Morphology and phylogeny of two species of Loxodes (Ciliophora, Karyorelictea), with description of a new subspecies, Loxodes striatus orientalis subsp. n.

Published by the International Society of Protistologists

The Journal of

Eukaryotic Microbiology

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE

Morphology and Phylogeny of Two Species of Loxodes (Ciliophora, Karyorelictea), with Description of a New Subspecies, Loxodes striatus orientalis subsp. n. Yuan Xua,b, Hongbo Panc, Miao Miaod, Xiaozhong Hub, Saleh A. Al-Farraje, Khaled A. S. Al-Rasheide & Weibo Songb a b c d e

State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, 200062, China Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao, 266003, China College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, 201302, China College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China Zoology Department, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia

Keywords Ciliates; freshwater; infraciliature; Remanella; SSU rRNA gene. Correspondence M. Miao, College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China Telephone number: +86 010 88256167; FAX number: +86 010 88256079; e-mail: [email protected] X. Hu, Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China Telephone number/FAX number: +86 532 82031610; e-mail: [email protected]

ABSTRACT The morphology and phylogeny of Loxodes vorax and L. striatus orientalis subsp. n. were investigated based on infraciliature and small subunit (SSU) rRNA gene sequence data. Loxodes striatus orientalis subsp. n. was separated from L. striatus striatus stat. n. by having fewer dikinetids in the intrabuccal kinety (35–55 vs. 50–70) and a variable number of macronuclei (2–4 vs. 2). In addition, the SSU rRNA gene sequence of the new subspecies differs in 13 and 11 nucleotides from that of two populations of the nominotypic subspecies. We also summarized the morphological differences between Loxodes and Remanella based on the data available. Phylogenetic analyses revealed that the genus Loxodes was monophyletic and nested within Remanella species. This study might, therefore, support the hypothesis that the freshwater genus Loxodes evolved from the marine genus Remanella.

Received: 10 March 2014; revised 30 June 2014; accepted July 21, 2014. doi:10.1111/jeu.12162

RECENTLY, several marine karyorelictid groups, including trachelocercids, Geleia, Kentrophoros and Remanella, have been studied using the protargol staining method and phylogenetic analysis based on the small subunit (SSU) rRNA gene (Gao et al. 2010; Xu et al. 2011a,b,c, 2012, 2013a; Yan et al. 2013). The sole freshwater genus, Loxodes, has been widely used as material for studying a wide range of different aspects of karyorelictids, including their nuclear division (Bobyleva et al. 1980; Raikov 1982, 1994, 1996), cytology, e.g. nitrate respiration, structure and function of €ller vesicles (Fenchel and Finlay 1986a; Finlay 1985), Mu phylogenetic position (Andreoli et al. 2009; Hammerschmidt et al. 1996), behaviour, e.g. predatory behaviour, defensive behaviour, geotaxis, and photo-behaviour (Buon-

anno 2005; Buonanno et al. 2005; Fenchel and Finlay 1984, 1986b; Goulder 1972) and the adaptation of ciliates to suboxic environments (Fenchel and Bernard 1996; Finlay et al. 1986). Since its initial establishment by Ehrenberg in 1830, six species have been assigned to this genus, namely, Loxodes kahli, Loxodes magnus, Loxodes rex, Loxodes rostrum, Loxodes striatus, and L. vorax, but detailed information on the oral ciliature is only available for two of these species (Foissner and Rieder 1983; Kim et al. 2009). In addition, detailed reinvestigation of Loxodes is required to separate it from Remanella (Foissner 1996). Indeed, since it was first established, the validity of the genus Remanella has long been questioned because the difference between it and Loxodes has not been shown at the

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–11

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infraciliature level (Foissner 1996). According to Foissner (1996), some of the uncertainties regarding the differences between these two genera may be caused by incomplete data and thus individual species need to be restudied. The molecular research into Loxodes represented the pioneering work on the phylogeny of ciliates and revealed the sister relationship between Karyorelictea and Heterotrichea (Hammerschmidt et al. 1996; Hirt et al. 1995). Since then, no new sequences of karyorelictids had been reported until Andreoli et al. (2009) developed the method of amplifying and sequencing the SSU rRNA gene from a single cell. Currently, there are only three sequences from L. magnus and two populations of L. striatus available. Recently, with more and more SSU rRNA gene sequences of Remanella having become available (Xu et al. 2012, 2013a), the monophyly of Remanella has begun to be questioned. In order to study the evolutionary relationship between Remanella and Loxodes, more molecular information on Loxodes needs to be supplied. In this paper, we report two Loxodes species isolated from freshwater lakes at two locations, Qingdao and Guangzhou, China, including one novel subspecies. In addition, we summarized the morphological differences between Loxodes and Remanella, and we analysed the evolutionary relationship between Loxodes and Remanella based on SSU rRNA gene sequence data.

