Comparative mitogenomics supports synonymy of the genera Ligula and Digramma (Cestoda: Diphyllobothriidae)

Comparative mitogenomics supports synonymy of the genera Ligula and Digramma (Cestoda:... Background: After observing differences in the number of reproductive complexes per proglottid within the genus Ligula, the genus Digramma was erected. However, the validity of Digramma has been previously questioned due to a low variability in the cox1, nad1 and ITS rDNA sequences between the two genera. We undertook a study to greatly increase the amount of sequence data available for resolution of this question by sequencing and characterizing the complete mitogenomes of Digramma interrupta and Ligula intestinalis. Results: The circular mtDNA molecules of Digramma interrupta and Ligula intestinalis are 13,685 bp and 13,621 bp in size, respectively, both comprising 12 PCGs, 22 tRNA genes, two rRNA genes, and two mNCRs. Both mitogenomes exhibit the same gene order and share 92.7% nucleotide identity, compared with 85.8–86.5% to the most closely related genus Dibothriocephalus.Eachgenefrom D. interrupta and L. intestinalis is almost of the same size, and the sequence identity ranges from 87.5% (trnD)to100%(trnH, trnQ and trnV). NCR2 sequences of D. interrupta and L. intestinalis are 249 bp and 183 bp in length, respectively, which contributes to the main difference in length between their complete mitogenomes. A sliding window analysis of the 12 PCGs and two rRNAs indicated nucleotide diversity to be higher in nad5, nad6, nad2, nad4and cox3, whereas the most conserved genes were rrnL and rrnS. Lower sequence identity was also found in nad2, nad4, nad5, nad6and cox3 genes between the two diphyllobothriids. Within the Diphyllobothriidae, phylogenetic analysis indicated Ligula and Digramma to be most closely related to one another, forming a sister group with Dibothriocephalus. Conclusions: Owing to higher nucleotide diversity, the genes nad2, nad4, nad5, nad6and cox3 should be considered optimal candidates to use as molecular markers for population genetics and species identification between the two closely related species. The phylogenetic results in combination with the comparative analysis of the two mitogenomes, consistently support the congeneric status of L. intestinalis and D. interrupta. Keywords: Mitogenome, Eucestoda, Diphyllobothriidea, Digramma, Ligula Background the Diphyllobothriidea and Bothriocephalidea Kuchta, Based upon its paraphyly, differences in the position of the Scholz, Brabec & Bray, 2008 were proposed [1–3]. genital pore, the presence of an external seminal vesicle The order Diphyllobothriidea includes 70 species and the absence of a uterine sac in the Diphyllobothriidea considered valid, classified into 18 genera across three Kuchta, Scholz, Brabec & Bray, 2008, the order Pseudo- families [2, 4]. Adult diphyllobothriideans are found only phyllidea van Beneden in Carus, 1863 was suppressed and in tetrapods, never having been recorded in fish [2], and the plerocercoids of groups such as Spirometra, Diphyllobothrium Cobbold, 1858 (syn. Diplogonoporus Lönnberg, 1892) and Dibothriocephalus Lühe, 1899 (a * Correspondence: gtwang@ihb.ac.cn Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, and recently resurrected genus including some species from State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Diphyllobothrium, i.e. Dib. dendriticus (Nitzsch, 1824), Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, People’s Dib. nihonkaiensis (Yamane, Kamo, Bylund & Wikgren, Republic of China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. cox2 cox2 6K 6K 5K 5K 4K 4K S1 S1 nad3 nad3 3K 3K 2K 2K 1K 1K atp6 atp6 Li et al. Parasites & Vectors (2018) 11:324 Page 2 of 11 1986) and Dib. latus (Linnaeus, 1758) belonging to the markers as a means to differentiate D. interrupta from L. family Diphyllobothriidae are often the principal agents of intestinalis. food-borne cestodosis [5]. Ligula spp. also belong to the Diphyllobothriidae; these species use copepods as first Methods intermediate hosts, freshwater fish as second intermediate Specimen collection and DNA extraction hosts and birds as definitive hosts [6]. Ligula intestinalis Plerocercoids of D. interrupta and L. intestinalis were (Linnaeus, 1758) is a tapeworm of veterinary importance isolated from the body cavity of Carassius auratus worldwide that reduces the fecundity of the cyprinid fishes collected from Liangzi Lake in Hubei Province and by parasitic castration [6, 7] and induces mass mortalities Gymnocypris selincuoensis from Siling Lake, Tibet, of the carp Chanodichthys erythropterus [8]. China, respectively. The tapeworms were preserved in Cholodkovsky [9]erected thegenus Digramma Cholod- 80% ethanol and stored at 4 °C. Total genomic DNA kovsky, 1915 after observing differences in the number of was extracted from the posterior region of a single reproductive complexes contained within each proglottid tapeworm using a TIANamp Micro DNA Kit (Tiangen when studying the genus Ligula Linnaeus, 1758. In China, Biotech, Beijing, China), according to the manufacturer’s Ligula spp. are distributed in the Qinghai-Tibet Plateau instructions. DNA was stored at -20 °C for subsequent with Schizothoracinae fishes serving as the primary second molecular analysis. The morphological identification of intermediate host. Digramma spp. are found across the rest specimens was confirmed using sequence data from the of China where the goldfish Carassius auratus acts as their ITS2 rDNA region [13, 14] and the cox1 gene [11]. common second intermediate host [10]. However, the val- idity of Digramma has been questioned due to a low level of difference between the species of two genera in the cox1, Amplification and DNA sequencing nad1 and ITS rDNA sequences [11–13]. Thus Digramma PCR was carried out as described previously [15, 16], is considered to be synonymous with Ligula [2]. Owing to with minor modifications. Five degenerate primer sets the fact that only one gene and a limited number of isolates (Additional file 1: Table S1) were designed to primarily were included in that study, more sequence data and a amplify partial sequences of the nad5, cytb, nad2, cox1 greater range of taxa from different genera are required for and rrnS genes. The sequenced fragments were subse- a more comprehensive phylogenetic analysis of the Diphyl- quently used to design primers specific for the amplifica- lobothriidea [13]. tion and sequencing of the whole mitogenome. PCR This study, therefore, aimed to sequence and characterize reactions were performed in a 20 μl reaction mixture, the complete mitogenomes of Digramma interrupta and containing 7.4 μl dd PCR grade H O, 10 μl2×PCR 2+ Ligula intestinalis and to perform phylogenetic analysis to buffer (2 mM Mg ,8 μl dNTP plus, Takara, Dalian, investigate whether or not these two diphyllobothriids are China), 0.6 μl of each primer (12.5 μM), 0.4 μlrTaq congeneric using mitogenomic data. Differences within the polymerase (250 U, Takara, Dalian, China), and 1 μl mitochondrial genes were also compared to determine genomic DNA template. Amplification was conducted which genes would be suitable for the design of molecular under the following conditions: initial denaturation at 98 0K 0K 13,621 nt 13 685 nt Ligula intestinalis Digramma interrupta Fig. 1 Circular map of the mitochondrial genome of Digramma interrupta and Ligula intestinalis. Protein-coding genes (12) are shown in red, tRNAs (22) in yellow, rRNAs (2) in green and non-coding regions in grey 13K 13K 12K 12K nad4L nad4L 11K cox3 cox3 11K 10K 10K 9K 9K L2 L2 S2 S2 8K 8K L1 L1 nad6 nad6 7K 7K Li et al. Parasites & Vectors (2018) 11:324 Page 3 of 11 Table 1 Annotated mitochondrial genomes of Digramma interrupta and Ligula intestinalis Gene Position Size (bp) Intergenic Codon Identity Sequence nucleotides (%) From To Start Stop Digramma