Accepted Manuscript

Title: Characterization of the mitochondrial genomesequences of the liver fluke Amphimerus sp. (Trematoda:Opisthorchiidae) from Ecuador and phylogenetic implications

Authors: Jun Ma, Jun-Jun He, Cheng-Yan Zhou, Miao-MiaoSun, William Cevallos, Hiromu Sugiyama, Xing-Quan Zhu,Manuel Calvopina

PII: S0001-706X(19)30220-7DOI: https://doi.org/10.1016/j.actatropica.2019.04.025Reference: ACTROP 5001

To appear in: Acta Tropica

Received date: 17 February 2019Revised date: 21 April 2019Accepted date: 22 April 2019

Please cite this article as: Ma J, He J-Jun, Zhou C-Yan, Sun M-Miao,Cevallos W, Sugiyama H, Zhu X-Quan, Calvopina M, Characterization of themitochondrial genome sequences of the liver fluke Amphimerus sp. (Trematoda:Opisthorchiidae) from Ecuador and phylogenetic implications, Acta Tropica (2019),https://doi.org/10.1016/j.actatropica.2019.04.025

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Characterization of the mitochondrial genome sequences of the liver fluke

Amphimerus sp. (Trematoda: Opisthorchiidae) from Ecuador and

phylogenetic implications

Jun Maa, Jun-Jun Hea, Cheng-Yan Zhoua, Miao-Miao Suna, William Cevallosb, Hiromu

Sugiyamac, Xing-Quan Zhua,*, Manuel Calvopiñad,*

aState Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary

Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of

Agricultural Sciences, Lanzhou, Gansu Province 730046, PR China

bCarrera de Medicina, Facultad de Ciencias Médicas, Universidad Central del Ecuador, Quito,

Ecuador

cDepartment of Parasitology, National Institute of Infectious Diseases, Tokyo 162-8640,

Japan

dOneHealth Group, Carrera de Medicina, Facultad de Ciencias de la Salud, Universidad de

las Américas (UDLA), Quito, Ecuador

_______________________

*Corresponding authors at: State Key Laboratory of Veterinary Etiological Biology, Lanzhou

Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province

730046, People’s Republic China; OneHealth Group, Carrera de Medicina, Facultad de Ciencias de la

Salud, Universidad de las Américas (UDLA), Quito, Ecuador

E-mail addresses: xingquanzhu1@hotmail.com (X.-Q. Zhu), manuel.calvopina@udla.edu.ec (M.

Calvopiña).

Research Highlights

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The complete mitochondrial (mt) genome sequences of the Ecuadorian Amphimerus were

determined

The Ecuadorian Amphimerus mt genome has similar gene content and arrangement to those

of other Opisthorchiidae

Phylogenetic analysis placed the Ecuadorian Amphimerus within Opisthorchiidae family,

but distinct from Opisthorchis

The Amphimerus mt genome provides a new resource for future systematic studies of

Opisthorchiidae trematodes

ABSTRACT

Amphimerus Barker, 1911 is a liver fluke infecting several animal species and humans. Being

a digenetic trematode of the Opisthorchiidae family, Amphimerus is closely related to the genera

Metorchis, Clonorchis and Opisthorchis. Recently, a high prevalence of Amphimerus infection

in humans, cats, and dogs had been demonstrated in a tropical Pacific region of Ecuador. Hence,

we determined and characterized the entire mt genome sequences of adult liver flukes,

morphologically identified as Amphimerus, collected in the endemic region of Ecuador, and

examined its phylogenetic relationships with flukes in the Opisthorchiidae family using

Bayesian inference (BI) based on the concatenated amino acid sequences and partial cox1

sequences. The complete mt genome sequence (15, 151 bp in length) of the Amphimerus sp.

contains 35 genes, including 12 protein-coding genes (PCGs, without atp8), two rRNAs (rrnL

and rrnS) and 21 tRNAs, lacking trnG. The gene content and arrangement of the Ecuadorian

Amphimerus mt genome was similar to those of other trematodes in the Opisthorchiidae family.

All genes in the circular mt genome of Amphimerus sp. are transcribed from the same strand in

one direction, with the A+T content of 60.77%. Genetic distances between Amphimerus sp. and

other genera in Opisthorchiidae were rather high, ranging from 26.86% to 28.75% at nucleotide

level and 29.37% to 31.12% at amino acid level. Phylogenetic analysis placed the Ecuadorian

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Amphimerus within the branch of Opisthorchiidae, but very distinct from Opisthorchis. Our

results indicate that the liver fluke Amphimerus from Ecuador does not belong to the genus

Opisthorchis, and that it should be assigned under the genus Amphimerus. The determination

of the mt genome of the Ecuadorian Amphimerus provides a new genetic resource for future

studies on taxonomy and molecular epidemiology of Opisthorchiidae trematodes.

