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Open Access

The complete mitochondrial DNA of three monozoic tapeworms in the Caryophyllidea: a mitogenomic perspective on the phylogeny of eucestodes

  • Wen X. Li1,
  • Dong Zhang1, 2,
  • Kellyanne Boyce3,
  • Bing W. Xi4,
  • Hong Zou1,
  • Shan G. Wu1,
  • Ming Li1 and
  • Gui T. Wang1Email author
Parasites & Vectors201710:314

https://doi.org/10.1186/s13071-017-2245-y

Received: 6 April 2017

Accepted: 12 June 2017

Published: 27 June 2017

Abstract

Background

External segmentation and internal proglottization are important evolutionary characters of the Eucestoda. The monozoic caryophyllideans are considered the earliest diverging eucestodes based on partial mitochondrial genes and nuclear rDNA sequences, yet, there are currently no complete mitogenomes available. We have therefore sequenced the complete mitogenomes of three caryophyllideans, as well as the polyzoic Schyzocotyle acheilognathi, explored the phylogenetic relationships of eucestodes and compared the gene arrangements between unsegmented and segmented cestodes.

Results

The circular mitogenome of Atractolytocestus huronensis was 15,130 bp, the longest sequence of all the available cestodes, 14,620 bp for Khawia sinensis, 14,011 bp for Breviscolex orientalis and 14,046 bp for Schyzocotyle acheilognathi. The A-T content of the three caryophyllideans was found to be lower than any other published mitogenome. Highly repetitive regions were detected among the non-coding regions (NCRs) of the four cestode species. The evolutionary relationship determined between the five orders (Caryophyllidea, Diphyllobothriidea, Bothriocephalidea, Proteocephalidea and Cyclophyllidea) is consistent with that expected from morphology and the large fragments of mtDNA when reconstructed using all 36 genes. Examination of the 54 mitogenomes from these five orders, revealed a unique arrangement for each order except for the Cyclophyllidea which had two types that were identical to that of the Diphyllobothriidea and the Proteocephalidea. When comparing gene order between the unsegmented and segmented cestodes, the segmented cestodes were found to have the lower similarities due to a long distance transposition event. All rearrangement events between the four arrangement categories took place at the junction of rrnS-tRNA Arg (P1) where NCRs are common.

Conclusions

Highly repetitive regions are detected among NCRs of the four cestode species. A long distance transposition event is inferred between the unsegmented and segmented cestodes. Gene arrangements of Taeniidae and the rest of the families in the Cyclophyllidea are found be identical to those of the sister order Proteocephalidea and the relatively basal order Diphyllobothriidea, respectively.

Keywords

MitogenomeCaryophyllidean tapewormParasitic PlatyhelminthesProglottizationSegmentation

Background

Scolex type, external segmentation and internal proglottization are all important evolutionary characters of the Cestoda. The Amphilinidea and Gyrocotylidea (Cestodaria) that do not possess a scolex are early divergent lineages in this class. Tapeworms of the order Caryophyllidea (Platyhelminthes: Eucestoda) are typified by a monozoic body (neither internal proglottization nor external segmentation). The Spathebothriidea are polyzoic but externally unsegmented, and all other eucestodes demonstrate classic proglottization (segmented body parts each with a set of reproductive organs). Morphological analysis shows the Caryophyllidea to be the earliest divergent lineage of Eucestoda [1] although phylogenetic analysis based on LSU rDNA and SSU rDNA have indicated that the Spathebothriidea may be the earliest diverging eucestodes [2, 3]. However, recently, topology constructed using large fragments of mtDNA supports the Caryophyllidea as the most primitive eucestodes [4]. These results indicate the Caryophyllidea to be a key group for studying evolutionary relationships within the Eucestoda as well as with other parasitic Monogenea, Aspidogastrea and Digenea.

