Morphological and Molecular Characterization of Choleoeimeria Pogonae N

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Yang, R., Brice, B. and Ryan, U. (2016) Morphological and molecular characterization of Choleoeimeria pogonae n. sp. coccidian parasite (Apicomplexa: Eimeriidae, 1989, Paperna and Landsberg) in a western bearded dragon (Pogona minor minor). Experimental Parasitology, 160 . pp. 11-16.

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Morphological and Molecular Characterization of Choleoeimeria pogonae n. sp. coccidian parasite (Apicomplexa:Eimeriidae, 1989, Paperna and Landsberg) in a Western bearded dragon (Pogona minor minor)

Rongchang Yang, Belinda Brice, Una Ryan

PII: S0014-4894(15)30059-X DOI: 10.1016/j.exppara.2015.11.001 Reference: YEXPR 7150

To appear in: Experimental Parasitology

Received Date: 18 June 2015 Revised Date: 22 October 2015 Accepted Date: 9 November 2015

Please cite this article as: Yang, R., Brice, B., Ryan, U., Morphological and Molecular Characterization of Choleoeimeria pogonae n. sp. coccidian parasite (Apicomplexa:Eimeriidae, 1989, Paperna and Landsberg) in a Western bearded dragon (Pogona minor minor), Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.11.001.

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60, 58, 65 JF419354 E. sp. WGK2346 JF419344 E. sp. WGK2534 KF225639 E. setonicis FJ829322 E. trichosuri clone G JF419351 E. sp. WGK2298 65, 67, 72 JF419336 E. sp. Euro38K JF419337 E. sp. RK38N 66,72, 87 JF419359 E. sp. WGK2336 66,72, 87AB243082 E. gruis AB205169 E. gruis 99, 100, 99 Eimeria spp. and Cyclospora spp. 100, 100, 100 AB544327 E. reichenowi 76 AB544326 E. reichenowi AF324215 E. pilarensis JX464223 E. cf. tenggilingi 75, 86, 60 AF307877 E. rioarribaensis JX839286 E. sp. 2 RCY-2012 AF111185 Cyclospora cercopitheci 77, 84, 82 AB769592 E. bukidnonensis JQ993661 E. sp. ex Apodemus sylvaticus JQ993664 E. sp. ex Gerbillus dasyurus JQ993663.1 MANUSCRIPT 100, 100, 100 JX839287 E. tiliquae n. sp. Clone 1 55, 60, 65 JX839288 E. tiliquae n. sp. Clone 2 AF324217 E. tropidura 78, 80, 82 KR360730 C. scincorum AY043207 C. sp. 100, 100, 100 KR779871 C. pogonae n. sp Choleoeimeria. spp . 66, 63, 58 KR360728 C. gallotiae 78, 79, 76 KR360733 C. wiegmanniana GU479650 G. balatonica GU479643 G. sp. BMR-2011d GU479671 G. balatonica 100, 99, 98 GU479655 E. subepithelialis GU479638 G. balatonica Goussia spp . and Eimeria. spp. GU479637 G. vargai GU479664 G. desseriACCEPTED 90, 86, 90 GU479666 G. desseri 88, 96, 96 GU479665 G. desseri EF472967 Toxoplasma gondii

0.01 ACCEPTED MANUSCRIPT 1 Morphological and Molecular Characterization of Choleoeimeria pogonae n. sp. 2 coccidian parasite (Apicomplexa:Eimeriidae, 1989, Paperna and Landsberg) in a Western 3 bearded dragon (Pogona minor minor ) 4 Rongchang Yang a* , Belinda Brice b and Una Ryan a

6 aSchool of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia, 6150.

7 bKanyana Wildlife Rehabilitation Centre, 120 Gilchrist Road, Lesmurdie, Western Australia 6076.

9 ______

10 *Corresponding author. Mailing address: School of Veterinary and Life Sciences, Murdoch

11 University, Murdoch, Western Australia, Australia, 6150. Phone: 61 89360 2495. Fax: 61 89310

12 4144.

