2urn:lsid:arphahub.com:pub:45048D35-BB1D-5CE8-9668-537E44BD4C7Eurn:lsid:zoobank.org:pub:91BD42D4-90F1-4B45-9350-EEF175B1727AZooKeysZK1313-29891313-2970Pensoft Publishers10.3897/zookeys.945.4968149681Research ArticleTrematodaGeneticsCenozoicAsiaCharacterization and comparative analysis of the complete mitochondrial genome of Azygiahwangtsiyui Tsin, 1933 (Digenea), the first for a member of the family AzygiidaeWuYuan-An1GaoJin-Weihttps://orcid.org/0000-0003-0551-13391ChengXiao-Feihttps://orcid.org/0000-0002-1294-264X1XieMin1YuanXi-Ping1LiuDong1SongRuiryain1983@163.com1Hunan Fisheries Science Institute, Changsha 410153, ChinaHunan Fisheries Science InstituteChangshaChina
Corresponding author: Rui Song (ryain1983@163.com)
Academic editor: D. Gibson
20200307202094511670FA0F2F-7193-5A9B-8E9A-16ECE40BEBE14EE745AE-3269-4266-8CAD-FF4B67F969FB39398952612201904052020Yuan-An Wu, Jin-Wei Gao, Xiao-Fei Cheng, Min Xie, Xi-Ping Yuan, Dong Liu, Rui SongThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.http://zoobank.org/4EE745AE-3269-4266-8CAD-FF4B67F969FB
Azygiahwangtsiyui (Trematoda, Azygiidae), a neglected parasite of predatory fishes, is little-known in terms of its molecular epidemiology, population ecology and phylogenetic study. In the present study, the complete mitochondrial genome of A.hwangtsiyui was sequenced and characterized: it is a 13,973 bp circular DNA molecule and encodes 36 genes (12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes) as well as two non-coding regions. The A+T content of the A.hwangtsiyui mitogenome is 59.6% and displays a remarkable bias in nucleotide composition with a negative AT skew (–0.437) and a positive GC skew (0.408). Phylogenetic analysis based on concatenated amino acid sequences of twelve protein-coding genes reveals that A.hwangtsiyui is placed in a separate clade, suggesting that it has no close relationship with any other trematode family. This is the first characterization of the A.hwangtsiyui mitogenome, and the first reported mitogenome of the family Azygiidae. These novel datasets of the A.hwangtsiyui mt genome represent a meaningful resource for the development of mitochondrial markers for the identification, diagnostics, taxonomy, homology and phylogenetic relationships of trematodes.
Wu Y-A, Gao J-W, Cheng X-F, Xie M, Yuan X-P, Liu D, Song R (2020) Characterization and comparative analysis of the complete mitochondrial genome of Azygia hwangtsiyui Tsin, 1933 (Digenea), the first for a member of the family Azygiidae. ZooKeys 945: 1–16. https://doi.org/10.3897/zookeys.945.49681
Introduction
The genus Azygia Looss, 1899 is an endoparasitic helminth found in the stomach and intestine of freshwater feral carnivorous fish (Frolova and Shcherbina 1975). This genus includes several species complexes and its type species is Azygialucii (Müller, 1776), which is a parasite of numerous, but especially esocid and percid, fishes in Europe. Many researchers have added to our knowledge of this cosmopolitan species. To date, species of Azygia are frequently reported from the esophagus, stomach, and intestine of a wide range of predatory fishes from Asia, Europe and North America, including China, Japan, India, Russia, Germany, and North America (Tubangui 1928; Tsin 1933; Moravec and Sey 1989; Marcogliese and Cone 1996; Besprozvannykh 2005; Jadhav et al. 2011; Pallewad et al. 2015; Womble et al. 2016; Nagasawa and Katahira 2017).
Azygiahwangtsiyui Tsin, 1933 is a member of the family Azygiidae Odhner, 1911 and is often overlooked; it is parasitic in the gastrointestinal tract of species of the family Channidae Fowler, 1934 but caused only slight clinical signs, including malnutrition and weight loss. In China, Azygiahwangtsiyui-infected freshwater predatory fishes have been described from Shandong, Heilongjiang, Jiangsu, Fujian, Sichuan and Hunan Provinces (Tsin 1933; Zmejev 1936; Ma 1958; Tang and Tang 1964; Kiang 1965; Chen 1973; Wang 1985; Cheng 2011). It has a mainly inland distribution and utilizes freshwater snail species (e.g. Viviparaquadrata (Benson, 1842)) as intermediate hosts (Tang and Tang 1964) and develops into adults in the gastrointestinal tract of predatory fish species such as Ophiocephalusargus Cantor, 1842 and Channaasiatica (Linnaeus, 1758) (Tsin 1933; Besprozvannykh 2005).
