Characterization of the complete mitochondrial genomes of the zoonotic parasites Bolbosoma nipponicum and Corynosoma villosum (Acanthocephala: Polymorphida) and the molecular phylogeny of the order Polymorphida

Abstract Abstract Acanthocephalans of the order Polymorphida mainly parasitic in birds and mammals, are of veterinary, medical and economic importance. However, the evolutionary relationships of its 3 families (Centrorhynchidae, Polymorphidae and Plagiorhynchidae) remain under debate. Additionally, some species of Polymorphida (i.e. Bolbosoma spp. and Corynosoma spp.) are recognized as zoonotic parasites, associated with human acanthocephaliasis, but the mitochondrial genomes for representatives of Bolbosoma and Corynosoma have not been reported so far. In the present study, the complete mitochondrial genomes B. nipponicum and C. villosum (Acanthocephala: Polymorphidae) are reported for the first time, which are 14 296 and 14 241 bp in length, respectively, and both contain 36 genes [including 12 PCGs, 22 tRNA genes and 2 rRNA genes] and 2 non-coding regions (NCR1 and NCR2). The gene arrangement of some tRNAs in the mitogenomes of B. nipponicum and C. villosum differs from that found in all other acanthocephalans, except Polymorphus minutus. Phylogenetic results based on concatenated amino acid (AA) sequences of the 12 protein-coding genes (PCGs) strongly supported that the family Polymorphidae is a sister to the Centrorhynchidae rather than the Plagiorhynchidae, and also confirmed the sister relationship of the genera Bolbosoma and Corynosoma in the Polymorphidae based on the mitogenomic data for the first time. Our present findings further clarified the phylogenetic relationships of the 3 families Plagiorhynchidae, Centrorhynchidae and Polymorphidae, enriched the mitogenome data of the phylum Acanthocephala (especially the order Polymorphida), and provided the resource of genetic data for diagnosing these 2 pathogenic parasites of human acanthocephaliasis.

In the present study, the complete mitochondrial genomes of Bolbosoma nipponicum Yamaguti, 1939 and C. villosum (Acanthocephala: Polymorphidae) are sequenced and annotated for the first time, based on specimens collected from the northern fur seal Callorhinus ursinus (Linnaeus) (Mammalia: Carnivora) and the Pacific halibut Hippoglossus stenolepis (Schmidt) (Pleuronectiformes: Pleuronectidae) in Alaska, USA, which also represented the first mitogenome from the genera Bolbosoma and Corynosoma (Polymorphida: Echinorhynchidae).Additionally, in order to further clarify the evolutionary relationships of the 3 families Plagiorhynchidae, Centrorhynchidae and Polymorphidae in the order Polymorphida, phylogenetic analyses based on concatenated amino acid (AA) sequences of the 12 protein-coding genes (PCGs) of all available acanthocephalan mitogenomes were performed using Bayesian inference (BI) and maximum likelihood (ML), respectively.

Species identification
The acanthocephalan specimens of B. nipponicum and C. villosum were collected from the intestine of subadult northern fur seals Callorhinus ursinus (Linnaeus) (Mammalia: Carnivora) and Hippoglossus stenolepis (Schmidt) (Pleuronectiformes: Pleuronectidae) in St. Paul Island, Alaska, USA, fixed and stored in 70% ethanol.The specimens were identified as B. nipponicum and C. villosum based on morphological features according to previous studies (Van Cleave, 1953;Margolis, 1956;Ru et al., 2022).Voucher specimens were deposited in the College of

Molecular procedures
The total genomic DNA of each individual of B. nipponicum and C. villosum was extracted using the Magnetic Universal Genomic DNA Kit (DP705) (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions: (1) cut the sample tissue into small pieces, add 300 μL tissue digestives GHA and 20 μL Proteinase K, and grind the tissue thoroughly; (2) transfer the above-treated sample solution of 300 μL into a new 1.5 mL centrifuge tube; (3) add 300 μL lysate GHL and 300 μL isopropyl alcohol, shake and mix well; (4) add 15 μL magnetic bead suspension GH, shake and mix for 1 min, stand for 9 min in total, shake and mix for 1 min each 3 mins; (5) the centrifuge tube was placed on the magnetic rack and stood for 30 s.After the magnetic bead was completely absorbed, the liquid was carefully absorbed, and the DNA solution was transferred to the new centrifuge tube.The genomic DNA sample was fragmented by sonication to a size of 350 bp in preparation for genomic library.

