Morphology, taxonomy and molecular phylogeny of three marine peritrich ciliates, including two new species: Zoothamnium apoarbuscula n. sp. and Z. apohentscheli n. sp. (Protozoa, Ciliophora, Peritrichia)

Introduction

The subclass Peritrichia Stein, 1859, is one of the largest groups of ciliates with over 1000 nominal species. It has a global distribution and its members occupy a wide range of marine, freshwater and terrestrial habitats (Entz 1884; Foissner et al. 1992, 2010; Hu et al. 2019; Kahl 1935; Kent 1880–1882; Lynn 2008; Penard 1922; Wilbert and Song 2005; Zhou et al. 2019). Peritrichia are divided into two orders, the Sessilida Kahl, 1933 and the Mobilida Kahl, 1933. Among the sessilids, some are solitary (e.g., Vorticella, Scyphidia); whereas, others are colonial (e.g., Epistylis, Opercularia, Zoothamnium).

Members of the genus Zoothamnium are colonial and have a continuous spasmoneme that runs throughout the entire colony (although sometimes detached from stalk base) causing the stalk to contract in a "zig-zag" fashion (Corliss 1979; de St. Vincent 1824; Lynn 2008). Nearly, 140 nominal species are included in this genus but most are poorly characterized due to their lack of information based on modern criteria, i.e., silverline system, infraciliature, and molecular data (Kahl 1933, 1935; Nenninger 1948; Sommer 1951; Stiller 1971). A combination of morphological similarity and inaccurate or incomplete descriptions renders it difficult to separate and identify species of Zoothamnium (Ji et al. 2006, 2011; Kahl 1933, 1935; Lu et al. 2020; Precht 1935; Sommer 1951; Stiller 1953a, b; Stiller and Stevčić 1967). Furthermore, new species of Zoothamnium are continuously reported suggesting that there is a large, undiscovered diversity (Ji et al. 2015; Lu et al. 2020; Schuster and Bright 2016; Shen et al. 2017; Sun et al. 2015; Wang and Ji 2019).

In the present study, we describe three marine Zoothamnium species collected from a marine fish aquarium in Qingdao, China, based on the observations of both living and silver-stained specimens. After comparison with known congeners, two were identified as new species, i.e., Z. apoarbuscula n. sp. and Z. apohentscheli n. sp. Another species was identified as Zoothamnium alternans Claparède & Lachmann, 1858. In addition, we analyze the molecular phylogeny of these three species based on SSU rDNA sequence data.

Results

Class: Oligohymenophorea de Puytorac et al., 1974.

Subclass: Peritrichia Stein, 1859.

Order: Sessilida Kahl, 1933.

Genus: Zoothamnium Bory de St. Vincent, 1824.

Zoothamnium apoarbuscula n. sp. (Fig. 2; Table 1)

Figure  2.  Morphology of Zoothamnium apoarbuscula n. sp. in vivo (a, b, d, g–p), after protargol staining (c, f, q–v) and after "wet" silver nitrate staining (e, w). a, b, h–k, m, n Different individuals, showing the variation of zooid shape, arrows mark the contractile vacuole. (c) Showing the variation of macronucleus shape. d, g Mature colony, arrows mark the macrozooid. e, w Detail of silverline system and pellicular pores. f Oral ciliature. l Upper portion of a relatively immature colony. o Part of primary stalk, showing the reticulate surface of spasmoneme. p Posterior portion of stalk, arrow marks the end of spasmoneme. q A stained colony, arrows mark macrozooids. r, s Two protargol-stained zooids showing ciliature, arrow in r marks P3, arrowhead in r marks trochal band, arrow in s marks the end of P3, arrowhead in s marks EM2. t Part of oral ciliature, arrow marks EM1, arrowhead marks EM2. u, v Infundibular polykineties, arrow in u marks P3, arrow in v marks P2. EM1, 2 epistomial membrane 1, 2, G germinal kinety, H haplokinety, Po polykinety, P1–3 infundibular polykineties 1–3. Scale bars: 25 μm in (a, b); 400 μm in (d); 300 μm in (g); 20 μm in (h–k, m, n); 15 μm in (r, s)

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Table  1.  Morphometrical characterization of Zoothamnium apoarbuscula n. sp. (upper line), Zoothamnium apohentscheli n. sp. (middle line) and Zoothamnium alternans (lower line)

Character	Species	Max	Min	Mean	SD	CV	n
Body length in vivo	Z. apoarbuscula n. sp.	50	25	37.0	6.75	18.2	10
	Z. apohentscheli n. sp.	65	40	54.5	8.96	16.4	10
	Z. alternans	45	35	40.2	3.19	7.9	10
Body width in vivo	Z. apoarbuscula n. sp.	30	20	24.0	3.94	16.4	10
	Z. apohentscheli n. sp.	40	25	31.0	4.59	14.8	10
	Z. alternans	25	15	18.7	2.75	14.7	10
Diameter of peristome lip in vivo	Z. apoarbuscula n. sp.	35	30	31.5	2.42	7.7	10
	Z. apohentscheli n. sp.	40	35	37.0	2.58	6.9	10
	Z. alternans	40	30	35.0	4.26	12.1	12
Height of colony	Z. apoarbuscula n. sp. Z. apohentscheli n. sp. Z. alternans	900 1700 1200	385 700 570	639.5 1166.4 804.0	336.13 257.55	31.4 32.3	2 5 5
Number of silverlines, peristome to aboral trochal banda	Z. apoarbuscula n. sp.	–	–	–	–	–	–
	Z. apohentscheli n. sp.	79	63	71.8	7.97	11.1	4
	Z. alternans	35	33	34.0	–	–	2
Number of silverlines, aboral trochal band to scopulaa	Z. apoarbuscula n. sp.	–	–	–	–	–	–
	Z. apohentscheli n. sp.	31 25	29 23	30.0 24.0	– –	– –	2 2
	Z. alternans
Measurements in μm Data based on live specimens CV coefficient of variation in %, Max maximum, Min minimum, Mean arithmetic mean, n number of specimens investigated, SD standard deviation aRough data