Keywords:

Amphimerus

Mitochondrial genome

Mitochondrial DNA

Phylogenetic analysis

Opisthorchiidae

1. Introduction

Clonorchis sinensis, Opisthorchis spp., Metorchis orientalis, and Amphimerus sp. are

platyhelminthes of the Trematoda class and belong to the Opisthorchiidae family, in which the

adult flukes reside within the bile ducts of a definitive host (Kaewpitoon et al., 2008; Calvopiña

et al., 2015; Lun et al., 2015). Infections by these liver flukes can occur through the

consumption of metacercariae in raw or undercooked infected freshwater fish, and are

considered important food-borne diseases and classified as neglected tropical diseases by the

WHO (WHO, 2011).

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Members of the genus Amphimerus Barker, 1911, are etiological agents of zoonotic disease

called amphimeriasis in the tropics (Calvopiña et al., 2011). To date, eleven species have been

reported including A. pseudofelineus, A. caudalitestis, A. pricei, A. lancea, A. parciovatus, A.

pseudofelineus minutus, A. bragai, A. minimus, A. neotropicalis, A. minutus and A. ruparupa

(Calvopiña et al., 2011). In Ecuador, a trematode resembling Amphimerus but identified as

Opisthorchis guayaquilensis was identified (Rodriguez et al., 1949). Amphimerus sp. parasitize

livers and pancreas of birds, reptiles and mammals including cats, dogs, marsupials, dolphins,

rodents and humans in Asia and the Americas (Eom et al., 1984; de Moraes Neto et al., 1998;

Calvopiña et al., 2011; Calvopiña et al., 2015).

The identification of Amphimerus species relies on morphologic features of the adult worms

(Calvopiña et al., 2011; Calvopiña et al., 2015). Given the similarities in morphological

characteristics, there have long been confusion in systematic relationship between certain flukes

in Amphimerus and Opisthorchis genera (Evans, 1963; Calvopiña et al., 2011). The

morphologic differences between adult flukes of Amphimerus, Clonorchis sinensis and

Opisthorchis spp. were reviewed by Calvopiña (Calvopiña et al., 2011), who also pointed out

that the assignment of species names was speculative, and the validity of some species in

Amphimerus was controversial simply based on morphological and morphometric features

(Calvopiña et al., 2015). Besides, a parasite resembling Amphimerus sp. found in the bile ducts

of dogs in Ecuador was described as Opisthorchis guayaquilensis (Rodriguez et al., 1949).

Afterwards, some authors considered this species to be Amphimerus guayaquilensis (de Moraes

Neto et al., 1998). Later, A. guayaquilensis was identified as synonym of A. pseudofelineus

(Artigas and Perez, 1964), while others believed A. guayaquilensis was distinct from A.

pseudofelineus and it was then regarded as a synonym of A. parciovatus (Thatcher, 1970). This

shows that the accuracy of reclassification regarding trematodes in Amphimerus is in dispute

and needs to be solved.

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Molecular identification, using genetic markers, is helpful in clarifying the ambiguities

among trematodes (Ma et al., 2015; Ma et al., 2017), including conspecific species of

Amphimerus (Park, 2007). The aminoacid sequences inferred from mitochondrial (mt) DNA,

as well as complete internal transcribed spacers ribosomal DNA (ITS rDNA) have been proven

useful in exploring the phylogenetic status among trematodes in Digenea (Ma et al., 2015; Ma

et al., 2016a; Ma et al., 2016b; Yang et al., 2016; Ma et al., 2017). However, the available

molecular information of Amphimerus sp. is rather limited until now.

Herein, we determined the entire mt genome sequences of Amphimerus adult worms

collected in Ecuador and reconstructed the phylogenetic relationships with all available mt

genome sequences from parasites in the Opisthorchiidae family, to illustrate its taxonomic

status and to test the hypothesis that Amphimerus from Ecuador does not belong to the genus

Opisthorchis.

