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Marine Life Science & Technology https://doi.org/10.1007/s42995-020-00046-y

RESEARCH PAPER

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)

Tong Wu1 · Yuqing Li1 · Borong Lu1 · Zhuo Shen2,3 · Weibo Song1 · Alan Warren4

Received: 14 February 2020 / Accepted: 9 April 2020 © Ocean University of China 2020

AbstractZoothamnium is a speciose genus, most species of which have incomplete morphological data based on modern criteria. In the present study, the morphology of three species of Zoothamnium, i.e., Z. apoarbuscula n. sp., Z. apohentscheli n. sp., and Z. alternans, collected in Qingdao, China, was revealed using living observation and silver staining. In addition, the SSU rDNA of each species was sequenced for phylogenetic analyses. Zoothamnium apoarbuscula n. sp. is characterized by its umbellate colony which is up to 900 μm high, dichotomously branched stalk, differentiation of zooids, and infundibular polykinety 3 comprising three equal-length ciliary rows. Zoothamnium apohentscheli n. sp is characterized by its large colony up to 1700 μm high, alternately branched stalk, and infundibular polykinety 3 comprising three equal-length ciliary rows. A population of Z. alternans is described in detail. Phylogenetic analyses based on SSU rDNA sequence data revealed that species with an alternately branched stalk cluster together in gene trees and probably represent an independent lineage within the genus Zoothamnium.

Keywords Classification · Novel species · Peritrichia · Sessilida · SSU rDNA

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., Vorti-cella, Scyphidia); whereas, others are colonial (e.g., Episty-lis, 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 char-acterized due to their lack of information based on modern criteria, i.e., silverline system, infraciliature, and molecular

Edited by Jiamei Li.

* Borong Lu boronglu@126.com

* Zhuo Shen shenzhuo@mail.sysu.edu.cn

1 Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China

2 Institute of Microbial Ecology and Matter Cycle, School of Marine Sciences, Sun Yat-sen University, Zhuhai 519082, China

3 Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai 519000, China

4 Department of Life Sciences, Natural History Museum, Cromwell Rd, London SW7 5BD, UK

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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 diver-sity (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 Zootham-nium species collected from a marine fish aquarium in Qingdao, China, based on the observations of both liv-ing 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 molecu-lar 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)

Diagnosis Colony inverted dome-like in outline with dichot-omously branched accessory branches all of which radiate from apical end of main stalk. With micro- and macrozoo-ids; 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 con-tractile vacuole apically located on dorsal wall of infundibu-lum. Macronucleus C-shaped, transversely oriented. Polyki-nety 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 tem-perature was 28 °C 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

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

Measurements in μmData based on live specimensCV coefficient of variation in   %, Max maximum, Min minimum, Mean arithmetic mean, n number of specimens investigated, SD standard deviationa Rough data

Character Species Max Min Mean SD CV n

Body length in vivo Z. apoarbuscula n. sp. 50 25 37.0 6.75 18.2 10Z. apohentscheli n. sp. 65 40 54.5 8.96 16.4 10Z. 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 10Z. apohentscheli n. sp. 40 25 31.0 4.59 14.8 10Z. 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 10Z. apohentscheli n. sp. 40 35 37.0 2.58 6.9 10Z. alternans 40 30 35.0 4.26 12.1 12

Height of colony Z. apoarbuscula n. sp.Z. apohentscheli n. sp.Z. alternans

90017001200

385700570

639.51166.4804.0

336.13257.55

31.432.3

255

Number of silverlines, peristome to aboral trochal banda Z. apoarbuscula n. sp. – – – – – –Z. apohentscheli n. sp. 79 63 71.8 7.97 11.1 4Z. alternans 35 33 34.0 – – 2

Number of silverlines, aboral trochal band to scopulaa Z. apoarbuscula n. sp. – – – – – –Z. apohentscheli n. sp. 31

252923

30.024.0

––

––

22Z. alternans

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deposited in the Laboratory of Protozoology, Ocean Univer-sity 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 lay-ered 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 dor-sal wall of infundibulum (Fig. 2a, b, j, m). Macronucleus C-shaped, transversely oriented (Fig. 2a–c, r, s). Micronu-cleus 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 dichoto-mously branched: all branches extend upwards collectively forming an inverted dome-like outline (Fig. 2d, g). Spasmo-neme 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. Hap-lokinety and polykinety make approximately one circuit around the peristome before entering the infundibulum where they make a further circuit (Fig. 2f, r, s). Three infun-dibular 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 abs-tomally 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 ads-tomally (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 haploki-nety and polykinety (Fig. 2f, s, t). Germinal kinety lies paral-lel 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)

Diagnosis Colony up to 1700  μm high. Stalk alter-nately 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 ori-ented. P3 consists of three equal-length rows of kinetosomes, and ends above P1 adstomally. Number of transverse silver-lines 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 tem-perature was 20 °C 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 Protozool-ogy, 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, num-bering 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. Hap-lokinety and polykinety make approximately 1.5 circuits around peristome and a further turn within infundibulum

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(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 infun-dibulum (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)

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

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 (devel-opment 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 sev-eral 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; Sum-mers 1938a, b). The descriptions of different populations are necessary in order to better understand its intraspecific varia-tion. 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 lay-ered 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 num-bers: 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

Fig. 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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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 bun-dles of fibrils (stalk myonemes) within a transparent sheath, with sparsely distributed mitochondria (Fig. 5h, i).