13 E-mail: [email protected]

14 15 MANUSCRIPT

ACCEPTED ACCEPTED MANUSCRIPT 16 ABSTRACT

17 A new species, Choleoeimeria pogonae n. sp. is described from a Western bearded dragon

18 (Pogona minor minor ) in Western Australia . Sporulated oocysts (n = 48) were cylindroidal in

19 shape. Oocyst length, 27.0 (26.0 - 28.3) m, oocyst width, 15.2 (14.0 -16.5) m, oocyst

20 length/width ratio (L/W) 1.8 (1.6-1.9), each with 4 sporocysts ( Eimeria -like) and a polar granule,

21 but lacking a micropyle and oocyst residuum. Sporocysts are ovoidal in shape, sporocyst length,

22 10.0 (9.0-11.0) m, sporocyst width 8.5 (7.0-9.5) m, sporocyst L/W ratio, 1.2 (1.1-1.3). Stieda,

23 substieda and parasubstieda bodies were all absent. Molecular analysis was conducted at the 18S

24 rRNA and cytochrome c oxidase I (COI) loci. Phylogenetic analysis of 18S sequences revealed that

25 C. pogonae n. sp. grouped together with another four Choleoeimeria spp. and exhibited 99.1% to

26 99.4% genetic similarity. At the COI locus, C. pogonae n. sp. was in an independent clade and had

27 the highest similarity (80.4%) to Eimeria cf. mivati from a chicken ( Gallus gallus domesticus ).

28 According to the morphological and molecular data, this isolate is a new species of coccidian 29 parasite. This study further supports the taxonomy of MANUSCRIPT Choleoeimeria spp . as a new genus based on 30 molecular phylogenetic analysis.

31 Keywords: 18S rRNA ; Cytochrome c oxidase I; Choleoeimeria pogonae n. sp; morphology;

32 molecular characterization.

34 ACCEPTED

2 ACCEPTED MANUSCRIPT 35 1. Introduction

36 The bearded dragon ( Pogona minor) is a species of lizard (Family: Agamidae) found on the

37 southwest coast and interior of Western Australia and includes the sub-species, the western bearded

38 dragon, (Pogona minor minor) and the Abrolhos bearded dragon (Pogona minor minima). The

39 latter is confined to the Abrolhos Islands, a chain of 122 islands, and associated coral reefs, in the

40 Indian Ocean off the west coast of Australia (Wilson and Swan, 2004). The Western bearded

41 dragon lives in a variety of habitats including woodlands, heath, arid desert and even coastal dunes.

42 It is regarded as semi- arboreal, and is often observed basking on rocks or fallen trees (Ziring,

43 1999).

44 Choleoeimeria is a coccidian genus, proposed by Paperna and Landsberg (1989) that includes

45 tetrasporocystic, dizoic and Eimeria –like coccidians infecting reptilian gall bladder epithelium (El–

46 Toukhy et al., 2014). Choleoeimeria species possess bivalved sporocysts and lack a Stieda body and

47 have cylindroidal or ellipsoidal oocysts. So far, over 40 Choleoeimeria species have been described 48 morphologically and four species have been reported MANUSCRIPT from Australia; C. egerniae from White's 49 skink (Egernia whitii) (Modrý et al., 2006), C. allogehyrae from the house gecko (Gehyra

50 australis), C. boulii from the Tree Dtella (Gehyra variegata) and C. heteronotis from Bynoe's

51 gecko ( Heteronotia binoei) (Paperna, 2007). To date no Choleoeimeria species have been reported

52 from the Western bearded dragon or from any other animal in Western Australia. Molecular

53 characterization of Choleoeimeria species is in its infancy and therefore only four partial 18S rRNA

54 sequences from Choleoeimeria species were available in GenBank, one from the gall bladder of a

55 diadem snake ( Spalerosophis diadema ) from the Czech Republic (accession number: AY043207).

