Spirostomum subtilis, BOSCARO ET AL., 2014

Chi, Yong, Duan, Lili, Luo, Xiaotian, Cheng, Ting, Warren, Alan, Huang, Jie & Chen, Xiangrui, 2020, A new contribution to the taxonomy and molecular phylogeny of three, well-known freshwater species of the ciliate genus Spirostomum (Protozoa: Ciliophora: Heterotrichea), Zoological Journal of the Linnean Society 189 (1), pp. 158-177 : 164-169

publication ID	https://doi.org/10.1093/zoolinnean/zlz115
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scientific name	Spirostomum subtilis
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SPIROSTOMUM SUBTILIS BOSCARO ET AL., 2014

( FIGS 4A–J View Figure 4 , 5A–J; TABLE View Figure 5 2)

Spirostomum subtilis was first reported by Boscaro et al. (2014) who provided features of the morphology in vivo, the macronucleus and gene sequence data, but without details of the infraciliature based on silver staining. Thus, an improved diagnosis, based on the original description and new data, is supplied here.

All measurements in μ m. Abbreviations: CV, coefficient of variation in %; M, Median; Ma, macronucleus; Max., maximum; Mean, arithmetic mean; Min., minimum; N, number of specimens investigated; SD, standard deviation. Ma nodules were selected randomly in each individual.

* data based on living cells; ** data based on protargol-stained specimens.

Improved diagnosis: Large, freshwater Spirostomum , body vermiform when fully extended, about 700–1100 μ m long in vivo, contracted body lanceolate in outline; peristome length variable, about 30–50% of body length; adoral zone with 145–195 membranelles; 18–28 somatic kineties; cortical granules greyish, arranged in a single row between adjacent ciliary rows; macronucleus moniliform with 10–25 nodules, 1–6 micronucleus; conspicuous contractile vacuole caudally positioned, up to one-third of body length, with a long canal that extends anteriorly.

Voucher specimens: Three protargol slides (registration no. CY2018050201-01, 02, 03) with voucher specimens are deposited in the Laboratory of Protozoology, Ocean University of China (OUC).

D e s c r i p t i o n o f t h e Q i n g d a o p o p u l a t i o n: B o d y flexible and contractile, when extended about 680–1100 × 30–50 μ m in vivo, slender and band-like, length to width ratio about 21–35:1 ( Figs 4A, B View Figure 4 , 5A– C View Figure 5 ); when contracted, lanceolate in shape ( Fig. 5E View Figure 5 ). Peristome about 30–40% of body length ( Figs 4B View Figure 4 , 5A, B, G View Figure 5 ). Adoral zone of membranelles conspicuous, cilia of membranelles 15–20 μ m long ( Fig. 5A, F View Figure 5 ). Somatic cilia about 10 μ m long. Pellicle soft and transparent with spherical, greyish cortical granules (0.5–0.7 μ m in diameter) distributed in rows, almost single line between each adjacent pair of somatic kineties ( Figs 4F View Figure 4 , 5H, I View Figure 5 ). Cytoplasm somewhat opaque at low magnification due to numerous dark-grey inclusions and food vacuoles containing bacteria and organic debris. Macronucleus moniliform, with 10–25 nodules, located in the middle portion of the body ( Figs 4A–C, F View Figure 4 , 5D, J View Figure 5 ). Three or four micronuclei, inconspicuous, invariably closely associated with macronuclear nodules ( Fig. 5J View Figure 5 , arrowheads). Contractile vacuole up to one-third body length, terminally positioned, with a long collecting canal on the dorsal side that extends nearly to the anterior end of the body ( Figs 4A, B View Figure 4 , 5A– G View Figure 5 ). Cells relatively active, gliding slowly on substrate, occasionally swims slowly while rotating about the main body axis.

Adoral zone of membranelles makes about one turn of the body (when observed in spirally contracted individuals), beginning at the apical end and terminating in the mid-body region ( Fig. 4C, D, G View Figure 4 ). About 145–195 adoral membranelles, each composed of one short and two long rows of basal bodies ( Fig. 4E, H View Figure 4 ). Paroral short and lies parallel to the right margin of the proximal portion of the adoral zone ( Fig. 4D, E, H View Figure 4 ). Circumoral kinety comprises a single row of kinetids and lies parallel to the right margin of the distal portion of the adoral zone of membranelles, connects with the anterior end of the paroral and continues a short distance beyond the posterior end of the paroral ( Fig. 4E, I View Figure 4 ).

Nineteen to 28 longitudinal somatic kineties spirally twisted in contracted cells, most commence at the apical end of the adoral zone of membranelles and terminate at the posterior end of the cell, but some originate near the left margin of the adoral zone of membranelles or below the proximal end of the adoral zone; such shortened rows occur on both ventral and dorsal sides ( Fig. 4C, D, J View Figure 4 ; Table 2). All somatic kineties are composed of dikinetids, only one basal body of each dikinetid is ciliated.

SPIROSTOMUM TERES CLAPARÈDE & LACHMANN, 1858

( FIGS 6A–F View Figure 6 , 7A–M; TABLE View Figure 7 2)

Spirostomum teres View in CoL was originally reported by Claparède & Lachmann (1858) and is commonly found in freshwater habitats worldwide. Although S. teres View in CoL has been reported many times in recent decades, there are still doubts about its identification ( Repak & Isquith, 1974; Foissner et al., 1992; Jang et al., 2012; Fernandes & da Silva Neto, 2013; Boscaro et al., 2014). An improved diagnosis is supplied, based on previous reports and new data.