Owing to its maternal inheritance, a lack of recombination and a fast rate of evolution [5], the haploid mitochondrial genome has proven to be a useful marker for population studies, species identification and phylogenetics [6, 7]. Its genome-level characteristics, gene arrangements and the positions of mobile genetic elements also enable it to be a powerful tool for reconstructing evolutionary relationships [810]. Using gene sequences and gene arrangements from the complete mt genome, the phylogenies of some parasitic Platyhelminthes have been reconstructed [1113]. However, due to a paucity of complete mt genomic information from these groups, very few parasitic flatworms have been included in these phylogenetic analyses. From the 16 orders of cestodes that exist, only four (Diphyllobothriidea, Bothriocephalidea, Proteocephalidea and Cyclophyllidea) are currently represented in the GenBank database, and as the ancestral taxa of the Eucestoda, no complete mitogenome from the Caryophyllidea has been sequenced.

Khawia sinensis Hsü, 1935, and Atractolytocestus huronensis Anthony, 1958, belong to the family Lytocestidae and are very common caryophyllideans in the intestine of the common carp (Cyprinus carpio). Both invasive tapeworms have a worldwide distribution and are translocated with the introduction of the common carp into countries around the world [14, 15]. Breviscolex orientalis Kulakovskaya, 1962, the only member of the family Capingentidae, is typically recorded in the cyprinids Hemibarbus barbus [16]. In addition, the Asian fish tapeworm Schyzocotyle acheilognathi (syn. Bothriocephalus acheilognathi), a segmented tapeworm of the Bothriocephallidea, is also an invasive parasite found worldwide.

This study has therefore generated the complete mitogenomes of three caryophyllideans, in addition to the Asian fish tapeworm in order to analyse the phylogenetic relationships of eucestodes and the differences in the gene arrangement between unsegmented and segmented eucestodes.

Methods

Specimen collection and DNA extraction

The following cestodes, K. sinensis and A. huronensis from the common carp (Cyprinus carpio), B. orientalis from Hemibarbus maculates and S. acheilognathi from the grass carp (Ctenopharyngodon idella), were collected from a fishery (29°59′10.47″N, 115°47′37″E) in Hubei Province, China. The parasites were preserved in 80% ethanol and stored at 4 °C. Specimens were stained with carmine and identified morphologically using the scolex and testis [16]. Total genomic DNA was extracted from the posterior region of a single tapeworm using a TIANamp Micro DNA Kit (Tiangen Biotech, Beijing, China), according to the manufacturer’s instructions. DNA was stored at -20 °C for subsequent molecular analysis. The morphological identification of specimens was verified by sequence analysis of the complete ITS1 rDNA region [17] and partial sequence of cox1 gene [18].

PCR and DNA sequencing

Partial sequences of the mtDNA from the four cestodes were initially amplified by PCR using degenerate primers (Additional file 1: Table S1). Using these fragments, specific primers were designed for subsequent PCR amplification (Additional file 1: Table S1). PCR reactions were conducted in a 20 μl reaction mixture, containing 7.4 μl molecular grade water, 10 μl 2 × PCR buffer (Mg2+, dNTP plus, Takara, Dalian, China), 0.6 μl of each primer, 0.4 μl rTaq polymerase (250 U/μl, Takara), and 1 μl DNA template. Amplification was performed under the following conditions: initial denaturation at 98 °C for 2 min, followed by 40 cycles at 98 °C for 10 s, 48–60 °C for 15 s, 68 °C for 1 min/kb, and a final extension at 68 °C for 10 min. PCR products were sequenced bidirectionally at Sangon Company (Shanghai, China) using the primer walking strategy.

