CC BY
4
Protistology 17(3): 149-159 (2023) | doi:10.21685/1680-0826-2023-17-3-3 Pl'OtiStOlO&y
Original article
Ciliates from faeces of kiang Equus kiang kept in a zoo, with analysis of interspecific differences of 18S sequences among trichostomatids (Litostomatea)
Olga Kornilova1, Maria Skazina2, Maria Belokon2, Natalia Egorova3, and Ludmila Chistyakova4*
1 Herzen State Pedagogical University of Russia, 191186 St. Petersburg, Russia
2 Saint-Petersburg University, 199034 St. Petersburg, Russia
3 Separate Structural Subdivision of the SAI "Moscow Zoo" - "Center for the Reproduction of Rare Animal Species", 123242 Moscow, Russia
4 Zoological Institute RAS, 199034 St. Petersburg, Russia
| Submitted August 20, 2023 | Accepted September 24, 2023 |
Summary
The species composition of ciliates - endobionts of the digestive tract of kiang Equus kiang was studied for the first time. In total, 21 ciliate species were found; all of them are characteristic representatives of the fauna of endobiotic ciliates of equids. Paraisotrichopsis composita and Triadinium magnum were previously found mainly in horses of the Asian region. The level of differences in 18S RNA sequences was established in representatives of various genera and families of ciliates, endobionts of the digestive tract of herbivorous mammals, primarily odd-toed ungulates and ruminants.
Key words: endobiotic ciliates, Equus kiang, equids, evolutionary distances analysis
Introduction
The modern species diversity of ciliates - endobionts of vertebrates was formed mainly as a result of protists adaptation to life in various parts of the digestive tract of herbivorous mammals (Kornilova, 2004a, 2006; Newbold et al., 2015). In these unique habitats, complex microbiomes are formed that are characterized by a rich species composition of both pro- and eukaryotes involved in the digestion of plant matter (Newbold et al., 2015). The species composition of communities of endobiotic ciliates differs among representatives of different orders of vertebrates (Vd'acny, 2018). The vast majority of
https://doi.org/10.21685/1680-0826-2023-17-3-3
© 2023 The Author(s)
Protistology © 2023 Protozoological Society Affiliated with RAS
endobiotic ciliates do not form cysts and remain viable in the external environment for a very short time, and the transfer of endobionts from one host to another occurs mainly due to grooming and coprophagy (Kornilova, 2004a).
In this regard, the most important factors determining the structure of communities of endobiotic ciliates include the taxonomic position of the host and the intensity of intra- and interspecific interactions of host individuals. In addition, endobiont communities appear to be somewhat influenced by the host's diet. In connection with the peculiarities of the lifestyle and distribution of ciliates — endobionts of different vertebrates, the study of the processes
Corresponding author: Ludmila Chistyakova. Zoological Institute RAS, Universitetskaya Emb. 1, 199034 St. Petersburg, Russia; pelomixa@mail.ru
Fig. 1. Ciliates from the faeces of kiang Equus kiang (Buetschlidae, Allantosomatidae); DIC, staining with methyl green. A — Bundleia postciliata; B — B. nana; C — B. triangularis; D — Hemiprorodon gymnoposthium; E — Polymorphella ampulla; F — B. inflata; G — B. dolichosoma; H — B. benbrooki; I — Paraisotrichopsis composita; J — Allantosoma intestinale. Abbreviations: n — macronucleus, v — concrement vacuole. Scale bars:10 ^m.
of interpopulation isolation and speciation in this group of protists is of considerable interest. One of the possible approaches in research in this area could be to determine the structure of ciliate-endobiont communities of closely related host species. It should be noted, however, that the fauna of endobiotic ciliates of many herbivorous mammals has not yet been studied. For example, the genus Equus (the only currently existing genus in the family Equidae) includes several modern equine species: Equusferus, E. caballus, E. hemionus, E. asinus, E. quagga, E. zebra, E. grevi, E. kiang, and E. africanus. The ciliate fauna of some ofthem was studied well enough (e.g., E. caballus), but others were studied incompletely (e.g., E. grevi), or even not investigated at all (e.g., E. kiang) (Kornilova, 2004a; Cedrola et al., 2019). Of undoubted interest in this regard is the study of the species diversity of ciliates — endobionts of rare animal species from hard-to-reach habitats kept in zoos.
