Parasitology Research (2019) 118:3043–3051 https://doi.org/10.1007/s00436-019-06397-5

PROTOZOOLOGY - ORIGINAL PAPER

Isospora svecica sp. n. (: ), a new species of coccidium from the white-spotted bluethroat Luscinia svecica cyanecula (Aves: Passeriformes: Muscicapidae)

Aneta Trefancová1 & Jana Kvičerová1

Received: 10 April 2019 /Accepted: 8 July 2019 /Published online: 30 August 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract Using a combination of morphological and molecular data, we describe a new apicomplexan parasite, svecica sp. n., from the white-spotted bluethroat, Luscinia svecica cyanecula, from the Czech Republic. Oocysts were found in its intestinal tract. Sporulation was exogenous and took 1–3 days. The oocysts were slightly ellipsoidal, of average size 26.17 × 20.33 μm, with a smooth bilayered wall. Micropyle, oocyst residuum, and polar granules were absent. Sporocysts were bottle-shaped, of an average size of 18.82 × 8.82 μm, with a thin, colourless wall. A conspicuous knob-like was present. Substieda body was barely visible. Sporocyst residuum was present in the form of granules of various sizes. Sporozoites were banana-shaped and contained large anterior and small posterior refractile bodies. Partial DNA sequences of three genes were obtained from oocysts of Isospora svecica sp. n., being most closely related to other isosporans described from passerines. Little is known about the parasites of the avian family Muscicapidae, including , a highly prevalent parasitic protist group in all classes. Only six species of the genus Isospora have so far been described in Muscicapidae, together with several “Isospora sp.” that in fact most likely represent Isospora lacazei. The newly described Isospora svecica sp. n. differs morphologically from other coccidia reported from muscicapid , and represents the first coccidian species described from Luscinia svecica.

Keywords Coccidia . Isospora . Aves . Muscicapidae . Morphology . Phylogeny

Introduction revealed that the Isospora-type oocysts with SB found in the faeces of some carnivores and are only The oocysts of Isospora (Apicomplexa: Coccidia: pseudoparasites, probably originating in avian hosts ) are often found in the faeces of various species (Barnard et al. 1974;Ernstetal.1969;Ghimire2010; of birds. For a long time, the origin of Isospora-type oocysts Levine and Mohan 1960; Trefancová et al. 2019). has been the subject of much discussion and controversy. About 150 species of Isospora have so far been described Several decades ago, the genus Isospora was split into the from passerine birds. The majority of these descriptions are genera Isospora (Eimeriidae) characterized by possessing a based solely on oocysts found in avian faeces. For most of Stieda body (SB) and infecting mainly birds and reptiles, them, molecular data are lacking. and () lacking a SB and infecting Luscinia svecica (Linnaeus, 1758) (Aves: Passeriformes: mainly mammals (Frenkel 1977; Carreno and Barta 1999; Muscicapidae) includes 10 subspecies, occurring mainly in Franzen et al. 2000; Barta et al. 2005). Moreover, it has been Asia and Europe. Two subspecies have been reported to nest in the Czech Republic, L. svecica svecica (Linnaeus, 1758) and L. svecica cyanecula (Meisner, 1804). The white- Section Editor: Panagiotis Karanis spotted bluethroat L. svecica cyanecula is a small songbird that inhabits moist areas around ponds, reed beds, sedges, * Aneta Trefancová and willows of central and southern Europe. It is a migratory [email protected] returning in March and April after overwintering on the Iberian Peninsula and in northern and central Africa 1 Department of Parasitology, Faculty of Science, University of South (Correia and Neto 2013). This subspecies nests twice a year, Bohemia, Branišovská 1760, 370 05 České Budějovice, Czech Republic and feeds mainly on insects and invertebrates from the 3044 Parasitol Res (2019) 118:3043–3051 ground or in clumps of vegetation, and sometimes also on Molecular and phylogenetic analyses berries. It is an endangered species within the Czech Republic, according to Czech legislation (no. 114/1992 Sb). In compar- Isosporan genomic DNA was extracted from the faecal mate- ison to its biology and ecology, little is known about its para- rial using the FastDNA® SPIN for Soil Kit (MP Biomedicals, sites. Studies on the parasitofauna not only of L. svecica but LLC, Santa Ana, CA, USA) following the manufacturer’s also of other members of the family Muscicapidae— protocol. One-step PCR was performed using the coccidia- representing an assemblage of 275 species in 48 genera—are specific primers ssuF 5′-GAAACTGCGAATGGCTCATT-3′ limited to a few reports, mostly concerning blood protists and ssuR 5′-CTTGCGCCTACTAGGCATTC-3′ amplifying ( and )(Kučera 1981; Paperna the gene encoding the small subunit of 18S rRNA, coiF 5′- et al. 2010; Sebaio et al. 2012;Peevetal.2016; Valkiūnas GGTTCAGGTGTTGGTTGGAC-3′ together with coiR 5′- et al. 2017), or helminths (Georgiev et al. 2004;Mutafchiev ATCCAATAACCGCACCAAGAG-3′ amplifying the mito- et al. 2013, 2014). To date, neither species of Isospora nor any chondrial gene for cytochrome c oxidase subunit I (COI), other coccidium have been described from L. svecica. and coiiiF 5′-AGAAAACCTAAAATCATCATGT-3′ together This study presents a morphological description of the with coiiiR 5′-AAGTGAGTTCGCATGTTTAC-3′ amplify- infective oocyst stage, molecular sequence data, and phy- ing the mitochondrial gene for cytochrome c oxidase subunit logenetic analyses of the Isospora from the faeces of the III (COIII). Primers for 18S rDNA and COI were adopted White-spotted Bluethroat L. svecica cyanecula from the from Schwarz et al. (2009) and Kvičerová et al. (2008), re- Czech Republic, which we conclude is a new species. spectively. Sequences of primers amplifying the COIII region were provided by John R. Barta (University of Guelph, Ontario, Canada). Each PCR reaction contained 2.5 μlof