dao, China (36°030 N, 120°210 E), where the water temperature was 16 °C (Fig. 1). Loxodes striatus orientalis subsp. n. was taken using a plankton net (mesh size 20 lm) on 14 April 2009 from a freshwater fish pond in Guangzhou, China (22°530 N, 113°320 E) where the water temperature was 23 °C (Fig. 1). Cells were isolated and observed in vivo using an oil immersion objective and differential interference microscopy. The infraciliature was revealed by using the protargol impregnation method (Wilbert 1975). Counts and measurements on impregnated specimens were conducted at a magnification of 1,000X. Drawings were based on free-hand sketches or with the help of a camera lucida. Terminology and systematics are according to Foissner (1996) and Lynn (2008) respectively. DNA extraction, polymerase chain reaction amplification, and gene sequencing Genomic DNA extraction was performed using the DNeasy Tissue Kit (Qiagen, Shanghai, China) as described by Gao et al. (2013). The SSU rRNA gene was amplified by the polymerase chain reaction (PCR) using the universal eukaryotic primers 82 Forward (50 -GAAACTGCGAATGG CTC-30 ) and Euk B (50 -TGATCCTTCTGCAGGTTCACCT AC-30 ) (Elwood et al. 1985; Medlin et al. 1988). The PCR amplification and sequencing of the SSU rRNA gene were performed according to Xu et al. (2013c).

MATERIALS AND METHODS Phylogenetic analyses Sampling and morphological investigations Loxodes vorax was collected on 12 May 2011 from the sediments in a freshwater lake in Zhongshan park, Qing-

Alignment of the SSU rRNA sequences was conducted using CLUSTAL W, ver. 1.80 (Thompson et al. 1994) and refined using BioEdit v. 7.0.5 (Hall 1999) to excise highly

Figure 1 Sampling location. A. A lake in the Zhongshan park, Qingdao (36°030 N, 120°210 E). B. A fish-farming pond in Guangzhou (22°530 N, 113°320 E). Scale bar 500 km.

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variable regions. The resulting curated alignment included 1,572 characters and 47 taxa. Spirostomum ambiguum, Eufolliculina uhligi, Blepharisma americanum, Stentor amethystinus, and S. roeseli were included as outgroup taxa for phylogenetic analyses. Bayesian inference (BI) was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using the GTR + I + G model as selected by AIC in MrModeltest v.2.0 (Nylander 2004). The chain length of Bayesian analyses was 1,000,000 generations and sampled every 100 generations. The first 25% of sampled trees were considered burn-in trees. Maximum likelihood (ML) analysis was carried out with 1,000 replicates online on the CIPRES Portal V 2.0 (http://www.phylo.org) using RAxML-HPC BlackBox using GTR + G model (Stamatakis et al. 2008). RESULTS AND DISCUSSION Description of Qingdao population of L. vorax (Table 1 and Fig. 2,3) Live cells about 150 9 30 lm in size, with buccal field occupying 1/4 of body length (Fig. 2A, B, 3A–C). Cilia about 8 lm long. Cells transparent and colourless with only buccal area brownish at low magnification (Fig. 3A–C). Brown cortical granules, ca. 0.5 lm in diam., mainly packed in the buccal area, around the pharyngeal tube, and distributed between ciliary rows and on the left side

Table 1. Morphometric data from Qingdao populations of Loxodes vorax (upper rows) and L. striatus orientalis subsp. n. (lower rows) Character

Min

Max

Mean

SD

CV

n

Body, length (lm)

82 123 30 68 27 43 8 9 65 65 24 20 5 2 35 35 2 2 1 2

136 192 45 103 46 57 8 11 90 100 38 49 10 4 43 55 2 4 1 2

104.5 167.3 36.0 85.5 35.0 51.3 8.0 10.3 77.3 86.1 29.2 34.9 6.4 3.2 38.1 47.5 2.0 2.5 1.0 2.0

