Moniezia benedeni and Moniezia expansa are distinct cestode species based on complete mitochondrial genomes

Accepted Manuscript Title: Moniezia benedeni and Moniezia expansa are distinct cestode species based on complete mitochondrial genomes Author: Aijiang Guo PII: DOI: Reference:

S0001-706X(16)30576-9 http://dx.doi.org/doi:10.1016/j.actatropica.2016.11.032 ACTROP 4125

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Please cite this article as: Guo, Aijiang, Moniezia benedeni and Moniezia expansa are distinct cestode species based on complete mitochondrial genomes.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.11.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Moniezia benedeni and Moniezia expansa are distinct cestode species based on complete mitochondrial genomes

Aijiang Guoa,b* Given name: Aijiang; Family name: Guo a

State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of

Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, Gansu Province, People’s Republic of China b

Jiangsu Co-innovation Center for Prevention and Control of Important Animal

Infectious Diseases and Zoonoses, Yangzhou 225009, Jiangsu Province, People’s Republic of China

*Corresponding author at: Lanzhou Veterinary Research Institute, CAAS, Xujiaping 1, Yanchangbu, Lanzhou 730046, Gansu, China. E-mail addresses: [email protected]

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Graphical abstract

Highlights   

Mitochondrial genomes of Moniezia benedeni and Moniezia expansa were sequenced. Phylogenetic analyses indicated that they had clear genetic distinctiveness. The concept for the existence of cryptic species in the two species was discussed.

ABSTRACT Moniezia spp. parasitize the intestines of ruminants, causing monieziasis. In this study, the complete mitochondrial (mt) genomes of M. benedeni and M. expansa have been determined, characterized and employed to test the hypothesis that M. benedeni and M. expansa are distinct species by phylogenetic analysis based on the concatenated amino acid sequences derived from 12 protein-coding genes, inferred with Bayesian and Maximum-likelihood methods. The complete mt genomes of M. benedeni and M. expansa were 13,958 bp and 13,934 bp in size, respectively. Nucleotide sequence identity between the two mt genomes was 83.4%. Each of the two circular mt genomes encodes 36 genes including two ribosomal RNA genes, 22 transfer RNA 2

genes and 12 protein-coding genes, which are transcribed from the same direction. The gene orders of the two mt genomes are identical to those of Anoplocephala spp. (Anoplocephalidae), Hymenolepis spp. (Hymenolepididae) and Dipylidium caninum (Dipylidiidae), but distinct from the species of the Taeniidae family. Phylogenetic analysis confirmed that M. benedeni and M. expansa are taxonomically valid species and have a sister relationship, regardless of the analytical method employed. Furthermore, comparing the cox1 gene sequences of Moniezia spp. from the NCBI deposited sequences and the ones obtained in the present study revealed that the nucleotide sequence differences were 12.5% for M. benedeni and 6.2 % for M. expansa, respectively, suggesting the existence of cryptic species in these parasites. The complete mt genome sequences reported herein will be valuable in further studies of diagnoses, molecular ecology and population genetics of Moniezia spp. of socio-economic importance.

Abbreviations: mt, mitochondrial; nad1-6, NADH dehydrogenase subunit 1-6; cox1-3, cytochrome c oxidase subunit 1-3; cytb, cytochrome b; atp6, ATPase subunit 6; trn, transfer RNA; rrnL, large subunit ribosomal RNA; rrnS, small subunit ribosomal RNA; Mb, Moniezia benedeni; Me, M. expansa; Am, Anoplocephala magna; Ap, Anoplocephala perfoliata

