Introduction
Species of Contracaecum Railliet & Henry, 1912 (Ascaridoidea: Anisakidae) usually parasitise the digestive tract of various piscivorous birds and pinniped marine mammals. They represent an important group of parasitic nematodes of veterinary, medical, and economic importance (Anderson Reference Anderson2000; Fagerholm and Overstreet Reference Fagerholm, Overstreet, Atkinson, Thomas and Hunter2009; Mozgovoi Reference Mozgovoi and Skrjabin1953). Recently, some nuclear and mitochondrial sequence data have been used for the molecular identification and phylogeny of Contracaecum spp. (Caffara et al. Reference Caffara, Tedesco, Davidovich, Locke, Gustinelli, King, Nuytten, Nuzzo and Fioravanti2023; Garbin et al. Reference Garbin, Servián, Fuentes, Morgenthaler, Millones, Fuchs, Diaz and Navone2024; Mattiucci et al. Reference Mattiucci, Paoletti, Solorzano and Nascetti2010, Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020; Presswell and Bennett Reference Presswell and Bennett2022; Shamsi et al. Reference Shamsi, Gasser, Beveridge and Shabani2008, Reference Shamsi, Norman, Gasser and Beveridge2009, Reference Shamsi, Nelson, Gordon, Markham, Francis, Suthar and Zhu2024). Additionally, some previous studies have demonstrated that mitochondrial genomes are valuable and significant for the DNA taxonomy, systematics, and phylogeny of zooparasitic nematodes (Chen et al. Reference Chen, Qiao, Zhao, Gu and Li2025; Gu et al. Reference Gu, Guo, Chen, Sitko, Li, Guo and Li2023, Reference Gu, Mu, Chen and Li2025; Liu et al. Reference Liu, Nadler, Liu, Podolska, D’Amelio, Shao, Gasser and Zhu2016; Sun et al. Reference Sun, Yousaf, Harras, Gibson, Chen, Elmahy and Li2025; Yousaf et al. Reference Yousaf, Sun, Chen, Khan and Li2025; Zeng et al. Reference Zeng, Chen, Ni, Kang and Li2024a,Reference Zeng, Chen, Xu and Lib). However, the current mitogenomic database for Contracaecum spp. is very limited. To date, complete mitogenomes have been reported only for C. rudolphii, C. osculatum, C. ogmorhini, and an unidentified species of Contracaecum (Deng et al. Reference Deng, Wang, Zhu, Zhang, Li, Zhang and Zhao2022; Lin et al. Reference Lin, Liu, Zhang, D’Amelio, Zhou, Yuan, Zou, Song and Zhu2012; Liu et al. Reference Liu, Nadler, Liu, Podolska, D’Amelio, Shao, Gasser and Zhu2016; Mohandas et al. Reference Mohandas, Jabbar, Podolska, Zhu, Littlewood, Jex and Gasser2014).
Contracaecum ovale (Linstow, 1907) is an obligate parasite of podicipediform birds, which was originally described from Podiceps cristatus in Germany but is now reported worldwide (Galeano and Tanzola Reference Galeano and Tanzola2012; Li et al. Reference Li, Xu and Zhang2013, Reference Li, Gibson and Zhang2016; Mozgovoi Reference Mozgovoi1950; Presswell and Bennett Reference Presswell and Bennett2022; Shen and Wu Reference Shen and Wu1964). Presswell and Bennett (Reference Presswell and Bennett2022) sequenced the partial nuclear small ribosomal subunit (18S), large ribosomal subunit (28S), and internal transcribed spacer 1 (ITS-1) regions of C. ovale based on specimens from the Australasian crested grebe P. cristatus australis (Gould) in New Zealand. However, data on mitochondrial cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 2 (cox2), and small subunit ribosomal RNA gene (12S) of C. ovale are still unavailable.
In this study, several specimens of C. ovale were collected from Tachybaptus ruficollis (Pallas) (Podicipediformes: Podicipedidae) in China. In order to investigate the genetic variation between the two different populations of C. ovale, i.e. from T. ruficollis in China and P. cristatus australis in New Zealand, the 18S, 28S, ITS, cox1, cox2, and 12S data for the present samples were sequenced and analysed. The mitogenome of C. ovale was also sequenced and annotated for the first time to further characterise the mitogenomes of Contracaecum species and enrich the mitogenomic database for zooparasitic nematodes. Moreover, preliminary molecular phylogenetic analyses, including currently limited representatives of Contracaecum, were undertaken in order to investigate the evolutionary relationships of C. ovale and its congeners.
