Published by the International Society of Protistologists
The Journal of
Eukaryotic Microbiology
Journal of Eukaryotic Microbiology ISSN 1066-5234
ORIGINAL ARTICLE
Two New Brackish Ciliates, Amphileptus spiculatus sp. n. and A. bellus sp. n. from Mangrove Wetlands in Southern China, with Notes on the Molecular Phylogeny of the Family Amphileptidae (Protozoa, Ciliophora, Pleurostomatida) Lei Wua, Zhenzhen Yia, Jiqiu Lia, Alan Warrenb, Hualin Xuc & Xiaofeng Lina a Laboratory of Protozoology, Key Laboratory of Ecology and Environment Science in Guangdong Higher Education, South China Normal University, Guangzhou 510631, China b Department of Life Sciences, The Natural History Museum, Cromwell Rd, London SW7 5BD, United Kingdom c Neilingding-Futian National Nature Reserve Administration Bureau of Guangdong, Shenzhen 518040, China
Keywords Ciliary pattern; new species; SSU rDNA; taxonomy. Correspondence X. Lin, School of Life Sciences, South China Normal University, Guangzhou 510631, China Telephone/FAX number: + 86-20-8521-0644; e-mail: [email protected]
ABSTRACT Two new brackish pleurostomatid ciliates, Amphileptus spiculatus sp. n. and A. bellus sp. n. were collected from mangrove wetlands of southern China and their morphology and molecular phylogeny were studied. Amphileptus spiculatus sp. n. can be distinguished from congeners by the presence of 11–14 right and 6–8 left kineties, two macronuclear nodules and a conspicuous beak-like anterior body end. Amphileptus bellus sp. n. is characterized by the presence of 2–4 macronuclear nodules, 31–35 right and 6 or 7 left kineties and two types of extrusomes. Phylogenetic analyses based on SSU rDNA sequences data indicate that the family Amphileptidae is paraphyletic.
Received: 28 September 2014; revised 1 January 2015; accepted February 12, 2015. doi:10.1111/jeu.12225
MANGROVE wetlands are unique ecosystems that interact strongly with marine, freshwaters, and terrestrial ecosystems and thus support a high biodiversity (Tam and Wong 2002). All the major groups of macroscopic organisms in mangroves, from fungi and vascular plants to benthic invertebrates and birds, have been investigated to some extent (Gopal and Chauhan 2006; Lu and Wang 1996). By comparison, unicellular microorganisms such as ciliated protozoa are poorly studied in this special biotope. Pleurostomatids are a large group of free-living ciliates. Recent studies have suggested that their diversity is high in mangrove wetlands and other brackish habitats (Chen et al. 2011; Pan et al. 2010, 2013; Wu et al. 2013, 2014). The earliest established genus of the order Pleurostomatida, Amphileptus Ehrenberg, 1830, has over 50 nominal species reported from aquatic habitats worldwide. The vast majority of Amphileptus species are from freshwaters (Canella 1960; Curds 1982; Song and Wilbert 1989; Stokes 1886; Vuxanovici 1960; Wang 1934, 1940; Wang and Nie 1933), most of the others being marine (Lin et al. 2005a,b, 2007; Song 1991; Song et al. 2004; Wilbert and Song
662
2005). To date, only two species have been isolated from brackish waters (Chen et al. 2011; Pan et al. 2014). To further explore the diversity of pleurostomatid ciliates in brackish habitats, we investigated mangrove wetlands in southern China. During this survey, two unusual pleurostomatid ciliates were collected. Detailed investigations of specimens in vivo and following protargol staining, and analysis of their small subunit (SSU) rDNA sequences, revealed them to be new members of the genus Amphileptus. In this article, we describe the two new species in detail. In addition, the molecular phylogeny of the family Amphileptidae is analyzed based on SSU rDNA sequences data that indicate the family Amphileptidae is paraphyletic. MATERIALS AND METHODS Sample collection and identification Amphileptus spiculatus sp. n. and A. bellus sp. n. were isolated from Futian mangrove wetland, Shenzhen (22°380 N, 114°060 E) on 13 April 2011 and from Daya Bay mangrove wetland, Huizhou (22°410 N, 114°230 E) on 10
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
Two New Brackish Ciliates
Wu et al.
December 2010, respectively, both in southern China (Fig. 1A, B). Samples were collected using 20-lm mesh plankton nets and were transported to the laboratory in 250-ml wide-mouth bottles containing water from the collection site. Ciliates were maintained in the laboratory in Petri dishes containing habitat water with rice grains to enrich the growth of bacteria, both as a food resource on which the Amphileptus fed directly and as food resource for other protists on which the Amphileptus fed as well. Observations of living cells were carried out with bright field and differential interference contrast microscopy. The protargol staining method according to Wilbert (1975) was used to reveal the ciliary pattern. Counts and measurement were made at 20–1,000X magnifications. Drawings of live specimens were based on in vivo observations and photomicrographs. Drawings of protargol-stained specimens were made with the help of a camera lucida at 1,000X. Terminology and systematics are according to Lynn (2008). SSU rDNA sequence For each species, one or more cells were isolated, repeatedly rinsed with filtered habitat water in order to remove contaminants, and transferred into 45 ll ATL buffer for DNA extraction. Genomic DNA extraction, gene amplification, and gene sequencing were conducted according to the method described by Wu et al. (2013). Phylogenetic analyses SSU rDNA sequences of Amphileptus bellus sp. n. and A. spiculatus sp. n., along with those of another 29 related species obtained from GenBank (including all available SSU rDNA sequences of the order Pleurostomatida), were used in our analyses. The GenBank accession numbers of Loxophyllum species were as follows: L. jini EF123708, L. rostratum DQ190465, L. spirellum
Figure 1 Sites from which samples were taken. A. Futian mangrove wetland, Shenzhen. B. Daya Bay mangrove wetland, Huizhou.
GU574810, L. sp. EU242511, L. chinense JN974455, L. caudatum JN974453, L. rugosum JN974454, L. meridionale KC469985, L. salinum KC476181, L. planum KC476180 and L. perihoplophorum KC493570. The accession numbers of all other species used in the analyses are shown in Fig. 5. Metopus contortus and M. palaeformis (class Armophorea) were used as the out-group taxa. The SSU rDNA sequences were aligned using CLUSTAL W implemented in Bioedit 7.0 (Hall 1999). The alignment was then further modified manually by removing both the primers and the highly variable regions. The final alignment of 1,587 characters was used to construct phylogenetic trees. Maximum likelihood (ML) analysis was performed online (http://www.phylo.org) on the CIPRES Portal V 1.15 (Miller et al. 2010), using the RAxML and the parameters according to Stamatakis et al. (2008). The reliability of internal branches was assessed by a nonparametric bootstrap method with 1,000 replicates. A Bayesian inference (BI) analysis was carried out with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using the Markov chain Monte Carlo algorithm and the best model indicated by MrModeltest v.2 (Nylander 2004). The program was run for 1,500,000 generations with a sample frequency of 100 and a burn-in of 3,750. The approximately unbiased (AU) test (Shimodaira 2002) was used to test the hypothesis that the family Amphileptidae is monophyletic. The ML tree was generated with a constraint block, enforcing the constraint of focal group monophyly in PAUP under the same GTR + I + G model. The site-wise likelihoods were calculated using PAUP (Swofford 2002) for the resulting constrained and nonconstrained ML topologies. The scores were then subjected to the AU test as implemented in Consel 0.1 (Shimodaira and Hasegawa 2001). RESULTS Amphileptus spiculatus sp. n. The cell is about 85–150 lm 9 90–150 lm in vivo, usually 100–125 lm in length. The body is pyriform, laterally compressed, approximately 3:1, flexible, and moderately contractile. The anterior tip is sharply pointed and curved to form a conspicuous beak, the posterior end is broadly rounded, the neck region comprises approximately 20% of the length of the cell (Fig. 2A, G). There are two ovoid macronuclear nodules located in the central region of the cell, each about 15–30 lm 9 10–25 lm in size after fixation (Fig. 2D, F, K). The single micronucleus is spherical, about 3–5 lm in length, and located between the two macronuclear nodules (Fig. 2D, F, K). There are two or three contractile vacuoles, about 5–10 lm in diam., located near the ventral margin in the cell posterior (Fig. 2A, J). The extrusomes are pyriform, straight to slightly curved, 5– 7 lm long (Fig. 2B), some of them are evenly arranged along the oral slit, and others are scattered in the cytoplasm (Fig. 2D, K, L). The pellicle is thin and with numerous small (< 0.5 lm across), grayish, dot-like cortical granules which are densely distributed between the ciliary
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
663
Two New Brackish Ciliates
Wu et al.