Sequence analyses

The complete mt sequences were assembled manually and aligned against the mitogenome sequences of other published cestodes using the program MAFFT 7.149 [19] to determine the gene boundaries. Protein-coding genes (PCGs) were inferred with the help of BLASTX [20] and SeqBuilder module in the Lasergene7 software package (DNASTAR), employing the genetic code 9, the echinoderm and flatworm mitochondrial. The majority of tRNAs were identified by comparing the results of tRNAscan-SE [21], ARWEN [22], MITOs [23] and DOGMA [24]. However, tRNA Phe and tRNA Gln from B. orientalis and tRNA Gln from A. huronensis were visually compared with the sequences from other cestodes. The location of the two ribosomal RNA genes, rrnL and rrnS, were explored through alignment with other available mt cestodes sequences, and their ends were assumed to extend to the boundaries of their flanking genes. The 5′ end of the rrnL gene in S. acheilognathi however, was determined by the result of alignments. MitoTool [25], a home-made program, was primarily used to parse the annotated mt genome into a Word document format, and generate *.sqn file for GenBank submission and a *.csv file for Table 1. Mitotool was furthermore employed to unify the name of all 36 genes (12 PCGs, 2 rRNAs and 22 tRNAs) and locate all NCR positions (setting threshold of 50 bp) within the mitogenomes of the selected cestodes. Finally, the fasta file containing the nucleotide sequences and gene order for all 36 genes (12 PCGs, 2 rRNAs and 22 tRNAs) was extracted from the GenBank files, processed and used to generate Additional file 2: Table S2 and Additional file 3: Table S3. Repetitive regions within the NCRs were found using a local version of a Tandem Repeats Finder [26]. The alignments located in highly repetitive regions (HRRs) were shaded and labelled using TEXshade software [27]. The secondary structure of each consensus repeat unit was predicted by Mfold software [28], and codon usage and relative synonymous codon usage (RSCU) were computed with MEGA 5 [29]. CREx program [30] was then utilised to calculate the rearrangement events and to conduct pairwise comparisons of gene orders from all of the cestodes using common intervals measurement.
Table 1

The annotated mitochondrial genome of the four cestodes

Gene

Position

Size

Intergenic nucleotides

Codon

Anti-codon

Gene

Position

Size

Intergenic nucleotides

Codon

Anti-codon

From

To

Start

Stop

From

To

Start

Stop

(A) Atractolytocestus huronensis

(B) Breviscolex orientalis

cox3

1

643

643

 

ATG

T

  

1

643

643

 

ATG

T

 

tRNA-His(H)

644

705

62

   

GTG

 

644

707

64

   

GTG

cytb

707

1792

1086

1

ATG

TAA

  

708

1793

1086

 

ATG

TAA

 

nad4L

1792

2052

261

-1

ATG

TAG

  

1793

2053

261

-1

ATG

TAA

 

nad4

2013

3245

1233

-40

ATG

TAG

  

2014

3246

1233

-40

ATG

TAG

 

tRNA-Gln(Q)

3247

3307

61

1

  

TTG

 

3247

3313

67

   

TTG

tRNA-Phe(F)

3304

3367

64

-4

  

GAA

 

3306

3369

64

-8

  

GAA

tRNA-Met(M)

3364

3425

62

-4

  

CAT

 

3364

3424

61

-6

  

CAT

atp6

3427

3942

516

1

ATG

TAA

  

3427

3942

516

2

ATG

TAG

 

nad2

3943

4818

876

 

GTG

TAG

  

3942

4814

873

-1

GTG

TAG

 

tRNA-Val (V)

4819

4879

61

   

TAC

 

4817

4876

60

2

  

TAC

tRNA-Ala (A)

4878

4938

61

-2

  

TGC

 

4875

4936

62

-2

  

TGC

tRNA-Asp(D)

4942

5002

61

3

  

GTC

 

4940

5002

63

3

  

GTC

nad1

5003

5896

894

 

ATG

TAG

  

5005

5898

894

2

ATG

TAG

 

tRNA-Asn(N)

5896

5959

64

-1

  

GTT

 

5898

5960

63

-1

  

GTT

tRNA-Pro(P)

5962

6021

60

2

  

TGG

 

5963

6024

62

2

  

TGG

tRNA-Ile(I)

6021

6084

64

-1

  

GAT

 

6024

6087

64

-1

  

GAT

tRNA-Lys(K)

6085

6143

59

   

CTT

 

6088

6147

60

   

CTT

nad3

6144

6491

348

 

GTG

TAG

  

6148

6498

351

 

GTG

TAG

 

tRNA-Ser(S1)

6489

6545

57

-3

  

GCT

 

6497

6552

56

-2

  

GCT

tRNA-Trp(W)

6547

6608

62

1

  

TCA

 

6555

6618

64

2

  

TCA

cox1

6613

8166

1554

4

ATG

TAG

  

6625

8166

1542

6

ATG

TAG

 

tRNA-Thr(T)

8157

8217

61

-10

  

TGT

 

8157

8219

63

-10

  

TGT

16S

8218

9171

954

     

8220

9166

947

    

tRNA-Cys(C)