Keywords: Moniezia benedeni; M. expansa; Mitochondrial genome; Phylogeny

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1. Introduction Moniezia species are the most common intestinal tapeworms in ruminants, with a worldwide distribution (Jyoti et al., 2014; Diop et al., 2015). The completion of the life cycle of Moniezia spp. requires oribatids as the intermediate hosts and ruminants as the definitive hosts. Infection of the latter hosts is initiated by ingesting the infected mites containing Moniezia cystercoids (Diop et al., 2015). The larvae develop to adult worms in the small intestine of ruminants and are responsible for the onset of monieziasis, resulting in gastrointestinal disorders (Diop et al., 2015), causing significant economic losses to the global buffalo and sheep industries (Cedillo et al., 2015; Prchal et al., 2015). In spite of the importance of these parasites, little is known about their ecology, evolutionary biology or population genetics. So far, at least 12 Moniezia species have been described in domestic and wild ruminants based on their relatively narrow set of morphological features (Ohtori et al., 2015), which are often convergent, causing controversy about the taxonomy of this genus (Diop et al., 2015). Based on the shapes of their interproglottidal glands and eggs, Moniezia benedeni and M. expansa (2-4 m) have been identified as distinct species (Diop et al., 2015; Ohtori et al., 2015) with the former species having a short continuous linear-pattern of interproglottidal glands and the latter species possessing a row of small circles-pattern of the interproglottidal glands. In addition, eggs present as tetragons for M. benedeni and triangles for M. expansa, respectively. However, if the worms lack the interproglottidal glands and sometimes the eggs are altered in shape, then morphological identification became unreliable (Diop et al., 2015; Ohtori et al., 2015). 4

Interesting, a study using multilocus enzyme electrophoresis reported the existence of cryptic species in M. benedeni (Chilton et al., 2007), further questing the value of only using morphological characters in identifying and differentiating Moniezia cestode species. As the limitation of using morphological characters in species identification of Moniezia cestodes, DNA sequence data (ITS1 and 5.8S) (Nguyen et al., 2012; Ohtori et al., 2015) have been used recently as genetic markers to distinguish these two species. An increasing number of complete mitochondrial (mt) genome sequences provides the opportunity to optimize the choice of molecular markers for studies of ecology, evolutionary biology and population genetics (von Nickisch-Rosenegk et al., 2001; Jeon et al., 2005; Littlewood et al., 2006; Jeon et al., 2007; Jia et al., 2010). The complete mt genomes provide individual markers with different levels of sequence variation as well as combined mtDNA molecular markers for within- and between-species studies (Zarowiecki et al., 2007). The metazoan genome sequences published to date usually contain 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes (Littlewood et al., 2006) with the triploblastic animals lacking the ATPase 8-coding gene in their mt genomes (von Nickisch-Rosenegk et al., 2001). All of these genes are transcribed from the same strand for the flatworms, nematodes and annelids. All other mtDNAs have these genes distributed either in the same strand or in both strands (von Nickisch-Rosenegk et al., 2001). The mt genomes of cestodes are similar to those of other eumetazoans regarding gene composition, length and the structures of their

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rRNA and tRNA molecules (Jeon et al., 2007). Currently, 42 complete mt genome sequences of cestode species in the order Cyclophyllidea are available in GenBank. In the family Anoplocephalidae, however, only two mt genome sequences from the genus Anoplocephala (Anoplocephala perfoliata and A. magna) are available (Guo, 2015; Guo, 2016b). In the present study, the complete mt genome sequences for M. benedeni and M. expansa have been determined and used to confirm that M. benedeni and M. expansa are distinct species. Additionally, the amino acid sequences derived from the 12 protein-coding genes for the M. benedeni and M. expansa mt genomes were used together with those of the published cestode species to assess the phylogeny of the genus Moniezia among major families in the order Cyclophyllidea.

2. Materials and methods 2.1. Parasites and genomic DNA extraction Moniezia benedeni and M. expansa tapeworms were taken from the feces of buffalo calves treated with anthelmintic albendazole in the Guangxi Autonomous Region, China. The tapeworms were washed with 0.75% NaCl and identified as M. benedeni and M. expansa based on their morphological characters of interproglottidal glands and eggs (Diop et al., 2015; Ohtori et al., 2015). Genomic DNA was extracted from the proglottides of individual tapeworms using a Genomic DNA Purification Kit (AXYGEN, USA) according to the manufacturer’s instructions. The study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute,

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Chinese Academy of Agricultural Sciences (No. LVRIAEC2010-002). 2.2. Long-PCR amplification and sequencing The complete mt genomes of M. benedeni and M. expansa were amplified by polymerase chain reaction (PCR) in three overlapping fragments with three pairs of primers (Additional file 1: Table S1) using genomic DNA extracted from a section of individual worms as the template. The primers were the same as those used in amplifying the mt genomes of other tapeworms (Jia et al., 2010; Guo, 2015; Guo, 2016a), which were designed based on the conserved regions from the available complete mtDNA sequences of cestodes in GenBank. PCR reactions were performed in a 50 μL total volume containing 25 μL 2×LA Taq Premix (Takara Biotechnology Co. Ltd., Dalian, China), 2 μM Primer of each primer, and 40 ng of genomic DNA. PCR cycle conditions were: 94˚C for 5 min (initial denaturation), 94˚C for 30 s, 50˚C for 30 s, 35 cycles at 68˚C for 8 min, and a final extension at 68˚C for 10 min. PCR products were examined by agarose gel electrophoresis and purified using a DNA Gel Extraction Kit (QIAquick PCR purification kit; QIAGEN, USA). Purified PCR products were sent to Sangon Company (Shanghai, China) for sequencing in both directions using the primer walking strategy. 2.3. Sequence analyses The complete mt genome sequences of M. benedeni and M. expansa were assembled manually and aligned against that of A. perfoliata using Clustal X 1.83 to identify gene boundaries. The protein-coding regions were identified using BLAST 7