This work for the first time presents the results of studying the species composition of ciliates isolated from the faeces of kiangs from the Moscow Zoo, in comparison with the ciliate fauna of other equids. We also assessed for the first time the level of differences in 18S RNA sequences in representatives ofvarious genera and families of ciliates, endobionts
of the digestive tract of herbivorous mammals. Such an analysis, in our opinion, is an important and necessary component in studies of this group of protists with the use of methods of molecular phylogeny.
Material and methods
Samples of faeces were collected in June 2023 from two female eastern kiangs (E. kiang holdereri Matschie, 1911) kept in Moscow Zoological Park. One ofthem was born in Berlin Tierpark in 1999 and moved to Moscow Zoo in 2002. The other was born in Moscow Zoo in 2000. Since 2002, they have been living together in one corral. The samples of their faeces were put in 50 ml test tubes and fixed with 96% ethanol immediately after defaecation to prevent the destruction of intestinal ciliates. After a short time, the fixative in the test tubes was completely changed. Additional samples were preserved in formaldehyde at a final concentration 4%. The ciliates were stained with methyl green 1% solution in 1% acetic acid to visualize the nuclei. Ciliates were observed and photographed on glass object slides using a Leica DM 2500 microscope equipped with differential interference contrast (DIC) and digital camera Leica DFC495 (8.0MP).
Fig. 2. Ciliates from the faeces of kiang Equus kiang (Blepharocorythidae); DIC, staining with methyl green. A — Blepharocorys microcorys; B — B. curvigula; C — B. angusta; D — Circodinium minimum. Abbreviation: n — macronucleus. Scale bars: 20 ^m.
The total number of ciliates in a fixed volume of liquid (100 ^l) was counted on the slides. Since the number of ciliates in the samples was very low, we quantified the results in the following manner: + about 10 cells per ml, ++ about 100 cells per ml, +++ many more than 100 cells per ml. Identification and taxonomy of ciliate species and genera were mainly based on the studies of Gassovsky (1919), Hsiung (1930), Strelkow (1931, 1939), and Lynn (2008).
For evolutionary distances analysis,we chose 18S sequences of Trichostomatia available from NCBI Genbank. The analysis included most of the groups identified in the traditional morphological system, whose composition was confirmed by the results of molecular phylogenetic analysis. We combined the selected sequences into three types of groups: sequences from the same family (e.g., Isotrichidae, Cycloposthiidae), sequences from the same genus (Ostracodinium sp., Blepharocorys sp., etc.), and those from the same species (Isotricha intestinalis, Spirodinium equi). Cycloposthiidae were analyzed three times: as a single group, and separa-tely as a group including mainly Cycloposthium sp. and as a group including mostly Triplumaria sp.The detailed information of sequences included and NCBI accession numbers are given in Table S1. Nucleotide alignment was created for each group of sequences with the online tool Clustal Omega (Goujon et al., 2010). When compiling the summary Table 2, the values for specimens, whose systematic position was uncertain based on the results of the analysis, were not taken into account. Sequence information for geographic isolates of the same species is available
only for several ciliates; therefore, we considered it unnecessary to dwell on the discussion of this information.
The pairwise (for family groups) and mean (species and genus groups) evolutionary distances were estimated in MEGA X (Kumar et al., 2018). Two types of distances were calculated: (1) the number of base differences per sequence (divided by the number of total positions of final alignment), and (2) the number of base substitutions per site using the Maximum Composite Likelihood model. The parameters for both estimates were as follows: inclusion of transitions and transversions, gamma-distributed rates among sites (gamma shape parameter 1), and partial deletion of missing sites (cutoff 95%). Standard error estimates were obtained by a bootstrap procedure (100 replicates). The resulted distances and standard error estimates are provided in the Table S1.
Results and discussion
The species composition of endobiotic ciliates in different representatives of the genus Equus is generally the same; the vast majority of ciliate species were found in all studied species ofthis group ofhosts (Cedrola et al., 2019). Some species of endobiotic ciliates, however, have a certain specificity in relation to their hosts. Thus, Trifascicularia cycloposthium, Triadinium elongatum, Ditoxum hamulus, and Spirodinium ferrumequinum were found only in the zebra E. quagga (Strelkov, 1931; Kornilova et al.,
Table 1. List of species of intestinal ciliates of different equids.