9172

9230

59

   

GCA

 

9167

9230

64

   

GCA

12S

9231

9931

701

     

9231

9937

707

    

tRNA-Leu(L1)

9932

9995

64

   

TAG

 

9938

10,002

65

   

TAG

tRNA-Ser(S2)

9998

10,059

62

2

  

TGA

 

10,009

10,072

64

6

  

TGA

tRNA-Leu(L2)

10,060

10,123

64

   

TAA

 

10,075

10,136

62

2

  

TAA

NCR1

10,124

10,996

873

     

10,137

10,344

208

    

cox2

10,997

11,569

573

 

GTG

TAA

  

10,345

10,920

576

 

ATG

TAG

 

tRNA-Glu(E)

11,570

11,640

71

   

TTC

 

10,921

10,987

67

   

TTC

nad6

11,641

12,099

459

 

GTG

TAG

  

10,988

11,446

459

 

GTG

TAG

 

tRNA-Tyr(Y)

12,106

12,171

66

6

  

GTA

 

11,454

11,517

64

7

  

GTA

tRNA-Arg(R)

12,173

12,226

54

1

  

TCG

 

11,519

11,574

56

1

  

TCG

nad5

12,227

13,783

1557

 

GTG

TAG

  

11,577

13,124

1548

2

GTG

TAA

 

tRNA-Gly(G)

13,784

13,847

64

   

TCC

 

13,124

13,186

63

-1

  

TCC

NCR2

13,848

15,130

1283

     

13,187

14,011

825

    

(C) Khawia sinensis

(D) Schyzocotyle acheilognathi (CN)

cox3

1

637

637

 

ATG

T

  

1

655

655

 

ATG

T

 

tRNA-His(H)

638

699

62

   

GTG

 

656

730

75

   

GTG

cytb

701

1822

1122

1

ATG

TAA

  

734

1831

1098

3

ATG

TAG

 

nad4L

1804

2064

261

-19

ATG

TAG

  

1833

2093

261

1

GTG

TAG

 

nad4

2025

3257

1233

-40

ATG

TAA

  

2054

3304

1251

-40

GTG

TAG

 

tRNA-Gln(Q)

3258

3318

61

   

TTG

 

3304

3367

64

-1

  

TTG

tRNA-Phe(F)

3315

3378

64

-4

  

GAA

 

3363

3426

64

-5

  

GAA

tRNA-Met(M)

3374

3436

63

-5

  

CAT

 

3423

3486

64

-4

  

CAT

atp6

3440

3955

516

3

ATG

TAA

  

3490

4005

516

3

ATG

TAG

 

nad2

3960

4832

873

4

ATG

TAG

  

4006

4878

873

 

ATG

TAG

 

tRNA-Val (V)

4835

4894

60

2

  

TAC

 

4883

4948

66

4

  

TAC

tRNA-Ala (A)

4893

4954

62

-2

  

TGC

 

4958

5019

62

9

  

TGC

tRNA-Asp(D)

4960

5024

65

5

  

GTC

 

5025

5087

63

5

  

GTC

nad1

5025

5918

894

 

ATG

TAG

  

5092

5985

894

4

ATG

TAA

 

tRNA-Asn(N)

5918

5984

67

-1

  

GTT

 

5991

6055

65

5

  

GTT

tRNA-Pro(P)

5988

6047

60

3

  

TGG

 

6060

6122

63

4

  

TGG

tRNA-Ile(I)

6047

6110

64

-1

  

GAT

 

6128

6193

66

5

  

GAT

tRNA-Lys(K)

6117

6177

61

6

  

CTT

 

6198

6259

62

4

  

CTT

nad3

6178

6523

346

 

ATG

T

  

6264

6608

345

4

ATG

TAA

 

tRNA-Ser(S1)

6524

6578

55

   

TCT

 

6607

6665

59

-2

  

GCT

tRNA-Trp(W)

6579

6641

63

   

TCA

 

6666

6729

64

   

TCA

cox1

6646

8196

1551

4

ATG

TAG

  

6742

8328

1587

12

ATG

TAG

 

tRNA-Thr(T)

8187

8247

61

-10

  

TGT

 

8342

8404

63

13

  

TGT

        

