNA gene sequence was identified by multiple sequence alignment.

Figures

FIGURE 48. Phylogenetic tree of 18 S rRNA gene sequences showing the position of Pseudourostyla cristatoides n. sp. based on maximum likelihood (ML) and Bayesian inference (BI). Both bootstrap values for ML and posterior probability values for BI are represented on branches. Nodes with a solid circle show fully supported branches (100 % ML, 100 % BI).

FIGURES 1 – 5. Morphology of Pseudourostyla cristatoides n. sp., from life (1, 2) and after protargol impregnation (3 – 5). 1. Ventral view of a representative individual, arrows indicate contractile vacuoles. 2. Ventral views of the process of forming contractile vacuoles. 3 – 5. Ventral (3) and dorsal views (4, 5) of the holotype specimen, arrow denotes the anteriormost midventral pair. AZM, adoral zone of membranelles; BC, buccal cirrus; DK, dorsal kineties 1; EM, endoral membrane; FC, frontal cirri forming bicorona; FTC, frontoterminal cirri; LMR, outermost left marginal row; Ma, macronucleus; Mi, micronucleus; MP, midventral pair; PM, paroral membrane; RMR, outermost right marginal row; TC, transverse cirri. Scale bars: 100 μm.

FIGURES 6 – 14. Pseudourostyla cristatoides n. sp., dividers after protargol impregnation. 6, 7. Ventral view of early dividers, the buccal cirrus (arrowheads) is dedifferentiated, arrows mark basal body patches left of some postoral midventral cirri, and proter’s oral primordium (asterisk) develops from a “ pocket ” of buccal cavity. 8. Ventral view of late early divider, showing forming adoral membranelles, fronto-ventral-transverse cirral (asterisks) and marginal cirral anlagen (left, arrows; right, arrowheads). The posterior cirri of rear corona participate in the fronto-ventral-transverse cirral anlagen. 9 – 12. Ventral and dorsal views of middle dividers, the leftmost frontal cirrus develops from the anterior end of undulating membranes and migrates posteriorly (asterisks), the dorsal kineties originate by intrakinetal formation (arrows), and the micronuclei are slightly inflated. 13, 14. Ventral and dorsal view of late divider, arrows denote anteriorly migrating frontoterminal cirri. Ma, macronucleus; Mi, micronucleus. Scale bars: 100 μm.

FIGURES 15 – 18. Pseudourostyla cristatoides n. sp., dividers after protargol impregnation. 15 – 18. Ventral and dorsal views of late dividers, the micronuclei are finished division and arrows denote anteriorly migrating frontoterminal cirri. Mi, micronuclei. Scale bars: 100 μm.

FIGURES 24 – 31. Photomicrographs of Pseudourostyla cristatoides n. sp. from life (bright field illumination, 24 – 29) and methyl green-pyronin impregnation (30, 31). 24 – 26. Dorsal views, arrows mark contractile vacuoles. 27. Ventral view to show arrangement of cirri. 28, 29. Dorsal views, arrow denotes narrowly spaced extrusomes, arrowheads in 29 indicate dorsal bristles. 30, 31. Extruded cortical granules (trichocyst type). Scale bars: 100 μm

FIGURES 32 – 42. Photomicrographs of Pseudourostyla cristatoides n. sp. during interphase (32 – 34, holotype specimen) and morphogenesis (35 – 42) after protargol impregnation. 32, 34. Ventral views, with arrow and arrowhead indicating macronucleus and micronucleus, respectively. 33. Dorsal view showing complete dorsal kineties and each arrow marks the leftmost and the rightmost dorsal kineties. 35, 36. Ventral views of individuals in early dividing stage, arrows indicate oral primordium patches, arrowhead denotes dedifferentiated buccal cirrus. 37, 38. Ventral views of middle dividers, arrows and arrowheads indicate left and right marginal anlagen, respectively. 39 – 42. Ventral (39, 40, 42) and dorsal view (41) of late dividers showing migrating buccal cirrus (arrow in 39), frontoterminal cirri (arrows in 40, 42), pretransverse cirri (arrowheads in 40), and dorsal kineties anlagen 1 and 11 (arrows in 41). Scale bars: 100 μm.

FIGURES 43 – 47. Reorganization in Pseudourostyla cristatoides n. sp. after protargol impregnation. 43, 44. Ventral (43) and dorsal (44) view of middle reorganizer showing left marginal row anlagen (arrow in 43), dorsal kineties anlagen (arrow in 44), extrusomes (arrowhead in 44). 45 – 47. Ventral views of late reorganizers displaying migrating buccal cirrus (arrow in 45), migrating frontoterminal cirri (arrow in 46, 47). Scale bars: 100 μm.

