Mitogenomic phylogenies suggest the resurrection of the subfamily Porrocaecinae and provide insights into the systematics of the superfamily Ascaridoidea (Nematoda: Ascaridomorpha), with the description of a new species of Porrocaecum
Parasites & Vectors volume 16, Article number: 275 (2023)
The family Toxocaridae is a group of zooparasitic nematodes of veterinary, medical and economic significance. However, the evolutionary relationship of Porrocaecum and Toxocara, both genera currently classified in Toxocaridae, and the monophyly of the Toxocaridae remain under debate. Moreover, the validity of the subgenus Laymanicaecum in the genus Porrocaecum is open to question. Due to the scarcity of an available genetic database, molecular identification of Porrocaecum nematodes is still in its infancy.
A number of Porrocaecum nematodes collected from the Eurasian marsh harrier Circus aeruginosus (Linnaeus) (Falconiformes: Accipitridae) in the Czech Republic were identified using integrated morphological methods (light and scanning electron microscopy) and molecular techniques (sequencing and analyzing the nuclear 18S, 28S and ITS regions). The complete mitochondrial genomes of the collected nematode specimens and of Porrocaecum (Laymanicaecum) reticulatum (Linstow, 1899) were sequenced and annotated for the first time. Phylogenetic analyses of ascaridoid nematodes based on the amino acid sequences of 12 protein-coding genes of mitochondrial genomes were performed using maximum likelihood and Bayesian inference.
A new species of Porrocaecum, named P. moraveci n. sp., is described based on the morphological and genetic evidence. The mitogenomes of P. moraveci n. sp. and P. reticulatum both contain 36 genes and are 14,517 and 14,210 bp in length, respectively. Comparative mitogenomics revealed that P. moraveci n. sp. represents the first known species with three non-coding regions and that P. reticulatum has the lowest overall A + T content in the mitogenomes of ascaridoid nematodes tested to date. Phylogenetic analyses showed the representatives of Toxocara clustered together with species of the family Ascarididae rather than with Porrocaecum and that P. moraveci n. sp. is a sister to P. reticulatum.
The characterization of the complete mitochondrial genomes of P. moraveci n. sp. and P. reticulatum is reported for the first time. Mitogenomic phylogeny analyses indicated that the family Toxocaridae is non-monophyletic and that the genera Porrocaecum and Toxocara do not have an affinity. The validity of the subgenus Laymanicaecum in Porrocaecum was also rejected. Our results suggest that: (i) Toxocaridae should be degraded to a subfamily of the Ascarididae that includes only the genus Toxocara; and (ii) the subfamily Porrocaecinae should be resurrected to include only the genus Porrocaecum. The present study enriches the database of ascaridoid mitogenomes and provides a new insight into the systematics of the superfamily Ascaridoidea.
The superfamily Ascaridoidea comprises a large group of parasitic nematodes that commonly occur in the digestive tract of all major lineages of vertebrates [1,2,3,4,5,6]. The Ascaridoidea is currently divided into six major families, namely Heterocheilidae, Acanthocheilidae, Anisakidae, Ascarididae, Toxocaridae and Raphidascarididae . Among them, the family Toxocaridae (Ascaridomorpha: Ascaridoidea) contains only two genera, Porrocaecum and Toxocara [4, 8, 9], with over 50 nominal species parasitizing birds and mammals worldwide [1, 2, 10]. Nematodes of the family Toxocaridae cause diseases in wildlife, domestic animals and humans and are therefore of veterinary, medical and economic significance [2, 11,12,13]. However, the evolutionary relationship of Porrocaecum and Toxocara, and the monophyly of the Toxocaridae remain under debate. Results from a number of earlier phylogenetic studies indicated that Porrocaecum and Toxocara have no close relationship and that Toxocaridae is not monophyletic [14,15,16,17] while, in contrast, the results of another molecular phylogeny study supported the monophyly of the Toxocaridae and showed an affinity between Porrocaecum and Toxocara .
Nematodes of the genus Porrocaecum are common parasites that mainly occur in the digestive tract of various species of birds worldwide [2, 18,19,20,21]. In 1953, Mozgovoi proposed dividing the genus Porrocaecum into two subgenera, Laymanicaecum and Porrocaecum, based on the presence or absence of the gubernaculum in the male . However, this proposal was rejected by Hartwich . To date, the validity of the subgenus Laymanicaecum has never been tested based on molecular phylogeny due to the scarcity and inaccessibility of suitable material or genetic data.