Table 1. Morphological characteristics of Amphileptus spiculatus sp. n. (1st line) and A. bellus sp. n. (2nd line and 3rd line for “Length of Ex”) Characters
Min
Max
Mean
SD
CV
n
Body length
70 300 25 40 11 31 6 6 2 2 15 15 10 10 6 6 6
140 500 45 100 14 35 8 7 2 4 30 65 25 30 8 20 16
112.7 399.5 34.4 60.9 11.8 31.9 6.5 6.8 2.0 2.5 21.4 39.9 15.5 18.3 6.9 16.4 12.1
18.23 67.56 5.35 14.76 1.06 1.21 0.58 0.35 0 0.57 4.23 14.31 4.33 5.95 0.59 3.32 2.33
16.2 17.0 15.6 24.3 9.0 3.8 9.0 5.1 0 23.1 19.8 35.8 28.1 32.4 8.5 20.2 19.3
26 29 26 29 26 29 26 29 26 29 26 29 26 29 26 29 29
Body width Number of RSKa Number of LKDb Number of Ma Length of Ma Width of Ma Length of Ex Type 1 Type 2
Figure 2 Amphileptus spiculatus sp. n. from live (A–C, G, H, J) and after protargol impregnation (D–F, I, K, L). A. Right view of a typical individual. B. Extrusomes. C. Cortical granules. D. Extrusome pattern and nuclear apparatus. E, F. Ciliary pattern of right (E) and left (F) sides. G. Right side view, arrow points to the beak-like anterior end. H. Anterior portion of left side, to show the bristles of dorsal brush (arrowheads) and cortical granules (arrows). I. Anterior portion of right side, arrows indicate the suture. J. Right side view, arrowheads mark the contractile vacuoles. K. Nuclear apparatus, arrow marks the micronucleus. L. Anterior portion of left side, arrowheads refer to the dorsal brush. DB = dorsal brush; PK1, 2 = perioral kinety 1, 2. Ma = macronuclear nodules. Scale bars, 50 lm in (A, D–F, G, J); 10 lm in (B); 20 lm in (H, I, K, L).
rows on the both sides of cell (Fig. 2C, H). The right side is densely ciliated with cilia about ~8 lm long arranged in rows that are located within conspicuous longitudinal furrows and which form a distinct anterior suture that is detectable also in vivo (Fig. 2G, J). The left somatic cilia are sparsely distributed and difficult to detect in vivo. The cytoplasm is slightly grayish, often with numerous refringent globules (2–5 lm across) that render the main body region more or less opaque (Fig. 2G, J). Locomotion is by gliding moderately fast on the substrate or by swimming while rotating clockwise about the longitudinal axis. The ciliary pattern is shown in Fig. 2E, F, I, L. There are 11–14 right kineties, including perioral kinety 2; the intermediate kineties are shortened forming a distinct anterior suture on the right side (Fig. 2E, I). The left side with 6–8 ciliated somatic kineties including perioral kinety 1 and dorsal brush kinety (DB), which extends to about anterior 2/5 of the cell-length and is composed of regularly spaced dikinetids (Fig. 2F, L). There are two perioral kineties located along the cytostome. Perioral kinety 1 (PK1), to the left of the oral slit, composed of dikinetids in the anterior 2/3 and of monokinetids in the posterior 1/3 (Fig. 2F). Perioral kinety 2 (PK2),
664
All measurements in lm. Data based on protargol-impregnated specimens. CV = coefficient of variation in %; Ex = extrusomes; LSK = left somatic kineties; Min = minimum; Max = maximum; Mean = arithmetic mean; n = sample size; RSK = right somatic kineties; SD = standard deviation. a Perioral kineties 2 included. b Perioral kineties 1 and dorsal brush kinety included.
Figure 3 Amphileptus bellus sp. n. drawn from live (A–C) and after protargol impregnation (D–G). (A) Right view of a typical individual. (B) Extrusomes. (C) Cortical granules. (D) Macronuclear pattern. (E) Extrusome pattern and nuclear apparatus. (F, G) Ciliary pattern of left (F) and right (G) side, arrowheads indicate the suture. DB = dorsal brush; PK1, 2 = perioral kinety 1, 2. Ma = macronuclear nodules. Scale bars, 100 lm in (A, E, F, G); 10 lm in (B).
to the right of the oral slit, consists of narrowly spaced dikinetids in the anterior half and continues to the posterior as a row of monokinetids (Fig. 2E).
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
Two New Brackish Ciliates
Wu et al.
Figure 4 Photomicrographs of Amphileptus bellus sp. n. from live (A–G) and after protargol impregnation (H–O). (A–C, F) Shape variation among different individuals, arrowheads mark the contractile vacuoles, arrows show the macronuclear nodules. (D) Anterior part of right side, arrow indicates the extrusomes, arrowhead points to the cilia. (E) Middle part of right side, arrows indicate the cortical granules. (G, O) Posterior part of cell, to show the bar-shaped extrusomes (arrowheads). (H, I) Ciliary pattern of right (H) and left (I) side, arrowheads refer to basal bodies. (J) Anterior part of left side, arrowheads mark the perioral kinety 1, arrows show the dorsal brush, double arrowheads indicate the extrusomes. (K) Ventral side view, to show perioral kinety 1 (arrow) and perioral kinety 2 (arrowhead). (L) Anterior part of right side, arrows point to the suture. (M) Nuclear apparatus, arrow indicates the micronucleus. (N) To show the spindle-shaped extrusomes (arrowheads). Scale bars, 150 lm in (A–C, F), 50 lm in (J).