56 The other three speciesACCEPTED reported from Spain were Choleoeimeria gallotiae from a Tenerife lizard

57 (Gallotia galloti ) (accession number: KR360728), Choleoeimeria scincorum from a skink, Mabuya

58 (s. l.) sp. Fitzinger 1826 (accession number: KR360730) and Choleoeimeria wiegmanniana from a

59 checkerboard worm lizard ( Trogonophis wiegmanni ) (accession number: KR360733). In the present

60 study, we characterized a novel Choleoeimeria sp . in a western bearded dragon from Western

3 61 Australia , both at morphological andACCEPTED molecular levels MANUSCRIPT and propose the name Choleoeimeria

62 pogonae n. sp . This is the first report of Choleoeimeria from lizard hosts of the family Agamidae.

65 2. Materials and methods

67 2.1. Sample collection

68 The Western bearded dragon ( Pogona minor minor ) came into care at the Kanyana Wildlife

69 Rehabilitation Centre (KWRC), Perth. On admission it was noted that it had a depressed demeanor,

70 was extremely dehydrated and was reluctant to open it’s eyes. A diagnosis of conjunctivitis was

71 made and the appropriate treatment implemented. A faecal sample was obtained a few days after

72 admission and microscopy (wet mount) revealed large numbers of amoeba, coccidian oocysts and 73 occasional nematodes. The lizard was subsequently treatedMANUSCRIPT with Metronidazole and Toltrazuril. 74 This lizard was euthanased three months later as it remained inactive and would not self-feed. It had

75 lost 19% of its body weight over this period, despite being force fed daily. A faecal sample obtained

76 a few days prior to euthanasia showed no evidence of coccidian oocysts, either by wet mount or

77 faecal flotation.

78 Faecal flotation was done using a standard protocol as described by Yang et al. (2014).

79 Sporulated oocysts were observed using an Olympus DP71 digital micro-imaging camera and

80 images were taken using Nomarski contrast imaging system with a 100x oil immersion objective.

81 ACCEPTED

82 2.2. DNA isolation

83 Total DNA was extracted from 200 mg of faecal sample using a Power Soil DNA Kit

84 (MolBio, Carlsbad, California) with some modifications as described by Yang et al. (2012). Briefly,

85 the faeces for DNA extraction were subjected to four cycles of freeze/thaw (liquid nitrogen

4 86 followed by boiling water) to ensureACCEPTED efficient lysi MANUSCRIPTs of oocysts before being processed using the

87 manufacturer’s protocol. A negative control (no faecal sample) was used in each extraction group.

89 2.3. PCR amplification and sequencing

91 A partial Choleoeimeria 18S rRNA sequence (1,285 bp) was amplified using a nested PCR

92 with the following primers EiF1 (Power et al., 2010) and EiR3 (Yang et al., 2012) for the external

93 reaction and EiF4: 5’ -CTAT GGC TAA TAC ATG CGC AAT -3’ (This study) and EiR4 – ACT

94 CAA AAG ATT ACC TAG AC-3 (This study) for the internal reaction. The PCR reaction

95 contained 2.5 µL of 10 × Kapa PCR buffer, 3 µL of 25 mM MgCl 2, 1.5 µL of 10nM dNTP’s, 10

96 pM of each primer, 1 unit of KapaTaq (Geneworks, Adelaide, SA), 1 µL of DNA (about 50 ng) and

97 14.9 µL of H 2O. PCR cycling conditions were 1 cycle of 94 ºC for 3 min, followed by 45 cycles of

98 94 ºC for 30 sec, 55 ºC for 30 sec and 72 ºC for 2 min and a final extension of 72 ºC for 5 min. The 99 external and internal PCR cycling conditions were identical.MANUSCRIPT 100 The partial Choleoeimeria COI gene sequence (723 bp) was amplified using a nested PCR

101 with the following primers COIF1 (Ogedengbe et al., 2011) and COXR1 (Dolnik et al., 2009) for

102 the external reaction and COIF2 (Yang et al., 2013a) and COXR2 (Dolnik et al., 2009) for the

103 internal reaction. The PCR reaction contained 2.5 µL of 10 × Kapa PCR buffer, 2 µL of 25 mM

104 MgCl 2, 1.0 µL of 10nM dNTP’s, 10 pM of each primer, 1 unit of KapaTaq (Geneworks, Adelaide,

105 SA), 1 µL of DNA (about 50ng) and 13.4 µL of H 2O. PCR cycling conditions were 1 cycle of 94 ºC

106 for 3 min, followed by 40 cycles of 94 ºC for 30 sec, 58 ºC for 30 sec and 72 ºC for 1 min and a 107 final extension of 72ACCEPTED ºC for 5 min. The external and internal PCR cycling conditions were identical.