Host species Family/genus/subgenus/species 1 2 3 4 5 6 7 8
Buetschliidae Poche, 1913
Alloiozona Hsiung, 1930
1 A. trizona Hsiung, 1930 + + +
Blepharoconus Gassovsky, 1919
2 B. cervicalis Hsiung, 1930 +
3 B. hemiciliatus Gassovsky, 1919 + +
4 Blepharoconus sp. +
Blepharosphaera Bundle, 1895
5 B. intestinalis Bundle, 1895 + + +
6 B. ellipsoidalis Hsiung, 1930 + +
7 B. citriformis Strelkow, 1939 +
8 B. ceratotherii Van Hoven et al., 1998 + + +
Holophryoides Gassovsky, 1919
9 H. ovalis (Fiorentini, 1890) + + + +
10 H. macrotricha Strelkow, 1939 + + + + +
Polymorphella Corliss, 1960
11 P. ampulla (Dogiel, 1929) abot 10 cells per ml + + +
Blepharozoum Gassovsky, 1919
12 B. zonatum Gassovsky, 1919 + +
Hemiprorodon Strelkow, 1939
13 H. gymnoposthium Strelkow, 1939 about 10 cells per ml + +
Blepharoprosthium Bundle, 1895
14 B. pireum Bundle, 1895 + + + +
15 B. polytrichum Strelkow, 1939 +
Bundleia Cunha & Muniz, 1928
subgen. Bundleia Strelkow, 1939
16 B. postciliata (Bundle, 1895) about 10 cells per ml + + + + + +
17 B. piriformis Strelkow, 1939 + + + +
18 B. nana Strelkow, 1939 about 10 cells per ml + + + +
19 B. vorax Strelkow, 1939 + +
20 B. asymmetrica Strelkow, 1939 + +
subgen. Fibrillobundleia Strelkow, 1939
21 B. benbrooki (Hsiung, 1930) about 10 cells per ml + + + +
22 B. inflata Strelkow, 1939 many more than 100 cells per ml + + + + +
23 B. dolichosoma Strelkow, 1939 about 10 cells per ml + +
subgen. Chlamydobundleia
24 B. elongata Strelkow, 1939 + + +
25 B. triangularis Strelkow, 1939 many more than 100 cells per ml + + +
Prorodonopsis Gassovsky, 1919
26 P. coli Gassovsky, 1919 + +
Sulcoarcus Hsiung, 1935
27 S. pellucidulus Hsiung, 1935 + + +
Table 1. Continuation.
Host species Family/genus/subgenus/species 1 2 3 4 5 6 7 8
Paraisotrichopsis Gassovsky, 1919
28 P. composita Gassovsky, 1919 about 10 cells per ml + +
Fiorentinus Jankowski, 1986
29 F. ovalis (Fiorentini, 1890) + + +
Didesmis Fiorentini, 1890
30 quadrata Fiorentini, 1890 + + +
31 spiralis Hsiung, 1929 +
Wolskana Ito, Ogimoto, Nakahara, 1996
32 W. tokarensis Ito, Ogimoto, Nakahara, 1996 +
Paraisotrichidae da Cuncha, 1917
Paraisotricha Fiorentini, 1890
33 P. minuta Hsiung, 1930 + + + +
34 P. colpoidea Fiorentini, 1890 + + +
Rhizotricha Wolska, 1964
35 R. beckeri (Hsiung, 1930) + +
Blepharocorythidae Hsiung, 1929
Blepharocorys Bundle, 1895
36 B. uncinata (Fiorentini, 1890) +
37 B. curvigula Gassovsky, 1919 about 100 cells per ml + + + + + +
38 B. angusta Gassovsky, 1919 many more than 100 cells per ml + + + + + + +
39 B. microcorys Gassovsky, 1919 about 100 cells per ml + + + + + +
40 B. valvata (Fiorentini, 1890) + + +
41 B. cardionucleata Hsiung, 1930 + + +
42 B. jubata Bundle, 1895 + + + +
Ochoterenaia Chavarria, 1933
43 O. appendiculata Chavarria, 1933 + + + +
Circodinium Wolska, 1971
44 C. minimum (Gassovsky, 1919) about 100 cells per ml + + + + + +
Charonnautes Strelkow, 1939
45 C. equi (Hsiung, 1930) + + + +
Cycloposthiidae Poche, 1913
Cycloposthium Bundle, 1895
46 C. bipalmatum (Fiorentini, 1890) about 10 cells per ml + + + + +
47 C. edentatum Strelkow, 1928 many more than 100 cells per ml + + + + +
48 C. dentiferum Gassovsky, 1919 + + +
49 C. affine Strelkow, 1929 + + +
50 C. corrugatum Hsiung, 1930 +
51 C. hemioni Kornilova, 2001 +
52 C. ponomarevi Kornilova, 2001 +
53 C. piscicauda Strelkow, 1928 +
4
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
Table 1. Continuation.
