The acanthocephalans were collected on 24 September 2018 from the intestine of 36 bullhead catfish Tachysurus fulvidraco (Richardson, 1846) from east Dongting Lake in Yueyang (29°22'N, 113°06'E), Hunan Province, China. Brentisentis yangtzensis was identified by morphology (e.g., Yu 1989) using a stereomicroscope and a light microscope. The parasites were preserved in 100% ethanol and stored at 4 °C. The total genomic DNA was extracted from an entire acanthocephalan using a TIANamp Micro DNA Kit (Tiangen Biotech, Beijing, China) according to manufacturer’s recommended protocol, and stored at -20 °C. Eleven acanthocephalans were collected in total.

Partial sequences of rrnL, cytb, nad1, and rrnS genes were amplified via a polymerase chain reaction (PCR) using four primer pairs. Based on these fragments, we designed specific primers for subsequent PCR amplification (Suppl. material 1). PCR reactions were conducted in a 50 ml reaction mixture, containing 18.5 ml double-distilled water (dd H₂O), 25 ml 2×PCR buffer (Mg²⁺, dNTP plus, Takara, China), 1.5 ml of each primer, 1 ml rTaq polymerase (250U, Takara, China) and 2.5 ml DNA template. Amplification was performed under the following conditions: initial denaturation at 98 °C for 2 min, followed by 40 cycles at 98 °C for 10 s, 48–60 °C for 15 s, 68 °C for 1 min/kb, and a final extension at 68 °C for 10 min. PCR products were sequenced bidirectionally at Sangon Biotech (Shanghai) Co., Ltd. (China) using the primer walking strategy.

The mitogenome of B. yangtzensis were assembled manually in a stepwise manner with the help of the DNAstar v7.1 program (Burland 2000), after quality-proofing of the obtained fragment. The mitogenome was annotated mainly following the procedures described previously (Zou et al. 2017; Zhang et al. 2017a; Li et al. 2017). In detail, protein-coding genes (PCGs) were inferred with the help of BLAST and ORF Finder tools (both available from the National Center for Biotechnology Information (NCBI)), employing the invertebrate mitochondrial code (Codon Table 5), and checking the nucleotide alignments against the reference genomes in acanthocephalan Leptorhynchoides thecatus (Linton, 1891) Kostylew, 1924 (NC_006892). A majority of the tRNAs were identified using the results of ARWEN (Laslett and Canback 2008) and MITOS web server (Bernt et al. 2013), the rest were found by alignment with other acanthocephalans (Suppl. material 2). Two ribosomal RNA genes (rrnL and rrnS) were found by alignment with other published acanthocephalan mitogenomes, and their ends were assumed to extend to the boundaries of their flanking genes. Codon usage and relative synonymous codon usage (RSCU) for 12 protein-encoding genes (PCGs) of the B. yangtzensis and L. thecatus (NC_006892) were computed and sorted using PhyloSuite (Zhang et al. 2018), and finally the RSCU figure drawn using ggplot2 plugin (Wickham 2016). The circular map of B. yangtzensis mitogenome was drawn with the mitochondrial visualization tool MTVIZ (http://pacosy.informatik.uni-leipzig.de/mtviz/).

Phylogenetic analyses were carried out on the newly sequenced mitogenome of B. yangtzensis and the 12 acanthocephalan mitogenomes available in GenBank (Suppl. Table S2). Two species of the Bdelloidea, Rotaria rotatoria (Pallas, 1766) (NC013568.1) and Philodina citrina Ehrenberg, 1832 (FR856884.1), were used as outgroups. Fasta files with the amino acid sequences for all 12 PCGs were extracted from the GenBank files using PhyloSuite. All the genes were aligned in batches with MAFFT (Katoh et al. 2002) integrated in PhyloSuite, using normal-alignment mode. PhyloSuite was then used to concatenate these alignments into a single alignment and generate phylip and nexus format files for the phylogenetic analyses, conducted using maximum likelihood (ML) and Bayesian inference (BI) methods. The selection of the best-fit partition strategy and models was carried out using PartitionFinder2 (Lanfear et al. 2017). ML analysis was performed using IQ-TREE (Nguyen et al. 2015) with 50, 000 Ultrafast bootstraps (Minh et al. 2013). BI analysis was performed in MrBayes 3.2.6 (Ronquist et al. 2012) with the default settings, and 3 × 10⁶ metropolis-coupled Markov Chain Monte Carlo generations.

