Rubrioxytricha guamensis

Kouser, Farzana, Liao, Lijian & Hu, Xiaozhong, 2024, Morphology and molecular phylogeny of a Chinese population of Rubrioxytricha guamensis Kumar et al., 2018 (Ciliophora: Hypotrichia), Journal of Natural History 58 (21 - 24), pp. 688-701 : 691-696

Morphology of Chinese population of Rubrioxytricha guamensis

Body size highly variable in long-term cultures, 60–125 × 20–35 μm (n = 15) in vivo and 61–186 × 20–63 μm (n = 48) after protargol staining, on average 109 × 32 μm ( Table 1 ). Body flexible and not contractile, generally long elliptical in outline, both anterior and posterior ends rounded and both margins slightly convex; ratio of length to width 2–5:1 and dorsoventrally flattened about 2:1 ( Figure 1A,K,L ). Buccal field broad, about half body width; adoral zone occupying approximately 35% of body length in live cells with cilia of membranelles at apical part about 11 μm long ( Figure 1A,B,K,M,O ). Cortical granules spherical, about 0.5 μm across, yellowish to greenish, arranged in lines or small groups and then in long lines on both ventral and dorsal sides ( Figure 1D,E ). Cytoplasm colourless with numerous irregular granules 0.5 μm across and food vacuoles containing diatoms and bacteria, which render cells yellow-brown at low magnification. Single contractile vacuole located at the left margin of the body and near mid-body, 12 μm in diameter and the contraction circle is 20s ( Figure 1A,J ). Two macronuclear nodules, positioned in the middle portion of body and more or less left of midline, with the anterior one close to the buccal vertex, elongate ellipsoidal, on average 16 × 6 μm after protargol preparations ( Figure 1A,C,I,M ). We did not recognize micronuclei in either live observation or protargol staining. Locomotion is by slowly crawling on the substrate or swimming forward by rotating around the longitudinal axis of the body.

Adoral zone composed of 23–35 membranelles ( Figure 1A,B,F,K,M,O ; Table 1 ). Undulating membranes in Australocirrus pattern, at the midline of the body, distinctly curved, paroral membrane extending slightly anterior to endoral membrane and almost equal in length, the two membranes optically intersecting at middle of paroral, cilia about 5 μm long. Eighteen frontal-ventral-transverse (FVT) cirri: three slightly enlarged frontal cirri with the right one close to the distal end of the adoral zone of membranelle; four frontoventral cirri arranged in a V-shaped pattern; single buccal cirrus near the intersection of undulating membranes; five ventral cirri distributed in two groups: three postoral ventral cirri and two pretransverse ventral cirri; five slightly enlarged transverse cirri, about 20 μm long and protruding distinctly behind the rear end of the body in vivo, arranged in a hooked pattern ( Figure 1B,M,O–Q ). One left and one right marginal cirral row with 16–35 and 17–34 cirri, respectively; rows confluent posteriorly.

Invariably, four DK with bristles about 4 μm long in protargol preparations. Kineties almost bipolar with about 14 dikinetids in DK 1 and 2; 13 dikineties in DK 3 and dorsomarginal kinety 4. Single caudal cirrus located at posterior end of DK 3, about 6 μm long in stained cells ( Figure 1C,N,R ).

18S rRNA gene sequence and phylogenetic analyses ( Fig. 2 )

The 18S rRNA gene sequence of Rubrioxytricha guamensis (excluding both primers) has been deposited in GenBank with accession number PP893155. It has a length of 1657 bp and a G + C content of 45.44%. Phylogenetic trees inferred from the SSU rDNA sequences using BI and ML share similar topologies; therefore, only the ML tree is shown here. All the species of Rubrioxytricha with data available cluster together within a clade that also includes Polystichotrix monilata (KT192639) and Pseudogastrostyla flava (KP266627). Five species of Rubrioxytricha with data available fall within a well-supported assemblage (ML/ BI, 100%/1.00). The new sequence of R. guamensis (PP893155) clusters with that of R. guamensis (KY947508) with high support (ML/BI, 99%/0.86), which is closely related to a cluster including three congeners, R. haematoplasma (MT364898), R. tsinlingensis (KR817675) and R. ferruginea (AF370027). Rubrioxytricha guangzhouensis (KJ645977) occupies a basal position within the genus. Polystichotrix monilata (KT192639) and Pseudogastrostyla flava (KP266627) cluster together with full support (ML/BI, 100%/1.00), which is sister to the Rubrioxytricha clade (ML/BI, 100%/1.00). These two clades then cluster with Ponturostyla enigmatica (KC896649) with full support (ML/BI, 100%/1.00).

Discussion

Identification of the Chinese population of R. guamensis

The genus Rubrioxytricha comprises six morphospecies, namely R. haematoplasma ( Blatterer and Foissner, 1990) Berger, 1999 (type species), R. guamensis Kumar et al., 2018a , R. ferruginea ( Stein, 1859) Berger, 1999 , R. guangzhouensis Luo et al., 2021 , R. indica Naqvi et al., 2006 , and R. tsinlingensis Chen et al., 2017 , which have all been described in detail (for details, see Table 2 ). Among them, R. guamensis can be easily distinguished by the colourless cytoplasm and yellowish cortical granules arranged in short rows and in small groups ( Kumar et al. 2018a). Our population, collected from Qingdao, corresponds well with the Guam population of R. guamensis in terms of ciliature and the colour and arrangement of cortical granules. Nevertheless, the Qingdao population has a relatively larger body length range both in vivo and after protargol staining when compared with the type population. We think that geographical variation could account for the abovementioned deviations in body size between the two populations. Guam is at a lower latitude than Qingdao and has a higher average annual temperature ( Guo et al. 2018; Yeo et al. 2023), and temperature is likely to cause differences in body size between populations of the same species ( Kumar et al. 2018a). The Qingdao population invariably has one caudal cirrus at the end of DK 3, while the Guam population sometimes possesses two caudal cirri. Additionally, the Guam population was collected from fresh water, while the Qingdao population was isolated from a limnetic biotope with a salinity of about 2‰. However, these divergences could be considered intraspecific variation.

