Novel Contributions to the Peritrich Family Vaginicolidae

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Zoological Journal of the Linnean Society, 2019, 187, 1–30. With 13 figures.

Novel contributions to the peritrich family Vaginicolidae (Protista: Ciliophora), with morphological and Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 phylogenetic analyses of poorly known species of Pyxicola, Cothurnia and Vaginicola

BORONG LU1, LIFANG LI2, XIAOZHONG HU1,5,*, DAODE JI3,*, KHALED A. S. AL-RASHEID4 and WEIBO SONG1,5

1Institute of Evolution and Marine Biodiversity, & Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China 2Marine College, Shandong University, Weihai 264209, China 3School of Ocean, Yantai University, Yantai 264005, China 4Zoology Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia 5Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China

Received 29 September 2018; revised 26 December 2018; accepted for publication 13 February 2019

The classification of loricate peritrich ciliates is difficult because of an accumulation of several taxonomic problems. In the present work, three poorly described vaginicolids, Pyxicola pusilla, Cothurnia ceramicola and Vaginicola tincta, were isolated from the surface of two freshwater/marine algae in China. In our study, the ciliature of Pyxicola and Vaginicola is revealed for the first time, demonstrating the taxonomic value of infundibular polykineties. The small subunit rDNA, ITS1-5.8S rDNA-ITS2 region and large subunit rDNA of the above species were sequenced for the first time. Phylogenetic analyses based on these genes indicated that Pyxicola and Cothurnia are closely related. The present study suggested that the loricate species probably represent a distinct lineage in peritrich evolution and both genera Cothurnia and Thuricola are monophyletic. Pyxicola pusilla, Cothurnia ceramicola and Vaginicola tincta are recircumscribed.

ADDITIONAL KEYWORDS: ciliates – Loricate sessilids – rDNA.

INTRODUCTION advanced the understanding of the taxonomy of sessilids (Lom, 1964; Foissner & Schiffmann, 1974, One of the largest components of the global ciliate 1975), most studies have focused on the aloricate biota is the peritrich order Sessilida, with more than forms, leaving loricate taxa relatively under- 800 species inhabiting various aquatic ecosystems researched (Ji & Kusuoka, 2009; Utz et al., 2014; Ji (Kahl, 1935; Foissner et al., 1992; Lynn, 2008; Sun et al., 2015; Jiang et al., 2016; Kühner et al., 2016; Liu et al., 2009; Foissner et al., 2010). They commonly feed et al., 2017;Shen et al., 2017; Sun et al., 2017; Zhuang on small-sized particles and contribute significantly et al., 2018). to the water quality (Pillai, 1952; Azam et al., 1983; The family Vaginicolidae is a species-rich group of Weber et al., 2007). Most are sedentary and attach to loricate sessilids, with nearly 200 nominal species substrates via the scopula, stalk or lorica. Although (Kahl, 1935; Corliss, 1979). However, among these the application of silver staining methods to reveal names are many synonyms and misidentifications, the infraciliature and silverline system has greatly because: (1) several genera have a similar appearance (e.g. Pyxicola Kent, 1882, was for a long *Corresponding authors. E-mail: [email protected]; time treated as Cothurnia Ehrenberg, 1831, because [email protected] of their similar stalked lorica); (2) the congeners of

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 1 2 B. LU ET AL. each genus share an almost identical body shape MATERIAL AND METHODS with each other (e.g. thuricolas all exhibit a trumpet- Sample collection shaped body when fully extended); (3) although the presence of a lorica is the key character for Pyxicola pusilla (Fig. 1E1) was isolated from the identifying vaginicolids, some species descriptions freshwater alga Vaucheria sp., collected together with are incomplete, causing difficulties in distinguishing in situ water on 16 March 2017 from Hangzhou Bay, Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 species (e.g. the narrow and wide side views of the 30°22′ N; 121°11′ E (Fig. 1C, E), Ningbo (Fig. 1A), lorica in some species are very different, but this China. The water temperature was 9.5 °C and the has largely been ignored in previous reports; i.e. salinity was 2 psu. Cothurnia ceramicola (Fig. 1D1) Kahl, 1935; Trueba, 1978, 1980; Warren & Paynter, was collected from the surface of Enteromorpha sp. 1991). It is now widely accepted that molecular data on 14 April 2017 at Tangdao Bay, 35°56′ N; 121°13′ can help to elucidate some of the problems in the E (Fig. 1B, D), Qingdao (Fig. 1A), China. The water taxonomy and systematics of ciliates (Li et al., 2008; temperature was 16 °C and the salinity was 32 psu. Sun et al., 2012, 2013; Li et al., 2015; Gao et al., 2017; Vaginicola tincta (Fig. 1F1) was also obtained from Yan et al., 2017), but sequence data remain scarce Vaucheria sp. in Ri Lake, 29°54′ N; 121°34′ E (Fig. for vaginicolids (Zhuang et al., 2016). 1C, F), Ningbo (Fig. 1A), China, on 29 March 2017. The genus Pyxicola was erected by Kent in 1882 The water temperature was 15 °C. In each case, the for operculated ciliates that had previously been specimens were directly isolated from the subsamples regarded as species of Cothurnia. Trueba (1978) for subsequent studies in the laboratory. The three revised the genus to include eight species and species of Thuricola discussed in the present work two forms. Thereafter, only one new species found were previously described by Lu et al. (2018). in China was added to the genus: Pyxicola ovata Xu, 1987. Unfortunately, details of the ciliature and molecular information for the genus are still Morphological study lacking. Living cells were investigated using a compound Cothurnia, the largest genus of Vaginicolidae, microscope equipped with a high-power oil-immersion includes approximately 100 species (Warren & objective and differential interference contrast optics. Paynter, 1991), but ciliary data are available for The ciliature was revealed using the protargol staining only two of them (Song, 1992; Zhuang et al., 2016) method (Wilbert, 1975). The protargol powder was and only three gene sequences have been deposited manually synthesized, following the method prescribed in GenBank (one of which is dubious). Furthermore, by Pan et al. (2013). Counts and measurements were some species of Cothurnia with cursory descriptions performed at 400 and 1000× magnifications. Drawings are difficult to separate from each other. Overall, an of living organisms were based on in vivo observation investigation of this genus based on modern criteria and photomicrographs, whereas those of stained is urgently needed. specimens were made with the help of a camera lucida. Vaginicola Lamarck, 1816 is also a species-rich Terminology is according to Trueba (1978) and Warren genus, containing nearly 50 nominal species (Corliss, & Paynter (1991). 1979; Foissner et al., 1992). Compared with the two genera mentioned above, the structure of members of this genus is relatively simple, which renders DNA extraction, amplification and sequencing species identification more difficult. This makes Genomic DNA was extracted according to the methods the ciliature more important for species separation described in Luo et al. (2017). The SSU rDNA was but, so far, no ciliature data are available for this amplified using the primers 82F (5′ -GAA ACT GCG genus. Meanwhile, most of the literature is too old AAT GGC TC-3′; Jerome et al., 1996) and 18SR (5′- to contain photomicrographs to exhibit details of the TGA TCC TTC TGC AGG TTC ACC TAC-3′; Medlin lorica morphology (Kent 1882; Stiller, 1971; Küsters, et al., 1988). A fragment of approximately 500 bp 1974). containing the ITS1, 5.8S ribosomal gene and ITS2 In this study we investigate and redescribe three was amplified using primers 5.8SF (5′-GTA GGT GAA species belonging to the three above mentioned genera, CCT GCG GAA GGA TC-3′) and 5.8SR (5′-CTG ATA based on the observation of specimens in vivo and TGC TTA AGT TCA GCG G-3′; Huang et al., 2018). The after protargol staining. The ciliature of Pyxicola and PCR amplifications of LSU rDNA were performed with Vaginicola is thus described here for the first time. the primers F3 (5′-ACG/C CGC TGA/G AT/CT TAA Phylogenetic analyses are performed based on the GCA T-3′) and R2 (5′-AAC CTT GGA GAC CTG AT-3′) small subunit (SSU) rDNA, ITS1-5.8S rDNA-ITS2 from Moreira et al. (2007).Q5 Hot Start High-Fidelity regions and the large subunit (LSU) rDNA sequences. DNA Polymerase (New England BioLabs, USA) was

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 MORPHOLOGY AND PHYLOGENY OF VAGINICOLIDS 3 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019