FIGURES 19 – 23. Pseudourostyla cristatoides n. sp., reorganizers after protargol impregnation. 19 – 21. Ventral and dorsal views of middle reorganizers, the marginal rows originate by common anlage (left, arrow; right, arrowhead), the dorsal kinety anlagen form intrakinetally, and asterisk marks posteriorly migrating buccal cirrus. 22, 23. Ventral and dorsal view of late reorganizer, showing the anteriorly migrating frontoterminal cirri (arrow). Scale bars: 100 μm.

Tables

TABLE 1. Morphometric data on the protargol-impregnated specimens of Pseudourostyla cristatoides n. sp.

Characteristics	Min	Max	Mean	SD	CV	n
Body, length Body, width Adoral zone of membranelles, length Adoral membranelles, number	210 65 75 84	290 125 115 115	243.8 89.0 99.0 97.4	20.5 13.2 11.5 8.3	8.4 14.8 11.6 8.5	25 24 25 25
Buccal cirri, number Frontal cirri, number Frontoterminal cirri, number	1 20 2	1 30 2	1.0 24.0 2.0	0.0 2.4 0.0	0.0 9.9 0.0	18 25 25
Midventral pairs, number Transverse cirri, number Macronucleus nodules, length Macronucleus nodules, width Macronuclear nodules, number	17 6 6.4 2.4 30	25 12 20 6.4 106	20.9 9.4 9.8 4.1 77.7	2.3 1.3 3.2 1.1 17.3	10.8 13.4 32.3 25.6 22.2	25 25 25 25 18
Micronuclei, length Micronuclei, with Micronuclei, number Posterior end of cell to rear most TC, distance	3.2 3.2 3 17.6	4.8 4.8 5 35.2	3.9 3.5 4.1 27.9	0.9 0.6 0.7 4.4	22.0 17.8 16.7 15.7	7 7 7 25
Dorsal kineties, number DK 1, number of bristles* DK 2, number of bristles*	10 38 34	13 63 58	11.3 51.4 43.5	0.8 6.5 6.1	7.5 12.7 14.0	25 25 25
DK 3, number of bristles* DK 4, number of bristles* DK 5, number of bristles* DK 6, number of bristles*	22 24 23 23	39 38 36 37	30.8 29.5 28.9 29.3	4.2 4.1 3.9 4.0	13.7 13.8 13.4 13.7	25 25 25 25
DK 7, number of bristles* DK 8, number of bristles* DK 9, number of bristles*	22 22 23	36 35 39	28.8 29.0 30.3	3.8 3.6 4.3	13.3 12.4 14.1	25 25 25
DK 10, number of bristles* DK 11, number of bristles* DK 12, number of bristles* DK 13, number of bristles*	23 28 22 36	47 48 43 38	32.2 33.8 34.0 -	5.4 5.3 5.8 -	16.9 15.6 17.0 -	25 21 9 2
Left marginal rows, number Left marginal row 1, number of cirri** Left marginal row 2, number of cirri** Left marginal row 3, number of cirri** Left marginal row 4, number of cirri**	5 32 31 30 22	7 47 46 45 37	6.0 36.9 37.9 36.9 29.9	0.4 3.5 3.8 4.2 3.8	6.8 9.5 10.0 11.4 12.8	25 25 25 25 25
Left marginal row 5, number of cirri** Left marginal row 6, number of cirri** Left marginal row 7, number of cirri**	12 4 7	30 20 9	21.3 11.9 -	4.7 4.1 -	21.9 34.3 -	25 23 2
Right marginal rows, number Right marginal row 1, number of cirri** Right marginal row 2, number of cirri** Right marginal row 3, number of cirri**	4 8 27 35	5 31 41 44	4.2 21.3 33.1 38.3	0.4 6.3 3.6 2.5	10.3 29.7 10.9 6.6	25 25 25 25
Right marginal row 4, number of cirri** Right marginal row 5, number of cirri**	37 39	48 49	41.0 43.4	2.9 3.8	7.0 8.9	25 5

TABLE 3. Pairwise similarity (%) based on the Kimura (1980) two-parameter distance option of the 18 S rRNA gene sequence between Pseudourostyla cristatoides n. sp. and the congeneric species with Hemicycliostyla franzi.

P. cristata (China, DQ019318 )	Pseudourostyla cristatoides n. sp. 99.00	P. cristata (China)	P. cristata (Japan)	P. cristata (Korea) *
P. cristata (Japan, FJ598608 )	99.00	99.88
P. cristata (Korea, GU942569 ) H. franzi ( AM412765 )	99.07 95.59	99.93 95.46	99.93 95.46	95.33