108 The amplicons from the second round PCRs were gel purified using an in house filter tip

109 method as previously described (Yang et al., 2013b). All the PCR products were sequenced using

110 forward and reverse primers in duplicate using amplicons from different PCR runs. An ABI

5 111 PrismTM Dye Terminator Cycle SequencingACCEPTED kit MANUSCRIPT(Applied Biosystems, Foster City, California) was

112 used for Sanger sequencing according to the manufacturer’s instructions.

113

114 2.4 . Phylogenetic analysis

115

116 Phylogenetic trees were constructed for Eimeria spp. at the 18S rRNA and COI loci with

117 additional isolates from GenBank. Phylogenies were conducted using MEGA (Molecular

118 Evolutionary Genetics Analysis software, version 6, Arizona State University, Tempe, Arizona,

119 USA). Maximum likelihood (ML), neighbor-joining (NJ) and maximum parsimony (MP) analyses

120 were conducted with the Tamura-Nei model based on the most appropriate model selection using

121 ModelTest in MEGA 6. Bootstrap analyses were conducted using 1,000 replicates to assess the

122 reliability of inferred tree topologies.

123 124 3. Results MANUSCRIPT 125 3.4 Description

126 3.4.1 C. pogonae n. sp. (Fig. 1a and 1b).

127 Diagnosis: Sporulated oocysts (n = 48) are cylindroidal, 27.0 × 15.2 (26.0-28.3 × 14.0-16.5) m, 128 length/width ratio (L/W) 1.8 (1.6-1.9), each with 4 sporocysts ( Eimeria -like) and a polar granule, 129 but lacking a micropyle and oocyst residuum. Sporocysts are ovoidal in shape, 10.0 × 8.5 (9.0-11.0 130 × 7.0-9.5) m, L/W 1.2 (1.1-1.3), and Stieda, substieda and parasubstieda bodies are all absent (Fig. 131 1 and Table 1). 132 Type hosts: Western bearded dragon ( Pogona minor minor ). 133 Type locality: Perth,ACCEPTED Western Australia. 134 Prevalence: Unknown . 135 Other hosts: Unknown . 136 Prepatent period: Unknown . 137 Patent period: Unknown . 138 Site of infection: Unknown.

6 ACCEPTED MANUSCRIPT 139 Sporulation time: 48-72 hours. 140 Material deposited: Oocysts in 10% formalin and oocyst photosyntypes were deposited in the 141 Western Australian Museum under the reference number WAM Z68780. DNA sequences have 142 been deposited in GenBank under accession numbers KR779871 and KR779872 for the 18S rRNA 143 and COI genes respectively . 144 Etymology : The species name of the parasite was derived from the species name of the host. 145 146 147 3.2 Phylogenetic analysis of C. pogonae n. sp at the 18S locus

148 A 1,285 bp 18S PCR product from C. pogonae n. sp . was successfully amplified and the

149 sequence was obtained. Phylogenetic analyses showed that C. pogonae n. sp. grouped in a clade

150 with three Choleoeimeria spp. (C. wiegmanniana - KR360733, C. gallotiae - KR360728 and C.

151 scincorum sp - KR360730 ) (data not shown) and shared a genetic similarity of 99.4%, 99.3% and

152 99.3%, respectively. One more partial 18S rRNA sequence (926 bp sequence overlapping with C.

153 pogonae n. sp .) was available from an unnamed Choleoeimeria sp. from the gall bladder of a

154 diadem snake (AY043207). Phylogenetic analysis based on this shorter 18S sequence, grouped C. 155 pogonae n. sp. in the same clade as the four CholeoeimeriaMANUSCRIPT spp. (Fig. 2) (99.1% similarity with this 156 Choleoeimeria sp. from a diadem snake).