Morphology is the most commonly used method for species identification and differentiation of metazoans and is widely adopted globally by parasitologists and taxonomists. A huge disadvantage of using morphological criteria, however, is that it is difficult to identify and distinguish closely related and cryptic species. Although the family Azygiidae was erected more than a century ago, its situation, and that of several species of Azygia, is still controversial and uncertain. Manter (1926) pointed out that Azygia is the only genus in the family then presenting systematic confusion: Azygialonga (Leidy, 1851) in North America may be a synonym of A.lucii in Europe (Manter 1926), and Van Cleave and Mueller (1934) reported that Azygiaacuminata Goldberger, 1911 and A.longa should be considered conspecific. Nevertheless, due to the discovery of some life histories of members of Azygia, A.lucii and A.longa have been recognized as two distinct species (Szidat 1932; Sillman 1953; Sillman 1962).
Mitochondrial (mt) genome and nuclear ribosomal DNA sequences are effective molecular tools for taxonomic identification, phylogeny and biogeographical research (Bernt et al. 2013; Le et al. 2019). However, only a partial cytochrome oxidase subunit 1 protein sequence (AIY67834) of Proterometramacrostoma Horsfall, 1933 (Azygiidae) is currently available in GenBank. None of the mitochondrial genome data have been sequenced for a member of the family Azygiidae. Therefore, we determined the complete mitochondrial genome sequence of A.hwangtsiyui as a basis for the future definition of strain- and species-specific markers, and for assessing mitogenomics in resolving the interrelationships of trematodes.
Materials and methodsSampling and DNA extraction
The specimens of flatworms were isolated from the stomach of their definitive host, in this case snakehead fish (Ophiocephalusargus (Cantor, 1842)) obtained from east Dongting Lake in Yueyang, Hunan province, China (29°22'N, 113°06'E). Azygiahwangtsiyui was morphologically identified according to the original and other descriptions (Tsin 1933; Tang and Tang 1964; Zhang et al. 1999; Besprozvannykh 2005), using a stereomicroscope and a light microscope. Furthermore, single samples were confirmed molecularly as A.hwangtsiyui based on sequencing of 1370 bp 28S rDNA sequence. The parasites were completely washed in water, preserved in 99% ethanol, and stored at 4 °C until genomic DNA extraction. Total genomic DNA extraction was performed from an intact specimen with the TIANamp Micro DNA Kit (Tiangen Biotech, Beijing, China), according to the manufacturer’s instructions.
DNA amplification and sequencing
According to conserved regions of mitochondrial genes in other available digenea mitogenomes, six partial gene fragments for cytb, nad4, nad1, 16S, 12S and cox2 were amplified using six generic primers sets HWF1/HWR1 (for cytb), HWF3/HWR3 (for nad4), HWF5/HWR5 (for nad1), HWF7/HWR7 (for 16S), HWF9/HWR9 (for 12S), and HWF11/HWR11 (for cox2), respectively. On the basis of these obtained nucleotide sequences, A.hwangtsiyui-specific primers were designed for amplification and sequencing of the remaining mitogenome (Suppl. material 1: Table S1). All primers were designed to produce amplicons with overlaps of approximately 100 bp. PCR reactions were performed in a 50 µl reaction solution with the ingredient of 18.5 µl ddH2O, 25 µl 2×PCR buffer (Mg2+, dNTP plus, Takara, Dalian, China), 1.5 µl of each primer (0.2–1.0 μM), 1 µl EX Taq polymerase (250U, Takara), and 2.5 µl DNA template. PCR amplification was compliant to the following amplification protocol: initial denaturation at 98 °C for 2 min, followed by 40 cycles 10 s at 98 °C, 15 s at 50 °C, 68 °C for 1 min/kb, and 10 min at 68 °C for a final extension. The amplified PCR products were purified with TIANgel Purification Kit (Tiangen Biotech, Beijing, China), and sequenced bidirectionally at Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) based on the primer walking method using several specific primers (Suppl. material 1: Table S1).