Figure 1. Map and sampling sites. A, map of the four seas of China, red asterisks indicate the locations of Qingdao and Ningbo. B, satellite map of southwest Qingdao, red asterisk indicates Tangdao Bay. C, satellite map of north-east Ningbo, red asterisks indicate the two sampling areas (wetland of Hangzhou Bay, Ri Lake). D, coast of Tangdao Bay. D1, Cothurnia ceramicola collected from site D. E, a pool in wetland of Hangzhou Bay. E1, Pyxicola pusilla collected from site E. F, Ri Lake, a freshwater lake in Ningbo. F1, Vaginicola tincta collected from site F. used to minimize the possibility of PCR amplification two hymenostomatians (Ichthyophthirius multifiliis errors. Sequencing was performed bidirectionally by DQ270016; Tetrahymena pyriformis KX832097); the Tsingke Biological Technology Company (Beijing, (3) LSU rDNA sequences of 14 sessilids and two China). hymenostomatians (Ichthyophthirius multifiliis EU185635; Tetrahymena pyriformis X54004). The hymenostomatians mentioned above were used as Molecular phylogeny methodology out-group taxa. All new sequences are deposited in GenBank, with Sequences were aligned using the GUIDANCE2 the accession numbers, lengths and GC contents of algorithm online (http://guidance.tau.ac.il/ver2/) with each summarized in Table 8. Besides these 15 newly its default parameters (Landan & Graur, 2008; Sela obtained sequences (including three ITS1-5.8S-ITS2 et al., 2015). The ends of the resulting alignments were region and three LSU rDNA of Thuricola), other trimmed manually using the program BioEdit v.7.0 sequences used in the present phylogenetic analyses (Hall, 1999). The final alignments of the SSU rDNA, were downloaded from GenBank. In order to compare ITS1-5.8S-ITS2 region and LSU rDNA sequences were morphologies, only species that had already been 1767, 544 and 1795 positions, respectively. definitively identified were chosen, including: (1) SSU Maximum likelihood (ML) analysis with 1000 rDNA sequences of 45 sessilids, three mobilids and bootstrap replicates was computed at the CIPRES four hymenostomatians (Glaucoma chattoni X56533; Science Gateway (http://www.phylo.org), using the Ichthyophthirius multifiliis U17354; Tetrahymena GTR + gamma model performed by RAxML-HPC2 corlissi U17356; Tetrahymena pyriformis EF070254); v.8.2.10 on XSEDE (Stamatakis, 2014). Bayesian (2) ITS1-5.8S-ITS2 region sequences of 29 sessilids and inference (BI) analysis was carried out using MrBayes

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 4 B. LU ET AL. v.3.2.6 on XSEDE (Ronquist et al., 2012) with the 19–22 μm in diameter. Lorica stalk highly variable GTR+ I+ G model selected by MrModeltest v.2.2 in length. Zooid about 55–85 × 10–25 μm in vivo, (Nylander, 2004) via the Akaike Information Criterion. peristomial lip single-layered, protrudes just above Markov chain Monte Carlo simulations were run for the aperture. Contractile vacuole located in anterior 6 000 000 generations with a sample frequency of 100 quarter of zooid. Infundibular polykinety 3 composed generations and a burn-in of 6000 trees (10%). All the of three equally long rows of kinetosomes. About 60 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 remaining trees were used to calculate the posterior striations from the peristome to the scopula. Fresh or probability using a 50% majority rule consensus. Tree brackish water. topologies were visualized using MEGA v.7.0 (Kumar et al., 2016). Systematic classification follows Gao et al. Description based on Ningbo population (2016) and Sun et al. (2012). Zooid is solitary, about 60–75 × 10–20 μm in vivo and usually convex at mid-body (Figs 2A, B, D, Topology testing 3A–D, F) although sometimes trumpet-shaped To assess the monophyly of the genus Vaginicola and the (Figs 2C, 3E). The single-layered peristomial lip family Vaginicolidae, the approximately unbiased (AU) is 21–28 μm in diameter, thin but everted strongly, test (Shimodaira, 2002) was used. Four constrained ML and is situated just beyond the aperture when the trees were generated by PAUP (Swofford, 2002) with zooid is fully extended. An operculum adheres to the enforced constraints (Table 9) and then compared with pellicle below the peristomial lip and is adjacent to the best (unconstrained) ML topologies implemented the lower edge of the lorica aperture (Figs 2A–D, in CONSEL (Shimodaira & Hasegawa, 2001). Internal 3A–G). The operculum is 16–18 μm in diameter relationships in the constrained group and among the and concave in its central part (Figs 2K, 3H–L). remaining taxa were unspecified. The colour of the operculum varies with age. The operculum is colourless when the cell is young and yellow to dark brown when the cell is old (Fig. 3A–G). The operculum closes the aperture of the lorica when RESULTS the cell is contracted (Figs 2E, 3H, I). The peristomial disc is obliquely elevated. The pellicle is very elastic, Subclass Peritrichia Stein, 1859 with about 62 distinct striations from the peristome Order Sessilida Kahl, 1933 to the scopula (counted from one live cell). The trochal Family Vaginicolidae Fromentel, 1874 band is unrecognizable in vivo. Genus Pyxicola Kent, 1882 The cytoplasm is colourless, with numerous small globular granules and several food vacuoles. No species of Pyxicola have previously been investigated A contractile vacuole is located at the anterior successfully using silver staining methods (Trueba, quarter of the body, about 10 μm in diameter. The 1978). This genus is characterized by the operculum macronucleus is vermiform, extends longitudinally attached to the zooid just below the peristomial lip. The throughout almost the whole body in vivo (Fig. 2F) and lorica is inhabited by a single zooid that is attached becomes twisted in stained cells (Figs 2C, 3P, Q). The to a non-contractile stalk. The anterior portion of the micronucleus was not observed. lorica is usually oblique and neck-like. The endostyle, mesostyle and stalk are contiguously connected with longitudinal striae. This combination Pyxicola pusilla (Wrześniowski, 1866) penetrates the lorica wall via a tube-like structure Kent, 1882 (Figs 2A–D, M, 3M, N). The zooid sits on top of the endostyle and the tube. The endostyle is 1.0–1.5 μm (Figs 2A–P, 3A–U; Tables 1–3) long and the mesostyle is 2.0–2.6 μm long, knot-like Since its original description, Cothurnia pusilla were and flared at the equator (Figs 2M, 3M, N). The lorica reported repeatedly (Wrześniowski, 1866, 1867, 1870). attaches to the substrate via a non-contractile stalk However, the ciliature of P. pusilla has never been that is continuous with the mesostyle, attaching to the revealed. An improved diagnosis based on previous substrate via a basal disc (Figs 2A–E, 3N). The stalk reports and new data is supplied. itself varies significantly in length (between 2 and 19 μm), but this variance is not correlated with age. Like the lorica, the basal disc becomes more coloured Improved diagnosis with age (Fig. 3A–G). Lorica about 50–80 × 25–40 μm in size, urceolate The lorica is urceolate, about 60–70 × 30–35 μm in with a short and oblique neck, aperture circular and size and with a colour the same as the operculum (Fig.

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 MORPHOLOGY AND PHYLOGENY OF VAGINICOLIDS 5 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019

Figure 2. Pyxicola pusilla in vivo (A–N) and after protargol staining (O, P). A–D, lateral views of different individuals, showing the varieties of body shape, lorica transparency and stalk length. E, a contracted individual inside the lorica. F, longitudinally oriented macronucleus. G–J, varieties of lorica shape. K, lateral view of operculum. L, apical view of operculum. M, details of lorica base, mesostyle and endostyle. N, detail of zooid, showing pellicular striations. O, macronucleus, the trochal band and the scopula. P, oral ciliature. Abbreviations: AR, annular ridge; BD, basal disc; CV, contractile vacuole; En, endostyle; G, germinal kinety; H, haplokinety; LS, longitudinal striae; Ma, macronucleus; Me, mesostyle; Op, operculum; Po, polykinety; P1–3, infundibular polykineties 1–3; Sc, scopula; St, stalk; TB, trochal band; Tu, tube. Scale bars = 30 μm.

