Parasites were collected from the rumen of 2 naturally infected cattle sacrificed at a local slaughter house (coordinates: 14.0390°N, 100.6207°E) in Pathum Thani province, Thailand on the 5th of August 2014 and the 15th of February 2016. The flukes were washed several times in 1xPBS, pH 7.4. The worms were either stored in liquid nitrogen until extraction of DNA or RNA or used for preparation of stained whole mounts and paraffin embedded tissue as described in the following sections.

Images of parasites in cattle rumen and 6-well culture dishes (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were acquired with an ASUS ZenFone ZD551KL (ASUSTeK Computer Inc., Taiwan). Parasites collected in 2014 were flattened in 1×PBS, pH 7.4 between 2 microscopic slides and images were taken under a Nikon SMZ445 stereo microscope with a C-LEDS Hybrid LED Stand (Nikon Corporation, Japan) at 0.8X objective magnification using an iPhone 7 (Apple Inc., California, USA) with an iDu LabCam microscope adapter for iPhone 7/8 with multi-fit wide field 10X ocular (iDu Optics LLC, New York, USA). The COX1 sequence of these specimens was subsequently determined from extracted DNA as described below.

PBS-washed mature and immature flukes collected in 2014 and 2016, respectively, were flattened between 2 glass slides and fixed in alcohol-formal acetic acid (AFA) fixative overnight. The fixed flukes were dehydrated in 70% ethanol for 30 min and immersed in Semichon’s carmine at room temperature, overnight. The flukes were dehydrated in 80–95% serial ethanol, 30 min each step, and counterstained with 0.02% fast green for 2–3 sec. The stained flukes were dehydrated in absolute ethanol, 30 min and cleared in xylene, 1 hr. They were mounted in Permount (Thermo Fisher Scientific) and examined under a light microscope.

PBS-washed immature flukes collected in 2016 were fixed in Bouin’s fixative solution (Sigma, St. Louis, Missouri, USA) at room temperature, overnight. The fixed flukes were gradually dehydrated in 50–100% ethanol with each step repeated 3 times, 20 min each step. Absolute ethanol was replaced with xylene 3 times, 10 min each. The flukes were incubated in xylene: paraplast mixed at ratios 2:1, 1:1, 1:2 at 60°C, 1 hr each step. The flukes were then incubated in pure paraplast at 60°C, 3 times, 1 hr each step. The embedded tissue was cut into 8-μm serial cross-sections and the sections were placed on gelatin coated microscopic glass slides and dewaxed and rehydrated. Basophilic substances were stained with progressive hematoxylin for 7 min. Nuclear coloration was converted from reddish purple to a crisp blue by bluing solution for 1 min. Acidophilic substances were stained with eosin for 15 sec. The stained tissue was dehydrated in graded ethanol series from 95% to 100% for each 1 min, and preserved in xylene, 10 min. The tissue was mounted in xylene based mounting medium and observed under a light microscope.

Total RNA of 5 immature Fischoederius sp. 1 (Thailand) collected in 2016 was separately extracted in TRIzol reagent (Ambion^TM Life Technologies, Waltham, Massachusetts, USA). The RNA was dissolved in RNase-free water and purified using a QIAgen RNeasy^® Plus Mini Kit (QIAgen, Hilden, Germany) to remove contaminating DNA. RT-PCR with primer pair 15/18 (Table 1) was used to amplify a 1,536 bp COX1 fragment from each RNA sample. The PCR products were sequenced to confirm sequence identity between the samples. RNA quality was analyzed by 1.2% (w/v) denaturing agarose gel electrophoresis. The concentration of RNA was measured on a NanoDrop ND-2000 UV-Vis spectrophotometer (Thermo Fisher Scientific). RNA at an amount of 14 μg (461 ng/μl) was mixed with GenTegra-RNA matrix in a RNAstable tube (Biomatrica, San Diego, California, USA) and dried in a vacuum desiccator, 4 hr. Following drying, the pooled RNA was shipped to Vishuo Biomedical, Singapore for NGS sequencing on an Illumina HiSeq instrument. Remaining total RNA was kept at −80°C. The Bridger de novo transcriptome assembler [13] was used to assemble the raw data. Mitochondrial sequences among the transcripts were identified by NCBI BLASTN searches using the mitochondrial genomes of F. cobboldi (GenBank: NC_030529), F. elongatus (GenBank: NC_02800) as query sequences.

