J. Ocean Univ. China (Oceanic and Coastal Sea Research) https://doi.org/10.1007/s11802-019-3897-3 ISSN 1672-5182, 2019 18 (3): 643-653 http://www.ouc.edu.cn/xbywb/ E-mail:[email protected]

Morphology and Molecular Phylogeny of Two Little-Known Species of Loxodes, L. kahli Dragesco & Njiné, 1971 and L. rostrum Müller, 1786 (Protist, Ciliophora, Karyorelictea)

WANG Lun, QU Zhishuai, LI Song, and HU Xiaozhong*

Institute of Evolution and Marine Biodiversity, Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China

(Received April 26, 2018; revised May 2, 2018; accepted December 5, 2018) © Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2019

Abstract The morphology and phylogeny of two little-known species, Loxodes kahli Dragesco & Njiné, 1971 and L. rostrum Müller, 1786, isolated from freshwater muddy sediments in China, were investigated based on live features, infraciliature, and small subunit ribosomal DNA (SSU rDNA) sequence data. Loxodes kahli is distinguished from its congeners mainly by the num- ber and arrangement of macronuclei (6–17 in one row) and the number of right somatic ciliary rows (11–26). The Chinese popula- tions of L. kahli also exhibit differences with other populations in terms of the body size and the number of right ciliary rows. The characteristics of L. rostrum are consistent with those of previous studies except for the number of right ciliary rows (9–10). The studied species were redefined based on the new information and previous descriptions. This study also gave a brief morphological summary of the species in the genus Loxodes by an identification key. SSU rDNA sequence-based phylogenetic analyses revealed that both species are grouped with their congeners, supporting the monophyly of the genus Loxodes.

Key words anaerobe; benthic ciliate; ciliature; Loxodida; SSU rDNA

chel and Finlay, 1984; Finlay, 1985; Fenchel and Finlay, 1 Introduction 1986; Finlay et al., 1986; Buonanno, 2005; Buonanno et al., 2005) or L. magnus Stokes, 1887 (Goulder, 1972; Since the introduction of a new fixative, and the com- Bobyleva et al., 1980; Raikov, 1982, 1994, 1996). More- bination of Wilbert’s protargol and Fernandez-Galiano’s over, the detailed information on the ciliature is only silver carbonate staining methods, the infraciliature data available for these species (Foissner and Rieder, 1983; of more than 40 species of karyorelicteans have been Kim et al., 2009) and L. vorax Stokes, 1885 (Xu et al., revealed (e.g., Foissner and Al-Rasheid, 1999; Song et al., 2015). For the remaining species of Loxodes, i.e., L. kahli 2009; Xu et al., 2012, 2013a, b, c, 2014, 2015, 2017; Yan Dragesco and Njiné, 1971, L. rostrum Müller, 1786, and et al., 2013, 2015a, b, 2016a, b, 2017; Liu et al., 2017). L. rex Dragesco, 1970, more detailed information re- Similar to most other marine karyorelicteans, members of garding the oral and somatic ciliature is still lacking. the genus Loxodes Ehrenberg, 1830 are often laterally Loxodes kahli was firstly discovered in Cameroon, Af- flattened and live in various freshwater environments, rica by Dragesco and Njiné (1971), who provided a brief such as muddy sediments and anaerobic water columns (Lynn, 2008). As the only freshwater representative of description, including the size, the number of macronu- karyorelicteans, Loxodes species have been widely used clei and micronuclei, and a sketch of ciliature. From then in the study of the nuclear division (Bobyleva et al., 1980; on, no other descriptions were available until the organ- Raikov, 1982, 1994, 1996), structure and function of ism was identified in South Korea (Kim et al., 2009). Müller vesicles (Fenchel and Finlay, 1986), perception However, in these investigations there was not statistic and reaction to oxygen and nitrate (Finlay, 1985; Finlay information about the detailed ciliature and DNA se- et al., 1986), defensive behavior (Buonanno, 2005; Buo- quences. L. rostrum was firstly identified by Müller (1786). nanno et al., 2005), geotaxis (Fenchel and Finlay, 1984; Then Raikov (1959) provided the related nuclear infor- Neugebauer et al., 1998), and grazing behavior (Goulder, mation, and Dragesco (1970), Dragesco and Dragesco- 1972). However, these investigations mainly focused on Kernéis (1986), and Foissner (1995) all gave similar de- L. striatus Penard, 1917 (Raikov, 1982, 1994, 1996; Fen- scriptions of L. rostrum. However, there was not detailed ciliature and molecular information in these descriptions. * Corresponding author. Tel: 0086-532-82031610 In this paper, we studied L. kahli collected from two E-mail: [email protected] freshwater habitats in Qingdao and Shenzhen, and L.