NCR1

8405

8528

124

    

16S

8248

9193

946

     

8529

9494

966

    

tRNA-Cys(C)

9194

9251

58

   

GCA

 

9495

9555

61

   

GCA

12S

9252

9960

709

     

9556

10,285

730

    

tRNA-Leu(L1)

9961

10,023

63

   

TAG

cox2

10,286

10,858

573

 

ATG

TAA

 

tRNA-Ser(S2)

10,025

10,087

63

1

  

TGA

E

10,862

10,924

63

3

  

TTC

tRNA-Leu(L2)

10,089

10,150

62

1

  

TAA

nad6

10,928

11,383

456

3

ATG

TAA

 

NCR1

10,151

10,699

549

    

L1

11,402

11,465

64

18

  

TAG

cox2

10,700

11,273

574

 

ATG

T

 

L2

11,468

11,531

64

2

  

TAA

tRNA-Glu(E)

11,272

11,332

61

-2

  

TTC

Y

11,539

11,602

64

7

  

GTA

nad6

11,333

11,791

459

 

ATG

TAA

 

S2

11,620

11,685

66

17

  

TGA

tRNA-Tyr(Y)

11,797

11,859

63

5

  

GTA

NCR2

11,686

11,851

166

    

tRNA-Arg(R)

11,872

11,925

54

12

  

TCG

 

11,852

11,909

58

   

TCG

nad5

11,926

13,476

1551

 

ATG

TAA

  

11,913

13,478

1566

3

ATG

TAA

 

tRNA-Gly(G)

13,476

13,537

62

-1

  

TCC

 

13,484

13,547

64

5

  

TCC

NCR2

13,538

14,620

1083

    

NCR3

13,548

14,046

499

    

Phylogenetic analyses

Phylogenetic analysis was carried out using the mitogenomes generated from the four cestodes as part of this study as well as those of the 50 cestodes available from GenBank (Additional file 2: Table S2). Two trematodes, Dicrocoelium chinensis (NC_025279) and Dicrocoelium dendriticum (NC_025280), were used as outgroups. Another program written in-house, BioSuite [31], was employed to align all of the genes in batches using integrated MAFFT, wherein codon-alignment mode was used for the 12 PCGs, and normal alignment mode for the remaining genes (2 rRNAs and 22 tRNAs). The alignments were then concatenated to generate well-supported Phylip and nexus format files for use in the phylogenetic analysis software. Both the maximum likelihood (ML) and Bayesian inference (BI) were used to reconstruct phylogenetic trees, and selection of the most appropriate evolutionary models for the dataset was carried out using ModelGenerator v0.8527 [32]. Based on the Akaike information criterion, GTR + I + G was chosen as the optimal model for nucleotide evolution. ML analysis was performed by RaxML GUI [33] using an ML + rapid bootstrap algorithm with 1000 replicates. BI analysis was performed in MrBayes 3.2.1 [34] with default settings and 1 × 107 Metropolis-coupled MCMC generations. The tree was then annotated using iTOL (a web-based tool) [35] with the help of several dataset files generated by MitoTool.

Results

Genome organisation and base composition

The mitogenomes of A. huronensis (GenBank accession number: KY486754), B. orientalis (KY486752), K. sinensis (KY486753) and S. acheilognathi (CN) (KX589243) are circular double-stranded DNA molecules. The size of these mitogenomes was 15,130 bp in A. huronensis, 14,620 bp in K. sinensis, 14,011 bp in B. orientalis, and 14,046 bp in S. acheilognathi (CN) (Fig. 1). The mitogenome of A. huronensis was the largest of all those available for cestodes (Additional file 2: Table S2, Fig. 2). The length of the S. acheilognathi (CN) mitogenome was about 140 bp longer than previously published due to the presence of a longer NCR between nad5 and cox3 [36]. Similar to other flatworm mitogenomes [11], which lacked the atp8 gene, and encoded all the genes on the same strand, all of those generated in this study contained the standard 36 elements: 12 PCGs (atp6, cytb, cox1–3, nad1–6 and nad4L), 22 tRNA genes and two rRNA genes (Fig. 1). Intriguingly, A-T content of the three Caryophyllidea species (K. sinensis, A. huronensis and B. orientalis) was the lowest of all published cestode mitogenomes (Fig. 2).
Fig. 1