2. Materials and Methods

2.1. Sampling and DNA extraction

The study was performed on six adult worms collected in 2010. Two specimens from

humans were collected in feces after praziquantel treatment, two from livers of sacrificed dogs

and cats. All hosts were naturally infected along the Cayapas River of Esmeraldas Province,

Ecuador. Details on collection, preservation and maintenance are given in Calvopiña et al

(2011). Each specimen was washed with normal saline, fixed in 75% (v/v) ethanol and

preserved at -20 °C. Total genomic DNA of each worm was extracted using sodium dodecyl

sulfate (SDS)/proteinase K treatment (Gasser et al., 2007) with column-purification (Wizard®

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SV Genomic DNA Purification System, Promega, Madison, USA), based on the protocol

provided by the manufacturer.

2.2. Amplification and sequencing of ITS rDNA and mt genome

The ITS rDNA region of each sample was amplified using primers BD1 (forward; 5′-GTC

GTA ACA AGG TTT CCG TA-3′) and BD2 (reverse; 5′-ATG CTT AAA TTC AGC GGG T-

3′) (Morgan and Blair, 1995) (spanning partial 18S rDNA, the complete ITS-1, 5.8S rDNA,

ITS-2, and partial 28S rDNA).

Thirteen pairs of primers were used to amplify the entire mt genome of Amphimerus from

one specimen (Additional file 1: Table S1), which were designed based on mtDNA sequences

from Clonorchis sinensis (FJ381664) (Shekhovtsov et al., 2010), M. orientalis (KT239342) (Na

et al., 2016), O. viverrini (JF739555) (Cai et al., 2012) and O. felineus (EU921260)

(Shekhovtsov et al., 2010).

PCR reactions were performed in a total volume of 50 μl, using 25 μl PrimeStar Max DNA

polymerase premix (Takara, Dalian, China), 30 pmol of each primer (synthesized in Genewiz,

Beijing, China), 1.5 μl DNA templates, and 22 μl H2O, in a thermocycler (Biometra, Göttingen,

Germany). PCR conditions for amplifying ITS rDNA were started with a denaturation at 98 °C

for 1.5 min, followed by 35 cycles of denaturation at 92 °C for 20 s, annealing at 55 °C for 20

s and extension at 60 °C for 1 min, with a final extension for 10 min at 72 °C. Long-PCR

conditions for amplifying mt genome sequences were started with a denaturation at 98 °C for

1.5 min, followed by 18 cycles of denaturation at 92 °C for 20 s, annealing at 53–66 °C for 20

s and extension at 62 °C for 2–6 min, followed by 98 °C denaturation for 1 min, plus 18 cycles

of 92 °C for 20 s (denaturation), 55–63 °C for 20 s (annealing) and 66 °C for 2–6 min, with a

final extension step for 10 min at 62 °C. A negative control, using double distilled water and

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without DNA, was included in each amplification run. Positive amplicons were selected and

sent to Genewiz Company (Beijing, China) for sequencing from both directions.

2.3. Assembly, annotation and bioinformatics analysis

The complete mt genome sequences of Amphimerus was assembled manually and aligned

against those of C. sinensis and O. felineus. Gene boundaries and arrangement of 12 protein-

coding genes (PCGs) and two ribosomal RNA genes were determined by comparing with those

of C. sinensis and O. felineus using Clustal X (1.83) (Table 2) (Shekhovtsov et al., 2010).

Amino acid sequences of 12 PCGs were inferred using MEGA v.6.06 or NCBI Trematode

Mitochondrial Code (Ma et al., 2017). The tRNA genes were identified using the programs

tRNAscan-SE (Lowe and Eddy, 1997) or by comparison with those of other opisthorchiid

trematodes (Shekhovtsov et al., 2010).

A comparative analysis of mt sequences in the 12 PCGs, was conducted among Amphimerus

and other four species from Opisthorchioidea at both nucleotide (nt) and amino acid (aa) levels,

including C. sinensis [Opisthorchiidae, Clonorchis, FJ381664, (Shekhovtsov et al., 2010)], O.

felineus [Opisthorchiidae, Opisthorchis, EU921260, (Shekhovtsov et al., 2010)], and O.

viverrini [Opisthorchiidae, Opisthorchis, JF739555, (Shekhovtsov et al., 2010)], M. orientalis

[Opisthorchiidae, Metorchis, KT239342, (Na et al., 2016)] and Haplorchis taichui

[Heterophyidae, Haplorchis, KF214770, (Lee et al., 2013)] (Table 3). The comparison of

sequences in each gene were also performed among those flukes in Opisthorchiidae (Table 4).