Although approximately 40 species of Porrocaecum have been described, the validity of some species is still questionable due to their high morphological similarities . Moreover, molecular identification of Porrocaecum nematodes using various nuclear and mitochondrial DNA (mtDNA) markers [large ribosomal DNA (28S), internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 (cox1) or 2 (cox2)] remains in its infancy due to a scarcity of available genetic databases. To date, there have been only eight species of Porrocaecum with their genetic data recorded in the GenBank database [7, 22]. Among these, only one unidentified species, Porrocaecum sp., has been sequenced for the complete mitochondrial genome .
In the present study, a number of Porrocaecum nematodes were collected from the Eurasian marsh harrier (Circus aeruginosus (Linnaeus); Falconiformes: Accipitridae) in the Czech Republic. In order to accurately identify these Porrocaecum nematodes to species level, we observed the detailed morphology of the present specimens using light and scanning electron microscopy, and the nuclear 18S, 28S and ITS regions were sequenced and analyzed. The complete mitochondrial genomes of the collected Porrocaecum nematodes and a representative of the subgenus Laymanicaecum, Porrocaecum (Laymanicaecum) reticulatum (Linstow, 1899), were also sequenced and annotated for the first time to reveal the patterns of mitogenomic evolution in this group. Moreover, in order to test the monophyly of the Toxocarinae/Toxocaridae and determine the systematic status of the subgenus Laymanicaecum in Porrocaecum, phylogenetic analyses of ascaridoid nematodes based on the amino acid sequences of 12 protein-coding genes (PCGs) of mitochondrial genomes and phylogeny of Porrocaecum based on 18S + ITS and 28S were performed using maximum likelihood (ML) and Bayesian inference (BI), respectively.
Parasite collection and species identification
Nematode specimens of Porrocaecum were collected from the intestine of the Eurasian marsh harrier C. aeruginosus (Falconiformes: Accipitridae) during a helminthological survey of birds in Czech Republic. The collected specimens were washed in saline, then stored in 70% ethanol until studied. For the light microscopy studies, nematodes were cleared in lactophenol, and drawings were made with the aid of a Nikon microscope drawing attachment (Nikon Corp., Tokyo, Japan). For the scanning electron microscopy (SEM) studies, specimens were post-fixed in 1% OsO4, dehydrated through an ethanol and acetone series and then critical point dried. The specimens were then coated with gold and examined using a Hitachi S-4800 scanning electron microscope at an accelerating voltage of 20 kV (Hitachi Ltd., Tokyo, Japan). In this article, measurements (the range, with the mean in parentheses) are presented in micrometers unless otherwise stated. For study of the mitochondrial genome, specimens of P. (Laymanicaecum) reticulatum were collected from the great egret [Ardea alba (Linnaeus); Ciconiiformes: Ardeidae] in Hustopeče and Bečvou, Czech Republic.
The mid-body of two nematode specimens (1 male, 1 female) was used for molecular analyses. Genomic DNA from each sample was extracted using a Column Genomic DNA Isolation Kit [Sangon Biotech （Shanghai） Co., Ltd., Shanghai, China] according to the manufacturer's instructions. The primers used for amplifying the target sequences of 18S, ITS and 28S were: primers 18SF and 18SR for the partial 18S ; primers SS1 and SS2R for the partial ITS region ITS-1 region ; primers NC13 and NC2 for ITS-2 ; and primers 28SF and 28SR for the partial 28S ribosomal DNA (rDNA) . The cycling conditions were as described previously . PCR products were checked on GoldView-stained 1.5% agarose gels and purified with Column PCR Product Purification Kit [Sangon Biotech （Shanghai） Co., Ltd.]. Sequencing of each sample was carried out for both strands. Specifically, sequences were aligned using ClustalW2. The DNA sequences obtained herein were compared (using the algorithm BLASTn) with those available in the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). The 18S, 28S and ITS sequence data obtained herein have been deposited in the GenBank database (http://www.ncbi.nlm.nih.gov).