software, ORF Finder software and compared with other sequences of cestodes available in the GenBank database. Gene sequences were translated into amino acid sequences with the genetic code set for Flatworm (Translation Table 9). Nucleotide composition (%) of the complete mt genomes and pairwise distance (%) of the homologous amino acid sequences among M. benedeni, M. expansa, A. perfoliata and A. magna were calculated using Lasergene. Putative transfer RNA (trn) genes were identified using tRNAscan-SE. Then trn genes were confirmed further by inferring their potential secondary structures and recognizing anticodon sequences by visual inspection. The two ribosomal RNA genes (rrn) for M. benedeni and M. expansa mtDNA were identified by sequence comparisons with those of previously published sequences from other cestodes. Palindromes and inverted sequences in the non-coding regions were detected using Einverted and Palindrome in the EMBOSS software (Rice et al., 2000). Secondary structures were predicted by Mfold software (Zuker, 2003). Tandem repetitive elements were identified using Tandem Repeats Finder (Benson, 1999). 2.4. Phylogenetic analyses Phylogenetic trees were constructed based on a total of 44 mtDNA genome sequences from 44 cestode species, including M. benedeni and M. expansa identified, two from the family Anoplocephalidae, twenty-nine from the family Taeniidae, one from the family Dipylidiidae, four from the family Hymenolepididae, six from Pseudophyllidea, and the mt genome from Schistosoma japonicum (Trematode) was

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used as an outgroup. The accession numbers of these 44 mt genome sequences were provided in Table S2. Inferred amino acid sequences of the 12 protein-coding genes were aligned by MAFFT 7.122 and poorly aligned positions were excluded using Gblocks server (Talavera and Castresana, 2007) using the option for a less stringent selection. Phylogenetic analyses for the concatenated dataset of amino acid sequences were conducted using Bayesian Inference (BI) and Maximum likelihood (ML) methods. BI analysis was performed using MrBayes 3.2 (Ronquist et al., 2012) with mtZoa model (rates = gamma, ngammacat = 5), as proposed by Rota-Stabelli et al. (Rota-Stabelli et al., 2009), and run for 5,000,000 generations and sampled every 1,000 generations with four chains. The first 25% of the trees were discarded as burn-in and the Bayesian posterior probability was calculated for the remaining tree. ML analysis was performed in Treefinder October using a model of MtArt+I+G, the best fit model selected from ProtTest software based on the Akaike information criterion. Bootstrap support was determined using 100 bootstrap replicates.

3. Results 3.1. General features of M. benedeni and M. expansa The complete mt genome sequences for M. benedeni and M. expansa were 13,958 bp and 13,934 bp in length, respectively, obtained by combination of the three assembled fragments and were deposited in GenBank (accession nos. KX121040 and KX121041). Each of the two circular mt genomes contains 36 genes, including 12 protein-coding genes (atp6, cox1-3, cytb, nad1-6 and nad4L), 22 transfer RNA genes