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 6 B. LU ET AL. Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019

Figure 3. Photomicrographs of Pyxicola pusilla in vivo (A–O) and after protargol staining (P–U). A–G, lateral views of different individuals, showing the varieties of body shape, lorica shape and colour, and stalk length; arrows indicate the contractile vacuole. H, I, two constricted individuals. J, apical view of a contracted individual, showing the aperture of lorica and frontal view of operculum. K, L. lateral view of operculum; arrows indicate the depression of operculum border. M, N. posterior portion of individuals; arrows indicate the mesostyle, arrowhead marks the basal disc. O, showing the striations of pellicle. P, Q. two specimens; arrows and arrowheads indicate macronucleus and the trochal band, respectively. R–T, oral ciliature. U, infundibular polykineties 1–3. Abbreviations: G, germinal kinety; H, haplokinety; P1–3, infundibular polykineties 1–3. Scale bars = 30 μm.

3A–G). The anterior portion of the lorica is constricted turn on opposite walls. Each of the three infundibular and bent, forming a short neck (Figs 2G–J, 3A–G), polykineties (P1–3) is composed of three rows of which is about one-eighth of the lorica in length. The kinetosomes (Figs 2P, 3R–U). The three rows of P1 are aperture is circular and oblique, about 19–22 μm across equal in length and extend to the cytostome (Figs 2P, (Figs 2G–J, 3A–G, J). There are three distinct annular 3R–U). P2 terminates at the convergence of P1 and P3, ridges at the main part of the lorica (Figs 2A–J, 3A–G) row 1 commences slightly before the other two rows that reaches its greatest width at the lowest ridge. The and row 3 being detached from the inner two rows at bottom of the lorica is double-layered and holds the the abstomal end (Figs 2P, 3R–U). P3 is shorter than mesostyle and endostyle within a tube extension (Figs P1 and P2 and terminates slightly behind P2, but 2G–J, M, 3H, I, M, N). clearly before P1. The rows in P3 lie parallel to each The oral ciliature is of the usual sessilid type. other and are equal in length (Figs 2P, 3R–U). The A haplokinety is accompanied by a polykinety and germinal kinety lies parallel to the haplokinety in circles about 1.75 turns around the peristomial disc. the abstomal two-thirds of the infundibulum (Figs 2P, Then they enter the infundibulum and make a further 3R–T). An epistomial membrane was not observed.

Table 1. Morphometric characterization of Chinese populations of Pyxicola pussila (upper line), Cothurnia ceramicola (middle line) and Vaginicola tincta (lower line) based on specimens in vivo (measurements in μm)

Characters Min Max Mean SD CV N

Body length 64 74 67.86 3.34 4.7 7 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 113 158 133.71 16.55 12.4 7 157 243 190.20 26.04 13.7 10 Body width 12 21 18.07 2.83 15.7 7 13 17 14.71 1.25 8.5 7 23 35 26.70 4.06 15.2 10 Diameter of peristomial lip 21 28 23.14 2.41 10.4 7 25 29 28.00 1.22 4.4 9 48 60 53.10 3.73 7.0 10 Lorica length 60 67 62.00 2.50 4.0 7 95 105 100.00 3.96 4.0 7 130 175 148.86 15.35 10.3 7 Lorica width 31 34 32.43 0.98 3.0 7 30 33 31.71 1.11 3.5 7 47 61 55.00 4.16 7.6 7 Diameter of aperture 19 22 20.00 0.96 4.8 7 30 34 32.14 1.35 4.2 7 49 64 57.14 4.98 8.7 7 Lorica length-width ratio 1.82 2.03 1.91 0.07 3.7 7 2.94 3.28 3.15 0.11 3.5 7 2.33 3.13 2.71 0.27 10.0 7 Endostyle length 1.0 1.5 1.33 0.19 14.3 7 2.0 3.0 2.30 0.35 15.2 6 – – – – – – Mesostyle length 2.0 2.6 2.39 0.26 10.9 7 1.5 2.5 2.12 0.37 17.5 6 – – – – – – Stalk length 2.0 19.0 10.29 6.47 62.9 7 3.0 5.0 4.08 0.74 18.1 6 – – – – – – Diameter of operculum 16 18 17.14 0.69 4.0 7 – – – – – – – – – – – – No. of striations from peristome to TBa – – – – – – 50 53 51.00 1.22 2.4 5 74 83 79.00 3.74 4.7 4 No. of striations from TB to scopulaa – – – – – – 56 73 64.75 5.68 8.8 8 76 84 80.20 3.35 4.2 5 No. of total striationsa 62 62 62.00 0 0 1 107 119 113.40 5.03 4.4 5 150 163 158.25 5.91 3.7 4 aRough values. CV, coefficient of variation in %; Max, maximum; Mean, arithmetic mean; Min, minimum; N, number of specimens; TB, trochal band; –, data not available.

Since the zooid is highly contractile and resides in the Genus Cothurnia Ehrenberg, 1831 lorica, the silverline system was unsuccessfully stained Species of Cothurnia usually have one or two zooids in by the silver nitrate method. Nevertheless, the pattern a lorica that lacks any closure apparatus. Zooids attach and number of silverlines could be inferred by the to the lorica base directly or via an endostyle, with transverse striations of the pellicle when viewed in vivo. transverse striations on the pellicle. The shape of lorica

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 8 B. LU ET AL. Tucolesco (1962) (1970) Nusch Trueba (1978) Shen & Gu (2016) Hutton (1878) Kent (1882) Kent (1882) Kent (1882) Stokes (1895) Sommer (1951) Data source original Wrze ś niowski (1866 , 1867 1870 ) Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 BW FW FW FW FW FW FW FW BW FW Habitat FW FW b b b b 1/3 short – 4–60 4–12 short 1/10 1/3–1/2 short Stalk length 2–19 3 1/6 – – – short short – – – inconspicuous Neck of lorica Neck short – – – brown – colourless to brown – colourless – colourless to brown brown Colour of lorica colourless to brown colourless to brown colourless to brown proportion of lorica length. b – annulated – annulated – – – – annulated Feature of Feature lorica annulated annulated commonly annulated 1.9 2.o 2.0 1.6 2.0 2.0 2.0 2.0 2.1 – – c. c. c. c. c. c. c. Shape quotient c. c. – proportion of body length; proportion of body length; a – – 12–14 21–23 (n = 2) 19–28 – – – 17–20 (n = 2) Diameter of aperture 19–22 – – = 2), = – – 25–29 31–38 (n 28–30 – – – 31–34 (n = 2) Lorica width 31–34 22 – 50 75–80 54–58 66–77 (n = 2) 50–55 50 50 80 60–77 (n = 2) Lorica length 60–67 46 50 a a – little – little – little 1/4 little to more less than 1/2 Body protrudes out little little – – – – – – – – 27–38 (n = 2) – Diameter of PL 21–28 16 – – – – – – – – 14–17 (n = 2) – Body width 12–21 – – – – – – – – – 78–82 (n = 2) – Body length 64–74 58 – , measurements in μ m ) Morphometric comparison of different populations Pyxicola pusilla (based on specimens in vivo ,

Pyxicola carteri forma constricta Pyxicola pusilla Pyxicola pusilla Pachytrocha Pachytrocha cothurnoides Pyxicola pusilla Pyxicola affinis Caulicola valvata Pyxicola carteri Pyxicola eforiana Table 2. Table Populations based on original study. The bold indicates these data was data not available; –, peristomial lip; PL, water; brackish BW, fresh water; FW, Abbreviations: Pyxicola pusilla Cothurnia pusilla Cothurnia furcifer

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 MORPHOLOGY AND PHYLOGENY OF VAGINICOLIDS 9 Kent (1869); Trueba (1978) Kent (1882) ; Trueba (1978) Data source Hadži, Hadži, 1940 ; Trueba (1978) List 1 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 Cordylophora or aquatic plant aquatic plant Substrate Microlistra spinosissima aquatic plant 12–130, 12–130, average 80 1–15 Stalk length 15–40 2–60 ** Trueba (1978) ; Shen & Gu (2016) . conspicuous conspicuous Neck of lorica Neck inconspicuous short and conspicuous colourless to brown colourless to brown Colour of lorica – colourless to brown inferred from the drawing. ** never clearly annulated slightly and gentle annulated Feature of Feature lorica slightly and gentle annulated commonly clearly annulated Nusch (1970) ; Tucolesco (1962) ; Nusch 1.7–2.6 2.2–3.0 Shape quotient 2.4–3.3 1.9–2.1 – – – Diameter of aperture 12–23 22–35 20–33 Lorica width 23–35 25–38 45–70 65–96 Lorica length 75–85 45–80 a a proportion of body length; *based on fixed specimens; proportion of body length; a Body protrudes out – 1/6–1/3 commonly just beyond PL 1/4–1/3 Diameter of PL – – 21–38 – Body width – – 12–21 – Body length – – 58–82 – Wrze ś niowski (1866 , 1867 1870 ); Hutton (1878) ; Kent (1882) ; Stokes (1895) Sommer (1951) , measurements in μ m) related congeners (based on specimens in vivo , Morphometric comparison of Pyxicola pusilla with closely

operculigera . Abbreviations: PL, peristomial lip; –, data not available; data not available; –, peristomial lip; PL, Abbreviations: Species Table 3. Table P . psammata * List 1: Original; Original; List 1: P P . pusilla P . carteri

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 10 B. LU ET AL. in this genus is highly diverse. The incomplete data of Germany, and redescribed several times thereafter. most Cothurnia species necessitate a reinvestigation However, its ciliature data is still unavailable. An of this species-rich genus based on modern criteria. improved diagnosis based on previous reports and new data is hereby supplied. Cothurnia ceramicola Kahl, 1933 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 (Figs 4A–L, 5A–R; Tables 1, 4, 5) Improved diagnosis Cothurnia ceramicola was originally reported by Kahl Lorica about 60–130 × 20–45 μm in size, wall in 1933 as an epibiont of the alga Ceramium from Kiel, transparent and colourless, cross-section circular.

