Available online at www.sciencedirect.com
ScienceDirect European Journal of Protistology 50 (2014) 134–152
Bozasella gracilis n. sp. (Ciliophora, Entodiniomorphida) from Asian elephant and phylogenetic analysis of entodiniomorphids and vestibuliferids Akira Itoa,∗ , Miki Ishiharab , Soichi Imaib a b
Ookusa Animal Clinic, Ookusa 503, Matsue, Shimane 690-0032, Japan Department of Parasitology, Nippon Veterinary and Life Science University, Musashino, Tokyo 180-8602, Japan
Received 1 May 2013; received in revised form 17 January 2014; accepted 20 January 2014 Available online 25 January 2014
Abstract Bozasella gracilis n. sp. in the order Entodiniomorphida was found in fecal samples of an Asian elephant kept in a zoo. The ciliate has general and infraciliary similarities to the families Ophryoscolecidae and Cycloposthiidae. Phylogenetic trees were inferred from 18S rRNA gene sequences of B. gracilis, 45 entodiniomorphids, 10 vestibuliferids, 5 macropodiniids, and an outgroup, using maximum likelihood, Bayesian inference, and neighbor joining analyses. Of them, there were 32 new sequences; 26 entodiniomorphid species in the genera, Bozasella, Triplumaria, Gassovskiella, Ditoxum, Spirodinium, Triadinium, Tetratoxum, Pseudoentodinium, Ochoterenaia, Circodinium, Blepharocorys, Sulcoarcus, Didesmis, Alloiozona, Blepharoconus, Hemiprorodon, and Prorodonopsis, and 6 vestibuliferid species in the genera, Buxtonella, Balantidium, Helicozoster, Latteuria, and Paraisotricha. Thirty additional sequences were retrieved from the GenBank database. Phylogenetic trees revealed non-monophylies of the orders Entodiniomorphida and Vestibuliferida, the suborders Entodiniomorphina and Blepharocorythina, and the families Cycloposthiidae and Paraisotrichidae. Bozasella gracilis was sister to Triplumaria. In addition, to avoid homonymy, we propose Gilchristinidae nom. nov., Gilchristina nom. nov. and Gilchristina artemis (Ito, Van Hoven, Miyazaki & Imai, 2006) comb. nov. © 2014 Elsevier GmbH. All rights reserved. Keywords: Bozasella gracilis n. sp.; 18S rRNA gene sequence; Elephant; Entodiniomorphida; Gilchristina artemis (Ito, Van Hoven, Miyazaki & Imai, 2006) comb. nov.; Vestibuliferida
Introduction Intestinal ciliates in the order Entodiniomorphida and the order Vestibuliferida have been described from African and Asian elephants (Buisson 1923; Eloff and Van Hoven 1980; Kofoid 1935; Ito et al. 2010, 2011; Latteur 1958, 1966, 1967;
∗ Corresponding
author. Tel.: +81 852 237780. E-mail address: [email protected] (A. Ito).
0932-4739/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ejop.2014.01.003
Latteur and Bousez 1970; Latteur and Dartevelle 1971; Latteur et al. 1970; Mandal and Choudhury 1983a,b, 1984; Timoshenko and Imai 1995, 1997; Wolska 1967, 1968, 1970, 1986). In our study on the phylogeny of entodiniomorphids and vestibuliferids, we found a unique new entodiniomorphid species from Asian elephants, which has morphological characters of both the family Ophryoscolecidae and the family Cycloposthiidae in its general appearance and infraciliature. In the following, we describe this new species, Bozasella gracilis n. sp. and its infraciliary bands. Further, we sequenced
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
the 18S rRNA gene of B. gracilis, 25 entodiniomorphids, and six vestibuliferids and discuss the phylogenies of the orders Entodiniomorphida and Vestibuliferida.
Material and Methods Sampling Fecal samples containing Bozasella gracilis n. sp. for morphological study were collected from a female Asian elephant (Elephas maximus maximus) which was born in Sri Lanka and was kept in Tokushima zoo, Tokushima prefecture, Japan. The samples were immediately fixed in five times the volume of 10% formalin solution within five minutes after defecation and stored in a dark place after they were filtered through two layers of gauze into a bottle in order to remove plant and feed material. We collected 12 samples for the 18S rRNA gene sequences and isolated 1–7 species from each sample. Cells of B.
135
gracilis, 25 entodiniomorphid ciliates and six vestibuliferid ciliates were obtained from these samples of five elephants, four horses, and a cattle listed in Table 1. Fecal samples were fixed within five minutes after defecation. Intestinal contents of a horse were fixed immediately after euthanasia due to limb disorder. A rectal content of a cattle was fixed just after rectal examination. All samples were fixed in five times the volume of 80% ethanol and preserved in the refrigerator (4 ◦ C) after they were filtered through two layers of gauzes; the supernatant was replaced with 100% ethanol. All samples were also fixed in five times the volume of 10% formalin solution to determine their species composition. Micrographs of 25 entodiniomorphid ciliates and six vestibuliferid ciliates fixed in formalin solution were shown in Figs 13–43.
Morphology and silver impregnation The infraciliary bands of the new species were stained using the pyridinated silver carbonate impregnation method,
Table 1. List of entodiniomorphid and vestibuliferid ciliates analyzed in this study, including specimen, host animal, and location. Species
Specimen
Host animal (birth place)
Location
Order Entodiniomorphida Bozasella gracilis n. sp. Triplumaria solea Triplumaria sukuna Triplumaria dvoinosi Triplumaria fulgora Triplumaria harpagonis Gassovskiella galea Ditoxum funinucleum Spirodinium equi Triadinium caudatum Tetratoxum parvum Tetratoxum unifasciculatum Tetratoxum excavatum Pseudoentodinium elephantis Ochoterenaia appendiculata Circodinium minimum Blepharocorys microcorys Blepharocorys angusta Blepharocorys jubata Blepharocorys uncinata Sulcoarcus pellucidulus Didesmis ovalis Alloiozona trizona Blepharoconus hemiciliatus Hemiprorodon gymnoprosthium Prorodonopsis coli
Feces Feces Feces Feces Feces Feces Feces Feces Feces Dorsal colon Feces Feces Dorsal colon Feces Feces Feces Feces Feces Feces Cecum Feces Ventral colon Cecum Feces Ventral colon Dorsal colon
Asian elephant (Sri Lanka) Asian elephant (Sri Lanka) Asian elephant (India) Asian elephant (Sri Lanka) Asian elephant (India) Asian elephant (India) Yonaguni horse Yonaguni horse Yonaguni horse Pony Yonaguni horse Riding horse Pony Asian elephant (India) Riding horse Yonaguni horse Riding horse Yonaguni horse Kiso horse Pony Yonaguni horse Pony Pony Riding horse Pony Pony
Tokushima Zoo Tokushima Zoo Kobe Oji Zoo Tokushima Zoo Kobe Oji Zoo Kobe Oji Zoo Yonaguni island Yonaguni island Yonaguni island Matsue Yonaguni island Yasugi Matsue Kobe Oji Zoo Yasugi Yonaguni island Yasugi Yonaguni island Matsue Matsue Yonaguni island Matsue Matsue Yasugi Matsue Matsue
Order Vestibuliferida Buxtonella sulcata Balantidium coli Helicozoster indicus Latteuria polyfaria Latteuria media Paraisotricha minuta
Rectum Feces Feces Feces Feces Ventral colon
Japanese black beef cattle Asian elephant (India) Asian elephant (Thailand) African bush elephant (South Africa) Asian elephant (India) Pony
Matsue Japan Miyazaki Phoenix Zoo Himeji Central Park Kobe Oji Zoo Matsue
136
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
Figs 1–6. Schematic figures of Bozasella gracilis n. sp. 1, 3–6. Cells from left side. 2. Cell from right side. 5, 6. Cells after silver impregnation. ACZ, adoral ciliary zone; ADC, anterior dorsal caudalium; AP, adoral polybrachykinety; CP, cytoproct; CV, contractile vacuole; DLG, dorsal left groove; LG, left median groove; MA, macronucleus; MI, micronucleus; OP, operculum; PAD, polybrachykinety of anterior dorsal caudalium; PDC, posterior dorsal caudalium; PK, paralabial kineties; PPD, polybrachykinety of posterior dorsal caudalium; PPV, polybrachykinety of posterior ventral caudalium; PVC, posterior ventral caudalium; PVP, perivestibular polybrachykinety; RG, right median groove; TF, tail flap; VP vestibular polybrachykinety; VS, vestibulum. Bar = 10 m.
following Ito and Imai (1998). The orientation of ciliates used by Dogiel (1927) was adopted; the side beneath which the macronucleus lies was termed the dorsal side, the opposite one the ventral side, thus defining the right and left sides. Cell measurements and morphometric ratios were determined by measuring a sample of 20 fixed cells using a calibrated micrometer; measurements are reported as mean ± S. D. (minimum–maximum). Body length was determined as the distance between the anterior and posterior ends of the body.