Mitogenome annotation and analysis
According to sequence chromatograms, all raw fragments were quality-proofed using CHROMAS (https://www.technelysium.com.au) to remove ambiguity codes and low-quality bases. Whenever the quality was sub-optimal, sequencing was repeated until the amplicon is the consensus sequence. Before manual assembly of the entire mitochondrial genomic sequence, identification of all amplicons was performed by BLASTN check (Altschul et al. 1990). The mt genome of A.hwangtsiyui was aligned against the mt genome sequences of other promulgated digenean mitogenomes utilizing multiple sequence alignment software MAFFT version 7.149 (Katoh and Standley 2013) to identify genetic boundary. Protein-coding genes (PCGs) were predicted with Open Reading Frame Finder (https://www.ncbi.nlm.nih.gov/orffinder/) adopting echinoderm and flatworm mitochondrial codes, and examining the nucleotide alignment against the reference mtDNA in trematode Dicrocoeliumchinense Tang et Tang, 1978 (NC_025279.1). Whole tRNAs were inferred with the detection results of ARWEN (Laslett and Canback 2008) and MITOS web server (Bernt et al. 2013). Two rRNA (rrnL and rrnS) were founding by comparison with those of published fluke mitogenomes. Codon usage and relative synonymous codon usage (RSCU) for 12 PCGs of A.hwangtsiyui were computed by PHYLOSUITE (Zhang et al. 2020), and its operation results were imported into GGPLOT2 program (Wickham 2016) to make figures of the RSCU. Tandem repeats in the non-coding regions were determined with Tandem Repeats Finder software version 4.09 (Benson 1999), and the prediction of their secondary structures were performed by the MFOLD web server (Zuker 2003). The annular diagram of A.hwangtsiyui mitogenome was plotted with mitochondrial genome data visualization tool MTVIZ (http://pacosy.informatik.uni-leipzig.de/mtviz/mtviz).
Phylogenetic analysis
For phylogenetic analyses, we utilized translated and concatenated amino acid sequences of twelve protein-coding genes for 49 Platyhelminthes including A.hwangtsiyui mitogenome determined in this study. Two tapeworm species, Cloacotaeniamegalops (Nitzsch in Creplin, 1829) (NC_032295.1) and Dibothriocephaluslatus (Linnaeus, 1758) (NC_008945.1) were included as outgroup taxa representing two different families. Species information including systematic positions and GenBank accession numbers is provided in Suppl. material 2: Table S2. The PHYLOSUITE program was used to extract twelve PCGs from the GenBank files, export fasta files with translated amino acid datasets, and align datasets in bulk using integrated applet MAFFT with normal-alignment mode. Phylogenetic analyses were performed using Bayesian Inference (BI) and Maximum Likelihood (ML) methods. Assessment of the best-fit evolutionary model for dataset was conducted via ModelGenerator v0.8527 (Keane et al. 2006). BI in MrBayes version 3.2.6 (Ronquist et al. 2012) was carried out under the MtRev matrix of amino acid substitution, and was analyzed with 1 × 107 metropolis-coupled Monte Carlo Markov Chain (MCMC) generations. Two independent runs with four simultaneous MCMC chains (one cold and three heated chains) were conducted for 1 × 107 million generations, sampling every 10,000 generations and discarding the initial 25% generations as burn-in. ML analysis in PHYLOSUITE was performed using MtART+I+G matrix with 1000 bootstrap replicates.
Results and discussionGeneral traits of the Azygiahwangtsiyui mitogenome
The entire A.hwangtsiyui mtDNA is 13,973 bp in length (GenBank accession number: MN844889) and comprised of 12 protein-coding genes (cox1-3, nad1-6, nad4L, cytb, and atp6), 22 tRNA genes, two rRNA genes (rrnL and rrnS), and two non-coding regions. The 12 protein-coding gene order arrangement is cox3-cytb-nad4L-nad4-atp6-nad2-nad1-nad3-cox1-cox2-nad6-nad5 (Fig. 1), which is identical to those of Clinostomumcomplanatum (Rudolphi, 1814), Echinostomahortense Asada, 1926, and some species of the Fasciolidae (Fasciolahepatica Linneuus, 1758, Fasciolagigantica Cobbold, 1856, and Fasciola sp. GHL-2014) (Liu et al. 2014a; Chen et al. 2016; Liu et al. 2016); the gene atp8 is similarly missing, as usual in trematode species. All genes are transcribed in the anticlockwise direction and encoded by H strand (Table 1), which is in accordance with other digeneans. The mt genome of A.hwangtsiyui has 22 intergenic spacers ranging from 1 to 15 bp and contains two overlapping nucleotides ranging from 1 to 40 bp (Table 1). Noteworthily, a 40 bp overlap between the nad4 and nad4L genes exists in the A.hwangtsiyui mitogenome, which is consistent with most helminths such as Eurytremapancreaticum Janson, 1889 (Chang et al. 2016), Hypoderaeumconoideum (Bloch, 1782) (Yang et al. 2015), but shorter than that of Schistosomamekongi Voge, Bruckner & Bruce, 1978 (64 bp; Littlewood et al. 2006). The nucleotide contents of T, C, A, G, in A.hwangtsiyui mitogenome are 42.8%, 12.0%, 16.8%, and 28.5%, respectively (Table 2). The whole A+T content of the mitogenome is 59.6%, which was markedly biased toward T over A (AT skew: –0.437), and G over C (GC skew: 0.408).
