Vaginicola tincta

Lu, Borong, Li, Lifang, Hu, Xiaozhong, Ji, Daode, Al-Rasheid, Khaled A S & Song, Weibo, 2019, Novel contributions to the peritrich family Vaginicolidae (Protista: Ciliophora), with morphological and phylogenetic analyses of poorly known species of Pyxicola, Cothurnia and Vaginicola, Zoological Journal of the Linnean Society 187 (1), pp. 1-30 : 25-27

COMMENTS ON VAGINICOLA TINCTA

( FIG. 12 ; TABLES 6, 7)

This species is commonly found in freshwater habitats and was originally reported by Ehrenberg (1830) and redescribed in 1838 by himself ( Fig. 12V ) ( Ehrenberg, 1830, 1838). Kahl (1935) made a revision of Vaginicola with a fine description of V. tincta ( Fig. 12W ). Later, Sommer (1951) and Stiller (1971) ( Fig. 12X ) reported two populations of this form. Vucetich & Escalante (1979) described a form that has a much slimmer lorica than V. tincta ( Fig. 12Y ). However, the form they described as V. lagena Kahl, 1935 fits better with V. tincta ( Fig. 12Z 2 ). Foissner et al. (1992) made an overview of V. tincta and provided an elaborate diagnosis. Recently, Shen & Gu (2016) reported that this species has been found many times in China ( Fig. 12Z ). Our population corresponds well with the original description and Foissner’s diagnosis ( Foissner et al., 1992), thus we think it is a population of V. tincta .

This species is characterized by its relatively large body and large, cylindrical lorica. There are three very similar congeners, namely Vaginicola ceratophylli ( Penard, 1922) Kahl, 1935 , Vaginicola plicata Shen, 1980 and Vaginicola festivus Li, 2016 . They all have a nearly cylindrical lorica with a flared aperture, weakly narrowed portion below the aperture and brown colour. Furthermore, all three species have a similar body size and a similar lorica size. Vaginicola ceratophylli differs from V. tincta by the plate on which the lorica rests ( Fig. 12Z 1 ), although it may be that they are conspecific if the plate is an optical illusion caused by an adhering layer ( Penard, 1922; Kahl, 1935; Warren & Paynter, 1991). Vaginicola plicata is distinguished from V. tincta by the wrinkled posterior portion of the lorica and the flared aperture. These characters are present in our population and perhaps also in other populations of V. tincta , but were overlooked by the researchers ( Fig. 12Z 3 ) ( Shen, 1980). So, V. ceratophylli and V. plicata are probably synonymous. Vaginicola festivus possesses a finger-like projection at the border of the peristomial disc that is absent in V. tincta ( Fig. 12Z 4 ) ( Shen & Gu, 2016).

PHYLOGENETIC ANALYSES

Among the phylogenetic trees of Peritrichia, the loricate sessilids tend to form a separate clade from aloricate sessilids, although the family Vaginicolidae is polyphyletic in the BI tree based on the SSU rDNA and the trees based on the ITS1-5.8S-ITS2 region. Although they are divided into two clades in the ML tree based on ITS1- 5.8S rDNA-ITS2 tree, the possibility that they cluster together is not rejected by the topology test (AU test, P = 0.777> 0.05).

Focusing on the loricate species, the relationship between them is basically in accordance with their morphology ( Fig. 13 ). These species can be divided into four groups based on morphology: Thuricola and Vaginicola attach to the substrate directly, although Vaginicola lacks a stalk and Thuricola possesses an internal stalk and a closure apparatus (valve); Cothurnia and Pyxicola attach to the substrate via a stalk outside the lorica, but Cothurnia lacks the operculum; while Pyxicola has an operculum attached to the border of the peristomial lip ( Kahl, 1935; Trueba, 1978, 1980; Clamp, 1991; Warren & Paynter, 1991; Lu et al. 2018).

Species of Cothurnia cluster in a clade with good support in SSU rDNA and concatenated trees ( Figs 8 , 9 ), indicating that this genus is probably monophyletic. Pyxicola pusilla is relatively closely related to Cothurnia in most trees. This is supported by their morphology in that they have a lorica that is attached to a substrate via a stalk ( Fig. 13 ). In contrast, Thuricola lacks a stalk to attach to the substrate, a feature clearly separating it from Pyxicola and Cothurnia . Three thuricolas are grouped in a clade in all trees with low to full support, showing the monophyly of this genus. Although the length of Thuricola branches is short in all trees, their morphologies show consistent and distinct differences. For example, there is a single valve in the lorica of T. obconica , compared with two spineless valves in the lorica of T. folliculata and one bigger spiniferous valve together with a smaller valve in the lorica of T. kellicottiana ( Fig. 13 ).

