Clevelandella sp. 4

Kotyk, Michael, Bourland, William A., Soviš, Matyáš, Méndez-Sánchez, Daniel, Škaloud, Pavel, Varadínová, Zuzana Kotyková & Čepička, Ivan, 2024, Morphology maưers: congruence of morphology and phylogeny in the integrative taxonomy of Clevelandellidae (Ciliophora: Armophorea) with description of six new species, Zoological Journal of the Linnean Society 202 (1), pp. 1-51 : 30-32

Clevelandella sp. 4

Clevelandella sp. 4 was detected in both dissected STR hosts in which it was highly abundant.

Description based on STR population: Large-sized Clevelandella (in vivo on average 152 v 20 × 63 v 10 µm, range 127–185 µm, N = 9), spade-shaped, and dorsoventrally flattened. Left cell margin conspicuously notched at the base of peristomial projection, the lobe does not overhang the peristomial projection. The peristomial projection constituted approx. one-third to onequarter of cell length.

Morphological analyses of cells

A NMDS (non-parametric multi-dimensional scaling) analysis of 455 morphologically characterized protargol-stained cells showed a significant grouping of cells based on their affiliation to particular species ( Fig. 35 ). Two clusters were clearly separated along the first NMDS axis, based on the presence/absence of the peristomial projection. The first cluster consisted of Anteclevelandella constricta , C. klobasa , P. brevis in ‘brevis’ form and P. brevis in ‘simplex’ form; the second cluster contained remaining species. The second NMDS axis was related to peristomial projection length, macronucleus length, and body width (variable scores -0.054, 0.047, and 0.040, respectively). The linear discriminant analysis (LDA) resulted in the model accuracy of 0.892, meaning that 89.2% of the cells were correctly assigned to the appropriate species based on the morphological traits only. The mean accuracy for all the species was 85%. According to the classification matrix (Supporting Information, Table S49), the lowest correct discrimination levels were recovered in Rhynchoclevelandella sp. 2 (14.3%) and C. kidderi (55.6%).

The mean shrinking during protargol preparations for all Clevelandellidae species combined is 18.6 v 7.4% for length and 16.6 v 10.9% for width. However, close observation on shrinking of individual species (Supporting Information, Table S50) uncovered the striking difference among species, with length shrinkage ranging from 4.2% in R. hastula to 29.2% in the long morph of C. hromadkai and width shrinkage ranging from 1.1% in C. fryntai to 31.9% in the long morph of C. hromadkai . In addition, there is an apparent disparity between shrinkage of the cell length and cell width in some species. The most notable example is C. elongata , whose length decreases in protargol preparations by 22.9%, but its width increases by 12.5%. The GLM model revealed the significant effect of species (F16,840 = 361.26, P <0.001), cell condition (F1,840 = 343.92, P <0.001), and their interaction (F16,840 = 4.5, P <0.001) on the cell length. The effect of species (F16,827 = 143.98, P <0.001), cell condition (F1,827 = 232.94, P <0.001), and their interaction (F16,827 = 4.85, P <0.001) on the cell width were also significant. As shrinkage is calculated directly from the cell measurements (i.e. cell length and width), the GLM model clearly indicates that the rate of shrinkage differs among species considerably. RMA regression did not reveal significant relationship between shrinkage and cell size either in length (R2 = 0.036, P = 0.466) or width (R2 = 0.143, P = 0.133). Nevertheless, a weak positive relationship between shrinkage and cell length (Supporting Information, Fig. S6 ) and a weak negative relationship between shrinkage and cell width (Supporting Information, Fig. S7 ) was found.

Figures

Figure 6. Schematic drawings of Clevelandella philipi sp. nov. from life (A) and after protargol impregnation (B–D). A, ventral view of a typical individual.B, ventral view showing the oral apparatus and nuclear structures. C, ventral infraciliature.D, dorsal infraciliature.AZM, adoral zone of membranelles; CPK, cilia of circumperistomial kineties; CV, contractile vacuole; HL, hyaline subcortical layer; Ma, macronucleus; Mi, micronucleus; POM, paroral membrane; POV, peristomial overture; SK, somatic kineties. Scale bars:20 µm.