The organization of the mitochondrial genome of Azygiahwangtsiyui.
Gene
Position
Size
Intergenic nucleotides
Codon
Anti-codon
Strand
From
To
Start
Stop
cox3
1
660
660
–
ATG
TAG
–
H
trnH
666
729
64
+5
–
–
GTG
H
cytb
732
1841
1110
+2
ATG
TAG
–
H
nad4L
1848
2108
261
+6
ATG
TAG
–
H
nad4
2069
3340
1272
–40
ATG
TAG
–
H
trnQ
3345
3409
65
+4
–
–
TTG
H
trnF
3423
3488
66
+13
–
–
GAA
H
trnM
3490
3555
66
+1
–
–
CAT
H
atp6
3556
4068
513
–
ATG
TAG
–
H
nad2
4072
4932
861
+3
GTG
TAG
–
H
trnV
4946
5009
64
+13
–
–
TAC
H
trnA
5013
5076
64
+3
–
–
TGC
H
trnD
5081
5146
66
+4
–
–
GTC
H
nad1
5149
6054
906
+2
GTG
TAG
–
H
trnN
6070
6134
65
+15
–
–
GTT
H
trnP
6148
6212
65
+13
–
–
TGG
H
trnI
6216
6279
64
+3
–
–
GAT
H
trnK
6280
6348
69
–
–
–
CTT
H
nad3
6349
6708
360
–
ATG
TAA
–
H
trnS1
6712
6770
59
+3
–
–
GCT
H
trnW
6781
6842
62
+10
–
–
TCA
H
cox1
6843
8396
1554
–
TTG
TAG
–
H
trnT
8410
8474
65
+13
–
–
TGT
H
rrnL
8475
9449
975
–
–
–
–
H
trnC
9450
9506
57
–
–
–
GCA
H
rrnS
9507
10246
740
–
–
–
H
cox2
10247
10828
582
–
GTG
TAA
–
H
nad6
10834
11277
444
+5
GTG
TAG
–
H
trnY
11284
11352
69
+6
–
–
GTA
H
trnL1
11352
11416
65
–1
–
–
TAG
H
trnS2
11421
11490
70
+4
–
–
TGA
H
trnL2
11491
11555
65
–
–
–
TAA
H
trnR
11558
11617
60
+2
–
–
TCG
H
nad5
11626
13225
1600
+8
GTG
T
–
H
trnE
13226
13288
63
–
–
–
TTC
H
trnG
13604
13669
66
–
–
–
TCC
H
Nucleotide contents of genes and the non–coding region within the mitochondrial genome of Azygiahwangtsiyui.
An annular diagram of the Azygiahwangtsiyui mitochondrial genome.