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(trn), 2 ribosomal RNA genes (rrnS and rrnL), and two non-coding regions (NC) (Fig. 1), similar to those of other cestodes. All genes are encoded in the same strand and transcribed from the same direction. The gene orders of these two mt genomes are identical to those of Anoplocephala spp. (Anoplocephalidae), Hymenolepis spp. (Hymenolepididae) and Dipylidium caninum (Dipylidiidae), but different from those of members of the Taeniidae owing to the position change between the tRNA (L1) and tRNA (S2) genes. The A+T content were 71.35% and 70.46% for mt genomes of M. benedeni and M. expansa, respectively, and these high A+T contents are consistent with those of other cestodes. There is 83.4% sequence identity between the entire mtDNA sequences of the two species. The complete mt genomes of M. benedeni and M. expansa have 793 bp and 785 bp of intergenic sequences, ranging from 1 bp to 288 bp and from 1 bp to 363 bp, respectively. 3.2. Protein-coding genes The mt genome of M. benedeni or M. expansa encodes 12 protein-coding genes (Table 1). A total of 3,352 and 3,348 amino acids are encoded in the mt genomes of M. benedeni and M. expansa, respectively. Comparing the lengths of each of the protein-coding genes between the two species, 7 of 12 protein-coding genes have different lengths (Table 1), including cox1-3, nad6, cytb, atp6 and nad2. The 12 protein-coding genes in the two mt genomes use ATG, GTG or ATT as their initiation codons, and use TAG, TAA or T as termination codons (Table 1). Comparison of each of the protein-coding genes between M. benedeni and M. expansa revealed the

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nucleotide sequence identities range from 75.9% to 87.2% (Table 2). The amino acid sequences deduced from individual mt protein-coding genes were compared and their identities ranged from 70.4% to 91.5% (Table 2), where cox1 is the most conserved protein and atp6 is the least conserved protein. 3.3. Transfer RNA genes and ribosomal RNA genes In the mt genomes of M. benedeni and M. expansa, the 22 tRNA genes ranged in size from 60 bp to 72 bp and from 61 bp to 75 bp (Table 1), respectively. The secondary structures predicted for the 22 tRNAs were similar to the corresponding mt tRNA genes from A. perfoliata and A. magna, respectively, where 18 of the 22 tRNAs were predicted to form standard secondary structures, and the remaining four tRNAs (trnS1(AGN), trnS2(UCA), trnC, and trnR) were predicted to have lost the DHU arms. Of the ribosomal RNA genes in the two mt genomes, rrnS was located between trnC and cox2, and rrnL was located between trnT and trnC. The lengths of rrnS in the M. benedeni and M. expansa mt genomes were 731 bp and 722 bp, respectively, and the lengths of rrnL are 967 bp and 968 bp, respectively (Table 1). Nucleotide identity was determined for rrnS (91.7%) and rrnL (88.8%), suggesting that rrnS is more conserved than rrnL. 3.4. Non-coding regions In the mt genomes of M. benedeni and M. expansa, there are two non-coding regions designated NC1 and NC2. In M. benedeni mt genome, NC1 (197 bp) was found between trnY and trnS2 and NC2 (288 bp) was located between nad5 and trnG. 11

In the mt genome of M. expansa, NC1 and NC2 were found to be 219 bp and 363 bp in length, respectively, and they are at the same relative location as those in the mt genome of M. benedeni (Table 1). The NCs show stem-loop structures and contain tandem repeats. In the M. benedeni mt genome, NC1 was predicted to form a stable secondary structure containing 28 canonical base pairs for the stem. Additionally, a tandem repeat element was also found. NC2 is mainly composed of seven 32 bp repeat elements and the remaining NC2 sequences which were predicted to form a stable potential secondary structure containing seven canonical base pairs for the stem. In the M. expansa mt genome, NC2 is mainly composed of nine 32 bp repeat elements and the remaining NC2 sequences which were predicted to form a stable potential secondary structure containing 26 canonical base pairs for the stem and a loop of 16 bases in length. Previous studies demonstrated that the stem-loop structures and repeat regions in the intergenic regions of the mt genomes of metazoans represent signals for transcription initiation and replication (Wolstenholme, 1992). 3.5. Phylogenetic analysis The tree inferred from the concatenated amino acid sequence dataset for the 12 mt proteins is shown in Fig. 2. The phylogenetic tree showed that the sister relationships between the Anoplocephalidae and Hymenolepididae, and between the Taeniidae and Dipylidiidae, are supported. Analyses also revealed that species of the genus Anoplocephala is close to the species of the genus Moniezia, where they formed a monophyletic sister group with strong bootstrap support. Meanwhile, the

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tree shows that M. benedeni and M. expansa are valid species, where they have clear genetic distinctiveness with maximum nodal support. The phylogenetic relationships of the selected taeniids are consistent with previous studies, where the Taeniidae consists of four valid genera and maintains monophyletic clade within the order Cyclophyllidea (Nakao et al., 2013).