Phylogenetic analyses
Phylogenetic analyses were conducted based on concatenated amino acid (AA) sequences of the 12 PCGs using BI and ML, respectively.Two species of Bdelloidea, Rotaria rotatoria (NC013568.1)and Philodina citrina (FR856884.1)were chosen as the out-group.The in-group included the newly sequenced B. nipponicum and C. villosum and the other 29 species of acanthocephalans with mitogenomic data.Detailed information on representatives included in the present phylogeny was provided in Table 1.For phylogenetic analyses, PhyloSuite was used to collect all mitogenomic sequences from GenBank files, standardize annotation and extract mitogenomic data (Zhang et al., 2020).The extracted amino acid sequences of all 12 PCGs were aligned in MAFFT v7.313 under iterative refinement method of E-INS-I (Katoh and Standley, 2013).The AAs dataset was concatenated into a single alignment by PhyloSuite, respectively.Substitution models were compared and selected according to the Bayesian Information Criterion (BIC) by using ModelFinder (Kalyaanamoorthy et al., 2017).The VT + F + I + G4 was identified as the optimal amino acid substitution model for both partitions (partition1: cox1, cox2, nad1; partition2: all other genes).Bayesian Information Criterion analysis settings were lser nst = 6, rates = invgamma, mcmc ngen = 5 000 000, printfreq = 1000, samplefreg = 100, nchains = 4, nruns = 2.The analysis continued until the average standard deviation of split frequencies was lower than 0.01.The first 25% of trees were  Dai-Xuan Li et al.  (Golombek et al., 2015;Minh et al., 2020).The iTOL v6.1.1 was used to visualize the phylogeny and architecture using files generated by PhyloSuite (Letunic and Bork, 2021).

Results
General characterization of the complete mitogenomes of B. nipponicum and C. villosum

Phylogenetic analyses
In the present study, phylogenetic trees based on concatenated amino acid sequences of the 12 PCGs using ML and BI methods have nearly same topologies, except the supported value of some branches, which all showed that the representatives of the phylum Acanthocephala were divided into 3 large monophyletic clades (
Recent mitogenomic phylogenies brought substantial changes to the traditional classification of Acanthocepha.However, phylogenetic relationships within many lineages of the class Palaeacanthocephala remain insufficiently resolved, due to large numbers of taxa (i.e. the order Heteramorphida, and the families of llliosentidae, Isthmosacanthidae, Heteracanthocephalidae, Fessisentidae, Diplosentidae, Transvenidae, Spinulacorpidae Hypoechinorhynchidae and Gymnorhadinorhynchidae of the order Echinorhynchida) that have not been included yet.Although all of the 3 family-level taxa (Plagiorhynchidae, Centrorhynchidae and Polymorphidae) of the order Polymorphida have been included in some previous mitogenomic phylogenetic studies, very limited genus/species-level taxa have been covered in each family.The evolutionary relationships of the 3 families in Polymorphida and its included genera of each family remain unsolved.The present mitogenomic phylogenies showed that the order Polymorphida is a monophyletic group, but rejected the monophyly of the order Echinorhynchida, in agreement with the previous studies (García-Varela and Nadler, 2006; García-Varela and de León, 2008;Verweyen et al., 2011;García-Varela et al., 2013;Braicovich et al., 2014).The previous molecular phylogenetic results using single or several concatenated genetic markers (i.Dai-Xuan Li et al.

2013
) and some recent mitogenomic phylogenies (Muhammad et al., 2020a(Muhammad et al., , 2020b(Muhammad et al., , 2020c;;Sarwar et al., 2021) indicated that the Plagiorhynchidae is a sister to the Polymorphidae or Centrorhynchidae.However, the present phylogenetic results strongly displayed that the Polymorphidae and Centrorhynchidae are more closely related to each other than to the Plagiorhynchidae (Gazi et al., 2016;Muhammad et al., 2019aMuhammad et al., , 2019b;;Song et al., 2019;Dai et al., 2022;Gao et al., 2022;Zhao et al., 2023).Although Zhao However, the present phylogenetic study including 2 additional genus-level taxa of the Polymorphidae, showed strong support for the close affinity between the Polymorphidae and Centrorhynchidae in both ML (BS = 98) and BI (BPP = 1).
Our phylogenetic results also revealed the genus Bolbosoma has a sister relationship with Corynosoma based on the mitogenomic data for the first time, in concordance with previous studies based on single or several concatenated genetic markers (García-Varela et al., 2013;Presswell et al., 2018;Ru et al., 2022).Moreover, the present findings further clarified the phylogenetic relationships of the 3 families Plagiorhynchidae, Centrorhynchidae and Polymorphidae, enriched the mitogenome data of the phylum Acanthocephala (especially the order et al., 2016 Bolbosoma nipponicum and Corynosoma villosum in the present study are indicated in bold.Parasitology Life Sciences, Hebei Normal University, Hebei Province, China (B.nipponicum: HBNU-A-2022M002L; C. villosum: HBNU-A-2022F003L).