10 × PCR buffer (containing 15 mM MgCl2;Qiagen, Materials and methods Hilden, Germany), 2 μl of extracted DNA, 1 μl of dNTPs (10 mM solution), 0.5 μl of each primer (25 pmol/μl; Generi Origin of the sample and coprological examination Biotech, Hradec Králové, Czech Republic), 0.2 μlof HotStarTaq Plus DNA Polymerase (Qiagen, Hilden,

A single, deceased individual of the white-spotted Germany), and 18.3 μl of PCR H2O. After visualization on bluethroat, L. svecica cyanecula, was recently found in 1% agarose gel, the PCR products of expected sizes were the vicinity of the village Lužnice, 49.0772158 N, enzymatically purified with alkaline phosphatase and exonu- 14.7550169 E (South Bohemia, Czech Republic). The clease I (Thermo Fisher Scientific, Waltham, MA, USA), and bird was dissected and its intestinal tract (small intestine sequenced via the Sanger sequencing method in SEQme, s.r.o. to cloaca) was removed. Subsequently, this part of intes- (Dobříš, Czech Republic). tine with its faecal contents was placed in a 4% potassium The sequences of Isospora svecica sp. n. were examined by dichromate (K2Cr2O7) solution. The sample was stored at the BLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast. room temperature with access to air for 3 days. It was cgi) and further edited using the Sequence Scanner v2.0 then examined for the presence of coccidia by (Applied Biosystems), EditSeq 5.05, and SeqMan 5.05 centrifugation-flotation technique using a modified (DNASTAR, Inc., Madison, WI, USA) programmes. Sheather’s sugar solution (specific gravity 1.3) (Sheather Alignments were created in Geneious v9.1.3 (http://www. 1923; Modrý et al. 2015) and light microscopy. geneious.com;Kearseetal.2012). The coccidian sequences of 18S rRNA, COI, and COIII genes (mainly sequences of Isospora spp.) obtained from the NCBI GenBank database Morphological and morphometrical analyses (https://www.ncbi.nlm.nih.gov/genbank/) and during our previous studies, together with the newly obtained Detected oocysts of Isospora svecica sp.n.were sequences of our sample, were used in phylogenetic examined and described based on the morphological and analyses. The accession numbers of all sequences used in morphometrical analyses of sporulated oocysts according the analyses are shown on the resultant phylogenetic trees. to Duszynski and Wilber (1997)andBertoetal.(2014). Phylogenetic relationships were reconstructed using Fully sporulated oocysts were measured and photographed using Nomarski interference contrast on an Olympus BX53 light microscope equipped with an Fig. 1 Phylogenetic relationships inferred by the BI analysis of the 18S„ Olympus DP73 digital camera and Olympus cellSens rDNA sequences. Numbers at the nodes show posterior probabilities Standard 1.13 imaging software. A line drawing was cre- under the BI analysis/bootstrap values derived from ML analysis; poste- rior probabilities and bootstrap supports lower than 0.50 or 50%, respec- ated using the CorelDRAW X7 graphics suite version tively, are marked with dash (-). The family Sarcocystidae is used as an v17.0.0.491. outgroup Parasitol Res (2019) 118:3043–3051 3045