Mitochondrial genome sequencing, assembly and annotation
A total of 30 Gb of clean genomic data of each species was generated using the Pair-End 150 sequencing method on the Illumina NovaSeq 6000 platform (Illumina, Inc., San Diego, CA, USA) by Novogene Technology Co., Ltd. (Tianjin, China). The complete mitochondrial genome was assembled using GetOrganelle v1.7.2a . PCGs, ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) were annotated using the MitoS web server (http://mitos2.bioinf.uni-leipzig.de/index.py) and the MitoZ v2.4 toolkit . The open reading frame (ORF) of each PCG was confirmed manually through the web version of ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/). The “lost” tRNA genes ignored by both MitoS and MitoZ were identified using BLAST based on a database of the existing tRNA sequences of nematodes (CNP0003131, NC_010690, NC_070176). The secondary structures of tRNAs were predicted by the ViennaRNA module , building on MitoS2  and the RNAstructure v6.3 software package , followed by a manual correction. The MitoZ v2.4 toolkit was used to visualize and depict gene element features . The base composition, amino acid usage and relative synonymous codon usage (RSCU) were calculated by Python script, which refers to the Codon Adaptation Index (CAI) . The total length of the base composition included ambiguous bases. Base skew analysis was used to describe the base composition of nucleotide sequences. The complete mitochondrial genomes of P. moraveci n. sp. and P. reticulatum obtained herein were deposited in the GenBank database (http://www.ncbi.nlm.nih.gov).
Phylogenetic analyses of ascaridoid nematodes were performed based on the amino acid sequences of 12 PCGs of mitochondrial genomes using ML and BI, respectively. Caenorhabditis elegans (Rhabditida: Rhabditoidea) and Heterakis gallinarum (Ascaridomorph: Heterakoidea) were chosen as the outgroup. The ingroup included 32 representatives of the superfamily Ascaridoidea. Detailed information on the representatives included in the present phylogeny analysis is provided in Table 1. The phylogenetic trees were re-rooted on C. elegans. Genes were aligned separately using the MAFFT v7.313 multiple sequence alignment program under the iterative refinement method of E-INS-I . Ambiguous sites and poorly aligned positions were eliminated using the BMGE v1.12 program (m = BLOSUM90, h = 0.5) . The aligned and eliminated sequences were concatenated into a matrix by the PhyloSuite v1.2.2 desktop platform . The mtMet + F + R4 model was identified as the optimal nucleotide substitution model for the ML inference. The partitioning schemes and the optimal nucleotide substitution model selected for each combination of partition for the BI inference are shown in Additional file 1: Table S1. Reliabilities for ML inference were tested using 1000 bootstrap (BS) replications, and BIC analysis was run for 5 × 106 Markov chain Monte Carlo (MCMC) generations.
Phylogenetic analyses of Porrocaecum species were performed based on the 18S + ITS and 28S sequence data using the ML method with IQTREE v2.1.2  and BI with MrBayes 3.2.7 , respectively. Toxocara cati (Ascaridida: Ascaridoidea) was chosen as the out-group. Detailed information on the Porrocaecum species included in the present phylogeny analysis is provided in Table 2. Three partitions and their models were selected for ML analyses: K2P + FQ + I (18S); K2P + FQ + G4 (ITS-1 + 5.8S + ITS-2); and TPM3 + F + G4 (28S). Similarly, three partitions were used for BI analyses: K80 + I (18S); K80 + G (ITS-1 + 5.8S + ITS-2); and HKY + G (28S). Reliabilities for ML inference were tested using 1000 BS replications, and BIC analysis was run for 5 × 106 MCMC generations.
In the ML tree, BS values ≥ 90 were considered to constitute strong branch support, whereas bootstrap values ≥ 70 and < 90 were considered to constitute moderate branch support. In the BI tree, Bayesian posterior probabilities (BPP) values ≥ 0.90 were considered to constitute strong branch support, whereas BPP values ≥ 0.70 and < 0.90 were considered to constitute moderate branch support. BS values ≥ 70 and BPP values ≥ 0.70 are shown in the phylogenetic trees.
Superfamily Ascaridoidea RailIiet & Henry, 1912
Family Ascarididae Baird, 1853
Subfamily Porrocaecinae Osche, 1958
Genus Porrocaecum Railliet & Henry, 1912
Porrocaecum (Porrocaecum) moraveci sp. n.
Type-host: Circus aeruginosus (Linnaeus) (Falconiformes: Accipitridae).
Type-locality: Přerov, Czech Republic.
Site in host: Intestine.
Type specimens: Holotype, male (HBNU–N–B20220021GL); allotype, female (HBNU–N–B20220022GL); paratype: 1 male (HBNU–N–B20220023GL); deposited in the College of Life Sciences, Hebei Normal University, Hebei Province, China.
Representative DNA sequences: Representative nuclear ribosomal and mitochondrial genome sequences were deposited in the GenBank database under the accession numbers OQ858491, OQ858492 (18S), OQ858562, OQ858563 (28S), OQ858560, OQ858561 (ITS), OQ863051 (mitochondrial genome).
ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN), details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn: lsid: zoobank.org: pub: 6F8AE7EE-67E8-41BF-AB2C-4EA4874D8843. The LSID for the new name Porrocaecum moraveci is urn: lsid: zoobank.org: act: 09174C82-DAF4-4C78-B47B-AA82C9B8FC74.
Etymology: The species is named in honor of Dr. František Moravec (Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Czech Republic), who has made great contributions to the taxonomy of ascaridoid nematodes.
Large-sized, whitish nematodes with transversely striated cuticle. Maximum width at about mid-body. Anterior extremity with three roughly hexagonal lips, postlabial grooves and lateral membranous flanges conspicuous (Figs. 1a–d, 2a, 3a, b). Dorsal lip with one pair of large double papillae (Figs. 1b, 2a); subventral lips each with single double papilla, small papilla and amphid (Figs. 1c, 3c). Single median superficial ditch and pair of small, submedial pores present on each lip (Figs. 2a, 3a, b). Anterior and lateral margins of each lip armed with about 100–120 acuminate denticles (Figs. 2a, 3b, c). Interlabia small, triangular, about one third of length of lips (Figs. 1a, d, 2a, 3a, b). Cervical alae absent. Cervical papillae not observed. Esophagus muscular, distinctly broader posteriorly than anteriorly (Fig. 1a). Ventriculus longer than wide (Fig. 1a). Intestinal caecum long, about two thirds of esophageal length (Fig. 1a). Ventricular appendix absent. Nerve-ring at about one fifth of esophageal length. Excretory pore just posterior to the nerve-ring (Fig. 1a). Tail of both sexes conical, with a very small finger-like mucron (Figs. 1e, g, h, 2b, d, 3f).
Based on one mature specimen. Body 62.0 mm long; maximum width 781 μm. Dorsal lip 213 μm long, 203 μm wide. Interlabia 73 μm long. Esophagus 3.61 μm long, 300 μm in maximum width, representing 5.80% of body length. Nerve-ring and excretory pore 820 and 855 μm, respectively, from anterior extremity. Ventriculus 290 μm long, 210 μm wide. Intestinal caecum 2.66 mm long, 235 μm wide, representing 73.7% of esophageal length. Posterior end of body distinctly curved ventrally. Spicules alate, unequal in length, sub-rounded at distal end, left spicule 792 μm long, representing 1.30% of body length; right spicule 696 μm long, representing 1.10% of body length (Fig. 1h). Gubernaculum absent. Caudal papillae 25 pairs in total: 20 pairs precloacal, one pair double paracloacal (slightly posterior to cloaca) and four pairs postcloacal (2 pairs ventral, 2 pairs lateral) (Figs. 1g, 2b–d). Single medio-ventral precloacal papilla unconspicuous (Fig. 2d). Tail 330 μm long. Caudal alae weak (Fig. 2b). Lateral phasmids present (Fig. 2, d).
Based on two mature specimens (measurements presented as the range with the mean in parentheses). Body 117.0–138.0 (127.5) mm long; maximum width 1.39–1.44 (1.41) mm. Dorsal lip 295–375 (335) μm long, 365–510 (438) μm wide. Interlabia 114–116 (115) μm long. Esophagus 4.68–6.61 (5.65) mm long, 490–510 μm (500) in maximum width, representing 4.00–4.80 (4.40)% of body length. Nerve-ring and excretory pore 970–1140 (1055) μm and 1.12–1.36 (1.24) mm, respectively, from anterior extremity. Ventriculus 330–450 (390) μm long, 240–380 (310) μm wide. Intestinal caecum 2.85–4.52 (3.69) mm long, 210–230 (220) μm in maximum width, representing 60.9–68.5 (64.7)% of esophageal length. Vulva slit-like, pre-equatorial, 36.8–43.5 (40.2) mm from anterior extremity, at 31.5% of body length (Figs. 1f, 3d). Vagina muscular, directed posteriorly from vulva. Eggs oval, thick-shelled, with punctate surface, 105–165 (129) × 80–120 (99) μm (n = 25) (Fig. 1i). Tail 500–598 (549) μm long (Figs. 1f, 3e). Caudal alae weak (Fig. 3e, f). Lateral phasmids present (Figs. 1e, 2f, g).