The term polybrachykinety refers to infraciliary bands composed of numerous, short, parallel kineties (Ito and Imai 1998; Fernández-Galiano et al. 1985). Somatic ciliary zones were termed the caudalium and caudalia. Taxonomy of the orders, suborders and families follows Lynn (2008). Tetratoxum parvum has three morphotypes based on the longitudinal grooves on the surface of the body (Strelkow 1939). The cell of T. parvum examined was the type without grooves. Prorodonopsis coli and Hemiprorodon gymnoprosthium closely
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
137
Figs 7, 8. Infraciliary bands of the adoral ciliary zone in Bozasella gracilis. 7. From left side. 8. From right side. AP, adoral polybrachykinety; DF, dorsal vestibular fibers; PK, paralabial kineties; PVP, perivestibular polybrachykinety; VF, ventral vestibular fibers; VP, vestibular polybrachykinety. Bar = 10 m.
resemble each other (Strelkow 1939). P. coli is holotrichous whereas H. gymnoprosthium has a small naked area on the body surface around the cytoproct. Cells with cilia flowing posteriorly could not be identified. Only the cells of these species with cilia flowing anteriorly was examined. Both the samples for Balantidium coli and Buxtonella sulcata were determined to be composed of single species by studying their parallel samples which were fixed in formalin solutions. Latteuria polyfaria and L. media closely resemble each other. Each sample for these two Latteuria species contained either of them.
DNA extraction and amplification Twelve parallel samples fixed in formalin solution were stained using the pyridinated silver carbonate impregnation method (Ito and Imai 1998) to correctly determine their species composition in detail. Samples fixed in ethanol were diluted with water in a dish; 10–100 l of ethanol sample was pipetted into 400 l of distilled water in the dish. The dilution rate depended on the densities of ciliates and fecal or intestinal particles. Center-well organ culture dishes (Falcon 353037) were used to easily pick and wash individual cells. A single cell was picked from a Falcon dish in 1 l diluted material using a micropipette under an inverted microscope and put into 400 l distilled water in another Falcon dish.
This process was repeated more than five times to isolate and wash the single cell. Single cells after washing were drawn up in a volume of 1 l distilled water and transferred to a PCR tube. The single cells of each entodiniomorphid species had typical morphological features in their body, nuclei, and ciliary distribution. They were not filled up with food particles in the endoplasm and were without fecal or intestinal particles attached to the buccal and/or somatic ciliary zones. DNA extraction of Bozasella gracilis, 25 entodiniomorphids, and six vestibuliferids was performed using the freeze-thawing single-cell PCR method of Honma et al. (2007). The PCR tube was frozen at −80 ◦ C for 3 min and thawed at 60 ◦ C for 30 s to facilitate cell breakage. After five rounds of this freeze-thawing step, 7 l of reaction buffer (10 mM Tris-HCl, 1.5 mM Mg Cl2 , 50 mM KCl, 0.01% Proteinase-K, 0.01% sodium dodecyl sulfate) was added to the PCR tube. The PCR tube was then incubated at 37 ◦ C for 60 min and heated at 95 ◦ C for 10 min. PCR amplifications were performed in 20 l vol., containing template DNA in 1 l of reaction mixture, 0.25 M of both the forward primer 82F (5 -GAA ACT GCG AAT GGC TC-3 ; Elwood et al. 1985) and the reverse primer EkyB (5 TGA TCC TTC TGC AGG TTC ACC TAC-3 ; Medlin et al. 1988), 0.2 mM each dNTP Mixtures, 2 mM MgCl2 , and 0.5 U of Takara Ex Taq DNA polymerase (Takara Bio) with the following PCR program: 1 cycle at 95 ◦ C for 5 min; 35 cycles at 98 ◦ C for 5 s, 50 ◦ C for 30 s, 72 ◦ C for 2 min; 1 cycle
138
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
Figs 9–12. Micrographs of B. gracilis. After silver impregnation. 9–11. Cells from left side. 12. Cell from right side. ACZ, adoral ciliary zone; ADC, anterior dorsal caudalium; AP, adoral polybrachykinety; DF, dorsal vestibular fibers; LG, left median groove; MA, macronucleus; OP, operculum; PAD, polybrachykinety of anterior dorsal caudalium; PDC, posterior dorsal caudalium; PK, paralabial kineties; PVC, posterior ventral caudalium; PVP, perivestibular polybrachykinety; TF, tail flap; VF, ventral vestibular fibers; VP vestibular polybrachykinety. Bar = 10 m.
at 72 ◦ C for 10 min. The PCR products were evaluated by electrophoresis in a 1.0% agarose gel followed by staining with ethidium bromide solution and by visualization on an ultraviolet transilluminator. The PCR products were purified using ExoSAP-IT (USB). The purified PCR products were sequenced in both directions using the BigDye Terminator Cycle Sequencing Kit on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems) according to the manufacture’s instructions with four primers; two forward primers (82F and either primer (5 -CCA TTT CAG TAC CTT ATG AG-3 ) or (5 -TTT GCC AAG GAT GTT TTC-3 )) and two reverse primers (EkyB and (5 -CTT GGC AAA TGC TTT CGC-3 )).
Sequence availability and phylogenetic analysis For the phylogenetic analysis of 18S rRNA gene sequences from 46 entodiniomorphids, 10 vestibuliferids, five macropodiniids and an outgroup, 30 gene sequences were retrieved from the GenBank database and were listed in Table 2 along with the 32 sequences examined in this study. Multiple sequence alignment was performed using Clustal W 1.83 in the software package MEGA 5 (Tamura et al. 2011). Phylogenetic trees were constructed using three different analyses, maximum likelihood (ML) (Felsenstein 1981), Bayesian inference (BI), and neighbor joining (NJ) (Saitou
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
139
Table 2. List of entodiniomorphid, vestibuliferid and macropodiniid ciliates and outgroup analyzed in this study, 32 new sequences in boldface and 30 sequences retrieved from GenBank database, including their taxonomic classification, GenBank accession number of 18S rRNA gene sequence, and reference. Order, Family, Species Order Entodiniomorphida Reichenow in Doflein & Reichenow, 1929 Suborder Entodiniomorphina Reichenow in Doflein & Reichenow, 1929 Family Ophryoscolecidae Stein, 1859 Entodinium longinucleatum Dogiel, 1925 Entodinium caudatum Stein, 1858 Epidinium caudatum (Fiorentini, 1889) Ophryoscolex purkynjei Stein, 1858 Eudiplodinium rostratum (Fiorentini, 1889) Diplodinium dentatum (Stein, 1858) Eudiplodinium maggii (Fiorentini, 1889) Metadinium medium Awerinzew & Mutafowa, 1914 Polyplastron multivesiculatum (Dogiel & Fedorowa, 1925) Ostracodinium gracile (Dogiel, 1925) Family Troglodytellidae Corliss, 1979 Troglodytella abrassarti Brumpt & Joyeux, 1912 Family Cycloposthiidae, Poche, 1913 Cycloposthium ishikawai Gassovsky, 1919 Cycloposthium edentatum Strelkow, 1929 Cycloposthium bipalmatum (Fiorentini, 1890) Bozasella gracilis n. sp. Triplumaria solea Ito, Mishima, Nataami, Ike & Imai, 2011 Triplumaria sukuna Ito, Mishima, Nataami, Ike & Imai, 2011 Triplumaria selenica Latteur, Tuffrau & Wespes, 1970 Triplumaria dvoinosi Timoshenko & Imai, 1995 Triplumaria fulgora Ito, Mishima, Nataami, Ike & Imai, 2011 Triplumaria harpagonis Ito, Mishima, Nataami, Ike & Imai, 2011 Tripalmaria dogieli Gassovsky, 1919 Family Spirodiniidae Strelkow, 1939 Gassovskiella galea (Gassovsky, 1919) Ditoxum funinucleum Gassovsky, 1919 Spirodinium equi Fiorentini, 1890 Triadinium caudatum Fiorentini, 1890 Cochliatoxum periachtum Gassovsky, 1919 Tetratoxum parvum Hsiung, 1930 Tetratoxum unifasciculatum (Fiorentini, 1890) Tetratoxum excavatum Hsiung, 1930 Family Parentodiniidae Ito, Miyazaki & Imai, 2002 Parentodinium sp Family Pseudoentodiniidae Wolska, 1986 Pseudoentodinium elephantis Wolska, 1986 Suborder Blepharocorythina Wolska, 1971 Family Blepharocorythidae Hsiung, 1929 Raabena bella Wolska, 1967 Ochoterenaia appendiculata Chavarria, 1933 Circodinium minimum Wolska, 1967 Blepharocorys microcorys Gassovsky, 1919 Blepharocorys angusta Gassovsky, 1919 Blepharocorys curvigula Gassovsky, 1919 Blepharocorys jubata Bundle, 1895 Blepharocorys uncinata (Fiorentini, 1890) Suborder Archistomatina de Puytorac et al., 1974 Family Buetschliidae Poche, 1913 Sulcoarcus pellucidulus Hsiung, 1935 Didesmis ovalis Fiorentini, 1890 Alloiozona trizona Hsiung, 1930
GenBank acc. no.
Reference
AB481099 U57765 U57763 U57768 AB536716 U57764 U57766 AB535215 U57767 AB535662
Ito et al. (2010) Wright et al. (1997) Wright et al. (1997) Wright and Lynn (1997b) Ito et al. (2010) Wright and Lynn (1997b) Wright and Lynn (1997b) Ito et al. (2010) Wright et al. (1997) Ito et al. (2010)
AB437346
Irbis et al. (2008)
EF632076 EF632077 AB530165 AB793744 AB793745 AB793777 AB533538 AB793778 AB793781 AB793782 EF632074
Strüder-Kypke et al. (2007) Strüder-Kypke et al. (2007) Miyazaki (2005)
AB793783 AB794091 AB794092 AB794968 EF632078 AB794969 AB794970 AB794971 AB530164
Ito et al. (2010)
Strüder-Kypke et al. (2007)
Strüder-Kypke et al. (2007)
Miyazaki (2005)
AB794972
AB534183 AB794973 AB794974 AB794975 AB794976 AB534184 AB794977 AB794978
AB795024 AB795025 AB795026
Ito et al. (2010)
Ito et al. (2010)
140
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
Table 2 (Continued) Order, Family, Species
GenBank acc. no.