16.1 22.7 4.4 10.0 5.5 4.4 0 0.8 7.3 10.3 4.7 9.8 1.7 0.6 2.8 6.4 0 0.6 0 0

15.4 13.6 12.2 11.7 15.6 8.5 0 7.9 9.4 12.0 16.1 28.0 25.7 19.8 7.4 13.4 0 25.9 0 0

15 15 15 15 15 10 15 15 15 15 15 15 10 10 15 10 15 15 15 15

Body, width (lm) Buccal area, length (lm) Right lateral ciliary rows, number Dikinetids in right buccal kinety, number Dikinetids in left outer buccal kinety, number Dikinetids in left inner buccal kinety, number Dikinetids in intrabuccal kinety, number Macronuclei, number Micronuclei, number

All data are based on protargol-impregnated specimens. Measurements in lm. CV = coefficient of variation in %; Max = maximum; Mean = arithmetic mean; Min = minimum; n = number of specimens investigated; SD = standard deviation of the mean.

€ller vesicles, 7 lm in (Fig. 3D–F). About three to four Mu diam., with globular content ca. 3 lm in diam. (Fig. 2A, C, 3G). No cytoplasmic spicules. In some individuals, a permanent vacuole was located at the posterior end with a diam. of about 7–15 lm (Fig. 2A, 3A). Locomotion by gliding between sand grains and on bottom of petri dish. Infraciliature consisting of dikinetids (Fig. 2E, F, 3O). Always eight right lateral ciliary rows (Fig. 2E, 3O). Dorsolateral kinety with only anterior basal body ciliated, extending to posterior end of cell and curving around rear end to ventral side which encloses ca. 6 right lateral ciliary rows (Fig. 2E, 3M, O). Left lateral ciliary row extending around cell as in Remanella with only one basal body ciliated (Fig. 2F, 3L). Right buccal kinety consisting of about 65–90 tightly spaced dikinetids (Fig. 2D, E, 3I, O). Left outer buccal kinety comprising 24–38 dikinetids (Fig. 2D, E, 3I, J). Left inner buccal kinety extending to right of and along anterior half of outer buccal kinety and consisting of 5–10 widely spaced dikinetids (Fig. 2D, E, 3I, J). Intrabuccal kinety extending from anterior end to posterior end of pharyngeal tube, consisting of 35–43 dikinetids (Fig. 2D, 3N). Two macronuclei with one micronucleus in between (Fig. 2C, F, 3K, O). Remarks According to Stoke (1885) and Kahl (1931), the principal characteristics of this species are: body in vivo 125– 135 lm long and yellow to brown-coloured; permanent vacuole located at the posterior end whose function is unclear; two macronuclei with a single micronucleus in between. The Qingdao population corresponds well with these characteristics from the original report, including the permanent vacuole located to the rear, which we observed in some individuals and which, according to Foissner (1995), may be a defecation vacuole (Fig. 2A, 3A). Thus, we have no doubt that these populations are conspecific. This species is most similar to L. rostrum in terms of body length and the two macronuclei. The main difference between them is the number of right lateral ciliary rows (8 in L. vorax vs. 10–16 in L. rostrum), spindle-shaped symbiotic green algae (absent in L. vorax vs. present in L. rostrum) and the permanent vacuole at the posterior end (present in L. vorax vs. absent in L. rostrum). Although we do not consider the permanent vacuole as a classification characteristic, L. vorax still can be distinguished from L. rostrum. Description of L. striatus orientalis subsp. n. (Table 1 and Fig. 4, 5) Live cells about 220 9 60 lm in size, with the buccal field occupying ca. 1/4 of body length (Fig. 4A, 5A–C). Cilia about 10 lm long. Cells dark at low magnification due to many food vacuoles, ca. 15 lm in diam., including digested algae (Fig. 4C, 5A–C, E). Brown cortical granules, round, ca. 0.5 lm in diam., mainly packed in buccal area, around pharyngeal tube, and distributed between ciliary €ller rows and on left side (Fig. 4D, 5D). About 6–8 Mu


