Taxonomy and phylogeny of Pseudovorticella littoralis sp. n. and P. alani sp. n. (Ciliophora: Peritrichia) from coastal waters of southern China

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ScienceDirect European Journal of Protistology 71 (2019) 125635

Taxonomy and phylogeny of Pseudovorticella littoralis sp. n. and P. alani sp. n. (Ciliophora: Peritrichia) from coastal waters of southern China Yong Zhanga , Zhuo Shena,b,c , Fan Zhanga , Ying Yua , Jiqiu Lia , Xiaofeng Lina,∗ a

Laboratory of Protozoology, Guangzhou Key Laboratory of Subtropical Biodiversity and Biomonitoring, School of Life Science, South China Normal University, Guangzhou, China b Institute of Microbial Ecology and Matter Cycle, School of Marine Sciences, Sun Yat-sen University, Zhuhai 519082, China c Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai 519000, China Received 1 March 2019; received in revised form 27 July 2019; accepted 6 August 2019 Available online 10 September 2019

Abstract The morphology, infraciliature, and silverline system of two peritrich ciliates, Pseudovorticella littoralis sp. n. and P. alani sp. n., isolated from coastal waters of southern China, were investigated based on both living and silver-stained specimens. Pseudovorticella littoralis sp. n. is characterized by the following combination of characters: cell inverted cone-shaped; contractile vacuole ventral; J-shaped macronucleus; infundibular polykinety 3 with two kinetosome rows of equal length; 19–26 silverlines from peristome to trochal band and 5–14 from trochal band to scopula. Pseudovorticella alani sp. n. is characterized by: cell inverted bell-shaped; contractile vacuole ventral; J-shaped macronucleus recurved almost forming a loop; infundibular polykinety 3 with three kinetosome rows, outer two rows longer than inner one; 48–61 silverlines between peristome and aboral trochal band, and 12–20 between aboral trochal band and scopula. The SSU rDNA sequences of both new species are reported and their genetic distances with congeners and phylogenetic relationships are investigated. Pseudovorticella and Epicarchesium cluster into two subclades with low support values. One subclade contains nearly all the available sequences of Pseudovorticella and Epicarchesium. Another one contains P. monilata and E. pectinatum. This calls on the need of a generic re-classification of Pseudovorticella and Epicarchesium based on more morphological and molecular data. © 2019 Elsevier GmbH. All rights reserved.

Keywords: Biodiversity; Ciliate; Mangrove wetland; New species; Vorticellidae

Introduction The peritrich genus Pseudovorticella Foissner and Schiffmann, 1975 is morphologically similar to Vorticella (i.e. solitary cell borne upon a non-branching contractile stalk), but differs from the latter in having a reticulate (vs. transverse) pellicular silverline system with lines running vertically as well as horizontally (Foissner and Schiffmann

∗ Corresponding

author. E-mail address: [email protected] (X. Lin).

https://doi.org/10.1016/j.ejop.2019.125635 0932-4739/© 2019 Elsevier GmbH. All rights reserved.

1975; Sun et al. 2013). Jankowski (2007) divided Pseudovorticella into three subgenera, namely P. (Pseudovorticella), P. (Danicella), and P. (Kiricella), however no subsequent study followed this suggestion. To date, Pseudovorticella comprises more than 60 species (H. Berger, personal communication), most of which were transferred from the genus Vorticella (Foissner and Schiffmann 1979; Gómez et al. 2018; Hu et al. 2019; Ji et al. 2011; Jiang et al. 2019; Leitner and Foissner 1997; Liang et al. 2019; Song 2012; Song et al. 2009; Sun et al. 2017; Warren 1987). Nevertheless, it is likely that the diversity of Pseudovorticella is underestimated because the morphologies of many vorticellid species are very similar

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in vivo and the silverline system of many nominal Vorticella species is unknown (Clamp 2006; Sun et al. 2013). Furthermore, most Pseudovorticella species have yet to be sequenced and less than half of Pseudovorticella sequences in public databases lack morphological data or voucher specimens. This combination of factors prevents the identification of species based on gene sequence data (Li et al. 2008; Gao et al. 2016; Sun et al. 2016). Further studies combining morphology and molecular data are therefore needed in order to apply DNA-barcoding for species identification of Pseudovorticella. In this study, two Pseudovorticella species were identified based on live observation, protargol staining to reveal the infraciliature, and silver nitrate staining to reveal the silverline system. The results show that both represent new members of the genus Pseudovorticella. In addition, their phylogenetic placements were analyzed based on SSU rDNA sequence data.