Amphileptus bellus sp. n. The cell size is highly variable, about 250–400 lm 9 25– 80 lm in vivo, usually 280–350 lm in length. The body is elongate, pyriform, laterally compressed, approximately
3–4:1, and moderately contractile. The anterior tip is bluntly pointed, and the posterior end is acuminate. The neck region comprises 25–35% of the length of the cell and is usually curved slightly dorsad (Fig. 3A, 4A–C). The two to four macronuclear nodules are ovoid to elliptical,
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
665
Two New Brackish Ciliates
Wu et al.
about 20–45 lm 9 15–30 lm in size in vivo, and located in the mid-region of the cell, usually seen in life as slightly transparent areas (Fig. 3D, 4B, C). The single ellipsoidal micronucleus is about 3–6 lm 9 1–3 lm after impregnation, and situated near the macronuclear nodules (Fig. 4M). There are two to four contractile vacuoles, the largest one is about 15–20 lm in diam. and subterminally positioned, and the others are 10–15 lm in diam. and distributed along the ventral margin in the anterior half of the cell (Fig. 3A, 4A–C, F). There are two kinds of extrusomes (Fig. 3B, 4D, G, N): type I, about 6–20 lm long after protargol staining, long bar shaped, some are densely arranged in the oral area, some form a bundle in the posterior region of the cell and some scattered in the cytoplasm (Fig. 3E, 4J, O); type II, spindle-shaped, about 6– 16 lm 9 2–4 lm in size after protargol staining, scattered in the cytoplasm (Fig. 4N). The pellicle is thin with small (< 0.5 lm across), densely spaced, grayish, dot-like cortical granules between ciliary rows on both sides of the cell (Fig. 3C, 4E). The right side is flat and densely ciliated, cilia on the right side about ~10 lm long (Fig. 4D, H); the left side is slightly humped in the central region of the body, cilia sparsely distributed and difficult to detect in life (Fig. 4F, I). The cytoplasm is colorless to grayish, often with numerous tiny, shining globules (2–5 lm across) that render the main part of the body opaque (Fig. 4C). The locomotion is by gliding moderately fast on the substrate or by swimming with a slow anticlockwise rotation about the longitudinal axis. The ciliary pattern is shown in Fig. 3F, G and 4H–L. There are 31–35 right somatic kineties including perioral kinety 2; the intermediate kineties shortened forming a distinct suture in the anterior part of the body (Fig. 3G, 4L). The left side with six- or seven-ciliated kineties including perioral kinety 1 and dorsal brush kinety (DB), which extends to about 1/3 of the cell length and is composed of narrowly spaced dikinetids (Fig. 3F, 4J). There are two perioral kineties along the cytostome. Perioral kinety 1 (PK1), to the left of the oral slit, comprises dikinetids in the anterior half and continues posteriorly as a row of monokinetids (Fig. 3F, 4K, J). The perioral kinety 2 (PK2), to the right of the oral slit, comprises regularly spaced dikinetids in the anterior 2/5 and monokinetids in the posterior 3/5 (Fig. 3F, 4K). SSU rDNA sequence analyses The two new SSU rDNA sequences have been deposited in GenBank database with length, GC content, and accession numbers as follows: A. bellus sp. n., 1,535 bp, 42.00%, KM025128; A. spiculatus sp. n., 1,534 bp, 42.00%, KM025129. Phylogenetic trees constructed using ML and BI analyses had almost identical topologies, therefore, only the ML tree is shown (Fig. 5). Epiphyllum shenzhenense is basal within the order Pleurostomatida. All members of Amphileptus and Pseudoamphileptus form a well-supported clade within the paraphyletic family Amphileptidae (93% ML, 1.00BI). Within the Amphileptus–Pseudoamphi-
666
Figure 5 Phylogenetic tree (ML) based on 31 SSU rDNA sequences of litostomatean ciliates. Newly sequenced species in present study are shown in bold. GenBank accession numbers are given after names of species. Numbers at nodes indicate bootstrap values of maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BI). –, represents disagreement between ML and BI topologies. The scale bar corresponds to five substitutions per 100 nucleotide positions.
leptus subclade, A. bellus occupies a basal position with moderate support values (82% ML, 0.93BI), A. spiculatus clusters with A. aeschtae with maximal support (100% ML, 1.00BI) and A. dragescoi clusters with Pseudoamphileptus macrostoma with low support values (30%ML, 0.85BI) forming a clade that is sister to A. procera. DISCUSSION Comparison of Amphileptus spiculatus sp. n. and A. bellus sp. n. with similar species It is widely accepted that the most important criteria for identification and separation of species in the genus Amphileptus include characteristics of the somatic kineties, contractile vacuoles, cortical granules, macronuclear nodules, and extrusomes (Foissner et al. 1995; Lin and Song 2004; Lin et al. 2007; Song et al. 2004). Amphileptus spiculatus sp. n. and A. bellus sp. n. both resemble the species A. salignus Chen et al. 2011; A. gui Lin et al. 2005a; and A. songi Pan et al. 2014; however, each can be distinguished from all three congeners by a combination of differences in numbers of somatic kineties, location of contractile vacuoles, and form/distribution of extrusomes (Table 2). Beyond its resemblance to the three species just cited, A. spiculatus sp. n. also resembles four other species of Amphileptus that have similar body sizes, numbers of somatic kineties, and numbers of macronuclear nodules (Table 2) but can be distinguished from them by differences in the number/distribution of contractile vacuoles and the form/distribution of the extrusomes (Table 2). Finally, A. bellus sp. n. shows some similarity to three species of Amphileptus with similar body sizes or
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
Two New Brackish Ciliates
Wu et al.
Table 2. Comparison of Ampileptus spiculatus sp. n. and A. bellus sp. n. with one another and with similar species
Species Amphileptus spiculatus sp. n. Amphileptus bellus sp. n.
Body length in vivo (lm)
Number of Ma
85–145 250–400
RSK/LSK
2
11–14/6–8
2–4
31–35/6–7
Number and position of contractile vacuoles
Shape and distribution of extrusomes
3, by posterior 1/4 of VM 2–4; 1 subterminal, rest by anterior 1/3
Biotope
Source of data
Pyriform, oral
B
Present work
Type 1: long BS, oral and tail; Type 2: SS, entire cytoplasm
B
Present work
B
Chen et al. (2011)
M
Lin et al. (2005a)
Species resembling both new species Amphileptus saligus
180–360
2
24–29/4
2–7; 1 dorsal, rest by VM
Amphileptus gui Amphileptus songi
150–300
2
37–50/7–11
3–7, by VM
Type 1: long BS, oral; Type 2: short BS, under pellicle BS, apical
200–450
2
21–28/11–13
3–7, by VM
BS, ventral
M
Song et al. (2004)
100–200
2
14–18/6–9
5–15, by VM
BS, along OS
M
Lin et al. (2007)
100–200
2
18–22/4
1, terminal
Clavate, apical
M
Lin et al. (2005a)
150–300
2
13–21/5–8
7, by VM
Fusiform, along OS
M
Pan et al. (2014)
180–210
2
15–19/7–8
3; by VM
BS, along OS
B
Pan et al. (2014)
29–34/7–10
5–13, by VM
BS, along OS
M
Lin et al. (2007)
–/–
6–10, by VM
–, oral
M
Wang (1934)
30–34/~5
many, by cell margins
BS, oral
M
Dragesco and (1971) Njine
Species resembling A. spiculatus only Amphileptus eigneri Amphileptus yuianus Amphileptus marinus Amphileptus ilberti
Species resembling A. bellus only Amphileptus aeschtae Amphileptus multinucleatus Amphileptus quadrinucleatus
150–350
200–300
150–250
40–70
400–650
4
B = brackish water; BS = bar-shaped; M = marine; Ma = macronucleus; OS = oral silt; RSK/LSK = right somatic kineties/left somatic kineties; SS = spindle-shaped; VM = ventral margin; – = data not available.
numbers of macronuclear nodules but can be distinguished from them clearly by marked differences in other characteristics (Table 2). Most of the species of Amphileptus that resemble one or both new species have been reported from marine habitats, and only two species, A. salignus and A. wilberti, have been reported from a similar environment (brackish water) as the one in which the new species were found. Phylogenetic relationships within the family Amphileptidae Consistent with previous reports (Gao et al. 2008; Pan et al. 2010, 2014; Wu et al. 2013, 2014; Zhang et al. 2012), our trees showed the family Amphileptidae as a paraphyletic taxon (AU test rejected monophyly; p = 0.019 < 0.5).