The nucleotide composition and skews of each PCG, ribosomal RNA genes, tRNA genes as

well as the non-coding region (NCR) were also summarized (Additional file 2, Table S2).

2.4. Phylogenetic analysis

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The concatenated amino acid sequences of Amphimerus mt genome, conceptually translated

from individual genes, were aligned with those sequences from selected flukes including O.

felineus [Opisthorchiidae, Opisthorchis, EU921260, (Shekhovtsov et al., 2010)], C. sinensis

[Opisthorchiidae, Clonorchis, FJ381664, (Shekhovtsov et al., 2010)], M. orientalis

[Opisthorchiidae, Metorchis, KT239342, (Na et al., 2016)], H. taichui [Heterophyidae,

Haplorchis, KF214770, (Lee et al., 2013)] and Metagonimus yokogawai [Heterophyidae,

Metagonimus, KC330755, (Chen et al., 2016)]. Sequence of Clinostomum complanatum

[Heterophyidae, Clinostomum, NC027082, (Chen et al., 2016)] is chosen as an outgroup.

Sequences of inferred amino acid of these seven trematodes were aligned using MAFFT 7.122.

The poorly aligned regions were removed using Gblocks Server v. 0.91b

(http://molevol.cmima.csic.es/castresana/Gblocks_server.html) using default settings, selecting

the option of less strict conservation of flanking positions. Then the whole alignment was

converted into nexus format and subjected to phylogenetic analysis using Bayesian inference

(BI) using MrBayes 3.1.2, using the mixed model in inferred amino acid matrix (Castoe and

Parkinson, 2006). Four independent Markov chains were run for 10, 000, 000 metropolis-

coupled MCMC generations, sampling trees every 10, 000 generations. The first 250 trees were

discarded as 'burn-in', with the remaining trees used for counting the Bayesian posterior

probabilities (BPP). The analysis was kept running until the calculated average standard

deviation of split frequencies was below 0.01 while the potential scale reduction factors were

close to 1. Phylograms were visualized using FigTree v. 1.42 (Chen et al., 2014).

Although only four species in Opisthorchiidae (M. orientalis, O. felineus, O. viverrini and

C. sinensis) had their complete mt genome sequences decoded; still, a few flukes had their

partial cox1 sequences reported, such as Pseudamphistomum truncatum (KP869078-KP869079)

(Sherrard-Smith et al., 2016), M. xanthosomus (FJ423740 (Shekhovtsov et al., 2009) and

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KT740974 (Sitko et al., 2016)) and O. lobatus (HQ328539) (Aung et al., 2017). Thus, a

truncated alignment of a 268 bp in cox1 was used as a molecular marker for BI phylogenetic

analysis with these parasites, choosing the nucleotide substitution model (4by4) (Ma et al.,

2017), with Markov chains running for 10, 000, 000 generations, sampling trees every 1, 000

generations and discarded first 2, 500 trees. The sequence of Schistosoma japonicum

[KU196417 (Yin et al., 2015), Schistosomatidae, Digenea] served as an outgroup.

3. Results and discussion

3.1. Species confirmation using ITS sequences

The adult worms, considered Amphimerus sp. according to the existing morphological keys

(Calvopiña et al., 2011). The ITS amplicons were all approximately 1,239 bp in length [partial

18S (43 bp), ITS-1 (720 bp), 5.8S (160 bp), ITS-2 (281 bp) and partial 28S (35 bp)]. The

amplified ITS sequences of six specimens were identical. The boundaries of ITS-1 sequences

were ascertained by comparison with those of Amphimerus sp. occurring in Ecuador

(LC440341). However, the sequences were merely released recently, so no references can be

found. Both ITS-1 and ITS-2 sequences of our samples of Amphimerus sp. were 100% identical

to those in LC440341. In the present study, the complete ITS sequence of Amphimerus sp. from

Ecuador was deposited in GenBank database (Accession No. MK238505).

The sequence differences in ITS rDNA between Amphimerus with M. orientalis

(Choudhary and Agrawal, 2017), O. felineus (Sun et al., 2011), O. viverrini (Kang et al., 2008)

and C. sinensis (Tatonova et al., 2017) were compared and summarized (Table 1). The

differences of ITS-1 sequences between Amphimerus and other four flukes from

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Opisthorchiidae ranged from 21.75% - 27.06%, with the sequence differences in ITS-2 ranging

from 11.81% - 16.00%. The comparative results were much higher than those among C. sinensis,

M. orientalis, O. felineus and O. viverrini (5.82% - 10.71% in ITS-1, 5.56% - 8.00% in ITS-2),

demonstrating that trematodes from these three genera may have closer genetic relationship

with each other than with Amphimerus. Meanwhile, among the five parasites, the sequence

differences in ITS-1 were obviously higher than in ITS-2, proving that, comparing to ITS-1, the

sequences in ITS-2 are more conservative and more suitable for the genetic studies of closely

related species.