Material and Methods Collection, observation and identification Pseudovorticella littoralis sp. n. was collected on 10 October 2010 from coastal waters of Daya Bay, Huizhou, China, when the water temperature was 27 ◦ C, salinity 30‰, and pH 7.4 at sampling site. Pseudovorticella alani sp. n. was collected on 10 December 2010 from Futian mangrove wetland, Shenzhen, China, when the water temperature was 22 ◦ C, salinity 13‰, and pH 7.3 at sampling site. All specimens were collected on glass slides (as artificial substrates) which were fixed to a frame (Song and Wibert 1995) and suspended at the sampling sites at a depth of about 0.5 m for one week to allow colonization by the peritrichs. A dissecting needle was used to remove stalked solitary peritrichs from the glass slides. These were then observed using bright field and differential interference contrast microscopy (Nikon 80i). The infraciliature (oral ciliature) was revealed by the protargol method according to Wilbert (1975), and the silverline system was revealed by the Chatton-Lwoff silver nitrate staining method according to Song and Wilbert (1995). Drawings of live specimens are based on in vivo observations and photomicrographs. Drawings of stained specimens were made with the help of a camera lucida at a magnification of 1000×. Systematics and terminology are mainly according to Lynn (2008) and Warren (1987).

The SSU rDNA sequence was amplified using ExTaq DNA polymerase and universal eukaryotic forward primer EukA (5 -AACCTGGTTGATCCTGCCAGT-3 ) and reverse primer EukB (5 -TGATCCTTCTGCAGGTTCACCTAC-3 ) (Medlin et al. 1988) with the following conditions: initial denaturation at 94 ◦ C for 5 min; 5 cycles of 1 min at 94 ◦ C, 1 min at 56 ◦ C, and 2 min at 72 ◦ C; followed by 30 cycles of annealing at 60 ◦ C; and a final extension at 72 ◦ C for 10 min. The SSU rDNA was sequenced by Shanghai Majorbio Biopharm Technology Co., Ltd. (Guangzhou branch).

Phylogenetic analyses Apart from the two newly sequenced species, all SSU rDNA sequences used to construct phylogenetic trees were obtained from GenBank database (for accession numbers, see Fig. 5). All SSU rDNA sequences of the genera Pseudovorticella and Epicarchesium available so far were included. The hymenostomes Tetrahymena corlissi and Glaucoma chattoni were used as the outgroup. Sequences were aligned using Clustal W and further modified manually in BioEdit 7.2.5 (Hall 1999). The length of the final alignment was 1620 bp. Maximum likelihood (ML) and Bayesian Inference (BI) trees were constructed. ML analysis was conducted by RaxML-HPC2 (Stamatakis 2014) at the CIPRES website (http://www.phylo.org/) using the GTRGAMMA + I nucleotide substitution model (Irwin and Lynn 2015). Statistical support was computed using 1000 bootstrap replicates. BI analysis was performed with MrBayes v3.1.2, using GTR + I+G evolutionary model as the best model selected by Akaike Information Criterion (Ronquist and Huelsenbeck 2003). The program was run for 1,000,000 generations with sampling every 100 generations and the first 2500 trees were discarded as burn-in. The Approximately Unbiased (AU) test was used to test the monophyly of the genus Pseudovorticella (Shimodaira 2002). Constrained ML trees were generated at the CIPRES website (http://www.phylo.org/) and internal relationships within the constrained groups and among the remaining taxa were unspecified. The constrained and unconstrained ML topologies were tested with standard parameters in CONSEL v.0.1 (Shimodaira and Hasegawa 2001).