644 WANG et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2019 18: 643-653 rostrum isolated from freshwater habitat in Zhanjiang, 2016 from the sediments of Buji River, Shenzhen, south- China with the detailed description of its morphology. ern China (22˚32΄N, 114˚06΄E), where the water tem- With our knowledge, this is the first time to study these perature was about 22℃ (Fig.1). L. rostrum was col- species. In addition, we summarized the morphological lected on April 14, 2014 from the sediments in a fresh- characters of the six species within this genus and ana- water lake in Huguangyan scenic spot in Zhanjiang, Chi- lyzed the evolutionary position of Loxodes based on na (21˚08΄N, 110˚17΄E), where the water temperature was small subunit ribosomal DNA (SSU rDNA) sequence about 23℃ (Fig.1). The cells were isolated by micro- data. pipettes and observed in vivo using bright-field and dif- ferential interference microscopy. The ciliature was re- 2 Materials and Methods vealed by the protargol staining method (Wilbert, 1975). The counts and measurements on stained specimens were 2.1 Sampling and Morphological Investigations performed at a magnification of 1000 × (Microscope The first population of L. kahli was collected on May Olympus BX53). The line drawings were made by free- 25, 2016 from the sediments in a freshwater pond in hand sketches or with the help of the camera lucida. The Baihuayuan Park, Qingdao, northern China (36˚03΄N, terminology and systematics are mainly in accordance 120˚21΄E), where the water temperature was about 20℃ with the works of Foissner (1996) and Gao et al. (2016), (Fig.1). The second one was collected on December 15, respectively.

Fig.1 Sampling location. A, A pond in Baihuayuan Park, Qingdao (36˚03΄N, 120˚21΄E); B, Buji River, Shenzhen (22˚32΄N, 114˚06΄E); C, A lake in Huguangyan Park, Zhanjiang (21˚08΄N, 110˚17΄E).

karyotic universal reverse primer 18SR (5’-TGA TCC 2.2 DNA Extraction, Polymerase Chain Reaction TTC TGC AGG TTC ACC TAC-3’) (Medlin et al., 1988). (PCR), and Gene Sequencing The PCR products were sequenced bidirectionally in the The genomic DNA extraction, PCR, and sequencing of Tsingke Biological Technology Company (Qingdao, China). the SSU rDNA gene were performed as described in the The contigs were assembled by Seqman (DNAStar). work of Gao et al. (2013). One or more cells were iso- lated from the environmental samples and washed for 2.3 Phylogenetic Analyses several times with filtered water from the sampled envi- The phylogenetic analyses, which were performed ac- ronment (0.22 µm) to remove contaminants. The cells cording to the study of Wang et al. (2017), included three were then transferred to a 1.5 mL Eppendorf tube with a new sequences in the present study and another 46 se- minimum volume of water. The genomic DNA was ex- quences that were downloaded from the National Center tracted using DNeasy Blood & Tissue Kit (Qiagen, Hil- for Biotechnology Information GenBank database (ac- den, Germany) following the manufacturer’s instructions. cession numbers shown in Fig.6). All the sequences were The PCR amplification of SSU rDNA was performed by aligned using the GUIDANCE2 Server (Sela et al., 2015) Q5 PremixTaq with the forward primer (5’-GCC AGT with default settings, and the resulting alignment was AGT SAT ATG CTT GTC T-3’) designed by our col- manually modified using BioEdit v.7.2.5 (Hall, 1999). league, Mr. Weibo Zheng (Yan et al., 2016b) and the eu- The maximum likelihood (ML) analyses were performed