Blepharoconus hemiciliatus Gassovsky, 1919 Hemiprorodon gymnoprosthium Strelkow, 1939 Prorodonopsis coli Gassovsky, 1919 Order Vestibuliferida de Puytorac et al., 1974 Family Isotrichidae Bütschli, 1889 Dasytricha ruminantium Schuberg, 1888 Isotricha prostoma Stein, 1859 Isotricha intestinalis Stein, 1859 Family Balantidiidae Reichenow, 1929 Balantidium coli (Malmsten, 1857) Family Pycnotrichidae Poche, 1913 Buxtonella sulcata Jameson, 1926 Family Paraisotrichidae Cunha, 1917 Helicozoster indicus Latteur, 1967 Latteuria polyfaria Timoshenko & Imai, 1997 Latteuria media Timoshenko & Imai, 1997 Paraisotricha colpoidea Fiorentini, 1890 Paraisotricha minuta Hsiung, 1930 Order Macropodiniidae Lynn, 2008 Family Macropodiniidae Dehority, 1996 Macropodinium ennuensis Dehority, 1996 Family Polycostidae Cameron & O’Donoghue, 2003 Polycosta turniae Cameron & O’Donoghue, 2003 Family Amylovoracidae Cameron & O’Donoghue, 2002 Amylovorax dogieli Cameron, O’Donoghue & Adlard, 2000 Bandia cribbi Cameron & O’Donoghue, 2002 Bitricha tasmaniensis Cameron, O’Donoghue & Adlard, 2000 Outgroup Didinium nasutum (Muller, 1786)
AB795027 AB795028 AB795029
and Nei 1987); ML analysis was performed with MEGA 5 by bootstrapping 1000 replicates. The best combination of model and rates for the ML tree was calculated in MEGA 5. Bayesian inference analysis was performed with MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003). The model used to construct the BI tree was the same model as calculated in MEGA 5 for the ML tree. Marcov Chain Monte Carlo (MCMC) were run with two sets of four independent chains (three heated chains and a cold chain). The temperature for heating the chains was 0.2. A total of 5,000,000 generations was calculated with trees sampled every 100 generations. An average standard deviation of the split frequencies below 0.05 was used to determine convergence. Neighbor joining analysis was performed with MEGA 5 by bootstrapping 1000 replicates. The evolutionary distances among Bozasella and Triplumaria species were calculated using the maximum composite likelihood method (Tamura et al. 2004). To test whether the family Isotrichidae is monophyletic or paraphyletic and to test the branching order among three clades in the suborder Entodiniomorphina, Ophryoscolecidae, (Cycloposthium + Troglodytella) and (Triplumaria + Bozasella), the Approximately Unbiased (AU) test (Shimodaira 2002), Kishino-Hasegawa (KH) test (Kishino and Hasegawa 1989), and Shimodaira-Hasegawa
U27814 AF029762 U57770
Reference
Embley et al. (1995) Strüder-Kypke et al. (2006) Wright and Lynn (1997a)
AB794980 AB794979 AB794981 AB794982 AB794983 EF632075 AB794984
Strüder-Kypke et al. (2007)
AF298820
Cameron et al. (2003)
AF298817
Cameron and O’Donoghue (2004)
AF298825 AF298824 AF298821
Cameron et al. (2001) Cameron and O’Donoghue (2004) Cameron et al. (2001)
U57771
Wright and Lynn (1997a)
(SH) test (Shimodaira and Hasegawa 1999) were performed with TREE-PUZZLE v5.2 (Schmidt et al. 2002) and CONSEL v0.1k (Shimodaira and Hasegawa 2001). A Maximum likelihood tree was constructed in MEGA 5 using the dataset consisting of 44 18S rRNA gene sequences, 40 entodiniomorphid species except for the family Buetschliidae, three vestibuliferids species in the family Isotrichidae, and an outgroup. The model used to construct the ML tree was calculated in MEGA 5. Based on the topology of this ML tree, we defined 6 constraint trees. Using the dataset and the trees file, a site-likelihood file was generated in TREE-PUZZLE with the command–line option -wsl. P-values of AU, KH and SH tests were calculated from the site-likelihood file by the commands, makermt, consel and catpv, in CONSEL.
Results A new entodiniomorphid species was found in the fecal sample of an Asian elephant that was kept in a zoo in Japan. The ciliate has morphological characters of the families Cycloposthiidae and Ophryoscolecidae. However, we conclude that this species is a member of the genus Bozasella Buisson, 1923 in the family Cycloposthiidae Poche,
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
141
Figs 13–24. Micrographs of cells fixed in formalin solution and examined in this study. 13. Triplumaria solea. 14. T. sukuna. 15. T. dvoinosi. 16. T. fulgora. 17. T. harpagonis. 18. Gassovskiella galea. 19. Ditoxum funinucleum. 20. Spirodinium equi. 21. Triadinium caudatum. 22. Tetratoxum parvum. 23. T. unifasciculatum. 24. T. excavatum.
142
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
Figs 25–35. Micrographs of cells fixed in formalin solution and examined in this study. 25. Pseudoentodinium elephantis. 26. Ochoterenaia appendiculata. 27. Circodinium minimum. 28. Blepharocorys microcorys. 29. B. angusta. 30. B. jubata. 31. B. uncinata. 32. Buxtonella sulcata. 33. Balantidium coli. 34. Helicozoster indicus. 35. Latteuria polyfaria.
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
143
Figs 36–43. Micrographs of cells fixed in formalin solution and examined in this study. 36. Latteuria media. 37. Paraisotricha minuta. 38. Sulcoarcus pellucidulus. 39. Didesmis ovalis. 40. Alloiozona trizona. 41. Blepharoconus hemiciliatus. 42. Hemiprorodon gymnoprosthium. 43. Prorodonopsis coli.
1913, because it has three caudalia with slit-like openings and no skeletal plate. Bozasella gracilis n. sp.
Description The body is slender and laterally compressed with an operculum at the anterior end and a tail flap at the posterior end (Figs 1, 2, 9). The adoral ciliary zone is retractable deep into the anterior end of the body (Fig 3). There are three caudalia, one each at the dorsal side of the operculum, the dorsal and ventral bases of the tail flap. These caudalia do not protrude from the surface of the body and their openings are slit-like. Ciliary tufts arise from each caudalium. These ciliary tufts are retractable considerably deep into the body and are invisible when completely retracted (Figs 1, 2, 9, 10). There are three longitudinal grooves on the surface of the body. One is the dorsal left groove extending from the anterior dorsal caudalium to the posterior dorsal caudalium (Fig. 1). The other two are the right and left median grooves, each extending
along the midline of the right and left surfaces of the body in a slight curve (Figs 1, 2, 10). The macronucleus lies beneath the dorsal surface of the body (Figs 1, 2). The macronucleus is rod-shaped when the anterior dorsal ciliary tuft is drawn from the caudalium (Figs 4, 6) whereas the macronucleus often bends into a boomerang shape when the anterior dorsal ciliary tuft is deeply retracted into the body between the vestibulum and the macronucleus (Figs 3, 5). The micronucleus is small and ovoid, adhering to the dorsal or the dorsal left side at the middle of the macronucleus (Figs 1, 2). The vestibulum is long, tubular, and curving (Figs 3, 4). The cytoproct is located behind the posterior ventral caudalium. Two contractile vacuoles lie beneath the dorsal surface of the body. The anterior vacuole is located near the anterior end of the macronucleus and the posterior vacuole near the micronucleus (Figs 1, 2).
Dimensions Body length, 72.9 ± 17.6 (48.5–109.7) m. Body width, 39.3 ± 9.1 (25.5–63.8) m.
144
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
Body length/body width, 1.87 ± 0.24 (1.36–2.40). Macronuclear length, 28.8 ± 5.9 (17.9–43.4) m. length/body length, 0.40 ± 0.04 Macronuclear (0.30–0.48). Distance from anterior end of the macronucleus to the micronucleus, 14.8 ± 4.8 (7.7–28.1) m. Distance from anterior end of the macronucleus/macronuclear length, 0.51 ± 0.08 (0.33–0.67).
Habitat, type of host, locality The intestine of the Asian elephant, Elephas maximus maximus, from Tokushima Zoo in Tokushima prefecture, Japan.
Etymology Bozasella gracilis is named according to its slender body.
Type material and GenBank accession number The slide containing the holotype specimen (MPM Coll. No. 20,897) was deposited in Meguro Parasitological Museum, Tokyo, Japan. GenBank accession number of 18S rRNA gene sequence of Bozasella gracilis is AB793744.
Remarks This new species has three caudalia and no skeletal plate, and could, therefore, be assigned to different genera of the family Cycloposthiidae, i.e. either the genus Trifascicularia Strelkow, 1931 or the genus Bozasella Buisson, 1923. Only one species in the genus Trifascicularia, T. cycloposthium, has three cylinder- or button-shaped caudalia, which are similar to those found in many Cycloposthium and Triplumaria species. Such caudalia are quite different from the caudalia with slit-like openings of this new species. Two Bozasella species, B. rhinoceros Buisson, 1923 and B. elephantis Mandal & Choudhury, 1983, have been described. The B. rhinoceros species has no skeletal plate whereas B. elephantis has a relatively large skeletal plate on the left body surface. A new genus may be created to accommodate B. elephantis. Otherwise B. elephantis may be transferred to the genus Triplumaria Strelkow, 1931 or the genus Tricaudalia Buisson, 1923. These two known Bozasella species have caudalia with slit-like openings, not protruding from the surface of the body, which are also found in the new species. Accordingly, the new species belongs to the genus Bozasella.