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Diagnosis Colony inverted dome-like in outline with dichotomously branched accessory branches all of which radiate from apical end of main stalk. With micro- and macrozooids; microzooids inverted bell-shaped, 25–50 × 20–30 μm in vivo; macrozooids globular, located on lower portions of accessory branches. Peristomial lip single layered, strongly everted. Peristomial disc conspicuously elevated. One contractile vacuole apically located on dorsal wall of infundibulum. Macronucleus C-shaped, transversely oriented. Polykinety 3 (P3) consists of three equal-length rows, terminates above polykinety 1 (P1). Marine habitat.

Type locality Marine fish aquarium (300 × 50 × 70 cm) in Laboratory of Protozoology (N36°03′44″; E120°19′52″), Ocean University of China, Qingdao, China. The water temperature was 28 ℃ and the salinity was 30 ‰.

Deposition of slides One protargol slide (registration number: WT-20190819-01-01) containing the holotype specimen and a silver nitrate slide (registration number: WT-20190819-01-03) containing paratype specimens were deposited in the Laboratory of Protozoology, Ocean University of China (OUC), Qingdao, China. One protargol slide (registration number: NHMUK 2020.4.23.1) containing paratype specimens was deposited in the Natural History Museum, London, UK.

Etymology The species-group name apoarbuscula is a composite of the Greek prefix "apo-" (away from) and the species-group name arbuscula reflecting its similarity to the well-known species Zoothamnium arbuscula Ehrenberg, 1838 in terms of its colony shape.

Description Colony with micro- and macrozooids, often densely covered with detritus. Microzooids about 25–50 × 20–30 μm in vivo, inverted bell-shaped (Fig. 2a, b, g–n). Peristomial lip about 30–35 μm in diameter, single layered and strongly everted (Fig. 2a, b, h–n). Peristomial disc conspicuously elevated in fully extended zooids (Fig. 2a, b, h, i). Macrozooids globular, located on lower portions of accessory branches (Fig. 2d, g, q). Pellicular striations transverse, easily recognizable above 1000 × magnification. Oral cilia about 13 μm in length.

Cytoplasm colorless, usually full of yellow or gray food granules. Single contractile vacuole located at the center of peristomial disc, slightly above peristomial lip and at dorsal wall of infundibulum (Fig. 2a, b, j, m). Macronucleus C-shaped, transversely oriented (Fig. 2a–c, r, s). Micronucleus not observed.

Colony up to 900 μm tall, mostly contains fewer than 50 zooids, with accessory branches that radiate from apical end of main stalk. Accessory branches equal length and dichotomously branched: all branches extend upwards collectively forming an inverted dome-like outline (Fig. 2d, g). Spasmoneme sturdy, comprising bundles of fibrils (stalk myonemes) within a transparent sheath and with a reticulate surface, detached from stalk base (Fig. 2o, p).

Oral ciliature of basic type for sessilid peritrichs. Haplokinety and polykinety make approximately one circuit around the peristome before entering the infundibulum where they make a further circuit (Fig. 2f, r, s). Three infundibular polykineties (P1–P3) each composed of three rows of kinetosomes (Fig. 2f). Rows of P1 are nearly equal in length. P2 and P3 are nearly equal in length, about half length of P1 (Fig. 2f, r, s, u, v). P2 ends adstomally at convergence of P1 and P3 (Fig. 2f, r, s, u). Row 1 of P2 converges abstomally with P1 (Fig. 2f, v). Row 3 of P2 is abstomally detached from rows 1 and 2 (Fig. 2f, v). P3 consists of three equal-length rows of kinetosomes, and ends above P1 adstomally (Fig. 2f, r, s, u). Two epistomial membranes (EM 1 and EM2): EM 1 located at the entrance of infundibulum (Fig. 2f, t); EM 2 located in front of distal ends of haplokinety and polykinety (Fig. 2f, s, t). Germinal kinety lies parallel to haplokinety in the upper half of infundibulum (Fig. 2f). Trochal band consists of dikinetids, located about two-thirds down length of zooid (Fig. 2r, s).

Silverline system consists of closely spaced transverse silverlines (Fig. 2e, w).

Zoothamnium apohentscheli n. sp. (Figs. 3, 4; Table 1)

Figure  3.  Zoothamnium apohentscheli n. sp. in vivo (a–f) and after protargol staining (g). a–d Different individuals, showing the variation of zooid shape. e Schematic of a zooid, showing pellicular striations and position of the trochal band. f A mature colony. g Oral ciliature. EM1, 2 epistomial membrane 1, 2, G germinal kinety, H haplokinety, Po polykinety, P1–3 infundibular polykineties 1–3, TB trochal band. Scale bars: 30 μm in (a–e); 500 μm in (f)

