applyparastyle “fig//caption/p[1]” parastyle “FigCapt” 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).