Characterization of the mitochondrial genome of Tetrameres grusi and insights into the phylogeny of Spirurina.

Tetrameres grusi is a significant parasitic nematode of cranes that is classified into suborder Spirurina. However, for more than a century, this classification has been controversial. Mitochondrial genomes are valuable resources for parasite taxonomy, population genetics and systematics studies. Here, the mitochondrial genome of T. grusi was determined and subsequently compared with those from Spirurina species using concatenated datasets of amino acid sequences predicted from mitochondrial protein-coding genes. The complete mitochondrial genome of T. grusi is circular with 13,709 bp, and it contains 12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one non-coding region. All of the protein-coding genes are transcribed in the same direction. There were 18 intergenic spacers of 1-44 bp, and six locations with gene overlaps, ranging from 1 bp to 28 bp, in the mitochondrial genome of T. grusi. The AT content of this mitochondrial genome was 71.56%. This was similar to mitochondrial genomes of other Spirurina species, which also exhibited strong AT content bias, not only in the nucleotide composition but also in codon usage. The sequenced mitogenomes of the 25 Spirurina nematodes showed three classes of gene arrangements based on the 12 protein-coding genes, and the gene arrangement of the T. grusi mitochondrial genome belonged to the Class I. Phylogenetic analyses using mitochondrial genomes of 25 Spirurina nematodes revealed that T. grusi (Habronematoidea) was closer to Gongylonema pulchrum (Spiruroidea) than Spirocerca lupi (Thelazioidea). The availability of the complete mitochondrial genome sequence of T. grusi provides new and useful genetic markers for further studies on Spirurina nematodes.

Introduction

Suborder Spirurina nematodes belong to order Spirurida, which is divided into 10 superfamilies, including Acuarioidea, Aproctoidea, Camallanoidea, Diplotriaenoidea, Filarioidea, Habronematoidea, Lucionematoidea, Rictularioidea, Spiruroidea, and Thelazioidea (de Ley and Blaxter, 2004; Hodda, 2011). Due to the species-rich in suborder Spirurina, including a heterogeneous group of nematodes. The phylogeny of suborder Spirurina nematodes are controversial and have been revised several times in recent years, especially the application of rDNA in molecular classification (Honisch and Krone, 2008).

Tetrameres nematodes are the important members of suborder Spirurina nematodes, which known as the ‘avian stomach nematodes’. Tetrameres nematodes are the etiological agents of tetrameriosis in poultry, and they may also infest wild birds, including penguin (Fink et al., 2004; González-Acuña et al., 2020; Werneck et al., 2020). Mature female worms are blood-red colored with a special subspherical morphology, as they lie embedded in the crypts of Lieberkühn while feeding on blood. In contrast, the male worms are white and live freely in the lumen of the proventriculus (Soulsby, 1982). Tetrameriosis can cause severe proventriculitis and reduces the secretion of digestive juices. It manifests into a debilitating state that is characterized by emaciation and anemic conditions. In heavily infected poultry the condition may lead to debilitation and wasting, and it may be fatal in migratory wild birds (Soulsby, 1982). The Tetrameres spp. have been frequently reported as being distributed worldwide (Agüero et al., 2016).

Although there are many reported Tetrameres spp., such as T. grusi (Mowlavi et al., 2006), T. mothedai (Schou et al., 2007), T. wetzeli (Diaz et al., 2013), T. pattersoni (Olsen et al., 2016), T. striata (Amundson et al., 2016), T. globosa (Królaczyk et al., 2018), T. americana (Mukaratirwa and Khumalo, 2010), and T. fissipina (Padilla-Aguilar et al., 2020), only the partial nuclear small subunit (SSU) rRNA gene sequences of T. grusi and T. fissipina have been deposited in the NCBI GenBank nucleic acid sequence database. Moreover, the classification of Tetrameres spp. nematodes has been controversial, primarily at the genus level. Nadler et al. (2007) first reported the SSU rRNA sequences of T. fissipina and discussed the taxonomy and phylogenies of Clade III nematodes on the basis of the SSU rRNA sequences (Nadler et al., 2007). In phylogenetic trees, T. fissipina is closer to the Filariata than Spirurina nematodes, which result is inconsistent with morphological taxonomy. Thus, the phylogenetic relationships among genus Tetrameres nematodes remains unclear, and phylogenetic analyses are needed to reconstruct its evolutionary history.

