A comparative fine structural and phylogenetic analysis of resting cysts in oligotrich and hypotrich Spirotrichea (Ciliophora)

Abstract

So far, neither morphology nor gene sequences have provided a reliable classification of halteriid and hypotrichid spirotrichs. Thus, we performed a comparative study on the fine structure of the resting cysts in some representative species, viz., the oligotrichs Halteria grandinella and Pelagostrombidium fallax and the oxytrichid hypotrichs Laurentiella strenua, Steinia sphagnicola, and Oxytricha granulifera. Main results include: (i) there are three different, very likely non-homologous cyst surface ornamentations, viz., spines (generated by the ectocyst), thorns (generated by the mesocyst), and lepidosomes (produced in the cytoplasm); (ii) Halteria has a perilemma; (iii) Halteria, Meseres and Pelagostrombidium have fibrous lepidosomes, while those of Oxytricha are tubular; (iv) the cyst wall structure of Pelagostrombidium and Strombidium is distinctly different from that of halteriids and oxytrichids, which are rather similar in this respect; (v) cyst ornamentation does not provide a reliable phylogenetic signal in oxytrichid hypotrichs because ectocyst spines occur in both flexible and rigid genera. The new observations and literature data were used to investigate the phylogeny of the core Spirotrichea. The Hennigian argumentation scheme and computer algorithms showed that the spirotrichs are bound together by the macronuclear reorganization band, the subepiplasmic microtubule basket, and the apokinetal stomatogenesis. The Hypotrichida and Oligotrichida are united by a very strong synapomorphy, viz., the perilemma, not found in any other member of the phylum. Halteriid and oligotrichid spirotrichs form a sister group supported by as many as 13 apomorphies. Thus, the molecular data, which classify the halteriids within the core hypotrichs, need to be reconsidered.

Figs 1–10

Resting cysts (1–7) and encystment (8–10) in some oligotrich (1–5, 8–10) and hypotrich (6, 7) spirotrichs. 1–4: Halteria grandinella in the light (1–3) and electron (4) microscope. 5–7: Schemes of the origin of the spines in Halteria, Laurentiella and Steinia. 8–10: Halteria grandinella, early (8, 9) and middle (10) phases of encystment.

Figs 11–20

Halteria grandinella, encystment (11–15) and resting cysts (16–20) in the light (11–19) and scanning (20) microscope (11–17, Venezuelan specimens; 18–20, Austrian specimens). 11–14: The globular trophic cells (see Fig. 21) become oblong, dislocating the equatorial ciliary rows (Fig. 21) subterminally (arrows). The arrowheads mark the head-like separated peristomial area. 15: When encysting specimens (12) are squashed, many conical lepidosomes become recognizable in the cytoplasm (arrows). 16: A young cyst with a very hyaline mucous coat (arrowheads), becoming recognizable due to adhering bacteria (B). 17: When squashed, the lepidosomes (L) become detached from the ectocyst. 18, 19: Optical sections. The cyst wall (opposed arrowheads) is distinct under bright field illumination (18), while the mucous coat and the minute lepidosomes are recognizable only with interference contrast (19). 20: A cyst with mucous coat opened, showing the lepidosomes. AM – adoral membranelles, B – bacteria, C – ciliate cortex, CV – contractile vacuole, L – lepidosomes, M – mucous coat, MA – macronucleus, PC – pericyst. Scale bars 5 μm (15), 10 μm (20), 20 μm (11–14, 16–19).

Figs 21–28

Halteria grandinella, trophic (21) and cystic (22–28) specimens in the scanning (21, 23, 25–27) and transmission (22, 24, 28) electron microscope (21–27, Austrian specimens; 28, Dominican specimen). 21: Ventrolateral overview with buccal adoral membranelles marked by arrowhead. 22: The cytoplasm contains globular “curious structures”. 23: The mucous envelope consists of fibres. Arrowhead marks the top of a lepidosome. 24: A late stage of cyst wall formation, showing that the mucous coat is made of fibres about 13 nm thick that are attached to, or even form, the ectocyst. 25–27: When the mucous coat is removed, the conical lepidosomes become recognizable. The lepidosomes are attached to the ectocyst by mucous fibres forming an irregular reticulum on the ectocyst; some are fallen down (arrows), showing that the contact is weak. 28: A decaying cyst, showing that the ectocyst is composed of two sheets of polygonal platelets (flocks). AM – adoral membranelles, BR – bristle (ciliary) rows, EC – ectocyst, L – lepidosomes, M – mucous fibres, PE – peristomial field. Scale bars 10 μm (21, 25), 2 μm (23, 26–28), 200 nm (22, 24).