Material and methods
Parasite collection and species identification
A total of 4 nematode individuals were collected from the alimentary canal of the little grebe T. ruficollis, which were washed in clear water and stored in 70% ethanol prior to study. The specimens were identified as C. ovale based on morphological characters according to previous studies (Li et al. Reference Li, Xu and Zhang2013; Mozgovoi Reference Mozgovoi1950), and using the genetic data provided by Presswell and Bennett (Reference Presswell and Bennett2022). Voucher specimens of C. ovale were deposited in the College of Life Sciences, Hebei Normal University, China.
Molecular procedures
The mid-body region of two specimens of C. ovale (1 male, 1 female) was used for molecular analyses. The total genomic DNA was extracted using the Magnetic Universal Genomic DNA Kit (DP705) [Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China], following the manufacturer’s instructions. The primers used for amplifying the partial 18S, ITS, 28S, cox1, cox2, and 12S target sequences are provided in Table 1. The cycling conditions for the PCR were as described previously (Li et al. Reference Li, Lü, Nadler, Gibson, Zhang, Chen, Zhao and Guo2018). PCR products were checked on GoldView-stained 1.5% agarose gels and purified with a Column PCR Product Purification Kit (Shanghai Sangon, China). Sequencing for each amplification product was carried out from both directions. Sequences were aligned using ClustalW2. All of the sequences obtained herein were deposited in the GenBank database (http://www.ncbi.nlm.nih.gov) under the following accession numbers: 18S: PX803984, PX803985; ITS: PX803982, PX803983; 28S: PX803986, PX803987; cox1: PX804687, PX804688; cox2: PX852397, PX852398; 12S: PX853353, PX853354.
The primers for amplifying different target regions by polymerase chain reaction (PCR) in the present study
Table 1. Long description
The table contains three columns: Primer, Sequence (5 prime to 3 prime), and Source.
* Primer 18S:
- 18S-F (5 prime-CCCGATTGATTCTGTCGGC-3 prime)
- 18S-R (5 prime-TGATCCTTCTGCAGGTTCACCTAC-3 prime)
- Source: Floyd et al. (2005)
* Primer 28S:
- 28S-F (5 prime-AGCGGAGGAAAAGAAACTAA-3 prime)
- 28S-R (5 prime-ATCCGTGTTTCAAGACGGG-3 prime)
- Source: Nadler and Hudspeth (1998)
* Primer I T S:
- S S 1 (5 prime-GTTTCCGTAGGTGAACCTGCG-3 prime)
- S S 2 R (5 prime-AGTGCTCAATGTGTCTGCAA-3 prime)
- N C 13 (5 prime-ATCGATGAAGAACGCAGC-3 prime)
- N C 2 (5 prime-TTAGTTTCTTTTCCTCCGCT-3 prime)
- Source: Zhu et al. (2000)
* Primer C O I:
- C O I-F (5 prime-TTTTTTGGTCATCCTGAGGTTTAT-3 prime)
- C O I-R (5 prime-ACATAATGAAAATGACTAACAAC-3 prime)
- Source: Lazarova et al. (2006)
* Primer C O I I:
- C O I I-F (5 prime-AATTTTAATTGTAGTCTTTTGTTTGG-3 prime)
- C O I I-R (5 prime-CTATGATTAGCACCACAAATC-3 prime)
- Source: Nadler and Hudspeth (2000)
* Primer 12S:
- 12S-F (5 prime-GTTCCAGAATAATCGGCTA-3 prime)
- 12S-R (5 prime-ATTGACGGATGGTTTGTACC-3 prime)
- Source: Li et al. (2012)
Mitochondrial genome sequencing, assembly and annotation
The genomic DNA library was constructed according to the protocol of Novogene (Tianjin, China), and a total of 50 GB of clean data were generated using the paired-end 150 bp sequencing method on the Illumina NovaSeq 6000 platform by Novogene (Tianjin, China). The methods and procedures for assembly and annotation of the complete mitochondrial genome of C. ovale were based on previous studies (Chen et al. Reference Chen, Qiao, Zhao, Gu and Li2025; Gu et al. Reference Gu, Guo, Chen, Sitko, Li, Guo and Li2023, Reference Gu, Mu, Chen and Li2025; Zeng et al. Reference Zeng, Chen, Ni, Kang and Li2024a, Reference Zeng, Chen, Xu and Lib). The complete mitochondrial genome of C. ovale obtained in this study has been deposited in the GenBank database (https://www.ncbi.nlm.nih.gov) under the accession number PX804114.