Mitochondrial genomes (mitogenomes) have maternal inheritance, an apparent lack of recombination, rapid evolutionary rates and comparatively conserved genomic structures (Pyziel et al., 2020). Recently, sequences of whole mitogenomes have been used to infer phylogenetic relationships among parasites at different taxonomic levels (Gao et al., 2021). For example, mitogenome dataset-based phylogenetic analyses showed that Parascaris equorum and Parascaris univalens are very closely related compared with other ascarids, and these mitogenome datasets provide genetic evidence that P. equorum and P. univalens represent the same species (Gao et al., 2019). In addition, Bunostomum trigonocephalum and Bunostomum phlebotomum are significantly similar in morphology and biological characteristics, but whether they represent different species or strains has been controversial. Gao et al. (2014) used mitogenome datasets to show that B. trigonocephalum and B. phlebotomum represent distinct but closely-related species (Gao et al., 2014). However, the mitogenome-based phylogenetic position of Tetrameres nematodes was unknown until now.

Therefore, the objectives of the present study were to determine the complete mitogenome of T. grusi, the first representative species of the Habronematoidea, and to compare this mitogenome with those of other nematodes in suborder Spirurina. Finally, its phylogenetic relationships were determined on the basis of the mitogenome amino acid sequence data to assess the systematics of T. grusi and its phylogenetic position within the Spirurina.

Materials and methods

Parasites and total genomic DNA isolation

A one-year-old female red-crowned crane died in September 2020 in Heilongjiang Zhalong National Natural Reserve, Qiqihar, China (47°13′42″N, 124°14′17″E), an autopsy was performed on the dead red-crowned crane to determine the causes of death. Adult female specimens of T. grusi were collected from the glandular stomach of red-crowned crane follow the wildlife protection law of the People's Republic of China (a draft of an animal protection law in China released on 2018). Specimens were washed in physiological saline and identified morphologically to the species level (Mowlavi et al., 2006). They were then fixed in 75% ethanol and stored at −20 °C until used. Total genomic DNA was isolated from individual specimen using a sodium dodecyl sulphate/proteinase K treatment, followed by spin-column purification (Wizard® Genomic DNA Purification System, Promega, WI, USA). To independently verify the identity of the specimen, the partial region of the SSU rRNA sequence was amplified from the genomic DNA using PCR with previously reported primers (Nadler et al., 2007). The region had a 100% identity with previously published sequences of T. grusi from Grus canadensis isolated in the USA (GenBank accession number ).

Sequencing and mitogenome assembly

Illumina paired-end shotgun libraries were prepared using the standard protocol of the Nextera™ DNA Sample Prep Kit (Epicentre®) and sequenced using an Illumina NovaSeq sequencing platform (Shanghai Personal Biotechnology Co. Ltd. China) using 2 × 100 cycles. Raw sequences data were deposited into the Short Read Archive database (https://www.ncbi.nlm.nih.gov/sra/) under the accession number . Clean data without sequencing adapters were de novo assembled using NOVOPlasty software (Dierckxsens et al., 2017). The completeness of the mitogenome assembly was further verified by PCR and Sanger sequencing using four pairs of primers that were designed on the basis of conserved regions (Table S1; Fig. S1).