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Figure  4.  Photomicrographs of Zoothamnium apohentscheli n. sp. in vivo (a–j), after protargol staining (k–n) and after DAPI staining (o). a A mature colony. b–d, g–i Different zooids, showing the variation of zooid shape, arrows mark the contractile vacuole. (e, f) Parts of stalk, arrows mark the spasmoneme. j Pellicular striations, arrow marks the trochal band. k, l Two protargol-stained zooids, showing ciliature, arrow in k marks polykinety 3, arrowhead in k marks epistomial membrane 1, double arrowhead in k marks the trochal band, arrow in l marks the germinal kinety, arrowhead in l marks the haplokinety. m Part of oral ciliature, arrow marks epistomial membrane 2, arrowhead marks epistomial membrane 1. n Infundibular polykineties. o DAPI-stained zooid, showing the macronucleus. P1–3 infundibular polykineties 1–3. Scale bars: 500 μm in (a); 25 μm in (c, d, g–i); 20 μm in (k, l)

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Diagnosis Colony up to 1700 μm high. Stalk alternately branched. Zooids inverted bell-shaped, usually 40–65 × 25–40 μm in vivo. Peristomial lip single layered and slightly everted. Peristomial disc moderately elevated. Single contractile vacuole at same level as peristomial lip, dorsally located. Macronucleus C-shaped, transversely oriented. P3 consists of three equal-length rows of kinetosomes, and ends above P1 adstomally. Number of transverse silverlines is 63–79 from peristome to trochal band, 29–31 from trochal band to scopula. Marine habitat.

Type locality Marine fish aquarium (300 × 50 × 70 cm) in Laboratory of Protozoology (N36°03′44″; E120°19′52″), Ocean University of China, Qingdao, China. The water temperature was 20 ℃ and the salinity was 30 ‰.

Deposition of slides One protargol slide (registration number: WT-20181224-01-01) containing the holotype specimen was deposited in the Laboratory of Protozoology, OUC. Another protargol slide (registration number: NHMUK 2020.4.23.2) containing paratype specimens was deposited in the Natural History Museum, London, UK.

Etymology The species-group name apohentscheli is a composite of the Greek prefix "apo-" (away from) and the species-group name hentscheli, reflecting the superficial similarity of this species to Z. hentscheli Kahl, 1935.

Description Zooids about 40–65 × 25–40 μm in vivo and inverted bell-shaped (Figs. 3a–e, 4b–d, g–i). Peristomial lip about 35–40 μm in diameter, single layered, moderately thickened and everted (Figs. 3a–e, 4c, d, g–i). Peristomial disc convex, clearly elevated above peristomial lip in fully extended zooids (Figs. 3a–e, 4b–d, g–i). Pellicular striations conspicuously recognizable at 1000 × magnification, numbering 63–79 from peristome to trochal band, 29–31 from trochal band to scopula (Figs. 3e, 4j). Oral cilia about 13 μm in length.

Cytoplasm colorless, usually containing several yellow or colorless food granules. Contractile vacuole situated at the dorsal wall of infundibulum, at same level as peristomial lip (Figs. 3a, b, 4d, g, i). Macronucleus C-shaped, transversely oriented (Figs. 3a–e, 4o). Micronucleus not observed.

Colony up to 1700 μm tall, usually with over 50 zooids. Stalk alternately branched, branches progressively narrowed and shortened from primary stalk to terminal branches (Figs. 3f, 4a). Stalk sheath colorless, with inconspicuous longitudinal striations (Fig. 4e, f).

Oral ciliature of usual type for sessilid peritrichs. Haplokinety and polykinety make approximately 1.5 circuits around peristome and a further turn within infundibulum (Figs. 3g, 4k, l). Three infundibular polykineties (P1–P3) each composed of three rows of kinetosomes (Figs. 3g, 4k, l, n). Three rows of P1 nearly equal length. P2 ends adstomally at convergence of P1 and P3 (Figs. 3g, 4k, l, n). Abstomal ends of rows 1 and 2 in P2 converge with P1 and diverge from row 3 (Figs. 3g, 4k, l). Two epistomial membranes (EM 1 and EM2): EM 1 located at entrance of infundibulum (Figs. 3g, 4k, m); EM 2 close to distal ends of haplokinety and polykinety (Figs. 3g, 4m). Germinal kinety lies parallel to haplokinety in upper half of infundibulum (Figs. 3g, 4l). Trochal band consists of dikinetids, located about two-thirds down length of zooid (Figs. 3e, 4j, k, l).

Zoothamnium alternans Claparède & Lachmann, 1858 (Fig. 5; Table 1)

Figure  5.  Morphology of Zoothamnium alternans in vivo (a–d, h–q), after protargol staining (f, r–v), after "wet" silver nitrate staining (e, w), after DAPI staining (x) and a mature colony of Zoothamnium alternans (g). a, c, d, j–p Showing the variation of zooid shape and macronucleus shape, arrows in a, l and p mark the contractile vacuole. b A relatively larger zooid at the top of primary stalk. e Silverline system. f Oral ciliature. g Mature colony of Z. alterans, arrows mark macrozooids (from Grell, 1968). h A mature colony the top of which is damaged: arrows mark macrozooids. i An immature colony, arrows mark macrozooids, arrowhead marks the end of the spasmoneme, double arrowhead marks the zooids at the top of primary stalk. q A macrozooid. r, s Two protargol-stained zooids showing ciliature, arrow in r marks P3, arrowhead in r marks the germinal kinety, double arrowhead in r marks the the trochal band, arrow in s marks the haplokinety, arrowhead in s marks EM1. t–v Infundibular polykineties, arrow in t marks P1, arrowhead in t marks P2, arrow in u marks P3, asterisk in v marks the adstomal end of P3. w Silverline system and pellicular pores. x DAPI-stained zooid, showing the macronucleus. EM1, 2 epistomial membrane 1, 2, G germinal kinety, H haplokinety, Po polykinety, P1–3 infundibular polykineties 1–3, Ma macronucleus. Scale bars: 20 μm in (a, c, d, k, m, n, q); 30 μm in (b); 600 μm in (g); 500 μm in (h, i); 15 μm in (r, s)

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1858 Zoothamnium alternans—Claparède & Lachmann, Mém. Inst. natn. Génev. 5 (year 1857): 103–104, Pl. Ⅱ, Figs. 1, 2, 3 and 4 (original description).