Figure 7. Clevelandella philipi sp. nov., in vivo (A, B) and after protargol impregnation (C–I). From STH (A–G) and ASS (H, I). A, ventral view showing thick, hyaline cortical layer (between black arrowheads), cilia of the circumperistomial kineties (black arrow) and adoral membranelles (white arrow). B, ventral view showing posterior extent of somatic ciliation (white arrowheads).Note variability of macronuclear shape (A, B). C, ventral infraciliature with detail (inset) showing right sutural kinetofragments (white arrowheads). D, dorsal view showing infraciliature, ciliated circumperistomial kineties (black arrow), looping kineties of peristomial projection (white arrow), and cytoproct (black arrowhead).E, ventral view showing left-hand spiralling ventral somatic kineties (black asterisk) and right sutural kinetofragments (black arrowhead).F, dorsal view showing straight somatic kineties (white asterisk), disordered basal bodies on left protuberance (white arrow), and paroral membrane (black arrowhead). G, dorsal view showing micronucleus (white arrowhead). H, dorsal view showing straight dorsal somatic kineties (asterisk), proximal margin of the peristomial overture (white arrow), and looping kineties of peristomial projection (black arrowhead). I, detail view showing diplostichomonad paroral membrane composed of shorter right file (black arrowhead) and longer left file (white arrowhead) of basal bodies. Ma, macronucleus. Scale bars:20 µm (A–H) 10 µm (I).

Figure 23. Paraclevelandia brevis, from life (A–C, H, I) and after protargol impregnation (D–G, J–P). Small ‘brevis’ morphotype (A–G), large ‘simplex’ morphotype (H–P); from ASS (A, H, I), SRU (J–M, K, O, P), STH (B, N), PAA (C), PGW (D–G). A, ventral view (optical section) showing the apical sac-like structure (black arrowhead) and adoral membranelles (white arrow). B, ventral view showing the subcortical layer of globules (probable mucocysts, black arrow), contractile vacuole (white arrowhead), adoral membranelles (white arrow), and circumperistomial cilia (black arrowhead). C, dorsal view showing relatively straight course of somatic kineties (asterisk). D, Ventral view (optical section) showing the micronucleus (white arrowhead) and adoral membranelles (white arrow). E, right ventrolateral view showing sutural kinetofragments (black arrowhead).F, right lateral view showing sutural kinetofragments (black arrowhead), ventral (white asterisk) and dorsal (black asterisk) kineties.G, detail view showing long (white arrowhead) and short (black arrowhead) files of the paroral membrane, asterisk indicates direction to anterior end of cell.H, ventral view (optical section) showing adoral membranelles (white arrow) and apical sac-like sructure (black arrowhead).I, ventral view showing left-hand spiralling somatic kineties (asterisk) and apical sac-like structure (black arrowhead). J–M, the same cell in ventral view: J, optical section showing inconspicuous anterior and posterior karyophore (white arrows), the separate apical sac-like structure (black arrowhead), and elliptical excretory antrum (white arrowheads); K, surface view showing sutural kinetofragmments (black arrowhead), left-hand spiralling ventral somatic kineties (asterisk), and peristomial overture (black arrow); L, excretory antrum (asterisk); M, elliptical cytoproct on left protuberance (black arrowhead). N, detail view showing long (white arrowhead) and short (black arrowhead) files of the diplostichomonad paroral membrane. Asterisk indicates anterior end. O, detail of macrunucleus and micronucleus (white arrowhead) and karyophore (black arrowhead). P, kinetofragments in right suture (black arrowheads).Ma, macronucleus. Scale bars:5 µm (A–F), 1.25 µm (G), 10 µm (H–K, O, P), 2 µm (L–N).

Figure 35. Non-metric multidimensional scaling (NMDS) plot based on the morphological analysis of traits measured on 455 scales. The cells are colour-coded and clustered based on their affiliation to 16 investigated strains.