Sequence analysis and gene annotation

Mitogenome sequences served as query for BLAST search of the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast) (Altschul et al., 1997). The protein-coding genes (PCGs) were initially identified using “ORF Finder” through NCBI and the MITOS Web Server (Rombel et al., 2002) to specify the mitochondrial genetic code of invertebrates. The MITOS Web Server was used to calculate the potential stem-loop secondary structures within these tRNA gene sequences (Bernt et al., 2013). The codon usage of the 12 PCGs was analyzed using the invertebrate genetic code and the Codon Usage web server (http://www.bioinformatics.org/sms2/codon_usage.html). An analysis of compositional skews was conducted using the formulae (Perna and Kocher, 1995): AT-skew = (A − T)/(A + T) and GC-skew = (G − C)/(G + C). A gene map of the mitogenome was constructed using the online mitochondrial visualization tool OrganellarGenomeDRAW (Lohse et al., 2013).

Phylogenetic analyses

Phylogenies were constructed using the data from the 12 PCGs of representative Spirurina nematodes available in GenBank. In addition to the newly sequenced mitogenome of T. grusi, it contained the mitogenomes of Brugia malayi (NC_004298), Brugia timori (AP017686), Brugia pahangi (AP017680), Wuchereria bancrofti (NC_016186), Loa loa (NC_016199), Acanthocheilonema viteae (NC_016197), Chandlerella quiscali (NC_014486), Mansonella perstans (MT361687), Setaria digitata (NC_014282), Setaria labiatopapillosa (NC_044071), Thelazia callipaeda (NC_018363), Heliconema longissimum (GQ332423), Gongylonema pulchrum (NC_026687), Spirocerca lupi (KC305876), Dirofilaria immitis (NC_005305), Dirofilaria repens (NC_029975), Onchocerca ochengi (NC_031891), Onchocerca volvulus (NC_001861), Onchocerca lupi (MW266120), Onchocerca flexuosa (NC_016172), Physaloptera rara (MH931178), Philometroides sanguineus (NC_024931), Dracunculus medinensis (NC_016019), and Camallanus cotti (NC_036308) with Bunostomum phlebotomum (NC_012308) as an outgroup. Nucleotide sequences of each gene and corresponding deduced amino acid sequences were aligned using MUSCLE 3.8 (Edgar, 2004) in MEGA 11.0 (Tamura et al., 2021). The amino acid sequences for each of the 12 PCGs from the mitogenomes were aligned using the default settings and then concatenated. On the basis of the constructed phylogenetic trees, the taxonomic status of T. grusi within Spirurina was estimated.

Phylogenetic analyses were performed using the concatenated set of amino acid sequences. Bayesian Inference (BI) and Maximum Likelihood (ML) phylogenetic trees were obtained by using MrBayes v3.2.6 (Huelsenbeck and Ronquist, 2001) and RaxML v8.2.11 (Stamatakis, 2014), respectively. The best-fit substitution model for phylogenetic analysis of the amino acid alignment was determined, using jModeltest under AIC was the SYM+I+G model. (Darriba et al., 2012). BI analysis was conducted for 10,000,000 generations, in two simultaneous runs, with four chains (three heated and one cold), to catalyze swapping among the Markov-chain Monte Carlo chains. Trees were sampled every 1,000 generations. We used the software Tracer v1.6 (http://tree.bio.ed.ac.uk/software/tracer/) to investigate the convergence of sampled parameters and potential autocorrelation (effective sample size (ESS) for all parameters >200). In addition, we checked the average standard deviations of the split frequencies between both runs (<0.01). Bayesian posterior probabilities were obtained from the 50% majority-rule consensus of the post-burn-in trees sampled at stationarity after removing the first 25% of trees as a “burn-in” stage. ML analysis was conducted for the GTRGAMMAI model and 1000 rapid bootstrapping replications were used to evaluate the bootstrap support values and search for the best ML tree. The final phylogenetic tree was graphically visualized and edited using FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