Figure  1.  Map and sample site. a Red dot indicates the location of Qingdao, China. b Red dot indicates the location of sample site. c The marine water aquarium in Laboratory of Protozoology, where Zoothamnium apoarbuscula n. sp., Zoothamnium apohentscheli n. sp. and Zoothamnium alternans were collected

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1930 Zoothamnium alternans Claparède & Lachmann, 1858—Fauré-Fremiet, Biol. Bull., 58: 28–51, Figs. 1–15 (growth and differentiation).

1933 Zoothamnium alternans Claparède & Lachmann, 1858—Kahl, Tierwelt N.- und Ostsee 23 (Teil Ⅱ. c3): 132, Fig. 23 (2, 2a).

1935 Zoothamnium alternans Claparède & Lachmann, 1858—Kahl, Tierwelt Dtl., 30: 748, Fig. 139 (9, 10).

1938 Zoothamnium alternans Claparède & Lachmann, 1858—Summers, Biol. Bull., 74: 117–129, Fig. 1 (development of colony).

1938 Zoothamnium alternans Claparède & Lachmann, 1858—Summers, Biol. Bull., 74: 130–159 (regulative development).

2001 Zoothamnium chlamydis n. sp.—Hu & Song, Acta Protozool., 40: 215–220, Figs. 1–20 (synonym).

2006 Zoothamnium alternans Claparède & Lachmann, 1858–Ji et al., Acta Protozool., 45: 28–32, Figs. 1a–i, 2a–h (redescription and revision).

2009 Zoothamnium alternans Claparède & Lachmann, 1858–Song, Warren & Hu, Free-living ciliates in the Bohai and Yellow Seas, China., 260–261, Figs. 8.1E–G, 8.2C, D (redescription).

Since the original record of Zoothamnium alternans from the North Sea, Germany, this species has been reported several times, including two based on silver-stained specimens (Claparède and Lachmann 1858; Fauré-Fremiet 1930; Hu and Song 2001; Ji et al. 2006, 2009; Kahl 1933, 1935; Summers 1938a, b). The descriptions of different populations are necessary in order to better understand its intraspecific variation. A detailed redescription and an improved diagnosis are supplied here based on the present population.

Improved diagnosis Colony feather-like in appearance, stalk alternately branched. With micro- and macrozooids; microzooids inverted bell-shaped, about 35–60 × 15–32 μm in vivo; macrozooids about 55–120 × 30–60 μm in vivo, located only on primary stalk. Peristomial lip single layered and distinctly everted. Peristomial disc moderately elevated. Contractile vacuole dorsally located, at the same level as peristomial lip. Macronucleus J-shaped. P3 consists of three approximately equal-length rows, ends adstomally above P1. Number of transverse silverlines is about 27–55 from peristome to trochal band, 19–30 from trochal band to scopula. Marine habitat.

Voucher slides Two protargol slides (registration numbers: LBR-20180413-01, LBR-20180413-02), and one "wet" silver nitrate slide (registration number: LBR-20180413-01-03), with voucher specimens were deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China.

Description based on the present population Colony with micro- and macrozooids. Microzooids usually inverted bell-shaped, 30–45 × 15–30 μm in vivo (Fig. 5a, c, d, j–o). Peristomial lip about 30–40 μm in diameter, single layered and distinctly everted (Fig. 5a, c, d, j–o). Peristomial disc moderately elevated above peristomial lip in fully extended zooids (Fig. 5a, c, d, j–o). Macrozooids about 55–65 × 30–40 μm in vivo (Fig. 5g–i, q), located only on primary stalk. Pellicular striations conspicuous above 1000 × magnification. Oral cilia about 15 μm in length. In some colonies, zooids at the top of primary stalk are larger than microzooids (Fig. 5i).

Cytoplasm colorless, includes several gray or colorless granules. Single contractile vacuole dorsally located at the same level as peristomial lip (Fig. 5a, l, p). Macronucleus J-shaped (Fig. 5a, c, d, r, s, x). Micronucleus not observed.

Colony up to 1200 μm tall. Stalk alternately branched forming a feather-like outline. Lengths of accessory branches increase progressively from two ends towards middle portion of colony (Fig. 5g–i). Spasmoneme sturdy, comprising bundles of fibrils (stalk myonemes) within a transparent sheath, with sparsely distributed mitochondria (Fig. 5h, i).

Oral ciliature of usual type for sessilid peritrichs. Haplokinety and polykinety make approximately 1.5 circuits around peristome before entering infundibulum (Fig. 5f, r, s). All three infundibular polykineties (P1–P3) three-rowed (Fig. 5f, v). Three rows of P1 nearly equal length (Fig. 5f, t). Adstomal end of P2 ends at convergence of P1 and P3 (Fig. 5f, v). Abstomal end of row 1 in P2 converges with P1 (Fig. 5f, t, v). Abstomal end of row 3 in P2 detached from rows 1 and 2 (Fig. 5f, t, v). Three rows of P3 equal length and end adstomally above P1. In abstomal half part of P3, there is a gap between rows 1 and 2 (Fig. 5f, r, u). Two epistomial membranes (EM 1 and EM2): EM 1 located at entrance of infundibulum (Fig. 5f, s); EM 2 located near distal ends of haplokinety and polykinety (Fig. 5f). Germinal kinety lies parallel to haplokinety in upper half of infundibulum (Fig. 5f, r). Trochal band consists of dikinetids, located two-thirds down length of zooid (Fig. 5e, r, s).