Two distinct subclades can be recognized within the family Amphileptidae s. l. (Fig. 5), with members of Amphileptus and Pseudoamphileptus forming one and E. shenzhenense comprising the other by itself. Amphileptus bellus sp. n. occupied a moderately supported (82% ML, 0.93BI), basal position within the Amphileptus– Pseudoamphileptus subclade (Fig. 5); however, none of its morphological characters can be identified as plesiomorphic in nature until sequences of more species of Amphileptus are added to phylogenetic analyses. Amphileptus spiculatus sp. n. and A. aeschtae form a maximally supported, divergent subclade of highly similar sequences differing by just 12 nt, but they have radically different morphological characteristics (Table 2). This suggests that relatively large-scale evolutionary diversification may have happened extremely rapidly in Amphileptus and that inclusion of many more sequences in phylogenetic analyses will
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
667
Two New Brackish Ciliates
Wu et al.
Table 1. Morphological characteristics of Amphileptus spiculatus sp. n. (1st line) and A. bellus sp. n. (2nd line and 3rd line for “Length of Ex”) Characters
Min
Max
Mean
SD
CV
n
Body length
70 300 25 40 11 31 6 6 2 2 15 15 10 10 6 6 6
140 500 45 100 14 35 8 7 2 4 30 65 25 30 8 20 16
112.7 399.5 34.4 60.9 11.8 31.9 6.5 6.8 2.0 2.5 21.4 39.9 15.5 18.3 6.9 16.4 12.1
18.23 67.56 5.35 14.76 1.06 1.21 0.58 0.35 0 0.57 4.23 14.31 4.33 5.95 0.59 3.32 2.33
16.2 17.0 15.6 24.3 9.0 3.8 9.0 5.1 0 23.1 19.8 35.8 28.1 32.4 8.5 20.2 19.3
26 29 26 29 26 29 26 29 26 29 26 29 26 29 26 29 29
Body width Number of RSKa Number of LKDb Number of Ma Length of Ma Width of Ma Length of Ex Type 1 Type 2
Figure 2 Amphileptus spiculatus sp. n. from live (A–C, G, H, J) and after protargol impregnation (D–F, I, K, L). A. Right view of a typical individual. B. Extrusomes. C. Cortical granules. D. Extrusome pattern and nuclear apparatus. E, F. Ciliary pattern of right (E) and left (F) sides. G. Right side view, arrow points to the beak-like anterior end. H. Anterior portion of left side, to show the bristles of dorsal brush (arrowheads) and cortical granules (arrows). I. Anterior portion of right side, arrows indicate the suture. J. Right side view, arrowheads mark the contractile vacuoles. K. Nuclear apparatus, arrow marks the micronucleus. L. Anterior portion of left side, arrowheads refer to the dorsal brush. DB = dorsal brush; PK1, 2 = perioral kinety 1, 2. Ma = macronuclear nodules. Scale bars, 50 lm in (A, D–F, G, J); 10 lm in (B); 20 lm in (H, I, K, L).
rows on the both sides of cell (Fig. 2C, H). The right side is densely ciliated with cilia about ~8 lm long arranged in rows that are located within conspicuous longitudinal furrows and which form a distinct anterior suture that is detectable also in vivo (Fig. 2G, J). The left somatic cilia are sparsely distributed and difficult to detect in vivo. The cytoplasm is slightly grayish, often with numerous refringent globules (2–5 lm across) that render the main body region more or less opaque (Fig. 2G, J). Locomotion is by gliding moderately fast on the substrate or by swimming while rotating clockwise about the longitudinal axis. The ciliary pattern is shown in Fig. 2E, F, I, L. There are 11–14 right kineties, including perioral kinety 2; the intermediate kineties are shortened forming a distinct anterior suture on the right side (Fig. 2E, I). The left side with 6–8 ciliated somatic kineties including perioral kinety 1 and dorsal brush kinety (DB), which extends to about anterior 2/5 of the cell-length and is composed of regularly spaced dikinetids (Fig. 2F, L). There are two perioral kineties located along the cytostome. Perioral kinety 1 (PK1), to the left of the oral slit, composed of dikinetids in the anterior 2/3 and of monokinetids in the posterior 1/3 (Fig. 2F). Perioral kinety 2 (PK2),
664
All measurements in lm. Data based on protargol-impregnated specimens. CV = coefficient of variation in %; Ex = extrusomes; LSK = left somatic kineties; Min = minimum; Max = maximum; Mean = arithmetic mean; n = sample size; RSK = right somatic kineties; SD = standard deviation. a Perioral kineties 2 included. b Perioral kineties 1 and dorsal brush kinety included.
Figure 3 Amphileptus bellus sp. n. drawn from live (A–C) and after protargol impregnation (D–G). (A) Right view of a typical individual. (B) Extrusomes. (C) Cortical granules. (D) Macronuclear pattern. (E) Extrusome pattern and nuclear apparatus. (F, G) Ciliary pattern of left (F) and right (G) side, arrowheads indicate the suture. DB = dorsal brush; PK1, 2 = perioral kinety 1, 2. Ma = macronuclear nodules. Scale bars, 100 lm in (A, E, F, G); 10 lm in (B).
to the right of the oral slit, consists of narrowly spaced dikinetids in the anterior half and continues to the posterior as a row of monokinetids (Fig. 2E).
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
Two New Brackish Ciliates
Wu et al.
Figure 4 Photomicrographs of Amphileptus bellus sp. n. from live (A–G) and after protargol impregnation (H–O). (A–C, F) Shape variation among different individuals, arrowheads mark the contractile vacuoles, arrows show the macronuclear nodules. (D) Anterior part of right side, arrow indicates the extrusomes, arrowhead points to the cilia. (E) Middle part of right side, arrows indicate the cortical granules. (G, O) Posterior part of cell, to show the bar-shaped extrusomes (arrowheads). (H, I) Ciliary pattern of right (H) and left (I) side, arrowheads refer to basal bodies. (J) Anterior part of left side, arrowheads mark the perioral kinety 1, arrows show the dorsal brush, double arrowheads indicate the extrusomes. (K) Ventral side view, to show perioral kinety 1 (arrow) and perioral kinety 2 (arrowhead). (L) Anterior part of right side, arrows point to the suture. (M) Nuclear apparatus, arrow indicates the micronucleus. (N) To show the spindle-shaped extrusomes (arrowheads). Scale bars, 150 lm in (A–C, F), 50 lm in (J).