4. Discussion Moniezia spp. are common parasites which affect ruminants, including cattle, sheep, goats and other species (Prchal et al., 2015). Currently, at least 12 species have been reported (Ohtori et al., 2015). Additionally, Chilton et al. demonstrated the existence of cryptic species of M. benedeni in Australia (Chilton et al., 2007). Therefore, it is important to study the genetic variation among Moniezia spp. for the reevaluation of conventional taxonomy and for the establishment of reliable identification methodologies. The mitochondrial genomes provide a useful source of molecular markers for investigating phylogenetic and ecological studies among distantly related taxa or closely related species, including taxonomy at the species level (Le et al., 2002). Comparative analysis of mt genomes has also been widely used to study phylogeny and molecular ecology (Jeon et al., 2007; Zarowiecki et al., 2007). In the present study, the complete mt genome sequences of M. benedeni and M. expansa were identified and comparative analyses between the two genomes were carried out. Nucleotide sequence variations were determined between the homologous mt genes and non-coding regions from the two species. The results support the

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hypothesis that M. benedeni and M. expansa represent distinct taxa. Some cox1 gene sequences for both M. benedeni and M. expansa are available in the GenBank database deposited from previous studies (Diop et al., 2015; Ohtori et al., 2015). These sequences were obtained from Moniezia spp. in Senegal, where M. benedeni species were collected from cattle and M. expansa species were collected from sheep/goats (Diop et al., 2015; Ohtori et al., 2015). Nucleotide sequence differences in cox1 gene region between the deposited cox1 sequences and the cox1 sequences obtained in the present study were at least 12.5% for M. benedeni and 6.2% for M. expansa, which are higher than within-species level variation (Diop et al., 2015; Ohtori et al., 2015). The high sequence variation in M. benedeni and M. expansa suggests the existence of cryptic species in these parasites. As such, phylogenetic and ecological studies of Moniezia species require further studies based on a more extensive taxonomic sampling of Moniezia spp. from different hosts and locations. Phylogenetic analyses using the concatenated amino acid sequences provided further evidence that M. benedeni and M. expansa represent close but distinct taxa, in accordance with the hypothesis based on morphology estimates of the sister group status between them. Furthermore, this study confirmed the taxonomical validity of the genus Anoplocephala and Moniezia. The sister-group relationships between the Anoplocephalidae and Hymenolepididae, and between the Taeniidae and Dipylidiidae, are in accordance with the studies using Cyclophyllidean mitochondrial 12S rDNA by the maximum-parsimony or ML method (von Nickisch-Rosenegk et al., 1999) and with studies using 18S rDNA gene sequences by the NJ distance-based method

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(Taleb-Hossenkhan and Bhagwant, 2012). These are also consistent with what has been observed in an early morphological study (Hoberg et al., 1999). The sister-group relationship of the Anoplocephalidae and Hymenolepididae obtained here is different from what is observed using 12S rDNA by the Fitch-Margoliash method (von Nickisch-Rosenegk et al., 1999), where the Anoplocephalidae is phylogenetically closer to the Taeniidae than to the Hymenolepididae. In conclusion, this study identified and compared the complete mt genome sequences of M. benedeni and M. expansa. Phylogenetic analysis using the concatenated amino acid sequences confirmed that M. benedeni and M. expansa represent close but distinct taxa and provided further support that Anoplocephala spp. and Moniezia spp. have a sister-relationship. Additionally, the data support the opinion for the existence of cryptic species in M. benedeni and M. expansa. The complete mt genomes of these two species will be useful not only for inferring the phylogenetic relationships among cestodes, but also for identifying suitable molecular markers for further studies regarding diagnostics, population genetics and molecular ecology in Moniezia species.