Results and discussion

General features of the mitogenome

The total T. grusi mitogenome (13,709 bp) is a closed circular molecule (GenBank accession number ) that contains 12 PCGs (cox1–3, nad1–6, nad4L, cytb, and atp6), 22 tRNA genes (one for each amino acid and two each for leucine and serine), two rRNA genes (rrnL and rrnS), and a major non-coding region (NCR), but lacks an atp8 gene (Fig. 1; Table 1). All the genes are transcribed in the same direction. There are 18 intergenic spacers, ranging from 1 to 44 bp, and six locations with gene overlaps, ranging from 1 to 28 bp, in the T. grusi mitogenome (Table 1). There is only one NCR, which is consistent with other related Spirurina species (Yatawara et al., 2010; Hu et al., 2003; McNulty et al., 2012).

Fig. 1

Gene map of the mitogenome of Tetrameres grusi. PCGs are colour-coded (cox: lavender; nad: yellow; atp: green; cyt: purple); rRNAs are in red; tRNAs are in dark blue. Abbreviations of PCGs are: atp6 for ATP synthase subunits 6, cox1–3 for cytochrome oxidase subunits 1–3, cytb for cytochrome b, nad1–6 and nad4L for NADH dehydrogenase subunits 1–6 and 4L, rrnL and rrnS for large and small rRNA subunits, 22 tRNAs are designated by the one-letter code for the corresponding amino acid, with numerals differentiating each of the two leucine and serine-specifying tRNAs (L1 and L2 for codon families CUN and UUR, respectively; S1 and S2 for codon families UCN and AGN, respectively), NCR refers to Non-coding region. All genes are transcribed in the clockwise direction.

Table 1

Features of the mitogenome of Tetrameres grusi.

Genes	Location	Length (bp)	Initiation codons	Termination codons	Anticodons	Intergenic spacers (bp)
cox1	1–1656	1656	ATG	TAG		5
trnW	1662–1717	56			TCA	44
nad6	1762–2206	445	ATT	T		0
trnR	2207–2260	54			ACG	1
trnQ	2262–2316	55			TTG	32
cytb	2349–2404	1056	TTG	TAG		5
trnL1	3409–3465	57			TAG	2
cox3	3468–4244	777	ATT	TAG		0
NCR	4245–4611	367				0
trnA	4612–4672	61			TGC	2
trnL2	4675–4728	54			TAA	0
trnN	4729–4786	58			GTT	1
trnM	4788–4845	58			CAT	2
trnK	4848–4904	57			CTT	3
nad4l	4908–5138	231	TTG	TAA		0
rrnS	5139–5830	687				0
trnY	5826–5880	55			GTA	0
nad1	5881–6753	873	ATA	TAG		1
trnF	6755–6814	60			GAA	3
atp6	6818–7393	576	TTG	TAG		5
trnI	7399–7451	53			GAT	2
trnG	7454–7512	59			TCC	0
cox2	7513–8205	693	TTG	TAA		0
trnH	8206–8262	57			GTG	0
rnnL	8262–9223	961				0
nad3	9224–9563	340	TTG	T		0
trnC	9564–9621	58			GCA	0
trnS2	9622–9678	57			TGA	0
trnP	9679–9739	61			AGG	19
trnD	9759–9814	56			GTC	8
trnV	9823–9878	56			TAC	9
nad5	9888-11,472	1585	TTG	T		0
trnE	11,473–11,529	57			TTC	0
trnS1	11,530–11,580	51			TCT	18
nad2	11,599–12,415	817	TTG	T		0
trnT	12,416–12,473	58			TGT	17
nad4	12,491–13,708	1218	TTG	TAA		1

As observed in other Spirurina species, the nucleotide composition of the entire mitogenome of T. grusi is biased toward A and T, with an overall AT content of 71.56%. The low GC contents in these mitogenomes may be indicative of the high conservation of this genic structure. Genic structures with the fastest evolutionary rates have high GC contents, with the predominant replacements being G and C nucleotide insertions, resulting in divergence from the ancestral structure in metazoans (de Lima et al., 2017). The AT- and GC-skews of the selected complete mitogenome were calculated as −0.489 and 0.582, respectively. The negative AT-skew indicated a higher incidence of T than A nucleotides. The positive GC-skew indicated that G are more abundant than C. The AT-/GC-skews of the PCGs, rRNA and tRNAs were calculated for the entire T. grusi mitogenome (Table 2).