Map of the mitochondrial genomes of Atractolytocestus huronensis, Breviscolex orientalis, Khawia sinensis and Schyzocotyle acheilognathi (China, CN). The 12 protein-coding genes (PCGs), 22 tRNA and two rRNA genes are depicted as well as the non-coding regions (NCRs)

Fig. 2

Maximum-likelihood tree inferred from 36 genes (12 protein-coding genes, 2 rRNAs and 22 tRNAs) of mitochondrial genomes of 54 cestode species from five orders, using two trematoda species as outgroups. Scale-bar represents the estimated number of substitutions per site. Bootstrap/posterior probability support values of ML/BI analysis are shown above the nodes. The bar graph (corresponding to tip labels in the tree) of the mitogenome length and A-T content are shown on the right of the tree

Protein-coding genes and codon usage

The size of the 12 PGCs ranged from 258 bp (nad4L) to 1554 bp (nad5) for the three caryophyllideans, but from 258 bp (nad4L) to 1584 bp (cox1) for S. acheilognathi (CN) (Additional file 3: Table S3). Only two types of start codons (ATG and GTG) were inferred from the sequence data of the four cestodes. GTG was used as a start codon for the following genes: nad2, nad3, cox2, nad5 and nad6 in A. huronensis, nad2, nad3, nad5 and nad6 in B. orientalis and nad4, nad4L in S. acheilognathi (CN). The rest of the PCGs of the aforementioned cestodes and all of the PCGs of K. sinensis used ATG as a start codon. From the three predicted stop codons, TAG, TAA and the abbreviated stop codon T, TAG was the most frequently occurring stop codon, followed by TAA and finally T. The unusual stop codon T encoded for cox3 in A. huronensis, B. orientalis and S. acheilognathi (CN) and cox2, cox3 and nad3 in K. sinensis (Table 1). RSCU for the four cestode mtDNAs calculated using the echinoderm mt genetic code are presented in Additional file 4: Figure S1. Overall, the three most commonly used T-rich codons for the three Caryophyllidea cestodes (A. huronensis, B. orientalis and K. sinensis) were Val (GTT), Leu (TTG) and Phe (TTT) compared with Tyr (TAT), Leu (TTG) and Phe (TTT) for S. acheilognathi (CN).

Transfer and ribosomal RNA genes

All 22 tRNAs from the mt genome of each Caryophyllidea species were concatenated. This created a total concatenated length of 1363 bp, 1378 bp, 1354 bp and 1404 bp for A. huronensis, B. orientalis, K. sinensis and S. acheilognathi (CN), respectively (Additional file 3: Table S3). Each tRNA identified from these four species, could be folded into the traditional cloverleaf structure, with the exception of tRNA Ser(AGN) and tRNA Arg in B. orientalis, K. sinensis and S. acheilognathi (CN) and tRNA Ser(AGN) , tRNA Arg and tRNA Cys in A. huronensis, which all lacked DHU arms (Additional file 5: Figure S2). All tRNAs had the standard anti-codons found in flatworms (Table 1), except tRNA Ser(AGN) in K. sinensis which had an anti-codon of TCT. The two ribosomal RNA genes, rrnL and rrnS were flanked by tRNA Thr and cox2 and separated by tRNA Cys . This was identical in all the cestodes for which a mitogenome was available (Additional file 6: Figure S3). The boundary of the rrnL gene for S. acheilognathi (CN) was redefined, being approximately 100 bp shorter than that of previously published mitogenomes. This is due to the difference in defining the boundary (Additional file 7: Figure S4) [36]. Thus, there was an additional 124 bp NCR located between tRNA Thr and rrnL. Additionally, to conduct phylogenetic analysis and linear order comparison (see later), we proposed a reasonable tRNA Gln annotation to a recently reported mitogenome from Testudotaenia sp. WL-2016 (KU761587) based upon alignments with other cestodes.