Silverline system consists of densely transverse silverlines, numbering 33–35 from peristome to trochal band (n = 2), 23–25 from trochal band to scopula (n = 2) (Fig. 5e, q, w).

Molecular data and phylogenetic trees (Fig. 6)

Figure  6.  Maximum likelihood tree inferred from SSU rDNA sequences, revealing the phylogenetic positions of Zoothamnium apoarbuscula n. sp., Z. apohentscheli n. sp. and Z. alternans. Numbers near nodes denote maximum likelihood (ML) bootstrap value and Bayesian inference (BI) posterior probability, respectively. Species identity of sequences (marked with asterisks) called "Zoothamnium plumula" (KY675162 and DQ662854) and "Zoothamnium alternans" (DQ662855) could be from misidentified materials and need to be confirmed. The scale bar indicates two substitutions per 100 nucleotide positions

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The newly obtained SSU rDNA sequences of the three Zoothamnium species are deposited in the GenBank database with length (bp), GC content and accession numbers as follows: Z. apoarbuscula n. sp.—1686, 43.12%, MT03 1923; Z. apohentscheli n. sp.—1686, 43.18%, MT03 1924; Z. alternans—1684, 41.75%, MT031922.

Phylogenetic trees based on SSU rDNA sequence data using Bayesian inference (BI) and maximum likelihood (ML) analyses had almost identical topologies, therefore only the ML tree is shown here. Zoothamnium apoarbuscula n. sp. and Z. apohentscheli n. sp. group into one of the three clades (clade Ⅰ) of the family Zoothamniidae. Within clade Ⅰ, Zoothamnium apoarbuscula n. sp. groups with Z. pararbuscula, Z. intermedium, Z. arbuscula, Z. plumula, Z. wangi, and Z. apohentscheli n. sp. with low support (53% ML, 0.51BI). This is sister to the other main group within clade Ⅰ which comprises Z. hentscheli and Z. parahentscheli. Zoothamnium alternans groups within clade Ⅲ of the family Zoothamniidae with moderate support (77% ML, 1.00 BI).

Discussion

Zoothamnium apoarbuscula n. sp.

Branching pattern of colony

We found numerous colonies of Zoothamnium apoarbuscula n. sp., most of which had fewer than 30 zooids. The largest colony we observed had about 45 zooids. All colonies had an umbellate shape, dichotomously branched stalk and macrozooids on lower portions of accessory branches. By contrast, its morphologically most similar species, Z. arbuscula and Z. pararbuscula Ji et al. 2005, typically form very large colonies with hundreds of zooids. Therefore, mature colonies of Z. apoarbuscula n. sp. may not have been observed. Based on these observations, colonies of Zoothamnium apoarbuscula n. sp. most likely branch in a dichotomous pattern.

Comparison with Zoothamnium pararbuscula

Zoothamnium apoarbuscula n. sp. is characterized by its umbellate colony shape, differentiated zooids and marine habitat. These features distinguish it from most other congeners except Z. pararbuscula from which it can be separated by: (1) the relatively smaller size of microzooids (25–50 × 20–30 μm vs. 36–62 × 32–38 μm); (2) the uneven diameter of the primary stalk which is conspicuously narrowed in the rear region above the base (vs. even diameter); (3) the reticulated surface of the spasmoneme (vs. smooth surface with granules) in the primary stalk; and (4) polykinety 2 about the same length as polykinety 3 (vs. about twice as long as polykinety 3) (Ji et al. 2005a). The separation of these two species is further supported by the molecular data (Fig. 6).

Zoothamnium apohentscheli n. sp.

Comparison with congeners in similar morphology (Fig. 7a–h; Table 2)

Figure  7.  Morphologically similar congeners (a–h) of Zoothamnium apohentscheli n. sp., illustrations from historical reports (i, j) and morphologically similar congeners (k–r) of Zoothamnium alternans. a Z. kentii (from Grenfell 1884). b Z. hentscheli (from Hentschel 1916). c Z. commune (from Kahl 1933). d Z. commune (from Ji et al. 2006). e Z. sinense (from Ji et al. 2006). f Z. wangi (from Ji et al. 2011, copyrighted). g Z. xuianum (from Sun et al. 2005, copyrighted). h Z. parahentscheli (from Ji et al. 2015). i Z. alternans (from Claparède and Lachmann 1858). j Z. alternans (from Ji et al. 2009). k Z. alternans sensu Greeff (1870). l Z. alternans sensu Kent (1880–1882). m Z. niveum (from Bauer-Nebelsick et al. 1996). n Z. perejaslawzewae (from Perejaslawzewa 1886). o Schematic of Z. perejaslawzewae (from Kahl 1933). p Z. plumula (from Perejaslawzewa 1886). q Z. plumula (from Ji et al. 2011, copyrighted). r Z. ignavum (from Schuster and Bright 2016). Scale bars: 400 μm for colony; 40 μm for zooid

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Table  2.  Morphometric comparison of Zoothamnium apohentscheli n. sp. with morphologically similar congeners (based on specimens in vivo, measurements in μm)