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Figure 2 Loxodes vorax in vivo (A–C) and after protargol impregnation (D, F). A. Right side of a typical individual, noting M€ uller vesicles; arrow €ller vesicles, macronuclei, micronucleus marks vacuole at posterior end. B. Different body shape. C. Detailed view of middle region to show Mu and digested algae (arrow). D. Infraciliature of buccal area, showing the right buccal kinety, left outer buccal kinety, left inner buccal kinety, intrabuccal kinety and right lateral ciliary row. E, F. General infraciliature of right (E) and left (F) side of the holotype specimen, noting dorsolateral kinety, right and left lateral ciliary rows; the dikinetids of left lateral ciliary row having only the anterior basal body ciliated; arrowheads mark several dikinetids behind the buccal area. IK = intrabuccal kinety; LC = left lateral ciliary row; LIK = left inner buccal kinety; LK = dorsolateral kinety; €ller vesicles; Ma = macronuclei; Mi = micronucleus; RC = right lateral ciliary rows; RK = right buccal kinLOK = left outer buccal kinety; M = Mu ety. Scale bars 75 lm (A, B) and 50 lm (E, F).

vesicles, 8 lm in diam. with globular content ca. 3 lm in diam. (Fig. 4A–C, 5F). No cytoplasmic spicules. Locomotion by gliding between sand grains and on bottom of Petri dish. Infraciliature consisting of dikinetids (Fig. 4F, G). 9–11 right lateral ciliary rows (Fig. 4F). Dorsolateral kinety with only anterior basal body ciliated, extending to posterior end of cell and curving around rear end to ventral side which encloses ca. 5 right lateral ciliary rows (Fig. 4F). Left lateral ciliary row extending around cell as in Remanella (Fig. 4G). Right buccal kinety consisting of about 65–100 tightly spaced dikinetids (Fig. 4E, 5I). Left outer buccal kinety comprising 20–49 dikinetids (Fig. 4E, F, 5I, J). Left inner buccal kinety extending to right of and along anterior half of outer buccal kinety and consisting of 2–4 widely spaced dikinetids (Fig. 4E, F, 5J). Intrabuccal kinety extending from anterior end to posterior end of pharyngeal tube, consisting of 35–55 dikinetids (Fig. 4E, G). Two nuclear groups, separated from each other, and with each including 1–2 macronuclei accompanied by one micronucleus, (Fig. 4A– C, G, 5E, G, H). Several conspicuous black inclusions after

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staining, 8–12 in diam., which might be food vacuoles (Fig. 5G, H). Many round black dots, ca. 2 lm in diam., distributed along the dorsal margin of the cell (Fig. 5H). Discussion Engelmann (1862) first described L. striatus and assigned it to the genus Drepanostoma. Then, Penard (1917) transferred it to the genus Loxodes and subsequently there have been a number of redescriptions (e.g. Dragesco and is 1986; Foissner and Rieder 1983; Kahl Dragesco-Kerne 1931; Klindworth and Bardele 1996; Puytorac and Njine 1970). The most remarkable character of L. striatus is its nuclear apparatus, which consists of two sets of macronuclei and a micronucleus. This feature is shared with L. striatus orientalis subsp. n. and L. striatus striatus stat. n. (Foissner and Rieder 1983). Within the genus, only L. vorax and L. rostrum have a similar number of macronuclei as L. striatus. However, the two macronuclei and one micronucleus of L. vorax and L. rostrum form a single is 1986). nuclear group (Dragesco and Dragesco-Kerne


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Figure 3 Loxodes vorax in vivo (A–H) and after protargol impregnation (I–O). A. Right side of a typical individual; arrow marks vacuole at posterior end. B, C. Different body shape. D. Cortical granules (arrowhead) densely packed in pharyngeal tube. E, F. Right (E) and left side (F), noting cortical granules (arrowheads) packed in oral area, between ciliary rows and scattered on left side. G. Detailed view of mid-body region, indicating €ller vesicles (arrows) and macronuclei. H. Digested algae. I, J. Infraciliature of buccal area, showing the right buccal kinety, left outer buccal Mu kinety, left inner buccal kinety and right lateral ciliary row. K. Macronuclei and micronucleus. L. Left side of anterior region, showing left lateral ciliary row. M. Right side of posterior region, marking dorsolateral kinety and right lateral ciliary rows. N. Intrabuccal kinety. O. Infraciliature of right side, noting several dikinetids behind the buccal area (arrowheads). IK = intrabuccal kinety; LC = left lateral ciliary row; LIK = left inner buccal kinety; LK = dorsolateral kinety; LOK = left outer buccal kinety; Ma = macronuclei; Mi = micronucleus; RC = right lateral ciliary rows; RK = right buccal kinety. Scale bars 75 lm (A–C), 25 lm (I) and 50 lm (O).