The circular duplex molecule mitogenome of B. yangtzensis is 13,864bp in size (GenBank accession number MK651258) and contains all 36 of the typical metazoan genes: 22 tRNA genes, 2 rRNA genes and 12 protein-encoding genes (PCGs) (lacking atp8) (Fig. 1). All genes are transcribed from the same strand, and 13 overlapping regions were found in the genome (Table 1). The lack of the gene atp8 is common in acanthocephalans (Gazi et al. 2016; Song et al. 2016) with one exception; in L. thecatus two putative atp8 genes have been suggested (Steinauer et al. 2005).

Figure 1. 

Map of the complete mitochondrial genome of Brentisentis yangtzensis.

The total length of the concatenated 12 protein-coding genes is 10,355 bp, with the average A+T content of 68.0%, ranging from 65.9% (nad3) to 69.5% (atp6 and nad4) (Suppl. material 3). ATG (for 6 PCGs) is the most commonly used start codon, whereas nad6, nad4, cox1 and cox2 used GTG, nad1 and nad3 used TTG and ATA, respectively. The most frequent terminal codons are TAG (for 7 PCGs), followed by T (4 PCGs) (Table 1).

Codon usage, relative synonymous codon usage (RSCU) and codon family proportion (corresponding to the amino acids usage) of B. yangtzensis and L. thecatus (NC_006892) is presented (Suppl. material 4). Leucine (16.28%), valine (11.92%) and serine (10.94%) are the most frequent amino acids in the PCGs of B. yangtzensis, whereas glutamine (0.81%), arginine (1.36%) and histidine (1.44%) are relatively scarce. A higher T content (42.2%) in 12 PCGs corresponds to a relatively high frequency of T-rich codons: TTA for leucine (9.9%), TTT for phenylalanine (7.2%), ATT for isoleucine (6.1%) and GTT for valine (5.0%).

All 22 commonly found tRNAs are present in the mitogenome of B. yangtzensis, ranging from 50 bp (trnS2) to 70 bp (trnT) in size, with a concatenated length of 1,247 bps (Table 1).

The genes rrnL and rrnS are 912 bp and 574 bp in size, with 71.8% and 69.8% A+T content, respectively (Suppl. Material 3). The location of rrnL is between trnY and trnL1, and rrnS is located between trnM and trnF; this is the same arrangement reported for other acanthocephalans (Fig. 2), except for Hebesoma violentum (Van Cleave, 1928) Salgado-Maldonado, 1978 in which rrnS is located between trnS1 and trnF (Pan and Nie 2014).

Figure 2. 

Phylogenetic tree of acanthocephalans inferred from maximum likelihood analysis with concatenated nucleotide sequence of all 36 genes (12 PCGs, 2 rRNAs, and 22 tRNAs). Bootstrap (BS)/Bayesian posterior probability (BPP) support values are shown above the nodes, only BS < 100 and BPP < 1 are displayed.

BI and ML yielded phylograms with identical topology and strong statistical support for all nodes (BP ≥ 75, BPP ≥ 0.96) (except the branch of Centrorhynchus aluconis (Müller, 1780) Lühe 1911 and Southwellina hispida (Van Cleave, 1925) Witenberg, 1932 52/0.96). Since both phylograms have an identical topology, only the latter was shown (Fig. 2). The resulting combined phylogenetic tree depicted almost the same results found in previous mitogenomic studies (Gazi et al. 2016), except for the position of B. yangtzensis (Palaeacanthocephala, Illiosentidae), as this was included for the first time in the present study. Within the Acanthocephala, tree topology indicates the existence of two major clades: class Archiacanthocephala (monophyletic and the most basal clade) and the other two classes (Eoacanthocephala + Palaeacanthocephala), resulting in the three monophyletic clades corresponding to the three classes (Archiacanthocephala, Eoacanthocephala, and Palaeacanthocephala) in the most widely accepted classification of the Acanthocephala.