ZooBank registration The ZooBank registration number of the present work is: :pub:1AB867B7-CE1D-4B9A-9685-B72AD0F60AEF

DNA extraction and gene sequencing

Results For each species a single individual of the raw culture was harvested, washed several times with filtered (0.22 ␮m pore size) in situ water, and placed in a 1.5 ml centrifuge tube. Genomic DNA was extracted using RED Extract-N-Amp Tissue PCR Kit (Sigma, USA).

Order Sessilida Kahl, 1933 Family Vorticellidae Ehrenberg, 1838

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Genus Pseudovorticella Foissner and Schiffmann, 1975

Pseudovorticella littoralis sp. n. Diagnosis: Cell inverted cone-shaped, 40–60 ␮m × 50–70 ␮m in vivo. Macronucleus J-shaped. Contractile vacuole ventral. Infundibular polykinety 3 with two kinetosome rows of equal length. 19–26 and 5–14 transverse silverlines above and below aboral trochal band, respectively. Type locality: Coastal waters of Daya Bay near Aotou dock (22◦ 43 53 N; 114◦ 34 18 E) at Huizhou, Guangdong Province, China. Type material: The protargol slide (registration number SZ2010-1010-01-3) with the holotype (Fig. 2h) circled in ink, and a paratype slide (registration number SZ-2010-1010-014) with silver nitrate-stained specimens, were deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China. Etymology: The species-group name littoralis refers to the type material was isolated from coastal waters.

Morphology (Figs. 1a–e, 2 a–i; Table 1) Cell (= zooid) inverted cone-shaped when expanded, 40–60 ␮m × 50–70 ␮m in vivo with length to width ratio about 1:1 (n = 10); widest at the peristomial lip which is single-layered and 5 ␮m thick (Figs. 1a, 2 c). Peristomial disc (= epistomial disc) flat, slightly elevated above peristomial lip (Figs. 1a, 2 b). Oral cilia about 13 ␮m long. Pellicle with fine striations, visible in vivo when viewed at a magnification of 400× (Fig. 2e). Cytoplasm slightly grayish, usually containing 2–5 food vacuoles, 10–13 ␮m in diameter (Figs. 1a, 2 e). Single contractile vacuole located near ventral wall of infundibulum in anterior third of body, up to 20 ␮m in diameter (Fig. 2b, c). Macronucleus usually J-shaped, anterior portion horizontally oriented and surrounding the infundibulum, posterior portion extends to aboral quarter of body (Fig. 2h). Micronucleus not observed. Stalk 250–350 ␮m in length, about 5 ␮m in diameter, with smooth surface (Fig. 1b). Stalk spasmoneme about 2 ␮m in diameter, with gray mitochondrial granules (about 0.2 ␮m in diameter) sparsely distributed (Fig. 2f). Telotroch not observed. Oral ciliature comprises epistomial membrane, germinal kinety, haplokinety, and polykinety (Figs. 1d, e, 2 g). Haplokinety and polykinety circle about one turn around peristome in parallel to each other before entering infundibulum where they separate and make a further turn. At lower half of infundibulum, polykinety separates into three infundibular polykineties (P1, P2, and P3). P1 and P2 composed of three kinetosome rows each, P3 consists of two rows which are parallel and equal in length. P1 and P2 much longer than P3.

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P2 terminates above P1 and P3. Upper half of P3 close to P2, lower half extends beyond adstomal end of P2 and terminates at same level as P1. Germinal kinety parallel to haplokinety within upper half of infundibulum. Epistomial membrane located near opening of infundibulum. Aboral trochal band composed of a double row of kinetostomes encircling cell about 80% down length of cell (Fig. 1c, e). Silverline system reticulate, transverse lines evenly spaced with many sparsely distributed pellicular pores (Figs. 1c; 2 i). 19–26 transverse silverlines from peristome to aboral trochal band, and 5–14 from aboral trochal band to scopula.