Other Loxodes forms have many more macronuclei and right lateral ciliary rows, and thus cannot be confused with L. striatus (Table 2). The two subspecies of L. striatus differ from each other in the number of dikinetids in the intrabuccal kinety (35–55 in L. striatus orientalis subsp. n. vs. 50–70 in L. striatus striatus stat. n.) and the number of macronuclei (2–4 in L. striatus orientalis vs. constant 2 in L. striatus striatus stat. n.) (Foissner and Rieder 1983). In addition, the SSU rRNA gene sequence of L. striatus orientalis subsp. n. differs in 13 and 11 nucleotides, respectively, from that of the two

populations of L. striatus in Genbank (Fig. 6). Unfortunately, there is no morphological description of these two populations of L. striatus and L. magnus in the original reports (Andreoli et al. 2009; Hammerschmidt et al. 1996; Hirt et al. 1995). According to personal communications, however, there is no evidence these species could be misidentified. Therefore, we think the two sequences of L. striatus were from L. striatus striatus stat. n., which explains the difference between the SSU rRNA gene sequences of L. striatus orientalis subsp. n. and L. striatus striatus stat. n.


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Figure 4 Loxodes striatus orientalis subsp. n. in vivo (A–D) and after protargol impregnation (E–G). A. Right side of a typical individual. B. Show€ller vesicles, macronuclei and micronuclei. C. Detailed view of middle region to indicate Mu €ller vesicles, macronuclei, miing the distribution of Mu cronuclei (arrows) and food vacuoles including digested algae (arrowheads). D. Distribution of cortical granules between ciliary rows (arrowheads). E. Infraciliature of buccal area, marking the right buccal kinety, left outer buccal kinety, left inner buccal kinety, intrabuccal kinety and right lateral ciliary row; arrowheads point to several dikinetids behind the buccal area. F, G. General infraciliature of right (F) and left (G) side of the same specimen, noting dorsolateral kinety, right and left lateral ciliary rows, and the nuclear apparatus. IK = intrabuccal kinety; LC = left lateral €ller vesicles; Ma = macronuclei; ciliary row; LIK = left inner buccal kinety; LK = dorsolateral kinety; LOK = left outer buccal kinety; M = Mu Mi = micronuclei; RC = right lateral ciliary rows; RK = right buccal kinety. Scale bars 100 lm (A, B) and 85 lm (F, G).

Molecular phylogeny based on sequences of SSU rRNA gene (Fig. 6, 7) The new SSU rRNA gene sequences of the two isolates have length (bp), GC content and Genbank accession numbers as follows: L. vorax – 1,556, 48.26%, KJ524909; L. striatus orientalis subsp. n. – 1,555, 47.72%, KJ524910. Loxodes striatus orientalis subsp. n. differs in 13, 11, 22 and 23 nucleotides from L. striatus striatus stat. n. (U24248), L. striatus striatus stat. n. (AM946031), L. magnus, and L. vorax respectively. L. vorax, meanwhile, differs in 26, 24, and 35 nucleotides from L. striatus striatus stat. n. (U24248), L. striatus striatus stat. n. (AM946031) and L. magnus respectively (Fig. 6). The ML and BI trees have similar topologies and thus only the topology of the ML tree is presented (Fig. 7). The only difference between these two analyses is that the topology of the BI tree includes a few parallel branches; these are marked by an asterisk in Fig. 7. As described in previous studies (Xu et al. 2013b), the class Karyorelictea is subdivided into four groups corresponding to four families: Loxodidae, Geleiidae, Kentrophoridae, and Trachelocercidae (Lynn 2008). Of these, Loxodidae is a