Vaginicola crystallina ( AF401521 ) clusters with Thuricola in all trees with full support ( Figs 8–11 ). The morphological feature that the lorica is directly attached to the substrate without a stalk supports this cluster ( Fig. 13 ). However, V. tincta is separated from V. crystallina in all trees, exhibiting a 140 nucleotide difference in the SSU rDNA sequence. This indicates that the genus Vaginicola is polyphyletic. In addition, the topology testing results strongly reject the grouping of them (AU test, P = 2e-004 in concatenated tree, P = 2e-004 in SSU rDNA tree, P = 0.012 in ITS1- 5.8S rDNA-ITS2 tree). We cannot find morphological support for the separation, because the morphological data of the sequenced V. crystallina is absent. Vaginicola tincta is placed at a basal position relative to Pyxicola and Cothurnia in the concatenated genes trees and to other genera in Vaginicolidae in the SSU rDNA tree with ML analyses ( Figs 8 , 9 ). This suggests that the stalkless lorica may represent the ancestral state and the stalked Pyxicola and Cothurnia are likely to have evolved from stalkless ancestors.

In conclusion, the loricate sessilids diverged from the aloricate sessilids and probably represent an independent lineage in the sessilids. The genera Cothurnia and Pyxicola are closely related, which is supported by both the molecular data and morphology. The genera Cothurnia and Thuricola are both monophyletic, whereas Vaginicola seems to be polyphyletic. The stalkless vaginicolids may be the ancestral group of Vaginicolidae . Additionally, morphological and molecular data for the loricate species are still seriously insufficient; therefore, the systematic relationships within the loricate group remain uncertain, pending a re-evaluation following the acquisition of additional data.

Figures

Figure 1. Map and sampling sites. A, map of the four seas of China, red asterisks indicate the locations of Qingdao and Ningbo. B, satellite map of southwest Qingdao, red asterisk indicates Tangdao Bay. C, satellite map of north-east Ningbo, red asterisks indicate the two sampling areas (wetland of Hangzhou Bay, Ri Lake). D, coast of Tangdao Bay. D1, Cothurnia ceramicola collected from site D. E, a pool in wetland of Hangzhou Bay. E1, Pyxicola pusilla collected from site E. F, Ri Lake, a freshwater lake in Ningbo. F1, Vaginicola tincta collected from site F.

Figure 2. Pyxicola pusilla in vivo (A–N) and after protargol staining (O, P). A–D, lateral views of different individuals, showing the varieties of body shape, lorica transparency and stalk length. E, a contracted individual inside the lorica. F, longitudinally oriented macronucleus.G–J, varieties of lorica shape. K, lateral view of operculum.L, apical view of operculum. M, details of lorica base, mesostyle and endostyle. N, detail of zooid, showing pellicular striations. O, macronucleus, the trochal band and the scopula. P, oral ciliature. Abbreviations: AR, annular ridge; BD, basal disc; CV, contractile vacuole; En, endostyle; G, germinal kinety; H, haplokinety; LS, longitudinal striae; Ma, macronucleus; Me, mesostyle; Op, operculum; Po, polykinety; P1–3, infundibular polykineties 1–3; Sc, scopula; St, stalk; TB, trochal band; Tu, tube. Scale bars = 30 μm.

Figure 3. Photomicrographs of Pyxicola pusilla in vivo (A–O) and after protargol staining (P–U). A–G, lateral views of different individuals, showing the varieties of body shape, lorica shape and colour, and stalk length; arrows indicate the contractile vacuole. H, I, two constricted individuals. J, apical view of a contracted individual, showing the aperture of lorica and frontal view of operculum. K, L. lateral view of operculum; arrows indicate the depression of operculum border. M, N. posterior portion of individuals; arrows indicate the mesostyle, arrowhead marks the basal disc. O, showing the striations of pellicle. P, Q. two specimens; arrows and arrowheads indicate macronucleus and the trochal band, respectively. R–T, oral ciliature. U, infundibular polykineties 1–3. Abbreviations: G, germinal kinety; H, haplokinety; P1–3, infundibular polykineties 1–3. Scale bars = 30 μm.