Host species Family/genus/subgenus/species
C. scutigerum Strelkow, 1928
C. plicatocaudatum Strelkow, 1939
Tripalmaria Gassovsky, 1919
T. dogieli Gassovsky, 1919
about 100 cells per ml
Trifascicularia Strelkow, 1931
T. cycloposthium Strelkow, 1931
Spirodiniidae Strelkow, 1939
Ditoxum Gassovsky, 1919
D. funinucleum Gassovsky, 1919
about 10 cells per ml
D. brevinucleatum Strelkow, 1931
D. hamulus Strelkow, 1931
D. gravinucleatum Hsiung, 1935
Triadinium Fiorentini, 1890
T. caudatum Fiorentini, 1890
T. magnum Hsiung, 1930
about 10 cells per ml
T. elongatum Strelkow, 1939
Gassovskiella Grain, 1994
G. galea (Gassovsky, 1919)
Cochliatoxum Gassovsky, 1919
C. periachtum Gassovsky, 1919
Tetratoxum Gassovsky, 1919
T. parvum Hsiung, 1930
about 10 cells per ml
T. unifasciculatum Fiorentini, 1890
about 10 cells per ml
T. excavatum Hsiung, 1930
Spirodinium Fiorentini, 1890
S. nanum Strelkow, 1931
S. equi Fiorentini, 1890
S. confusum Hsiung, 1935
S. ferrumequinum Strelkow, 1931
S. uncinucleatum Hsiung, 1935
S. magnum Ike, Imai, Ishii, 1983
Allantosomatidae Jankowski, 1967
Allantosoma Gassovsky, 1919
A. intestinale* Gassovsky, 1919
about 10 cells per ml
A. cucumis Strelkow, 1939
Arcosoma Jankowski, 1967
A. lineare (Strelkow, 1939)
A. dicorniger (Hsiung, 1928)
A. brevicorniger (Hsiung, 1928)
Allantoxena Jankowski, 1978
A. biseriale (Strelkow, 1939)
A. japonensis (Imai, 1979)
Strelkowella Kornilova, 2004
1
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Notes: 1 - E. kiang (this paper), 2 - E. caballus (Cedrola et al., 2019), 3 - E. asinus (Strelkow, 1939, Kornilova, 2003, Gurelli, Goçmen, 2010, Gurelli, 2012), 4 - E. ferus (Kornilova, 2003), 5 - E. hemionus (Kornilova, 2003), 6 - E. zebra (Kornilova et al., 2020), 7 - E. quagga (Kornilova et al., 2021), 8 - E. grevi (Kornilova, 2003).
The number of ciliates is indicated only for E. kiang, for other equids only the presence or absence of a certain species of ciliates is indicated.
* Due to the neuter gender of the name Allantosoma we use species name "intestinale" according to the Article 30.1.2 of the International Code of Zoological Nomenclature (Aescht, 2001).
Table 1. Continuation.
