Tracheloraphis, DRAGESCO, 1960

Yan, Ying, Xu, Yuan, Al-Farraj, Saleh A., Al-Rasheid, Khaled A. S. & Song, Weibo, 2016, Morphology and phylogeny of three trachelocercids (Protozoa, Ciliophora, Karyorelictea), with description of two new species and insight into the evolution of the family Trachelocercidae, Zoological Journal of the Linnean Society 177 (2), pp. 306-319 : 311-314

GENUS TRACHELORAPHIS DRAGESCO, 1960

TRACHELORAPHIS SIMILIS RAIKOV & KOVALEVA, 1968

( FIGS 6 , 7 ; TABLE 1)

This species was reported by Raikov & Kovaleva (1968) and no redescriptions have been made since then. Furthermore, in the original report it was insufficiently described and no details about the live morphology, or drawings, are available. Hence, an improved definition and a redescription, based mainly on the Chinese population, are presented here.

Improved diagnosis: Body size in vivo 800–1500 × 20– 40 μm; 13–16 and 18–21 somatic kineties on head and trunk, respectively; two nuclear groups, each of which is composed of about four macronuclei and two micronuclei; glabrous stripe corresponding to area occupied by five or six somatic kineties; cortical granules yellowish and c. 0.5 μm in diameter.

Deposition of voucher material: A voucher slide with protargol-impregnated specimens is deposited in the Laboratory of Protozoology, Ocean University of China (No. YY2012121301).

Redescription based on the Chinese population: Fully extended cells about 1200 × 30 μm in vivo; body flexible and contractile with cross-section elliptical; cell distinctly tripartite, with neck, tail and trunk regions ( Figs 6A , 7A ). Head conspicuously claviform; tail wedgeshaped ( Figs 6A, B , 7A, C ). Endoplasm greyish and opaque due to multiple refractile inclusions ( Figs 6B, C , 7B–D ). Two nuclear groups, each of which contains about four macronuclei, 9–15 μm in diameter, and two micronuclei, c. 4–5 μm in diameter ( Figs 6A, C, H , 7B, G, I, J ). Small yellowish cortical granules, c. 0.5 μm in diameter, distributed between ciliary rows and in glabrous stripe ( Figs 6D , 7E, F ). Locomotion by gliding between sand grains and organic debris.

Cell surface densely ciliated with unciliated zone, glabrous stripe, about as wide as five or six somatic kineties ( Figs 6E, G, H , 7G, H, L ). Entire infraciliature consisting of dikinetids with cilia about 10 μm long ( Figs 6G, H , 7G ). About 15 and 19 somatic kineties on head and trunk, respectively. Anterior and posterior secant system formed on left side of glabrous stripe where some kineties abut to bristle kinety ( Figs 6F, H , 7L ). Oral ciliature consisting of circumoral kinety, which is interrupted by three inserted brosse kineties ( Figs 6E–H , 7H, L ).

Remarks: Based on the original description given by Raikov & Kovaleva (1968), this species has an elongated spindle-shaped body, a pointed tail that forms a slight hook and a glabrous stripe as wide as six kineties. Given these characteristics, this Guangzhou population corresponds well to the original report. Raikov & Kovaleva (1968) describe Tracheloraphis similis as colourless but provide no information on its cortical granules. Based on our study, however, this species has yellowish cortical granules but they are so small (c. 0.5 μm in diameter) that the whole cell looks colourless at low magnification. The other minor differences between these two populations are the body length in vivo (600–800 μm vs. 800–1500 μm in Guangzhou population) and the number of somatic kineties (16 vs. 13–16 on head, 18–21 on trunk in Guangzhou population). These differences are probably populationdependent because these values overlap with each other. Consequently, we identified the Guangzhou isolate as a population of T. similis .

MOLECULAR PHYLOGENY BASED ON SSU RRNA GENE SEQUENCES

The length (bp), GC content and GenBank accession numbers of the two species are as follows: Trachelolophos quadrinucleatus sp. nov. – 1554, 47.04%, KT361660 ; Tracheloraphis similis – 1628, 47.91%, KT361661 .