interrupta/Ligula intestinalis cox3 1/1 643/643 643/643 GTG/GTG T/T 91.29 trnH 644/644 710/710 67/67 100 GAP1 711/711 713/713 3/3 100 gaa/gaa cytb 714/714 1820/1820 1107/1107 ATG/ATG TAA/TAA 93.41 GAP2 1821/1821 1821/1821 1/1 100 c/c nad4L 1822/1822 2082/2082 261/261 ATG/ATG TAA/TAA 94.64 nad4 2043/2043 3293/3293 1251/1251 -40/-40 ATG/ATG TAG/TAG 91.37 trnQ 3293/3293 3357/3357 65/65 -1/-1 100 trnF 3353/3353 3419/3419 67/67 -5/-5 97.01 trnM 3416/3416 3482/3482 67/67 -4/-4 98.51 GAP3 3483/3483 3485/3485 3/3 66.67 gtt/att atp6 3486/3486 3995/3995 510/510 ATG/ATG TAA/TAA 93.33 GAP4 3996/3996 3997/3997 2/2 100 tc/tc nad2 3998/3998 4876/4876 879/879 ATG/ATG TAG/TAG 91.24 GAP5 4877/4877 4877/4877 1/1 100 t/t trnV 4878/4878 4941/4941 64/64 100 GAP6 4942/4942 4949/4949 8/8 87.5 gtcttaag/gttttaag trnA 4950/4950 5010/5010 61/61 98.36 GAP7 5011/5011 5013/5013 3/3 100 tgg/tgg trnD 5014/5014 5077/5077 64/64 87.5 nad1 5078/5078 5968/5968 891/891 ATG/ATG TAG/TAG 93.04 trnN 5968/5968 6033/6033 66/66 -1/-1 96.97 GAP8 6034/6034 6040/6040 7/7 100 tatgggt/tatgggt trnP 6041/6041 6105/6105 65/65 95.38 GAP9 6106/6106 6112/6113 7/8 62.5 cgcatta/tagtatta trnI 6113/6114 6177/6178 65/65 93.85 GAP10 6,178/6,179 6194/6195 17/17 82.35 taaagaaggaaaggata/taaagaaggaaaaggtg trnK 6195/6196 6258/6259 64/64 98.44 GAP11 6259/6260 6261/6260 3/1 33.33 aat/a nad3 6262/6261 6618/6617 357/357 ATG/ATG TAG/TAG 94.96 trnS1 6608/6607 6666/6665 59/59 -11/-11 96.61 GAP12 6667/6666 6668/6667 2/2 50 tc/tt trnW 6669/6668 6731/6730 63/63 93.65 GAP13 6732/6731 6739/6738 8/8 87.5 aatataaa/agtataaa cox1 6740/6739 8305/8304 1566/1566 ATG/ATG TAG/TAG 93.49 trnT 8296/8295 8357/8356 62/62 -10/-10 98.39 rrnL 8358/8357 9324/9323 967/967 96.38 trnC 9325/9324 9388/9387 64/64 98.44 rrnS 9389/9388 10130/10130 742/743 95.83 cox2 10131/10131 10700/10700 570/570 ATG/ATG TAA/TAA 94.39 GAP14 10701/10701 10701/10701 1/1 100 a/a Li et al. Parasites & Vectors (2018) 11:324 Page 4 of 11 Table 1 Annotated mitochondrial genomes of Digramma interrupta and Ligula intestinalis (Continued) Gene Position Size (bp) Intergenic Codon Identity Sequence nucleotides (%) From To Start Stop trnE 10702/10702 10765/10765 64/64 98.44 GAP15 10766/10766 10770/10770 5/5 100 ttagc/ttagc nad6 10771/10771 11229/11229 459/459 ATG/ATG TAG/TAG 91.29 GAP16 11230/11230 11232/11232 3/3 100 ata/ata trnY 11233/11233 11298/11297 66/65 96.97 NCR1 11299/11298 11521/11521 223/224 91.07 trnL1 11522/11522 11588/11588 67/67 91.04 GAP17 11589/11589 11600/11601 12/13 84.62 tgcggggggttt/ttgtggggggttt trnS2 11601/11602 11665/11667 65/66 96.97 GAP18 11666/11668 11676/11678 11/11 72.73 tagttaaaaga/cagttaaataa trnL2 11677/11679 11740/11742 64/64 95.31 trnR 11741/11743 11795/11797 55/55 92.73 GAP19 11796/11798 11798/11800 3/3 100 ttt/ttt nad5 11799/11801 13367/13369 1569/1569 ATG/ATG TAA/TAA 90.12 NCR2 13368/13370 13616/13552 249/183 67.86 trnG 13617/13553 13682/13618 66/66 96.97 GAP20 13683/13619 13685/13621 3/3 100 aag/aag genome 1/1 13685/13621 13,685/13,621 92.72 °C for 2 min, followed by 40 cycles at 98 °C for 10 s, 48– L. intestinalis and the results were sorted and imported 60 °C for 15 s, 68 °C for 1 min/kb, and a final extension into ggplot2 [22] to draw the RSCU figure. A Tandem at 68 °C for 10 min. PCR products were sequenced on Repeats Finder [23] was used to predict tandem repeats an ABI 3730 automatic sequencer using the Sanger (TR) in the major non-coding regions (mNCRs), and the method at Sangon Company (Shanghai, China) using the secondary structures of NCR1 and TR were folded by primer walking strategy. Mfold software [24]. Non-synonymous (dN) and syn- onymous (dS) mutation rates among the 12 PCGs of D. Sequence annotation and analyses interrupta and L. intestinalis were computed using KaKs_- The mitogenome was annotated broadly following the Calculator [25] utilising a modified Yang-Nielsen algo- procedure described previously [15, 16]. The amplified rithm. DnaSP v5 [26] was adopted to conduct sliding fragments were initially checked by BLASTN [17], before window analyses. A sliding window of 500 bp and step being assembled manually in a stepwise manner. The size of 25 bp was implemented to estimate nucleotide annotation was recorded in a Word document with the divergence Pi (π) between the alignments of the mitogen- help of the Geneious program [18], using the mitogenome omes of D. interrupta and L. intestinalis. of Dibothriocephalus latus (syn. Diphyllobothrium latum) (NC_008945) as the reference sequence. PCGs were found Phylogenetic analyses by searching for ORFs (employing genetic code 9, echino- The mitogenomes of 35 cestodes, covering five orders and derm mitochondrial; flatworm mitochondrial) and the ten families (Additional file 2: Table S2) were obtained nucleotide alignments against the selected reference from GenBank and were used, along with the two new genome in Geneious. rrnL and rrnS were annotated in a mitogenomes generated in this study, to create the phylo- similar way, via comparison with homologs using genetic reconstruction. Two trematodes, Dicrocoelium Geneious. ARWEN [19] and MITOS [20] web servers chinensis (NC_025279) and Dicrocoelium dendriticum were employed to identify and fold all tRNAs. Similarly, (NC_025280), were used as outgroups. All 36 genes (12 the NCBI submission file (*.sqn) and tables of statistics for PCGs, 2 rRNAs and 22 tRNAs) were used for phylogen- mitogenomes were generated using a home-made etic inference and were extracted from GenBank files GUI-based program, MitoTool [21]. MitoTool was also using MitoTool. PCGs were aligned in batches using used to calculate codon usage and relative synonymous MAFFT and integrated into our own in-house GUI-based codon usage (RSCU) for the 12 PCGs of D. interrupta and program, BioSuite [27], adopting codon-alignment mode. Li et al. Parasites & Vectors (2018) 11:324 Page 5 of 11 All RNA genes (rRNA and tRNA) were aligned using a were visualised and annotated by iTOL [33] with the help structural alignment algorithm Q-INS-i incorporated into of several dataset files generated by MitoTool. MAFFT-with-extensions software [28]. Gaps and ambigu- ous sites were deleted using GBlocks [29] integrated by Results BioSuite with default settings. BioSuite was subsequently Genome organization and base composition used to concatenate the sequences into a single alignment The length of the circular mtDNA molecules of D. and generate phylip and nexus format files. interrupta (GenBank accession number: MF671697) and L. GTR+I+G was chosen as the optimal model of nucleo- intestinalis (GenBank accession number: MF671696) was tide evolution for all datasets based on the Akaike infor- 13,685 bp and 13,621 bp, respectively. Both mitogenomes mation criterion by ModelGenerator [30]. Two analytical were composed of 12 PCGs, 22 tRNA genes, two rRNA methods were performed: maximum likelihood (ML) and genes and two mNCRs (major non-coding regions), all of Bayesian inference (BI). The ML analysis was performed which were transcribed from the same strand (Fig. 1). As in RAxML GUI [31] using a ML+rapid bootstrap (BP) commonly reported for flatworms [34], the two mitogen- algorithm with 1000 replicates. BI analysis was performed omes lacked the atp8gene. Thegeneorder ofthetwo in MrBayes 3.2.6 [32] with default settings, 6 × 10 mitogenomes was identical, conforming to the synapo- metropolis-coupled MCMC generations, and 1000 sample morphic gene arrangement of the order Diphyllobothrii- frequency. Stationarity was considered to have been dea [35]. The A+T content of the mitogenomes of D. reached when the average standard deviation of split interrupta and L. intestinalis were 67.9% and 67%, respect- frequencies was below 0.01, ESS (estimated sample size) ively, which is in accordance with that of other cestodes was above 200, and PSRF (potential scale reduction factor) (Additional file 2: Table S2). approached 1. Bayesian