Host species Family/genus/subgenus/species 1 2 3 4 5 6 7 8
83 S. urunbasiensis Kornilova, 2004 +
Total number of species 21 75 41 17 58 13 36 9
2021; Gurelli, 2023). Spirodinium nanum was also found mainly in zebras, but sporadic findings of this species in the domestic horse are also known (Ike et al., 1985; Kornilova et al., 2021, 2022; Gurelli, 2023). Cycloposthium hemioni and C. ponomarevi were first isolated and described from the intestines of the wild ass E. hemionus and have so far been found only in this host (Kornilova, 2003). The largest number of ciliate species was registered in the domestic horse E. caballus (Table 1). This is probably due to the worldwide distribution ofthis host species and frequent interactions of horses from different populations. At the same time, some species of ciliates were found only within certain isolated populations ofhorses. For example, Wolskana toka-rensis was found only in Tokara ponies (native Japanese horses living on the Tokara islands) and horses from Iceland, and Strelkowella urunbasiensis — in endemic Yakut horses exclusively (Ito et al., 1996; Kornilova, 2004b; Kornilova et al., 2019).
In the faeces of kiangs, we found 21 species of ciliates, representatives ofthe families Buetschliidae, Spirodiniidae, Cycloposthiidae, Bleharocorythidae, and Allantosomatidae (Table 1; Figs 1—3). The number of ciliates in the samples was generally small and amounted to 1400 ciliates per ml. The most numerous species were Bundleia inflata, B. triangularis, Blepharocorys angusta, and Cycloposthium edentatum.
All ciliates found in the faeces of kiangs from the Moscow Zoo are typical representatives of the fauna of equine endobiotic ciliates. It is interesting to note the finding of Paraisotrichopsis composita, previously discovered mainly in horses of the Asian region (Cedrola et al., 2019). The same applies to the Triadinium magnum. It should be emphasized that when keeping the vertebrate hosts in zoos, a depletion of their ciliate composition is sometimes
observed (Modry et al., 2009). Some species of endobiotic ciliates, such as Triadinium elongatum and Spirodinium ferrumequinum, were found in their specific hosts (the zebra E. quagga) only in natural habitats (Strelkow, 1931; Kornilova et al., 2021). Thus, to obtain a complete picture of the ciliate composition, it is certainly important to investigate endobiont samples from free-grazing kiangs in their natural habitat (Tibet and adjacent regions).
Studying the genetic variability of the isolated populations of endobiotic ciliates from different host species is impossible without investigating the level of interspecific differences between representatives of various genera and families of trichostomatid ciliates. Ofcourse, to obtain a more or less complete and adequate idea of the nature of the differences between taxa ofvarious ranks and the significance of the morphological differential characters observed, it is necessary to use a multigene phylogeny. However, the analysis using the 18S RNA gene sequence (the only one possible based on the available data) also seems relevant; its results are presented in Table 2, Fig. 4, and Table S1.
Endobiotic ciliates of mammals can be divided into two large groups, depending on the nature of their localization in the digestive tract of the host: hindgut (predominantly inhabitants ofthe intestines of odd-toed ungulates, elephants, damans, primates, and rodents) and foregut (inhabitants of the foregut fermentation chambers of ruminants, hippos, camels, and marsupials) (Kornilova, 2004a; New-bold et al., 2015). Among the hindgut ciliates included in the analysis, the species of the genus Bundleia (Buetschliidae) stand apart — the level of differences between them is much higher than the differences between the representatives of one genus, and even family, of other trichostomatids. The differences between other representatives of the
* > I
Fig. 3. Ciliates from the faeces of kiang Equus kiang (Spirodiniidae, Cycloposthiidae); DIC, staining with methyl green. A — Triadinium magnum; B — Ditoxum funinucleum; C — Tripalmaria dogieli; D — Cycloposthium edentatum; E — C. bipalmatum; F — Tetratoxumparvum; G — T. unifasciculatum. Scale bars: 20 ^m.
family Buetschliidae are also generally higher than between representatives of other families of hindgut ciliates. A situation similar to Bundleia sp. is observed for species of the genus Blepharocorys, although the differences between them are not so great. The range of differences between representatives of the families Blepharocorythidae (except the genus Blepharocorys), Spirodiniidae and Cycloposthiidae is approximately the same. It is interesting to note that the level of differences between Troglo-dytella abrassarti (Troglodytellidae) and different cycloposthiids appears to be approximately the same as between representatives ofthe genera Cycloposthium and Triplumaria (Cycloposthiidae). Moreover, according to the results of molecular phyloge-netic analysis, species of the genus Triplumaria form a clade, which isseparate from other cyclopostiids on the phylogenetic tree (Fregulia et al., 2021; Kor-nilova et al., 2023). Evolution divergence between species within the genera Cycloposthium and Triplumaria turns out to be approximately two times less than between representatives of different genera
of cyclopostiids. The species C. compressum stands out; evolutionary differences for it are approximately twice as large as that of other representatives of the group. Notably, the level of differences between Cycloposthium from equids and rodents turns out to be approximately the same.