Phylogenetic analyses
Phylogenetic trees of Contracaecum spp. were constructed using Bayesian inference (BI) with MrBayes v3.2.7 based on the cox1, cox2, 12S, and ITS-1 data. Anisakis pegreffii was chosen as the outgroup. The ingroup included all species of Contracaecum with the genetic data (Supplementary Tables S1–S5). The nucleotide sequences were aligned in batches using MAFFT with the iterative refinement method of E-INS-I. Ambiguous sites and poorly aligned regions were excluded using BMGE v1.12 (h = 0.4). Substitution models were compared and selected according to the Bayesian information criterion (BIC) by ModelFinder. Optimal nucleotide substitution models (i.e. HKY + F + I model for cox1, HKY + F + I + G4 model for cox2, HKY + F + G4 model for 12S, and K2P + G4 model for ITS-1 data) were identified. Bayesian inference analysis was run for 5 × 106 Markov Chain Monte Carlo (MCMC) generations.
A phylogenetic tree of the Anisakidae was constructed based on the concatenated amino acid sequences of the 12 mitochondrial protein-coding genes (PCGs) using BI with MrBayes 3.2.7. Toxocara canis was chosen as the outgroup. The ingroup included 4 species of Contracaecum and 5 species of other anisakid nematodes with mitogenomic data (Supplementary material, Table S1–S5). The optimal amino acid substitution model selected for mitogenomic phylogeny was BLOSUM62 + I + G.
Results
Molecular identification
The 18S (OL470519), 28S (OL470528), and ITS-1 (OL470520) sequences for C. ovale are registered in the GenBank database, but there are some uncertain nucleotide loci (i.e. K, Y) in the 28S sequence (OL470528). Pairwise comparison of the 18S, 28S, and ITS-1 sequences of the present specimens with those of C. ovale available in GenBank showed 100% similarity in the 18S, 100% (excluding the nucleotide loci of K and Y) in the 28S, and 99.8% in the ITS-1 region. This study also provided the mitochondrial cox1, cox2, and 12S of C. ovale for the first time. Molecular analyses of two different individuals of C. ovale collected from T. ruficollis in China showed no genetic divergence with respect to the cox1 and 12S regions, but 0.73% nucleotide divergence in the cox2 sequences.
Characterisation of the complete mitogenome of Contracaecum ovale
The complete mitogenome of C. ovale is 13,769 bp in length, and includes 36 genes, i.e. 12 PCGs (cox1–3, nad1–6, nad4L, cytb, and atp6; missing atp8), 22 tRNA genes, and 2 ribosomal RNAs (rrnS and rrnL), plus two non-coding regions (NCR1 is 421 bp, located between trnS2 and trnN; NCR2 is 127 bp, located between nad4 and cox1) (Figure 1, Table 2). All genes are encoded on the same strand and in the same direction. The overall A + T content in the mitogenome of C. ovale is 71.5%, displaying a strong A + T bias. The nucleotide content of the mitogenome of C. ovale is provided in Table 3.
Gene map of the complete mitochondrial genome of Contracaecum ovale. All 22 tRNA genes are designated by one-letter amino acid code, with numbers differentiating the two serine and two leucine tRNA genes.
Figure 1. Long description
At the center of the diagram is the text Contracaecum ovale mitochondrion genome 13,769 b p.
Moving outward, the first track is a scale bar marked from 1 k b p to 13 k b p in a clockwise direction.
The second track is a green area plot representing G C Skew plus and G C Skew minus. The green peaks are concentrated between 1 k b p and 5 k b p, and again between 11 k b p and 13 k b p.