Sequence divergence and phylogenetic position of Rubrioxytricha guamensis

There are only two nucleotide differences in the 18S rRNA gene between the Qingdao population and the Guam population, which corresponds to 99.9% similarity and supports the conspecificity of the two populations. Within the genus, phylogenetic grouping and morphological classification of species are consistent. Rubrioxytricha ferruginea and R. tsinlingensis clustered into a branch, which then grouped with Rubrioxytricha haematoplasma at full confidence level. This can perhaps be explained by the colour of the cytoplasm: all three species have a brown to orange cytoplasm ( Song and Wilbert 1989; Blatterer and Foissner 1990; Shin and Kim 1993; Berger 1999; Chen et al. 2017; Luo et al. 2021). The branch of these three species grouped together with the branch consisting of two populations of Rubrioxytricha guamensis , which may be due to their yellowish to greenish cortical granules ( Kumar et al. 2018a). Independent branching of Rubrioxytricha guangzhouensis corresponds to its reddish-brown granules. We hypothesise that this may be related to the fact that Rubrioxytricha guangzhouensis is isolated from marine or brackish water and only Rubrioxytricha guangzhouensis has mitochondria-like granules.

Figures

Figure 1. (A–R) Morphology of Chinese population of Rubrioxytricha guamensis from life (A, D–L) and after protargol staining (B, C, M–R). (A) Ventral view of a representative individual. (B, C) Ventral (B) and dorsal (C) view of the same specimen showing the ciliary pattern and nuclear apparatus, arrowhead shows buccal cirrus. (D) Distribution of cortical granules (arrowheads). (E) Shows flexibility of the body; arrowheads represent the spherical yellow cortical granules at the body margin. (F) Ventral view of anterior cell part. (G) Posterior cell part, arrow shows the slightly enlarged transverse cirri. (H) Various cytoplasmic inclusions, ie crystals, lipid droplets. (I) Mid-portion of a slightly squeezed cell, arrows show two macronuclear nodules.(J) Arrow marks the contractile vacuole. (K, L) Ventral views of different cells, showing body shape. (M, N) Ventral (M) and dorsal (N) view of the same specimen, showing ciliature and nuclear apparatus. (O) Ventral view of the anterior end of body. (P) Ventral view of the mid-portion of body, showing left and right marginal rows and postoral ventral cirri. (Q) Ventral view of the posterior portion of cell. (R) Dorsal view of the posterior end of cell, showing caudal cirrus. AZM, adoral zone of membranelles; CC, caudal cirrus; E, endoral membrane; FC, frontal cirri; FVC, frontoventral cirri; LMR, left marginal row; Ma, macronuclear nodules; P, paroral membrane; PTVC, pretransverse ventral cirri; PVC, postoral ventral cirri; RMR, right marginal row; TC, transverse cirri; 1–4, dorsal kineties 1–4. Scale bars = 30 µm.

Figure 2. Maximum likelihood (ML) phylogenetic tree inferred from the 18S rRNA gene sequences. Numbers near nodes represent the ML bootstrap values and Bayesian posterior probabilities. For both trees, the scale bar corresponds to 0.01 expected substitutions per site. The red box (arrow) indicates the clade of genus Rubrioxytricha.

Tables

Table 1. Morphological characteristics of the Chinese population of Rubrioxytricha guamensis. Data based on protargol-stained specimens. Measurements in µm.

Character	Min	Max	Mean	M	SD	CV	n
Body length	61	186	109.0	105	30.75	28.2	48
Body width	20	63	32.4	31	9.94	30.7	48
Adoral zone of membranelles, length	20	58	36.3	35	7.60	21.0	48
Adoral membranelles, number	23	35	28.7	29	2.8	9.9	48
Buccal cirrus, number	1	1	1	1	0	0	48
Frontal cirri, number	3	3	3	3	0	0	48
Frontoventral cirri, number	4	4	4	4	0	0	48
Postoral ventral cirri, number	3	3	3	3	0	0	48
Pretransverse ventral cirri, number	2	2	2	2	0	0	48
Transverse cirri, number	5	5	5	5	0	0	48
Caudal cirrus, number	1	1	1	1	0	0	48
Left marginal cirri, number	16	35	27.0	28	3.87	14.4	48
Right marginal cirri, number	17	34	25	25	4.17	16.7	48
Dorsal kineties, number	4	4	4	4	0	0	48
Dikinetids in DK1, number	8	20	13.6	14	2.65	19.5	48
Dikinetids in DK2, number	8	24	14.0	15	3.20	22.9	48
Dikinetids in DK3, number	8	21	12.9	13	2.98	23.1	48
Dikinetids in DK4, number	7	20	12.8	13	3.22	25.2	48
Macronuclear nodules, number	2	2	2	2	0	0	48
Macronuclear nodule, length	9	29	16.1	15	4.98	31.0	48
Macronuclear nodule, width	4	9	6.4	6	1.44	22.7	48

Abbreviations:CV,coefficient of variation in %; DK, dorsal kinety; Max,maximum; Mean, arithmetic mean; Min, minimum; n, number of cells measured; SD, standard deviation.