Hammondia hammondi KT184369 XR_001974441 1/100 H. heydorni JX220987 1/53 Cystoisospora sp. ex Meles meles KU198329 Cystoisospora canis KT184362 0,9/- C. felis KT184364 Sarcocysdae 1/92 Cystoisospora sp. ex Felis sylvestris f. catus AB519675 EU200792 (Cystoisospora spp., 1/99 C. moni C. suis U97523 spp., 0,8/51 Cystoisospora sp. ex Nyctereutes procyonoides AB519674 Toxoplasma gondii) 1/80 Cystoisospora sp. ex Panthera gris altaica FJ357797 C. ohioensis GU292304 C. belli JX025650 0,8/- C. orlovi AY365026 subepithelialis GU479655 E. siliculiformis GU479657 E. ruli GU479667 Eimeria spp. 0,6/55 E. anguillae GU479633 1/100 1/100 E. percae GU479663 bigeneca AF060975 1/- Isospora wiegmanniana KU180242 0,9/61 0,9/98 I. gekkonis KU180246 0,9/- I. tarentolae KU180245 1/100 I. amphiboluri KU180241 1/100 Isospora spp. I. chafarinensis KU180244 1/50 I. abdallahi KU180240 0,9/76 0,9/73 I. fahdi KU180239 1/73 I. albogularis KU180243 1/76 I. takydromi KU180238 Isospora sp. ex Talpa europaea JQ993671 E. lancasterensis KT360976 0,9/53 E. sciurorum KT360994 0,9/50 E. vilasi JQ993653 0,6/- Eimeria sp. ex Gerbillus dasyurus JQ993664 1/100 0,6/- Eimeria sp. ex Cricetus cricetus JQ993663 1/77 E. telekii AF246717 0,7/- Eimeria sp. ex M. agress iz. 93MAG_FR KU174483 Eimeria spp. E. caviae JQ993649 0,5/- E. nafuko JQ993665 1/55 E. chinchillae JQ993650 0,7/- E. falciformis AF080614 E. burdai JQ993666 E. myoxi JF304148 1/65 E. nkaka JQ993651 E. alorani JQ993659 0,9/61 Eimeria sp. ex Mastomys natalensis JQ993667 1/- 1/60 Isospora sp. ex Columbia livia f. domesca AB757863 1/94 E. innocua HG793045 0,7/50 E. dispersa KT184338 1/52 cayetanensis KX618190 E. gallopavonis KT184344 1/- 1/95 E. necatrix KT184349 Eimeria spp. 1/78 E. acervulina FJ236372 1/87 Legend E. brune KT184337 1/99 E. maxima U67117 coccidia of mammals I. manorinae KT224379 0,7/- (Sarcocysdae) Isospora sp. ex Troglodytes troglodytes MK559090 I. robini AF080612 1/96 ex Turdidae eimerians of fish 0,6/- Isospora sp. ex Turdus falcklandii JX984668 Isospora sp. ex Parus major MK559088 1/50 (Eimeriidae) Isospora svecica sp. n. MK559092 0,7/50 ex Muscicapidae ,,“ sp. ex Passer melanurus AY331571 isosporans of reples Isospora sp. ex Lamprotornis superbus KF648870 0,5/- ,,Atoxoplasma“ sp. ex Psarocolius decumanus AY331569 Isospora spp. (Eimeriidae) 0,8/53 I. neochmiae KT224380 0,9/62 eimerians of rodents I. butcherae KY801685 sp. ex AY331566 0,7/51 ,,Atoxoplasma“ Creatophora cinerea (Eimeriidae) Isospora cf. erithaci MK559091 Isospora sp. ex Emberiza citrinella MK559089 0,6/- eimerians of ruminants Isospora sp. ex Emberiza citrinella MK559087 ex Emberizidae I. gryphoni KF854254 (Eimeriidae) 1/75 Isospora sp. ex Serinus canaria KT184357 0,8/100 eimerians of poultry E. alabamensis AF291427 E. arloingi AF291427 (Eimeriidae) E. hirci KX845685 1/- 1/100 Eimeria spp. E. zuernii KT184356 eimerians of rabbits 1/78 E. bovis U77084 (Eimeriidae) 1/55 E. irresidua HQ173832 isosporans of birds (passerines) 1/97 E. flavescens HQ173830 1/99 E. piriformis HQ173836 1/98 (Eimeriidae) E. vejdovskyi HQ173838 Eimeria spp. E. intesnalis HQ173831 1/86 E. magna HQ173833 1/71 E. media HQ173834