Acknowledgements This work was supported by the “National Key Basic Research Program (973 Program) of China” (Grant No. 2015CB150300). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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from cattle, goats and sheep in central Vietnam. J Helminthol 86, 426-9. Ohtori, M., Aoki, M. and Itagaki, T., 2015. Sequence differences in the internal transcribed spacer 1 and 5.8S ribosomal RNA among three Moniezia species isolated from ruminants in Japan. J Vet Med Sci 77, 105-7. Prchal, L., Bartikova, H., Becanova, A., Jirasko, R., Vokral, I., Stuchlikova, L., Skalova, L., Kubicek, V., Lamka, J., Trejtnar, F. and Szotakova, B., 2015. Biotransformation of anthelmintics and the activity of drug-metabolizing enzymes in the tapeworm Moniezia expansa. Parasitology 142, 648-59. Rice, P., Longden, I. and Bleasby, A., 2000. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16, 276-7. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Hohna, S., Larget, B., Liu, L., Suchard, M.A. and Huelsenbeck, J.P., 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61, 539-42. Rota-Stabelli, O., Yang, Z. and Telford, M.J., 2009. MtZoa: a general mitochondrial amino acid substitutions model for animal evolutionary studies. Mol Phylogenet Evol 52, 268-72. Talavera, G. and Castresana, J., 2007. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56, 564-77. Taleb-Hossenkhan, N. and Bhagwant, S., 2012. Molecular characterization of the parasitic tapeworm Bertiella studeri from the island of Mauritius. Parasitol Res 110, 759-68. von Nickisch-Rosenegk, M., Brown, W.M. and Boore, J.L., 2001. Complete sequence of the mitochondrial genome of the tapeworm Hymenolepis diminuta: gene arrangements indicate that Platyhelminths are Eutrochozoans. Mol Biol Evol 18, 721-30. von Nickisch-Rosenegk, M., Lucius, R. and Loos-Frank, B., 1999. Contributions to the phylogeny of the Cyclophyllidea (Cestoda) inferred from mitochondrial 12S rDNA. J Mol Evol 48, 586-96. Wolstenholme, D.R., 1992. Animal mitochondrial DNA: structure and evolution. Int Rev Cytol 141, 173-216. Zarowiecki, M.Z., Huyse, T. and Littlewood, D.T., 2007. Making the most of mitochondrial genomes--markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes: Digenea). Int J Parasitol 37, 1401-18. Zuker, M., 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31, 3406-15.

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Fig. 1. Structures of the mitochondrial genomes of Moniezia benedeni and M. expansa. The complete mitochondrial genomes of the two species are circular DNA molecules, which consist of 36 genes. All genes are encoded by the same strand and transcribed from the same direction. The arrow indicates transcriptional orientation. NC refers to non-coding region. Twenty-two tRNA genes are designated with one letter amino acid codes. Gene scaling is approximate.

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Fig.

2.

Phylogenetic

Hymenolepididae

and

relationships Taeniidae.

among

Anoplocephalidae,

Phylogenetic

tree

Dipylidiidae,

constructed

by

the

maximum-likelihood method based on a concatenated amino acid sequence of 12

protein-coding genes for 44 cestode mitochondrial genomes is shown. Sequence from Schistosoma japonicum (Trematode) is used as outgroup. The bootstrap values are 19

shown close to the branches. Accession numbers for the 44 mitochondrial genomes are available in Table S2.

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Table 1 Sequence information for the complete mitochondrial genomes of Moniezia benedeni (Mb) and M. expansa (Me). Genes

Positions

Size (bp)

Initiation codons

Termination codons

cox1 tRNA-Thr (T) rrnL tRNA-Cys (C) rrnS cox2 tRNA-Glu (E) nad6 tRNA-Tyr (Y) Non-coding region (NC1) tRNA-SerUCN (S2) tRNA-LeuCUN (L1) tRNA-LeuUUR (L2) tRNA-Arg (R) nad5 Non-coding region (NC2) tRNA-Gly (G) cox3 tRNA-His (H) cytb nad4L nad4 tRNA-Gln (Q) tRNA-Phe (F) tRNA-Met (M) atp6 nad2 tRNA-Val (V) tRNA-Ala (A) tRNA-Asp (D) nad1 tRNA-Asn (N) tRNA-Pro (P) tRNA-Ile (I) tRNA-Lys (K) nad3 tRNA-SerAGN (S1) tRNA-Trp (W)

Mb/Me

Mb/Me

Mb/Me

Mb/Me

1-1611/1-1612 1586-1651/1591-1654 1652-2618/1655-2622 2619-2678/2623-2685 2679-3409/2686-3407 3410-3985/3408-3986 3991-4056/3993-4054 4061-4519/4059-4511 4567-4638/4543-4617 4639-4835/4618-4836

1611/1612 66/64 967/968 60/63 731/722 576/579 66/62 459/453 72/75 197/219

ATG/ATG – – – – ATG/ATG – ATG/ATG – –

TAG/T – – – – TAG/TAA – TAA/TAA – –

4836-4896/4837-4897 4992-5059/4924-4991 5072-5138/5021-5088 5188-5248/5099-5159 5250-6827/5164-6741 6828-7115/6742-7104