Figure 1 .
Figure 1.Gene maps of the mitochondrial genomes of Bolbosoma nipponicum and Corynosoma villosum.All genes are transcribed in the clockwise direction on the same strand, and 22 tRNA genes are designated by the 1-letter code with numbers differentiating each of the 2 tRNAs serine and leucine.The outermost circle shows the GC content and the innermost circle shows the GC skew. 48 cod: initial/terminal codons, Int seq: intergenic sequences.Bolbo.50 Dai-Xuan Li et al. trnL1; rrnS is 585 bp in B. nipponicum vs 584 bp in C. villosum, both located between trnM and trnF) (Fig. 1, Table Figs 6).Clade I includes Moniliformis sp., Macracanthorhynchus hirudinaceus and Oncicola luehei, which represents the class Archiacanthocephala at the most basal position of the phylogenetic trees.Clade II contains the representatives of the class Eoacanthocephala (Acanthogyrus bilaspurensis, A. cheni, Hebesoma violentum, Paratenuisentis ambiguus and Pallisentis celatus) and Polyacanthorhynchus caballeroi (belonging to the class Polyacanthocephala). Clade III is composed of the representatives of the class Palaeacanthocephala, of which the order Echinorhynchida (including Cavisoma magnum, Echinorhynchus truttae, Pseudoacanthocephalus bufonis, Brentisentis yangtzensis, Pomphorhynchus spp., Leptorhynchoides thecatus and Micracanthorhynchina dakusuiensis) is a non-monophyletic group, but the order Polymorphida (including Plagiorhynchus transversus, Polymorphus minutus, Southwellina hispida, Centrorhynchus spp., Sphaerirostris spp., Bolbosoma nipponicum and Corynosoma villosum) is a monophyletic group.In the order Polymorphida, the family Polymorphidae is a sister to the family Centrorhynchidae.Bolbosoma nipponicum and Corynosoma villosum clustered together with strong nodal support (BPP = 1, ML-BP = 100) in all BI and ML trees.

Figure 2 .
Figure 2. Relative synonymous codon usage (RSCU) of Bolbosoma nipponicum and Corynosoma villosum.Codon families (in alphabetical order) are provided below the horizontal axis.Values on the top of each bar represent amino acid usage in percentage.

Figure 3 .
Figure 3.The predicted secondary structures of 22 tRNAs in the mitogenome of Bolbosoma nipponicum (Watson-Crick bonds indicated by lines, GU bonds indicated by dots, red bases representing anticodons).The tRNAs are labelled with the abbreviations of their corresponding amino acids according to the IUPAC-IUB code.

Figure 4 .
Figure 4.The predicted secondary structures of 22 tRNAs in the mitogenome of Corynosoma villosum (Watson-Crick bonds indicated by lines, GU bonds indicated by dots, red bases representing anticodons).The tRNAs are labelled with the abbreviations of their corresponding amino acids according to the IUPAC-IUB code. 54 et al.' (2023) study also suggests a close affinity between the Polymorphidae and Centrorhynchidae, there are only 2 representatives of the Polymorphidae in their phylogeny, and the supported value for the close phylogenetic relationship of the Polymorphidae and Centrorhynchidae is weak.

Figure 5 .
Figure 5.Comparison of the linearized mitochondrial genome arrangement for acanthocephalans species.All genes are transcribed in the same direction from left to right.The tRNAs are labelled by single-letter code for the corresponding amino acid.Bolbosoma nipponicum and Corynosoma villosum are indicated using asterisk (*).

Figure 6 .
Figure 6.Phylogenetic analyses of Acanthocephala inferred from ML and BI methods based on concatenated amino acid sequences of 12 PCGs of mitochondrial genomes.Rotaria rotatoria and Philodina citrina were chosen as the out-group.Bootstrap values ⩾70 and Bayesian posterior probabilities values ⩾0.70 are shown in the phylogenetic trees.Bolbosoma nipponicum and Corynosoma villosum are indicated using asterisk (*).

Table 1 .
Detailed information of representatives with their mitogenomic data included in the present phylogeny (Sarwar et al., 2021)

Table 2 .
Organization of the mitochondrial genomes of Bolbosoma nipponicum and Corynosoma villosum Parasitology treated as 'burn-in'.For ML analysis, 1000 bootstrap replicates were used to calculate the bootstrap of the program in IQTREE v2.1.2,keep the default values for other parameters

Table 2
).Two rRNAs (rrnL and rrnS) were identified in the mitogenomes of B. nipponicum and C. villosum (rrnL is 913 bp in B. nipponicum vs 914 bp in C. villosum, both located between trnY and

Table 3 .
Base composition and skewness in the mitogenomes of Bolbosoma nipponicum and Corynosoma villosum Bolbosoma nipponicum and Corynosoma villosum in the present study are indicated in bold.