Non-coding regions

The position of the NCR in all cestodes was identified with a threshold value of 50 bp. The majority of cestodes contained two NCRs, except for Pseudanoplocephala crawfordi [37], Taenia crocutae [38], Taenia solium [39] and S. acheilognathi (CN) all of which had three NCRs, and Hydatigera taeniaeformis which has just one NCR. These NCRs occurred in the junctions of rrnS-tRNA Arg (P1) and nad5-cox3 (P2) (Additional file 6: Figure S3). The length of the major NCRs were 873 bp (NCR1) and 1283 bp (NCR2) in A. huronensis, 549 bp (NCR1) and 1083 bp (NCR2) in K. sinensis, 208 bp (NCR1) and 825 bp (NCR2) in B. orientalis and 124 bp (NCR1), 166 bp (NCR2) and 499 bp (NCR3) in S. acheilognathi (CN). The concatenated size (2156 bp) of all NCRs from A. huronensis was the longest of all the cestodes (Additional file 3: Table S3). Various highly repetitive regions (HRRs) were detected in NCRs from the four cestode species, and the consensus repeats were capable of forming stem loop structures (Fig. 3).
Fig. 3

Highly repetitive regions (HRRs) and their secondary structures of the consensus repeat units in the major non-coding regions (NCRs) of the mitochondrial genomes of Atractolytocestus huronensis (a), Khawia sinensis (b), Breviscolex orientalis (c) and Schyzocotyle acheilognathi (China, CN) (d). Thermodynamic value (dG) is shown under the secondary structure

Phylogeny and gene order

Both phylogenetic trees (BI and ML) demonstrated high statistical support for branch topology, especially on the order level (BP ≥ 85, BPP = 1). Since the two trees had the same topology, only the latter was shown (Fig. 2). The most derived Cyclophyllidea cestodes, together with the Proteocephalidea (represented by Testudotaenia sp. WL-2016), constitute a reciprocal monophyletic group with the Bothriocephalidea. This clade formed a sister-group to the Diphyllobothriidea, and all clades exhibited a sister-group relationship with the basal Caryophyllidea (Fig. 2). Breviscolex orientalis belonging to the family Capingentidae clustered into a well-supported clade with A. huronensis from the family Lytocestidae inferred by a maximum possible nodal support (BP = 100, BPP = 1) which formed a sister-group relationship with another Lytocestidae species, K. sinensis.

Amongst the 54 mitogenomes across the five orders, each order had a unique arrangement except for the Cyclophyllidea which had two types: group 1 (represented by the Taeniidae) was identical to the Diphyllobothriidea, and group 2 (represented by the Hymenolepididae, Anoplocephalidae, Dipylidiidae and Paruterinidae) was identical to the Proteocephalidea. These corresponded to four mt gene arrangement categories: I, Caryophyllidea; II, Diphyllobothriidea and group 1; III, Bothriocephalidea; IV, Proteocephalidea and group 2 (Fig. 4). Pairwise analysis between the four gene arrangement categories indicated similarities (common intervals algorithm) in the gene order between unsegmented and segmented cestodes to be lower than within segmented cestodes (Table 2).
Fig. 4

Rearrangement events predicted by CREx to explain gene order changes among the four mitogenome arrangements categories, Caryophyllidea (I), Diphyllobothriidea and Cyclophyllidea group 2 (II), Bothriocephalidea (III), Proteocephalidea and Cyclophyllidea group 1 (IV). L1, tRNA Leu(CUN) ; L2, tRNA Leu(UUR) , S2, tRNA Ser(UCN) ; E, tRNA Glu ; Y, tRNA Tyr ; TDRL, tandem-duplication-random-loss

Table 2

Pairwise comparisons of mitochondrial DNA gene orders among the four categories of mitogenome arrangements (see Fig. 4)

 

I

II

III

IV

I

1254

   

II

832

1254

  

III

818

992

1254

 

IV

828

1122

996

1254

Scores indicate the similarity between gene orders, where “1254” represents an identical gene order

Discussion

In the phylogenetic analysis employed in this study, the Caryophyllidea was resolved as the sister taxon to all other eucestodes in line with previous studies. Although only five orders of cestodes are included in the phylogenetic analysis, the evolutionary relationships remain consistent with the results generated through morphological examination [1] and sequence data obtained from large fragments of mtDNA [4].