https://binary.pensoft.net/fig/428055Protein-coding genes and non-coding regions
A total of 3364 amino acids was encoded by the A.hwangtsiyui mtDNA. The full scale of 12 concatenated protein-coding genes was 10126 bp, composed of 45.2% T, 11.5% C, 14.7% A, and 28.6% G. Average A+T content of concatenated 12 protein-coding genes was 59.9%, varying from 57.7% (cox2) to 64.8% (nad4L) (Table 2 and Suppl. material 2: Table S2). All 12 protein-coding genes of A.hwangtsiyui mt genome have a lower A+T percentage than those of Trichobilharziaszidati Neuhaus, 1952, Calicophoronmicrobothrioides Price & McIntosh, 1944, and some members of the Schistosomatidae Poche, 1907, but possess a higher A+T percentage than those of Metagonimusyokogawai Katsurada, 1912, and Paragonimuswestermani Kerbert, 1878 (Suppl. material 3: Table S3) (Lee et al. 1987; Littlewood et al. 2006; Biswal et al. 2014; Semyenova et al. 2017; Oey et al. 2019). There is an obvious bias towards T over A (AT skew = –0.509), and G over C (GC skew = 0.425), and the coding strand is enriched with T and poor with A and especially C. For A.hwangtsiyui, the length of protein-coding genes was followed in the order: nad5 (1600 bp) > cox1 (1554 bp) > nad4 (1272 bp) > cytb (1110 bp) > nad1 (906 bp) > nad2 (861 bp) > cox3 (660 bp) > cox2 (582 bp) > atp6 (513 bp) > nad6 (444 bp) > nad3 (360 bp) > nad4L (264 bp). There are two non-coding regions (NCR1 and NCR2) in A.hwangtsiyui mitogenome, while the mt genome of Paragonimusheterotremus Chen et Hsia (1964), C.complanatum, Fascioloidesmagna (Bassi, 1875), and T.szidati have a single non-coding region (Chen et al. 2016; Ma et al. 2016; Semyenova et al. 2017; Qian et al. 2018). NCR1 and NCR2 of the A.hwangtsiyui mitogenome is partitioned into two parts by trnG, and accompanied by 70.5% and 57.6% A+T content, respectively. NCR1 and NCR2 have similar chemical base counts, 315 bp and 317 bp in size, respectively. While the NCR1 lacks distinguishing features and any tandem repeats, the NCR2 contains two typical tandem repeats, and each of tandem repeats sequence (120 bp) forms a hairpin-like secondary structure including a whole set of stems and loops (Suppl. material 4: Figure S1). Although tandem repeats are a segment of function-deficiency mitochondrial genome sequences, its hairpin-like secondary structures are widely perceived as regulating the replication and transcription of mitochondrial genome.
Codon usage, transfer RNAs, and ribosomal RNAs
For the A.hwangtsiyui mitogenome, codon ends in G or T were more continual than those ending in A or C. The most frequently used start codon in protein-coding genes was ATG (for six PCGs), secondly was GTG (for five PCGs), which resembles that of the most frequent extrapolated start codons for mitogenome protein-encoding genes of digenean species (Chen et al. 2016). The least-used start codon was TTG (only one PCGs). Likewise, the most-used terminal codon was TAG (for nine PCGs), followed by TAA (for two PCGs). Only one of 12 protein-coding sequence (nad5) was terminated with abbreviated T stop codon (Table 1). Although incomplete stop codons (T or TA) frequently occur in cestodes and nematodes, they were rarely presented in flukes other than D.chinensis, Dicrocoeliumdendriticum (Rudolphi, 1819), and Postharmostomumcommutatum (Dietz, 1858) (Liu et al. 2014b; Fu et al. 2019). The codon UUU (Phe, 10.17%), UUG (Leu, 8.17%), and GUU (Val, 7.19%) were the most frequently occurring codons in protein-coding genes. Leucine, valine, and phenylalanine are the most-used amino acids, with frequency of 15.96%, 13.38%, and 11.09%, respectively. The least-used codons were CGA (Arg, 0.06%) and GAC (Asp, 0.15%), and the least frequent utilized amino acid was glutamine (1.01%) (Suppl. material 5: Figure S2). As most of digenea mitogenome sequences, the mitogenome of A.hwangtsiyui possessed 22 commonly found tRNAs, with the exception of that of P.westermani Korean isolate (23 tRNAs), and P.westermani Indian isolate (24 tRNAs) (Biswal et al. 2014). In A.hwangtsiyui, tRNA-Gly (trnG) is located between NCR1 and NCR2 (Fig. 1). The size of ribosomal RNA genes (rrnL and rrnS) in mitochondrial DNA of A.hwangtsiyui are 975 bp and 740 bp, respectively (Table 2). The upstream and downstream of rrnL and rrnS are cascaded with trnT and cox2 genes, respectively, and are detached from each other by trnC, as in all reported platyhelminths to date (Littlewood et al. 2006, Le et al. 2016).
Gene arrangement
Comparative analysis of gene arrangement among 47 selected digenean taxa, two gene blocks (cox1-trnT-rrnL-trnC-rrnS-cox2-nad6 and cytb-nad4L-nad4-trnQ) are shared by all selected taxa (Fig. 2). Disregarding P.heterotremus and members of the family Schistosomatidae and Fasciolidae Raillet, 1895, the gene order of the remaining digenea taxa is virtually identical with the exception of the translocation of trnE and trnG among the remaining members of selected digenea representatives in family level. Intriguingly, there is the translocation of trnE and trnG within different species of family Fasciolidae. The translocations of three tRNAs (trnS1, trnS2 and trnS) can be discovered even between taxa of the same subgroup. Gene order of the Brachycladiumgoliath (Van Beneden, 1858) mt genome (the only representative of family Brachycladiidae Faust, 1929) is nearly same as that of P.westermani (Troglotrematidae Ward, 1918) except for the relocations of trnY between trnG and cox3, and trnE to the position between trnN and trnP. The groups of Schistosomatidae show a massive gene reorganization of protein-coding genes and tRNAs compared with other sequenced digenea mitogenome, which is in accord with previous finding reported by Littlewood et al. (2006).