posterior probability (BPP) values The mitogenomes of D. interrupta and L. intestinalis were calculated in a consensus tree, after discarding the shared 92.7% nucleotide identity (Table 1), compared with first 25% of samples as burn-in. Finally, the resultant trees 85.8% and 86.2% to Dib. latus, 86.1% and 86.5% to Dib. Table 2 Nucleotide composition and skewness of different elements of the mitochondrial genomes of Digramma interrupta and Ligula intestinalis Region Size (bp) T(U) C A G AT (%) GC (%) AT skew GC skew Digramma interrupta/Ligula intestinalis PCGs 10,062/10,062 45.6/45.1 12.3/13.1 21.9/21.5 20.2/20.3 67.5/66.6 32.5/33.4 -0.351/-0.354 0.244/0.216 1st codon position 3354/3354 41.2/41.4 11.2/11.3 23.6/23.4 24.0/23.9 64.8/64.8 35.2/35.2 -0.272/-0.277 0.366/0.358 2nd codon position 3354/3354 47.6/47.3 15.1/15.4 17.6/17.5 19.7/19.7 65.2/64.8 34.8/35.1 -0.461/-0.461 0.132/0.122 3rd codon position 3354/3354 47.9/46.5 10.6/12.6 24.5/23.6 17.0/17.4 72.4/70.1 27.6/30.0 -0.323/-0.327 0.231/0.159 atp6 510/510 47.6/47.3 13.3/13.3 21.6/20.2 17.5/19.2 69.2/67.5 30.8/32.5 -0.377/-0.401 0.134/0.181 cox1 1566/1566 45.0/44.0 12.6/13.8 22.3/21.8 20.1/20.4 67.3/65.8 32.7/34.2 -0.336/-0.338 0.229/0.194 cox2 570/570 40.2/38.9 12.8/14.2 23.9/24.7 23.2/22.1 64.1/63.6 36.0/36.3 -0.255/-0.223 0.288/0.217 cox3 643/643 46.8/47.1 12.4/12.4 20.5/19.4 20.2/21.0 67.3/66.5 32.6/33.4 -0.390/-0.416 0.238/0.256 cytb 1107/1107 43.8/43.9 13.7/14.1 22.1/21.6 20.3/20.4 65.9/65.5 34.0/34.5 -0.329/-0.341 0.194/0.183 nad1 891/891 45.3/44.9 11.0/11.4 20.4/21.1 23.2/22.6 65.7/66.0 34.2/34.0 -0.379/-0.361 0.357/0.327 nad2 879/879 48.9/48.9 10.5/11.1 21.0/19.8 19.6/20.1 69.9/68.7 30.1/31.2 -0.398/-0.424 0.303/0.287 nad3 357/357 51.3/49.9 7.8/9.2 21.8/20.7 19.0/20.2 73.1/70.6 26.8/29.4 -0.402/-0.413 0.417/0.371 nad4 1251/1251 47.2/46.9 13.7/14.2 19.4/19.0 19.6/19.8 66.6/65.9 33.3/34.0 -0.417/-0.423 0.175/0.164 nad4L 261/261 48.3/49.4 9.2/8.8 27.2/27.2 15.3/14.6 75.5/76.6 24.5/23.4 -0.279/-0.290 0.250/0.246 nad5 1569/1569 42.4/41.4 13.3/15.2 23.8/23.6 20.5/19.8 66.2/65.0 33.8/35.0 -0.281/-0.273 0.214/0.133 nad6 459/459 48.8/48.1 9.8/10.2 21.1/21.1 20.3/20.5 69.9/69.2 30.1/30.7 -0.396/-0.390 0.348/0.333 rrnL 967/967 40.1/39.7 12.0/12.2 28.3/28.2 19.5/19.9 68.4/67.9 31.5/32.1 -0.172/-0.169 0.239/0.239 rrnS 742/743 38.0/38.2 12.1/12.9 30.2/29.3 19.7/19.5 68.2/67.5 31.8/32.4 -0.115/-0.131 0.237/0.203 NCR1 223/224 44.4/41.5 8.5/10.3 34.5/33.5 12.6/14.7 78.9/75.0 21.1/25.0 -0.125/-0.107 0.191/0.179 NCR2 249/183 51.8/50.3 7.6/6.6 22.1/24.6 18.5/18.6 73.9/74.9 26.1/25.2 -0.402/-0.343 0.415/0.478 tRNAs 1410/1410 38.5/37.9 12.4/13.0 29.1/28.6 20.0/20.4 67.6/66.5 32.4/33.4 -0.140/-0.141 0.234/0.220 Full genome 13,685/13,621 44.1/43.6 12.1/12.9 23.8/23.4 20.0/20.2 67.9/67.0 32.1/33.1 -0.299/-0.301 0.245/0.221 Li et al. Parasites & Vectors (2018) 11:324 Page 6 of 11 nihonkaiensis, species of the most closely related genus, within the mitogenome of D. interrupta and L. intestina- respectively. Nucleotide identity was 92.3% between Dib. lis were identical to one another. GTG was identified as latus and Dib. nihonkaiensis, 99.2% between Diphyllobo- the initial codon for cox3, and ATG for the rest of the thrium grandis (Blanchard, 1894) and D. balaenopterae 12 PCGs (Table 1). For each PCG, however, all selected (Lönnberg, 1892), 99.3% between Spirometra decipiens Diphyllobothriidea species shared the same start codon (Diesing, 1850) and S. erinaceieuropaei (Rudolphi, 1819), (Additional file 3: Table S3), indicating that it may be a respectively. Amongst all 36 genes, the majority were synapomorphy within this order. Amongst the termin- equal in size between D. interrupta and L. intestinalis, ation codons, five out of 12 were identified as TAA, six with the exception of rrnS, trnY and trnS2 which had only as TAG, while cox3 used a truncated T stop codon. one base difference. Sequence identity ranged from 87.5% Codon usage, RSCU, and codon family proportion (cor- (trnD)to 100% (trnH, trnQ and trnV)(Table 1). Seven responding to the amino acid usage) among D. interrupta overlapping regions and 20 intergenic sequences (Gap1– and L. intestinalis were investigated (Additional file 4: 20) were found in both genomes, identical in size and Figure S1). The most abundant codon families were Phe, position, with the exception of GAP9, GAP11 and GAP17, Leu2, and Ile within the two mitogenomes, which show a which differed in size (Table 1). preference for the A+T-rich synonymous codons (Additional file 4: Figure S1). This corresponds to the high Protein-coding genes and codon usage A+T bias of the two diphyllobothriid mitogenomes. Concatenated PCGs of the mitogenome of D. interrupta We measured the selective pressure acted upon and L. intestinalis were both 10,062 bp in size, with an amino acid replacement mutations by the ratio of A+T content of 67.5% and 66.6%, respectively (Table 2). non-synonymous (dN) to synonymous (dS) substitu- The high A+T content was mainly concentrated on the tionsfor all12 PCGsof D. interrupta vs L. intestinalis. third codon position (72.4% for D. interrupta and 70.1% Although the values (dN/dS) of atp6 (0.113), nad5 for L. intestinalis). The start and termination codons (0.111) and nad2 (0.110) genes were higher than cox1 Fig. 2 a Ratios of non-synonymous (dN) to synonymous (dS) substitution rates estimated from individual protein-coding genes of Digramma interrupta and Ligula intestinalis. b Sliding window analysis of the alignment of complete mtDNAs of D. interrupta and L. intestinalis. The black line shows the value of nucleotide diversity in a sliding window analysis of window size 500 bp with step size 25 bp, and the value is inserted at its mid-point. Gene boundaries are indicated with a variation ratio per gene (below each gene) Li et al. Parasites & Vectors (2018) 11:324 Page 7 of 11 (0.007) and cox2 (0.008) genes (Fig. 2a), all PCGs were between nad5and trnG, respectively. The mNCRs under strong negative (purifying) selection (dN/dS < were situated in the same location as all diphyllobo- 0.12). thriideans surveyed to date (see Additional file 3 in our recent paper [35]). The NCR1 sequences of the Transfer and ribosomal RNA genes two mitogenomes of D. interrupta and L. intestinalis The sizes of rrnL in both the D. interrupta and L. were 223 and 224 bp in length with a heightened A intestinalis mitogenome was 967 bp, with 68.4% and +T bias of 78.9% and 75%, whereas the NCR2 67.9% A+T content, respectively. Similarly, the lengths sequences were 249 and 183 bp in size with 73.9% of rrnS were 742 and 743 bp, with an A+T content of and 74.9% A+T content, respectively (Table 2). The 68.2% and 67.5%, respectively (Table 2). All 22 NCR2 of D. interrupta contained six TRs (tandem commonly found tRNAs were present in the mito- repeats). Repeat units 1–5 were identical in nucleo- chondrial genome of D. interrupta and L. intestinalis, tide composition and size (34 bp). Repeat unit 6 was adding up to a 1,410 bp total concatenated length in truncated with 29 bp (Fig. 3). TRs were also found both mitogenomes (Table 2). All tRNAs could be in the NCR2 of L. intestinalis, and the consensus folded into the conventional cloverleaf structure, with repeat (35 bp) was almost identical to that of D. (AGN) the exception of trnS1 and trnR, which lacked interrupta, with an insertion of a single nucleotide A (AGN) DHU arms. The absence of DHU-arms in trnS1 at the 17th position. Only four repeat units, how- and trnR has also been reported in the Caryophyllidea ever, could be found in the mitogenome of L. intesti- [35] and the Anoplocephalidae [36]. nalis, which contributed to the main difference in length of the complete mitogenome between the two Non-coding regions genera. The last repeat unit was also truncated with The two major non-coding regions (mNCRs), NCR1 22 bp. Both NCR1 and the consensus repeat and NCR2, were