Infraciliature of Bozasella gracilis Buccal infraciliature Bozasella gracilis has an adoral polybrachykinety (AP), a perivestibular polybrachykinety (PVP), a vestibular
polybrachykinety (VP), and paralabial kineties (PK) in the adoral ciliary zone (Figs 5–8, 11, 12). The AP is wide and C-shaped, extending along the ventral side of the vestibular opening (Figs 5–8). The PVP is slender and C-shaped, extending along the vestibular opening opposite to the AP (Figs 5, 7, 8, 11). The PVP is wide at the right part and gradually narrows toward its left end. The VP is long and spindle-shaped, extending along the left vestibular wall (Figs 5–8). Numerous fibers arise from the VP. These dorsal fibers (DF) and ventral fibers (VF), both numbering between around 35 to 45, extend transversely on the vestibular wall in arches. Fibers in the posterior three fourths of the DF are bifurcated (Figs 7, 8). The PK is composed of more than four short transverse kineties which extend along the ventral right side of the AP. Kinetids in the PK are slightly larger than those in kineties of the polybrachykineties (Figs 7, 8, 12).
Somatic infraciliature Bozasella gracilis has somatic infraciliary bands (the polybrachykinety of the anterior dorsal caudalium (PAD), the polybrachykinety of the posterior dorsal caudalium (PPD), and the polybrachykinety of the posterior ventral caudalium (PPV)) in three caudalia (Figs 5–6, 12). These caudalial polybrachykineties are not kept in a fixed position. When the ciliary tufts are deeply retracted, the PAD is between the macronucleus and the vestibulum at the level of the anterior third of the body, and the PPD and PPV are located at the level of the posterior third of the body (Fig. 5). When the ciliary tufts completely come out of the caudalial opening the PAD is located anteriorly away from the macronucleus, and the PPD and PPV lie just beneath the surface of the basis of the tail flap (Fig. 6).
Phylogenetic analysis 18S rRNA gene sequences of B. gracilis and 31 species examined here were listed in Table 1 with specimens, host animals, and locations. Their GenBank accession numbers were listed in Table 2. Thirty sequences retrieved from the GenBank were also listed in Table 2. Didinium nasutum was selected as outgroup. A total of 1457 unambiguously aligned sites were retained using Clustal W to construct ML tree in MEGA 5, BI tree in MrBayes, and NJ tree in MEGA 5. The ML tree was drawn to scale in Fig. 44 with the bootstrap values at the nodes. The best combination of model and rates for ML tree was the General Time Reversible (GTR) model with gamma distribution and estimate of invariable sites. The posterior probability values at the nodes in the BI tree and the bootstrap values in the NJ tree were shown with the values of the ML tree in Fig. 44. Taxonomy of the orders, the suborders and the families follows Lynn (2008). Bozasella gracilis n. sp. was located in the clade consisting of six Triplumaria species. The pairwise divergences (p-distance) among Bozasella and Triplumaria species were
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
145
Fig. 44. Maximum likelihood phylogenetic tree inferred from 18S rRNA gene sequences by MEGA 5, based on the General Time Reversible (GTR) model with gamma distribution and estimate of invariable sites. The first numbers at the nodes are the bootstrap values (percent out of 1000 replicates) for the maximum likelihood tree and the second and third numbers are the posterior probability values for the Baysian inference tree and the bootstrap values (percent out of 1000 replicates) for the neighbor joining tree. The scale bar represents 2 change per 100 positions. New sequences are shown in boldface. Ar, Suborder, Archistomatina; Bal, Family Balantidiidae; Bl, Suborder Blepharocorythina; Ble, Family Blepharocorythidae; Bue, Family Buetschliidae; Cyc, Family Cycloposthiidae; EN, Order Entodiniomorphida; En, Suborder Entodiniomorphina; Iso, Family Isotrichidae; MA, Order Macropodiniida; Oph, Family Ophryoscolecidae; Par, Family Parentodiniidae; Pis, Family Paraisotrichidae; Pse, Family Pseudoentodiniidae; Pyc, Family Pycnotrichidae; Spi, Family Spirodiniidae; Tro, Family Troglodytellidae; VE, Order Vestibuliferida.
146
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
Table 3. P-distance in the clade of Bozasella gracilis and Triplumaria species. Species
B. gracilis
T. sukuna
T. solea
T. selenica
T. dvoinosi
T. fulgora
Bozasella gracilis Triplumaria sukuna Triplumaria solea Triplumaria selenica Triplumaria dvoinosi Triplumaria fulgora Triplumaria harpagonis
0.005 0.007 0.008 0.007 0.012 0.013
0.004 0.006 0.007 0.010 0.011
0.008 0.009 0.012 0.014
0.006 0.010 0.011
0.006 0.012
0.018
shown in Table 3. The p-distances between Bozasella gracilis and Triplumaria sukuna and between T. sukuna and T. solea were 0.005 and 0.004, respectively. These values were lower than those among other Triplumaria species. In the phylogenetic trees, Australian ciliates in the order Macropodiniida formed a clade, which was clearly monophyletic with very high support values for the node of the cluster (100% ML, 1.00 BI, 100% NJ). The other two orders, Entodiniomorphida and Vestibuliferida, were not monophyletic but formed the (Entodiniomorphina + Blepharocorythina + Isotrichidae) clade with moderate to high support values (86% ML, 1.00 BI, 71% NJ). However, (Vestibuliferida except for Isotrichidae + Archistomatina) was not supported as a clade (29% ML, 0.72 BI, 22% NJ). In the (Entodiniomorphina + Blepharocorythina + Isotrichidae) clade, Isotrichidae was monophyletic in ML and BI trees with low support values (60% ML, 0.65 BI) whereas it was paraphyletic in NJ tree. The sister group of Isotrichidae, the (Entodiniomorphina + Blepharocorythina) clade, was supported as monophyletic with high values (89% ML, 1.00 BI, 85% NJ) and was divided into two clades. These two clades did not correspond to suborders, Entodiniomorphina and Blepharocorythina. One clade supported with very high values (98% ML, 1.00 BI, 96% NJ) was composed of Entodiniomorphina except for the families Parentodiniidae and Pseudoentodiniidae, in addition to Raabena in Blepharocorythina. Another with high values (84% ML, 1.00 BI, 88% NJ) was composed of Blepharocorythina except for Raabena, in addition to the families Parentodiniidae and Pseudoentodiniidae in the Entodiniomorphina. The clade of the suborder Entodiniomorphina except for the families Parentodiniidae and Pseudoentodiniidae was monophyletic with support values (71% ML, 0.95 BI, 62% NJ). In this clade, the families Ophryoscolecidae and Spirodiniidae were monophyletic with support values (the Ophryoscolecidae clade, 95% ML, 1.00 BI, 100% NJ; the Spirodiniidae clade, 85% ML, 1.00 BI, 79% NJ) whereas the family Cycloposthiidae was non-monophyletic and divided into three. Tripalmaria was located in the Spirodiniidae clade. Cycloposthium was a sister group of Troglodytella. Triplumaria species and B. gracilis formed a monophyletic clade with very high support values (96% ML, 1.00 BI, 98% NJ).