The third track is a black line plot showing G C Content, which fluctuates around a baseline throughout the genome.
The fourth and outermost track is a thick grey ring containing colored arrows that indicate gene direction and type.
- C D S (blue arrows): Includes c o x 1, c o x 2, n a d 3, n a d 5, n a d 6, n a d 4 L, n a d 1, a t p 6, n a d 2, c y t b, c o x 3, and n a d 4.
- t R N A (pink arrows): Labeled with single letters C, M, D, G, H, A, P, V, E, W, S 2, N, Y, S 1, L 2, K, I, R, Q, F, L 1, and T.
- r R N A (green arrows): r r n L and r r n S.
- N C R (orange arrows): N C R 1 and N C R 2.
A legend in the bottom right corner defines the color coding: blue for C D S, pink for t R N A, green for r R N A, orange for N C R, black for G C Content, green for G C Skew plus, and purple for G C Skew minus.
Organisation of the mitochondrial genome of Contracaecum ovale. ‘Ini/Ter cod’ and ‘Int seq’ indicate initial/terminal codons and the length of intergenic sequences, respectively
Table 2. Long description
A circular genome map with genes color-coded by function.
* Protein-coding genes (green): cox1, cox2, nad3, nad5, nad6, nad4L, nad1, atp6, nad2, cytb, cox3, and nad4.
* Ribosomal R N A genes (blue): rrnL and rrnS.
* Transfer R N A genes (red): trnC, trnM, trnD, trnG, trnH, trnA, trnP, trnV, trnW, trnE, trnS2, trnN, trnY, trnK, trnL2, trnS1, trnI, trnR, trnQ, trnF, trnL1, and trnT.
* Non-coding regions (yellow): N C R 1 and N C R 2.
Clockwise gene order starting from the top:
cox1, trnC, trnM, trnD, trnG, cox2, trnH, rrnL, nad3, nad5, trnA, trnP, trnV, nad6, nad4L, trnW, trnE, rrnS, trnS2, N C R 1, trnN, trnY, nad1, atp6, trnK, trnL2, trnS1, nad2, trnI, trnR, trnQ, trnF, cytb, trnL1, cox3, trnT, nad4, and N C R 2.
All genes are transcribed in the same direction as indicated by a large clockwise arrow in the center of the circle.
Base composition and skewness of mitochondrial genome of Contracaecum ovale
Table 3. Long description
The table contains 11 rows and 9 columns. The columns are labeled: Gene/region, A percent, T percent, C percent, G percent, A T percent, A T-skew, G C-skew, and Total (b p).
* Whole mitochondrial genome: A 22.35, T 49.15, C 8.36, G 20.15, A T 71.49, A T-skew minus 0.37, G C-skew 0.41, Total 13769 b p.
* Protein coding genes (P C G s): A 19.55, T 51.08, C 8.45, G 20.91, A T 70.64, A T-skew minus 0.45, G C-skew 0.42, Total 10295 b p.
* 1st codon: A 26.71, T 41.25, C 8.45, G 23.59, A T 67.96, A T-skew minus 0.21, G C-skew 0.47, Total 3433 b p.
* 2nd codon: A 18.94, T 50.77, C 13.93, G 16.35, A T 69.72, A T-skew minus 0.46, G C-skew 0.08, Total 3431 b p.
* 3rd codon: A 13.00, T 61.24, C 2.97, G 22.79, A T 74.23, A T-skew minus 0.65, G C-skew 0.77, Total 3431 b p.
* t R N As: A 30.79, T 40.98, C 8.98, G 19.25, A T 71.77, A T-skew minus 0.14, G C-skew 0.36, Total 1247 b p.
* r R N As: A 29.15, T 43.99, C 8.23, G 18.63, A T 73.14, A T-skew minus 0.20, G C-skew 0.39, Total 1664 b p.
* r r n S: A 30.5, T 40.71, C 9.36, G 19.43, A T 71.21, A T-skew minus 0.14, G C-skew 0.35, Total 705 b p.
* r r n L: A 28.15, T 46.40, C 7.40, G 18.04, A T 74.56, A T-skew minus 0.24, G C-skew 0.42, Total 959 b p.