Pseudovorticella alani sp. n. Diagnosis: Cell inverted bell-shaped, about 70–90 ␮m × 50–65 ␮m in vivo. Macronucleus J-shaped, strongly recurved almost forming a loop, longitudinally oriented. Contractile vacuole ventral. Infundibular polykinety 3 three-rowed, outer two rows longer than inner row. 48–61 and 12–20 transverse silverlines above and below aboral trochal band, respectively. Type locality: A small body of standing water near Fengtang estuary at Futian mangrove wetland (22◦ 31 25 N; 114◦ 0 40 E) in Shenzhen, Guangdong Province, China. Type material: The protargol slide (registration number SZ2010-1210-01-1) with the holotype (Fig. 4c) circled in ink, and a paratype slide (registration number SZ-2010-1210-012) with silver nitrate-stained specimens, were deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China. Etymology: The species is named in honor of Dr. Alan Warren, Natural History Museum, UK, in recognition of his outstanding contribution to the taxonomy of ciliates.

Morphology (Figs. 3a-f, 4a-h; Table 1) Cell inverted bell-shaped when expanded, 70–90 ␮m × 50–65 ␮m in vivo, slightly constricted below peristomial lip which is single-layered, about 5 ␮m thick. Peristomial disc elevated about 6 ␮m above peristomial lip (Fig. 3a, b). Oral cilia about 12 ␮m long. Pellicle striations visible in vivo when viewed at a magnification of 400× (Fig. 4e). Surface of pellicle with numerous small granules, possibly bacteria (Fig. 4d). Cytoplasm slightly grayish, usually containing 3–6 food vacuoles about 10–13 ␮m in diameter (Figs. 3a, 4 b). Single contractile vacuole located near ventral wall of infundibulum in anterior third of body, up to about 13 ␮m across when fully expanded (Fig. 4b). Macronucleus J-shaped, unusual long and strongly recurved almost forming a loop, with its upper arm horizontally oriented surrounding infundibulum and posterior portion recurved and extending upwards, almost reaching anterior end (Figs. 3e, f, 4 c). Micronucleus not observed.

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Fig. 1. a–e. Morphology and infraciliature of Pseudovorticella littoralis sp. n. in vivo (a, b), after silver nitrate (c) and protargol (d, e) staining. (a) Typical cell (= zooid). (b) Showing length of stalk relative to cell. (c) Silverline system. (d) Oral infraciliature. (e) General infraciliature. ATB, aboral trochal band; EM, epistomial membrane; G, germinal kinety; H, haplokinety; Ma, macronucleus; Po, polykinety; P1-3, infundibular polykinety 1-3. Scale bars: 40 ␮m (a); 200 ␮m (b); 20 ␮m (e).

Fig. 2. a–i. Photomicrographs of Pseudovorticella littoralis sp. n. in vivo (a–f), after protargol (g, h) and silver nitrate (i) staining. (a) Typical individual, showing length of stalk relative to cell. (b) Cell at high magnification, arrow shows contractile vacuole. (c) To show the peristomial lip (arrow) and contractile vacuole (arrowhead). (d) Apical view of a cell, arrow indicates edge of peristomial lip. (e) To show pellicular striations (arrow). (f) Detail of stalk and spasmoneme (arrow). (g) Infundibular part of oral infraciliature, arrow shows infundibular polykinety 3. (h) The holotype specimen, showing macronucleus (arrows). (i) To show silverline system; arrowhead marks the aboral trochal band. Scale bars: 100 ␮m (a); 30 ␮m (b–f, h, i); 10 ␮m (g).

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Fig. 3. a–f. Morphology and infraciliature of Pseudovorticella alani sp. n. in vivo (a, b), after silver nitrate (d) and protargol (c, e–h) staining. (a) Typical cell. (b) Showing length of stalk relative to cell. (c) Infundibular part of oral infraciliature. (d) Silverline system. (e) General infraciliature and macronucleus. (f) To show the variable shape of macronucleus. ATB, aboral trochal band; EM, epistomial membrane; G, germinal kinety; H, haplokinety; Ma, macronucleus; Po, polykinety; P1-3, infundibular polykinety 1-3. Scale bars: 40 ␮m (a, f); 150 ␮m (b); 25 ␮m (e).