The resulting topologies generated using ML and BI are generally concordant and thus only a single topology with support values generated from both analyses is presented ( Fig. 8 ). As described in previous studies ( Yan et al., 2013, 2015), the family Trachelocercidae is a monophyletic group (88% ML, 1.00 BI), being a sister clade to the family Kentrophoridae (87% ML, 1.00 BI). Within Trachelocercidae , the genus Apotrachelocerca occupies a basal position. The topology then separates into two clades: Kovalevaia and Trachelolophos form one clade with low support (22% ML, 0.52 BI), while Prototrachelocerca , Trachelocerca and Tracheloraphis form the other clade with high support (98% ML, 1.00 BI).

Tracheloraphis and Trachelocerca are not monophyletic as Tracheloraphis similis branches sister to Prototrachelocerca with full support and three populations of Tracheloraphis huangi fall within the Trachelocerca clade. The SSU rRNA gene sequence of Tracheloraphis sp. ( L31520 ) was reported by Hirt et al. (1995), but without information on the morphology. It was only when Foissner & Dragesco (1996a) described the shape and structure of the oral ciliature that the generic classification based on morphology became clear. We therefore suggest that this sequence should be treated as a generic classification of an unknown environmental sequence within Trachelocercidae .

With or without Tracheloraphis sp. ( L31520 ), however, the hypothesis that Tracheloraphis is monophyletic is rejected by the AU test (P = 3e-033, <0.05 or P = 5e- 004, <0.05), while the hypothesis that Trachelocerca is monophyletic is not rejected (P = 0.209,> 0.05). A fuller picture of the relationship between Trachelocerca and Tracheloraphis will only be possible once more phylogenetic analyses based on multiple genetic and morphogenetic studies become available. Thus, further research needs to be performed focusing on these aspects.

Figures

Figure 6. Tracheloraphis similis from life (A–D) and after protargol staining (E–H). A, typical individual; B, wedgeshaped tail; C, four macronuclei and two micronuclei forming a nuclear group; D, distribution of cortical granules between somatic kineties and in glabrous stripe; E, F, infraciliature of anterior end, indicating circumoral kinety, brosse, bristle kinety, glabrous stripe and anterior secant system forming on left side of it (arrowheads); G, H, general infraciliature, noting two nuclear groups, each of which contains about four macronuclei; arrowheads point to secant system on left side of glabrous stripe. Abbreviations: B, brosse; BK, bristle kinety; CG, cortical granules; CK, circumoral kinety; GS, glabrous stripe; Ma, macronuclei; Mi, micronuclei; NG, nuclear groups; SK, somatic kineties. Scale bars: 400 μm in A, 150 μm in G, H.

Figure 7. Tracheloraphis similis from life (A–F) and after protargol staining (G–L). A, typical individual; B, four macronuclei and two micronuclei forming a nuclear group; C, wedge-shaped tail; D, detailed view of middle region to show cytoplasmic granules; E, F, distribution of cortical granules (arrowheads) between somatic kineties and in glabrous stripe; G, general infraciliature, to show two nuclear groups, each of which contains about four macronuclei; H, L, infraciliature of anterior end, indicating circumoral kinety, brosse, glabrous stripe and bristle kinety; I, J, to show nuclear group including four (I) or six macronuclei (J) and micronucleus (I); K, infraciliature of mid-body portion, noting glabrous stripe bordered by bristle kinety. Abbreviations: B, brosse; BK, bristle kinety; CK, circumoral kinety; GS, glabrous stripe; Ma, macronuclei; Mi, micronucleus; NG, nuclear groups; NU, nucleolus; SK, somatic kineties. Scale bars: 400 μm in A; 150 μm in G.

Figure 8. ML tree inferred from the SSU rRNA gene sequences showing the positions of two newly sequenced species (arrow). Nodal support for branches in the ML and BI trees are marked in order. Nodes marked by filled circles indicate full support in both analyses (100% ML, 1.00 BI). Clades with a different topology between the two analyses are shown by an asterisk. Heterotrichea are selected as outgroups. All branches are drawn to scale. The scale bar corresponds to 2 substitutions per 100 nucleotide positions.