Figure 4. Cothurnia ceramicola in vivo (A–J) and after protargol staining (K, L). A, lateral view of an individual with single zooid. B, showing macronucleus in vivo. C, lateral view of an individual with two zooids. D–G, varieties of lorica shape. H, striations of pellicle. I, apical view of zooid, showing the location of contractile vacuole. J, posterior portion of individual, showing lorica base, endostyle, mesostyle, stalk and basal disc. K, showing macronucleus and the trochal band. L, oral ciliature. Abbreviations: AR, annular ridge; BD, basal disc; CV, contractile vacuole; En, endostyle; EM1–2, epistomial membrane 1–2; G, germinal kinety; H, haplokinety; In, infundibular; Ma, macronucleus; Me, mesostyle; Po, polykinety; P1–3, infundibular polykineties 1–3; St, stalk; TB, trochal band; Tu, tube. Scale bars = 50 μm.

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 MORPHOLOGY AND PHYLOGENY OF VAGINICOLIDS 11 Precht Precht (1935) original Kahl (1933) Khal (1935) Precht (1935) Küsters (1974) Song (1992) Sun et al. (2009) Shen & Gu (2016) Data source Felinska Felinska (1965) Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 MW MW MW MW MW MW MW MW FW Habitat MW short 3–5 short short short, striated 7–9, striated 10, striated* short, striated* short, striated* Stalk short short 1.5–2.5 short, striated* short, striated* short, striated* short, striated short, striated* short, striated* short, striated* Mesostyle short, short, striated the span of annular ridges is very large. ** short 2–3, 2–3, striated short, striated* short, striated* short, striated* short, striated short, striated* short, striated* short, striated* Endostyle short ** – annulated slightly annulated* annulated annulated sometimes smooth* annulated smooth* slightly annulated* slightly annulated* Feature of Feature lorica cylindroid cylindroid cylindroid – cylindroid to elliptical elliptical cylindroid cylindroid cylindroid cylindroid Shape 2.4 2.5* 2.5* – 2.9–3.3 – – – – – c. c. c. Shape quotient proportion of body length; *inferred from the drawing; *inferred from the drawing; proportion of body length; a 21* 21* – 30–34 c. c. – – 23–31 – – 29–32 Diameter of aperture 24* 24* – 30–35 c. c. – – 29–35 24–33 20–45 29–32 Lorica width 74–77 95–105 60 60 78–82 65–75 67–102 50–65 110–130 69–76 Lorica length a a a a* a a a a a 1/5 c. 1/4–1/3 more than 1/3 much less than 1/4 1/3 less than 1/2 1/5 1/6–1/3 Body protrudes out 1/3–2/3 – – – – – – – 22–24 25–29 Diameter of PL 26–28 – – – – – – – 14 13–17 Body width 15–17 113–158 – – – – – – 140–220 71–76 Body length 100–150 , measurements in μ m) Morphometric comparison of different populations Cothurnia ceramicola (based on specimens in vivo ,

ceramicola ceramicola ceramicola ceramicola ceramicola ceramicola ceramicola ceramicola ...... C. ceramicola (?) C. C C C C C ceramicola C. C C Table 4. Table Populations data not available; –, peristomial lip; PL, marine water; MW, fresh water; FW, Abbreviations: C

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 12 B. LU ET AL. Data source List 1 Kahl (1933) ; Warren & Paynter (1991) Kahl (1933) ; Warren & Paynter (1991) Entz (1884) ; Warren & Paynter (1991) Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 Stalk 3–9, 3–9, striated 15, striated short short Mesostyle 1.5–2.5, 1.5–2.5, striated short, striated short, striated short Endostyle 2–3, 2–3, striated short, striated short short Feature Feature of lorica 3–5 annular ridges 3–4 annular ridges annular ridges in middle 3 annular ridges in middle Shape cylindroid cylindroid aperture narrowed aperture narrowed 2.3* 2.0 2.0 Shape quotient 2.5–3.3 c. c. c. Diameter of aperture 21–34 30 15 15 Lorica width 20–45 28–35 30 30 Lorica length 60– 130 75–88 60 60 Striations conspicuous conspicuous conspicuous inconspicuous a proportion of body length; *inferred from drawing. proportion of body length; a a a Body protrudes out 1/6–1/2 just beyond 1/3 1/6 Diameter of PL 22–29 – 15–20 20 Body width 13–17 8–10 10 17–20 60 90 105 Body length 71–220 , measurements in μ m) related congeners (based on specimens in vivo , Morphometric comparison of Cothurnia ceramicola with closely

ceramicola . Species data not available; –, peristomial lip; PL, Abbreviations: Table 5. Table List 1: original; Kahl (1933 , 1935 ); (1935) ; Küsters (1974) Sun et al. (2009) . Precht original; List 1: C C . curvula C . fibripes C . harpactici

Aperture about 20–45 μm in diameter, circular and the peristome to the trochal band, 55–75 striations distinctly everted. Zooid about 70–220 × 13–17 μm in from the trochal band to the scopula. vivo, when fully extended, protruding a quarter to a half out the lorica, with a single-layered peristomial lip. Contractile vacuole dorsally located. Endostyle Description based on Qingdao population and mesostyle knot-like and stalk with conspicuous One or two zooids reside in the lorica. The solitary Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 longitudinal striae. Three equally long kinetosomal zooid is relatively smaller and the two co-existing rows in infundibular polykinety 3, terminating before zooids are abreast and anisometric (Figs 4A, C, 5A–F). infundibular polykinety 1. About 50–55 striations from When fully extended, the zooid is 110–160 × 13–17 μm

Figure 5. Photomicrographs of Cothurnia ceramicola in vivo (A–L) and after protargol staining (M–R). A–F, lateral views of different individuals with single zooid (A, C, E, F) and two zooids (B, D); arrow indicates the contractile vacuole. G, the single-layered peristomial lip. H, arrow indicates the contractile vacuole. I, apical view of zooid; arrow indicates the contractile vacuole, arrowhead indicates the infundibulum. J, lateral view of lorica; arrows indicate the annular ridges on the lorica, arrowheads indicate the everted aperture. K, apical view of aperture. L, posterior portion of individual; arrow indicates the mesostyle, arrowhead indicates the lorica base, double-arrowheads indicate the basal disc. M, N, oral ciliature. O, P, two stained specimens. Q, R, oral ciliature; arrow indicates the epistomial membrane 2. Abbreviations: EM1–2, epistomial membrane 1–2; G, germinal kinety; H, haplokinety; P1–3, infundibular polykineties 1–3. Scale bars = 50 μm.