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Eimeria ranae MH698563 Caryospora bigeneca KF859856 Isospora amphiboluri KR108297 E. alabamensis KT184376 0,7/- E. arloingi KX857470 E. cylindrica KU351702 1/100 KX857469 0,7/74 E. hirci 1/60 E. ahsata KT184373 Eimeria spp. 1/76 KT184372 0,9/55 E. bovis 1/100 E. zuernii HM771687 0,8/54 E. innocua KR108296 1/99 E. dispersa KJ608416 E. acervulina FJ236428 0,6/- 0,7/- E. maxima FJ236459 1/87 E. miva EF174185 1/56 1/100 E. mis HM771681 E. pavonina JN596590 Eimeria sp. ex Phasianus colchicus KJ547708 Eimeria spp. 1/96 1/77 E. tenella KX094951 0,7/56 1/100 E. necatrix HM771680 0,9/61 E. gallopavonis HG793051 1/98 E. adenoeides KJ608415 1/100 E. meleagridis HG793047 E. myoxi JQ993696 E. nkaka JQ993697 0,9/62 E. callospermophili JQ993688 1/99 E. cahirinensis JQ993686

0,7/- E. tamiasciuri KT184375 1/86 KT361004 1/82 E. lancasterensis E. sciurorum KT361002 JX464221 1/94 E. apionodes 0,8/57 E. nafuko JQ993708 0,6/- JQ993709 Eimeria spp. 1/100 E. burdai 1/100 E. ferrisi KT361028 Eimeria sp. ex M. agress iz. 93MAG_FR KU215459 JQ993701 1/98 E. alorani 1/82 Eimeria apionodes KU215473 E. vermiformis HM771683 1/77 E. falciformis HM771682 0,9/51 I. manorinae KT224377 0,9/50 Isospora sp. ex Sylvia conspicillata KP688311 Isospora svecica sp. n. MK573841 0,5/- I. serinuse KR477879 1/82 Isospora sp. ex Parus major MK573833 E. irresidua JQ993694 0,6/- 1/97 E. flavescens JQ993692 1/92 E. piriformis JQ993698 1/96 Legend 1/63 E. vejdovskyi JQ993699 Eimeria spp. E. coecicola JQ993690 1/99 E. magna JQ993695 isosporans of reples 0,5/54 E. intesnalis JQ993693 (Eimeriidae) I. gryphoni KC346355 1/88 eimerians of rodents Isospora sp. ex Serinus canaria KC346356 Isospora sp. ex Emberiza citrinella MK573834 (Eimeriidae) 1/72 0,9/56 Isospora sp. ex Emberiza citrinella MK573829 ex Emberizidae 1/98 Isospora sp. ex Emberiza citrinella MK573831 0,7/- eimerians of ruminants 1/96 I. greineri KR108298 (Eimeriidae) Isospora sp. ex Lamprotornis superbus KF648868 1/51 1/98 Isospora sp. ex Apodemus flavicollis JQ993711 1/94 Isospora spp. eimerians of poultry I. butcherae KY801687 1/99 I. neochmiae KT224378 (Eimeriidae) Isospora sp. ex Troglodytes troglodytes MK573836 eimerians of rabbits Isospora sp. ex Erithacus rubecula MK573830 0,6/- Isospora sp. ex Phoenicurus ochruros MK573842 (Eimeriidae) Isospora sp. ex Erithacus rubecula MK573838 ex Muscicapidae 1/50 1/100 Isospora cf. erithaci MK573837 isosporans of birds (passerines) 1/100 Isospora cf. erithaci MK573839 (Eimeriidae) Isospora sp. ex Turdus merula MK573835 sp. ex MK573832 1/58 Isospora Turdus philomelos sp. ex MK573840 0,9/63 Isospora Turdus merula ex Turdidae Isospora sp. ex Turdus merula MK573843