Among foregut ciliates, 18S RNA sequence data are available primarily for endobionts of ruminants. For the family Isotrichidae, the level of differences between representatives of different genera (and species ofthe same genus Isotricha) is higher than the upper limit of the range of evolutionary divergence between representatives ofdifferent genera within the families Spirodiniidae and Cycloposthiidae. Within the family Ophryoscolecidae, three groups are clearly distinguished: a group of species from the genera Polyplastron-Ostracodinium, species of the genus Epidinium, and species of the genus Entodinium. The differences between the species ofthe first group correspond to the differences between representatives of the families Spirodiniidae and Cycloposthiidae. Evolution divergence between representatives
Fig. 4. Pairwise estimates (min/max) of the number ofsubstitutions per site for selected groups. 1 — Buetschliidae, Bundleia sp. intragenus; 2 — Buetschliidae, Bundleia sp. and species of other genera; 3 — Buetschliidae, other genera; 4 — Blepharocorythidae, Blepharocorys sp. intragenus; 5 — Blepharocorythidae, Blepharocorys sp. and species of other genera; 6 — other genera; 7 — Spirodiniidae; 8 — Cycloposthiidae/Troglodytellidae/ Rhinozetidae, all species without C. compressum; 9 — Cycloposthiidae/Troglodytellidae/Rhinozetidae, C. compressum with other species; 10 — Cycloposthium sp. (without C. compressum); 11 — Triplumaria sp.; 12 — Isotrichidae; 13 — Ophryoscolecidae, Polyplastron/Ostracodinium, 14 — Ophryoscolecidae, Ostracodinium sp., 15 — Ophryoscolecidae, Polyplastron/Ostracodinium — Epidinium; 16 — Ophryoscolecidae, Polyplastron/ Ostracodinium — Entodinium; 17 — Ophryoscolecidae, Epidinium — Entodinium; 18 — Ophryoscolecidae, Entodinium sp.
of the groups Polyplastron-Ostracodinium — Epidinium, Polyplastron-Ostracodinium — Entodinium and Epidinium — Entodinium is at least twice as large. At the same time, the range of differences between the species of the genus Ostracodinium turns out to be somewhat wider than between the species of the genera Cycloposthium and Triplumaria of hindgut ciliates. The differences between some species ofthe genus Entodinium significantly exceed the differen-
ces between representatives of different genera ofthe families Spirodiniidae, Cycloposthiidae, and even Blepharocorythidae.
Thus, to conclude, the ranges ofinterspecific and intergeneric differences vary among representatives of different families of hindgut and foregut ciliates. Meanwhile, for three out of the four analyzed families of hindgut ciliates, these ranges (numeric values for these ranges) is approximately the same.
Table 2. The range (max/min) of interspecific and intergeneric differences between representatives of different families of hindgut and foregut ciliates (for explanations see Material and methods). Full table presented in Supplement (Table S1).