The mitogenome gene order of the cestodes was extremely conservative. Amongst the 54 mitogenomes across the five orders, only four gene arrangement categories were found. With respect to the three types of gene arrangements (II, III and IV) in the segmented cestodes, all the rearrangement operations are acted on the four closely linked tRNA genes (tRNA Leu(CUN) -tRNA Ser(UCN) -tRNA Leu(UUR) -tRNA Tyr ) (Fig. 4). When compared with the category I in the unsegmented cestodes, there probably exists a long distance transposition event (the three tRNA genes tRNA Leu(CUN) -tRNA Ser(UCN) -tRNA Leu(UUR) translocate to the 3′ end of the four genes cox2-tRNA Glu -Nad6-tRNA Tyr ) (Fig. 4), which may be the main cause of the low similarity value. According to the results of CREx program, the gene rearrangements from category II to category III and IV undergo a tandem-duplication-random-loss (TDRL) event and a simple transposition event, respectively. A TDRL event can provide directional information, allowing the inference of the ancestral state from the comparison of only two taxa because reversing the rearrangement would require more than a single operation [40]. Based on this assumption on TDRL event (Fig. 4), category II may be the ancestral state of the two categories II and III. Two categories of mt gene order were also found in the most derived Cyclophyllidea owing to the transposition of two tRNA genes [41]. However, the two types of gene arrangements are identical to those of the sister order Proteocephalidea and the relatively basal order Diphyllobothriidea.

There are perhaps more gene arrangements in other orders of cestodes; however, due to the limited amount of mitogenome data available so far, we can only but speculate. The rearrangement events that have been observed among the four arrangement categories in this study all took place in P1 as mentioned above (Fig. 4), revealing a rearrangement hot spot. Interestingly, P1 is furthermore the position in which one or two NCRs frequently occurred, and in which highly repetitive regions (HRRs) also are found within the NCRs. Whether an association exists between the rearrangement hot spot and the NCRs is something that requires further investigation to ascertain whether they may be important in the evolution of cestodes.

The phylogenetic relationship between B. orientalis and A. huronensis was found to be closer than that of A. huronensis and K. sinensis, which conflicts with classic systematics. On the basis of the paramuscular position of the vitelline follicles, B. orientalis is placed into the family Capingentidae Kulakovskaya, 1962, being the only member of this family found in the Palaearctic region. However, the fibres of the longitudinal musculature are situated mostly in the inner region of the vitelline field or entirely medullary to it, which is similar to the topography present in the Lytocestidae which possess cortically situated vitelline follicles [42]. Breviscolex orientalis has a cuneiform scolex, as do both species of Caryophyllaeides Nybelin, 1922 in the Lytocestidae [16]. These results suggest that the morphological characters of B. orientalis are closer to those of the Lytocestidae. Despite the similar result found in this study, relocation of B. orientalis, the only member of the family Capingentidae, into the family Lytocestidae, needs more molecular support.

Conclusions

Among the four arrangement categories, the rearrangement events are detected in P1 where the NCRs with highly repetitive regions (HRRs) are common. A putative long-distance transposition event is detected between the unsegmented and segmented cestodes. The TDRL event suggests that the mt gene arrangement of the Diphyllobothriidea is the ancestral state relative to Bothriocephalidea. Gene arrangements of the Taeniidae and the rest of the families in the Cyclophyllidea are found to be identical to those of the sister order Proteocephalidea and the relatively basal order Diphyllobothriidea, respectively.

Declarations

Acknowledgements

The authors thank Prof. P. Nie for some suggestions to improve the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (31,272,695, 31,572,658, 31,302,222), the Earmarked Fund for China Agriculture Research System (CARS-46-08) and the major scientific and technological innovation project of Hubei Province (2015ABA045).

Availability of data and materials

The datasets supporting the conclusions of this article are available in the GenBank international nucleotide sequence repository under accession numbers KY486752– KY486754, KX589243.

Authors’ contributions

WXL designed the experiments, performed the analysis and wrote the manuscript. DZ performed the laboratory work and the phylogenetic analysis. KB analysed the data. All authors contributed to the interpretation of the findings. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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Authors’ Affiliations

(1)
Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, and State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences
(2)
University of Chinese Academy of Sciences
(3)
South Devon College University Centre
(4)
Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences

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