* Non-coding region 1: A 36.10, T 47.03, C 6.41, G 10.45, A T 83.13, A T-skew minus 0.13, G C-skew 0.24, Total 421 b p.
* Non-coding region 2: A 33.07, T 45.67, C 3.94, G 17.32, A T 78.74, A T-skew minus 0.16, G C-skew 0.63, Total 127 b p.
The 12 PCGs of C. ovale mitogenome comprise 10,295 bp (excluding termination codons) and encode 3,431 amino acids. The lengths of 12 PCGs vary from 234 bp (nad4L) to 1,587 bp (nad5) (Figure 1, Table 2). Among the 12 PCGs in the mitogenome of C. ovale, 7 genes (cox1, nad3–6, atp6, and cytb) use TTG as the start codon, and the remaining 5 genes (cox2, cox3, nad1, nad2, and nad4L) use ATT, TTG, ATG, GTG, and ATA as start codons, respectively. There are five genes (nad1–4 and cytb) using TAA as the termination codon, and five genes (cox2, nad5, nad6, nad4L, and atp6) using TAG as the termination codon. The remaining two genes (cox1 and cox3) are both inferred to terminate with an incomplete stop codon T (Table 2). The composition and usage of codons in the mitogenome of C. ovale are shown in Figure 2.
Relative synonymous codon usage (RSCU) of Contracaecum ovale. Codon families (in alphabetical order) are indicated below the horizontal axis. Values at the top of each bar represent amino acid usage in percentage.
Figure 2. Long description
A bar chart where the vertical Y axis represents R S C U values from 0 to 8 and the horizontal X axis lists amino acid codon families in alphabetical order. Each bar is composed of colored segments representing specific codons, with the total amino acid usage percentage printed above each bar.
From left to right, the data includes:
* Stop codon asterisk: 0.29 percent usage, segments U A G pink and U A A blue.
* Ala: 3.14 percent, segments G C G pink, G C A blue, G C U yellow, G C C orange.
* Arg: 0.96 percent, segments C G A pink, C G G blue, C G C yellow, C G U orange.
* Asn: 3.43 percent, segments A A C pink, A A U blue.
* Asp: 2.21 percent, segments G A C pink, G A U blue.
* Cys: 1.54 percent, segments U G C pink, U G U blue.
* Gln: 1.22 percent, segments C A G pink, C A A blue.
* Glu: 2.13 percent, segments G A A pink, G A G blue.
* Gly: 5.94 percent, segments G G G pink, G G A blue, G G C yellow, G G U orange.
* His: 1.63 percent, segments C A U pink, C A C blue.
* Ile: 6.15 percent, segments A U U pink, A U C blue.
* Leu: 15.35 percent, segments C U U pink, U U A blue, U U G yellow, C U C orange, C U G purple, C U A green.
* Lys: 2.79 percent, segments A A A pink, A A G blue.
* Met: 5.27 percent, segments A U G pink, A U A blue.
* Phe: 13.81 percent, segments U U U pink, U U C blue.
* Pro: 2.45 percent, segments C C A pink, C C U blue, C C G yellow, C C C orange.
* Ser: 10.70 percent, segments U C G pink, A G C blue, U C A yellow, U C U orange, U C C purple, A G G green, A G A magenta, A G U light orange.
* Thr: 3.35 percent, segments A C C pink, A C U blue, A C A yellow, A C G orange.
* Trp: 2.19 percent, segments U G G pink, U G A blue.
* Tyr: 5.22 percent, segments U A U pink, U A C blue.
* Val: 10.17 percent, segments G U A pink, G U C blue, G U U yellow, G U G orange.
The two ribosomal RNAs, rrnL and rrnS, are 959 bp and 705 bp in size, with 74.6% and 71.2% A + T content, respectively (Tables 2, 3). The rrnL is located between trnH and nad3, and the rrnS between trnE and trnS2 (Figure 1). In the complete mitogenome of C. ovale, 22 tRNAs were identified, ranging from 52 bp (trnS1) to 62 bp (trnK) in length (Table 2). Among them, three tRNAs (trnI, trnK, and trnM) are predicted to be folded into a typical cloverleaf secondary structure. Both tRNAs (trnS1 and trnS2) lack the dihydrouridine (DHU) arm. The remaining tRNAs lack the TψC (T) arm. The lengths of 22 tRNAs and their anticodon secondary structures are provided in Figure 3 and Table 2.