Species	Body length	Body width	Detritus coata	Branching pattern	Colony length	No. of silverlinesb	Habitat	No. ciliary rows in P3	Data source
Z. apohentscheli n. sp.	40–65	25–40	Presentc	Alternate	Up to 1700	29–31	MW	3	Present study
Z. kentii	90d	45e	Present	Regularly dichotomous	Up to 2300f	–	FW	–	Grenfell (1884)
Z. kentii	50–90	30–45	Present	Irregular, usually alternate	Up to 2300	–	FW	–	Foissner et al. (1992)
Z. hentscheli	63–84	35–40	Present	Irregular, usually alternate	Up to 1200	–	FW	–	Hentschel (1916), Khal (1935)
Z. commune	55–75	–	Absent	Irregularly dichotomous to alternate	–	–	MW	–	Khal(1933, 1935)
Z. commune	60–104	48–56	Absent	Alternate	Up to 1000	38–43	MW	3	Ji et al. (2006)
Z. sinense	36–48	30–40	Absent	Alternate	ca. 400	20–35	MW	3	Ji et al. (2006), Song (1991)
Z. wangi	65–90	45–55	Partly present	Alternate	Up to 1000	38–50	MW	2	Ji et al.(2005b, 2011)
Z. xuianum	30–50	25–35	Absent	Alternate	Up to 800	12–17	BW, MW	3	Ji et al. (2009), Sun et al. (2005)
Z. parahentscheli	50–75	30–40	–	Alternate	Up to 2000	18–40	MW	3	Ji et al.(2009, 2015)
BW brackish water, FW freshwater, MW marine water, – data not available aZooids and stalks covered with detritus bFrom trochal band to scopula cIn most specimens, but not all d1/285 inch in Grenfell (1884) eInferred from "the length being nearly twice the breadth" in Grenfell (1884) f1/11 inch in Grenfell (1884)

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Zoothamnium apohentscheli n. sp. is mainly characterized by the alternately branched stalk. Considering its stalk branching pattern and single-layered peristomial lip, six congeners should be compared with Z. apohentscheli n. sp., namely Z. hentscheli, Z. commune Kahl, 1933, Z. sinense Song, 1991, Z. wangi Ji et al., 2005, Z. xuianum Sun et al., 2005, and Z. parahentscheli Sun et al., 2009.

Zoothamnium hentscheli, which is the most morphologically similar species to Z. apohentscheli n. sp., was originally described by Hentschel (1916) as "Zoothamnium sp. a." and was renamed as Z. hentscheli by Kahl (1935). Foissner et al. (1992) synonymized Z. hentscheli with Z. kentii Grenfell, 1884 based on both species having a characteristic coat of detritus. However, the stalk of Z. hentscheli is usually alternately branched which is conspicuously different with the regularly dichotomously branched stalk of Z. kentii (Grenfell 1884; Hentschel 1916; Kahl 1935) (Fig. 7a, b). Furthermore, the mature colony of Z. hentscheli is shorter and has fewer zooids than that of Z. kentii, i.e., up to 1200 μm high with up to 78 zooids in Z. hentscheli vs. up to 2300 μm high with 80–90 zooids in Z. kentii (Grenfell 1884; Hentschel 1916; Kahl 1935). Thus, we consider these to be separate species. This is consistent with Ji et al.(2006, 2009) who did not accept the synonymy of Z. hentscheli and Z. kentii.

Like Z. hentscheli, Z. apohentscheli n. sp. is typically covered with detritus. However, the latter differs from Z. hentscheli by its relatively smaller zooid (40–65 μm vs. 63–84 μm in length) and marine (vs. freshwater) habitat (Hentschel 1916) (Fig. 7b).

Zoothamnium commune also has a marine habitat, but it differs from Z. apohentscheli n. sp. by its larger zooid size (55–104 × 48–56 μm vs. 40–65 × 25–40 μm), the size of its terminal zooids which are usually larger than (vs. the same size as) zooids on lateral branches, and having more pellicular striations (38–43 vs. 29–31) between the trochal band and the scopula (Ji et al. 2006; Kahl 1933, 1935) (Fig. 7c, d).

Zoothamnium sinense can be separated from Z. apohentscheli n. sp. by its smaller colony size (ca. 400 μm vs. up to 1700 μm high), relatively smaller zooid size (36–48 μm vs. 40–65 μm in length) and the abstomal half of the innermost row of P3 being separated from (vs. parallel with and adjacent to) the other two rows (Ji et al. 2006; Song 1991) (Fig. 7e).

Zoothamnium wangi can be easily separated from Z. apohentscheli n. sp. by its larger zooid size (65–90 × 45–55 μm vs. 40–65 × 25–40 μm), more pellicular striations (38–50 vs. 29–31) between trochal band and scopula, and the two-rowed (vs. three-rowed) P3 (Ji et al. 2005b, 2011) (Fig. 7f).

Zoothamnium xuianum can be separated from Z. apohentscheli n. sp. by its smaller colony size (up to 800 μm vs. up to 1700 μm), relatively stiff (vs. flexible) accessory branches, sparsely (vs. densely) distributed zooids on the accessory branches, fewer pellicular striations (12–17 vs. 29–31) between the trochal band and scopula and its brackish water (vs. marine) habitat (Ji et al. 2009; Sun et al. 2005) (Fig. 7g).

Zoothamnium parahentscheli differs from Z. apohentscheli n. sp. by its remarkably stronger primary stalk (20–28 μm vs. 14–19 μm in dia.) and its shorter accessory branches, i.e., mostly 50–200 μm long vs. mostly over 300 μm (Ji et al. 2009, 2015) (Fig. 7h).