Ciliates max min
Buetschliidae Bundleia sp. intragenus Bundleia sp. and species of other genera Other genera 0.3106/14.38 B. nana - B. benbrooki 0.2710/13.27 B. benbrooki - Sulcoarcus pellucidulus 0.1321/8.63 S. pellucidulus - D. ovalis (Fiorentinus ovalis) 0.2055/11.24 B. postciliata - B. benbrooki 0.0737/5.69 B. postciliata - Alloiozona trizona 0.0304/2.75 A. trizona - Blepharoconus hemiciliatus
Blepharocorythidae Blepharocorys sp. intragenus Blepharocorys sp. and species of other genera Other genera 0.0495/4.58 B. microcorys - B. curvigula 0.0398/3.74 B. microcorys - Circodinium minimum 0.0131/1.29 O. appendiculata - C. minimum 0.0125/1.23 B. curvigula - B. jubata 0.0296/2.84 B. jubata - Ochoterenaia appendiculata 0.0131/1.29 O. appendiculata - C. minimum
Spirodiniidae 0.033/4.27 Ditoxum funinucleum - Gassovskiella galea 0.0128/1.75 Cochliatoxum periachtum -Triadinium caudatum
Cycloposthiidae+Troglodytellidae+ Rhinozetidae All species without C. compressum C. compressum with other species Cycloposthium sp. (without C. compressum) Triplumaria sp. 0.0354/3.37 C. caudatum, C. cristatum, C elongatum - Triplumaria harpagonis 0.0566/5.16 C. compressum - T. fulgora, T. harpagonis 0.0154/1.5 C. bursa - C. cristatum 0.0185/1.8 T. fulgora - T. harpagonis 0.0043/0.43 Cycloposthium incurvum -Monoposthium cynodontum 0.0257/2.44 C. compressum - C. elongatum, C. caudatum 0.0007/0.07 C. lenticularis - C. minutum 0.0058/0.58 T. fulgora, T. selenica - T. dvoinosi, T. sukuna - T. solea
Isotrichidae 0.0620/5.7 Isotricha prostoma - Dasytricha ruminantium 0.0363/3.43 I. prostoma - I. intestinalis
Ophryoscolecidae Polyplastron/Ostracodinium Ostracodinium sp. Polyplastron/Ostracodinium - Epidinium Polyplastron/Ostracodinium - Entodinium Epidinium - Entodinium Entodinium sp. 0.0288/2.76 P. multivesiculatum - O. gracile 0.0176/1.71 O. mammosum - O. gracile 0.0811/7.17 P. multivesiculatum - E. ecaudatum 0.0978/8.42 P. multivesiculatum - E. caudatum 0.0656/5.92 E. ecaudatum - E. caudatum 0.0406/ E. caudatum - E. simplex 0.0217/2.1 P. multivesiculatum - O. clipeolum 0.0007/0.07 O. gracile - O. clipeolum 0.0659/5.92 O. clipeolum - E. ecaudatum, E. caudatum 0.0724/6.44 O. clipeolum. - E. furca monolobum 0.04/3.75 E. ecaudatum - E. furca monolobum 0.0066/0.66 E. dubardi - E. furca dilobum
Within individual families, the ranges of differences between members of different genera and species of the same genus have been reported to overlap substantially; the differences between some species ofthe same genus are sometimes significantly greater than between species belonging to different genera of the same family.
Acknowledgements
The work was supported by the Russian Science Foundation (grant No 23-24-00240, https://rscf.ru/ project/23-24-00240/). The research was carried out using the equipment of the Core Facilities Centre "Taxon" at the Zoological Institute, Russian Academy of Sciences (St. Petersburg, Russia).
References
Aescht E. 2001. Catalogue of the generic names ofciliates (Protozoa, Ciliophora). Denisia. 1: 1—350.
Cedrola F., Bordim S., D'Agosto M. and Dias R. 2019. Intestinal ciliates (Alveolata, Ciliophora) in Brazilian domestic horses (Equus caballus L.) and a review on the ciliate communities associated with
horses around the world. Zootaxa. 4585: 478—488. https://doi.Org/10.11646/zootaxa.4585.3.4
Fregulia P., Cedrola F., Senra M.V., D'Agosto M.and Dias R.J. 2021. New finds on the systematics of cycloposthiid ciliates (Ciliophora: Entodinio-morphida: Cycloposthiidae) based on new 18S-rDNA sequences from a Brazilian Capybara. Curr. Microbiol. 78: 3872-3876. https://doi.org/10.1007/ s00284-021-02651-1
Gassovsky G.N. 1919. On the microfauna of the intestine of the horse. Travaux de la Seciété des Naturalistes de Pétrograd 49: 20-37, 65-69.
Goujon M., McWilliam H., Li W., Valentin F., Squizzato S., Paern J. and Lopez R. 2010. A new bioinformatics analysis tools framework at EMBL-EBI. Nucl. Acid. Res. 38 (suppl_2), W695-W699.