Fig. 4. a–h. Photomicrographs of Pseudovorticella alani sp. n. in vivo (a, b, d–f), after protargol (c, g) and silver nitrate (h) staining. (a) Typical individual at low magnification. (b) Cell at high magnification, arrow shows contractile vacuole, arrowhead indicates a food vacuole. (c) The holotype specimen, showing macronucleus (arrow). (d) To show the granules (bacteria?) on pellicle surface (arrow). (e) To show pellicular striations. (f) Detail of stalk, arrow indicates the granules (mitochondria?). (g) Infundibular part of oral infraciliature, arrow points to infundibular polykinety 3. (h) To show silverline system, arrow indicates pellicular pore. Scale bars: 80 ␮m (a); 40 ␮m (b, c); 10 ␮m (d–h).

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Table 1. Morphometrical characterization and silverline system of peritrich ciliates Pseudovorticella littoralis sp. n. and Pseudovorticella alani sp. n. Measurements in ␮m. Characters Cell length in vivo Cell width in vivo Cell length after protargol Cell width after protargol Macronucleus length Macronucleus diameter No. of silverlinesa No. of silverlinesb

Species

Min

Max

Mean

SD

n

P. littoralis P. alani P. littoralis P. alani P. littoralis P. alani P. littoralis P. alani P. littoralis P. alani P. littoralis P. alani P. littoralis P. alani P. littoralis P. alani

40 70 50 50 33 40 32 30 80 100 4 5 19 48 5 12

60 90 70 65 50 56 50 49 120 119 6 6 26 61 14 20

51.15 80.80 59.68 58.16 38.33 49.38 38.60 39.69 98.20 110.25 5.00 5.50 22.50 54.59 11.17 16.00

6.81 5.96 5.45 3.78 4.75 5.47 6.06 5.38 16.95 7.85 0.82 0.58 2.28 3.90 2.79 2.62

13 15 13 15 15 13 15 13 5 4 10 4 12 8 12 8

Abbreviations: Max, maximum; Mean, arithmetic mean; Min, minimum; SD, standard deviation; n, number of samples. a No. of silverlines between peristome and aboral trochal band view based on silver nitrate-stained specimens. b No. of silverlines between aboral trochal band and scopula based on silver nitrate-stained specimens.

Stalk 300–400 ␮m long, about 5 ␮m across, surface smooth. Spasmoneme distinctly helical, about 2 ␮m across, surface sparsely lined with dark grey mitochondria (about 1 ␮m across) (Fig. 4f). Telotroch not observed. Oral infraciliature as shown in Figs. 3c, e, 4 g. Haplokinety and polykinety make about one turn around peristome in parallel to each other before entering infundibulum where they make a further circuit. Polykinety separates into three infundibular polykineties (P1, P2, and P3) in lower half of infundibulum, all of which are three-rowed. P1 and P2 significantly longer than P3. P2 terminates adstomally above P1 and P3. Inner row of P3 absent in adstomal half. Outer two rows of P3 terminate adstomally at end of P1. Germinal kinety within upper half of infundibulum and parallel to haplokinety. Epistomial membrane short, near beginning of haplokinety and polykinety. Aboral trochal band composed of a double-kinety that encircles cell about 65–75% down its length (Fig. 3b, d). Reticulate silverline system typical of genus, associated with numerous sparsely distributed pellicular pores (Fig. 4h). With 48–61 and 12–20 transverse silverlines above and below aboral trochal band, respectively (Fig. 3d).