Zoothamnium alternans Claparède & Lachmann, 1858

Identification

Apart from the relatively smaller size of its zooids, which could be interpreted as a population-dependent difference, our population closely matches Z. alternans in the alternately branched stalk, the presence of both micro- and macrozooids, location of macrozooids on the main stalk, the J-shaped macronucleus, and the oral ciliature (especially the gap between rows1 and 2 of P3) (Claparède and Lachmann 1858; Ji et al. 2006, 2009; Kahl 1935) (Figs. 5g, 7i). Thus, we identify our population to Z. alternans.

Comparison with congeners in similar morphology (Figs. 5g, 7k–r; Table 3)

Table  3.  Morphometric comparison of different populations of Zoothamnium alternans with other morphologically similar congeners (based on specimens in vivo, measurements in μm)

Species	MIZ length	MIZ width	MAZ length	MAZ width	Colony	No. of silverlinesa	Typical MAZ	Position of MAZ	Mab	Data source
Z. alternans	35–45	15–25	55–65	30–40	up to 1200	33–35	Present	Along primary stalk	J-shaped	Present study
Z. alternans	Up to 58	–	Up to 120	–	–	–	Present	Along primary stalk	–	Claparède and Lachmann (1858), Kahl (1935)
Z. chlamydis	50–60	25–30	70–90	50–60	Up to 600	27–47	Present	Along primary stalk	Band-like and longitudinalc	Hu and Song (2001)
Z. alternans	40–56	26–32	70–90	45–55	Up to 1200	40–55	Present	Along primary stalk	J-shaped	Ji et al.(2006, 2009)
Z. alternans	–	–	–	–	–	–	Present	On primary stalk and accessory branches	–	Greeff (1870)
Z. alternans	45–60	–	120	–	–	–	Absent	–	–	Kent (1880–1882)
Z. niveum	54–66	16–22	20–150	–	About 10, 000	70	Present	Along primary stalk	C-shaped	Bauer-Nebelsick (1996), Ehrenberg (1838)
Z. perejaslawzewae	–	–	–	–	–	–	Absent	–	–	Kahl(1933, 1935), Perejaslawzewa (1886)
Z. plumula	50–75	35–45	90–100	50–60	Up to 3000	50–60	Absent	–	C-shaped	Ji et al. (2011), Khal(1933, 1935), Perejaslawzewa (1886)
Z. ignavum	36–43d	26–32e	35–86	35–86	Up to 1800	–	Present	Seem to cluster together on primary stalkf	S-shaped	Schuster and Bright (2016)
Ma macronucleus, MAZ macrozooids, MIZ microzooids, – data not available aFrom peristome to trochal band bMacronucleus of microzooids cThe shape is not clear in the photomicrographs d39.4 ± 3 μm in Schuster and Bright (2016) eInferred from "oral width is 28.8 ± 3.1 μm" in Schuster and Bright (2016) fInferred from the drawing and description "restricted to the most proximal parts of the branches" in Schuster and Bright (2016)

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Superficially, Z. niveum Ehrenberg, 1838, Z. plumula Kahl, 1933, Z. perejaslawzewae Kahl, 1933, and Z. ignavum Schuster & Bright, 2016 resemble Z. alternans in terms of the feather-shaped colony. However, Z. niveum can be clearly separated from Z. alternans by its considerably larger colony (up to 1 cm high vs. up to 1.2 mm high) (Bauer-Nebelsick et al. 1996) (Fig. 7m). Zoothamnium plumula can be easily separated from Z. alternans by the absence (vs. presence) of macrozooids and the C-shaped (vs. J-shaped) macronucleus (Ji et al. 2011; Kahl 1933, 1935; Perejaslawzewa 1886; Song et al. 2002) (Fig. 7n, o). Zoothamnium perejaslawzewae can be separated from Z. alternans by its coupled (vs. individual, alternately attached) zooids on the accessory branches, the absence (vs. presence) of macrozooids and the uniformly decreasing (vs. increasing and then decreasing) length of the accessory branches with the height of the colony (Kahl 1933, 1935; Perejaslawzewa 1886) (Fig. 7p, q). Zoothamnium ignavum differs from Z. alternans by its clustered (vs. dispersed along primary stalk) macrozooids and S-shaped (vs. J-shaped) macronucleus (Schuster and Bright 2016) (Fig. 7r).

We agree with Ji et al. (2006) who concluded that Z. alternans sensu Greeff (1870) and Z. alternans sensu Kent (1880–1882) are misidentifications. These two forms are also easily separated from Z. alternans, i.e, Z. alternans sensu Greeff (1870) has conspicuously larger zooids on accessory branches which is not the case in Z. alternans (Fig. 7k), and Z. alternans sensu Kent (1880–1882) has several slender, elongated zooids on some of its accessory branches (vs. absent in Z. alternans), and lacks macrozooids (vs. present in Z. alternans) (Fig. 7l).

Wang and Nie (1932) reported Z. alternans found in Xiamen, China. However, the presence of macrozooids, which is a key character of Z. alternans, was not reported in their population. Thus, the Xiamen population needs to be reinvestigated to confirm its identity. Shen and Gu (2016) reported a population which they identified as Z. alternans but which needs to be reinvestigated because of the following characters which do not match with Z. alternans: 1) the maximum width of zooid nearly equal with (vs. consciously narrow than) the width of peristomial lip; and 2) the obliquely oriented C-shaped (vs. J-shaped) macronucleus.