WANG et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2019 18: 643-653 645 in CIPRES Science Gateway (URL: http://www.phylo.org/ sub_sections/portal) (Miller et al., 2010) using RAxML- HPC2 (Stamatakis et al., 2008) on XSEDE v.8.1.24 with 3 Results and Discussion the GTRGAMMA+I model. The reliability of internal 3.1 L. kahli Dragesco & Njiné, 1971 (Figs.2A–2F branches was assessed using a nonparametric bootstrap and 3; Table 1) method with 1000 replicates. The Bayesian inference (BI) analyses were carried out by MrBayes on XSEDE 3.2.6 This organism has been redescribed only once (Kim in CIPRES Science Gateway with the model GTR+I+G et al., 2009) since it was firstly reported by Dragesco and as selected by MrModeltest v.2.2 (Nylander, 2004). Njiné (1971), who were cited by Dragesco and Dragesco- Markov chain Monte Carlo simulations were run with Kernéis (1986). However, no detailed information is two sets of four chains for 6000000 generations with a available about its infraciliature, especially the oral ap- sampling frequency of 100 generations, and 25% of them paratus. On the basis of the new data, the discussion be- were discarded as burn-in. MEGA v.7.0.21 was used to low provides an improved diagnosis and redescription of visualize the tree topologies. the species.

Fig.2 L. kahli in vivo (A) and after protargol staining (B–F); L. rostrum in vivo (G) and after protargol staining (H–J). A, G, Right side of a representative individual. B, C, H, I, General ciliature of right (B, H) and left (C, I) side; arrowheads in B and C indicate the sharp posterior end of body in the Qingdao population. D, Typical distribution of macronuclei and micronuclei of the Shenzhen population, which are more densely arranged than those in C. E, J, Ciliature of buccal area, showing the right buccal kinety, left outer buccal kinety, left inner buccal kinety, and right lateral somatic ciliary row. F, Cortical granules in the anterior body, with a dense distribution in the buccal area and around the pharyngeal tube. IK = intrabuccal kinety; LC = left lateral ciliary row; LIK = left inner buccal kinety; LK = dorsolateral somatic ki- nety; LOK = left outer buccal kinety; Ma = macronucleus; Mi = micronucleus; RC = right lateral somatic ciliary rows; RK = right buccal kinety. Scale bars = 75 µm (A–D), 50 µm (G) and 80 µm (H, I).

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Fig.3 L. kahli in vivo (A–H) and after protargol staining (I–Q). D and B are from the Shenzhen population, the others from the Qingdao population. A. Right side of a representative individual, arrowheads indicate the Müller vesicles. B, C, Different body shapes; arrowheads and arrow indicate the Müller vesicles and the sharp posterior end, respectively. D, The buccal area showing the cortical granules (arrowheads) tightly arranged in the pharyngeal tube. E, Detailed view of the anterior body region indicate the Müller vesicles (arrowheads) and pharyngeal cortical granules (arrows). F, Showing brown cortical granules (arrowheads) and somatic cilia (arrows). G, Right lateral somatic cilia (arrowheads). H, Macronuclei (arrowheads). I, Ciliature of the right side of an individual. J, Ciliature of the buccal area showing the right buccal kinety (arrowheads). K, Ciliature of the buccal area indicating the sparsely placed left inner buccal kinety (arrowheads). L, Arrowheads indicating dorsolateral somatic kinety. M, Macronuclei and micronucleus N, Intrabuccal kinety (red lines) and LIK (arrowheads). O, Left outer buccal kinety (arrowheads). P, Left side of anterior region dem- onstrating the left lateral ciliary row (arrowheads). Q, Right side of posterior region, with the dorsolateral somatic ki- nety ending at a sharp tip of the posterior end of cell (arrowhead). LK = dorsolateral somatic kinety; RC = right lateral somatic ciliary rows. Scale bars = 80 µm (A, B, and I), 100 µm (C) and 20 µm (E).

Improved diagnosis matic ciliary row. The right buccal, left outer buccal, left

Cell size in vivo about (130–600) µm × (30–70) µm. inner buccal, and intrabuccal kineties composed of 50– Buccal field occupying about 1/7–1/5 of body length. 120, 27–60, 3–6 and 25–54 dikinetids, respectively. Six Five to nine Müller vesicles and 11–26 right lateral so- to 17 macronuclei and 5 to 15 micronuclei (ratio: about

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1:1). Brown cortical granules about 0.5 µm in diameter. 13 (Figs.2B and 3I). The dorsolateral somatic kinety com- Description of L. kahli Qingdao population mences at the anterior third, extends to the posterior end Live cells are highly variable in size, ranging from 130 of the cell, and curves around the rear end to the ventral