located between trnY and trnL1and sequence in NCR2 of the two mitogenomes were Fig. 3 Major non-coding regions (mNCRs) in the mitogenomes of Digramma interrupta and Ligula intestinalis. Tandem repeat units are shown on the right. The secondary structures of the mNCRs and consensus repeat sequence are illustrated Li et al. Parasites & Vectors (2018) 11:324 Page 8 of 11 capable of forming stem-loop structures (Fig. 3, clustered together with Diphyllobothrium, then forming predicted by Mfold web server). a sister group with Spirometra. Discussion Sliding window analyses and nucleotide diversity The ordinal topology of Caryophyllidea + (Diphyllobo- A sliding window analysis of the 12 PCGs and two thriidea + (Bothriocephalidea + (Proteocephalidea + rRNAs of D. interrupta and L. intestinalis indicated Cyclophyllidea))) was consistent with previously identi- the nucleotide diversity Pi (π)tobehigherin nad5 fied interordinal relationships of tapeworms, when (0.099), nad6 (0.088), nad2 (0.088), nad4 (0.087) and reconstructed from the dataset of nucleotide or amino cox3 (0.087), whereas the most conserved genes were acid sequences from partial mitogenomes, large and rrnL (0.036) and rrnS (0.04) (Fig. 2b). small subunit rRNA genes, and a combination of the former two [37]. Additionally, this relationship identified between tapeworm groups was congruent with latest Phylogeny mitochondrial phylogenomics [35]. However, a sister-group Both methods (BI and ML) produced phylograms with relationship between the orders Diphyllobothriidea and concordant branch topologies, thus only the latter was Bothriocephalidea has been suggested based on mitochon- shown (Fig. 4). The phylogenetic tree indicated the drial phylogenomics [38, 39]. This inconsistency may be ordinal topology to be Caryophyllidea + (Diphyllobo- due to the different methods of phylogenetics employed. thriidea + (Bothriocephalidea + (Proteocephalidea + Within the family Diphyllobothriidae, the phylogenetic Cyclophyllidea))). Within the family Diphyllobothriidae, relationship of the three genera Spirometra, Diphyllobo- Ligula and Digramma clustered with maximum nodal thrium and Dibothriocephalus was congruent with that support (BP = 100 and BPP = 1), which formed a sister of recent studies on the phylogenetics of Eucestoda group with the genus Dibothriocephalus. This clade based on mitogenomes, with the topology of Spirometra Fig. 4 Phylogeny of five cestode orders using maximum likelihood analysis inferred from concatenated nucleotide sequences of all 36 genes (12 PCGs, 2 rRNAs and 22 tRNAs). Bootstrap support values are shown above the nodes. The anti-codon of the trnR gene present in individual orders was denoted. The scale-bar represents the estimated number of substitutions per site Li et al. Parasites & Vectors (2018) 11:324 Page 9 of 11 Fig. 5 Sequence identity of 12 protein-coding genes and two rRNA genes between Digramma interrupta and Ligula intestinalis +(Dibothriocephalus + Diphyllobothrium)[35, 38–41]. nad5, nad6and cox3genes between D. interrupta and L. In the present study, however, Ligula and Digramma were intestinalis (90–92%), in comparison to the moderate vari- closely related to one another with maximum nodal ation seen between the cox1, cytb and nad1 genes (Fig. 5). support, then forming a sister group with the genus Dibo- Additionally, the relatively looser selection pressure of thriocephalus. Inferred by the ITS2 rDNA sequence [13] nad5 (0.111) and nad2 (0.110) may accelerate the accu- and the 18S rDNA gene [42], the complexes of Ligula and mulation of non-synonymous substitutions, which would Digramma have also been shown to be closely related to increase variation of the two genes [48]. These results Dibothriocephalus. Further studies using the concatenated suggest that the nad2, nad4, nad5, nad6and cox3genes nucleotide sequences of 18S rDNA + 28S rDNA + rrnL + should be considered as optimal candidates for genetic cox1, have again demonstrated the genus Dibothriocepha- markers to be used for population genetics and species lus to be the sister group of Ligula [5]. These phylogenetic identification studies between the two closely related results suggest that Dibothriocephalus is the most closely species, D. interrupta and L. intestinalis. related genus to Ligula and Digramma. However, mitogenome sequence identity between D. Conclusions interrupta and L. intestinalis is92.7%, whichismuchhigher The complete mitogenomes of Ligula intestinalis and than between either of these species and the represented Digramma interrupta were sequenced and characterized. members of Dibothriocephalus (85.8–86.5%). Furthermore, The mitogenomes of these two species show a higher high mitogenome sequence identity was also found be- identity to each other than to any species in closely related tween the congeners in Dibothriocephalus (92.3%), Diphyl- genera. The two mitogenomes consistently support D. lobothrium (99.2%) and Spirometra (99.3%). These results interrupta to be a congeneric species with L. intestinalis. suggest that sequence differences between D. interrupta High sequence variation in the nad2, nad4, nad5, nad6 and L. intestinalis are of a degree expected between mem- and cox3 genes between the two diphyllobothriids suggest bers of the same genus. that these five genes should be considered as optimal In one study, sequence identity of the cox1and nad1 candidates for genetic markers when studying population genes between D. interrupta and L. intestinalis has been genetics or looking to differentiate the two closely related shown to be 100% and 92.6% [11]; however, identity was species, D. interrupta and L. intestinalis. deemed at 93.5% and 93.0% in the present study, respect- ively (Table 1). This inconsistency may be due to the use Additional files of partial sequence of cox1and nad1 genes or resulting from the use of formalin-preserved specimens [12]. The Additional file 1: Table S1. Primers used to amplify and sequence the gene cox1 is considered to be a useful barcode for mitochondrial genome of Digramma interrupta and Ligula intestinalis. metazoans [43], and widely employed for cestode studies (DOCX 15 kb) [44–47]. The two mitochondrial genes cox1and cytb have Additional file 2: Table S2. The list of cestode species and outgroups also been used to study the population genetic structure used for comparative mitogenomic and phylogenetic analyses, and accession number, A+T content and skewness of different elements of of L. intestinalis on a local and global scale [14]. However, each mitogenome. (XLSX 19 kb) a lower sequence identity was found in the nad2, nad4, Li et al. Parasites & Vectors (2018) 11:324 Page 10 of 11 3. Waeschenbach A, Webster BL, Bray RA, Littlewood DTJ. Added resolution Additional file 3: Table S3. General statistics (length and codons) for among ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) mitochondrial protein-coding genes and rRNAs of 38 cestodes. Abbreviations with complete small and large subunit nuclear ribosomal RNA genes. Mol of species name are the initials of genus and species name combined. Phylogenet Evol. 2007;45:311–25. (XLSX 21 kb) 4. Kuchta R, Scholz T. Diphyllobothriidea. In: Caira JN, Jensen J, editors. Additional file 4: Figure S1. Relative Synonymous Codon Usage (RSCU) Planetary Biodiversity Inventory (2008–2017): Tapeworms from vertebrate of Digramma interrupta and Ligula intestinalis. Codon families are labelled bowels of the earth. The University of Kansas Natural History Museum on the x-axis. Values on the top of the bars denote amino acid usage. Special Publication No. 25; 2017. (PDF 37 kb) 5. Waeschenbach A, Brabec J, Scholz T, Littlewood DTJ, Kuchta R. The catholic taste of broad tapeworms-multiple routes to human infection. 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Comparative mitogenomics supports synonymy of the genera Ligula and Digramma (Cestoda: Diphyllobothriidae)