SSU rDNA sequence and phylogenetic analyses The SSU rDNA sequences of Pseudovorticella littoralis sp. n. and P. alani sp. n. have been deposited in GenBank database with length, GC content, and accession number as follows: P. littoralis sp. n. 1752 bp, 44.2%, ID MK627468; P. alani 1708 bp, 43.5%, ID MK627469. The SSU rDNA

sequences of the two new species and 18 Pseudovorticella species downloaded from GenBank were compared based on genetic distances calculated with MEGA7. The nucleotide sequences differed by 0 to 10.88% between species (Table S1). Both new species differ from known congeners by over 4.8%. The tree topologies inferred from Maximum likelihood (ML) and Bayesian inference (BI) analyses are generally congruent with each other, therefore only the ML tree is shown here (Fig. 5). In our phylogenetic analyses, the genus Pseudovorticella is non-monophyletic. All species of Pseudovorticella and Epicarchesium form a clade. Their members are separated into two groups, A and B (Fig. 5). Group B comprises E. pectinatum, P. monilata, the type species of Pseudovorticella, and seven unidentified Pseudovorticella species, namely P. sp. AU, P. sp. 4 PS, P. sp. rotund, P. sp. 7 PS, P. sp. JP, P. sp. 8 PS, P. sp. 15 PS. The last five were considered to be conspecific with P. monilata by Jiang et al. (2019). Our two new species are clustered in the large group A which includes all remaining species of the genera Pseudovorticella and Epicarchesium for which SSU rDNA sequence data are available. Group A comprises two subclades: sub-clade I which comprises “Pseudovorticella sp. Slender” and four Epicarchesium species, and sub-clade II which comprises E. corlissi and all remaining Pseudovorticella species sequenced so far. Pseudovorticella littoralis sp. n. and the unidentified P. sp. Guadeloupensis occupy the basal position within sub-clade II; however, their nucleotide sequences differed by 6.14% (Table S1). Pseudovorticella alani sp. n. is sister to P. sp. 1 LZ54 and differed by 2.66% between their nucleotide sequences.

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Fig. 5. SSU rDNA phylogenetic tree of peritrich ciliates with special focus on Pseudovorticella and Epicarchesium species. Newly sequenced species are shown in bold type. Numbers at the nodes represent support values in the following order: Maximum Likelihood (ML) bootstrap values/Bayesian inference (BI) posterior probabilities. “-” reflects value that are below 50% or 0.50. Black solid dots indicate full support (100%/1.00). The scale bar indicates five substitutions per 100 nucleotides.

The Approximately Unbiased (AU) test reveals that the monophyly of the genus Pseudovorticella (constraint 1, p = 1E-009) is rejected at 5% significance level.

line system (Foissner and Schiffmann 1975; Ji et al. 2003; Song and Wilbert 1989; Sun et al. 2007; Warren 1987). The two new species are compared with closely related congeners and morphologically similar species of Vorticella.

Discussion As noted in most previous studies, the critical features for the discrimination of Pseudovorticella morphospecies are the shape and size of the cell, the number and location of the contractile vacuoles, the shape and position of the macronucleus, the pattern of the oral ciliature, and the features of the silver-

Comparison of Pseudovorticella littoralis sp. n. with related species The congeners that most closely resemble P. littoralis sp. n. are P. jaerae (Precht, 1935) Sun et al., 2009, P. patellina (Müller, 1776) Song and Warren, 2000, P. parafornicata Sun

Present work Present work Sun et al. (2009) Song and Warren (2000) Sun et al. (2009) Sun et al. (2006) Sun et al. (2009) Sun et al. (2017) Ji et al. (2006) Sun et al. (2009) Foissner (1979) Sun et al. (2017) Marine Brackish Marine Marine Marine Marine Marine Marine Marine Marine Freshwater Marine 2 rows 3 rows 3 rows 3 rows 3 rows 3 rows 3 rows 3 rows 2 rows 3 rows – 3 rows J-shaped J-shaped J-shaped J-shaped J-shaped J-shaped J-shaped J-shaped J-shaped J-shaped J-shaped J-shaped –, data not available; ATB: aboral trochal band; Ma: macronucleus; OA: oral area; Sc: scopula.