Inferred secondary structures of 22 tRNAs in the mitogenome of Contracaecum ovale. Lines between bases indicate Watson–Crick bonds, dots indicate GU bonds, and bases in red represent anticodons. tRNA, Transfer RNA.
Figure 3. Long description
The diagram consists of 22 panels arranged in five rows. Each panel displays a t R N A cloverleaf structure with an acceptor stem at the top, a D-loop on the left, an anticodon loop at the bottom, and a T-psi-C loop on the right.
Row 1 contains t R N A-Ala, t R N A-Arg, t R N A-Asn, t R N A-Asp, and t R N A-Cys.
Row 2 contains t R N A-Gln, t R N A-Glu, t R N A-Gly, t R N A-His, and t R N A-Ile.
Row 3 contains t R N A-Leu 1, t R N A-Leu 2, t R N A-Lys, t R N A-Met, and t R N A-Phe.
Row 4 contains t R N A-Pro, t R N A-Ser 1, t R N A-Ser 2, t R N A-Thr, and t R N A-Trp.
Row 5 contains t R N A-Tyr and t R N A-Val.
In each structure, the anticodon triplet at the base of the bottom loop is highlighted in red text. For example, t R N A-Ala has the anticodon U G C, and t R N A-Arg has A C G. Watson-Crick base pairs are represented by short lines between letters, while G U wobble pairs are represented by dots. Most structures follow the standard cloverleaf model, though t R N A-Ser 1 and t R N A-Ser 2 lack a fully developed D-arm, appearing more simplified on the left side.
The mitogenome of C. ovale, the gene arrangements of which (12 PCGs, 22 tRNAs, and 2 rRNAs) are shown in Figure 2, belongs to the GA3 type of nematode mitogenome.
Molecular phylogenetic results
The mitogenomic phylogenetic results indicate that representatives of the Anisakidae divide into two monophyletic clades (Clades I and II) (Figure 4). Clade I, including four species of Contracaecum, represents the subfamily Contracaecinae, whereas Clade II, containing species of Anisakis, Pseudoterranova, and Phocanema, represents the subfamily Anisakinae. Within the genus Contracaecum, C. ovale is sister to C. rudolphii + C. ogmorhini.
Bayesian inference (BI) of Anisakidae based on the concatenated amino acid sequences of 12 protein-coding genes (PCGs) of mitogenomes. Toxocara canis was chosen as the outgroup. Bayesian posterior probability values ≥0.95 are shown in the phylogenetic trees.
Figure 4. Long description
A rectangular cladogram rooted on the left. A scale bar at the top left indicates a distance of 0.01.
At the top, a single red branch leads to the outgroup N C 0 1 0 6 9 0 Toxocara canis.
Below this, the main ingroup splits into two primary purple clades.
The upper purple clade contains the genus Contracaecum. It branches into N C 0 2 4 0 3 7 Contracaecum osculatum and a sub-clade containing Contracaecum ovale asterisk, followed by a cluster of N C 0 1 4 8 7 0 Contracaecum rudolphii B, K U 5 5 8 7 2 7 Contracaecum ogmorhini, and K U 5 5 8 7 2 6 Contracaecum ogmorhini. Bayesian posterior probability values of 1 and 0.99 are marked at these nodes.
The lower purple clade branches into two groups. The first group includes M K 8 2 0 6 7 9 Anisakis simplex and N C 0 3 4 3 2 9 Anisakis pegreffii. The second group contains N C 0 3 1 6 4 6 Phocanema krabbei, N C 0 3 1 6 4 5 Pseudoterranova decipiens, K U 5 5 8 7 2 2 Pseudoterranova decipiens, and N C 0 3 1 6 4 3 Phocanema bulbosa. All nodes in this lower section are marked with a posterior probability of 1.
Phylogenetic analyses of Contracaecum spp. based on the ITS-1, cox1, cox2, and 12S sequence data showed different evolutionary relationships between C. ovale and its congeners (Figure 5). The phylogenetic analysis of the ITS-1 data showed that the Chinese specimens clustered with the sample of C. ovale collected from New Zealand.