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Biomedicine; Parasitology; Entomology; Tropical Medicine; Infectious Diseases; Veterinary Medicine/Veterinary Science; Virology
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Abstract

Background: After observing differences in the number of reproductive complexes per proglottid within the genus Ligula, the genus Digramma was erected. However, the validity of Digramma has been previously questioned due to a low variability in the cox1, nad1 and ITS rDNA sequences between the two genera. We undertook a study to greatly increase the amount of sequence data available for resolution of this question by sequencing and characterizing the complete mitogenomes of Digramma interrupta and Ligula intestinalis. Results: The circular mtDNA molecules of Digramma interrupta and Ligula intestinalis are 13,685 bp and 13,621 bp in size, respectively, both comprising 12 PCGs, 22 tRNA genes, two rRNA genes, and two mNCRs. Both mitogenomes exhibit the same gene order and share 92.7% nucleotide identity, compared with 85.8–86.5% to the most closely related genus Dibothriocephalus.Eachgenefrom D. interrupta and L. intestinalis is almost of the same size, and the sequence identity ranges from 87.5% (trnD)to100%(trnH, trnQ and trnV). NCR2 sequences of D. interrupta and L. intestinalis are 249 bp and 183 bp in length, respectively, which contributes to the main difference in length between their complete mitogenomes. A sliding window analysis of the 12 PCGs and two rRNAs indicated nucleotide diversity to be higher in nad5, nad6, nad2, nad4and cox3, whereas the most conserved genes were rrnL and rrnS. Lower sequence identity was also found in nad2, nad4, nad5, nad6and cox3 genes between the two diphyllobothriids. Within the Diphyllobothriidae, phylogenetic analysis indicated Ligula and Digramma to be most closely related to one another, forming a sister group with Dibothriocephalus. Conclusions: Owing to higher nucleotide diversity, the genes nad2, nad4, nad5, nad6and cox3 should be considered optimal candidates to use as molecular markers for population genetics and species identification between the two closely related species. The phylogenetic results in combination with the comparative analysis of the two mitogenomes, consistently support the congeneric status of L. intestinalis and D. interrupta. Keywords: Mitogenome, Eucestoda, Diphyllobothriidea, Digramma, Ligula Background the Diphyllobothriidea and Bothriocephalidea Kuchta, Based upon its paraphyly, differences in the position of the Scholz, Brabec & Bray, 2008 were proposed [1–3]. genital pore, the presence of an external seminal vesicle The order Diphyllobothriidea includes 70 species and the absence of a uterine sac in the Diphyllobothriidea considered valid, classified into 18 genera across three Kuchta, Scholz, Brabec & Bray, 2008, the order Pseudo- families [2, 4]. Adult diphyllobothriideans are found only phyllidea van Beneden in Carus, 1863 was suppressed and in tetrapods, never having been recorded in fish [2], and the plerocercoids of groups such as Spirometra, Diphyllobothrium Cobbold, 1858 (syn. Diplogonoporus Lönnberg, 1892) and Dibothriocephalus Lühe, 1899 (a * Correspondence: gtwang@ihb.ac.cn Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, and recently resurrected genus including some species from State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Diphyllobothrium, i.e. Dib. dendriticus (Nitzsch, 1824), Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, People’s Dib. nihonkaiensis (Yamane, Kamo, Bylund & Wikgren, Republic of China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. cox2 cox2 6K 6K 5K 5K 4K 4K S1 S1 nad3 nad3 3K 3K 2K 2K 1K 1K atp6 atp6 Li et al. Parasites & Vectors (2018) 11:324 Page 2 of 11 1986) and Dib. latus (Linnaeus, 1758) belonging to the markers as a means to differentiate D. interrupta from L. family Diphyllobothriidae are often the principal agents of intestinalis. food-borne cestodosis [5]. Ligula spp. also belong to the Diphyllobothriidae; these species use copepods as first Methods intermediate hosts, freshwater fish as second intermediate Specimen collection and DNA extraction hosts and birds as definitive hosts [6]. Ligula intestinalis Plerocercoids of D. interrupta and L. intestinalis were (Linnaeus, 1758) is a tapeworm of veterinary importance isolated from the body cavity of Carassius auratus worldwide that reduces the fecundity of the cyprinid fishes collected from Liangzi Lake in Hubei Province and by parasitic castration [6, 7] and induces mass mortalities Gymnocypris selincuoensis from Siling Lake, Tibet, of the carp Chanodichthys erythropterus [8]. China, respectively. The tapeworms were preserved in Cholodkovsky [9]erected thegenus Digramma Cholod- 80% ethanol and stored at 4 °C. Total genomic DNA kovsky, 1915 after observing differences in the number of was extracted from the posterior region of a single reproductive complexes contained within each proglottid tapeworm using a TIANamp Micro DNA Kit (Tiangen when studying the genus Ligula Linnaeus, 1758. In China, Biotech, Beijing, China), according to the manufacturer’s Ligula spp. are distributed in the Qinghai-Tibet Plateau instructions. DNA was stored at -20 °C for subsequent with Schizothoracinae fishes serving as the primary second molecular analysis. The morphological identification of intermediate host. Digramma spp. are found across the rest specimens was confirmed using sequence data from the of China where the goldfish Carassius auratus acts as their ITS2 rDNA region [13, 14] and the cox1 gene [11]. common second intermediate host [10]. However, the val- idity of Digramma has been questioned due to a low level of difference between the species of two genera in the cox1, Amplification and DNA sequencing nad1 and ITS rDNA sequences [11–13]. Thus Digramma PCR was carried out as described previously [15, 16], is considered to be synonymous with Ligula [2]. Owing to with minor modifications. Five degenerate primer sets the fact that only one gene and a limited number of isolates (Additional file 1: Table S1) were designed to primarily were included in that study, more sequence data and a amplify partial sequences of the nad5, cytb, nad2, cox1 greater range of taxa from different genera are required for and rrnS genes. The sequenced fragments were subse- a more comprehensive phylogenetic analysis of the Diphyl- quently used to design primers specific for the amplifica- lobothriidea [13]. tion and sequencing of the whole mitogenome. PCR This study, therefore, aimed to sequence and characterize reactions were performed in a 20 μl reaction mixture, the complete mitogenomes of Digramma interrupta and containing 7.4 μl dd PCR grade H O, 10 μl2×PCR 2+ Ligula intestinalis and to perform phylogenetic analysis to buffer (2 mM Mg ,8 μl dNTP plus, Takara, Dalian, investigate whether or not these two diphyllobothriids are China), 0.6 μl of each primer (12.5 μM), 0.4 μlrTaq congeneric using mitogenomic data. Differences within the polymerase (250 U, Takara, Dalian, China), and 1 μl mitochondrial genes were also compared to determine genomic DNA template. Amplification was conducted which genes would be suitable for the design of molecular under the following conditions: initial denaturation at 98 0K 0K 13,621 nt 13 685 nt Ligula intestinalis Digramma interrupta Fig. 1 Circular map of the mitochondrial genome of Digramma interrupta and Ligula intestinalis. Protein-coding genes (12) are shown in red, tRNAs (22) in yellow, rRNAs (2) in green and non-coding regions in grey 13K 13K 12K 12K nad4L nad4L 11K cox3 cox3 11K 10K 10K 9K 9K L2 L2 S2 S2 8K 8K L1 L1 nad6 nad6 7K 7K Li et al. Parasites & Vectors (2018) 11:324 Page 3 of 11 Table 1 Annotated mitochondrial genomes of Digramma interrupta and Ligula intestinalis Gene Position Size (bp) Intergenic Codon Identity Sequence nucleotides (%) From To Start Stop Digramma interrupta/Ligula intestinalis cox3 1/1 643/643 643/643 GTG/GTG T/T 91.29 trnH 644/644 710/710 67/67 100 GAP1 711/711 713/713 3/3 100 gaa/gaa cytb 714/714 1820/1820 1107/1107 ATG/ATG TAA/TAA 93.41 GAP2 1821/1821 1821/1821 1/1 100 c/c nad4L 1822/1822 2082/2082 261/261 ATG/ATG TAA/TAA 94.64 nad4 2043/2043 3293/3293 1251/1251 -40/-40 ATG/ATG TAG/TAG 91.37 trnQ 3293/3293 3357/3357 65/65 -1/-1 100 trnF 3353/3353 3419/3419 67/67 -5/-5 97.01 trnM 3416/3416 3482/3482 67/67 -4/-4 98.51 GAP3 3483/3483 3485/3485 3/3 66.67 gtt/att atp6 3486/3486 3995/3995 510/510 ATG/ATG TAA/TAA 93.33 GAP4 3996/3996 3997/3997 2/2 100 tc/tc nad2 3998/3998 4876/4876 879/879 ATG/ATG TAG/TAG 91.24 GAP5 4877/4877 4877/4877 1/1 100 t/t trnV 4878/4878 4941/4941 64/64 100 GAP6 4942/4942 4949/4949 8/8 87.5 gtcttaag/gttttaag trnA 4950/4950 5010/5010 61/61 98.36 GAP7 5011/5011 5013/5013 3/3 100 tgg/tgg trnD 5014/5014 5077/5077 64/64 87.5 nad1 5078/5078 5968/5968 891/891 ATG/ATG TAG/TAG 93.04 trnN 5968/5968 6033/6033 66/66 -1/-1 96.97 GAP8 6034/6034 6040/6040 7/7 100 tatgggt/tatgggt trnP 6041/6041 6105/6105 65/65 95.38 GAP9 6106/6106 6112/6113 7/8 62.5 cgcatta/tagtatta trnI 6113/6114 6177/6178 65/65 93.85 GAP10 6,178/6,179 6194/6195 17/17 82.35 taaagaaggaaaggata/taaagaaggaaaaggtg trnK 6195/6196 6258/6259 64/64 98.44 GAP11 6259/6260 6261/6260 3/1 33.33 aat/a nad3 6262/6261 6618/6617 357/357 ATG/ATG TAG/TAG 94.96 trnS1 6608/6607 6666/6665 59/59 -11/-11 96.61 GAP12 6667/6666 6668/6667 2/2 50 tc/tt trnW 6669/6668 6731/6730 63/63 93.65 GAP13 6732/6731 6739/6738 8/8 87.5 aatataaa/agtataaa cox1 6740/6739 8305/8304 1566/1566 ATG/ATG TAG/TAG 93.49 trnT 8296/8295 8357/8356 62/62 -10/-10 98.39 rrnL 8358/8357 9324/9323 967/967 96.38 trnC 9325/9324 9388/9387 64/64 98.44 rrnS 9389/9388 10130/10130 742/743 95.83 cox2 10131/10131 10700/10700 570/570 ATG/ATG TAA/TAA 94.39 GAP14 10701/10701 10701/10701 1/1 100 a/a Li et al. Parasites & Vectors (2018) 11:324 Page 4 of 11 Table 1 Annotated mitochondrial genomes of Digramma interrupta and Ligula intestinalis (Continued) Gene Position Size (bp) Intergenic Codon Identity Sequence nucleotides (%) From To Start Stop trnE 10702/10702 10765/10765 64/64 98.44 GAP15 10766/10766 10770/10770 5/5 100 ttagc/ttagc nad6 10771/10771 11229/11229 459/459 ATG/ATG TAG/TAG 91.29 GAP16 11230/11230 11232/11232 3/3 100 ata/ata trnY 11233/11233 11298/11297 66/65 96.97 NCR1 11299/11298 11521/11521 223/224 91.07 trnL1 11522/11522 11588/11588 67/67 91.04 GAP17 11589/11589 11600/11601 12/13 84.62 tgcggggggttt/ttgtggggggttt trnS2 11601/11602 11665/11667 65/66 96.97 GAP18 11666/11668 11676/11678 11/11 72.73 tagttaaaaga/cagttaaataa trnL2 11677/11679 11740/11742 64/64 95.31 trnR 11741/11743 11795/11797 55/55 92.73 GAP19 11796/11798 11798/11800 3/3 100 ttt/ttt nad5 11799/11801 13367/13369 1569/1569 ATG/ATG TAA/TAA 90.12 NCR2 13368/13370 13616/13552 249/183 67.86 trnG 13617/13553 13682/13618 66/66 96.97 GAP20 13683/13619 13685/13621 3/3 100 aag/aag genome 1/1 13685/13621 13,685/13,621 92.72 °C for 2 min, followed by 40 cycles at 98 °C for 10 s, 48– L. intestinalis and the results were sorted and imported 60 °C for 15 s, 68 °C for 1 min/kb, and a final extension into ggplot2 [22] to draw the RSCU figure. A Tandem at 68 °C for 10 min. PCR products were sequenced on Repeats Finder [23] was used to predict tandem repeats an ABI 3730 automatic sequencer using the Sanger (TR) in the major non-coding regions (mNCRs), and the method at Sangon Company (Shanghai, China) using the secondary structures of NCR1 and TR were folded by primer walking strategy. Mfold software [24]. Non-synonymous (dN) and syn- onymous (dS) mutation rates among the 12 PCGs of D. Sequence annotation and analyses interrupta and L. intestinalis were computed using KaKs_- The mitogenome was annotated broadly following the Calculator [25] utilising a modified Yang-Nielsen algo- procedure described previously [15, 16]. The amplified rithm. DnaSP v5 [26] was adopted to conduct sliding fragments were initially checked by BLASTN [17], before window analyses. A sliding window of 500 bp and step being assembled manually in a stepwise manner. The size of 25 bp was implemented to estimate nucleotide annotation was recorded in a Word document with the divergence Pi (π) between the alignments of the mitogen- help of the Geneious program [18], using the mitogenome omes of D. interrupta and L. intestinalis. of Dibothriocephalus latus (syn. Diphyllobothrium latum) (NC_008945) as the reference sequence. PCGs were found Phylogenetic analyses by searching for ORFs (employing genetic code 9, echino- The mitogenomes of 35 cestodes, covering five orders and derm mitochondrial; flatworm mitochondrial) and the ten families (Additional file 2: Table S2) were obtained nucleotide alignments against the selected reference from GenBank and were used, along with the two new genome in Geneious. rrnL and rrnS were annotated in a mitogenomes generated in this study, to create the phylo- similar way, via comparison with homologs using genetic reconstruction. Two trematodes, Dicrocoelium Geneious. ARWEN [19] and MITOS [20] web servers chinensis (NC_025279) and Dicrocoelium dendriticum were employed to identify and fold all tRNAs. Similarly, (NC_025280), were used as outgroups. All 36 genes (12 the NCBI submission file (*.sqn) and tables of statistics for PCGs, 2 rRNAs and 22 tRNAs) were used for phylogen- mitogenomes were generated using a home-made etic inference and were extracted from GenBank files GUI-based program, MitoTool [21]. MitoTool was also using MitoTool. PCGs were aligned in batches using used to calculate codon usage and relative synonymous MAFFT and integrated into our own in-house GUI-based codon usage (RSCU) for the 12 PCGs of D. interrupta and program, BioSuite [27], adopting codon-alignment mode. Li et al. Parasites & Vectors (2018) 11:324 Page 5 of 11 All RNA genes (rRNA and tRNA) were aligned using a were visualised and annotated by iTOL [33] with the help structural alignment algorithm Q-INS-i incorporated into of several dataset files generated by MitoTool. MAFFT-with-extensions software [28]. Gaps and ambigu- ous sites were deleted using GBlocks [29] integrated by Results BioSuite with default settings. BioSuite was subsequently Genome organization and base composition used to concatenate the sequences into a single alignment The length of the circular mtDNA molecules of D. and generate phylip and nexus format files. interrupta (GenBank accession number: MF671697) and L. GTR+I+G was chosen as the optimal model of nucleo- intestinalis (GenBank accession number: MF671696) was tide evolution for all datasets based on the Akaike infor- 13,685 bp and 13,621 bp, respectively. Both mitogenomes mation criterion by ModelGenerator [30]. Two analytical were composed of 12 PCGs, 22 tRNA genes, two rRNA methods were performed: maximum likelihood (ML) and genes and two mNCRs (major non-coding regions), all of Bayesian inference (BI). The ML analysis was performed which were transcribed from the same strand (Fig. 1). As in RAxML GUI [31] using a ML+rapid bootstrap (BP) commonly reported for flatworms [34], the two mitogen- algorithm with 1000 replicates. BI analysis was performed omes lacked the atp8gene. Thegeneorder ofthetwo in MrBayes 3.2.6 [32] with default settings, 6 × 10 mitogenomes was identical, conforming to the synapo- metropolis-coupled MCMC generations, and 1000 sample morphic gene arrangement of the order Diphyllobothrii- frequency. Stationarity was considered to have been dea [35]. The A+T content of the mitogenomes of D. reached when the average standard deviation of split interrupta and L. intestinalis were 67.9% and 67%, respect- frequencies was below 0.01, ESS (estimated sample size) ively, which is in accordance with that of other cestodes was above 200, and PSRF (potential