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monophyletic group (94% ML, 1.00 BI), and within Loxodidae, the genus Loxodes forms another fully supported monophyletic clade which, however, nests in the genus Remanella. L. vorax occupies a basal position within Loxodes genus. The newly sequenced L. striatus orientalis subsp. n. branches with the group including both L. striatus striatus stat. n. and L. magnus. Although the validation of the genus Remanella is not supported by molecular results, Loxodes and Remanella can be separated morphologically (see the discussion below). We therefore support the hypothesis proposed in previous studies (Foissner 1996; Xu et al. 2013b) that the freshwater genus Loxodes evolved from its marine congener Remanella. Morphological comparison of Loxodes and Remanella According to previous studies (Foissner 1996; Xu et al. 2012, 2013a), the somatic and oral ciliature of Loxodes and Remanella are almost identical. The main differences between Loxodes and Remanella are: the absence and presence of cytoplasmic spicules, the granules contained €ller vesicles, the shape of the posterior body in the Mu end, and the habitat (Foissner 1996; Xu et al. 2012,


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Figure 5 Loxodes striatus orientalis subsp. n. in vivo (A–F) and after protargol impregnation (G–J). A. Right side of a typical individual. B, C. Different €ller vesicles (arrowbody shape. D. Distribution of cortical granules around oral area (arrowheads). E, F. Detailed view of middle region to indicate Mu heads), macronuclei and food vacuoles including digested algae (arrows). G, H. Infraciliature of middle region, showing macronuclei, micronuclei; arrows point to black inclusion (food vacuoles?); arrowheads mark black dots (extrusomes?) along the cell margin. I, J. Infraciliature of buccal area, marking the right buccal kinety, left outer buccal kinety, left inner buccal kinety, and right lateral ciliary row. LIK = left inner buccal kinety; LOK = left outer buccal kinety; Ma = macronuclei; Mi = micronuclei; RC = right lateral ciliary rows; RK = right buccal kinety. Scale bars 100 lm (A–C) and 30 lm (I).

2013a). Some other differences mentioned below between these two genera may be caused by incomplete data, and thus a reinvestigation of Loxodes is needed (Foissner 1996). Our studies, therefore, try to address some of the uncertainties identified by Foissner (1996): 1 As in Remanella, not all somatic dikinetids have both basal bodies ciliated. The dorsolateral kinety, first right lateral ciliary row, which is closest to the ventral side, and several obliquely arranged dikinetids at the anterior end of each right lateral ciliary row, have only their anterior basal body ciliated. 2 The special fibres associated with the dorsolateral kinety in Remanella, which have been mentioned in previous studies (Foissner 1996; Xu et al. 2012, 2013a), were not

seen in our protargol slides of either L. vorax or L. striatus orientalis subsp. n. It cannot be concluded for certain, however, whether Loxodes does or does not have this feature, since whether the fibres are revealed through protargol methods sometimes depends on the intensity of staining. 3 Although some light and electron microscopic studies have suggested the left outer buccal kinety consists of is 1986; monokinetids (Dragesco and Dragesco-Kerne 1970), based on our studies, the left Puytorac and Njine outer buccal kineties of both L. vorax and L. striatus orientalis subsp. n. are composed of dikinetids. 4 Klindworth and Bardele (1996) had shown, using electron microscopy, that the left ciliary row in Loxodes is actually composed of two kineties. In our studies, the left lateral


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row looks like a single row curving around the cell margin with only one basal body ciliated, as in Remanella (Fig. 2F). Therefore, the differences between Loxodes and Remanella can be summarized here as: (1) habitat (freshwater vs. marine); (2) cytoplasmic spicules (absent vs. present); (3) posterior body end (round vs. pointed); (4) number and €ller vesicle (many tiny barium grantype of granules in Mu ules vs. single or compound strontium granule). TAXONOMIC SUMMARY Order Loxodida Jankowski, 1980 €tschli, 1889 Family Loxodidae Bu Genus Loxodes Ehrenberg, 1830 IK = intrabuccal kinety; LIK = left inner buccal kinety; LOK = left outer buccal kinety; RC = right lateral ciliary rows; RK = right buccal kinety.