µm × 30 µm to 350 µm × 70 µm with buccal field occupy- side, enclosing about 6 rightmost lateral somatic ciliary ing 1/5–1/6 of body length (Figs.2A, 3A–3C). They are rows (Figs.2B, 3I, and 3Q). The left lateral ciliary row elongated and exhibit an elliptical outline, with pointed extends around the cell similar to that in the species of ends curved to ventral side. The somatic cilia are ap- genus Remanella (Xu et al., 2015) (Figs.2C and 3P). The proximately 8 µm in length. The cells are transparent and right buccal kinety consists of about 50–95 tightly ar- colorless in the buccal area, and several portions of the ranged dikinetids according to the length of the buccal margin appear brownish at low magnification (Figs.3A– area (Figs.2B, 2E, and 3J). The left outer buccal kinety 3C). The brown cortical granules (about 0.5 µm in di- consists of 27–42 dikinetids (Figs.2B, 2E, and 3O). The ameter) mainly concentrate in the buccal area, around the left inner buccal kinety comprises 3–5 discretely distrib- pharyngeal tube, and are distributed between the ciliary uted dikinetids (Figs.2B, 2E, 3K, and 3N) and is located rows and the left side (Figs.2F, 3D–3F). Müller vesicles at the right of and along the anterior half of the left outer number five to nine, are about 9 µm in diameter, with buccal kinety. Starting from the anterior end (Fig.2C) to barium granules (about 3 µm in diameter) (Figs.3A, 3E). the posterior end (Fig.3N) of the buccal area, the intra- Neither cytoplasmic spicules nor contractile vacuoles are buccal kinety consists of 25–38 dikinetids. The macronu- observed. Cells often glide on the bottom of the Petri clei and micronuclei vary in numbers from 6 to 12, fea- dish or other substances, sometimes slowly swim in the turing a numerical ratio of about 1:1 (Figs.2C and 3I). water. Cells often become twisted during swimming. Deposition of Qingdao specimens Ciliature is formed by dikinetids with only the anterior A voucher slide of protargol-stained specimens was basal body being ciliated (Figs.2B, 2C, and 3I). The right deposited in the Laboratory of Protozoology, OUC, Chi- lateral somatic ciliary rows vary in numbers from 11 to na (registration number: WL 2016052501). Table 1 Morphometric characteristics of Loxodes kahli (upper rows, Qingdao population; middle rows, Shenzhen population) and Loxodes rostrum (lower rows, Zhanjiang population)

Characters Min Max Mean SD CV n 144 243 193 23.3 12.1 26 Body, length 144 280 214 32.6 13.5 29 68 133 101.5 14.6 14.3 27 39 76 54.3 9.7 17.8 26 Body, width 50 125 66.6 14.8 17.6 29 31 47 40.2 5.4 13.4 27 26 52 39.4 6.9 17.4 26 Buccal area, length 25 58 38.9 7.1 18.3 28 26 39 31.7 4.5 14.1 27 11 13 12.4 0.6 5.1 26 Right lateral ciliary rows, number 11 13 11.9 0.6 4.9 28 9 10 9.2 0.4 4.5 28 50 95 67.3 9.9 14.7 19 Dikinetids in right buccal kinety, number 50 120 76.1 14.5 19.4 23 60 85 72.1 7.2 10 12 27 42 32.3 4.2 13 18 Dikinetids in left outer buccal kinety, number 28 60 44.4 9 18.4 15 26 39 31.5 4.4 13.9 11 3 5 3.3 0.6 19.4 10 Dikinetids in left inner buccal kinety, number 3 6 4.1 1.1 28.1 7 3 5 3.6 0.7 18.5 11 25 38 31.3 3.6 11.6 16 Dikinetids in intrabuccal kinety, number 27 51 40.4 7.4 16.2 20 30 39 34.4 4.7 13.6 11 5 12 8.9 1.6 17.5 26 Macronuclei, number 8 17 11.9 2.1 19.5 28 2 2 2 0 0 34 4 12 7.9 1.8 22.8 26 Micronuclei, number 6 15 10.6 2.7 27 28 1 1 1 0 0 34 Notes: Data are from protargol-stained specimens (measurements in μm). Abbreviations: CV = coefficient of variation in %, n = number of specimens investigated, SD = standard deviation.