Table 2

Nucleotide composition of 12 PCGs, rRNAs, and NCR of Tetrameres grusi. DNA base composition is shown as percentages.

	Size(bp)	A(%)	T(%)	G(%)	C(%)	A+T(%)	AT-skew	GC-skew
Mitogenome	13,709	18.29	53.26	21.73	6.71	71.56	−0.489	0.528
PCGs	10,267	15.56	54.23	23.02	7.19	69.8	−0.554	0.524
atp6	576	13.02	59.72	21.01	6.25	72.74	−0.642	0.541
cox1	1,656	18.54	46.07	24.46	10.93	64.61	−0.426	0.382
cox2	693	19.19	45.6	26.26	8.95	64.79	−0.408	0.492
cox3	777	17.76	49.03	25.48	7.72	66.8	−0.468	0.535
nad1	873	11.91	56.01	24.4	7.67	67.93	−0.649	0.522
nad2	817	14.44	57.41	22.77	5.39	71.85	−0.598	0.617
nad3	340	13.82	63.24	19.41	3.53	77.06	−0.641	0.692
nad4	1,218	14.70	55.67	23.07	6.57	70.36	−0.582	0.557
nad4L	231	13.42	61.9	21.21	3.46	75.32	−0.644	0.719
nad5	1,585	14.57	57.92	21.45	6.06	72.49	−0.598	0.559
nad6	445	13.03	62.25	22.92	1.8	75.28	−0.654	0.854
cytb	1,056	16.76	54.45	20.83	7.95	71.21	−0.529	0.448
rrnS	687	27.07	48.18	17.9	6.84	75.25	−0.281	0.447
rnnL	961	23.83	52.86	17.59	5.72	76.69	−0.379	0.509
tRNA	1,248	29.01	47.76	18.11	5.13	76.76	−0.244	0.559
NCR	367	29.97	48.23	17.98	3.81	78.2	−0.234	0.650

PCGs and codon usage

In the T. grusi mitogenome, the 12 PCGs accounted for 10,267 bp and encoded 3,413 amino acids, excluding the termination codons. The average AT content of the 12 PCGs was 69.8%. Moreover, the AT-skew of the PCGs was negative, whereas the GC-skew was positive (Table 2). A common feature of mitogenomes in most metazoans is a bias toward a higher representation of the A and T nucleotides, which leads to subsequent biases in the corresponding encoded amino acids (Hu et al., 2020).

The initiation and termination codons of the PCGs in the T. grusi mitogenome are listed in Table 1. The most common initiation codon for T. grusi was TTG (8 of 12 PCGs), followed by ATT (2 of 12 PCGs), ATG (1 of 12 PCGs), and ATA (1 of 12 PCGs). Eight of 12 PCGs were predicted to have TAG (5) or TAA (3) as termination codons, and the incomplete termination codon T were present in 4 PCGs, which is consistent with sequences of other Spirurina, including G. pulchrum, B. malayi, D. immitis, O. volvulus, W. bancrofti, L. loa, C. quiscali, and S. digitata, although different from S. lupi (Liu et al., 2013).

The codon usage analyses of the 12 PCGs in the mitogenome is summarized in Table 3. The most frequently employed amino acid was Phe (TTT: 18.33%), followed by Leu (TTG; 9.27%), Val (GTT; 7.86%), and Tyr (TAT; 5.73%). The least frequently used codons were Leu (CTC, 0%) and Arg (CGA; 0%) (Table 3), which is consistent with previously published data (Deng et al., 2021a).

Table 3

Codon usage analysis of 12 PCGs in the mitochondrial genome of Tetrameres grusis.