470–600 13–26 – – – – 6–8 6–8 6–8 Dragesco and is Dragesco-Kerne (1986) 500–1,200 79–84 – – – – 132–181 132–181 39–138 Dragesco and is Dragesco-Kerne (1986) 100–180 8 65–90 24–38 5–10 35–43 1 2 1 Present work 209–250 10–13 – ca. 40 4 50–70 2 2 2 Foissner and Rieder (1983) Body length, in vivo (lm) RC, number Dikinetids in RK, number Dikinetids in LOK, number Dikinetids in LIK, number Dikinetids in IK, number Nuclear groups, number Macronuclei, number Micronuclei, number Data source

150–300 9–11 65–100 20–49 2–4 35–55 2 2–4 2 Present work

340–440 25–32 – ca. 80 10–12 70–150 18–26 18–26 10–16 Foissner and Rieder (1983)

70–300 10–12 – – – – 1 2 1 Dragesco and is Dragesco-Kerne (1986)

L. kahli L. vorax L. magnus L. striatus striatus stat. n. L. striatus orientalis subsp. n. Characteristics

Table 2. Morphological information of two subspecies of Loxodes striatus and its congeners

L. rostrum

L. rex


Loxodes vorax Stoke 1885 Remarks. This organism has been redescribed several times (Hausmann 1983; Kahl 1931; P€ atsch 1974) since it was first reported by Stoke (1885). There is still no information on its infraciliature, however, especially its oral ciliature. Consequently, an improved diagnosis and a redescription of the species, based on the Qingdao population, are presented here. Improved diagnosis. Cell size in vivo about 100– 180 9 20–45 lm; buccal field occupying almost 1/4 of body length; 8 right lateral ciliary rows; right buccal, left outer buccal, left inner buccal and intrabuccal kineties composed of 65–90, 24–38, 5–10 and 35–43 dikinetids respectively; two macronuclei and one micronucleus; brown cortical granules, 0.5 lm in diam. Loxodes striatus (Engelmann 1862) Penard 1917 Remarks. We split this species into two subspecies, differing mainly in the number of dikinetids in the intrabuccal kinety, the number of macronuclei, and the SSU rRNA gene sequence. Improved diagnosis. Cell size in vivo about 150– 300 9 40–70 lm; buccal field occupying ca. 1/5–1/4 of body length; 9–13 right lateral ciliary rows; right buccal, left outer buccal, left inner buccal and intrabuccal kineties composed of 65–100, 20–49, 2–4 and 35–70 dikinetids respectively; 2–4 macronuclei and two micronuclei; brown cortical granules, 0.5 lm in diam. Loxodes striatus striatus stat. n Diagnosis and description: see Foissner and Rieder (1983). Loxodes striatus orientalis subsp. n Diagnosis. Cell size in vivo about 150–300 9 40–70 lm; buccal field occupying almost 1/4 of body length; 9–11 right lateral ciliary rows; right buccal, left outer buccal, left inner buccal and intrabuccal kineties composed of 65–100, 20–49, 2–4 and 35–55 dikinetids respectively; 2–4 macronuclei and two micronuclei; brown cortical granules, 0.5 lm in diam.


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Type location. A freshwater fish pond in Guangzhou, China (22°530 N, 113°320 E). Etymology. The subspecies-group name orientalis indicates that the present form was described first from oriental (Chinese) waters.

Figure 6 Unmatched sites from SSU rRNA gene sequence alignment of five Loxodes species. Numbers represent the positions of the nucleotides in alignment. Matched sites are marked with dots. The dashes represent deletions compared to other species. Asterisks mean this species should be changed to L. striatus striatus stat. n.


Type material. The holotype specimen marked with an ink circle is deposited in the Laboratory of Protozoology, OUC, China (no. PHB09041401). ACKNOWLEDGMENTS We are grateful to Dr. Denis H. Lynn (University of Guelph, Canada), Dr. David M. Roberts and Dr. Alan Warren (Natural History Museum, UK) for their help with checking the identification of Loxodes species whose SSU rRNA gene sequences have been published before. This work was supported by the Natural Science Foundation of China (Project numbers: 41376141 to XH, 31272285 to MM, 31201703 to HP, 41276139 to WS) and the International Research Group Program (IRG14-22), Deanship of Scientific Research, King Saud University.

Figure 7 Maximum likelihood (ML) tree inferred from the small subunit rRNA gene sequences showing the positions of two newly sequenced Loxodes isolates (arrows). Nodal support for branches in the ML and BI trees are marked in order. Black nodes indicate full support in both analyses (100% ML, 1.00 BI). Clades with a different topology between the two analyses are shown by an asterisk. Heterotrichs are selected as the out-group. Double asterisks mean both forms should be L. striatus striatus stat. n. All branches are drawn to scale. The scale bar corresponds to five substitutions per 100 nucleotide positions.


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