scale reduction factor) (Additional file 2: Table S2). approached 1. Bayesian posterior probability (BPP) values The mitogenomes of D. interrupta and L. intestinalis were calculated in a consensus tree, after discarding the shared 92.7% nucleotide identity (Table 1), compared with first 25% of samples as burn-in. Finally, the resultant trees 85.8% and 86.2% to Dib. latus, 86.1% and 86.5% to Dib. Table 2 Nucleotide composition and skewness of different elements of the mitochondrial genomes of Digramma interrupta and Ligula intestinalis Region Size (bp) T(U) C A G AT (%) GC (%) AT skew GC skew Digramma interrupta/Ligula intestinalis PCGs 10,062/10,062 45.6/45.1 12.3/13.1 21.9/21.5 20.2/20.3 67.5/66.6 32.5/33.4 -0.351/-0.354 0.244/0.216 1st codon position 3354/3354 41.2/41.4 11.2/11.3 23.6/23.4 24.0/23.9 64.8/64.8 35.2/35.2 -0.272/-0.277 0.366/0.358 2nd codon position 3354/3354 47.6/47.3 15.1/15.4 17.6/17.5 19.7/19.7 65.2/64.8 34.8/35.1 -0.461/-0.461 0.132/0.122 3rd codon position 3354/3354 47.9/46.5 10.6/12.6 24.5/23.6 17.0/17.4 72.4/70.1 27.6/30.0 -0.323/-0.327 0.231/0.159 atp6 510/510 47.6/47.3 13.3/13.3 21.6/20.2 17.5/19.2 69.2/67.5 30.8/32.5 -0.377/-0.401 0.134/0.181 cox1 1566/1566 45.0/44.0 12.6/13.8 22.3/21.8 20.1/20.4 67.3/65.8 32.7/34.2 -0.336/-0.338 0.229/0.194 cox2 570/570 40.2/38.9 12.8/14.2 23.9/24.7 23.2/22.1 64.1/63.6 36.0/36.3 -0.255/-0.223 0.288/0.217 cox3 643/643 46.8/47.1 12.4/12.4 20.5/19.4 20.2/21.0 67.3/66.5 32.6/33.4 -0.390/-0.416 0.238/0.256 cytb 1107/1107 43.8/43.9 13.7/14.1 22.1/21.6 20.3/20.4 65.9/65.5 34.0/34.5 -0.329/-0.341 0.194/0.183 nad1 891/891 45.3/44.9 11.0/11.4 20.4/21.1 23.2/22.6 65.7/66.0 34.2/34.0 -0.379/-0.361 0.357/0.327 nad2 879/879 48.9/48.9 10.5/11.1 21.0/19.8 19.6/20.1 69.9/68.7 30.1/31.2 -0.398/-0.424 0.303/0.287 nad3 357/357 51.3/49.9 7.8/9.2 21.8/20.7 19.0/20.2 73.1/70.6 26.8/29.4 -0.402/-0.413 0.417/0.371 nad4 1251/1251 47.2/46.9 13.7/14.2 19.4/19.0 19.6/19.8 66.6/65.9 33.3/34.0 -0.417/-0.423 0.175/0.164 nad4L 261/261 48.3/49.4 9.2/8.8 27.2/27.2 15.3/14.6 75.5/76.6 24.5/23.4 -0.279/-0.290 0.250/0.246 nad5 1569/1569 42.4/41.4 13.3/15.2 23.8/23.6 20.5/19.8 66.2/65.0 33.8/35.0 -0.281/-0.273 0.214/0.133 nad6 459/459 48.8/48.1 9.8/10.2 21.1/21.1 20.3/20.5 69.9/69.2 30.1/30.7 -0.396/-0.390 0.348/0.333 rrnL 967/967 40.1/39.7 12.0/12.2 28.3/28.2 19.5/19.9 68.4/67.9 31.5/32.1 -0.172/-0.169 0.239/0.239 rrnS 742/743 38.0/38.2 12.1/12.9 30.2/29.3 19.7/19.5 68.2/67.5 31.8/32.4 -0.115/-0.131 0.237/0.203 NCR1 223/224 44.4/41.5 8.5/10.3 34.5/33.5 12.6/14.7 78.9/75.0 21.1/25.0 -0.125/-0.107 0.191/0.179 NCR2 249/183 51.8/50.3 7.6/6.6 22.1/24.6 18.5/18.6 73.9/74.9 26.1/25.2 -0.402/-0.343 0.415/0.478 tRNAs 1410/1410 38.5/37.9 12.4/13.0 29.1/28.6 20.0/20.4 67.6/66.5 32.4/33.4 -0.140/-0.141 0.234/0.220 Full genome 13,685/13,621 44.1/43.6 12.1/12.9 23.8/23.4 20.0/20.2 67.9/67.0 32.1/33.1 -0.299/-0.301 0.245/0.221 Li et al. Parasites & Vectors (2018) 11:324 Page 6 of 11 nihonkaiensis, species of the most closely related genus, within the mitogenome of D. interrupta and L. intestina- respectively. Nucleotide identity was 92.3% between Dib. lis were identical to one another. GTG was identified as latus and Dib. nihonkaiensis, 99.2% between Diphyllobo- the initial codon for cox3, and ATG for the rest of the thrium grandis (Blanchard, 1894) and D. balaenopterae 12 PCGs (Table 1). For each PCG, however, all selected (Lönnberg, 1892), 99.3% between Spirometra decipiens Diphyllobothriidea species shared the same start codon (Diesing, 1850) and S. erinaceieuropaei (Rudolphi, 1819), (Additional file 3: Table S3), indicating that it may be a respectively. Amongst all 36 genes, the majority were synapomorphy within this order. Amongst the termin- equal in size between D. interrupta and L. intestinalis, ation codons, five out of 12 were identified as TAA, six with the exception of rrnS, trnY and trnS2 which had only as TAG, while cox3 used a truncated T stop codon. one base difference. Sequence identity ranged from 87.5% Codon usage, RSCU, and codon family proportion (cor- (trnD)to 100% (trnH, trnQ and trnV)(Table 1). Seven responding to the amino acid usage) among D. interrupta overlapping regions and 20 intergenic sequences (Gap1– and L. intestinalis were investigated (Additional file 4: 20) were found in both genomes, identical in size and Figure S1). The most abundant codon families were Phe, position, with the exception of GAP9, GAP11 and GAP17, Leu2, and Ile within the two mitogenomes, which show a which differed in size (Table 1). preference for the A+T-rich synonymous codons (Additional file 4: Figure S1). This corresponds to the high Protein-coding genes and codon usage A+T bias of the two diphyllobothriid mitogenomes. Concatenated PCGs of the mitogenome of D. interrupta We measured the selective pressure acted upon and L. intestinalis were both 10,062 bp in size, with an amino acid replacement mutations by the ratio of A+T content of 67.5% and 66.6%, respectively (Table 2). non-synonymous (dN) to synonymous (dS) substitu- The high A+T content was mainly concentrated on the tionsfor all12 PCGsof D. interrupta vs L. intestinalis. third codon position (72.4% for D. interrupta and 70.1% Although the values (dN/dS) of atp6 (0.113), nad5 for L. intestinalis). The start and termination codons (0.111) and nad2 (0.110) genes were higher than cox1 Fig. 2 a Ratios of non-synonymous (dN) to synonymous (dS) substitution rates estimated from individual protein-coding genes of Digramma interrupta and Ligula intestinalis. b Sliding window analysis of the alignment of complete mtDNAs of D. interrupta and L. intestinalis. The black line shows the value of nucleotide diversity in a sliding window analysis of window size 500 bp with step size 25 bp, and the value is inserted at its mid-point. Gene boundaries are indicated with a variation ratio per gene (below each gene) Li et al. Parasites & Vectors (2018) 11:324 Page 7 of 11 (0.007) and cox2 (0.008) genes (Fig. 2a), all PCGs were between nad5and trnG, respectively. The mNCRs under strong negative (purifying) selection (dN/dS < were situated in the same location as all diphyllobo- 0.12). thriideans surveyed to date (see Additional file 3 in our recent paper [35]). The NCR1 sequences of the Transfer and ribosomal RNA genes two mitogenomes of D. interrupta and L. intestinalis The sizes of rrnL in both the D. interrupta and L. were 223 and 224 bp in length with a heightened A intestinalis mitogenome was 967 bp, with 68.4% and +T bias of 78.9% and 75%, whereas the NCR2 67.9% A+T content, respectively. Similarly, the lengths sequences were 249 and 183 bp in size with 73.9% of rrnS were 742 and 743 bp, with an A+T content of and 74.9% A+T content, respectively (Table 2). The 68.2% and 67.5%, respectively (Table 2). All 22 NCR2 of D. interrupta contained six TRs (tandem commonly found tRNAs were present in the mito- repeats). Repeat units 1–5 were identical in nucleo- chondrial genome of D. interrupta and L. intestinalis, tide composition and size (34 bp). Repeat unit 6 was adding up to a 1,410 bp total concatenated length in truncated with 29 bp (Fig. 3). TRs were also found both mitogenomes (Table 2). All tRNAs could be in the NCR2 of L. intestinalis, and the consensus folded into the conventional cloverleaf structure, with repeat (35 bp) was almost identical to that of D. (AGN) the exception of trnS1 and trnR, which lacked interrupta, with an insertion of a single nucleotide A (AGN) DHU arms. The absence of DHU-arms in trnS1 at the 17th position. Only four repeat units, how- and trnR has also been reported in the Caryophyllidea ever, could be found in the mitogenome of L. intesti- [35] and the Anoplocephalidae [36]. nalis, which contributed to the main difference in length of the complete mitogenome between the two Non-coding regions genera. The last repeat unit was also truncated with The two major non-coding