157

158 3.3. Phylogenetic analysis of C. pogonae n. sp. at the COI locus

159

160 Direct sequencing of the partial COI gene (723 bp) fragment from this sample produced a

161 clean chromatogram. There were no COI sequences from other Choleoeimeria sp . available in

162 GenBank and few Eimeria COI sequences. The phylogenetic analysis placed C. pogonae n. sp . in a

163 separate clade (Fig.ACCEPTED 3) and exhibited 80.4% similarity with E. cf. mivati (FJ236441) from a chicken

164 (Gallus gallus domesticus ) and 80.2% with E. tiliquae (JX839284) from a shingleback skink.

165 166 4. Discussion

167

7 168 Sporulated oocysts of C. pogonaeACCEPTED n. sp . are MANUSCRIPT morphologically distinct from other

169 Choleoeimeria species from reptiles and they did not match any other existing documented

170 Choleoeimeria species from the order Squamata

171 (http://biology.unm.edu/biology/coccidia/passer1.html (Accessed on 18 Jun. 2015). For example,

172 oocysts of C. pogonae n. sp . are cylindroidal and measured 27.0 × 15.2 (26.0-28.3 × 14.0-16.5) µm

173 in size with a width to length ratio of 1.80. Oocysts contained a polar granule. Morphologically,

174 oocysts of C. pogonae n. sp . are most similar to C. tilburyi (Modrý et al. 2000), which has a similar

175 oocyst width to length ratio (1.8) to C. pogonae n. sp ., but C. tilburyi oocysts are cylindroidal and

176 measured 28.9 (26.0–33.0) × 16.0 (14.0–18.0) µm (Modrý et al., 2000). Choleoeimeria pogonae n.

177 sp . and C. tilburyi each have one polar granule. Sporocysts of C. pogonae n. sp. measure 10.0 × 8.5

178 (9.0-11 × 7.0–9.5) µm, while sporocysts of C. tilburyi measure 10.6 × 7.2 (9.0–12.0 × 6.0–8.0)

179 (Table 1). Although C. pogonae n. sp . and C. tilburyi share morphological similarities, E. tilburyi

180 was described from an East African chameleon while C. pogonae n. sp. is from an Australian 181 agamid lizard and therefore there are differences in hostMANUSCRIPT families as well as zoogeography. The 182 pathogenicity of C. pogonae n. sp. is unknown and further research is required to better understand

183 the pathogenic potential of C. pogonae n. sp.

184 Cannon (1967) described 5 new Eimeria species from Australian lizards. Of these, E.

185 egerniae oocysts, described from the gall bladder epithelium of White's skink, with a large L/W

186 ratio (1.88), most closely resembles those from C. pogonae n. sp. However, a polar granule is

187 present in in C. pogonae n. sp. but is absent in E. egerniae . The dimensions of their oocysts and

188 sporocysts also differ (Table 1). 189 At the molecularACCEPTED level, based on 18S sequences, C. pogonae n. sp. was most closely related to 190 C. wiegmanniana - KR360733. (99.4% similarity) from a checkerboard worm lizard, followed by

191 C. gallotiae. - KR360728 (99.3% similarity) from a Tenerife lizard and C. scincorum - KR360728

192 (99.3% similarity) from a skink , Mabuya (s. l.) sp . Fitzinger 1826.

8 193 Unfortunately, no CholeoeimeriaACCEPTED COI sequences MANUSCRIPT were available in GenBank. At this locus,

194 C. pogonae n. sp. formed a separate clade and exhibited only 80.4% similarity with E. cf. mivati

195 (FJ236441) from a chicken and 80.2% with E. tiliquae (JX839284) from a shingleback skink. As

196 more Choleoeimeria COI sequences become available in GenBank, phylogenetic analysis will be

197 able to give better resolution on the species level.