Bayesian inference (BI) of Contracaecum spp. based on four different genetic markers (A, cox1; B, cox2; C, 12S; D, ITS-1). Anisakis pegreffii was chosen as the outgroup. Bayesian posterior probability values ≥0.70 are shown in the phylogenetic trees.
Figure 5. Long description
A multi-panel figure containing four phylogenetic trees labeled A through D. Each tree includes a scale bar at the top left indicating a tree scale of 0.1. The outgroup Anisakis pegreffii is highlighted in red at the top of each tree.
Panel A (C O X 1 marker): The tree branches from the root into two main clades. The upper clade includes Contracaecum sp. and Contracaecum ovale. The lower clade contains Contracaecum osculatum, Contracaecum rudolphii, and Contracaecum ogmorhini. Bayesian posterior probability values range from 0.747 to 0.999 at the nodes.
Panel B (C O X 2 marker): This tree shows a more complex branching pattern. Major clusters include Contracaecum mirounga and Contracaecum radiatum at the top, followed by a large cluster containing Contracaecum osculatum, Contracaecum quadripapillatum, and Contracaecum ovale. The bottom half of the tree features a dense cluster including Contracaecum micropapillatum, Contracaecum gibsoni, Contracaecum bioccai, and Contracaecum ogmorhini.
Panel C (12 S marker): The tree splits into a small upper clade with Contracaecum ovale and a large lower clade. The lower clade further divides into groups containing Contracaecum osculatum, Contracaecum multipapillatum, Contracaecum septentrionale, and a bottom-most cluster featuring Contracaecum rudolphii and Contracaecum ogmorhini.
Panel D (I T S 1 marker): The tree displays a high density of terminal nodes. Key groupings include Contracaecum osculatum near the top, followed by clusters of Contracaecum quadripapillatum, Contracaecum overstreeti, and Contracaecum micropapillatum. The lower section contains Contracaecum ovale, Contracaecum bioccai, and a large terminal group of Contracaecum rudolphii and Contracaecum ogmorhini.
Discussion
Mozgovoi (Reference Mozgovoi1950) described Contracaecum spasskii from four podicipedid birds. Later, the detailed morphology of C. spasskii was further studied using light and scanning electron microscopy by Li et al. (Reference Li, Xu and Zhang2013) based on specimens collected from T. ruficollis (Podicipediformes: Podicipedidae) in China. Recently, Presswell and Bennett (Reference Presswell and Bennett2022) listed C. spasskii as a synonym of C. ovale, and provided partial 18S, 28S, and ITS-1 data for this species based on specimens from P. cristatus australis in New Zealand. In this study, we also sequenced the 18S, 28S, and ITS-1 regions based on newly collected material from T. ruficollis in China. Our sequences are identical to those of Presswell and Bennett for the 18S and 28S data [excluding the uncertain nucleotide loci (i.e. K, Y) in their 28S sequence]. Additionally, pairwise comparison of the ITS-1 sequences of our material from China with those of Presswell and Bennett’s (Reference Presswell and Bennett2022) from New Zealand showed only a single locus of nucleotide polymorphism. Consequently, the present results support that our specimens are conspecific with C. ovale.
To date, a total of 8 recognised anisakid species have been sequenced for their complete mitogenomes, including three nominal Contracaecum species (Deng et al. Reference Deng, Wang, Zhu, Zhang, Li, Zhang and Zhao2022; Lin et al. Reference Lin, Liu, Zhang, D’Amelio, Zhou, Yuan, Zou, Song and Zhu2012; Liu et al Reference Liu, Liu, Zhu and Weng2015, Reference Liu, Nadler, Liu, Podolska, D’Amelio, Shao, Gasser and Zhu2016; Mohandas et al. Reference Mohandas, Jabbar, Podolska, Zhu, Littlewood, Jex and Gasser2014; Yamada et al. Reference Yamada, Ikeda and Ono2017). This study reports for the first time the complete mitogenome of C. ovale (13,769 bp in length), which is the smallest of the currently known anisakid mitogenomes (13,823–14,022 bp in length). In fact, the mitogenome of C. ovale is also smaller than all of those reported for species of the superfamily Ascaridoidea, with the exception of Seuratascaris physalis bazhaiensis, the mitogenome of which has only 13,628 bp (Gu et al. Reference Gu, Mu, Chen and Li2025). The overall A + T content in the mitogenome of C. ovale is 71.5%, which represents the highest level of A + T content for any studied anisakid.