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 14 B. LU ET AL. in size and trumpet-like. The peristomial lip is single- rows in P1 are equally long and their adstomal ends layered, about 25–30 μm across and conspicuously terminate at the same level (Figs 4L, 5Q, R). P2 everted; the peristomial disc projects slightly from the terminates adstomally above the adstomal end of P1 peristomial lip and forms a convex contour (Figs 4A, C, and P3 and the abstomal ends of each row are diverged 5A, H). Pellicular striations can be recognized under a and staggered (Figs 4L, 5Q, R). The rows of P3 are magnification of 200× (Figs 4H, 5A, J). The numbers of parallel and equal in length, terminating adstomally Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 striations from the peristome to the trochal band and just beyond the adstomal ends of P2. Since the outer the trochal band to the scopula are 50–53 (N = 5) and two rows of P3 are very proximal, they sometimes 56–73 (N = 8), respectively. The trochal band is located overlapped in stained specimens (Figs 4L, 5N, Q, R). above mid-body and can be recognized as a high crest The germinal kinety lies parallel to the haplokinety in on the body surface (Figs 4A, C, 5A–F). the abstomal half of the infundibulum (Figs 4L, 5Q, R). The cytoplasm is hyaline without special features, Epistomial membrane 1 is located near the entrance with numerous reflective granules usually less than of the infundibulum (Figs 4L, 5M, Q). Epistomial 1 μm in diameter. Several spherical food vacuoles membrane 2 is shorter than epistomial membrane are scattered throughout the cytoplasm in the upper 1 and located slightly ahead of the distal end of the half of the body. The freshly detached food vacuoles haplo- and polykinety (Figs 4L, 5M). The silverline are generally fusiform. The contractile vacuole is system is in the Vorticella pattern and the number of approximately 10 μm across when fully expanded, silverlines can be determined by counting pellicular situated at the dorsal wall of the infundibulum and striations in cells observed in vivo. its centre is just lower than the peristomial lip (Figs 4A, C, I, 5A, H, I). A vermiform macronucleus stretches almost along the entire body length and is relatively Genus Vaginicola Lamarck, 1816 straight in vivo (Fig. 4B). Due to significant shrinkage Species of this genus have a comparatively simple in the fixation process, the macronucleus is twisted formation. Commonly, one or two zooids attach to the in protargol stained specimens (Figs 4K, 5O, P). The lorica floor via the scopula, with transverse striations micronucleus was not observed. on the pellicle. The lorica attaches to the substrate The attachment apparatus of the zooids and the directly without stalk. Although species of Vaginicola lorica consists of four parts that are continuous with are commonly found in a wide variety of habitats each other, namely endostyle, mesostyle, stalk and and some have been investigated many times, their basal disc. The endostyle is nearly cylindrical, 2–3 μm species identification is exclusively based on live long, while the mesostyle is widened, knot-like and specimens. 1.5–2.5 μm long (Figs 4J, 5L). Endo- and mesostyle pass through the lorica base via a tube extension around them. The non-contractile stalk is 3–5 μm long with Vaginicola tincta Ehrenberg, 1830 an expanded base (Figs 4J, 5L). Striae run lengthways (Figs 6A–I, 7A–U; Tables 1, 6, 7) throughout from mesostyle to stalk. The basal disc is Vaginicola tincta was originally described by colourless, considerably expanded and adheres to the Ehrenberg (1830) and was subsequently redescribed substrate (Figs 4J, 5L). several times (Ehrenberg 1838; Kahl, 1935; Sommer, The lorica is about 95–105 × 30–35 μm in size, with a 1951; Stiller, 1971; Vucetich & Escalante, 1979; ratio of length to width of 2.9–3.3, quasi-cylindrical, the Foissner et al., 1992; Kreutz & Foissner, 2006; Shen & anterior portion slightly narrower than the basal portion Gu, 2016). However, its ciliature is still unknown. We and circular in cross-section (Figs 4A–G, 5A–F, J). The here provide an improved diagnosis based on present aperture is distinctly everted and nearly as wide as the and previous observations. lorica (Fig. 5K). The wall of the lorica is transparent, with three to five continuous annular ridges (Figs 4D–G, 5A–F, J), mainly situated in the upper half. The lorica Improved diagnosis itself tapers to the base in a curve, with a bottom that is Lorica about 75–175 × 30–65 μm, cross-section double-layered and a tube that surrounds and holds the circular. Aperture about 45–65 μm in diameter, endostyle and mesostyle (Figs 4J, 5J, L). circular and everted. Fully extended zooids about The oral ciliature is broadly similar to that of 100–250 × 20–35 μm in vivo, peristomial lip single- most sessilids: the haplokinety and polykinety spiral layered. Contractile vacuole located dorsally at the through a 1.5 turn around the peristomial disc before same level as peristomial lip. Rows of P2 shortened plunging into the infundibulum and diverging within progressively from row 1 to row 3, row 1 merging with the infundibulum to lie on opposite walls (Figs 4L, P1 abstomally and terminates above other two rows, 5M–R). Each of the three infundibular polykineties row 3 divergent from other two rows abstomally. P3 is composed of three rows of kinetosomes. The three composed of three rows, commencing at the same level

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Figure 6. Vaginicola tincta in vivo (A–G) and after protargol staining (H, I). A, B, two individuals with single zooid (A) and two zooids (B). C, D, two contracted individuals. E, striations of pellicle. F, apical view of zooid, showing the location of contractile vacuole. G, macronucleus in vivo. H, showing macronucleus and the trochal band. I, oral ciliature. Abbreviations: CV, contractile vacuole; EM1–2, epistomial membrane 1–2; G, germinal kinety; H, haplokinety; In, infundibulum; Ma, macronucleus; Po, polykinety; P1–3, infundibular polykineties 1–3; TB, trochal band. Scale bars = 80 μm (A–G), 40 μm (H). and terminating slightly above adstomal end of P1; up to about 155–245 μm long and 20–35 μm wide inner row shorter than other two rows and terminates and trumpet-like in vivo. The peristomial lip is earliest. About 70–85 striations from the peristome to single-layered and thin and is significantly wider the trochal band and 75–85 striations from the trochal (48–60 μm in diameter) than the body (Figs 6A, B, band to the scopula. 7A–F). The peristomial disc is elevated above the peristomial lip and has a slightly convex surface (Figs 6A, B, 7I). Pellicular striations with convex ribbing Description based on Ningbo population are conspicuous at magnifications of 400× and higher Usually with a single zooid, sometimes coupled (Figs 6E, 7A, N). There are about 74–83 (N = 4) with a second anisometric zooid (Figs 6A, B, 7A– striations from the peristome to the trochal band F). When solitary, less than one-third of the zooid and 76–84 striations (N = 5) from the trochal band protrudes through the aperture. When two zooids to the scopula. The trochal band can be recognized are present, more than one-third of the larger as a high crest on the body surface in the mid-body zooid protrudes through the aperture. The zooid is region (Figs 6E, 7N).

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Figure 7. Photomicrographs of Vaginicola tincta in vivo (A–N) and after protargol staining (O–U). A–F, lateral views of different individuals with single zooid (A, C–E) and two zooids (B, F), showing varieties of body shape, lorica shape and colour. G, H, two contracted individuals. I, arrow indicates the contractile vacuole. J, apical view of zooid; arrow indicates the contractile vacuole, arrowhead indicates the infundibular. K, apical view of lorica. L, anterior portion of lorica; arrow indicates the everted aperture. M, posterior portion of lorica; arrow indicates the fluctuation of lorica. N, striations of pellicle; arrow indicates the trochal band. O–Q, three stained specimens; arrows indicate the trochal band. R, T, oral ciliature; asterisks indicate the beginning of haplokinety and polykinety. S, U, infundibular polykineties 1–3; asterisks indicate the ending of row1 within P2 and P3. Abbreviations: EM1–2, epistomial membrane 1–2; G, germinal kinety; H, haplokinety; Ma, macronucleus; Po, polykinety; P1–3, infundibular polykineties 1–3. Scale bars = 80 μm (A–H), 40 μm (O–Q).