Figs 29–34

Halteria grandinella, transmission electron micrographs of resting cysts. 29, 30: Overview and detail showing one of the many dark granules (30, arrow) contained in the cytoplasm. 31: The darkly stained ectocyst contains many minute, bright areas (arrows). 32–34: Structure of cyst wall and lepidosomes. The cyst wall consists of five layers (33): the mucous pericyst with lepidosomes, ectocyst, mesocyst, endocyst, and the metacyst. Fig. 32 is from an unstained section, showing the high osmium affinity of the lepidosomes, the ectocyst, and the endocyst. Note also the bright vacuoles (asterisks) which show a dark globule after uranyl acetate and lead citrate staining (Fig. 30). The lepidosomes (34) consist of strands of highly osmiophilic material. The strands, which have a width of about 11 nm (34, arrowheads), are not tubular or fibrous because ring-shaped or circular transverse profiles were very rarely observed. C – ciliate cortex, EC – ectocyst, EN – endocyst, L – lepidosomes, ME – metacyst, MS – mesocyst, R – cortical ridges. Scale bars 10 μm (29), 1 μm (32), 500 nm (30, 33), 100 nm (31, 34).

Figs 35–40

Transmission electron micrographs of trophic Halteria grandinella (35, 36) and cystic Oxytricha granulifera (37, 38), L. strenua (39), and S. sphagnicola (40). 35, 36: Both transverse sections of the body (35) and longitudinal sections of the cilia (36) show that Halteria has a perilemma. Opposed arrowheads mark regions where the cell membrane and the two membranes of the very flat alveoli are recognizable. 37, 38: The lepidosomes of O. granulifera are embedded in mucous material and have a diameter of 0.5–3 μm. They lack a surrounding membrane (37) and consist of highly wrinkled tubules with a diameter of about 14 nm (38, arrows). 39, 40: In hypotrichs, the conical processes of the cysts are formed by the mesocyst (39, Laurentiella) or by the ectocyst (40, Steinia), while the oligotrichs Halteria (Figs 29, 32) and Pelagostrombidium (Figs 42–46) produce processes by lepidosomes, that is, structures generated in the cytoplasm and then released by exocytosis. CM – ciliary membrane, EC – ectocyst, EN – endocyst, ME – metacyst, MS – mesocyst, MTU – cortical microtubules, PL – perilemma. Scale bars 4 μm (40), 1 μm (39), 500 nm (37), 200 nm (35, 36, 38).

Figs 41–50

Pelagostrombidium fallax, resting cysts in the light microscope (48) and in the scanning (41, 42) and transmission (43–47, 49, 50) electron microscope. All figures from poorly preserved specimens, as described in the material section. Thus, the brush-like distal end of the lepidosomes could be an artifact. 41, 42, 48: The flask-shaped cyst has 1–5 μm long processes (lepidosomes) and is covered with an only partially preserved, membrane-like material (41). The lepidosomes are attached to the ectocyst by fibrous processes forming a broadly conical base resembling the conditions in Halteria (Figs 25–27). 43: Pelagostrombidium has a several μm thick pericyst made of lepidosomes and a membrane-like coat. 44–47: A thick, compact layer, which usually shows alternating bright and dark zones (44), is underneath the thin ectocyst. Arrows mark the broadened lepidosome base (cp. Fig. 42). 49, 50: At high magnification, the ectocyst often appears to be composed of two dark layers separated by a narrow, bright zone. EC – ectocyst, L – lepidosomes, MH – membranous sheet(s), W – part of cyst wall (mesocyst ?). Scale bars 30 μm (41), 2 μm (42, 48), 1 μm (43), 500 nm (44–47), 100 nm (49, 50).

Figs 51, 52

Phylogenetic relationships of the core Spirotrichea, using a manually constructed Hennigian argumentation scheme (51) and a computer-generated PAUP* cladogram (52). The Hypotrichida and Oligotrichida form a monophylum, based on a single but very “strong” synapomorphy (10–1), viz., the perilemma. See Tables 2 and 3 for characters and character states. Asterisks indicate “convergences”.