3.2. Mitochondrial genome content, organization and annotation

The complete mt genome sequence of Amphimerus from Ecuador (GenBank accession No.

MK238506) was 15, 151 bp in length (Fig. 1) which contains 35 genes, including 12 PCGs

(cox1-3, nad1-6, nad4L, atp6 and cytb), two rRNA genes (rrnL and rrnS) and 21 tRNA genes

(Table 1). The gene organization and arrangement of the Amphimerus mt genome was in

accordance with other opisthorchiid parasites, except that trnG was missing. The total A+T

content for Amphimerus mtDNA was 60.77%, which was slightly higher than those of C.

sinensis (FJ381664, 60.15%) (Shekhovtsov et al., 2010), O. felineus (EU921260, 59.85%)

(Shekhovtsov et al., 2010) and Metorchis orientalis (NC028008, 60.60%) (Na et al., 2016). The

A+T content for each gene of Amphimerus mt genome ranged from 47.82% (trnR) to 68.66%

(trnM). The nucleotide composition of Amphimerus mt genome is biased towards T obviously

[15.76% A (2, 388 bp), 27.87% G (4, 223 bp), 45.01% T (6, 820 bp) and 11.35% C (1,720 bp)]

(Additional file 2: Table S2).

The mt genome of Amphimerus from Ecuador encoded 12 PCGs, which could be inferred

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into 3,383 aminoacids in total. In the mt genome of Amphimerus sp., 12 PCGs had ATG (cox3,

cytb, nad4, atp6, nad2, nad3, cox2 and nad6), GTG (in nad4L, nad1 and nad5) and TTG (in

cox1) as start codons, respectively, while TAG was the only stop codon in all PCGs (Table 2).

Non incomplete codons were used in Amphimerus sp. mt genome. The inferred 21 tRNA genes

of Amphimerus sp. mt genome ranged from 56 bp (trnC) to 69 bp (trnR) in length (Table 1),

with their structures similar to those of C. sinensis and O. felineus. More remarkable, trnG in

Amphimerus mt genome could not be detected neither by comparison with sequences from

corresponding trematodes nor by prediction of tRNA secondary structure nor anticodon seeking,

thus the tRNA was presumed to have been deleted. The boundaries of two rRNA genes (rrnL

and rrnS) were determined, with rrnL (986 bp) was located between trnT and trnC, and rrnS

(768 bp) situated between trnC and cox2 (Table 1).

The only NCR in mt genome of Amphimerus sp., 1,702 bp in length, was situated between

trnE and cox3, with the A+T content being 60.05%. The NCR contained six copies of a 270 bp

- complete direct repeat unit, which was located at positions 13, 450 - 15, 080, following by a

71 nt fragment of unique sequence (15, 081-15, 151).

3.3. Comparative analysis of mt genomes among trematodes in the Opisthorchioidea

superfamily

The genetic differences of 12 protein coding regions (PCGs) of mt genomes of

representative species in Opisthorchioidea (Amphimerus, C. sinensis, M. orientalis, O. felineus

O. viverrini, and H. taichui) were compared at both amino acid and nucleotide levels (Table 3).

The sequence differences between Amphimerus sp. and other four flukes in Opisthorchiidae (C.

sinensis, M. orientalis, O. viverrini and O. felineus) ranged from 26.86% to 28.75% in nt and

ranged from 29.35% to 31.12% in aa. These were higher than those among these four flukes

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(18.98% - 21.81% in nt, 15.81%-18.91% in aa), further indicating that these four species might

have closer genetic relationships with each other than with Amphimerus. The results of

sequence comparison also showed that Amphimerus from Ecuador had the similar genetic

differences with parasites from Opisthorchis, Metorchis and Clonorchis.

The sequence differences among five parasites in Opisthorchiidae (18.98% - 28.75% in nt,

15.87% - 31.12% in aa) were clearly lower than those with H. taichui (Heterophyidae:

Opisthorchioidea) (30.44% - 34.04% in nt, 31.01% - 37.54% in aa), clearly demonstrated that

the sequence differences among species in Opisthorchiidae (intergeneric differences, maybe) is

lower than those between different families within superfamily Opisthorchioidea (interfamily).