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Description of L. kahli Shenzhen population kahli from Cameroon, Africa (Fig.4B), whereas Kim et al. Live cells vary from 200 µm × 40 µm to 310 µm × 65 (2009) reported the organism from Taehwagang River in µm in size, with the buccal field occupying 1/6–1/7 of Ulsan, South Korea (Fig.4C). The most significant char- body length, and have an elongated bursiform outline. acters to distinguish this species from its congeners are The somatic cilia are approximately 5 µm in length. The the numbers of nuclei and the right lateral somatic ciliary Müller vesicles vary in numbers of 6–10, and each of rows and body size. them is 9–10 µm across, with the barium granules about 3 In terms of the body shape, biotope, and ciliature data, µm in diameter. L. kahli should be compared with its five congeners (Ta- Other infrastructure data of Shenzhen population are ble 2). L. kahli can be separated from L. vorax, L. striatus, highly similar to those of Qingdao population (Table 1). and L. rostrum by the number of macronuclei (no less Figs.2C and 2D show the differences in the nuclear ar- than 5 versus 2–4) (Dragesco and Dragesco-Kernéis, rangement. 1986; Foissner et al., 1995; Kim et al., 2009; Xu et al., Deposition of Shenzhen specimens 2015). Compared with L. kahli, L. rex shows a larger cell A voucher slide of protargol-stained specimens was size (500–1200 µm versus 130–600 µm) and higher deposited in the Laboratory of Protozoology, OUC, Chi- numbers of macronuclei (132–181 versus 6–17) and right na (registration number: WL 2016121501). lateral somatic ciliary rows (79–84 versus 11–26) (Dra- Remark: Dragesco and Njiné (1971) first identified L. gesco and Dragesco-Kernéis, 1986; Hines et al., 2016).

Fig.4 Previous images of L. kahli (A, B, and C) and L. rostrum (D). A, from Dragesco and Njiné (1971). B, C, from Kim et al. (2009). D, from Dragesco (1970). Scale bars = 100 µm (A–C), 50 µm (D).

Table 2 Morphological comparison of all known Loxodes species Loxodes Loxodes Loxodes Loxodes Loxodes Loxodes Loxodes Loxodes Loxodes Loxodes Loxodes Loxodes

kahli kahli kahli kahli magnus magnus rostrum rostrum rex striatus vorax rex Body length, 115–800 130–350 200–310 470–600 160–300 250–470 about 140 70–300 500–1200 150–300 100–180 550–1350 in vivo (400) Body width, Average 30–70 40–65 40–70 40–200 87–159 about 40 40–70 20–45 in vivo 205 Right ciliary 13–26 18–20 11–13 11–13 25–32 23–26 9–10 10–12 79–84 9–13 8 about 80 rows, number (24) (19) Müller’s vesi- 5–9 6–10 about 10 4–12 10–24 8–18 4–6 2–6 about 60 4–12 3–4 45–60 cle, number Macronuclear 3–31 nodules, 6–12 8–17 6–8 6–11 9–13 2 2 132–181 2–4 2 about 150 (11–23) number Micronuclei, 9–32 6–12 7–15 6–8 5–9 8–13 1 1 1–2 1 about 70 number (12) Present Present Present Reference work, work, a; b c b; d c work, b; d b e; f d; f g Qingdao Shenzhen Zhanjiang Notes: Measurements in µm. a, Dragesco and Njiné, 1971; b, Dragesco and Dragesco-Kernéis, 1986; c, Kim et al., 2009; d, Foissner et al., 1995; e, Foissner and Rieder, 1983; f, Xu et al., 2015; g, Hines et al., 2016.

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L. magnus is the most similar species with L. kahli. However, the African population possesses 13–26 right The former can be distinguished from the three Asian ciliary rows (Dragesco and Njine, 1971), which perfectly populations of the latter by the following characteristics: cover the range of the South Korean population and 1) the arrangement of macronuclei (two rows versus one overlap with that of the Chinese populations. The African row); 2) the number of Müller’s vesicles (mostly more population is considerably larger (470–600 µm versus no than 10 versus fewer than 10); 3) more right lateral so- longer than 350 µm). On the other hand, the numbers of matic ciliary rows (25–32 versus 11–26). The morpho- Müller’s vesicle, macronuclei, and micronuclei in the metric data of the African population of L. kahli (Dra- four populations match perfectly. In conclusion, the dif- gesco and Njiné, 1971) overlap with those of L. magnus; ferences in body sizes and numbers of right lateral so- however, they can be distinguished by the arrangement matic ciliary rows are population-dependent. of macronuclei (in one row versus in two rows) (Table 2). 3.2 L. rostrum Müller 1786 (Figs.2G–2J and 5; Table 1) Intraspecific variations exist among the four L. kahli The Zhanjiang population of L. rostrum matches well populations: the characters of the two Chinese popula- with that reported in the previous studies (Dragesco and tions match well except that the Shenzhen population Dragesco-Kernéis, 1986; Foissner et al., 1995). However, contains more macronuclei and micronuclei. The South the range of a significant characteristic, which is the Korean population possesses more right somatic ciliary number of right lateral somatic ciliary rows, is broader. rows than the Chinese populations (18–20 versus 11–13) We present more detailed information about ciliature (Kim et al., 2009), whereas their body sizes are similar. here.