0.03 Fig. 2 Phylogenetic relationships inferred by the BI analysis of the COI probabilities and bootstrap supports lower than 0.50 or 50%, respectively, sequences. Numbers at the nodes show posterior probabilities under the are marked with dash (-). Eimeria ranae is used as an outgroup BI analysis/bootstrap values derived from ML analysis; posterior

Bayesian inference (BI) in the MrBayes v3.2.6 (Ronquist et al. and exported in FigTree v1.4.2 (http://tree.bio.ed.ac.uk/), and 2012) programme and the maximum likelihood method (ML) edited in Adobe Illustrator CS6 v16.0.3 (Adobe Systems Inc.). in the online programme PHYML v3.0 (Guindon et al. 2010). The best fitting evolutionary model for all phylogenetic anal- yses was selected by the SMS: Smart Model Selection soft- Results ware (http://www.atgc-montpellier.fr/sms/, Lefort et al. 2017). BI analysis was conducted under the GTR + Г +I Microscopic examination and phylogenetic analyses evolutionary model for 10 million generations, and the trees were summarized after removing 25 % of burn-in. For the ML The coprological examination revealed massive infection with analysis, the GTR + Г + I evolutionary model was also coccidia of the genus Isospora. Apart from the oocysts of selected, and the bootstrap values were calculated by 1000 Isospora, no other stages of any other endoparasites were replicates. The resultant phylogenetic trees were visualized observed in the faecal material. Phylogenetic analyses of three Parasitol Res (2019) 118:3043–3051 3047

Lankesterella sp. ex Lithobates clamitans KT369005 Isospora amphiboluri KR108297 Eimeria mephidis KT203398 1/92 E. zuernii KX495130 0,9/82 E. dispersa KJ608416 1/50 E. innocua KR108296 KP796149 1/94 E. praecox KX094945 E. acervulina KX094948 1/85 1/99 0,9/61 E. maxima HQ702481 0,8/- JN864949 0,9/- E. mis 1/97 E. brune KX094959 E. tenella KX094951 Eimeria spp. 1/100 E. necatrix HQ702482 1/100 1/99 E. meleagrimis KJ608414 0,9/76 E. gallopavonis KJ608413 1/98 E. adenoeides KJ608415 1/99 E. meleagridis KJ608418 E. falciformis KX495129 1/100 Eimeria sp. ex M. agress iz. 93MAG_FR MH698511 I. serinuse KX276860 0,6/50 1/100 I. manorinae KX276861 Isospora sp. ex Parus major MK573848 0,9/51 Isospora svecica sp. n. MK573853 0,9/83 0,9/63 E. flavescens KP025693 1/100 E. irresidua KP025690 Legend 1/100 E. intesnalis KP009592 KF419217 isosporans of reples 1/100 E. magna Eimeria spp. KP025692 0,7/74 1/95 E. vejdovskyi (Eimeriidae) 1/50 E. media KP025691 Isospora sp. ex Garrulax chinensis KT203397 0,5/- eimerians of rodents Isospora sp. ex Troglodytes troglodytes MK573851 (Eimeriidae) Isospora sp. ex Emberiza citrinella MK573849 1/50 Isospora sp. ex Emberiza citrinella MK573844 1/84 1/100 ex Emberizidae eimerians of poultry Isospora sp. ex Emberiza citrinella MK573846 1/100 I. superbusi KT203396 (Eimeriidae) 0,9/- 1/100 I. greineri KR108298 Isospora spp. eimerians of rabbits Isospora sp. ex Erithacus rubecula MK573852 Isospora sp. ex Erithacus rubecula MK573845 0,9/73 ex Muscicapidae (Eimeriidae) 1/89 Isospora sp. ex Phoenicurus ochruros MK573854 Isospora sp. ex Turdus merula MK573850 isosporans of birds (passerines) 0,9/80 Isospora sp. ex Turdus philomelos MK573847 ex Turdidae (Eimeriidae) 0,6/66 Isospora sp. ex Turdus merula MK573855