It is noteworthy that the sequence differences between O. felineus and O. viverrini (21.65%

in nt, 18.91% in aa) is similar to those of intergeneric differences among Opisthorchis,

Metorchis and Clonorchis (18.98% - 21.81% in nt, 15.81% - 18.91% in aa). It was reported in

previous study that the genus Opisthorchis is paraphyletic (Dao et al., 2017). Although we can

tell from the comparison in ITS (Table 1) that these two opisthorchiid flukes had rather low

sequence differences (5.16% in ITS-1 and 5.84% in ITS-2).

Each gene or region of the Amphimerus sp. mt genome was compared with those from mt

genomes of C. sinensis, O. felineus, O. viverrini and M. orientalis (Table 4). At both nucleotide

and amino acid levels, the sequence comparisons among five mt genomes showed that cytb (nt:

21.51% - 23.03%, aa: 16.94% - 20.49%), cox1 (nt: 22.14% - 23.10%, aa: 17.15% - 18.88%),

cox2 (nt: 20.81% - 23.96%, aa: 18.40% - 24.30%) and nad1 (nt: 20.71% - 23.48%, aa: 18.67%

- 24.33%) were rather conservative genes. Large differences in sequences were detected in cox3

(nt: 36.00% - 38.62%, aa: 47.44% - 49.30%), nad6 (nt: 27.92% - 32.47%, aa: 26.14% - 30.07%)

and nad2 (nt: 27.55% - 31.24%, aa: 34.26% - 37.02%) (Table 2).

Differences in nucleotide sequences between Amphimerus sp. and four flukes in

Opisthorchiidae ranged from 20.48% to 24.42% in rrnL, and from 24.74% to 26.56% in rrnS,

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demonstrating that rrnL is more conserved than rrnS in Opisthorchiidae. Owing to the deletion

of trnG and the presence of only one NCR in mt genome of Amphimerus, the comparison did

not include NCR and tRNAs.

3.4. Phylogenetic analysis

The phylogeny of eight trematode species was reconstructed based on the concatenated

amino acid sequences of the 12 mitochondrial PCGs (Fig. 2). Clonorchis sinensis, O. felineus,

O. viverrini, M. orientalis and Amphimerus sp. were clustered into the group of Opisthorchiidae,

with Amphimerus sp. related to O. felineus based on present phylogenetic analysis. Among

these five trematodes, O. felineus and O. viverrini were not grouped together. According to this

evolutionary tree, Opisthorchis viverrini was closely related to M. orientalis, then C. sinensis

joined the group, with the node receiving the nodal value of 0.96. Then O. felineus was clustered

with these species, which was consistent with results of previous studies (Le et al., 2016; Na et

al., 2016). Results of the present BI tree support the hypothesis that the genus Opisthorchis

could be paraphyletic (Dao et al., 2017). Members of the family Heterophyidae (M. yokogawai

and H. taichui), in combination with parasites in Opisthorchiidae, represented the superfamily

Opisthorchioidea, which were well separated from the outgroup parasite in Clinostomidae. The

systematic status of the selected flukes were in concordance with those of previous studies (Lee

et al., 2004; Lee et al., 2013; Yang et al., 2015; Chen et al., 2016; Le et al., 2016; Liu et al.,

2016; Ma et al., 2016b; Na et al., 2016; Ma et al., 2017). Each node was supported by nodal

value with BPP > 0.91.

The evolutionary tree of selected trematodes from five genera based on partial cox1

sequences (Fig. 3) clearly showed that Amphimerus was neither grouped with species in the

Opisthorchis genus, nor with any flukes from Metorchis, Clonorchis or Pseudamphistomum,

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indicating that Amphimerus sp. from Ecuador did not belong to the genus Opisthorchis. The

nodal value received marginally support (BPP>0.8). All the other trematodes had the same

phylogenetic relationship as showed in previous reports (Shekhovtsov et al., 2009; Sherrard-

Smith et al., 2016; Sitko et al., 2016; Aung et al., 2017). However, the cox1 alignment was only

268 bp and could not represent the entire cox1 gene. Therefore, the phylogenetic analysis based

on partial cox1 sequences serves as a supporting material suggesting Ecuadorian Amphimerus

distinct from Opisthorchis.