1, ventral 1, ventral 1, ventral 2, dorsal – 1, ventral 1, ventral 1, ventral 1, dorsal 1, ventral 2, ventral and dorsal 1, ventral 5–14 12–20 8–11 13–16 – 9–11 11–14 12–16 15–20 13–16 8–10 10–14 19–26 48–61 20–26 19–22 – 20–24 35–39 35–39 30–37 22–27 26–27 33–37 40–60 70–90 40–55 50–110 20–30 75–90 50–60 75–85 40–54 70 40–50 35–50

50–70 50–65 25–45 50–100 20–30 55–65 40–45 70–80 45–62 30 – 35–50

Habitat Shape of Ma Structure of polykinety 3 No. and position of contractile vacuole(s) No. of silverlines from ATB to Sc No. of silverlines from OA to ATB Body width in vivo (␮m)

P. littoralis sp. n. P. alani sp. n. P. jaerae P. patellina P. parafornicata P. jiangi P. parakenti P. liangae P. punctata P. lima P. sphagni Vorticella chiangi

Pseudovorticella alani sp. n. bears a strong resemblance to P. punctata (Dons, 1918) Warren, 1987 in terms of its cell shape, number of contractile vacuoles, and arrangement of P3 (Table 2). However, P. alani sp. n. has a relatively larger cell size (70–90 ␮m × 50–65 ␮m vs. 40–55 ␮m × 45–65 ␮m), more silverlines above the trochal band (48–61 vs. 30–37) and a ventrally (vs. dorsally) located contractile vacuole (Ji et al. 2006; Warren 1987). Paravorticella lima (Kahl, 1933) Jankowski, 1976 has similar body shape and size with P. alani sp. n. (Table 2). However, P. lima can be separated from the new species by

Body length in vivo (␮m)

Comparison of Pseudovorticella alani sp. n. with congeners

Species

et al., 2009, P. jiangi Sun et al., 2006, P. parakenti Sun et al., 2009 and P. liangae Sun et al., 2017 (Table 2). Pseudovorticella jaerae resembles P. littoralis sp. n. in cell shape and size, characteristics of the contractile vacuole, numbers of silverlines, and the shape of the macronucleus. However, the former differs from the new species by the appearance of the pellicle (with a thin layer of pellicular blisters vs. only with fine striations) and the structure of P3 (3-rowed vs. 2-rowed) (Precht 1935; Sun et al. 2009). Pseudovorticella parakenti and P. littoralis sp. n have a similar cell shape and size, position of the contractile vacuole, appearance of the pellicle, and macronuclear shape. However, the former can be separated from the latter by the number of silverlines above the aboral trochal band (35–39 vs. 19–26) and the structure of P3 (3-rowed vs. 2-rowed) (Sun et al. 2009). Pseudovorticella littoralis sp. n. and P. patellina have a similar cell shape. Nevertheless, P. littoralis sp. n. can be clearly separated from P. patellina by the shape of the macronucleus (J-shaped vs. band-like), the number and position of the contractile vacuole (one ventral vs. two dorsal), and the structure of P3 (2-rowed vs. 3-rowed) (Kahl 1935; Müller 1776; Song and Warren 2000). Pseudovorticella parafornicata, like P. littoralis sp. n., has an inverted cone-shaped cell, but the former differs from the latter by having a smaller cell size (20–30 ␮m × 20–30 ␮m vs. 40–60 ␮m × 50–70 ␮m) (Sun et al. 2009). Pseudovorticella jiangi and P. liangae also have a similar cell shape but a markedly larger cell size compared with P. littoralis sp. n. (75–90 ␮m × 55–65 ␮m and 75–85 ␮m × 70–80 ␮m, respectively vs. 40–60 ␮m × 50–70 ␮m). Moreover, both have a 3-rowed P3 (vs. 2-rowed in P. littoralis sp. n.) (Sun et al. 2006, 2017). Pseudovorticella littoralis sp. n. also resembles Vorticella chiangi Sun et al., 2017 in several morphological characters (Table 2). However, it can be recognized clearly by the structure of P3 (2-rowed vs. 3-rowed), the number of silverlines above the trochal band (19–26 vs. 33–37) and in having a reticulate (vs. transverse) silverline pattern (Sun et al. 2017).

Data source

Y. Zhang et al. / European Journal of Protistology 71 (2019) 125635

Table 2. Comparison of Pseudovorticella littoralis sp. n. and P. alani sp. n. with closely related species.