The extraction of DNA from Fischoederius sp. 1 (Thailand) specimens was done as previously described [14]. The DNA was used in standard PCR for amplification of specific mitochondrial DNA fragments to either quality control transcriptome data or to add missing sequence data in the region between the ND2 and ND1 genes. The used primers and their details are listed in Table 1. PCR products were inserted into the pGEM^®-T Easy vector (Promega, Wisconsin, USA) by standard ligation and had their sequences determined by Sanger dideoxy sequencing (SolGent, Daejeon, Korea and Macrogen, Daejeon, Korea).

Genes and functional RNAs in the mitochondrial genome of Fischoederius sp. 1 (Thailand) were identified by sequence comparison with the mitochondrial genomes of F. cobboldi (GenBank: NC_030529) and F. elongatus (GenBank: NC_02800). Furthermore, MITOS [15] was used for verification of mitochondrial genome annotation. All identified gene and functional RNA sequences were carefully inspected and their 5′ and 3′ ends were manually confirmed and corrected if necessary.

Pairwise nucleotide sequence alignments of all mitochondrial genes (COX3, CYTB, ND4L, ND4, ATP6, ND2, ND1, ND3, COX1, COX2, ND6, and ND5) of Fischoederius sp. 1 (Thailand), F. cobboldi (GenBank: NC_030529), F. elongatus (GenBank: NC_02800) genes were done in EMBOSS needle [16] to obtain sequence identity values (Table 2). The gapopen penalty was set to 30 to suppress invalid gaps.

The deduced amino acid sequences of all mitochondrial genes of Fischoederius sp. 1 (Thailand), F. cobboldi (GenBank: NC_030529), F. elongatus (GenBank: NC_02800), Gastrothylax crumenifer (GenBank: NC_027833), Calicophoron microbothrioides (GenBank: NC_027271), Explanatum explanatum (GenBank: NC_027958), Fasciola hepatica (GenBank: NC_002546), Homalogaster paloniae (GenBank: NC_030530), Ogmocotyle sikae (GenBank: NC_027112), Ogmocotyle sp JM 2015 (GenBank: KR006935), Orthocoelium streptocoelium (GenBank: NC_ 028071), Paramphistomum cervi (GenBank: NC_023095) were concatenated for each species and aligned in Clustal Omega [17] (Supplementary Fig. S1). Regions containing gaps were then removed in trimal [18]. Phylogenetic analysis of the alignment was performed in MrBayes 3.2.5 [19] using a Bayesian approach. The following parameters were used in the analysis: outgroup Fasciola hepatica, prset aamodelpr=mixed; mcmc nchains=4 ngen=100,000.

Partial COX1 sequences from India (GenBank: JX518952–JX518954, JX518950, JX518951, JQ806364, and JQ806365) and Thailand (GenBank: MN437486–MN437488) were aligned in Clustal Omega with the corresponding region (base pairs 703–1,065) of the COX1 gene from Fischoederius sp. 1 (Thailand), F. cobboldi (China, GenBank: NC_030529), F. elongatus (China, GenBank: NC_02800) (Supplementary Fig. S2).

Fischoederius specimens were collected from a cow (Bos taurus indicus/Bos taurus hybrid) with a single infection spot in its rumen which suggested a single infection event. Moreover, molecular analysis of the mitochondrial COX1 gene from several worms showed the same sequence. The flukes in the rumen revealed a turgid, bottle-like appearance and reddish-brown color (Fig. 1A). The flukes were washed and kept in PBS and relaxed to a more elongated form (Fig. 1B). Microscopic examination revealed that all of the flukes were immature, i.e., the reproductive system was underdeveloped; thus eggs were absent from the flukes.