In the (Blepharocorythina except for Raabena + Parentodiniidae + Pseudoentodiniidae) clade, Blepharocorythina species except for Raabena formed a monophyletic group with high support values (89% ML, 1.00 BI, 88% NJ) and the (Parentodiniidae + Pseudoentodiniidae) clade was also monophyletic with very high support values (99% ML, 1.00 BI, 100% NJ). The Archistomatina species formed a monophyletic clade with high support values (95% ML, 1.00 BI, 86% NJ). The family Paraisotrichidae was not monophyletic. Helicozoster and Latteuria species in the Paraisotrichidae formed a monophyletic clade with very high support values (99% ML, 1.00 BI, 100% NJ) and two Paraisotricha species also formed a monophyletic clade with very high support values (100% ML, 1.00 BI, 100% NJ). But they were not sister. Six constraint trees were used for the statistical tests (AU, KH, and SH, Fig. 45). Based on the ML tree constructed in MEGA 5 with the GTR model with gamma distribution and estimate of invariable sites, six trees were manually written in MEGA 5 and their branch lengths were calculated in TREE-PUZZLE. The family Isotrichidae, represented by Dasytricha and Isotricha spp., was monophyletic in Trees 1, 3, and 5 but paraphyletic in Trees 2, 4, and 6. In Trees 1 and 2, Ophryoscolecidae and (Cycloposthium + Troglodytella) were sister. In Trees 3 and 4, Ophryoscolecidae and (Triplumaria + Bozasella) were sister. In Trees 5 and 6, (Cycloposthium + Troglodytella) and (Triplumaria + Bozasella) were sister. P-values of AU, KH and SH test of these six trees were shown in Table 4. P-values of the Tree 3 were the highest. P-values of Trees 1, 3 and 5 were higher than those of Trees 2, 4 and 6. P-values of Trees 3 and 4 were higher than those of Trees 1 and 2. P-values of Trees 5 and 6 were lower than those of other trees,
ScienceDirect European Journal of Protistology 50 (2014) 134–152
Bozasella gracilis n. sp. (Ciliophora, Entodiniomorphida) from Asian elephant and phylogenetic analysis of entodiniomorphids and vestibuliferids Akira Itoa,∗ , Miki Ishiharab , Soichi Imaib a b
Ookusa Animal Clinic, Ookusa 503, Matsue, Shimane 690-0032, Japan Department of Parasitology, Nippon Veterinary and Life Science University, Musashino, Tokyo 180-8602, Japan
Received 1 May 2013; received in revised form 17 January 2014; accepted 20 January 2014 Available online 25 January 2014
Abstract Bozasella gracilis n. sp. in the order Entodiniomorphida was found in fecal samples of an Asian elephant kept in a zoo. The ciliate has general and infraciliary similarities to the families Ophryoscolecidae and Cycloposthiidae. Phylogenetic trees were inferred from 18S rRNA gene sequences of B. gracilis, 45 entodiniomorphids, 10 vestibuliferids, 5 macropodiniids, and an outgroup, using maximum likelihood, Bayesian inference, and neighbor joining analyses. Of them, there were 32 new sequences; 26 entodiniomorphid species in the genera, Bozasella, Triplumaria, Gassovskiella, Ditoxum, Spirodinium, Triadinium, Tetratoxum, Pseudoentodinium, Ochoterenaia, Circodinium, Blepharocorys, Sulcoarcus, Didesmis, Alloiozona, Blepharoconus, Hemiprorodon, and Prorodonopsis, and 6 vestibuliferid species in the genera, Buxtonella, Balantidium, Helicozoster, Latteuria, and Paraisotricha. Thirty additional sequences were retrieved from the GenBank database. Phylogenetic trees revealed non-monophylies of the orders Entodiniomorphida and Vestibuliferida, the suborders Entodiniomorphina and Blepharocorythina, and the families Cycloposthiidae and Paraisotrichidae. Bozasella gracilis was sister to Triplumaria. In addition, to avoid homonymy, we propose Gilchristinidae nom. nov., Gilchristina nom. nov. and Gilchristina artemis (Ito, Van Hoven, Miyazaki & Imai, 2006) comb. nov. © 2014 Elsevier GmbH. All rights reserved. Keywords: Bozasella gracilis n. sp.; 18S rRNA gene sequence; Elephant; Entodiniomorphida; Gilchristina artemis (Ito, Van Hoven, Miyazaki & Imai, 2006) comb. nov.; Vestibuliferida
Introduction Intestinal ciliates in the order Entodiniomorphida and the order Vestibuliferida have been described from African and Asian elephants (Buisson 1923; Eloff and Van Hoven 1980; Kofoid 1935; Ito et al. 2010, 2011; Latteur 1958, 1966, 1967;
∗ Corresponding
author. Tel.: +81 852 237780. E-mail address: [email protected] (A. Ito).
0932-4739/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ejop.2014.01.003
Latteur and Bousez 1970; Latteur and Dartevelle 1971; Latteur et al. 1970; Mandal and Choudhury 1983a,b, 1984; Timoshenko and Imai 1995, 1997; Wolska 1967, 1968, 1970, 1986). In our study on the phylogeny of entodiniomorphids and vestibuliferids, we found a unique new entodiniomorphid species from Asian elephants, which has morphological characters of both the family Ophryoscolecidae and the family Cycloposthiidae in its general appearance and infraciliature. In the following, we describe this new species, Bozasella gracilis n. sp. and its infraciliary bands. Further, we sequenced
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
the 18S rRNA gene of B. gracilis, 25 entodiniomorphids, and six vestibuliferids and discuss the phylogenies of the orders Entodiniomorphida and Vestibuliferida.
Material and Methods Sampling Fecal samples containing Bozasella gracilis n. sp. for morphological study were collected from a female Asian elephant (Elephas maximus maximus) which was born in Sri Lanka and was kept in Tokushima zoo, Tokushima prefecture, Japan. The samples were immediately fixed in five times the volume of 10% formalin solution within five minutes after defecation and stored in a dark place after they were filtered through two layers of gauze into a bottle in order to remove plant and feed material. We collected 12 samples for the 18S rRNA gene sequences and isolated 1–7 species from each sample. Cells of B.
135
gracilis, 25 entodiniomorphid ciliates and six vestibuliferid ciliates were obtained from these samples of five elephants, four horses, and a cattle listed in Table 1. Fecal samples were fixed within five minutes after defecation. Intestinal contents of a horse were fixed immediately after euthanasia due to limb disorder. A rectal content of a cattle was fixed just after rectal examination. All samples were fixed in five times the volume of 80% ethanol and preserved in the refrigerator (4 ◦ C) after they were filtered through two layers of gauzes; the supernatant was replaced with 100% ethanol. All samples were also fixed in five times the volume of 10% formalin solution to determine their species composition. Micrographs of 25 entodiniomorphid ciliates and six vestibuliferid ciliates fixed in formalin solution were shown in Figs 13–43.
Morphology and silver impregnation The infraciliary bands of the new species were stained using the pyridinated silver carbonate impregnation method,
Table 1. List of entodiniomorphid and vestibuliferid ciliates analyzed in this study, including specimen, host animal, and location. Species
Specimen
Host animal (birth place)
Location
Order Entodiniomorphida Bozasella gracilis n. sp. Triplumaria solea Triplumaria sukuna Triplumaria dvoinosi Triplumaria fulgora Triplumaria harpagonis Gassovskiella galea Ditoxum funinucleum Spirodinium equi Triadinium caudatum Tetratoxum parvum Tetratoxum unifasciculatum Tetratoxum excavatum Pseudoentodinium elephantis Ochoterenaia appendiculata Circodinium minimum Blepharocorys microcorys Blepharocorys angusta Blepharocorys jubata Blepharocorys uncinata Sulcoarcus pellucidulus Didesmis ovalis Alloiozona trizona Blepharoconus hemiciliatus Hemiprorodon gymnoprosthium Prorodonopsis coli
Feces Feces Feces Feces Feces Feces Feces Feces Feces Dorsal colon Feces Feces Dorsal colon Feces Feces Feces Feces Feces Feces Cecum Feces Ventral colon Cecum Feces Ventral colon Dorsal colon
Asian elephant (Sri Lanka) Asian elephant (Sri Lanka) Asian elephant (India) Asian elephant (Sri Lanka) Asian elephant (India) Asian elephant (India) Yonaguni horse Yonaguni horse Yonaguni horse Pony Yonaguni horse Riding horse Pony Asian elephant (India) Riding horse Yonaguni horse Riding horse Yonaguni horse Kiso horse Pony Yonaguni horse Pony Pony Riding horse Pony Pony
Tokushima Zoo Tokushima Zoo Kobe Oji Zoo Tokushima Zoo Kobe Oji Zoo Kobe Oji Zoo Yonaguni island Yonaguni island Yonaguni island Matsue Yonaguni island Yasugi Matsue Kobe Oji Zoo Yasugi Yonaguni island Yasugi Yonaguni island Matsue Matsue Yonaguni island Matsue Matsue Yasugi Matsue Matsue
Order Vestibuliferida Buxtonella sulcata Balantidium coli Helicozoster indicus Latteuria polyfaria Latteuria media Paraisotricha minuta
Rectum Feces Feces Feces Feces Ventral colon
Japanese black beef cattle Asian elephant (India) Asian elephant (Thailand) African bush elephant (South Africa) Asian elephant (India) Pony
Matsue Japan Miyazaki Phoenix Zoo Himeji Central Park Kobe Oji Zoo Matsue
136
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
Figs 1–6. Schematic figures of Bozasella gracilis n. sp. 1, 3–6. Cells from left side. 2. Cell from right side. 5, 6. Cells after silver impregnation. ACZ, adoral ciliary zone; ADC, anterior dorsal caudalium; AP, adoral polybrachykinety; CP, cytoproct; CV, contractile vacuole; DLG, dorsal left groove; LG, left median groove; MA, macronucleus; MI, micronucleus; OP, operculum; PAD, polybrachykinety of anterior dorsal caudalium; PDC, posterior dorsal caudalium; PK, paralabial kineties; PPD, polybrachykinety of posterior dorsal caudalium; PPV, polybrachykinety of posterior ventral caudalium; PVC, posterior ventral caudalium; PVP, perivestibular polybrachykinety; RG, right median groove; TF, tail flap; VP vestibular polybrachykinety; VS, vestibulum. Bar = 10 m.
following Ito and Imai (1998). The orientation of ciliates used by Dogiel (1927) was adopted; the side beneath which the macronucleus lies was termed the dorsal side, the opposite one the ventral side, thus defining the right and left sides. Cell measurements and morphometric ratios were determined by measuring a sample of 20 fixed cells using a calibrated micrometer; measurements are reported as mean ± S. D. (minimum–maximum). Body length was determined as the distance between the anterior and posterior ends of the body.