61/61 68/68 67/68 61/61 1578/1578 288/363

– – – – ATG/ATG –

– – – – TAA/TAA –

7116-7182/7105-7167 7186-7839/7171-7821 7830-7895/7814-7886 7900-8997/7887-8981 9005-9265/8994-9254 9232-10479/9221-10468 10481-10542/10470-10531 10543-10603/10532-10592 10604-10674/10593-10660 10675-11181/10661-11176 11204-12079/11194-12057 12082-12145/12060-12123 12151-12216/12129-12194 12223-12285/12201-12261 12289-13179/12266-13156 13204-13274/13184-13253 13283-13345/13260-13329 13346-13406/13330-13390 13411-13473/13393-13455 13477-13824/13459-13806 13825-13890/13805-13865 13891-13955/13866-13931

67/63 654/651 66/73 1098/1095 261/261 1248/1248 62/62 61/61 71/68 507/516 876/864 64/64 66/66 63/61 891/891 71/70 63/70 61/61 63/63 348/348 66/61 65/66

– ATG/ATG – ATG/ATG ATG/ATG ATG/ATG – – – ATG/ATG ATG/ATT – – – GTG/ATG – – – – ATG/ATG – –

– TAG/TAG – TAG/TAG TAG/TAA TAG/TAG – – – TAA/TAG TAG/TAG – – – TAA/TAG – – – – TAG/TAG – –

21

Table 2 Identities in the mitochondrial nucleotide and deduced amino acid (aa) sequences of protein-coding and two ribosomal RNA genes among species for Moniezia benedeni (Mb), M. expansa (Me), Anoplocephala magna (Am) and Anoplocephala perfoliata (Ap). Gene/region Nucleotide length (bp) Mb

Me Ap

Nucleotide identity (%)

Number of aa

aa identity (%)

Am Mb/ Mb/ Mb/ Me/ Me/ Ap/ Mb Me Ap Am Mb/ Mb/ Mb/ Me/ Me/ Ap/ Me

Ap

Am Ap Am Am

Me

Ap

Am Ap

Am Am

atp6

507

516 516 516 75.9 67.9 69.2 65.7 67.4 85.1 169 171

171 171 70.4 59.8 59.8 59.9 61.6 89.5

nad1

891

891 891 891 86.3 76.2 74.7 76.4 75.0 85.3 296 296

296 296 87.9 73.7 73.4 74.4 74.1 89.6

nad2

876

864 876 876 83.0 77.9 77.4 74.3 75.5 88.5 291 287

291 291 80.2 71.1 71.1 66.9 67.2 88.4

nad3

348

348 348 348 83.0 74.1 76.4 76.4 76.4 85.3 115 115

115 115 77.6 69.8 69.8 75.0 69.8 83.6

nad4

1248 1248 1248 1248 80.7 69.8 71.8 69.4 70.0 84.9 415 415

415 415 79.3 65.1 64.7 65.4 64.7 88.7

nad4l

261

86 86 87.4 71.3 77.0 75.9 73.6 83.9

nad5

1578 1578 1581 1581 80.2 68.7 67.4 67.9 66.9 84.0 525 525

526 526 75.3 59.0 59.4 60.6 61.3 85.0

nad6

459

453 459 459 78.4 68.9 69.6 69.3 70.0 85.2 152 150

152 152 72.8 55.6 57.5 58.3 59.6 85.0

cox1

1591 1594 1593 1590 87.2 76.7 77.9 77.8 77.6 88.4 536 537

530 529 91.5 78.3 78.3 79.7 79.4 92.1

cox2

576

579 576 576 84.5 78.3 77.4 76.0 77.2 88.9 191 192

191 191 87.5 77.6 78.1 78.1 78.6 93.2

cox3

654

651 644 644 81.3 69.3 70.7 70.8 70.8 85.2 217 216

214 214 77.0 61.8 64.5 65.9 66.4 86.0

cytb

1098 1095 1101 1101 82.6 73.4 73.1 71.4 71.0 84.9 365 364

366 366 82.7 65.8 66.7 67.7 68.5 85.6

rrnS

731

–

–

–

–

–

–

–

–

–

rrnL

967 968

–

–

–

–

–

–

–

–

–

261 261 261 84.3 77.4 79.3 74.3 74.7 87.0 86

722 724 724 91.7 77.8 79.4 78.7 80.1 91.8 – 981 973 88.8 77.8 76.4 77.8 76.6 90.8 –

22

86