The Echinorhynchida is paraphyletic, with three species separated into two clades: Echinorhynchus truttae Schrank, 1788 (Echinorhynchidae) formed a sister clade with species of the Polymorphida and B. yangtzensis (Illiosentidae) formed a sister clade with L. thecatus (Rhadinorhynchidae). The result shows the relatively close relationship between the Illiosentidae and the Rhadinorhynchidae; however, as each family of the Echinorhynchida was represented by a single species in our study, this topology should be interpreted with some caution. Previous studies have shown the close relationship between Leptorhynchoides (Rhadinorhynchidae) and a genus of the Illiosentidae (Monks 2001; García-Varela and Nadler 2005; García-Varela and González-Oliver 2008). Two species of the Gyracanthocephala Van Cleave, 1936, one (Pallisentis celatus (Van Cleave, 1928) Baylis, 1933) formed a sister clade with Polyacanthorhynchus caballeroi Diaz-Ungria et Rodrigo, 1960 (Polyacanthorhynchida Amin, 1987), and another (Acanthosentis cheni Amin, 2005) nested with species of the Neoechinorhynchida. This result suggests paraphyly of the Gyracanthocephala and corresponds with previous phylogenetic analyses (Song et al. 2016). As the lack of the mitochondrial genome information on acanthocephalan species, more studies and sequences of acanthocephalan (Illiosentidae and Rhadinorhynchidae) species are needed to gain a clear understanding of the phylogenetic relationships of these acanthocephalans.

The mitochondrial gene arrangements of acanthocephalan species are relatively conserved (Fig. 2). Besides the incomplete mitochondrial genome of E. truttae, the gene arrangement of 12 protein coding genes and two rRNA genes are highly conserved, with only a few translocations of tRNAs (trnS1, trnS2, trnV, and trnK) detected. In mitochondrial genomes, conserved gene arrangement is considered to be a typical characteristic (Boore 2000; Li et al. 2012), but the arrangement of tRNAs shows more variability, and there are examples of extensive gene rearrangement (Hyman et al. 2011; Zhang et al. 2017b; Wang et al. 2017). Transfer RNA genes are more often translocated than other genes, probably because of their small size (Kilpert and Podsiadlowski 2006).

In many cases, the gene order of the mitochondrial genome can form useful information in inferring phylogenetic relationships of metazoans (Littlewood et al. 2006; Luo et al. 2008; Wang et al. 2017; Zhang et al. 2018); however, for the taxa in our study, a phylogeny inferred from gene order is incompatible with that based on amino acid sequence (Fig. 2). The translocations of four tRNAs (trnS2, trnV, trnk, trnS1) were detected between B. yangtzensis and L. thecatus (Echinorhynchida), which formed a monophyletic clade in the phylogenetic tree. However, only 1–4 translocations of tRNAs were detected among all of the acanthocephalan species. And these four tRNA (trnS2, trnV, trnk, trnS1) translocations were also detected within the Archiacanthocephala clade. Moreover, identical gene order was found both in a sister clade (C. aluconis and Plagiorhynchus transversus (Rudolphi, 1819) Travassos, 1926) and across different classes (B. yangtzenensis (Palaeacanthocephala), A. cheni (Eoacanthocephala), and Macracanthorhynchus hirudinaceus (Pallas, 1781) Travassos, 1917 (Archiacanthocephala); and Oncicola luehei (Travassos, 1917) Schmidt, 1972 (Archiacanthocephala) and L. thecatus (Palaeacanthocephala)).