Fig. 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, show-ing the variation of zooid shape, arrows mark the contractile vacu-ole. (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 sur-face 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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Oral ciliature of usual type for sessilid peritrichs. Hap-lokinety 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 infundibu-lum (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 silver-lines, 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)

The newly obtained SSU rDNA sequences of the three Zoothamnium species are deposited in the GenBank data-base with length (bp), GC content and accession numbers as follows: Z. apoarbuscula n. sp.—1686, 43.12%, MT031923; Z. apohentscheli n. sp.—1686, 43.18%, MT031924; Z. alter-nans—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 apoarbus-cula 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 mac-rozooids on lower portions of accessory branches. By con-trast, 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 colo-nies of Z. apoarbuscula n. sp. may not have been observed. Based on these observations, colonies of Zoothamnium

Fig. 3 Zoothamnium apohentscheli n. sp. in vivo (a–f) and after pro-targol 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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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 habi-tat. 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).

Fig. 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 varia-tion 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, show-ing 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 mem-brane 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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Zoothamnium apohentscheli n. sp.

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

Zoothamnium apohentscheli n. sp. is mainly character-ized 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 morphologi-cally 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 alter-nately 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

Fig. 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 vacu-ole. b A relatively larger zooid at the top of primary stalk. e Silver-line 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 spasmo-neme, 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, dou-ble arrowhead in r marks the the trochal band, arrow in s marks the haplokinety, arrowhead in s marks EM1. t–v Infundibular polykine-ties, 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 macronu-cleus. EM1, 2 epistomial membrane 1, 2, G germinal kinety, H hap-lokinety, 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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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. apo-hentscheli 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 inner-most 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. apo-hentscheli 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. apo-hentscheli 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 brack-ish water (vs. marine) habitat (Ji et al. 2009; Sun et al. 2005) (Fig. 7g).

Fig. 6 Maximum likelihood tree inferred from SSU rDNA sequences, revealing the phylogenetic positions of Zoothamnium apoarbus-cula n. sp., Z. apohentscheli n. sp. and Z. alternans. Numbers near nodes denote maximum likelihood (ML) bootstrap value and Bayes-ian inference (BI) posterior probability, respectively. Species iden-

tity of sequences (marked with asterisks) called “Zoothamnium plu-mula” (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 nucleo-tide positions

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Zoothamnium parahentscheli differs from Z. apo-hentscheli 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 alter-nately 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)

Superficially, Z. niveum Ehrenberg, 1838, Z. plumula Kahl, 1933, Z. perejaslawzewae Kahl, 1933, and Z. igna-vum 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 consid-erably larger colony (up to 1 cm high vs. up to 1.2 mm

Fig. 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 Clapa-

rède 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. pere-jaslawzewae (from Perejaslawzewa 1886). o Schematic of Z. pere-jaslawzewae (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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high) (Bauer-Nebelsick et al. 1996) (Fig. 7m). Zootham-nium 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 sep-arated from Z. alternans by its coupled (vs. individual, alternately attached) zooids on the accessory branches, the absence (vs. presence) of macrozooids and the uni-formly 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

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

BW brackish water, FW freshwater, MW marine water, – data not availablea Zooids and stalks covered with detritusb From trochal band to scopulac In most specimens, but not alld 1/285 inch in Grenfell (1884)e Inferred from “the length being nearly twice the breadth” in Grenfell (1884)f 1/11 inch in Grenfell (1884)

Species Body length Body width Detritus coata Branching pattern

Colony length

No. of silverlinesb

Habitat No. ciliary rows in P3

Data source

Z. apo-hentscheli n. sp.

40–65 25–40 Presentc Alternate Up to 1700 29–31 MW 3 Present study

Z. kentii 90d 45e Present Regularly dichoto-mous

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 dichoto-mous 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 pre-

sentAlternate 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. para-hentscheli

50–75 30–40 – Alternate Up to 2000 18–40 MW 3 Ji et al. (2009, 2015)

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Tabl

e 3

Mor

phom

etric

com

paris

on o

f diff

eren

t pop

ulat

ions

of Z

ooth

amni

um a

ltern

ans w

ith o

ther

mor

phol

ogic

ally

sim

ilar c

onge

ners

(bas

ed o

n sp

ecim

ens i

n vi

vo, m

easu

rem

ents

in μ

m)