Though Amphimerus sp. was related to O. felineus in Fig. 2 (BPP=1) and to the branch of

O. viverrini and O. lobatus in Fig. 3 (BPP=1) based on the phylogenetic analysis results in the

present study, it did not suggest that Amphimerus sp. belonged to the genus Opisthorchis. There

were only five trematodes with their decoded mt genome sequences available in GenBank.

Further studies are needed to assess the validity of the genus Amphimerus by providing more

detailed analyses of mtDNA sequences from a broader variety of taxa within Opisthorchiidae

family, which could shed more light on the interrelationships of this highly diverse major

digenean lineage.

4. Conclusions

The present study determined the complete ITS rDNA and mt genome sequences of

Amphimerus from Ecuador. The results of comparative sequence analyses showed that

Amphimerus from Ecuador has high sequence differences with flukes from other three genera

in the Opisthorchiidae (Metorchis, Clonorchis and Opisthorchis). The phylogenetic analysis

indicated that Amphimerus from Ecuador does not belong to the genus Opisthorchis, and that

it should be assigned under the genus Amphimerus. Future studies are required to better

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understand the phylogenetics and systematics of trematodes in Amphimerus by decoding more

mt genomes of the species in the superfamily Opisthorchioidea. The availability of the

Amphimerus mt genome provides useful genetic markers for future studies on taxonomy,

systematics and epidemiology of Amphimerus of humans and animals.

Competing interests

The authors declare that they have no conflicts of interest.

Acknowledgements

This study was funded in part by the grants from the Universidad Central del Ecuador (No.

CUP 91750000.0000.374072), the JSPS KAKENHI Grants (Nos. 25305011 and 16H05820),

the National Natural Science Foundation of China (Grant No. 31702225) and the International

Science and Technology Cooperation Project of Gansu Provincial Key Research and

Development Program (Grant No. 17JR7WA031).

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Fig. 1 Organization of the mitochondrial (mt) genome of the Ecuadorian Amphimerus. The

scales are approximate. All genes are transcribed in the clockwise direction, using standard

nomenclature. “NCR” refers to the non-coding region in the mt genome of the Ecuadorian

Amphimerus. The A+T content is shown for each gene or region of the mt genome and

represented by colour.

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Fig. 2 Phylogenetic relationships of the Ecuadorian Amphimerus and selected trematodes based

on mt amino acid sequences. Tree was inferred from the concatenated amino acid sequence

dataset for 12 protein-coding genes using Bayesian inference (BI), choosing Clinostomum

complanatum (Heterophyidae, Clinostomum, NC027082) as the outgroup.

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Fig. 3 Phylogenetic relationships of the Ecuadorian Amphimerus and selected flukes based on

partial cox1 nucleotides sequences. Tree was conducted using Bayesian inference (BI), with

Schistosoma japonicum (AM689524, Schistosomatidae, Digenea) serving as the outgroup.

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Table 1

Sequence differences in the internal transcribed spacers (ITS-1 and ITS-2) of ribosomal DNA

among five trematodes of the family Opisthorchiidae.

CS MO OF OV

ITS-1 ITS-2 ITS-1 ITS-2 ITS-1 ITS-2 ITS-1 ITS-2

Nos. MF319654 HM347227 EU038134 EF688142 EU038147 MG797539

AMP 25.33 16.00 27.06 11.81 21.75 13.75 23.19 12.85

CS 10.71 8.00 8.52 7.95 8.76 8.00

MO 5.82 6.87 8.05 5.56

OF 5.16 5.84

CS: Clonorchis sinensis; MO: Metorchis orientalis; OF: Opisthorchis felineus; OV: Opisthorchis viverrini;

Nos.: GenBank Accession Numbers; AMP: Amphimerus sp. All values in the table are given as

percentages.

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Table 2

The features of the mitochondrial genome of the Ecuadorian Amphimerus.