0.05 Fig. 3 Phylogenetic relationships inferred by the BI analysis of the COIII probabilities and bootstrap supports lower than 0.50 or 50%, respectively, sequences. Numbers at the nodes show posterior probabilities under the are marked with dash (-). Lankesterella sp. is used as an outgroup BI analysis/bootstrap values derived from ML analysis; posterior genes (18S rRNA, COI, COIII) placed I. svecica sp. n. close to Taxonomic summary other isosporans described from passerine birds, namely the common canary (Serinus canaria forma domestica), yellow- Type host: white-spotted bluethroat, Luscinia svecica throated miner (Manorina flavigula), and great tit (Parus cyanecula Meisner, 1804 (Aves: Passeriformes: major)(Figs.1, 2,and3). Both BI and ML trees shared the Muscicapidae). general topology. Type locality: Lužnice, 49.0772158 N, 14.7550169 E, South Bohemian Region, Czech Republic. Prevalence: Unknown; the sample was obtained from a Description of Isospora svecica sp.n.(Figs.4 and 5) single individual. Sporulation: Exogenous. Sporulated oocysts (n = 33) are slightly ellipsoidal in shape, Sporulation time: 1–3days. measuring 26.2 (24.5–28.2) × 20.3 (18.9–21.6) μm; shape- Site of infection: Unknown, oocysts obtained from intesti- index (length/width ratio) 1.3 (1.3–1.3), with a smooth, nal tract. bilayered oocyst wall of approximately 1 μm in thickness. Material deposited: Oocysts in absolute ethanol and Micropyle, oocyst residuum, and polar granule are absent. digital photomicrographs (photosyntypes) are deposited Sporocysts are bottle-shaped and markedly ellipsoidal, mea- at the Protistological collection of the Institute of suring 18.8 (17.0–20.4) × 8.8 (7.6–10.4) μm; shape-index 2.1 Parasitology, Biology Centre, Czech Academy of (2.0–2.2), with a thin colourless wall and a conspicuous knob- Sciences, České Budějovice, collection number IP CAS like Stieda body, ~ 1.9 × 1.3 μm. The substieda body is barely Prot 57. visible; the parastieda body is absent. Sporocyst residuum Type sequences: MF422202 (18S rDNA), MK573841 consists of granules of various sizes dispersed among the spo- (COI), and MK573853 (COIII). rozoites. Sporozoites are banana-shaped and possess a large Etymology: The name Isospora svecica sp. n. was de- anterior refractile body, a small posterior refractile body, and a rived from the generic name of the type host L. svecica nucleus located between them. cyanecula. 3048 Parasitol Res (2019) 118:3043–3051

phoenicurus and 1 from Luscinia megarhynchos (Table 1). Confusion of the new isosporan described herein with these six Isospora species is unlikely due to clear differential diag- nostic features, such as the absence of the polar granule and different oocyst shape and size, and character of the sporocyst residuum in the newly described species (see Table 1). Nonetheless, several old, substandard, descriptions of “Isospora sp.” exist from Ficedula spp., Oenanthe spp., Phoenicurus spp., Saxicola spp., Luscinia luscinia,and Luscinia megarhynchos (Table 1), that have later been sug- gested by others (Labbé 1896, 1899;Ryšavý 1954; Scholtyseck 1954; Rakhmatullina and Svanbaev 1972)torep- resent Isospora lacazei (Labbé, 1893), the taxonomic validity of which is, however, unclear. I. lacazei represents a contro- versial coccidian reported from the passeriform birds. It was first described from the goldfinch (Carduelis carduelis), and then reported to occur in over 50 species of passeriform birds, including Muscicapidae (at least nine isosporans, see Table 1), although its validity is uncertain and often discussed because of the substantial variability of oocyst morphology (subspherical, spherical, oval, or ellipsoidal oocysts with a size range of 18–29 × 17–27 μm). There is a doubt as to whether all described species under this name belong to the “ Fig. 4 Composite line drawing of sporulated oocyst of Isospora svecica same species, and Levine ( 1982) suggested that it may be a sp. n. ex Luscinia svecica cyanecula. Scale bar: 5 μm complex of several species.” Moreover, it was also described from mammals (e.g. from cattle) when its oocysts merely Discussion passed through their gut (Levine and Mohan 1960; Duszynski and Gutiérrez 1981). However, all of the descrip- Similarly to Eimeria, Isospora species have been described tions of I. lacazei were based solely on oocyst shape and size; based mainly on the morphology of their sporulated oocysts other morphological features/structures were not assessed. and the host identity. However, oocyst morphological features Thus, Levine and Mohan (1960), and later also many other may overlap among the species (Duszynski 1971;Longand coccidiologists, proposed that the validity of the species, Joyner 1984; Gardner and Duszynski 1990), and some of which closely resemble I. lacazei, should be confirmed by them can infect multiple host species, or even genera. Thus, detailed morphological studies and cross-transmission exper- a combination of morphological and molecular (sequential) iments, which is complicated in endangered species because data is optimal. of their conservation status. Another complication is the fact Based on both morphological (i.e. oocysts with 2 sporo- that no sequence of any of the numerous described I. lacazei cysts possessing SB) as well as molecular data (phylogenetic exists in the GenBank database. I. svecica sp. n. differs from affiliation to the genus Isospora and to isosporans parasitizing the original description of I. lacazei (Labbé, 1893) in oocyst other birds), we prove that this coccidium is an avian parasite. shape (slightly ellipsoidal vs. spherical) and size (24.5–28.2 × General knowledge on the occurrence of coccidia in 18.9–21.6 μmvs.23–25 × 16–18 μm). muscicapid birds is very poor; four species have been de- We obtained sufficiently long sequences of all three genes scribed from the genus Ficedula, 1 from Phoenicurus of Isospora svecica sp. n., and constructed reasonably long