Amphileptus bellus sp. n. The cell size is highly variable, about 250–400 lm 9 25– 80 lm in vivo, usually 280–350 lm in length. The body is elongate, pyriform, laterally compressed, approximately
3–4:1, and moderately contractile. The anterior tip is bluntly pointed, and the posterior end is acuminate. The neck region comprises 25–35% of the length of the cell and is usually curved slightly dorsad (Fig. 3A, 4A–C). The two to four macronuclear nodules are ovoid to elliptical,
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
665
The Journal of
Eukaryotic Microbiology
Journal of Eukaryotic Microbiology ISSN 1066-5234
ORIGINAL ARTICLE
Two New Brackish Ciliates, Amphileptus spiculatus sp. n. and A. bellus sp. n. from Mangrove Wetlands in Southern China, with Notes on the Molecular Phylogeny of the Family Amphileptidae (Protozoa, Ciliophora, Pleurostomatida) Lei Wua, Zhenzhen Yia, Jiqiu Lia, Alan Warrenb, Hualin Xuc & Xiaofeng Lina a Laboratory of Protozoology, Key Laboratory of Ecology and Environment Science in Guangdong Higher Education, South China Normal University, Guangzhou 510631, China b Department of Life Sciences, The Natural History Museum, Cromwell Rd, London SW7 5BD, United Kingdom c Neilingding-Futian National Nature Reserve Administration Bureau of Guangdong, Shenzhen 518040, China
Keywords Ciliary pattern; new species; SSU rDNA; taxonomy. Correspondence X. Lin, School of Life Sciences, South China Normal University, Guangzhou 510631, China Telephone/FAX number: + 86-20-8521-0644; e-mail: [email protected]
ABSTRACT Two new brackish pleurostomatid ciliates, Amphileptus spiculatus sp. n. and A. bellus sp. n. were collected from mangrove wetlands of southern China and their morphology and molecular phylogeny were studied. Amphileptus spiculatus sp. n. can be distinguished from congeners by the presence of 11–14 right and 6–8 left kineties, two macronuclear nodules and a conspicuous beak-like anterior body end. Amphileptus bellus sp. n. is characterized by the presence of 2–4 macronuclear nodules, 31–35 right and 6 or 7 left kineties and two types of extrusomes. Phylogenetic analyses based on SSU rDNA sequences data indicate that the family Amphileptidae is paraphyletic.
Received: 28 September 2014; revised 1 January 2015; accepted February 12, 2015. doi:10.1111/jeu.12225
MANGROVE wetlands are unique ecosystems that interact strongly with marine, freshwaters, and terrestrial ecosystems and thus support a high biodiversity (Tam and Wong 2002). All the major groups of macroscopic organisms in mangroves, from fungi and vascular plants to benthic invertebrates and birds, have been investigated to some extent (Gopal and Chauhan 2006; Lu and Wang 1996). By comparison, unicellular microorganisms such as ciliated protozoa are poorly studied in this special biotope. Pleurostomatids are a large group of free-living ciliates. Recent studies have suggested that their diversity is high in mangrove wetlands and other brackish habitats (Chen et al. 2011; Pan et al. 2010, 2013; Wu et al. 2013, 2014). The earliest established genus of the order Pleurostomatida, Amphileptus Ehrenberg, 1830, has over 50 nominal species reported from aquatic habitats worldwide. The vast majority of Amphileptus species are from freshwaters (Canella 1960; Curds 1982; Song and Wilbert 1989; Stokes 1886; Vuxanovici 1960; Wang 1934, 1940; Wang and Nie 1933), most of the others being marine (Lin et al. 2005a,b, 2007; Song 1991; Song et al. 2004; Wilbert and Song
662
2005). To date, only two species have been isolated from brackish waters (Chen et al. 2011; Pan et al. 2014). To further explore the diversity of pleurostomatid ciliates in brackish habitats, we investigated mangrove wetlands in southern China. During this survey, two unusual pleurostomatid ciliates were collected. Detailed investigations of specimens in vivo and following protargol staining, and analysis of their small subunit (SSU) rDNA sequences, revealed them to be new members of the genus Amphileptus. In this article, we describe the two new species in detail. In addition, the molecular phylogeny of the family Amphileptidae is analyzed based on SSU rDNA sequences data that indicate the family Amphileptidae is paraphyletic. MATERIALS AND METHODS Sample collection and identification Amphileptus spiculatus sp. n. and A. bellus sp. n. were isolated from Futian mangrove wetland, Shenzhen (22°380 N, 114°060 E) on 13 April 2011 and from Daya Bay mangrove wetland, Huizhou (22°410 N, 114°230 E) on 10
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
Two New Brackish Ciliates
Wu et al.
December 2010, respectively, both in southern China (Fig. 1A, B). Samples were collected using 20-lm mesh plankton nets and were transported to the laboratory in 250-ml wide-mouth bottles containing water from the collection site. Ciliates were maintained in the laboratory in Petri dishes containing habitat water with rice grains to enrich the growth of bacteria, both as a food resource on which the Amphileptus fed directly and as food resource for other protists on which the Amphileptus fed as well. Observations of living cells were carried out with bright field and differential interference contrast microscopy. The protargol staining method according to Wilbert (1975) was used to reveal the ciliary pattern. Counts and measurement were made at 20–1,000X magnifications. Drawings of live specimens were based on in vivo observations and photomicrographs. Drawings of protargol-stained specimens were made with the help of a camera lucida at 1,000X. Terminology and systematics are according to Lynn (2008). SSU rDNA sequence For each species, one or more cells were isolated, repeatedly rinsed with filtered habitat water in order to remove contaminants, and transferred into 45 ll ATL buffer for DNA extraction. Genomic DNA extraction, gene amplification, and gene sequencing were conducted according to the method described by Wu et al. (2013). Phylogenetic analyses SSU rDNA sequences of Amphileptus bellus sp. n. and A. spiculatus sp. n., along with those of another 29 related species obtained from GenBank (including all available SSU rDNA sequences of the order Pleurostomatida), were used in our analyses. The GenBank accession numbers of Loxophyllum species were as follows: L. jini EF123708, L. rostratum DQ190465, L. spirellum
Figure 1 Sites from which samples were taken. A. Futian mangrove wetland, Shenzhen. B. Daya Bay mangrove wetland, Huizhou.
GU574810, L. sp. EU242511, L. chinense JN974455, L. caudatum JN974453, L. rugosum JN974454, L. meridionale KC469985, L. salinum KC476181, L. planum KC476180 and L. perihoplophorum KC493570. The accession numbers of all other species used in the analyses are shown in Fig. 5. Metopus contortus and M. palaeformis (class Armophorea) were used as the out-group taxa. The SSU rDNA sequences were aligned using CLUSTAL W implemented in Bioedit 7.0 (Hall 1999). The alignment was then further modified manually by removing both the primers and the highly variable regions. The final alignment of 1,587 characters was used to construct phylogenetic trees. Maximum likelihood (ML) analysis was performed online (http://www.phylo.org) on the CIPRES Portal V 1.15 (Miller et al. 2010), using the RAxML and the parameters according to Stamatakis et al. (2008). The reliability of internal branches was assessed by a nonparametric bootstrap method with 1,000 replicates. A Bayesian inference (BI) analysis was carried out with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using the Markov chain Monte Carlo algorithm and the best model indicated by MrModeltest v.2 (Nylander 2004). The program was run for 1,500,000 generations with a sample frequency of 100 and a burn-in of 3,750. The approximately unbiased (AU) test (Shimodaira 2002) was used to test the hypothesis that the family Amphileptidae is monophyletic. The ML tree was generated with a constraint block, enforcing the constraint of focal group monophyly in PAUP under the same GTR + I + G model. The site-wise likelihoods were calculated using PAUP (Swofford 2002) for the resulting constrained and nonconstrained ML topologies. The scores were then subjected to the AU test as implemented in Consel 0.1 (Shimodaira and Hasegawa 2001). RESULTS Amphileptus spiculatus sp. n. The cell is about 85–150 lm 9 90–150 lm in vivo, usually 100–125 lm in length. The body is pyriform, laterally compressed, approximately 3:1, flexible, and moderately contractile. The anterior tip is sharply pointed and curved to form a conspicuous beak, the posterior end is broadly rounded, the neck region comprises approximately 20% of the length of the cell (Fig. 2A, G). There are two ovoid macronuclear nodules located in the central region of the cell, each about 15–30 lm 9 10–25 lm in size after fixation (Fig. 2D, F, K). The single micronucleus is spherical, about 3–5 lm in length, and located between the two macronuclear nodules (Fig. 2D, F, K). There are two or three contractile vacuoles, about 5–10 lm in diam., located near the ventral margin in the cell posterior (Fig. 2A, J). The extrusomes are pyriform, straight to slightly curved, 5– 7 lm long (Fig. 2B), some of them are evenly arranged along the oral slit, and others are scattered in the cytoplasm (Fig. 2D, K, L). The pellicle is thin and with numerous small (< 0.5 lm across), grayish, dot-like cortical granules which are densely distributed between the ciliary
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
663
Two New Brackish Ciliates
Wu et al.