The mitogenome of C. ovale includes 12 protein-coding genes, 22 tRNA genes, 2 ribosomal RNAs, and 2 non-coding regions, which is a typical composition for an ascaridoid mitogenome (Chen et al. Reference Chen, Qiao, Zhao, Gu and Li2025). All of the currently known ascaridoid mitogenomes have 36 genes, but the number of non-coding regions varies from one to four in ascaridoids (Ortleppascaris sinensis and S. physalis bazhaiensis have a single non-coding region, Porrocaecum moraveci and Anisakis simplex have three, Contracaecum osculatum has four, and the remainder all have two) (Gu et al. Reference Gu, Guo, Chen, Sitko, Li, Guo and Li2023, Reference Gu, Mu, Chen and Li2025; Mohandas et al. Reference Mohandas, Jabbar, Podolska, Zhu, Littlewood, Jex and Gasser2014; Zhao et al. Reference Zhao, Tu, Wu and Li2018). The 36-gene arrangement of the mitogenome of C. ovale belongs to type GA3 for the Nematoda, which is a typical and highly conservative gene order for ascaridoids (Chen et al. Reference Chen, Qiao, Zhao, Gu and Li2025). This study not only provided the mitogenomic characterisation of C. ovale for the first time, and also enriched the mitogenomic database for zooparasitic nematodes in general.
The mitogenomic phylogeny of the currently limited number of representatives of Contracaecum suggests that C. rudolphii and C. ogmorhini are more closely related to each other than either is to C. ovale, which is readily understandable when the similarity in morphological characters between C. rudolphii and C. ogmorhini is considered (i.e. both these species possess well-developed interlabia (almost as long as the lips) and very long spicules (>6 mm) vs C. ovale with very small interlabia (only c. 1/4–1/3 of lip length) and short spicules (<3 mm)) (Fagerholm and Gibson Reference Fagerholm and Gibson1987; Johnston and Mawson Reference Johnston and Mawson1941; Li et al. Reference Li, Xu and Zhang2013). The present phylogenetic results of the cox1 and 12S data both support a close affinity between C. rudolphii and C. ogmorhini, and are concordant with the mitogenomic phylogenetic results. BI analysis of the ITS-1 data also showed that the present samples clustered together with C. ovale reported from P. cristatus australis in New Zealand, which further confirmed that the present specimens are conspecific with C. ovale. Mattiucci et al. (Reference Mattiucci, Cianchi, Nascetti, Paggi, Sardella, Timi, Webb, Bastida, Rodríguez and Bullini2003) considered the existence of two distinct sibling species (C. ogmorhini (sensu stricto) and C. margolisi) in different populations of C. ogmorhini (sensu lato); this was based on the evidence of 18 enzyme loci and preliminary sequence analysis of the mitochondrial cytb gene. Our phylogenetic cox2 data results also support C. margolisi as representing a species distinct from C. ogmorhini (sensu stricto). The present molecular phylogenies, especially the mitogenomic phylogeny, included only a very limited number of Contracaecum species and hence did not provide a basal framework for evolutionary relationships within the genus. However, as genetic data increases (especially mitogenomic data) from a broader representation of the genus, a more rigorous molecular phylogenetic study will be possible in the future, giving better insights into the evolutionary relationships of Contracaecum species.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X2610203X.
Acknowledgements
The authors are grateful to Dr. David Gibson (Parasites and Vectors Division, Natural History Museum, UK) for improving the English of this paper.
Author contribution
L.-W.L. and L.L. contributed to the study design and wrote the manuscript. Z.-J.H. provided nematode specimens. L.-W.L. and Y.-Y.X. sequenced and analysed the genetic data and mitogenome. All authors read and approved the final manuscript.
Financial support
This study was supported by the Key Development Foundation of Hebei Normal University (L2024ZD17) for Dr. Liang Li.
Competing interests
The authors declare that they have no competing interests.