Gurelli G. 2012. Comparative study of hindgut ciliates in horses, mules and donkeys in Turkey. Glob. Vet. 9: 700-705. doi:10.5829/idosi.gv.2012. 9.6.66105
Gurelli G. 2023. Intestinal ciliates of plains zebra (Equus quagga Gmelin, 1788) in Turkey Zootaxa. 5239: 079-090. https://doi.org/10.11646/ zootaxa.5239.1.3
Gurelli G. and Goçmen B. 2010. Intestinal ciliate composition found in the feces of the Cypriot wild donkey, Equus asinus Linnaeus, 1758. Eur.
J. Protistol. 46: 38-42. https://doi.org/10.1016/). ejop.2009.09.001
Hsiung T.S. 1930. A monograph on the protozoan fauna of the large intestine of the horse.Iowa State College Journal of Science 4: 359-423.
Ike K., Imai S. and Ishii T. 1985. Establishment of intestinal ciliates in new-born horses. Jap. J. Vet. Sci. 47: 39-43. https://doi.org/10.1292/jvms 1939.47.39
Ito A., Imai S., Ogimoto K. and Nakahara M. 1996. Intestinal ciliates found in the feces of Japanese native Tokara pony, with the description of a new genus and a new species. J. Vet. Med. Sci. 58: 103-108.
Kornilova O.A. 2003. The fauna of ciliates from the intestine of Asiatic wild ass (kulan). St. Petersburg, TESSA (in Russian).
Kornilova O.A. 2004a. History of study of endobiotic ciliates of mammalia. St.Petersburg, TESSA (in Russian).
Kornilova O.A. 2004b. Strelkowella urunbasiensis gen. n. et sp. n. (Suctoria, Allantosomatidae) from the hindgut of the Yakut Horse. Vestnik Zoologii, 38 (6): 69-73.
Kornilova O.A. 2006. Endosymbiotic ciliates in mammals. Sci. J. Dept. Zool. Herzen. S.P. Univ. of Russia, 6: 21-78.
Kornilova O.A., L.V. Chistyakova L.V. and Ka-myshatskaya O.G. 2019. Report on ciliates from the hindgut of horses in Iceland. Amurian Zool. J. 11 (4): 375-383 https://doi.org/10.33910/2686-9519-2019-11-4-375-383
Kornilova O.A., Tsushko K. and Chistyakova L. 2020. The first record of intestinal ciliates from the Mountain Zebra (Equus zebra) in South Africa. Acta Protozool. 59: 149-155. https://doi.org/10.4 467/16890027AP.20.012.13267
Kornilova O.A., Radaev A.V. and Chistyakova L.V. 2021. Intestinal ciliates (Ciliophora) from the wild plains zebra (Equus quagga) in South Africa, with notes on the microtubule cytoskeleton organization. Eur. J. Protistol. 81: 125842. https:// doi.org/10.1016/j.ejop.2021.125842
Kumar S., Stecher G., Li M., Knyaz C., and Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35: 1547. https://doi.org/10.1093/ molbev/msy096
Lynn D. H. 2008. The ciliated protozoa. In: Characterization, Classification, and Guide to the Literature, 3rd ed. Springer Science+Business Media BV.
Modry D., Petrzelkova K.J., Pomajbikova K., Tokiwa T, et al. 2009. The occurrence and ape-to-ape transmission of the entodiniomorphid ciliate Troglodytella abrassarti in captive gorillas. J. Euk. Microbiol. 56: 83-87. https://doi.org/10.1111/ j.1550-7408.2008.00369.x
Strelkow A. 1931. Über die Fauna des Colons beim Zebra. Zoologischer Anzeiger. 94: 37-54.
Strelkow A. 1939. Parasitical infusoria from the intestine of Ungulata belonging to the family Equidae. Scientific notes of the Herzen Leningrad State Pedagogical Institute. 17: 1-262 (in Russian).
Vd'acny P. 2018. Evolutionary associations of endosymbiotic ciliates shed light on the timing of the marsupial-placental split. Mol. Biol. Evol. 35: 1757-1769. https://doi.org/10.1093/molbev/ msy071
Supplementary material
Table S1. Estimates of evolutionary divergence for Trichostomatia representatives.