Phylogenetic analyses

In the SSU rDNA tree, species of Zoothamnium grouped into three clades. The genus Zoothamnium is non-monophyletic which is consistent with the previous studies (Li et al. 2008; Zhuang et al. 2018). Both Z. apoarbuscula n. sp. and Z. apohentscheli n. sp. were grouped in clade Ⅰ. We failed to find morphological support for this clade. Zoothamnium apoarbuscula n. sp., Z. pararbuscula and Z. arbuscula do not group together in a single clade although they share similar morphologies. Zoothamnium apohentscheli n. sp. is most closely related with Z. wangi and Z. plumula (?, KT675162) with strong support (99% ML, 1.00 BI). Each of them has an alternately branched stalk. Our population of Z. alternans clusters with Z. alternans (?, DQ662855), Z. pelagicum, Z. plumula (?, DQ662854, deposited in GenBank database erroneously as Z. pluma), Z. ignavum and Z. niveum which collectively formed clade Ⅲ. This grouping is supported by a shared morphological character, i.e., their alternately branched stalk forming a feather-shaped colony, which clearly differentiates them from other species of Zoothamnium.

It is noteworthy that the SSU rDNA sequence of Z. alternans (?, DQ662855) differs from the sequence of our population by six base pairs, and no morphological information or voucher specimens are available for sequence DQ662855. Thus, the species identity of sequence DQ662855 needs to be confirmed. In addition, there is a marked difference between the two SSU rDNA sequences (DQ662854 and KT675162) of Z. plumula (?) and neither morphological information nor voucher specimens are available for these two sequences. Thus, the species identities of these two sequences also need to be confirmed.

Materials and methods

Sample collection and morphological methods

Samples were collected from a marine aquarium (300 × 50 × 70 cm; Fig. 1c) in the Laboratory of Protozoology, Ocean University of China, using glass microscope slides as artificial substrates. Briefly, the slides were fixed onto a frame that was immersed in the tank for about 7–10 days to allow colonization of peritrichs to occur (Small 1973).

Zoothamnium apoarbuscula n. sp. was collected on August 19, 2019 (water temperature 28 ℃, salinity 30‰). Zoothamnium apohentscheli n. sp. was collected on December 24, 2018 (water temperature 20 ℃, salinity 30‰). Zoothamnium alternans was collected on April 13, 2018 (water temperature 25 ℃, salinity 30‰).

Colonies were removed from the slide using acupuncture needles and collected using glass micropipettes. Live specimens were investigated using differential interference contrast microscopy at magnifications of 40 × to 1000 ×. The infraciliature was revealed by protargol staining according to Ji and Wang (2018). The silverline system was demonstrated using the "wet" silver nitrate method (Song and Wilbert 1995). Measurements and counts were performed under 400–1000 × magnifications. Drawings of live specimens were based on living observations, freehand sketches and photomicrographs. Drawings of stained specimens were made with a drawing device. Terminology is according to Foissner et al. (1992) and Warren (1986).

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from cleaned cells using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The small subunit (SSU) rDNA sequence was amplified by PCR according to Lian et al. (2019), using the primers: 18SF (5ʹ-AAC CTG GTT GAT CCT GCC AGT-3ʹ) (Medlin et al. 1988) and 5.8SR (5ʹ-CTG ATA TGC TTA AGT TGA GCG G-3ʹ) (Gao et al. 2012). To minimize the possibility of errors, Q5^® Hot Start High-Fidelity DNA Polymerase (New England BioLabs, USA) was used in PCR amplification. The fragments were sequenced bidirectionally by the Tsingke Biological Technology Company (Qingdao, China).

Phylogenetic analyses

Besides the three new sequences in present work, another 59 sequences of peritrichs used in the present phylogenetic analyses were acquired from GenBank. Four species of Hymenostomatia (Glaucoma chattoni X56533; Ichthyophthirius multifiliis U17354; Tetrahymena corlissi U17356; Tetrahymena pyriformis EF070254) were selected as the outgroup taxa. Sequences were aligned by the GUIDANCE2 algorithm online using the default parameters (Landan and Graur 2008; Sela et al. 2015). The resulting alignment was manually refined using the program BioEdit 7.0 (Hall 1999). The length of the final alignment was 1728 positions. Maximum likelihood (ML) bootstrapping analysis was carried out with 1000 replicates, using RAxML-HPC2 v.8.2.10 on XSEDE (Stamatakis 2014) on CIPRES Science Gateway (http://www.phylo.org), with the GTR + gamma model. Bayesian inference (BI) analysis was carried out using MrBayes v.3.2.6 on XSEDE (Ronquist et al. 2012) with the best fit model GTR + I + G selected by the Akaike Information Criterion in MrModeltest 2.2 (Nylander 2004). Markov chain Monte Carlo simulations were run for 6, 000, 000 generations, and trees were sampled every 100 generations with a burn-in of 6000 trees (10%). Tree topologies were manually formatted with MEGA 7.0 (Kumar et al. 2016). Systematic classification is based on Lynn (2008) and Gao et al. (2016).

Acknowledgements

This work was supported by the Natural Science Foundation of China (project number: 31801984, 31772440, 31970486) and the Fundamental Research Funds for the Central Universities (No. 201762017).

Author contributions

WS conceived and guided the study. TW conducted sampling and performed experiments. BL identified the species. YL analyzed the phylogeny and interpreted the results. TW and BL wrote the manuscript, and AW and ZS reviewed and edited the manuscript. All authors read and approved the final version of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal and human rights statements

We declare that all applicable international, national, and or institutional guidelines for sampling, care, and experimental use of organisms for the study have been followed and all necessary approvals have been obtained.