The term polybrachykinety refers to infraciliary bands composed of numerous, short, parallel kineties (Ito and Imai 1998; Fernández-Galiano et al. 1985). Somatic ciliary zones were termed the caudalium and caudalia. Taxonomy of the orders, suborders and families follows Lynn (2008). Tetratoxum parvum has three morphotypes based on the longitudinal grooves on the surface of the body (Strelkow 1939). The cell of T. parvum examined was the type without grooves. Prorodonopsis coli and Hemiprorodon gymnoprosthium closely
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
137
Figs 7, 8. Infraciliary bands of the adoral ciliary zone in Bozasella gracilis. 7. From left side. 8. From right side. AP, adoral polybrachykinety; DF, dorsal vestibular fibers; PK, paralabial kineties; PVP, perivestibular polybrachykinety; VF, ventral vestibular fibers; VP, vestibular polybrachykinety. Bar = 10 m.
resemble each other (Strelkow 1939). P. coli is holotrichous whereas H. gymnoprosthium has a small naked area on the body surface around the cytoproct. Cells with cilia flowing posteriorly could not be identified. Only the cells of these species with cilia flowing anteriorly was examined. Both the samples for Balantidium coli and Buxtonella sulcata were determined to be composed of single species by studying their parallel samples which were fixed in formalin solutions. Latteuria polyfaria and L. media closely resemble each other. Each sample for these two Latteuria species contained either of them.
DNA extraction and amplification Twelve parallel samples fixed in formalin solution were stained using the pyridinated silver carbonate impregnation method (Ito and Imai 1998) to correctly determine their species composition in detail. Samples fixed in ethanol were diluted with water in a dish; 10–100 l of ethanol sample was pipetted into 400 l of distilled water in the dish. The dilution rate depended on the densities of ciliates and fecal or intestinal particles. Center-well organ culture dishes (Falcon 353037) were used to easily pick and wash individual cells. A single cell was picked from a Falcon dish in 1 l diluted material using a micropipette under an inverted microscope and put into 400 l distilled water in another Falcon dish.
This process was repeated more than five times to isolate and wash the single cell. Single cells after washing were drawn up in a volume of 1 l distilled water and transferred to a PCR tube. The single cells of each entodiniomorphid species had typical morphological features in their body, nuclei, and ciliary distribution. They were not filled up with food particles in the endoplasm and were without fecal or intestinal particles attached to the buccal and/or somatic ciliary zones. DNA extraction of Bozasella gracilis, 25 entodiniomorphids, and six vestibuliferids was performed using the freeze-thawing single-cell PCR method of Honma et al. (2007). The PCR tube was frozen at −80 ◦ C for 3 min and thawed at 60 ◦ C for 30 s to facilitate cell breakage. After five rounds of this freeze-thawing step, 7 l of reaction buffer (10 mM Tris-HCl, 1.5 mM Mg Cl2 , 50 mM KCl, 0.01% Proteinase-K, 0.01% sodium dodecyl sulfate) was added to the PCR tube. The PCR tube was then incubated at 37 ◦ C for 60 min and heated at 95 ◦ C for 10 min. PCR amplifications were performed in 20 l vol., containing template DNA in 1 l of reaction mixture, 0.25 M of both the forward primer 82F (5 -GAA ACT GCG AAT GGC TC-3 ; Elwood et al. 1985) and the reverse primer EkyB (5 TGA TCC TTC TGC AGG TTC ACC TAC-3 ; Medlin et al. 1988), 0.2 mM each dNTP Mixtures, 2 mM MgCl2 , and 0.5 U of Takara Ex Taq DNA polymerase (Takara Bio) with the following PCR program: 1 cycle at 95 ◦ C for 5 min; 35 cycles at 98 ◦ C for 5 s, 50 ◦ C for 30 s, 72 ◦ C for 2 min; 1 cycle
138
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
Figs 9–12. Micrographs of B. gracilis. After silver impregnation. 9–11. Cells from left side. 12. Cell from right side. ACZ, adoral ciliary zone; ADC, anterior dorsal caudalium; AP, adoral polybrachykinety; DF, dorsal vestibular fibers; LG, left median groove; MA, macronucleus; OP, operculum; PAD, polybrachykinety of anterior dorsal caudalium; PDC, posterior dorsal caudalium; PK, paralabial kineties; PVC, posterior ventral caudalium; PVP, perivestibular polybrachykinety; TF, tail flap; VF, ventral vestibular fibers; VP vestibular polybrachykinety. Bar = 10 m.
at 72 ◦ C for 10 min. The PCR products were evaluated by electrophoresis in a 1.0% agarose gel followed by staining with ethidium bromide solution and by visualization on an ultraviolet transilluminator. The PCR products were purified using ExoSAP-IT (USB). The purified PCR products were sequenced in both directions using the BigDye Terminator Cycle Sequencing Kit on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems) according to the manufacture’s instructions with four primers; two forward primers (82F and either primer (5 -CCA TTT CAG TAC CTT ATG AG-3 ) or (5 -TTT GCC AAG GAT GTT TTC-3 )) and two reverse primers (EkyB and (5 -CTT GGC AAA TGC TTT CGC-3 )).
Sequence availability and phylogenetic analysis For the phylogenetic analysis of 18S rRNA gene sequences from 46 entodiniomorphids, 10 vestibuliferids, five macropodiniids and an outgroup, 30 gene sequences were retrieved from the GenBank database and were listed in Table 2 along with the 32 sequences examined in this study. Multiple sequence alignment was performed using Clustal W 1.83 in the software package MEGA 5 (Tamura et al. 2011). Phylogenetic trees were constructed using three different analyses, maximum likelihood (ML) (Felsenstein 1981), Bayesian inference (BI), and neighbor joining (NJ) (Saitou
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
139
Table 2. List of entodiniomorphid, vestibuliferid and macropodiniid ciliates and outgroup analyzed in this study, 32 new sequences in boldface and 30 sequences retrieved from GenBank database, including their taxonomic classification, GenBank accession number of 18S rRNA gene sequence, and reference. Order, Family, Species Order Entodiniomorphida Reichenow in Doflein & Reichenow, 1929 Suborder Entodiniomorphina Reichenow in Doflein & Reichenow, 1929 Family Ophryoscolecidae Stein, 1859 Entodinium longinucleatum Dogiel, 1925 Entodinium caudatum Stein, 1858 Epidinium caudatum (Fiorentini, 1889) Ophryoscolex purkynjei Stein, 1858 Eudiplodinium rostratum (Fiorentini, 1889) Diplodinium dentatum (Stein, 1858) Eudiplodinium maggii (Fiorentini, 1889) Metadinium medium Awerinzew & Mutafowa, 1914 Polyplastron multivesiculatum (Dogiel & Fedorowa, 1925) Ostracodinium gracile (Dogiel, 1925) Family Troglodytellidae Corliss, 1979 Troglodytella abrassarti Brumpt & Joyeux, 1912 Family Cycloposthiidae, Poche, 1913 Cycloposthium ishikawai Gassovsky, 1919 Cycloposthium edentatum Strelkow, 1929 Cycloposthium bipalmatum (Fiorentini, 1890) Bozasella gracilis n. sp. Triplumaria solea Ito, Mishima, Nataami, Ike & Imai, 2011 Triplumaria sukuna Ito, Mishima, Nataami, Ike & Imai, 2011 Triplumaria selenica Latteur, Tuffrau & Wespes, 1970 Triplumaria dvoinosi Timoshenko & Imai, 1995 Triplumaria fulgora Ito, Mishima, Nataami, Ike & Imai, 2011 Triplumaria harpagonis Ito, Mishima, Nataami, Ike & Imai, 2011 Tripalmaria dogieli Gassovsky, 1919 Family Spirodiniidae Strelkow, 1939 Gassovskiella galea (Gassovsky, 1919) Ditoxum funinucleum Gassovsky, 1919 Spirodinium equi Fiorentini, 1890 Triadinium caudatum Fiorentini, 1890 Cochliatoxum periachtum Gassovsky, 1919 Tetratoxum parvum Hsiung, 1930 Tetratoxum unifasciculatum (Fiorentini, 1890) Tetratoxum excavatum Hsiung, 1930 Family Parentodiniidae Ito, Miyazaki & Imai, 2002 Parentodinium sp Family Pseudoentodiniidae Wolska, 1986 Pseudoentodinium elephantis Wolska, 1986 Suborder Blepharocorythina Wolska, 1971 Family Blepharocorythidae Hsiung, 1929 Raabena bella Wolska, 1967 Ochoterenaia appendiculata Chavarria, 1933 Circodinium minimum Wolska, 1967 Blepharocorys microcorys Gassovsky, 1919 Blepharocorys angusta Gassovsky, 1919 Blepharocorys curvigula Gassovsky, 1919 Blepharocorys jubata Bundle, 1895 Blepharocorys uncinata (Fiorentini, 1890) Suborder Archistomatina de Puytorac et al., 1974 Family Buetschliidae Poche, 1913 Sulcoarcus pellucidulus Hsiung, 1935 Didesmis ovalis Fiorentini, 1890 Alloiozona trizona Hsiung, 1930
GenBank acc. no.
Reference
AB481099 U57765 U57763 U57768 AB536716 U57764 U57766 AB535215 U57767 AB535662
Ito et al. (2010) Wright et al. (1997) Wright et al. (1997) Wright and Lynn (1997b) Ito et al. (2010) Wright and Lynn (1997b) Wright and Lynn (1997b) Ito et al. (2010) Wright et al. (1997) Ito et al. (2010)
AB437346
Irbis et al. (2008)
EF632076 EF632077 AB530165 AB793744 AB793745 AB793777 AB533538 AB793778 AB793781 AB793782 EF632074
Strüder-Kypke et al. (2007) Strüder-Kypke et al. (2007) Miyazaki (2005)
AB793783 AB794091 AB794092 AB794968 EF632078 AB794969 AB794970 AB794971 AB530164
Ito et al. (2010)
Strüder-Kypke et al. (2007)
Strüder-Kypke et al. (2007)
Miyazaki (2005)
AB794972
AB534183 AB794973 AB794974 AB794975 AB794976 AB534184 AB794977 AB794978
AB795024 AB795025 AB795026
Ito et al. (2010)
Ito et al. (2010)
140
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
Table 2 (Continued) Order, Family, Species
GenBank acc. no.