Table 1. Morphological characteristics of Amphileptus spiculatus sp. n. (1st line) and A. bellus sp. n. (2nd line and 3rd line for “Length of Ex”) Characters
Min
Max
Mean
SD
CV
n
Body length
70 300 25 40 11 31 6 6 2 2 15 15 10 10 6 6 6
140 500 45 100 14 35 8 7 2 4 30 65 25 30 8 20 16
112.7 399.5 34.4 60.9 11.8 31.9 6.5 6.8 2.0 2.5 21.4 39.9 15.5 18.3 6.9 16.4 12.1
18.23 67.56 5.35 14.76 1.06 1.21 0.58 0.35 0 0.57 4.23 14.31 4.33 5.95 0.59 3.32 2.33
16.2 17.0 15.6 24.3 9.0 3.8 9.0 5.1 0 23.1 19.8 35.8 28.1 32.4 8.5 20.2 19.3
26 29 26 29 26 29 26 29 26 29 26 29 26 29 26 29 29
Body width Number of RSKa Number of LKDb Number of Ma Length of Ma Width of Ma Length of Ex Type 1 Type 2
Figure 2 Amphileptus spiculatus sp. n. from live (A–C, G, H, J) and after protargol impregnation (D–F, I, K, L). A. Right view of a typical individual. B. Extrusomes. C. Cortical granules. D. Extrusome pattern and nuclear apparatus. E, F. Ciliary pattern of right (E) and left (F) sides. G. Right side view, arrow points to the beak-like anterior end. H. Anterior portion of left side, to show the bristles of dorsal brush (arrowheads) and cortical granules (arrows). I. Anterior portion of right side, arrows indicate the suture. J. Right side view, arrowheads mark the contractile vacuoles. K. Nuclear apparatus, arrow marks the micronucleus. L. Anterior portion of left side, arrowheads refer to the dorsal brush. DB = dorsal brush; PK1, 2 = perioral kinety 1, 2. Ma = macronuclear nodules. Scale bars, 50 lm in (A, D–F, G, J); 10 lm in (B); 20 lm in (H, I, K, L).
rows on the both sides of cell (Fig. 2C, H). The right side is densely ciliated with cilia about ~8 lm long arranged in rows that are located within conspicuous longitudinal furrows and which form a distinct anterior suture that is detectable also in vivo (Fig. 2G, J). The left somatic cilia are sparsely distributed and difficult to detect in vivo. The cytoplasm is slightly grayish, often with numerous refringent globules (2–5 lm across) that render the main body region more or less opaque (Fig. 2G, J). Locomotion is by gliding moderately fast on the substrate or by swimming while rotating clockwise about the longitudinal axis. The ciliary pattern is shown in Fig. 2E, F, I, L. There are 11–14 right kineties, including perioral kinety 2; the intermediate kineties are shortened forming a distinct anterior suture on the right side (Fig. 2E, I). The left side with 6–8 ciliated somatic kineties including perioral kinety 1 and dorsal brush kinety (DB), which extends to about anterior 2/5 of the cell-length and is composed of regularly spaced dikinetids (Fig. 2F, L). There are two perioral kineties located along the cytostome. Perioral kinety 1 (PK1), to the left of the oral slit, composed of dikinetids in the anterior 2/3 and of monokinetids in the posterior 1/3 (Fig. 2F). Perioral kinety 2 (PK2),
664
All measurements in lm. Data based on protargol-impregnated specimens. CV = coefficient of variation in %; Ex = extrusomes; LSK = left somatic kineties; Min = minimum; Max = maximum; Mean = arithmetic mean; n = sample size; RSK = right somatic kineties; SD = standard deviation. a Perioral kineties 2 included. b Perioral kineties 1 and dorsal brush kinety included.
Figure 3 Amphileptus bellus sp. n. drawn from live (A–C) and after protargol impregnation (D–G). (A) Right view of a typical individual. (B) Extrusomes. (C) Cortical granules. (D) Macronuclear pattern. (E) Extrusome pattern and nuclear apparatus. (F, G) Ciliary pattern of left (F) and right (G) side, arrowheads indicate the suture. DB = dorsal brush; PK1, 2 = perioral kinety 1, 2. Ma = macronuclear nodules. Scale bars, 100 lm in (A, E, F, G); 10 lm in (B).
to the right of the oral slit, consists of narrowly spaced dikinetids in the anterior half and continues to the posterior as a row of monokinetids (Fig. 2E).
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
Two New Brackish Ciliates
Wu et al.
Figure 4 Photomicrographs of Amphileptus bellus sp. n. from live (A–G) and after protargol impregnation (H–O). (A–C, F) Shape variation among different individuals, arrowheads mark the contractile vacuoles, arrows show the macronuclear nodules. (D) Anterior part of right side, arrow indicates the extrusomes, arrowhead points to the cilia. (E) Middle part of right side, arrows indicate the cortical granules. (G, O) Posterior part of cell, to show the bar-shaped extrusomes (arrowheads). (H, I) Ciliary pattern of right (H) and left (I) side, arrowheads refer to basal bodies. (J) Anterior part of left side, arrowheads mark the perioral kinety 1, arrows show the dorsal brush, double arrowheads indicate the extrusomes. (K) Ventral side view, to show perioral kinety 1 (arrow) and perioral kinety 2 (arrowhead). (L) Anterior part of right side, arrows point to the suture. (M) Nuclear apparatus, arrow indicates the micronucleus. (N) To show the spindle-shaped extrusomes (arrowheads). Scale bars, 150 lm in (A–C, F), 50 lm in (J).