The endoplasm is transparent and comparatively contractile vacuole is approximately 7 μm in diameter featureless, but full of reflective granules (less than and is situated at the dorsal wall of the infundibulum, 1 μm across). Several comparatively large granules at the same level as the peristomial lip (Figs 6A, B, F, (2–3 μm across) are distributed at the posterior end of 7I, J). The macronucleus is vermiform and extends the the body (Figs 6A, 7A). A few fusiform or globular food entire length of the body, with several inconspicuous vacuoles are scattered throughout the cytoplasm. The curves in vivo (Fig. 6G). The zooid contracts strongly

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 MORPHOLOGY AND PHYLOGENY OF VAGINICOLIDS 17 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 Data source original Kahl (1935) Berger Foissner, & Kohmann (1992) Ehrenberg (1830) Shen & Gu (2016) Sommer (1951) Kreutz & (2006) Foissner Ehrenberg (1838) Stiller (1971) Vucetich & Vucetich Escalante (1979) Colour of lorica colourless to brown colourless brown brown – brown brown brown* brown brown Shape cylindroid cylindroid cylindroid cylindroid – – cylindroid cylindroid* cylindroid cylindroid Shape quotient 2.3–3.2 2.0–2.7 – – – – – – – – Diameter of aperture 49–64 – – – – – – – – 20 Lorica width 47–61 30–53 – 40 – – – – 48 28–30 Lorica length 130–175 75–133 85 85 – – – 110 85 87–105 inferred from drawing. * Striations fine fine – fine – – – – – – a a a a proportion of body length; a Body protrudes out 1/7–1/3 – – – – – 1/4–1/2 1/3 – 1/3 Diameter of PL 48–60 – – – – – 22–28 – – – Body width 23–35 – – – – – – – – – Body length 157–243 – – 100 – 128 155–178 – – 130 , measurements in μ m) tincta (based on specimens in vivo , Morphometric comparison of different populations Vaginicola

Table 6. Table Populations data not available; –, peristomial lip; PL, Abbreviations: V . tincta V . tincta V . tincta V . tincta V . tincta V . tincta V . tincta V . tincta V . tincta V . tincta

on fixation causing the macronucleus to become more twisted in protargol-stained specimens (Figs 6H, 7O, Shen & Gu (2016) List 1 Shen (1980) Penard Penard (1922) ; Shen & Gu (2016) Data source P). The micronucleus was not observed. The lorica is about 130–175 × 45–65 μm in size, nearly cylindrical in shape, with the middle portion

slightly convex (Figs 6A–D, 7A–H) and a circular Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 colourless colourless to brown brown brown Colour of lorica cross-section (Fig. 7K). The diameter of the flared aperture is nearly the same as the widest part of the lorica. Due to the gradual accumulation of ferric deposits on its surface (Kreutz & Foissner, 2006), the lorica changes colour from colourless to dark smooth* annular constriction annular constriction plate Posterior brownish with age (Fig. 7A–H). Sometimes, the ferric deposits are unevenly distributed, forming a relatively transparent area at the posterior and/or a comparatively light-coloured area at the anterior widened* weakly narrowed weakly narrowed weakly narrowed Below aperture (Fig. 7A, B). An inconspicuous annular depression exists at the rear end (Figs 6A–D, 7M). The bottom is moderately flat with a slight curve. Aperture not flared flared flared flared The oral ciliature basically accords with that of other sessilids. The haplokinety and polykinety make a 1.75 turn around the peristomial disc and an additional turn on the opposite walls in the infundibulum (Figs 6I, 7R– Shape truncated cone cylindroid cylindroid cylindroid U). Each infundibular polykinety (P1–3) is three-rowed. The rows in P1 are equally long and diverge slightly at the adstomal ends (Figs 6I, 7R–U). The arrangement 2.5 2.0–3.2 2.5–3.5 c. 1.8–2.0 Shape quotient of the rows in P2 is distinctive in that they become progressively shorter from inside to outside. Row 1 joins in P1 abstomally and terminates above the other two rows, while row 3 detaches from the other two rows Diameter of aperture 49–64 – – – abstomally and terminates at the same level as row 2 (Figs 6I, 7R–U). The abstomal end of P3 is nearly flush Lorica width 30–61 35–42 34–46 65–80 with row 1, ending adstomally above the adstomal ends of row 2 and row 3, which are approximately at the same level (Figs 6I, 7R–U). Occasionally, row 2 terminates Lorica length 75–175 120–150 100–106 115–145

slightly ahead of row 3. The adstomal end of P3 is slightly above that of P1. The germinal kinety accompanies the haplokinety in the abstomal half of the infundibulum – – fingerlike projection – Feature Feature of PD (Figs 6I, 7R, T). There are two epistomial membranes. Epistomial membrane 1 is near the opening of the proportion of body length; *inferred from the drawing. proportion of body length; a a infundibulum (Figs 6I, 7R, T). Epistomial membrane 2 a a a is ahead of the distal end of the haplokinety (Figs 6I, 7T). 1/7–1/2 Body protrudes out 1/3 1/2 1/4

Phylogenetic trees Diameter of PL 48–60 – 29 – Trees based on concatenated genes Both phylogenetic trees generated with ML and Body width 23–35 – 20–23 – BI methods are congruent with respect to major nodes, so only the ML tree is shown here, with nodal support provided from both algorithms (Fig. 8). The Body length 100–243 235–260 182 150–185 loricate vaginicolids are divided into two major clades, representing a distinct branch separated from the

, measurements in μ m) related congeners (based on specimens in vivo , tincta with closely Morphometric comparison of Vaginicola aloricate sessilids with relatively strong support (84% ML, 0.96 BI). Cothurnia ceramicola robustly groups with two cothurnids (92% ML, 0.99 BI). Then these cluster together with P. pusilla (69% ML, 1.00 BI) and these Species V . tincta data not available; –, peristomial disc; PD, Abbreviations: Kreutz & Foissner (2006) ; Shen & Gu (2016) . Berger & Kohmann (1992) ; Kreutz & Foissner Ehrenberg (1830 , 1838 ); Kahl (1935) ; Stiller (1971) Foissner, original; List 1: Table 7. Table V . ceratophylli V . festivus V. plicata V.

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Figure 8. Phylogenetic tree inferred from concatenated genes (SSU rDNA, ITS1-5.8S rDNA-ITS2, LSU rDNA), revealing the phylogenetic relationship within the family Vaginicolidae. Numbers near nodes denote maximum likelihood (ML) bootstrap values and Bayesian inference (BI) posterior probability, respectively. Asterisks indicate disagreements between ML and BI. The scale bar indicates five substitutions per 100 nucleotide positions. The out group includes Glaucoma chattoni, Ichthyophthirius multifiliis, Tetrahymena corlissi and Tetrahymena pyriformis. then form a clade with V. tincta. The other major clade is topology of the ML tree is broadly consistent with the fully supported (100% ML, 1.00 BI) and comprises three concatenated genes tree. Members of Vaginicolidae species of Thuricola and Vaginicola crystallina. form an independent branch, albeit with low support (42% ML). Cothurnia and Thuricola are both monophyletic. However, Vaginicola tincta occupies the Trees based on SSU rDNA basal position in the Vaginicolidae clade, although with The entire ML tree and the portion of the BI tree low support (37% ML). In the BI tree, the vaginicolids that has a different topology are shown in Fig. 9. The form a polytomy.

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Figure 9. Phylogenetic tree inferred from SSU rDNA sequences, revealing the phylogenetic relationship within the family Vaginicolidae. The topology of Vaginicolidae with the BI analyses is shown here because it different from the ML analyses. Numbers near nodes denote maximum likelihood (ML) bootstrap values and Bayesian inference (BI) posterior probability, respectively. Asterisks indicate disagreements between ML and BI. The scale bar indicates five substitutions per 100 nucleotide positions. The out group includes Glaucoma chattoni (X56533), Ichthyophthirius multifiliis (U17354), Tetrahymena corlissi (U17356) and Tetrahymena pyriformis (EF070254).

Trees based on ITS1-5.8S-ITS2 region the monophyly of Vaginicolidae. Vaginicola tincta The topologies of the ML and BI trees are almost clusters with Pyxicola pusilla (68% ML, 0.99 BI) that identical, therefore, only the ML tree is shown (Fig. together group with a branch uniting Astylozoidae, 10). Unlike the concatenated and SSU rDNA trees, Vorticellidae and Ophrydiidae. Cothurnia ceramicola Vaginicolidae is not monophyletic. The AU test could clusters with a group that includes three thuricolas not statistically significantly reject (P = 0.777 > 0.05) and V. crystallina (51% ML, 0.91 BI).

Table 8. List of species, for which SSU rDNA, ITS1-5.8S-ITS2 region and LSU rDNA sequences were newly obtained

Species SSU rDNA ITS1-5.8S rDNA-ITS2 LSU rDNA

Acc. No. Length of seq. GC% Acc. No. Length of seq. GC% Acc. No. Length of seq. GC% Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 P. pusilla MK184555 1571bp 42.71 MK182738 456 bp 31.58 MK182732 1767 bp 41.88 C. ceramicola MK184556 1615 bp 44.22 MK182739 445 bp 40.00 MK182733 1766 bp 43.60 V. tincta MK184557 2083 bp* 41.28 MK182740 473 bp 29.81 MK182734 1766 bp 41.62 T. obconica – – – MK182741 459 bp 33.33 MK182735 1765 bp 42.04 T. folliculata – – – MK182742 459 bp 33.33 MK182736 1759 bp 42.07 T. kellicottiana – – – MK182743 459 bp 33.12 MK182737 1765 bp 41.93

*containing an intron at 3’ end.