198 The genus Choleoeimeria proposed by Paperna and Landsberg (1989), was validated using

199 phylogenetic analysis of 18S rRNA sequences in 2002 by Jirk ů et al. (2002) and more recently by

200 Palma et al. (2015). The genus Choleoeimeria was also accepted as a new genus by other

201 researchers (Modrý and Jirk ů 2006; Sloboda and Modrý 2006; Abdel-Baki et al., 2008; 2009; 2013;

202 2014a, b; Jirk ů et al., 2009; Al-Quraishy 2011; McAllister 2012a, b and Abdel-Baki, 2014). In the

203 present study, molecular analysis at two loci (18S and COI) was conducted, which provided clearer

204 insights into the taxonomic status of C. pogonae n. sp. For example, at the 18S locus, C. pogonae n.

205 sp. grouped in a clade with another four Choleoeimeria spp. Phylogenetic analysis at the COI locus 206 clearly showed that C. pogonae n. sp. formed a separateMANUSCRIPT clade from all the Eimeria species, 207 providing further support for the importance of using multiple loci for coccidian species

208 delimitation (Barta, 2001; Tender et al., 2002). Further studies are required, including molecular

209 analysis on additional Choleoeimeria species, along with morphological characterization and

210 pathogenic investigations to further validate Choleoeimeria as a new genus.

211

212 Acknowledgements

213 The authors wish to thank June Butcher and the volunteers at the Kanyana Wildlife

214 Rehabilitation CentreACCEPTED for their commitment and dedication in caring for all the animals admitted to

215 the centre. We are also grateful to the staff at the Wattle Grove Veterinary Hospital, Perth for their

216 expert treatment and care of the wildlife treated at their clinic.

217

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288 Yang, R., Fenwick, S., Potter, A., Elliot, A., Power, M., Beveridge, I., Ryan, U., 2012. Molecular

289 characterisation of Eimeria species in Macropods. Exp. Parasitol. 132, 216-21. 290 Yang, R., Brice, B.,ACCEPTED Bennett, M. D., Ryan, U., 2013a. Novel Eimeria sp. isolated from a King’s 291 skink ( Egernia kingii ) in Western Australia. Exp. Parasitol. 133, 162-165.

292 Yang, R., Murphy, C., Song, Y., Ng-Hublin, J., Estcourt, A., Hijjawi, N., Chalmers, R., Hadfield,

293 S., Bath, A., Gordon C., Ryan, U.M., 2013b. Specific and quantitative detection and

12 294 identification of CryptosporidiumACCEPTED hominis andMANUSCRIPT C. parvum in clinical and environmental

295 samples. Exp. Parasitol. 135, 142-147.

296 Yang, R., Brice, B., Ryan, U., Bennett, M. D., 2013c. Eimeria tiliquae n. sp. (Apicomplexa:

297 Eimeriidae) from the shingleback skink ( Tiliqua rugosa rugosa ).Exp. Parasitol. 133, 144-149.

298 Yang, R., Brice, B., Ryan, U., 2014. Isospora anthochaerae n. sp. from a Red Wattlebird

299 (Anthochaera carunculata ) (Passeriformes: Meliphagidae) in Western Australia. Exp.

300 Parasitol. 140, 1-7.

301 Ziring, N., 1999. Sidney´s Overview of Bearded Dragon Species.

302 http://users.erols.com/ziring/dragon-species.html

303

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306 Fig. 1. a) Nomarski interference-contrast photomicrograph of C. pogonae n. sp. b) Line drawing of

307 the sporulated oocyst of C. pogonae n. sp. Scale bar = 20 µm.

308 Fig. 2. Evolutionary relationships of C. pogonae n. sp. inferred by maximum likelihood analysis of

309 partial 18S rRNA gene sequences (926 bp). Percentage support (>50%) from 1000 psudoreplicates

310 from maximum likelihood, neighbour-joining and maximum parsimony analysis, respectively, is

311 indicated at the left of the support node. (‘_’ = Not available)

312 Fig. 3. Evolutionary relationships of C. pogonae n. sp. inferred by distance analysis of the

313 mitochrondial cytochrome oxidase gene (COI). Percentage support (>50%) from 1000

314 pseudoreplicates from neighbor-joining analyses is indicated at the left of the supported node.

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Table 1 Comparative morphology of Choleoeimeria pogonae n. sp . from the Western bearded dragon with Choleoeimeria spp . recorded from Squamata and Eimeria egerniae from the White's skink.