Fig.5 L. rostrum in vivo (A–F) and after protargol staining (G–K). A, Right side of a cell showing the body shape. B, Food vacuoles and green algae (arrowheads). C, Detailed view of Müller vesicles, macronuclei (arrows), micronucleus (arrowhead), and lithosome. D, Cortical granules (arrowheads) and the right lateral somatic ciliary rows (arrows) around the buccal area. E, Cortical granules (arrowheads) inside the buccal area. F, Detailed view of the mid-body mar- gin, showing cortical granules on the right side along the ciliary rows and cilia (arrowheads). G, Intrabuccal kinety (ar- rowheads). H, Right side of the posterior end of the cell indicating the dorsolateral somatic kinety. I, Müller vesicles (arrowheads) and lithosome. J, Ciliature of the buccal area showing the right buccal kinety, left outer buccal kinety, left inner buccal kinety, and right lateral somatic ciliary rows. K, Ciliature of the right side of a representative individual. L = lithosome; LIK = left inner buccal kinety; LK = dorsolateral somatic kinety; LOK = left outer buccal kinety; M = Müller vesicles; Ma = macronucleus; RC = right lateral somatic ciliary rows; RK = right buccal kinety. Scale bars = 75 µm (A, K).

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Improved diagnosis middle of the cell. The main differences between these

Cell size in vivo 70–300 µm long. The buccal field oc- species are the number of right lateral somatic ciliary cupying about 1/3 of the body length. Nine to 12 right rows (8 in L. vorax versus 9–12 in L. rostrum), spindle- lateral somatic ciliary rows. The right buccal, left outer shaped symbiotic green algae (absent in L. vorax versus buccal, left inner buccal, and intrabuccal kineties com- present in L. rostrum), and the permanent vacuole at the posed of 65–85, 26–39, 3–5, and 30–45 dikinetids, re- posterior end of the cell (present in L. vorax versus ab- spectively. Two macronuclei and one micronucleus. sent in L. rostrum) (Table 2; Foissner, 1995; Xu et al.,

Brown cortical granules measuring 0.5 µm in diameter. 2015). Although the permanent vacuole is an invalid Description of Zhanjiang population classification character, L. vorax still can be well distin-