Blepharoconus hemiciliatus Gassovsky, 1919 Hemiprorodon gymnoprosthium Strelkow, 1939 Prorodonopsis coli Gassovsky, 1919 Order Vestibuliferida de Puytorac et al., 1974 Family Isotrichidae Bütschli, 1889 Dasytricha ruminantium Schuberg, 1888 Isotricha prostoma Stein, 1859 Isotricha intestinalis Stein, 1859 Family Balantidiidae Reichenow, 1929 Balantidium coli (Malmsten, 1857) Family Pycnotrichidae Poche, 1913 Buxtonella sulcata Jameson, 1926 Family Paraisotrichidae Cunha, 1917 Helicozoster indicus Latteur, 1967 Latteuria polyfaria Timoshenko & Imai, 1997 Latteuria media Timoshenko & Imai, 1997 Paraisotricha colpoidea Fiorentini, 1890 Paraisotricha minuta Hsiung, 1930 Order Macropodiniidae Lynn, 2008 Family Macropodiniidae Dehority, 1996 Macropodinium ennuensis Dehority, 1996 Family Polycostidae Cameron & O’Donoghue, 2003 Polycosta turniae Cameron & O’Donoghue, 2003 Family Amylovoracidae Cameron & O’Donoghue, 2002 Amylovorax dogieli Cameron, O’Donoghue & Adlard, 2000 Bandia cribbi Cameron & O’Donoghue, 2002 Bitricha tasmaniensis Cameron, O’Donoghue & Adlard, 2000 Outgroup Didinium nasutum (Muller, 1786)
AB795027 AB795028 AB795029
and Nei 1987); ML analysis was performed with MEGA 5 by bootstrapping 1000 replicates. The best combination of model and rates for the ML tree was calculated in MEGA 5. Bayesian inference analysis was performed with MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003). The model used to construct the BI tree was the same model as calculated in MEGA 5 for the ML tree. Marcov Chain Monte Carlo (MCMC) were run with two sets of four independent chains (three heated chains and a cold chain). The temperature for heating the chains was 0.2. A total of 5,000,000 generations was calculated with trees sampled every 100 generations. An average standard deviation of the split frequencies below 0.05 was used to determine convergence. Neighbor joining analysis was performed with MEGA 5 by bootstrapping 1000 replicates. The evolutionary distances among Bozasella and Triplumaria species were calculated using the maximum composite likelihood method (Tamura et al. 2004). To test whether the family Isotrichidae is monophyletic or paraphyletic and to test the branching order among three clades in the suborder Entodiniomorphina, Ophryoscolecidae, (Cycloposthium + Troglodytella) and (Triplumaria + Bozasella), the Approximately Unbiased (AU) test (Shimodaira 2002), Kishino-Hasegawa (KH) test (Kishino and Hasegawa 1989), and Shimodaira-Hasegawa
U27814 AF029762 U57770
Reference
Embley et al. (1995) Strüder-Kypke et al. (2006) Wright and Lynn (1997a)
AB794980 AB794979 AB794981 AB794982 AB794983 EF632075 AB794984
Strüder-Kypke et al. (2007)
AF298820
Cameron et al. (2003)
AF298817
Cameron and O’Donoghue (2004)
AF298825 AF298824 AF298821
Cameron et al. (2001) Cameron and O’Donoghue (2004) Cameron et al. (2001)
U57771
Wright and Lynn (1997a)
(SH) test (Shimodaira and Hasegawa 1999) were performed with TREE-PUZZLE v5.2 (Schmidt et al. 2002) and CONSEL v0.1k (Shimodaira and Hasegawa 2001). A Maximum likelihood tree was constructed in MEGA 5 using the dataset consisting of 44 18S rRNA gene sequences, 40 entodiniomorphid species except for the family Buetschliidae, three vestibuliferids species in the family Isotrichidae, and an outgroup. The model used to construct the ML tree was calculated in MEGA 5. Based on the topology of this ML tree, we defined 6 constraint trees. Using the dataset and the trees file, a site-likelihood file was generated in TREE-PUZZLE with the command–line option -wsl. P-values of AU, KH and SH tests were calculated from the site-likelihood file by the commands, makermt, consel and catpv, in CONSEL.
Results A new entodiniomorphid species was found in the fecal sample of an Asian elephant that was kept in a zoo in Japan. The ciliate has morphological characters of the families Cycloposthiidae and Ophryoscolecidae. However, we conclude that this species is a member of the genus Bozasella Buisson, 1923 in the family Cycloposthiidae Poche,
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
141
Figs 13–24. Micrographs of cells fixed in formalin solution and examined in this study. 13. Triplumaria solea. 14. T. sukuna. 15. T. dvoinosi. 16. T. fulgora. 17. T. harpagonis. 18. Gassovskiella galea. 19. Ditoxum funinucleum. 20. Spirodinium equi. 21. Triadinium caudatum. 22. Tetratoxum parvum. 23. T. unifasciculatum. 24. T. excavatum.
142
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
Figs 25–35. Micrographs of cells fixed in formalin solution and examined in this study. 25. Pseudoentodinium elephantis. 26. Ochoterenaia appendiculata. 27. Circodinium minimum. 28. Blepharocorys microcorys. 29. B. angusta. 30. B. jubata. 31. B. uncinata. 32. Buxtonella sulcata. 33. Balantidium coli. 34. Helicozoster indicus. 35. Latteuria polyfaria.
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
143
Figs 36–43. Micrographs of cells fixed in formalin solution and examined in this study. 36. Latteuria media. 37. Paraisotricha minuta. 38. Sulcoarcus pellucidulus. 39. Didesmis ovalis. 40. Alloiozona trizona. 41. Blepharoconus hemiciliatus. 42. Hemiprorodon gymnoprosthium. 43. Prorodonopsis coli.
1913, because it has three caudalia with slit-like openings and no skeletal plate. Bozasella gracilis n. sp.
Description The body is slender and laterally compressed with an operculum at the anterior end and a tail flap at the posterior end (Figs 1, 2, 9). The adoral ciliary zone is retractable deep into the anterior end of the body (Fig 3). There are three caudalia, one each at the dorsal side of the operculum, the dorsal and ventral bases of the tail flap. These caudalia do not protrude from the surface of the body and their openings are slit-like. Ciliary tufts arise from each caudalium. These ciliary tufts are retractable considerably deep into the body and are invisible when completely retracted (Figs 1, 2, 9, 10). There are three longitudinal grooves on the surface of the body. One is the dorsal left groove extending from the anterior dorsal caudalium to the posterior dorsal caudalium (Fig. 1). The other two are the right and left median grooves, each extending
along the midline of the right and left surfaces of the body in a slight curve (Figs 1, 2, 10). The macronucleus lies beneath the dorsal surface of the body (Figs 1, 2). The macronucleus is rod-shaped when the anterior dorsal ciliary tuft is drawn from the caudalium (Figs 4, 6) whereas the macronucleus often bends into a boomerang shape when the anterior dorsal ciliary tuft is deeply retracted into the body between the vestibulum and the macronucleus (Figs 3, 5). The micronucleus is small and ovoid, adhering to the dorsal or the dorsal left side at the middle of the macronucleus (Figs 1, 2). The vestibulum is long, tubular, and curving (Figs 3, 4). The cytoproct is located behind the posterior ventral caudalium. Two contractile vacuoles lie beneath the dorsal surface of the body. The anterior vacuole is located near the anterior end of the macronucleus and the posterior vacuole near the micronucleus (Figs 1, 2).
Dimensions Body length, 72.9 ± 17.6 (48.5–109.7) m. Body width, 39.3 ± 9.1 (25.5–63.8) m.
144
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
Body length/body width, 1.87 ± 0.24 (1.36–2.40). Macronuclear length, 28.8 ± 5.9 (17.9–43.4) m. length/body length, 0.40 ± 0.04 Macronuclear (0.30–0.48). Distance from anterior end of the macronucleus to the micronucleus, 14.8 ± 4.8 (7.7–28.1) m. Distance from anterior end of the macronucleus/macronuclear length, 0.51 ± 0.08 (0.33–0.67).
Habitat, type of host, locality The intestine of the Asian elephant, Elephas maximus maximus, from Tokushima Zoo in Tokushima prefecture, Japan.
Etymology Bozasella gracilis is named according to its slender body.
Type material and GenBank accession number The slide containing the holotype specimen (MPM Coll. No. 20,897) was deposited in Meguro Parasitological Museum, Tokyo, Japan. GenBank accession number of 18S rRNA gene sequence of Bozasella gracilis is AB793744.
Remarks This new species has three caudalia and no skeletal plate, and could, therefore, be assigned to different genera of the family Cycloposthiidae, i.e. either the genus Trifascicularia Strelkow, 1931 or the genus Bozasella Buisson, 1923. Only one species in the genus Trifascicularia, T. cycloposthium, has three cylinder- or button-shaped caudalia, which are similar to those found in many Cycloposthium and Triplumaria species. Such caudalia are quite different from the caudalia with slit-like openings of this new species. Two Bozasella species, B. rhinoceros Buisson, 1923 and B. elephantis Mandal & Choudhury, 1983, have been described. The B. rhinoceros species has no skeletal plate whereas B. elephantis has a relatively large skeletal plate on the left body surface. A new genus may be created to accommodate B. elephantis. Otherwise B. elephantis may be transferred to the genus Triplumaria Strelkow, 1931 or the genus Tricaudalia Buisson, 1923. These two known Bozasella species have caudalia with slit-like openings, not protruding from the surface of the body, which are also found in the new species. Accordingly, the new species belongs to the genus Bozasella.