Gene

Coding position

(5′–3′)

Length

(bp)

Numbers of

amino acids

Initial and ending

codons

Anticodon

Intergenic

nucleotides

cox3 1-648 648 215 ATG/TAG 11

trnH 660-724 65 GTG 17

cytb 742-1857 1116 371 ATG/TAG 12

nad4L 1870-2133 264 87 GTG/TAG -40

nad4 2094-3380 1287 428 ATG/TAG 1

trnQ 3382-3443 62 TTG 11

trnF 3455-3519 65 GAA 0

trnM 3520-3586 67 CAT 1

atp6 3588-4103 516 171 ATG/TAG 35

nad2 4139-5008 870 289 ATG/TAG 3

trnV 5012-5077 66 TAC 10

trnA 5088-5154 67 TGC 5

trnD 5160-5219 60 GTC 0

nad1 5220-6122 903 300 GTG/TAG 10

trnN 6133-6200 68 GTT 3

trnP 6204-6268 65 TGG -1

trnI 6268-6331 64 GAT 15

trnK 6347-6411 65 CTT 3

nad3 6415-6771 357 118 ATG/TAG 9

trnS1 6781-6840 60 GCT 12

trnW 6853-6916 64 TCA -3

cox1 6914-8458 1545 514 TTG/TAG 0

trnT 8459-8522 64 TGT 0

rrnL 8523-9508 986 0

trnC 9509-9564 56 GCA 0

rrnS 9565-10332 768 0

cox2 10333-10971 639 212 ATG/TAG 22

nad6 10994-11455 462 153 ATG/TAG 10

trnY 11466-11528 63 GTA 1

trnL1 11530-11592 63 TAG -3

trnS2 11590-11655 66 TGA 12

trnL2 11668-11732 65 TAA 9

trnR 11742-11810 69 TCG 0

nad5 11810-13387 1578 525 GTG/TAG -1

trnE 13387-13449 63 TTC 0

LNCR 13450-15151 1702 0

NCR: Non-coding region

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Table 3

Sequence differences (%) in the 12 mitochondrial protein-coding regions among flukes from

Opisthorchioidea.

Species Opisthorchioidea

Opisthorchiidae Heterophyidae

CS (nt/aa) MO (nt/aa) OF (nt/aa) OV (nt/aa) HT (nt/aa)

AMP 28.24/30.43 26.86 / 29.58 27.22 / 29.37 28.75 / 31.12 30.44 / 31.01

CS 20.21 / 17.37 18.98 / 15.87 21.81 / 18.75 33.74 / 37.37

MO 19.54 / 17.20 19.08 / 17.42 33.00 / 36.80

OF 21.65 / 18.91 33.93 / 37.06

OV 34.04 / 37.54

nt: nucleotide; aa: amino acid; AMP: Amphimerus sp.; CS: Clonorchis sinensis; MO: Metorchis orientalis;

OF: Opisthorchis felineus; OV: Opisthorchis viverrini; HT: Haplorchis taichui.

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Table 4

Differences in mitochondrial nucleotides and predicted amino acids sequences among five

species within the family Opisthorchiidae.

Gene Nucleotide identity (%) Amino acid identity (%)

AMP/CS AMP/MO AMP/OF AMP/OV AMP/CS AMP/MO AMP/OF AMP/OV

cox3 38.62 37.75 36.00 37.90 49.30 49.30 49.30 47.44

cytb 21.68 21.51 22.52 23.03 17.79 18.06 16.94 20.49

nad4L 26.52 23.48 25.38 25.00 19.54 16.09 19.54 18.39

nad4 29.06 27.54 28.05 30.77 31.47 30.61 30.61 32.24

atp6 27.52 26.55 25.39 25.97 26.74 32.75 30.99 30.99

nad2 31.24 28.97 27.55 30.92 37.02 34.26 35.29 37.02

nad1 20.71 22.81 21.15 23.48 22.33 23.00 18.67 24.33

nad3 24.09 24.65 24.93 25.77 27.97 28.81 27.12 29.66

cox1 23.10 22.96 22.61 22.14 18.88 17.15 17.88 18.30

cox2 23.00 20.81 21.28 23.96 22.64 22.17 18.40 24.30

nad6 32.47 27.92 31.39 31.60 30.07 26.14 30.07 29.41

nad5 37.86 33.48 35.85 39.03 29.92 29.05 28.75 30.59

rrnL 21.39 21.36 20.48 24.42

rrnS 25.38 25.76 24.74 26.56

12 PCGs 28.24 26.86 27.22 28.75 30.43 29.58 29.37 31.12

aa: amino acid; nt: nucleotide; AMP: Amphimerus sp.; CS: Clonorchis sinensis; MO: Metorchis

orientalis; OF: Opisthorchis felineus; OV: Opisthorchis viverrini.

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Supplementary material

Additional file 1: Table S1. Sequences of primers used to amplify fragments of the Ecuadorian

Amphimerus mitochondrial genome.

Additional file 2: Table S2. Nucleotide composition and skews of the Ecuadorian Amphimerus

mitochondrial genes.

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