Six dead bodies of White’s thrushes were dissected from January 2013 to August 2017 in the Daejeon city, Korea. These birds collected from the wildlife rescue center in Daejeon were immediately frozen after death and the flukes were subsequently recovered after thawing the bodies. One hundred and fifty-six flukes were harvested from the intestines of these birds. Twenty flukes were fixed with 10% formaldehyde without performing the flattening procedure for the light microscopic observation. The worms were stained with Semichon’s acetocarmine, cleared with xylene, and mounted with Canada balsam. Additionally, to observe their internal organs, 5 flukes were embedded in paraffin, sectioned into slices of 4 μm, and then stained with hematoxylin and eosin. Measurements of the specimens are presented in micrometer as the range, with the mean in parentheses.

For the SEM examination, 5 parasites were washed 5 times with 0.2 M cacodylate buffer (pH 7.3), fixed in 2.5% glutaraldehyde, and then post-fixed in 1% osmium tetroxide at 4°C. The specimens were dehydrated in a graded ethyl alcohol series, dried with a critical point CO₂ dryer, coated with gold and examined using SEM (S-4800, Hitachi) at 15 kV.

Twenty flukes were fixed in 100% ethyl alcohol for the DNA sequencing. Genomic DNA was extracted from the collected parasites according to the manufacturer’s instructions using a DNeasy^® Blood & Tissue Kit (Qiagen, Alameda, California, USA). Using 18S rRNA gene sequences of Leucochloridium, primers for common trematodes were designed using an online tool (Primer3Plus, http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). The oligonucleotide sequences of primers were employed to detect the L. paradoxum 18S rRNA gene (DNA) were 5′-AGGGAATGGGTGGATTTATT-3′ (left primer) and 5′-AGACACGACTGAAAGGTTGC-3′ (right primer). The primer sets were designed around 1,397 bp of the product, variable for all sequences aligned. Under standard conditions like 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 1 min, the polymerase chain reaction (PCR) was performed in a MyCycler Personal Thermal Cycler (Bio-Rad Laboratories, Hercules, California, USA) using the EmeraldAmp GT PCR Master Mix (Takara, Shiga, Japan) with a 1 μl aliquot of DNA. Next, the PCR products were visualized via electrophoresis on a 1.2% agarose gel and then purified using the QIAquick PCR Purification Kit (Qiagen). PCR amplicons were directly sequenced with the same primers as those used in PCR using the ABI Prism BigDye Terminator v.3.0 Ready Reaction Cycle Sequencing kits (Applied Biosystems, Foster City, California, USA). The sequencing data revealed around 726 bp of DNA. Furthermore, the sequence data were aligned using the Clustal Omega program (Clustal O 1.2.1). The phylogenetic tree was constructed based on the sequence analysis conducted using the neighbor-joining method by Basic Local Alignment Search Tool (BLAST) analysis. The neighbor-joining methods were based on a guide tree that provided pairwise and multiple alignment parameters [17].

Morphological characteristics observed in the collected specimens were as follows: the bodies were elongated, relatively wide with rounded extremities that were usually widest at the acetabular levels and 1,987–2,120 long by 819–831 wide (2,009×824) (Figs. 1, 3). Oral sucker subterminal, 351–432×308–425 (402×391). Pharynx muscular, 142–179×78–170 (163×150). The prepharynx and esophagus were almost invisible. The ratio of the lengths of the oral sucker to that of the pharynx was 3.9:1 (Fig. 1A, C). The intestinal ceca were somewhat uniform in diameter throughout their entire length, each branch extending into the posterior end of the body to the area of the cirrus pouch (Figs. 1A, 2B). The ventral suckers were approximately 1.5 times larger than the oral suckers, occupying the central parts of the bodies and they were 536–673×447–605 (629×553) in size (Figs. 1, 3A). Testes round or slightly elliptical, right testis 152–217×114–172 (183×152), left testis 170–235×99–145 (205×123) (Fig. 2). The cirrus pouches were somewhat spherical (Fig. 2B). The genital pores observed at the posterior ends of the bodies were median and ventro-terminal (Figs. 2, 3B). The reproductive glands occupied majority of the post-acetabular, intra-cecal areas and they were arranged in a triangle. The ovaries were spherical, 145–175×111–135 (161×129) in size, situated behind the ventral suckers and lay symmetrically to the left testes (Fig. 2). Uterine seminal receptacles were present; ootypes were surrounded by the Mehlis’ gland, present to the right of the ovaries and in the post-testicular regions. The Laurer’s canals opened posteriorly towards the dorsal side (Fig. 3C, D). The vitellaria folliculars were found to begin adjacent to the levels of the posterior margins of the oral suckers and end at or near the levels of the right testes (Fig. 1). The uteri were filled with eggs and confined to the intercecal areas. The eggs were oval, embryonated, and 19.6–26.0×12.2–15.8 (23.6×14.6) in size.

In SEM study, the worms were found to have flat leaf-like bodies with prominent oral and ventral suckers (Fig. 3A). The bodies were covered with a smooth cytoplasmic layer and no tegumental spines were observed (Fig. 3D). The 2 suckers were remarkably well-developed. The ventral suckers were approximately 1.5 times larger than the oral suckers and located at the center of the bodies (Fig. 3A). The genital pores were situated at the median and ventro-terminal regions. The unspined cirri appeared from round to cylindrical (as the fully evaginated cirrus) in outline (Fig. 3B). The Laurer’s canals opened on the dorsal surfaces of the bodies (Fig. 3C).

To identify the genotype of the Urogonimus specimens in relation to their phylogenetic relationship with Leucochloridiida, the genomic DNA samples of the specimens were compared by phylogenetic analysis (Fig. 4). The partial sequence of the 18S rRNA in Urogonimus specimens was identified and compared to those of the parasites such as Leucochloridium holostomum (KP903629), L. paradoxum (KP903632), and L. perturbatum (KP903641). As a PCR amplicon, partial sequences (540 bp) of the Urogonimus specimens contained 15 variable sites of a common sequence which belonged to the clades of L. holostomum, L. paradoxum, and L. perturbatum, respectively. The partial sequences of the 18S rRNA gene of the Urogonimus specimens showed high similarity with those of the parasites L. holostomum (97.6%), L. paradoxum (97.6%), L. perturbatum (97.4%), Michajlovia migrata (99.3%), Urotocus rossitensis (99.3%), and U. macrostomus (98.5%). The results of the phylogenetic analysis validate the fact that these samples belonged to the same clades as Urogonimus based on their key morphological features. After the sequence of Urogonimus specimens was compared with several genes, the sequence data was inferred using the neighbor-joining method (Fig. 4). The sequence of Urogonimus specimens was also identified to be of U. turdi, which is genetically close to M. migrata (99.3%), U. rossitensis (99.3%), and U. macrostomus (98.5%). Although the morphology of U. turdi was also found to match with the above Leucochloridium sp., the identification strongly supported the fact that the samples are closer to the clades of the Urogonimus species. Finally, we submitted the sequence data to GenBank and received the GenBank accession number for our nucleotide sequence (BankIt MK602324).