regions (mNCRs), NCR1 22 bp. Both NCR1 and the consensus repeat and NCR2, were located between trnY and trnL1and sequence in NCR2 of the two mitogenomes were Fig. 3 Major non-coding regions (mNCRs) in the mitogenomes of Digramma interrupta and Ligula intestinalis. Tandem repeat units are shown on the right. The secondary structures of the mNCRs and consensus repeat sequence are illustrated Li et al. Parasites & Vectors (2018) 11:324 Page 8 of 11 capable of forming stem-loop structures (Fig. 3, clustered together with Diphyllobothrium, then forming predicted by Mfold web server). a sister group with Spirometra. Discussion Sliding window analyses and nucleotide diversity The ordinal topology of Caryophyllidea + (Diphyllobo- A sliding window analysis of the 12 PCGs and two thriidea + (Bothriocephalidea + (Proteocephalidea + rRNAs of D. interrupta and L. intestinalis indicated Cyclophyllidea))) was consistent with previously identi- the nucleotide diversity Pi (π)tobehigherin nad5 fied interordinal relationships of tapeworms, when (0.099), nad6 (0.088), nad2 (0.088), nad4 (0.087) and reconstructed from the dataset of nucleotide or amino cox3 (0.087), whereas the most conserved genes were acid sequences from partial mitogenomes, large and rrnL (0.036) and rrnS (0.04) (Fig. 2b). small subunit rRNA genes, and a combination of the former two [37]. Additionally, this relationship identified between tapeworm groups was congruent with latest Phylogeny mitochondrial phylogenomics [35]. However, a sister-group Both methods (BI and ML) produced phylograms with relationship between the orders Diphyllobothriidea and concordant branch topologies, thus only the latter was Bothriocephalidea has been suggested based on mitochon- shown (Fig. 4). The phylogenetic tree indicated the drial phylogenomics [38, 39]. This inconsistency may be ordinal topology to be Caryophyllidea + (Diphyllobo- due to the different methods of phylogenetics employed. thriidea + (Bothriocephalidea + (Proteocephalidea + Within the family Diphyllobothriidae, the phylogenetic Cyclophyllidea))). Within the family Diphyllobothriidae, relationship of the three genera Spirometra, Diphyllobo- Ligula and Digramma clustered with maximum nodal thrium and Dibothriocephalus was congruent with that support (BP = 100 and BPP = 1), which formed a sister of recent studies on the phylogenetics of Eucestoda group with the genus Dibothriocephalus. This clade based on mitogenomes, with the topology of Spirometra Fig. 4 Phylogeny of five cestode orders using maximum likelihood analysis inferred from concatenated nucleotide sequences of all 36 genes (12 PCGs, 2 rRNAs and 22 tRNAs). Bootstrap support values are shown above the nodes. The anti-codon of the trnR gene present in individual orders was denoted. The scale-bar represents the estimated number of substitutions per site Li et al. Parasites & Vectors (2018) 11:324 Page 9 of 11 Fig. 5 Sequence identity of 12 protein-coding genes and two rRNA genes between Digramma interrupta and Ligula intestinalis +(Dibothriocephalus + Diphyllobothrium)[35, 38–41]. nad5, nad6and cox3genes between D. interrupta and L. In the present study, however, Ligula and Digramma were intestinalis (90–92%), in comparison to the moderate vari- closely related to one another with maximum nodal ation seen between the cox1, cytb and nad1 genes (Fig. 5). support, then forming a sister group with the genus Dibo- Additionally, the relatively looser selection pressure of thriocephalus. Inferred by the ITS2 rDNA sequence [13] nad5 (0.111) and nad2 (0.110) may accelerate the accu- and the 18S rDNA gene [42], the complexes of Ligula and mulation of non-synonymous substitutions, which would Digramma have also been shown to be closely related to increase variation of the two genes [48]. These results Dibothriocephalus. Further studies using the concatenated suggest that the nad2, nad4, nad5, nad6and cox3genes nucleotide sequences of 18S rDNA + 28S rDNA + rrnL + should be considered as optimal candidates for genetic cox1, have again demonstrated the genus Dibothriocepha- markers to be used for population genetics and species lus to be the sister group of Ligula [5]. These phylogenetic identification studies between the two closely related results suggest that Dibothriocephalus is the most closely species, D. interrupta and L. intestinalis. related genus to Ligula and Digramma. However, mitogenome sequence identity between D. Conclusions interrupta and L. intestinalis is92.7%, whichismuchhigher The complete mitogenomes of Ligula intestinalis and than between either of these species and the represented Digramma interrupta were sequenced and characterized. members of Dibothriocephalus (85.8–86.5%). Furthermore, The mitogenomes of these two species show a higher high mitogenome sequence identity was also found be- identity to each other than to any species in closely related tween the congeners in Dibothriocephalus (92.3%), Diphyl- genera. The two mitogenomes consistently support D. lobothrium (99.2%) and Spirometra (99.3%). These results interrupta to be a congeneric species with L. intestinalis. suggest that sequence differences between D. interrupta High sequence variation in the nad2, nad4, nad5, nad6 and L. intestinalis are of a degree expected between mem- and cox3 genes between the two diphyllobothriids suggest bers of the same genus. that these five genes should be considered as optimal In one study, sequence identity of the cox1and nad1 candidates for genetic markers when studying population genes between D. interrupta and L. intestinalis has been genetics or looking to differentiate the two closely related shown to be 100% and 92.6% [11]; however, identity was species, D. interrupta and L. intestinalis. deemed at 93.5% and 93.0% in the present study, respect- ively (Table 1). This inconsistency may be due to the use Additional files of partial sequence of cox1and nad1 genes or resulting from the use of formalin-preserved specimens [12]. The Additional file 1: Table S1. Primers used to amplify and sequence the gene cox1 is considered to be a useful barcode for mitochondrial genome of Digramma interrupta and Ligula intestinalis. metazoans [43], and widely employed for cestode studies (DOCX 15 kb) [44–47]. The two mitochondrial genes cox1and cytb have Additional file 2: Table S2. The list of cestode species and outgroups also been used to study the population genetic structure used for comparative mitogenomic and phylogenetic analyses, and accession number, A+T content and skewness of different elements of of L. intestinalis on a local and global scale [14]. However, each mitogenome. (XLSX 19 kb) a lower sequence identity was found in the nad2, nad4, Li et al. Parasites & Vectors (2018) 11:324 Page 10 of 11 3. Waeschenbach A, Webster BL, Bray RA, Littlewood DTJ. Added resolution Additional file 3: Table S3. General statistics (length and codons) for among ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) mitochondrial protein-coding genes and rRNAs of 38 cestodes. Abbreviations with complete small and large subunit nuclear ribosomal RNA genes. Mol of species name are the initials of genus and species name combined. Phylogenet Evol. 2007;45:311–25. (XLSX 21 kb) 4. Kuchta R, Scholz T. Diphyllobothriidea. In: Caira JN, Jensen J, editors. Additional file 4: Figure S1. Relative Synonymous Codon Usage (RSCU) Planetary Biodiversity Inventory (2008–2017): Tapeworms from vertebrate of Digramma interrupta and Ligula intestinalis. Codon families are labelled bowels of the earth. The University of Kansas Natural History Museum on the x-axis. Values on the top of the bars denote amino acid usage. Special Publication No. 25; 2017. (PDF 37 kb) 5. Waeschenbach A, Brabec J, Scholz T, Littlewood DTJ, Kuchta R. The catholic taste of broad tapeworms-multiple routes to human infection. 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