Live cells are about 140 µm × 40 µm in size, with the guished from L. rostrum. buccal field occupying about 1/3 of the body length. The body is elongated and flattened, with a pointed anterior 3.3 Molecular Phylogeny Based on SSU end curving to the ventral side and a rounded posterior rDNA Sequences end (Figs.2G and 5A). The cilia are about 8 µm long. The The newly characterized SSU rDNA sequences of the cells contain several green algae as symbionts, which three isolates were deposited in the GenBank database decrease quickly in number in the laboratory (Figs.2G with respectively the following length (bp), GC content, and 5B). Brown cortical granules are about 0.5 µm in and accession numbers: 1558, 47.24%, and MF988317 diameter, mainly concentrated in buccal area, around the for L. kahli from Qingdao; 1610, 47.27%, and MF98 pharyngeal tube, and distributed between the ciliary rows 8318 for L. kahli from Shenzhen; 1649, 47.91%, and and the left side (Figs.5D–5F). About four to six Müller FM988316 for L. rostrum. vesicles that are 12 µm in diameter, with the barium Given the similar topologies of the ML and BI trees, granules about 3 µm across (Figs.2G, 5C, and 5I). Each of only the topology of the ML tree is presented in this pa- these several vesicles includes a red round lithesome per (Fig.6). The only difference between these analyses is measuring about 13 µm across (Figs.5C and 5I). No cy- that the topology of the BI tree includes certain polyto- toplasmic spicules were noted. Locomotion is shown by mies. As described in previous studies (Xu et al., 2015), gliding on the bottom of the Petri dish or other sub- the class Karyorelictea is subdivided into four groups stances and slow swimming in the water. corresponding to four families, i.e., Loxodidae, Geleiidae, The ciliature consists of dikinetids with only the ante- Kentrophoridae, and Trachelocercidae (Lynn, 2008; Gao rior basal body being ciliated. Nine or ten right lateral et al., 2016). Of these families, Loxodidae is a mono- somatic ciliary rows were observed (Figs.2H and 5K). The dorsolateral somatic kinety extends to the posterior phyletic group (50% ML, 0.92 BI), and within the family, end of the cell and curves around the rear end to the ven- the genus Loxodes forms a fully supported clade that is tral side, which encloses about 6 rightmost lateral so- related to the genus Remanella. L. vorax (KJ524909) and matic ciliary rows (Figs.2H and 5H). The left lateral L. rostrum branch off firstly, followed by the clade of L. ciliary row surrounds the cell, as in Remanella species striatus orientalis and L. cf. striatus. The newly charac- (Fig.2I). The right buccal kinety contains 60–85 densely terized L. kahli branches within the group including L. arranged dikinetids (Figs.2H, 2J, and 5J). The left outer striatus (U24248), L. striatus (AM946031), L. vorax (KJ buccal kinety consists of 26–39 dikinetids (Figs.2H, 2J, 651829), and L. magnus (84% ML, 1.00 BI). Although and 5J). The left inner buccal kinety comprises three to the validation of the genus Remanella lacks supporting five discretely distributed dikinetids (Figs.2H, 2J, and 5J), molecular results, Loxodes and Remanella can be sepa- and is located at the right of and along the anterior half of rated morphologically (Xu et al., 2015). From the mo- the left outer buccal kinety. The intrabuccal kinety ex- lecular results, we discovered an interesting pheno- tends from the anterior end to the posterior end of the menon in which sequences of L. kahli are almost identi- pharyngeal tube, comprising 30–45 dikinetids (Figs.2I cal with South Korean population of L. vorax (KJ65 and 5G). 1829). Only one different nucleotide was observed. How- Deposition of Zhanjiang specimens ever, the South Korean population of L. vorax (KJ651829) A voucher slide of protargol-stained specimens was feature 26 different nucleotides from those of the Zhan- deposited in the Laboratory of Protozoology, OUC, jiang population of L. vorax (KJ524909), which provided China (registration number: QZS 2014041401). reliable morphological data. Thus, L. vorax (KJ651829) Remark: The Chinese population of L. rostrum is al- is likely a misidentified population of L. kahli especially most identical with that reported in previous studies (Ta- in the absence of reliable morphological data. During ble 2). The only difference is the number of right lateral sampling and sequence extraction, we noted that several somatic ciliary rows (9–10 versus 10–12) (Dragesco and species of Loxodes can be included in one sample, pos- Dragesco-Kernéis, 1986; Foissner et al., 1995). The other sibly resulting in misidentification. We suggest that characteristics perfectly match. Within the genus, L. microscopic examination should be imperative before vorax is most similar to L. rostrum according to the body DNA extraction and gene sequencing to avoid such size and the two macronuclei, with a micronucleus in the problems.

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Fig.6 ML tree inferred from the SSU rDNA sequences showing the positions of three newly sequenced Loxodes isolates (arrows). Nodal support for branches in the ML and BI trees are marked in order. Black dots indicate full support in both analyses (100% ML, 1.00 BI). Heterotrichs are selected as the outgroup taxa. All branches are drawn to scale. The scale bar corresponds to five substitutions per 100 nucleotide positions.

with the present work, we proposed an identification key 3.4 Identification Key of Genus Loxodes to distinguish the species within the genus Loxodes as According to the previous studies and combined them follows:

1. More than 100 macronuclei scattered within the body...... L. rex - Fewer than 100 macronuclei ranged in rows or groups...... 2 2. More than 5 macronuclei ranged in one or two rows...... 3 - Less than 5 macronuclei in one or two groups...... 4 3. 23–32 macronuclei ranged in two rows along the body...... L. magnus - 11–26 macronuclei ranged in one row along the body...... L. kahli 4. 2–4 macronuclei divided into two groups...... L. striatus - Two macronuclei with one micronucleus in the middle of the body...... 5 5. 8 right ciliary rows...... L. vorax - 9–12 right ciliary rows...... L. rostrum

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