Figure 4. Cothurnia ceramicola in vivo (A–J) and after protargol staining (K, L). A, lateral view of an individual with single zooid. B, showing macronucleus in vivo. C, lateral view of an individual with two zooids. D–G, varieties of lorica shape. H, striations of pellicle. I, apical view of zooid, showing the location of contractile vacuole. J, posterior portion of individual, showing lorica base, endostyle, mesostyle, stalk and basal disc. K, showing macronucleus and the trochal band. L, oral ciliature. Abbreviations: AR, annular ridge; BD, basal disc; CV, contractile vacuole; En, endostyle; EM1–2, epistomial membrane 1–2; G, germinal kinety; H, haplokinety; In, infundibular; Ma, macronucleus; Me, mesostyle; Po, polykinety; P1–3, infundibular polykineties 1–3; St, stalk; TB, trochal band; Tu, tube. Scale bars = 50 μm.

Figure 8. Phylogenetic tree inferred from concatenated genes (SSU rDNA, ITS1-5.8S rDNA-ITS2, LSU rDNA), revealing the phylogenetic relationship within the family Vaginicolidae. Numbers near nodes denote maximum likelihood (ML) bootstrap values and Bayesian inference (BI) posterior probability, respectively. Asterisks indicate disagreements between ML and BI. The scale bar indicates five substitutions per 100 nucleotide positions. The out group includes Glaucoma chattoni, Ichthyophthirius multifiliis, Tetrahymena corlissi and Tetrahymena pyriformis.

Figure 9. Phylogenetic tree inferred from SSU rDNA sequences, revealing the phylogenetic relationship within the family Vaginicolidae. The topology of Vaginicolidae with the BI analyses is shown here because it different from the ML analyses. Numbers near nodes denote maximum likelihood (ML) bootstrap values and Bayesian inference (BI) posterior probability, respectively. Asterisks indicate disagreements between ML and BI. The scale bar indicates five substitutions per 100 nucleotide positions. The out group includes Glaucoma chattoni (X56533), Ichthyophthirius multifiliis (U17354), Tetrahymena corlissi (U17356) and Tetrahymena pyriformis (EF070254).

Figure 12. Different isolates of Pyxicola pusilla (A–H) and its similar species (I–K), different isolates under the names of Cothurnia ceramicola (L–R) and its similar species (S–U), different isolates under the names of Vaginicola tincta (V–Z) and its similar species (Z1–Z4). A, Cothurnia pusilla sensu Wrześniowski, 1866. B, Cothurnia furcifer sensu Hutton, 1878. C, Pyxicola affinis sensu Kent, 1882. D, Pachytrocha cothurnoides sensu Kent, 1882. E, Caulicola valvata sensu Stokes, 1895. F, Pyxicola carteri sensu Sommer, 1951. G, Pyxicola eforiana sensu Tucolesco, 1962. H, after Shen & Gu, 2016. I, Pyxicola carteri (from Kent, 1882). J, Pyxicola operculigera (from Kent, 1869). K, Pyxicola psammata (from HadŽi, 1940). L, after Kahl, 1933. M, after Precht, 1935. N, after Felinska, 1965. O, after Küsters, 1974. P, after Song, 1992. Q, after Sun et al., 2009. R, after Shen & Gu, 2016. S, Cothurnia curvula (from Entz, 1884). T, Cothurnia harpactici (from Kahl, 1933). U, Cothurnia fibripes (from Kahl, 1933). V, Vaginicola tincta after Ehrenberg, 1838. W, after Kahl, 1935. X, after Stiller, 1971. Y, after Vucetich & Escalante, 1979. Z, after Shen & Gu, 2016. Z1, Vaginicola ceratophylli (from Penard, 1922). Z2, Vaginicola lagena (from Vucetich & Escalante, 1979). Z3, Vaginicola plicata (from Shen, 1980). Z4, Vaginicola festivus (from Shen & Gu, 2016). Scale bars = 30 μm.

Figure 13. Morphological differences and correlations of the six sequenced vaginicolids in this work.