Species Hosts/Locality References Oocysts Sporocysts Shape Size Shape index Wall Polar granule Shape Size

Choleoeimeria 30-37 × 20-26 (33 ovoidal 13 × 9.3 (11-14 × 9-10) Amphisbaena alba (Brazil) Lainson 2003 ellipsoidal-cylindroidal 1.50 bi-layered absent amphisbaenae × 22) 25.0 × 14.0 (24.0– ovoidal 9.4 × 6.5 (9–10 × 6–7) Choleoimeria bohemii Chamaeleo melleri (Tanzania ) Modrý et al.,2000 cylindroidal 1.79 bi-layered absent 26.0 × 13.0–15.0) Choleoeimeria Brookesia decaryi 25.6 × 15 (23.0– ovoidal to 10.1 × 6.9 (9–11 × 6–7) Modrý et al., 2001 cylindroidal 1.71 bi-layered absent brookesiae (Madagascar) 27.0 × 13.0–16.0) ellipsoidal Choleoeimeria Stenodactylus doriae (Saudi 24.0 ×17.0 (23.0– ellipsoidal 9.0 × 5.0 (8.0–10.9 × Abdel-Baki, 2014 ellipsoidal 1.41 bi-layered absent duszynskii Arabia) 25. 9 ×16.0–18.0) 4.0–6.0) 29.4×15.7 (26.7- ellipsoidal 12.2×7.3 (10.5- Choleoeimeria gallotiae Gallotia galloti (Spain) Palma et al., 2015 cylindroidal 1.87 bi-layered absent 31.1×15.2-16.3) 14.4×6.6-8.9) Abdel-Baki et al., tetrasporocystic, subspheroidal 8.0 × 6.0 Choleoeimeria ghaffari Eryx jayakari (Saudi Arabia) 23.9 × 14.0 1.71 bi-layered absent 2013 cylindroidal to ellipsoidal Modrý et al., cylindical to 27.7 × 18.4 (26.0– ovoidal 7.3 × 5.2 (6.5–8.0 × Choleoeimeria glawi FurciferPardalis (Madagascar) 1.51 bi-layered absent 2001 ellipsoidal 29.5 × 17.0–19.0) 5.0–5.5) Chamaeleo calyptratus (Czech Sloboda M. and 28.3 × 14.8 (25.0– ovoidal to 10.1 × 6.9 (9.0–11.0 × Choleoeimeria hirbayah cylindroidal 1.91 bi-layered absent Republic ) Modrý D. 2006 30.0 ×13.5–17.5)MANUSCRIPT ellipsoidal 6.0–7.5) 31.2 × 19.3 (29.5– ovoidal 10.2 × 7.6 (10.0–11.0 × Choleoeimeria largeni Chamaeleo gracilis (Kenya) Modrý et al., 2000 cylindroidal 1.56 bi-layered 1 to 3 34 ×18.5–20.0) 7.0–8.5) Pogona minor minor 27.0 × 15.2 (26.0 – ovoidal 10.0 × 8.5 (9.0-11.0 × Choleoeimeria pogonae This study cylindroidal 1.80 bi-layered occasionally 1 (Australia) 28.3 × 14.0 -16.5) 7.0–9.5) Choleoeimeria Cerastes gasperettii (Saudi Abdel-Baki et al. 23.0 × 15.0 (22.0– ellipsoidal 8.0 × 5.0 (7.0–9.0 × 5.0– ellipsoidal 1.53 bi-layered absent salaselensis Arabia) 2014 25.0 × 14.0–17.0) 6.0) Choleoeimeria Skink, Mabuya (s. l.) sp . 27.2×14 (25- ovoidal 8.8 ×7.0(8.2-10.1 ×6.2- Palma et al., 2015 cylindroidal 1.94 bi-layered occasionally 1 scincorum (Spain) 29.6×12.4-15.2) 7.5) 28.9 × 16 (26.0– ovoidal to 10.6 × 7.2 (9.0–12.0 × Choleoeimeria tilburyi Chamaeleo jacksoni (Kenya) Modrý et al. 2000 cylindroidal 1.81 bi-layered occasionally 1 33.0 × 14.0–18.0) ellipsoidal 6.0–8.0) Choleoeimeria Trogonophis wiegmanni 28.7×18.9 ( 26.1- spherical 8.7 ×7.9 (7.7-9.8 × 7.2- Palma et al., 2015 cylindroidal 1.51 bi-layered present, 1 wiegmanniana (Spain) 31.2×17.7-20.1) 8.9) 27.3×14.5 (25-34 ovoidal 8.9 × 6.6 (8.1-10.3 × Eimeria egerniae Cannon (1967) ellipsoidal 1.88 bi-layered absent Egernia whitii (Australia) ×11-14) 5.8-7.6) ACCEPTED ACCEPTED MANUSCRIPT