Amphileptus bellus sp. n. The cell size is highly variable, about 250–400 lm 9 25– 80 lm in vivo, usually 280–350 lm in length. The body is elongate, pyriform, laterally compressed, approximately
3–4:1, and moderately contractile. The anterior tip is bluntly pointed, and the posterior end is acuminate. The neck region comprises 25–35% of the length of the cell and is usually curved slightly dorsad (Fig. 3A, 4A–C). The two to four macronuclear nodules are ovoid to elliptical,
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
665
Two New Brackish Ciliates
Wu et al.
about 20–45 lm 9 15–30 lm in size in vivo, and located in the mid-region of the cell, usually seen in life as slightly transparent areas (Fig. 3D, 4B, C). The single ellipsoidal micronucleus is about 3–6 lm 9 1–3 lm after impregnation, and situated near the macronuclear nodules (Fig. 4M). There are two to four contractile vacuoles, the largest one is about 15–20 lm in diam. and subterminally positioned, and the others are 10–15 lm in diam. and distributed along the ventral margin in the anterior half of the cell (Fig. 3A, 4A–C, F). There are two kinds of extrusomes (Fig. 3B, 4D, G, N): type I, about 6–20 lm long after protargol staining, long bar shaped, some are densely arranged in the oral area, some form a bundle in the posterior region of the cell and some scattered in the cytoplasm (Fig. 3E, 4J, O); type II, spindle-shaped, about 6– 16 lm 9 2–4 lm in size after protargol staining, scattered in the cytoplasm (Fig. 4N). The pellicle is thin with small (< 0.5 lm across), densely spaced, grayish, dot-like cortical granules between ciliary rows on both sides of the cell (Fig. 3C, 4E). The right side is flat and densely ciliated, cilia on the right side about ~10 lm long (Fig. 4D, H); the left side is slightly humped in the central region of the body, cilia sparsely distributed and difficult to detect in life (Fig. 4F, I). The cytoplasm is colorless to grayish, often with numerous tiny, shining globules (2–5 lm across) that render the main part of the body opaque (Fig. 4C). The locomotion is by gliding moderately fast on the substrate or by swimming with a slow anticlockwise rotation about the longitudinal axis. The ciliary pattern is shown in Fig. 3F, G and 4H–L. There are 31–35 right somatic kineties including perioral kinety 2; the intermediate kineties shortened forming a distinct suture in the anterior part of the body (Fig. 3G, 4L). The left side with six- or seven-ciliated kineties including perioral kinety 1 and dorsal brush kinety (DB), which extends to about 1/3 of the cell length and is composed of narrowly spaced dikinetids (Fig. 3F, 4J). There are two perioral kineties along the cytostome. Perioral kinety 1 (PK1), to the left of the oral slit, comprises dikinetids in the anterior half and continues posteriorly as a row of monokinetids (Fig. 3F, 4K, J). The perioral kinety 2 (PK2), to the right of the oral slit, comprises regularly spaced dikinetids in the anterior 2/5 and monokinetids in the posterior 3/5 (Fig. 3F, 4K). SSU rDNA sequence analyses The two new SSU rDNA sequences have been deposited in GenBank database with length, GC content, and accession numbers as follows: A. bellus sp. n., 1,535 bp, 42.00%, KM025128; A. spiculatus sp. n., 1,534 bp, 42.00%, KM025129. Phylogenetic trees constructed using ML and BI analyses had almost identical topologies, therefore, only the ML tree is shown (Fig. 5). Epiphyllum shenzhenense is basal within the order Pleurostomatida. All members of Amphileptus and Pseudoamphileptus form a well-supported clade within the paraphyletic family Amphileptidae (93% ML, 1.00BI). Within the Amphileptus–Pseudoamphi-
666
Figure 5 Phylogenetic tree (ML) based on 31 SSU rDNA sequences of litostomatean ciliates. Newly sequenced species in present study are shown in bold. GenBank accession numbers are given after names of species. Numbers at nodes indicate bootstrap values of maximum likelihood (ML) and posterior probabilities of Bayesian analysis (BI). –, represents disagreement between ML and BI topologies. The scale bar corresponds to five substitutions per 100 nucleotide positions.
leptus subclade, A. bellus occupies a basal position with moderate support values (82% ML, 0.93BI), A. spiculatus clusters with A. aeschtae with maximal support (100% ML, 1.00BI) and A. dragescoi clusters with Pseudoamphileptus macrostoma with low support values (30%ML, 0.85BI) forming a clade that is sister to A. procera. DISCUSSION Comparison of Amphileptus spiculatus sp. n. and A. bellus sp. n. with similar species It is widely accepted that the most important criteria for identification and separation of species in the genus Amphileptus include characteristics of the somatic kineties, contractile vacuoles, cortical granules, macronuclear nodules, and extrusomes (Foissner et al. 1995; Lin and Song 2004; Lin et al. 2007; Song et al. 2004). Amphileptus spiculatus sp. n. and A. bellus sp. n. both resemble the species A. salignus Chen et al. 2011; A. gui Lin et al. 2005a; and A. songi Pan et al. 2014; however, each can be distinguished from all three congeners by a combination of differences in numbers of somatic kineties, location of contractile vacuoles, and form/distribution of extrusomes (Table 2). Beyond its resemblance to the three species just cited, A. spiculatus sp. n. also resembles four other species of Amphileptus that have similar body sizes, numbers of somatic kineties, and numbers of macronuclear nodules (Table 2) but can be distinguished from them by differences in the number/distribution of contractile vacuoles and the form/distribution of the extrusomes (Table 2). Finally, A. bellus sp. n. shows some similarity to three species of Amphileptus with similar body sizes or
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
Two New Brackish Ciliates
Wu et al.
Table 2. Comparison of Ampileptus spiculatus sp. n. and A. bellus sp. n. with one another and with similar species
Species Amphileptus spiculatus sp. n. Amphileptus bellus sp. n.
Body length in vivo (lm)
Number of Ma
85–145 250–400
RSK/LSK
2
11–14/6–8
2–4
31–35/6–7
Number and position of contractile vacuoles
Shape and distribution of extrusomes
3, by posterior 1/4 of VM 2–4; 1 subterminal, rest by anterior 1/3
Biotope
Source of data
Pyriform, oral
B
Present work
Type 1: long BS, oral and tail; Type 2: SS, entire cytoplasm
B
Present work
B
Chen et al. (2011)
M
Lin et al. (2005a)
Species resembling both new species Amphileptus saligus
180–360
2
24–29/4
2–7; 1 dorsal, rest by VM
Amphileptus gui Amphileptus songi
150–300
2
37–50/7–11
3–7, by VM
Type 1: long BS, oral; Type 2: short BS, under pellicle BS, apical
200–450
2
21–28/11–13
3–7, by VM
BS, ventral
M
Song et al. (2004)
100–200
2
14–18/6–9
5–15, by VM
BS, along OS
M
Lin et al. (2007)
100–200
2
18–22/4
1, terminal
Clavate, apical
M
Lin et al. (2005a)
150–300
2
13–21/5–8
7, by VM
Fusiform, along OS
M
Pan et al. (2014)
180–210
2
15–19/7–8
3; by VM
BS, along OS
B
Pan et al. (2014)
29–34/7–10
5–13, by VM
BS, along OS
M
Lin et al. (2007)
–/–
6–10, by VM
–, oral
M
Wang (1934)
30–34/~5
many, by cell margins
BS, oral
M
Dragesco and (1971) Njine
Species resembling A. spiculatus only Amphileptus eigneri Amphileptus yuianus Amphileptus marinus Amphileptus ilberti
Species resembling A. bellus only Amphileptus aeschtae Amphileptus multinucleatus Amphileptus quadrinucleatus
150–350
200–300
150–250
40–70
400–650
4
B = brackish water; BS = bar-shaped; M = marine; Ma = macronucleus; OS = oral silt; RSK/LSK = right somatic kineties/left somatic kineties; SS = spindle-shaped; VM = ventral margin; – = data not available.
numbers of macronuclear nodules but can be distinguished from them clearly by marked differences in other characteristics (Table 2). Most of the species of Amphileptus that resemble one or both new species have been reported from marine habitats, and only two species, A. salignus and A. wilberti, have been reported from a similar environment (brackish water) as the one in which the new species were found. Phylogenetic relationships within the family Amphileptidae Consistent with previous reports (Gao et al. 2008; Pan et al. 2010, 2014; Wu et al. 2013, 2014; Zhang et al. 2012), our trees showed the family Amphileptidae as a paraphyletic taxon (AU test rejected monophyly; p = 0.019 < 0.5).