Codon	Number	/1000	Fraction	Codon	Number	/1000	Fraction
UUU(Phe)	627	183.28	0.98	UCU(Ser1)	135	39.46	0.43
UUC(Phe)	12	3.51	0.02	UCC(Ser1)	4	1.17	0.01
UUA(Leu1)	99	28.94	0.22	UCA(Ser1)	3	0.88	0.01
UUG(Leu1)	317	92.66	0.69	UCG(Ser1)	9	2.63	0.03
CUU(Leu2)	20	5.85	0.04	CCU(Pro)	55	16.08	0.77
CUC(Leu2)	0	0	0	CCC(Pro)	2	0.58	0.03
CUA(Leu2)	3	0.88	0.01	CCA(Pro)	3	0.88	0.04
CUG(Leu2)	19	5.55	0.04	CCG(Pro)	11	3.22	0.15
AUU(Ile)	141	41.22	0.94	ACU(Thr)	58	16.95	0.89
AUC(Ile)	9	2.63	0.06	ACC(Thr)	1	0.29	0.02
AUA(Met)	37	10.82	0.26	ACA(Thr)	3	0.88	0.05
AUG(Met)	104	30.4	0.74	ACG(Thr)	3	0.88	0.05
GUU(Val)	269	78.63	0.71	GCU(Ala)	55	16.08	0.71
GUC(Val)	9	2.63	0.02	GCC(Ala)	9	2.63	0.12
GUA(Val)	29	8.48	0.08	GCA(Ala)	5	1.46	0.06
GUG(Val)	73	21.34	0.19	GCG(Ala)	9	2.63	0.12
UAU(Tyr)	196	57.29	0.92	UGU(Cys)	117	34.2	0.93
UAC(Tyr)	17	4.97	0.08	UGC(Cys)	9	2.63	0.07
UAA(*)	3	0.88	0.38	UGA(Trp)	23	6.72	0.28
UAG(*)	5	1.46	0.63	UGG(Trp)	58	16.95	0.72
CAU(His)	40	11.69	0.87	CGU(Arg)	47	13.74	0.85
CAC(His)	6	1.75	0.13	CGC(Arg)	1	0.29	0.02
CAA(Gln)	15	4.38	0.33	CGA(Arg)	0	0	0
CAG(Gln)	30	8.77	0.67	CGG(Arg)	7	2.05	0.13
AAU(Asn)	89	26.02	0.98	AGU(Ser2)	97	28.35	0.31
AAC(Asn)	2	0.58	0.02	AGC(Ser2)	8	2.34	0.03
AAA(Lys)	19	5.55	0.27	AGA(Ser2)	17	4.97	0.05
AAG(Lys)	51	14.91	0.73	AGG(Ser2)	40	11.69	0.13
GAU(Asp)	72	21.05	0.9	GGU(Gly)	144	42.09	0.61
GAC(Asp)	8	2.34	0.1	GGC(Gly)	17	4.97	0.07
GAA(Glu)	19	5.55	0.26	GGA(Gly)	15	4.38	0.06
GAG(Glu)	54	15.78	0.74	GGG(Gly)	62	18.12	0.26

Transfer RNAs, ribosomal RNAs, and the NCR

The 22 tRNA genes identified in the T. grusi mitogenome ranged from 51 to 61 bp. The total length was 1,248 bp, and the AT content was 76.76% (Table 2). RrnL was located between trnH and nad3, and rrnS was located between nad4L and trnY. The rrnL and rrnS genes were 961 bp and 687 bp in T. grusi, respectively (Table 1), and they had AT contents of 76.69% and 75.25%, respectively.

The predicted secondary structures of the 22 tRNAs in T. grusi were similar to those of all the Spirurina nematodes examined to date (Fig. S2). The locations of the tRNAs in T. grusi were consistent with a majority of Spirurina, except for those of C. quiscali, O. flexuosa, and H. longissimum (Park et al., 2011; McNulty et al., 2012).