Phylogenetic relationships and gene arrangement of Azygiahwangtsiyui with other selected digeneas based on translated mitochondrial proteins. The concatenated amino-acid sequence datasets of the 12 protein-coding genes were analyzed by Bayesian Inference (BI) and Maximum Likelihood (ML), utilizing Cloacotaeniamegalops (NC_032295.1) and Dibothriocephaluslatus (NC_008945.1) as the outgroups. Both ML and BI analyses constructed identical tree topologies.
To assess phylogenetic relationships among available flatworms, we utilized concatenated amino acid sequence dataset representing 12 protein-coding genes of A.hwangtsiyui, 46 other digenean representatives, and two tapeworm species (C.megalops and D.latus) for analyzing molecular-based phylogeny. In this study, the topological structure is divided into two large clades: one consists of seven members of the family Schistosomatidae; and the other clade comprises 40 members from 16 families including the family Azygiidae (A.hwangtsiyui) (Fig. 2). The topological structure shows that A.hwangtsiyui (Azygiidae) is identified as the most basal lineage of the Digenea, but separated from C.complanatum (Clinostomidae Lühe, 1901), and Cyathocotyleprussica Mühling, 1896 (Cyathocotylidae Poche, 1926). Phylogenetic analyses of all complete digenea mtDNAs confirmed taxonomic and previous phylogenetic assessments (Olson et al. 2003; Kostadinova and PéRez-del-Olmo 2014; Fu et al. 2019). The intricate structure and varying content of the family Azygiidae still awaits investigation of relationships based on a much wider taxon sampling and more mitogenome datasets.
Acknowledgments
This work was supported by the Earmarked Fund for China Agriculture Research System (CARS-45) and Important Research Project of Hunan Provincial Science and Technology Department (2016NK2176).
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Table S1
molecular data
Primers for amplification and sequencing mitochondrial genome of Azygiahwangtsiyui.
https://binary.pensoft.net/file/428057This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Yuan-An Wu, Jin-Wei Gao, Xiao-Fei Cheng, Min Xie, Xi-Ping Yuan, Dong Liu, Rui Song10.3897/zookeys.945.49681.suppl2393990318CEB78A-9321-5447-A2F1-D7BAA335AB98
Table S2
molecular data
Information of the Digenea and the outgroups for which complete mitogenomes are available in GenBank.
https://binary.pensoft.net/file/428058This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Yuan-An Wu, Jin-Wei Gao, Xiao-Fei Cheng, Min Xie, Xi-Ping Yuan, Dong Liu, Rui Song10.3897/zookeys.945.49681.suppl3393990569071E2A-C3CB-5B9A-ADF8-57EFBE1A8083
Table S3
molecular data
A+T content (%) for 12 protein-coding genes in the available 49 Platyhelminthes mitogenomes.
https://binary.pensoft.net/file/428059This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Yuan-An Wu, Jin-Wei Gao, Xiao-Fei Cheng, Min Xie, Xi-Ping Yuan, Dong Liu, Rui Song10.3897/zookeys.945.49681.suppl43939911706B0859-8AC5-5D54-870A-F96E6D170850
Figure S1
molecular data
Secondary structure of tandem repeats in non-coding region 2 (NCR2) of Azygiahwangtsiyui mitochondrial genome.
https://binary.pensoft.net/file/428060This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Yuan-An Wu, Jin-Wei Gao, Xiao-Fei Cheng, Min Xie, Xi-Ping Yuan, Dong Liu, Rui Song10.3897/zookeys.945.49681.suppl539399011EE62074-E6F3-5A97-81F4-BAB72E1CFB3E
Figure S2
molecular data
Relative synonymous codon usage (RSCU) of Azygiahwangtsiyui mitochondrial genome.
https://binary.pensoft.net/file/428061This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Yuan-An Wu, Jin-Wei Gao, Xiao-Fei Cheng, Min Xie, Xi-Ping Yuan, Dong Liu, Rui Song