Table 9. Approximately unbiased (AU) test results

Topology constraints AU value (P)

Concatenated tree SSU rDNA tree ITS1-5.8S rDNA-ITS2 tree

Vaginicola 2.00e–04 2.00e–04 0.012 Vaginicolidae – – 0.777

P < 0.05 refute the possibility of monophyly; P > 0.05 do not refute the possibility of monophyly.

Trees based on LSU rDNA lorica shape quotient (i.e. the length divided by the The topologies of the ML and BI trees are almost identical width) is about 2, the aperture is obliquely truncated, (Fig. 11). All six species of Vaginicolidae sequenced in the colour of the lorica changes with age, but is often this work form a branch (75% ML, 1.00 BI) and are brown, and the cell protrudes just beyond the aperture. separated from the aloricate species, which is consistent Our population corresponds with all these common with the concatenated tree. Pyxicola pusilla, V. pusilla features. Accordingly, we believe that the Ningbo and C.ceramicola group together, forming a sister clade isolate is a population of P. pusilla. to the Thuricola clade (77% ML, 1.00 BI). However, P. Before the genus Pyxicola was created, these pusilla clusters with V. tincta with low support (53%) in operculated loricate peritrichs were treated as the ML tree, whereas it clusters with C. ceramicola with cothurnids. Thus, some populations were placed moderate support (0.77) in the BI tree. in the genus Cothurnia at one time or other. The isolate reported by Hutton (1878), namely Cothurnia furcifer, matches perfectly with Wrześniowski’s form (Fig. 12B) and thus can be considered as P. pusilla. DISCUSSION Pyxicola affinis was first described by Kent (1882) and subsequently cited by Blochmann (1886) and Hickson Comments on Pyxicola pusilla (1903) as Cothurnia affinis and shows a larger lorica (Fig. 12; Tables 2, 3) (80 × 40 μm vs. 46 × 22 μm). It has a longer stalk than This species was first described as Cothurnia pusilla Wrześniowski’s form (27–40 μm vs. 3 μm) (Fig. 12C). (Wrześniowski, 1866). Kent (1882) established the Pachytrocha cothurnoides, reported by Kent (1882) and genus Pyxicola for the operculated loricate peritrichs also as Cothurnia cothurnoides by Blockmann (1886), and reassigned this species as P. pusilla. This species corresponds in all points with Pyxicola pusilla except has been described multiple times under different that the indurate operculum of Pyxicola is replaced by names and the descriptions for this form vary, a fleshy pad for this isolate (Blochmann, 1886) (Fig. especially in respect to the annular ridges of the lorica 12D). We agree with Kahl (1935) and Trueba (1978) who (inconspicuous to conspicuous) and the length of the suggested that this isolate was very likely a mutilated stalk (from 3–60 μm; Kent, 1882; Stokes, 1895; Finley Pyxicola pusilla with a fallen operculum. Stokes & Bacon, 1965; Nusch, 1970; Stiller, 1971; Trueba, (1895) reported a new genus and species, Caulicola 1978; Shen & Gu, 2016). Even so, there are some valvata, largely because the operculum adheres to the common features in these reports. For example, the aperture rather than the zooid, but this was probably

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Figure 10. Phylogenetic tree inferred from ITS1-5.8S-ITS2 region sequences, revealing the phylogenetic relationship within the family Vaginicolidae. Numbers near nodes denote maximum likelihood (ML) bootstrap values and Bayesian inference (BI) posterior probability, respectively. Asterisks indicate disagreements between ML and BI. The scale bar indicates ten substitutions per 100 nucleotide positions. The out group includes Ichthyophthirius multifiliis (DQ270016) and Tetrahymena pyriformis (KX832097). an optical illusion (which in fact also occurred in our out of the lorica (one-third vs. significantly less observations; Fig. 12E). So, we agree with Trueba’s than one-third in the original and our population). view in treating C. valvata as a junior synonym of P. Furthermore, it corresponds well with both the pusilla. Trueba (1978) considered that P. carteri sensu original description and present population (Fig. 12G). Sommer, 1951 is a population of P. pusilla. We follow Nusch (1970) described a form under the name of the proposal, because the shape quotient of its lorica is Pyxicola carteri forma constricta and considered that 2 and the contractile vacuole is situated at the same Pyxicola nolandi Finley & Bacon, 1965 is a synonym. position as in our population (from the drawing; Fig. However, the lorica of this form is plumper than in 12F). Pyxicola eforiana reported by Tucolesco (1962) the original population with a shape quotient about from brackish water is also a synonym of P. pusilla. 2. Thus, we agree with Trueba (1978) in regarding The lorica of this form is larger than in the original this as a population of P. pusilla. In addition to the population (75–80 vs. 46) and our population (75–80 above forms, three other populations of P. pusilla were vs. 60–67), and a larger proportion of the body projects described (Kent, 1882; Trueba, 1978; Shen & Gu, 2016).

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Figure 11. Phylogenetic trees inferred from LSU rDNA sequences, revealing the phylogenetic relationship within the family Vaginicolidae. Numbers near nodes denote bootstrap values. The scale bar indicates five substitutions per 100 nucleotide positions. The out group includes Ichthyophthirius multifiliis (EU185635) and Tetrahymena pyriformis (X54004).

These are similar to each other and correspond with outside the lorica, with a small contractile vacuole the original description. Unfortunately, some previous just below the peristomial lip (from Fig. 8 from pl. 2 in descriptions are cursory and the photomicrographs of Leidy, 1882) and its lorica shape quotient is 2.5–3.0; all P. pusilla are only available from Trueba’s paper. It these characters are divergent with P. pusilla. Thus, is possible, therefore, that there are some mistakes in we consider P. annulata to be a synonym of P. carteri. the above synonymy list. Pyxicola annulata described The other three species in the P. pusilla complex, by Leidy (1882) was divided into two forms by Trueba namely Pyxicola carteri Kent, 1882, Pyxicola operculigera (1978) and considered as synonyms of P. pusilla (Kent, 1869) Kent, 1882 and Pyxicola psammata Hadži, and P. carteri, respectively. However, in this case 1940, should be compared with P. pusilla, considering, in Trueba’s opinion is contestable: although the form particular, that P. pusilla possesses an urceolate lorica with comparatively conspicuous annular ridges was smaller than 100 μm with an annulated wall and a treated as P. pusilla, one-fifth of the body protrudes habitat in freshwater or brackish water. Pyxicola carteri

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Figure 12. Different isolates of Pyxicola pusilla (A–H) and its similar species (I–K), different isolates under the names of Cothurnia ceramicola (L–R) and its similar species (S–U), different isolates under the names of Vaginicola tincta (V–Z) and its similar species (Z1–Z4). A, Cothurnia pusilla sensu Wrześniowski, 1866. B, Cothurnia furcifer sensu Hutton, 1878. C, Pyxicola affinis sensu Kent, 1882. D, Pachytrocha cothurnoides sensu Kent, 1882. E, Caulicola valvata sensu Stokes, 1895. F, Pyxicola carteri sensu Sommer, 1951. G, Pyxicola eforiana sensu Tucolesco, 1962. H, after Shen & Gu, 2016. I, Pyxicola carteri (from Kent, 1882). J, Pyxicola operculigera (from Kent, 1869). K, Pyxicola psammata (from Hadži, 1940). L, after Kahl, 1933. M, after Precht, 1935. N, after Felinska, 1965. O, after Küsters, 1974. P, after Song, 1992. Q, after Sun et al., 2009. R, after Shen & Gu, 2016. S, Cothurnia curvula (from Entz, 1884). T, Cothurnia harpactici (from Kahl, 1933). U, Cothurnia fibripes (from Kahl, 1933). V, Vaginicola tincta after Ehrenberg, 1838. W, after Kahl, 1935. X, after Stiller, 1971. Y, after Vucetich & Escalante, 1979. Z, after Shen & Gu, 2016. Z1, Vaginicola ceratophylli (from Penard, 1922). Z2, Vaginicola lagena (from Vucetich & Escalante, 1979). Z3, Vaginicola plicata (from Shen, 1980). Z4, Vaginicola festivus (from Shen & Gu, 2016). Scale bars = 30 μm.