Fig. 5 DIC photomicrographs of sporulated oocysts of Isospora svecica n. sp. Abbreviations: arb, anterior refractile body; n, nucleus; prb, posterior refractile body; sb, Stieda body; ssb, substieda body; sr, sporocyst residuum. Scale bar: 5 μm aaio e 21)118:3043 (2019) Res Parasitol Table 1 Morphological features of isosporan oocysts (Isospora spp.) described from passerine birds of the genus Luscinia and related genera Ficedula, Oenanthe, Phoenicurus,andSaxicola.

Oocyst Sporocyst

Species of Isospora Type host Shape Average Polar granule Residuum Micropyle Shape Average SB sSB Residuum size (μm) size (μm)

I. ficedulae Schwalbach 1959 Ficedula Spherical 20.2 × 20.2 1–3 splinter-like; Absent Pear-shaped Present Six-edged Compact hypoleuca round – I. hypoleuca Dolnik, von Ronn Ficedula Spherical 19.4 × 19.3 Several small Absent Absent Slightly elongated 15.3 × 9.2 Prominent Absent Small 3051 & Bensch 2009 hypoleuca seed-shaped knob-like cap com- pact I. landauae Cringoli & Quesada Ficedula 20.9 × 18.5 2 rounded Absent 15.0 × 10.2 Present Triangular Diffuse 1990 hypoleuca I. luscinia Golemansky 1977 Luscinia Spherical 18.8 × 18.8 1–5 No record No record Elongate-ellipsoidal 16.1 × 8.0 Present No record Diffuse megarhynchos