A small number of mature parasites with identical COX1 sequence was found in another cow that suffered from multiple infections. The image of an unstained specimen that was subsequently used for DNA extraction and COX1 sequencing is shown in Fig. 1C. Natural pigmentation limited the resolution of its morphology. Other Fischoederius specimens collected from this animal had significant different COX1 sequences and will be reported elsewhere. Carmine-stained mature and immature specimens were prepared to reveal the details of their morphology (Fig. 1D). Parasite morphology was found to be in general as described for F. elongatus [4,5]. In the mature parasite the lateral vitellaria fields extended from the distal end of the uterus at the genital pore down to the anterior testis. The ventral pouch opened in the area of the pharynx and extended to the posterior end of the anterior testis. The ovary was located between the lobed testes and the egg-filled sinusoid uterus progressed midline between the ceca and ended at the genital pore in the region of the cecal bifurcation. The distal end of the pair of slightly sinusoid ceca was within the anterior half of the body (Fig. 1D). The immature parasite had the same overall morphology but lacked visible vitellaria and showed only underdeveloped reproductive organs (Fig. 1D). Cross sections of an immature specimen (Fig. 2) showed further details similar to historical descriptions of F. elongatus (F. siamensis/F. fischoederi) [7]. Triangular form of the ventral pouch, location of the genital pore at the cecal bifurcation, maximum body diameter in the first third of the body, testes located at the caudal end of the ventral pouch, one anterodorsal to the other with the former located above the ventral pouch and the latter behind it.

The total RNA of 5 worms with confirmed identical COX1 sequence was pooled and used in a transcriptome project (BioProject PRJNA723375). Assembled transcript sequences identified by BLASTN as mitochondrial confirmed that the data originated from a single species but also showed that the mitochondrial data between the ND2 and ND1 genes was incomplete. For this reason PCR with genomic DNA was used to amplify this part of the mitochondrial genome and the ND2, ND1 genes were included to validate the transcriptome data. PCR amplification proceeded without problems but subsequent sequencing reactions failed 3′ to ND2_tRNA-Val and 5′ to tRNA-Asp_ND1. Based on PCR product sizes a region of approximate 750 bp that should include the still missing tRNA-Ala sequence could not be resolved. Forward and reverse primers designed on a published Fischoederius tRNA-Ala sequence (GenBank: NC_028001) were then used to resolve sequences from inside this region together with primers binding outside. Tedious manual interpretation of the sequencing results allowed to resolve the full sequence. The difficulties were caused by 2 large inverted repeat units that obviously formed structures interfering with DNA synthesis during sequencing. The region including the repeats had a size of 746 bp and included a second tRNA-Asp and a partial ND1 gene (117 bp of the gene’s 5′-end) followed by the expected tRNA-Ala (Fig. 3). The complete 14,780 bp mitochondrial genome of Fischoederius sp. 1 (Thailand) contained 12 protein coding genes in the order COX3, CYTB, ND4L, ND4, ATP6, ND2, ND1, ND3, COX1, COX2, ND6, ND5, 23 tRNAs in the order H, Q, F, M, V, D, A, D, N, P, I, K, S, W, T, C, Y, L, S, L, R, G, E, and one each rRNA L and rRNA S (Fig. 3). Sequence comparison of the 12 protein coding genes of Fischoederius sp. 1 (Thailand), F. elongatus (China), F. cobboldi (China) showed significant differences ranging from 6.0–13.2% (Table 2) that were far beyond intraspecies variation (nucleotide polymorphisms) and supported classification as 3 different species. A phylogenetic analysis of the 12 mitochondrial proteins between 12 trematodes confirmed the taxonomic position of Fischoederius sp. 1 (Thailand) in the Gastrothylacidae (Fig. 4). The alignment of the concatenated sequences used in this analysis had a length of 3,312 amino acids and covered 67.23% (9,936 bp) of the 14,780 bp mitochondrial genome (Supplementary Fig. S1). Partial COX1 sequence data from India referring to F. elongatus (GenBank: JX518952–JX518954, JQ806365) was found to be identical with Fischoederius sp. 1 (Thailand) whereas partial F. elongatus COX1 sequence data recently reported from Thailand (GenBank: MN437486–MN437488) was different to the here reported data and the F. elongatus COX1 data from China and India (Supplementary Fig. S2).