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 MORPHOLOGY AND PHYLOGENY OF VAGINICOLIDS 25 differs from P. pusilla in having a slenderer lorica (shape cylindroid), has a smooth pellicle (vs. clearly striated quotient about 2.5 vs. 2.0) with a longer neck and a body pellicle) and a stalk of about 10 μm long (vs. 3–9; that protrudes up to one-third of its length outside the Song, 1992). Sun et al. (2009) recorded a population lorica (vs. just the peristomial lip beyond aperture; Fig. of C. ceramicola in the Yellow Sea, which corresponds 12I) (Kent, 1882; Trueba, 1978). Pyxicola operculigera closely with our population. They are conspecific, the possesses a long stalk, usually longer than the lorica only difference being that the P3 is composed of two Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 and even up to 150 μm, which is much longer than the rows of kinetosomes, although this is probably an 2–60 μm long stalk of P. pusilla. Moreover, its lorica is illusion caused by the overlapped row 2 and row 3 never clearly annulated, whereas it is commonly and (Fig. 12Q). Shen & Gu (2016) depicted a species as C. clearly annulated in P. pusilla (Fig. 12J) (Kent, 1882; ceramicola, but this was probably a different species, Trueba, 1978). The lorica of P. psammata is much because of its occurrence in freshwater (Fig. 12R). slenderer than that of P. pusilla (shape quotient from Cothurnia ceramicola is mostly characterized by its 2.4–3.3, average 2.9 vs. about 2.0) and the operculum of marine habitat, annulated wall and combination of the former is smaller than that of the latter (7 μm across endostyle–mesostyle-stalk. Based on these characters, vs. 12–18 μm across; Fig. 12K) (Hadži, 1940). three closely similar species should be compared, i.e. C. curvula Entz, 1884, C. harpactici Khal, 1933 and C. fibripes Kahl, 1933. Cothurnia curvula can be separated Comments on Cothurnia ceramicola from C. ceramicola by the curved anterior portion of the (Fig. 12; Tables 4, 5) lorica (vs. straight), the obviously narrowed aperture The lorica of our population is much larger than Kahl’s (vs. equal to the lorica width) and the small proportion (95–105 vs. 60) and with relatively distinct annular of the body that projects through the aperture (just the ridges compared to the slight ridges in Kahl’s form (from peristomial lip vs. 1/6–1/3 of the body length; Fig. 12S) the drawing). We think these are population-dependent (Entz, 1884). Cothurnia harpactici is close in size to differences, because unstriated, partly striated and C. ceramicola, but its stalk is much longer (15 μm vs. completely striated loricas were found in a single species 3–5 μm; Fig. 12T) (Kahl, 1933; Precht, 1935; Warren & in another vaginicolid genus, Platycola (Warren & Carey, Paynter, 1991). Cothurnia fibripes is different from C. 1983). Our population matches Kahl’s form with respect ceramicola in its distinctly shorter body length (60 μm to the marine habitat, having a lorica that is cylindroid vs. 95–100 μm) (Kahl, 1935). Furthermore, its lorica in shape, slightly narrower towards the top and 1/4 to 1/3 is plumper than C. ceramicola (shape quotient 2.0 vs. of the body projects outside the lorica (Kahl, 1933). It also 2.9–3.3) with a much narrower aperture (15 μm vs. shares an obviously striated endostyle, as well as a clearly 30–34 μm; Fig. 12U). striated and truncated-cone shaped mesostyle (Fig. 12L). Therefore, we identify it as Cothurnia ceramicola. Comments on Vaginicola tincta Precht (1935) described several populations, also collected from Kiel, that match perfectly with Kahl’s (Fig. 12; Tables 6, 7) population and they are clearly conspecific, except This species is commonly found in freshwater habitats for one form that was found from Gonothyraea and was originally reported by Ehrenberg (1830) and loveni (Allman, 1859) where Precht put a question redescribed in 1838 by himself (Fig. 12V) (Ehrenberg, mark after the specific name, because the double- 1830, 1838). Kahl (1935) made a revision of Vaginicola layered part of the lorica was considerably larger with a fine description of V. tincta (Fig. 12W). Later, and the body protruded less (Fig. 12M). We think Sommer (1951) and Stiller (1971) (Fig. 12X) reported these are population-dependent differences. Felinska two populations of this form. Vucetich & Escalante (1965) reported a form under C. ceramicola, but this (1979) described a form that has a much slimmer identification is suspect, because her population lorica than V. tincta (Fig. 12Y). However, the form differs distinctly from Kahl’s form by the narrowed they described as V. lagena Kahl, 1935 fits better aperture, the larger double-layered proportion (more with V. tincta (Fig. 12Z2). Foissner et al. (1992) made than 1/3 vs. less than 1/4) and the much larger span an overview of V. tincta and provided an elaborate of the annular ridges (Fig. 12N). The lorica of Küsters’ diagnosis. Recently, Shen & Gu (2016) reported that population is longer than Kahl’s (67–102 μm vs. this species has been found many times in China 60 μm) and our population is more similar to Küsters’ (Fig. 12Z). Our population corresponds well with the population than to that of Kahl in the lorica size (Fig. original description and Foissner’s diagnosis (Foissner 12O) (Küsters, 1974). Song (1992) described a form et al., 1992), thus we think it is a population of V. tincta. of C. ceramicola isolated from the surface of Penaeus This species is characterized by its relatively large chinensis (Osbeck, 1765), but the identification is body and large, cylindrical lorica. There are three very doubtful: its lorica is clearly elliptical (vs. invariably similar congeners, namely Vaginicola ceratophylli

(Penard, 1922) Kahl, 1935, Vaginicola plicata Shen, is polyphyletic in the BI tree based on the SSU rDNA 1980 and Vaginicola festivus Li, 2016. They all have a and the trees based on the ITS1-5.8S-ITS2 region. nearly cylindrical lorica with a flared aperture, weakly Although they are divided into two clades in the narrowed portion below the aperture and brown colour. ML tree based on ITS1-5.8S rDNA-ITS2 tree, the Furthermore, all three species have a similar body possibility that they cluster together is not rejected by size and a similar lorica size. Vaginicola ceratophylli the topology test (AU test, P = 0.777 > 0.05). Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 differs from V. tincta by the plate on which the lorica Focusing on the loricate species, the relationship rests (Fig. 12Z1), although it may be that they are between them is basically in accordance with their conspecific if the plate is an optical illusion caused by morphology (Fig. 13). These species can be divided an adhering layer (Penard, 1922; Kahl, 1935; Warren into four groups based on morphology: Thuricola and & Paynter, 1991). Vaginicola plicata is distinguished Vaginicola attach to the substrate directly, although from V. tincta by the wrinkled posterior portion of the Vaginicola lacks a stalk and Thuricola possesses lorica and the flared aperture. These characters are an internal stalk and a closure apparatus (valve); present in our population and perhaps also in other Cothurnia and Pyxicola attach to the substrate via populations of V. tincta, but were overlooked by the a stalk outside the lorica, but Cothurnia lacks the researchers (Fig. 12Z3) (Shen, 1980). So, V. ceratophylli operculum; while Pyxicola has an operculum attached and V. plicata are probably synonymous. Vaginicola to the border of the peristomial lip (Kahl, 1935; Trueba, festivus possesses a finger-like projection at the border 1978, 1980; Clamp, 1991; Warren & Paynter, 1991; Lu of the peristomial disc that is absent in V. tincta (Fig. et al. 2018). 12Z4) (Shen & Gu, 2016). Species of Cothurnia cluster in a clade with good support in SSU rDNA and concatenated trees (Figs 8, 9), indicating that this genus is probably monophyletic. Phylogenetic analyses Pyxicola pusilla is relatively closely related to Among the phylogenetic trees of Peritrichia, the Cothurnia in most trees. This is supported by their loricate sessilids tend to form a separate clade from morphology in that they have a lorica that is attached to aloricate sessilids, although the family Vaginicolidae a substrate via a stalk (Fig. 13). In contrast, Thuricola

Figure 13. Morphological differences and correlations of the six sequenced vaginicolids in this work.

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 1–30 MORPHOLOGY AND PHYLOGENY OF VAGINICOLIDS 27 lacks a stalk to attach to the substrate, a feature at King Saud University (Research Group Project No. clearly separating it from Pyxicola and Cothurnia. RGP-083). We especially thank Dr Feng Gao (OUC) Three thuricolas are grouped in a clade in all trees and Ms Tengteng Zhang (OUC) for their valuable help with low to full support, showing the monophyly of this with the phylogenetic analysis. genus. Although the length of Thuricola branches is short in all trees, their morphologies show consistent Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/1/1/5434147/ by Ocean University of China user on 08 October 2019 and distinct differences. 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