The mitogenome of T. grusi contains only one NCR, an AT-rich region, having 78.2% AT content (Table 2), which was located between cox3 and trnA (Fig. 1; Table 1). This result is consistent with some Spirurina, but differs from other nematodes, such as Ascaris suum (Liu et al., 2012) and B. phlebotomum (Gao et al., 2014), which have two significantly different sized NCRs.

Gene rearrangements

Gene rearrangements in nematode mitogenomes commonly occur and are used to study phylogenetic relationships. To determine the occurrence of arrangements in T. grusi, we compared the gene order of the T. grusi mitogenome with those of 24 Spirurina species. The circular mitogenomes were linearized at the 5’ end of their cox1 genes in the H−strand direction to determine the gene orders of Spirurina species. The sequenced mitogenomes of the 25 Spirurina nematodes showed three classes of gene arrangements based on the 12 PCGs (Fig. 2).

Fig. 2

Mitogenome arrangement in Tetrameres grusi compared with those in Spirurina nematodes. The circular mitogenomes were linearized at the 5′ end of cox1 gene for illustration purpose. Non-coding regions were not shown. Triangular markers of the same color represent the corresponding duplicated genes. The purple frames represent the duplicated gene fragments.

Among the gene arrangements, class I contained 22 Spirurina nematodes that shared an identical arrangement based on 12 PCGs (the order: cox1 > nad6 > cytb > cox3 > nad4L > rrnS > nad1 > atp6 > cox2 > rrnL > nad3 > nad5 > nad2 > nad4), but the gene rearrangement events occurred in transposed tRNAs. The gene arrangements in the mitogenomes of nematodes have previously been divided into 26 types (Liu et al., 2013; Deng et al., 2021b). Here, four types of gene arrangements were identified within Spirurina (Fig. 2). In total, 18 out of 24 Spirurina nematodes, T. grusi, S. digitata, S. labiatopapillosa, D. immitis, D. repens, W. bancrofti, L. loa, B. malayi, B. pahangi, B. timori, A. viteae, T. callipaeda, S. lupi, G. pulchrum, O. flexuosa, O. ochengi, O. lupi, and P. rara, had identical gene arrangements, classified as GA9 type. C. quiscali, O. volvulus, and H. longissimum belonged to the GA8, GA10, and GA11 types, respectively. The rearrangement events mainly occurred in two regions of the mitogenome, between cox3 and nad4L and between nad3 and nad5. In comparison with the GA9 type, four tRNAs (trnM, trnK, trnA and trnN) were transposed to between cox3 and nad4L in the GA8 type. In comparison with the GA10 type, trnK was transposed to between cox3 and nad4L in the GA11 type, whereas trnM, which is between cox3 and nad4L in the GA10 type, was transposed to between nad3 and nad5 in the GA11 type. Additionally, trnV, which is between nad3 and nad5 in the GA10 type, was transposed to between cox3 and nad4L in the GA11 type.

For the Class II gene arrangement, P. sanguineus and D. medinensis shared an identical arrangement based on 12 PCGs (the order: cox1 > cox2 > rrnL > nad3 > nad5 > nad6 > nad4L > rrnS > nad2 > cytb > cox3 > nad4 > nad1 > atp6). However, four tRNAs (trnS2, trnK, trnE, and trnV) existed, and the tRNA transposition phenomenon occurred between P. sanguineus and D. medinensis, resulting in gene rearrangements. Notably, six tRNAs were duplicated in the D. medinensis mitogenome, trnF (3), trnI (2), trnT (2), trnP (2), trnK (2), and trnC (2).