I. parvae Chatterjee & Ficedula parva Spherical 19.0 × 19.0 1–2 rounded Present Present Absent Compact Choudury 1976 I. phoenicuri Schwalbach 1959 Phoenicurus Ellipsoidal 30.6 × 20.0 1–2 Elongate-ellipsoidal Present Present Compact phoenicurus Isospora sp. Labbé 1896 Oenanthe Only detection of isosporan oocysts in faeces was reported. The author presumed it was Isospora lacazei. oenanthe Isospora sp. Labbé1899 Phoenicurus Only detection of isosporan oocysts in faeces was reported. The author presumed it was Isospora lacazei. phoenicurus Isospora sp. Labbé 1899 Luscinia Only detection of isosporan oocysts in faeces was reported. The author presumed it was Isospora lacazei. luscinia Isospora sp. Rakhmatullina and Oenanthe Only detection of isosporan oocysts in faeces was reported. The author presumed it was Isospora lacazei. Svanbaev 1972 pleschanka Isospora sp. Rakhmatullina and Phoenicurus Only detection of isosporan oocysts in faeces was reported. The author presumed it was Isospora lacazei. Svanbaev 1972 phoenicurus Isospora sp. Rakhmatullina and Saxicola Only detection of isosporan oocysts in faeces was reported. The author presumed it was Isospora lacazei. Svanbaev 1972 torquata Isospora sp. Ryšavý 1954 Luscinia Only detection of isosporan oocysts in faeces was reported. The author presumed it was Isospora lacazei. megarhynch- os Isospora sp. Ryšavý 1954 Ficedula Only detection of isosporan oocysts in faeces was reported. The author presumed it was Isospora lacazei. albicollis Isospora sp. Scholtyseck 1954 Luscinia Only detection of isosporan oocysts in faeces was reported. The author presumed it was Isospora lacazei. megarhynch- os Isospora sp. Schwalbach 1959 Ficedula Ellipsoidal 30.6 × 20.0 2 star-formed Absent Present Triangular Compact hypoleuca Isospora sp. Schwalbach 1959 Ficedula Spherical 21.6 × 21.6 4–8 stick-formed Absent Present Absent Diffuse hypoleuca Isospora sp. Svobodová 1994 Ficedula Only detection of isosporan oocysts in faeces was reported. No description, only a DIC photomicrograph of a poor quality. hypoleuca Isospora sp. Svobodová 1994 Phoenicurus Only detection of isosporan oocysts in faeces was reported. No description, only a DIC photomicrograph of a poor quality. 3049 ochruros 3050 Parasitol Res (2019) 118:3043–3051 alignments (1,411 bp for 18S rDNA, 774 bp for COI, and Brooks DR, McLennan DA (1991) Phylogeny, ecology, and behavior. 853 bp for COIII). All phylogenetic analyses were well- Chicago University Press, Chicago, USA Carreno RA, Barta JR (1999) An eimeriid origin of isosporoid coccidia resolved and highly supported, and placed this new species with stieda bodies as shown by phylogenetic analysis of small sub- to other isosporans described from passerine birds, namely the unit ribosomal RNA gene sequences. J Parasitol 85:77–83 common canary (Serinus canaria forma domestica), yellow- Correia E, Neto JM (2013) Migration strategy of white-spotted throated miner (Manorina flavigula), and great tit (Parus bluethroats (Luscinia svecica cyanecula and L. s. nammetum)along – major), as well as to eimerians infecting rabbits. Quite a big the eastern Atlantic route. Ardeola: Rev Ibérica Ornitología 60:245 259 cluster (comprising 12 sequences of 18S rDNA, 20 sequences Duszynski DW (1971) Increase in size of Eimeria separata oocysts dur- of COI, and 13 sequences of COIII) of isosporans reported ing patency. J Parasitol 57:948–952 from other passeriform birds formed its sister group. Our data Duszynski DW, Gutiérrez RJ (1981) The coccidia of quail in the United further support the hypothesis that the genus Isospora is not States. J Wildl Dis 17:371–379 monophyletic, which was previously proposed also by Barta Duszynski DW, Wilber PG (1997) A guideline for the preparation of species descriptions in the Eimeriidae. J Parasitol 83:333–336 et al. (2005), Ogedengbe et al. (2018), or Trefancová et al. Ernst JV,Cooper C Jr, Chobotar B (1969) Isospora boughtoni Vo lk , 1 93 8 (2019). The phylogeny of some parasites tends to mirror their and Isospora sp. (Protozoa: Eimeriidae) from an opossum Didelphis hosts’ phylogeny (Brooks and McLennan 1991; Paterson and marsupialis. J Wildl Dis 5:406–409 Banks 2001). In this case, the revealed clustering slightly mir- Franzen C, Müller A, Bialek R, Diehl V, Salzberger B, Fätkenheuer G rors the host phylogeny regarding the basal position of (2000) Taxonomic position of the human intestinal protozoan para- site Isospora belli as based on ribosomal RNA sequences. 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Syst Parasitol 57:68–75 Acknowledgements We thank Christopher Mark Steer for language cor- rection of this manuscript. We are grateful to ornithologists Jakub Vlček Ghimire TR (2010) Redescription of genera of family Eimeriidae – and Petr Veselý for capture of birds. We also thank Jana Říhová for help Minchin, 1903. Int J Life Sci 4:26 47 during the field studies. We thank John R. Barta (University of Guelph, Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O Ontario, Canada) for providing sequences of primers amplifying the (2010) New algorithms and methods to estimate maximum- COIII region. likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:3071–32 Funding information This work was supported by the Czech Science Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Foundation (grant number 17-19831S). 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