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having fewer silverlines above the trochal band (22–27 vs. 48–61) (Kahl 1933; Sun et al. 2009). In view of the unusual shape of its macronucleus, P. alani sp. n. should be compared with P. sphagni Foissner and Schiffmann, 1975 (Table 2). The latter can, however, be separated from the new species by its freshwater (vs. brackish) habitat, and in having more (two vs. one) contractile vacuoles and fewer silverlines above (26–27 vs. 48–61) and below (8–10 vs. 12–20) the trochal band (Foissner, 1979).

Phylogeny of the genus Pseudovorticella Until recently, only three SSU-rDNA sequences of Pseudovorticella were available and these form a clade in phylogenetic trees implying that the genus is monophyletic (Li et al. 2008; Sun et al. 2012). Sun et al. (2016) added ten new sequences from clone libraries and 20 environmental sequences from high-throughput sequencing and produced a phylogenetic tree of the peritrichs which revealed the nonmonophyly of Pseudovorticella. Jiang et al. (2019) reported the SSU-rDNA sequences of five Pseudovorticella species and analyzed the genetic distances of all known SSU-rDNA sequences of Pseudovorticella. Five undefined Pseudovorticella species, namely P. sp. 15 PS, P. sp. 8 PS, P. sp. 7 PS, P. sp. rotund, and P. sp. JP are considered to be conspecific with P. monilata (Jiang et al. 2019). The present study uses all sequences of Pseudovorticella available so far in GenBank and the two newly sequenced species in the phylogenetic analyses of the genus. The tree topologies support the establishment of the two new species (Fig. 5). Furthermore, the SSU rDNA sequences differed by 4.84–7.45% between P. littoralis sp. n. and other Pseudovorticella species and differed by 2.07–9.1% between P. alani sp. n. and other Pseudovorticella species (Table S1). Pseudovorticella alani sp. n. is sister to P. sp. 1 LZ54 which together group with P. sp. 5 PS. However, no morphological data are available for P. sp. 5 PS or P. sp. 1 LZ54.

Phylogeny of the genus Epicarchesium The genus Epicarchesium Jankowski, 1985 is typically characterized by the colonial zooids and reticulate silverline pattern (Leitner and Foissner 1997). Previous phylogenetic studies have disclosed the close and complex relationship between Epicarchesium and Pseudovorticella and hypothesized that the silverline system probably is a key character for determining genus-level evolutionary relationships within the Vorticellidae (Ji et al. 2015; Li et al. 2008; Sun et al. 2016; Zhuang et al. 2017). Epicarchesium abrae, E. variabile and two unidentified congeners form a fully supported clade with Pseudovorticella sp. (no morphological description available) as the distal lineage of Pseudovorticella sub-clade A in our analysis based on the SSU rDNA sequences. Epicarchesium corlissi locates in the main group of sequences of Pseudovorticella. The other one, E. pectinatum forms a group

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with Pseudovorticella species in sub-clade B. This result is consistent with previous studies and supports the hypothesis mentioned above. So far, there are only six SSU rDNA sequences of Epicarchesium species recorded in GenBank. Even so, no gene sequence is available for the type species, E. granulatum (Kellicott, 1887) Jankowski, 1985. More molecular and morphological data (in particular the infraciliature and silverline system), are needed for a wider range of congeners and of related genera in order to verify this inference or make a generic re-classification of Pseudovorticella and Epicarchesium.

Author contributions Zhang Y., Lin X., and Shen Z. wrote and revised the manuscript. Shen Z. collected samples and performed staining. Yu Y. drew the illustrations. Zhang F. and Li J. analyzed data.

Acknowledgements This work was supported by the “National Natural Science Foundation of China” (project numbers: 41576148, 31971519, 31761133001) and the Guangdong MEPP Fund (NO. GDOE 2019 A23). We acknowledge Dr. Alan Warren, Natural History Museum, UK for his kind help in language polishing and the comments of reviewers.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.ejop.2019.125635.

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