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60, 58, 65 JF419354 E. sp. WGK2346 JF419344 E. sp. WGK2534 KF225639 E. setonicis FJ829322 E. trichosuri clone G JF419351 E. sp. WGK2298 65, 67, 72 JF419336 E. sp. Euro38K JF419337 E. sp. RK38N 66,72, 87 JF419359 E. sp. WGK2336 66,72, 87AB243082 E. gruis AB205169 E. gruis 99, 100, 99 Eimeria spp. and Cyclospora spp. 100, 100, 100 AB544327 E. reichenowi 76 AB544326 E. reichenowi AF324215 E. pilarensis JX464223 E. cf. tenggilingi 75, 86, 60 AF307877 E. rioarribaensis JX839286 E. sp. 2 RCY-2012 AF111185 Cyclospora cercopitheci 77, 84, 82 AB769592 E. bukidnonensis JQ993661 E. sp. ex Apodemus sylvaticus JQ993664 E. sp. ex Gerbillus dasyurus JQ993663.1 MANUSCRIPT 100, 100, 100 JX839287 E. tiliquae n. sp. Clone 1 55, 60, 65 JX839288 E. tiliquae n. sp. Clone 2 AF324217 E. tropidura 78, 80, 82 KR360730 C. scincorum AY043207 C. sp. 100, 100, 100 KR779871 C. pogonae n. sp Choleoeimeria spp . 66, 63, 58 KR360728 C. gallotiae 78, 79, 76 KR360733 C. wiegmanniana GU479650 G. balatonica GU479643 G. sp. BMR-2011d GU479671 G. balatonica 100, 99, 98 GU479655 E. subepithelialis GU479638 G. balatonica Goussia spp . and Eimeria. spp. GU479637 G. vargai GU479664 G. desseriACCEPTED 90, 86, 90 GU479666 G. desseri 88, 96, 96 GU479665 G. desseri EF472967 Toxoplasma gondii

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FJ236388 Eimeria maxima clone 113-68

FJ236456 Eimeria maxima clone 161-22

61 FJ236450 Eimeria maxima clone 113-95

FJ236415 Eimeria maxima clone 113-82

FJ236394 Eimeria maxima clone 158-49

FJ236436 Eimeria maxima clone 61-6

59 FJ236448 Eimeria maxima clone 113-92

100 FJ236407 Eimeria maxima clone 60-2

99 MANUSCRIPTFJ236386 Eimeria maxima clone 41-15.1

FJ236441 Eimeria cf. mivati clone 86-92 100

HM117019 Eimeria sp. Phasianus colchicus/24-6.10-s3 100 98 JQ993709 Eimeria burdai 83 JX839284 Eimeria tilique 100 KR779872 Choleoeimeria pogonae n. sp.

ACCEPTED JX473252 Neospora caninum strain NC-Liverpool 100

JX473251 Hammondia heydorni isolate HhCanis-2009

AB354574 Plasmodium fieldi ACCEPTED MANUSCRIPT Highlights

• a new Choleoeimeria species (C. pogonae n. sp. ) in a Western bearded dragon • Morphology study distinct to other Choleoeimeria species from reptiles. • Genetic study: 98.3% similar to Choleoeimeria sp. from the gall bladder at 18S locus. • First COI gene sequence obtained from Choleoeimeria.

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