Infraciliature of Bozasella gracilis Buccal infraciliature Bozasella gracilis has an adoral polybrachykinety (AP), a perivestibular polybrachykinety (PVP), a vestibular
polybrachykinety (VP), and paralabial kineties (PK) in the adoral ciliary zone (Figs 5–8, 11, 12). The AP is wide and C-shaped, extending along the ventral side of the vestibular opening (Figs 5–8). The PVP is slender and C-shaped, extending along the vestibular opening opposite to the AP (Figs 5, 7, 8, 11). The PVP is wide at the right part and gradually narrows toward its left end. The VP is long and spindle-shaped, extending along the left vestibular wall (Figs 5–8). Numerous fibers arise from the VP. These dorsal fibers (DF) and ventral fibers (VF), both numbering between around 35 to 45, extend transversely on the vestibular wall in arches. Fibers in the posterior three fourths of the DF are bifurcated (Figs 7, 8). The PK is composed of more than four short transverse kineties which extend along the ventral right side of the AP. Kinetids in the PK are slightly larger than those in kineties of the polybrachykineties (Figs 7, 8, 12).
Somatic infraciliature Bozasella gracilis has somatic infraciliary bands (the polybrachykinety of the anterior dorsal caudalium (PAD), the polybrachykinety of the posterior dorsal caudalium (PPD), and the polybrachykinety of the posterior ventral caudalium (PPV)) in three caudalia (Figs 5–6, 12). These caudalial polybrachykineties are not kept in a fixed position. When the ciliary tufts are deeply retracted, the PAD is between the macronucleus and the vestibulum at the level of the anterior third of the body, and the PPD and PPV are located at the level of the posterior third of the body (Fig. 5). When the ciliary tufts completely come out of the caudalial opening the PAD is located anteriorly away from the macronucleus, and the PPD and PPV lie just beneath the surface of the basis of the tail flap (Fig. 6).
Phylogenetic analysis 18S rRNA gene sequences of B. gracilis and 31 species examined here were listed in Table 1 with specimens, host animals, and locations. Their GenBank accession numbers were listed in Table 2. Thirty sequences retrieved from the GenBank were also listed in Table 2. Didinium nasutum was selected as outgroup. A total of 1457 unambiguously aligned sites were retained using Clustal W to construct ML tree in MEGA 5, BI tree in MrBayes, and NJ tree in MEGA 5. The ML tree was drawn to scale in Fig. 44 with the bootstrap values at the nodes. The best combination of model and rates for ML tree was the General Time Reversible (GTR) model with gamma distribution and estimate of invariable sites. The posterior probability values at the nodes in the BI tree and the bootstrap values in the NJ tree were shown with the values of the ML tree in Fig. 44. Taxonomy of the orders, the suborders and the families follows Lynn (2008). Bozasella gracilis n. sp. was located in the clade consisting of six Triplumaria species. The pairwise divergences (p-distance) among Bozasella and Triplumaria species were
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
145
Fig. 44. Maximum likelihood phylogenetic tree inferred from 18S rRNA gene sequences by MEGA 5, based on the General Time Reversible (GTR) model with gamma distribution and estimate of invariable sites. The first numbers at the nodes are the bootstrap values (percent out of 1000 replicates) for the maximum likelihood tree and the second and third numbers are the posterior probability values for the Baysian inference tree and the bootstrap values (percent out of 1000 replicates) for the neighbor joining tree. The scale bar represents 2 change per 100 positions. New sequences are shown in boldface. Ar, Suborder, Archistomatina; Bal, Family Balantidiidae; Bl, Suborder Blepharocorythina; Ble, Family Blepharocorythidae; Bue, Family Buetschliidae; Cyc, Family Cycloposthiidae; EN, Order Entodiniomorphida; En, Suborder Entodiniomorphina; Iso, Family Isotrichidae; MA, Order Macropodiniida; Oph, Family Ophryoscolecidae; Par, Family Parentodiniidae; Pis, Family Paraisotrichidae; Pse, Family Pseudoentodiniidae; Pyc, Family Pycnotrichidae; Spi, Family Spirodiniidae; Tro, Family Troglodytellidae; VE, Order Vestibuliferida.
146
A. Ito et al. / European Journal of Protistology 50 (2014) 134–152
Table 3. P-distance in the clade of Bozasella gracilis and Triplumaria species. Species
B. gracilis
T. sukuna
T. solea
T. selenica
T. dvoinosi
T. fulgora
Bozasella gracilis Triplumaria sukuna Triplumaria solea Triplumaria selenica Triplumaria dvoinosi Triplumaria fulgora Triplumaria harpagonis
0.005 0.007 0.008 0.007 0.012 0.013
0.004 0.006 0.007 0.010 0.011
0.008 0.009 0.012 0.014
0.006 0.010 0.011
0.006 0.012
0.018
shown in Table 3. The p-distances between Bozasella gracilis and Triplumaria sukuna and between T. sukuna and T. solea were 0.005 and 0.004, respectively. These values were lower than those among other Triplumaria species. In the phylogenetic trees, Australian ciliates in the order Macropodiniida formed a clade, which was clearly monophyletic with very high support values for the node of the cluster (100% ML, 1.00 BI, 100% NJ). The other two orders, Entodiniomorphida and Vestibuliferida, were not monophyletic but formed the (Entodiniomorphina + Blepharocorythina + Isotrichidae) clade with moderate to high support values (86% ML, 1.00 BI, 71% NJ). However, (Vestibuliferida except for Isotrichidae + Archistomatina) was not supported as a clade (29% ML, 0.72 BI, 22% NJ). In the (Entodiniomorphina + Blepharocorythina + Isotrichidae) clade, Isotrichidae was monophyletic in ML and BI trees with low support values (60% ML, 0.65 BI) whereas it was paraphyletic in NJ tree. The sister group of Isotrichidae, the (Entodiniomorphina + Blepharocorythina) clade, was supported as monophyletic with high values (89% ML, 1.00 BI, 85% NJ) and was divided into two clades. These two clades did not correspond to suborders, Entodiniomorphina and Blepharocorythina. One clade supported with very high values (98% ML, 1.00 BI, 96% NJ) was composed of Entodiniomorphina except for the families Parentodiniidae and Pseudoentodiniidae, in addition to Raabena in Blepharocorythina. Another with high values (84% ML, 1.00 BI, 88% NJ) was composed of Blepharocorythina except for Raabena, in addition to the families Parentodiniidae and Pseudoentodiniidae in the Entodiniomorphina. The clade of the suborder Entodiniomorphina except for the families Parentodiniidae and Pseudoentodiniidae was monophyletic with support values (71% ML, 0.95 BI, 62% NJ). In this clade, the families Ophryoscolecidae and Spirodiniidae were monophyletic with support values (the Ophryoscolecidae clade, 95% ML, 1.00 BI, 100% NJ; the Spirodiniidae clade, 85% ML, 1.00 BI, 79% NJ) whereas the family Cycloposthiidae was non-monophyletic and divided into three. Tripalmaria was located in the Spirodiniidae clade. Cycloposthium was a sister group of Troglodytella. Triplumaria species and B. gracilis formed a monophyletic clade with very high support values (96% ML, 1.00 BI, 98% NJ).
In the (Blepharocorythina except for Raabena + Parentodiniidae + Pseudoentodiniidae) clade, Blepharocorythina species except for Raabena formed a monophyletic group with high support values (89% ML, 1.00 BI, 88% NJ) and the (Parentodiniidae + Pseudoentodiniidae) clade was also monophyletic with very high support values (99% ML, 1.00 BI, 100% NJ). The Archistomatina species formed a monophyletic clade with high support values (95% ML, 1.00 BI, 86% NJ). The family Paraisotrichidae was not monophyletic. Helicozoster and Latteuria species in the Paraisotrichidae formed a monophyletic clade with very high support values (99% ML, 1.00 BI, 100% NJ) and two Paraisotricha species also formed a monophyletic clade with very high support values (100% ML, 1.00 BI, 100% NJ). But they were not sister. Six constraint trees were used for the statistical tests (AU, KH, and SH, Fig. 45). Based on the ML tree constructed in MEGA 5 with the GTR model with gamma distribution and estimate of invariable sites, six trees were manually written in MEGA 5 and their branch lengths were calculated in TREE-PUZZLE. The family Isotrichidae, represented by Dasytricha and Isotricha spp., was monophyletic in Trees 1, 3, and 5 but paraphyletic in Trees 2, 4, and 6. In Trees 1 and 2, Ophryoscolecidae and (Cycloposthium + Troglodytella) were sister. In Trees 3 and 4, Ophryoscolecidae and (Triplumaria + Bozasella) were sister. In Trees 5 and 6, (Cycloposthium + Troglodytella) and (Triplumaria + Bozasella) were sister. P-values of AU, KH and SH test of these six trees were shown in Table 4. P-values of the Tree 3 were the highest. P-values of Trees 1, 3 and 5 were higher than those of Trees 2, 4 and 6. P-values of Trees 3 and 4 were higher than those of Trees 1 and 2. P-values of Trees 5 and 6 were lower than those of other trees,