For the Class III gene arrangement, C. cotti was the only member (the order: cox1 > cox2 > rrnL > nad3 > nad5 > cytb > cox3 > nad4 > nad6 > nad4L > nad2 > nad1 > nad5 > cytb > cox3 > nad4 > nad2 > nad1 > atp6). The rearrangement in the C. cotti mitogenome contained 46 genes, including six PCGs (cox3, cytb, nad1, nad2, nad4, and nad5) and six tRNAs (trnA, trnI, trnQ, trnT, trnW, and trnY) that were duplicated, and these duplicated genes were concentrated in two fragments (nad5−trnA−trnQ−trnI−cytb−cox3−trnT−nad4 and nad2−trnY−nad1), except trnW. Additionally, trnF, trnL1, and trnS2 were absent from the C. cotti mitogenome.

In the present study, BI and ML approaches were used to estimate the phylogenetic position of T. grusi within 24 spirurid nematodes on the basis of the concatenated amino acid sequences of 12 PCGs. Both the methods produced nearly identical tree topologies (Fig. 3).

Fig. 3

Phylogenetic relationships of Tetrameres grusi with other 24 Spirurina species based on concatenated amino acid sequences of 12 PCGs analyzed by BI and ML using Bunostomum phlebotomum as outgroup. Posterior probability values are indicated.

The topological structures of the phylogenetic trees divided the Spirurina into two clades. Filarioidea, Spiruroidea, Habronematoidea, Thelazioidea, and Physalopteroidea were grouped together form a major clade, whereas Dracunculoidea and Camallanoidea form the other clade. Our results revealed that Filarioidea, including Onchocercidae and Setariidae, formed a clade with a high nodal support value (ML/BI = 100/1.00). T. grusi (Habronematoidea) was sister to G. pulchrum (Spiruroidea), and together they formed a clade with S. lupi (Thelazioidea). In spite of S. lupi and T. callipaeda belongs to the superfamily Thelazioidea, bur T. callipaeda did not clustered together with S.lupi instead of it showed an early diverging position to S.lupi in our study. The results were identical to those previously reported using a smaller number of mitogenome and SSU rRNA sequences (Nadler et al., 2007; Liu et al., 2015).

Zou et al. (2017) conducted structural and phylogenomic comparative analyses on Spirurina utilizing complete mitogenome datasets. Their topology of the Spirurina clade indicated that S. lupi is closely related to G. pulchrum and therefore the authors concluded that it should belong to the family Gongylonematidae (Zou et al., 2017). Interestingly, the controversial taxonomy within Spirurina was also observed in present study, that G. pulchrum formed a sister group to T. grusi, and together they formed a clade with S. lupi. Based on our results, one can either allocate the three species (G. pulchrum, T. grusi, and S. lupi) to the same family, or allocate G. pulchrum and T. grusi to the same family, sister to S. lupi, or allocate each species to its own family.

The phylogenetic relationships of the genus Tetrameres (T. fissipina) have been previously investigated using nuclear SSU rRNA sequences, showing that T. fissipina was closer to the Onchoceridae sp., a kind of free-living filarial worm belong to Filarioidea, than Thelazia lacrymalis (Thelazioidea) and S. lupi (Thelazioidea) (Nadler et al., 2007). Whereas, the clade formed by G. pulchrum, T. grusi, and S. lupi were more closely related to the Filarioidea family than T. callipaeda (Thelazioidea) using mitogenome sequences in present study. Thus, the evolutionary relationship of the genus Tetrameres within Spirurina remains unclear absolutely owing to the scarcity of full mitogenome sequences, and more nematode mitogenomes need to be sequenced and then, the evolutionary relationships among these nematodes should be reexamined.

Conclusions

In conclusion, we reported the complete mitogenome sequence of T. grusi and used the characterization of this mitogenome to reassess the systematic relationships within the suborder Spirurina. Our phylogenetic analyses suggested that T. grusi (Habronematoidea) was more closely related to G. pulchrum (Spiruroidea) than S. lupi (Thelazioidea). It is anticipated that additional taxonomic samplings will provide more reliable information for determining mitogenome evolution and developing better phylogenetic hypotheses for spirurid nematodes evolution.

Declaration of competing interest

The authors report no conflicts of interests.