Two distinct subclades can be recognized within the family Amphileptidae s. l. (Fig. 5), with members of Amphileptus and Pseudoamphileptus forming one and E. shenzhenense comprising the other by itself. Amphileptus bellus sp. n. occupied a moderately supported (82% ML, 0.93BI), basal position within the Amphileptus– Pseudoamphileptus subclade (Fig. 5); however, none of its morphological characters can be identified as plesiomorphic in nature until sequences of more species of Amphileptus are added to phylogenetic analyses. Amphileptus spiculatus sp. n. and A. aeschtae form a maximally supported, divergent subclade of highly similar sequences differing by just 12 nt, but they have radically different morphological characteristics (Table 2). This suggests that relatively large-scale evolutionary diversification may have happened extremely rapidly in Amphileptus and that inclusion of many more sequences in phylogenetic analyses will
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
667
Two New Brackish Ciliates
Wu et al.
Table 1. Morphological characteristics of Amphileptus spiculatus sp. n. (1st line) and A. bellus sp. n. (2nd line and 3rd line for “Length of Ex”) Characters
Min
Max
Mean
SD
CV
n
Body length
70 300 25 40 11 31 6 6 2 2 15 15 10 10 6 6 6
140 500 45 100 14 35 8 7 2 4 30 65 25 30 8 20 16
112.7 399.5 34.4 60.9 11.8 31.9 6.5 6.8 2.0 2.5 21.4 39.9 15.5 18.3 6.9 16.4 12.1
18.23 67.56 5.35 14.76 1.06 1.21 0.58 0.35 0 0.57 4.23 14.31 4.33 5.95 0.59 3.32 2.33
16.2 17.0 15.6 24.3 9.0 3.8 9.0 5.1 0 23.1 19.8 35.8 28.1 32.4 8.5 20.2 19.3
26 29 26 29 26 29 26 29 26 29 26 29 26 29 26 29 29
Body width Number of RSKa Number of LKDb Number of Ma Length of Ma Width of Ma Length of Ex Type 1 Type 2
Figure 2 Amphileptus spiculatus sp. n. from live (A–C, G, H, J) and after protargol impregnation (D–F, I, K, L). A. Right view of a typical individual. B. Extrusomes. C. Cortical granules. D. Extrusome pattern and nuclear apparatus. E, F. Ciliary pattern of right (E) and left (F) sides. G. Right side view, arrow points to the beak-like anterior end. H. Anterior portion of left side, to show the bristles of dorsal brush (arrowheads) and cortical granules (arrows). I. Anterior portion of right side, arrows indicate the suture. J. Right side view, arrowheads mark the contractile vacuoles. K. Nuclear apparatus, arrow marks the micronucleus. L. Anterior portion of left side, arrowheads refer to the dorsal brush. DB = dorsal brush; PK1, 2 = perioral kinety 1, 2. Ma = macronuclear nodules. Scale bars, 50 lm in (A, D–F, G, J); 10 lm in (B); 20 lm in (H, I, K, L).
rows on the both sides of cell (Fig. 2C, H). The right side is densely ciliated with cilia about ~8 lm long arranged in rows that are located within conspicuous longitudinal furrows and which form a distinct anterior suture that is detectable also in vivo (Fig. 2G, J). The left somatic cilia are sparsely distributed and difficult to detect in vivo. The cytoplasm is slightly grayish, often with numerous refringent globules (2–5 lm across) that render the main body region more or less opaque (Fig. 2G, J). Locomotion is by gliding moderately fast on the substrate or by swimming while rotating clockwise about the longitudinal axis. The ciliary pattern is shown in Fig. 2E, F, I, L. There are 11–14 right kineties, including perioral kinety 2; the intermediate kineties are shortened forming a distinct anterior suture on the right side (Fig. 2E, I). The left side with 6–8 ciliated somatic kineties including perioral kinety 1 and dorsal brush kinety (DB), which extends to about anterior 2/5 of the cell-length and is composed of regularly spaced dikinetids (Fig. 2F, L). There are two perioral kineties located along the cytostome. Perioral kinety 1 (PK1), to the left of the oral slit, composed of dikinetids in the anterior 2/3 and of monokinetids in the posterior 1/3 (Fig. 2F). Perioral kinety 2 (PK2),
664
All measurements in lm. Data based on protargol-impregnated specimens. CV = coefficient of variation in %; Ex = extrusomes; LSK = left somatic kineties; Min = minimum; Max = maximum; Mean = arithmetic mean; n = sample size; RSK = right somatic kineties; SD = standard deviation. a Perioral kineties 2 included. b Perioral kineties 1 and dorsal brush kinety included.
Figure 3 Amphileptus bellus sp. n. drawn from live (A–C) and after protargol impregnation (D–G). (A) Right view of a typical individual. (B) Extrusomes. (C) Cortical granules. (D) Macronuclear pattern. (E) Extrusome pattern and nuclear apparatus. (F, G) Ciliary pattern of left (F) and right (G) side, arrowheads indicate the suture. DB = dorsal brush; PK1, 2 = perioral kinety 1, 2. Ma = macronuclear nodules. Scale bars, 100 lm in (A, E, F, G); 10 lm in (B).
to the right of the oral slit, consists of narrowly spaced dikinetids in the anterior half and continues to the posterior as a row of monokinetids (Fig. 2E).
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
Two New Brackish Ciliates
Wu et al.
Figure 4 Photomicrographs of Amphileptus bellus sp. n. from live (A–G) and after protargol impregnation (H–O). (A–C, F) Shape variation among different individuals, arrowheads mark the contractile vacuoles, arrows show the macronuclear nodules. (D) Anterior part of right side, arrow indicates the extrusomes, arrowhead points to the cilia. (E) Middle part of right side, arrows indicate the cortical granules. (G, O) Posterior part of cell, to show the bar-shaped extrusomes (arrowheads). (H, I) Ciliary pattern of right (H) and left (I) side, arrowheads refer to basal bodies. (J) Anterior part of left side, arrowheads mark the perioral kinety 1, arrows show the dorsal brush, double arrowheads indicate the extrusomes. (K) Ventral side view, to show perioral kinety 1 (arrow) and perioral kinety 2 (arrowhead). (L) Anterior part of right side, arrows point to the suture. (M) Nuclear apparatus, arrow indicates the micronucleus. (N) To show the spindle-shaped extrusomes (arrowheads). Scale bars, 150 lm in (A–C, F), 50 lm in (J).
Amphileptus bellus sp. n. The cell size is highly variable, about 250–400 lm 9 25– 80 lm in vivo, usually 280–350 lm in length. The body is elongate, pyriform, laterally compressed, approximately
3–4:1, and moderately contractile. The anterior tip is bluntly pointed, and the posterior end is acuminate. The neck region comprises 25–35% of the length of the cell and is usually curved slightly dorsad (Fig. 3A, 4A–C). The two to four macronuclear nodules are ovoid to elliptical,
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 662–669
665