Ma

mac

ronu

cleu

s, M

AZ m

acro

zooi

ds, M

IZ m

icro

zooi

ds, –

dat

a no

t ava

ilabl

ea Fr

om p

erist

ome

to tr

ocha

l ban

db M

acro

nucl

eus o

f mic

rozo

oids

c The

shap

e is

not

cle

ar in

the

phot

omic

rogr

aphs

d 39.4

± 3 

μm in

Sch

uste

r and

Brig

ht (2

016)

e Infe

rred

from

“or

al w

idth

is 2

8.8 ±

3.1 

μm”

in S

chus

ter a

nd B

right

(201

6)f In

ferr

ed fr

om th

e dr

awin

g an

d de

scrip

tion

“res

trict

ed to

the

mos

t pro

xim

al p

arts

of t

he b

ranc

hes”

in S

chus

ter a

nd B

right

(201

6)

Spec

ies

MIZ

leng

thM

IZ w

idth

MA

Z le

ngth

MA

Z w

idth

Col

ony

No.

of s

ilver

lines

aTy

pica

l MA

ZPo

sitio

n of

MA

ZM

abD

ata

sour

ce

Z. a

ltern

ans

35–4

515

–25

55–6

530

–40

up to

120

033

–35

Pres

ent

Alo

ng p

rimar

y st

alk

J-sh

aped

Pres

ent s

tudy

Z. a

ltern

ans

Up

to 5

8–

Up

to 1

20–

––

Pres

ent

Alo

ng p

rimar

y st

alk

–C

lapa

rède

and

Lac

h-m

ann

(185

8), K

ahl

(193

5)Z.

chl

amyd

is50

–60

25–3

070

–90

50–6

0U

p to

600

27–4

7Pr

esen

tA

long

prim

ary

stal

kB

and-

like

and

long

itudi

nalc

Hu

and

Song

(200

1)

Z. a

ltern

ans

40–5

626

–32

70–9

045

–55

Up

to 1

200

40–5

5Pr

esen

tA

long

prim

ary

stal

kJ-

shap

edJi

et a

l. (2

006,

200

9)Z.

alte

rnan

s–

––

––

–Pr

esen

tO

n pr

imar

y st

alk

and

acce

ssor

y br

anch

es–

Gre

eff (1

870)

Z. a

ltern

ans

45–6

0–

120

––

–A

bsen

t–

–K

ent (

1880

–188

2)Z.

niv

eum

54–6

616

–22

20–1

50–

Abo

ut 1

0,00

070

Pres

ent

Alo

ng p

rimar

y st

alk

C-s

hape

dB

auer

-Neb

elsi

ck (1

996)

, Eh

renb

erg

(183

8)Z.

per

ejas

law

zewa

e–

––

––

–A

bsen

t–

–K

ahl (

1933

, 193

5), P

ere-

jasl

awze

wa

(188

6)Z.

plu

mul

a50

–75

35–4

590

–100

50–6

0U

p to

300

050

–60

Abs

ent

–C

-sha

ped

Ji et

 al.

(201

1), K

hal

(193

3, 1

935)

, Per

eja-

slaw

zew

a (1

886)

Z. ig

navu

m36

–43d

26–3

2e35

–86

35–8

6U

p to

180

0–

Pres

ent

Seem

to c

luste

r to

geth

er o

n pr

imar

y st

alkf

S-sh

aped

Schu

ster a

nd B

right

(2

016)

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but which needs to be reinvestigated because of the follow-ing characters which do not match with Z. alternans: 1) the maximum width of zooid nearly equal with (vs. con-sciously 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-mono-phyletic 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. Zootham-nium 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 Gen-Bank database erroneously as Z. pluma), Z. ignavum and Z. niveum which collectively formed clade III. This group-ing 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. alter-nans (?, DQ662855) differs from the sequence of our popu-lation 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 Protozo-ology, 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 °C, salinity 30‰). Zoothamnium apohentscheli n. sp. was collected on Decem-ber 24, 2018 (water temperature 20  °C, salinity 30‰). Zoothamnium alternans was collected on April 13, 2018 (water temperature 25 °C, salinity 30‰).

Colonies were removed from the slide using acupunc-ture 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 demon-strated using the “wet” silver nitrate method (Song and Wilbert 1995). Measurements and counts were performed under 400–1000 × magnifications. Drawings of live speci-mens 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) fol-lowing 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 Bio-Labs, 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; Ichthyoph-thirius 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

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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 topolo-gies 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 Sci-ence Foundation of China (project number: 31801984, 31772440, 31970486) and the Fundamental Research Funds for the Central Uni-versities (No. 201762017).

Author contributions WS conceived and guided the study. TW con-ducted sampling and performed experiments. BL identified the spe-cies. 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 fol-lowed and all necessary approvals have been obtained.

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