Improved diagnosis: Body contractile and flexible, extended specimens 150–600 × 20–75 μ m in vivo, length:width ratio about 5–16:1, contracted body lanceolate in outline; peristome length variable about 30–50% of body length; adoral zone with about 79–92 membranelles, 10–30 somatic kineties; cortical granules arranged in 2–5 rows between adjacent somatic kineties; single ellipsoidal macronucleus positioned near the middle portion of the body; contractile vacuole positioned at the posterior end, up to 25% of body length with a long canal that extends anteriorly.

Voucher specimens: Three protargol slides (registration no. CY2018043002-01, 02, 03) with voucher specimens are deposited in the Laboratory of Protozoology, Ocean University of China (OUC).

Morphology description of Qingdao population: Body about 225–370 × 30–50 μ m in vivo, ratio of length to width about 5–10:1 ( Figs 6A View Figure 6 , 7A–D View Figure 7 ); shape somewhat variable when extended ( Fig. 6B View Figure 6 ); lanceolate when contracted. Pellicle thin and transparent, with numerous spherical, greyish cortical granules (0.6– 0.8 μ m in diameter) densely distributed in 3–5 rows between adjacent somatic kineties ( Figs 6D View Figure 6 , 7F, G View Figure 7 ). Peristome about 30–50% of body length, adoral zone of membranelles conspicuous ( Fig. 7A, B View Figure 7 ). Cytoplasm usually greyish, typically packed with small granules and food vacuoles ( Fig. 7A, C View Figure 7 ). Single ellipsoidal macronucleus in the middle portion of the body ( Figs 6A, B, E View Figure 6 , 7D, E, H, I View Figure 7 ); micronuclei inconspicuous and invariably closely associated with macronucleus ( Fig. 7K View Figure 7 ). Contractile vacuole up to 25% of body length, located at the posterior end of the body with a long collecting canal ( Figs 6A View Figure 6 , 7D, E View Figure 7 ). Locomotion mainly by gliding over substratum, sometimes swims while rotating about the main body axis.

Adoral zone of membranelles makes one turn of the body (when observed in spirally contracted individuals), beginning at the apical end and terminating in the mid-body region ( Figs 6E, F View Figure 6 , 7I, M View Figure 7 ). About 79–88 adoral membranelles, each consists of three rows of basal bodies, two long and one short ( Figs 6C View Figure 6 , 7J, M View Figure 7 ). Paroral short and lies parallel to the right margin of the proximal portion of the adoral zone ( Figs 6C, F View Figure 6 , 7I, M View Figure 7 ). Circumoral kinety comprises a single row of kinetids and lies parallel to the right margin of the distal portion of the adoral zone of membranelles, connects to the anterior end of the paroral and continues a short distance beyond the posterior end of the paroral ( Figs 6C, E, F View Figure 6 , 7I, M View Figure 7 ).

Sixteen to 18 longitudinal somatic kineties spirally twisted in contracted cells. Most kineties extend complete length of cell, some shortened kineties originate from the left margin of the adoral zone of membranelles or below the oral cavity ( Figs 6E, F View Figure 6 , 7L View Figure 7 ; Table 2). All somatic kineties composed of dikinetids.

MOLECULAR DATA AND PHYLOGENETIC ANALYSES

The three new SSU rRNA gene sequences obtained in this study were deposited in GenBank. Their lengths, G+C contents and accession numbers are as follows: Spirostomum minus , 1597 bp, 48.34%, MK929559 View Materials ; S. subtilis , 1597 bp, 47.46%, MK929558 View Materials ; S. teres , 1597 bp, 48.53%, MK929560 View Materials .

The ML and BI analyses based on the SSU rDNA sequences generated phylogenetic trees with similar topologies. Therefore, only the ML tree is shown here ( Fig. 8 View Figure 8 ). In the class Heterotrichea, there are nine monophyletic families and one paraphyletic family ( Blepharismidae ). All species of Spirostomum group together with strong support (ML 95%, BI 1.00) in a clade that is sister to Anigsteinia . Spirostomum is divided into two subclades: ( S. minus + S. semivirescens + S. ambiguum + S. subtilis ) and ( S. yagiui + S. dharwarensis + S. teres + S. caudatum ).

The Qingdao population of Spirostomum subtilis ( MK929558 View Materials ) clusters with two other populations of S. subtilis ( HG939549 View Materials , HG939550 View Materials ) with maximum support ( Fig. 8 View Figure 8 ). The populations of S. minus form two paraphyletic clades because a sequence under the name of S. semivirescens is nested within clade II of S. minus . Clade I is strongly supported (ML 99%, BI 1.00), whereas clade II is poorly supported (ML 62%, BI 0.80). The Qingdao population of S. minus ( MK929559 View Materials ) is nested in clade II, forming a polytomy with other populations of that species. One sequence of S. teres ( HG939538 View Materials ) groups with S. dharwarensis , and the remaining populations group together with variable support values (ML 39%, BI 0.96). The Qingdao population of S. teres ( MK929560 View Materials ) is closely related to a Korean population ( KU848235 View Materials ).