Partial 18S region
Two 18S sequences of P. moraveci sp. n. obtained herein are 1717 bp in length, with no nucleotide divergence detected. In the genus Porrocaecum, the 18S sequence data are available in GenBank for P. angusticolle (Molin, 1860) (EU004820), P. depressum (Zeder, 1800) (U94379), P. reticulatum (Linstow, 1899) (MF072700), Porrocaecum sp. (MT141136) and P. streperae Johnston & Mawson, 1941 (EF180074). Pairwise comparison of the 18S sequences of P. moraveci with those of Porrocaecum spp. showed 0.17% (Porrocaecum sp.) to 0.47% (P. depressum and P. streperae) of nucleotide divergence.
Partial 28S region
Two 28S sequences of P. moraveci sp. n. obtained herein are 746 bp in length, with no nucleotide divergence detected. In the genus Porrocaecum, the 28S sequences are only available in GenBank for P. angusticolle (MW441213-MW4412136) and P. depressum (U94765). Pairwise comparison of the 28S sequences of P. moraveci with those of P. angusticolle and P. depressum showed 1.88% (P. angusticolle) and 10.8% (P. depressum) of nucleotide divergence.
Partial ITS (ITS-1 + 5.8S + ITS-2) region
Two ITS sequences of P. moraveci sp. n. obtained herein are 988 bp in length, with no nucleotide divergence detected. In the genus Porrocaecum, the ITS-1 + 5.8S + ITS-2 sequences are available in GenBank for P. angusticolle (MW447303–MW447305), P. crassum (Deslongchamps, 1824) (AY603533), P. depressum (AY603534), P. ensicaudatum (Zeder, 1800) (AY603532), P. reticulatum (MF061688), P. streperae (AJ007460) and Porrocaecum sp. (LC666446). Pairwise comparison of the ITS-1 + 5.8S + ITS-2 sequences of P. moraveci with those of P. angusticolle, P. crassum, P. depressum, P. ensicaudatum, P. reticulatum, P. streperae and Porrocaecum sp. showed 8.30% (P. angusticolle) to 30.1% (P. crassum) of nucleotide divergence.
We assigned the present specimens to the genus Porrocaecum based on the combination of morphological characters, including the lips possessing dentigerous ridges, the presence of interlabia, the ventriculus and ventricular appendage, the excretory pore just posterior to nerve ring and the absence of an intestinal caecum. In Porrocaecum, P. moraveci sp. n. is similar to the following species in having short interlabia (c. 1/3 length of lips), long intestinal caecum (c. 2/3 length of esophagus) and short spicules (0.60–1.00 mm), including P. angusticolle (Molin, 1860), P. depressum (Zeder, 1800), P. circum Wang, 1965 and P. phalacrocoracis Yamaguti, 1941 [2, 19, 20, 22, 36, 37].
Porrocaecum moraveci sp. n. differs from P. phalacrocoracis and P. circum by its distinctly shorter esophagus in both sexes (male 3.61 mm, female 4.68–6.61 mm in the new species vs male 2.08–3.20 mm, female 2.60–3.84 mm in P. phalacrocoracis and P. circum), unequal spicules (vs spicules equal in length in the latter two species), slightly less number of precloacal papillae (20 pairs vs 21–23 pairs in P. phalacrocoracis and P. circum) and much smaller body length of female (117.0–138.0 mm in P. moraveci sp. n. vs 50.0–65.0 mm in the latter two species).
The new species can be differentiated from P. angusticolle by having no cervical alae (vs cervical alae starting at base of subventral lips in P. angusticolle) and distinctly unequal spicules (vs spicules almost equal in length in the latter). Porrocaecum depressum has been reported from various birds of Accipitriformes, Falconiformes, Strigiformes worldwide, and there are considerable morphological variations in the lengths of the body, esophagus and spicules, the number and arrangement of caudal papillae and the morphology of the tail tip [2, 18,19,20, 38, 39]. Although the new species is rather similar to P. depressum, it is different from P. depressum by distinctly unequal spicules (vs spicules almost equal in length in P. depressum). Moreover, pairwise comparison of the genetic data of P. moraveci with those of P. angusticolle and P. depressum showed 1.88% (P. angusticolle) and 10.8% (P. depressum) of nucleotide divergence in the 28S region, 8.30% (P. angusticolle) to 14.5% (P. depressum) of nucleotide divergence in the ITS region and 7.98–8.18% (P. angusticolle) to 10.1% (P. depressum) of nucleotide divergence in the cox2 region, respectively, which strongly supports the new species being different from P. angusticolle and P. depressum.
General characterization of the complete mitogenomes of Porrocaecum (Porrocaecum) moraveci sp. n. and P. (Laymanicaecum) reticulatum
The circular mitogenomes of P. moraveci sp. n. and P. reticulatum are 14,517 bp and 14,210 bp in length, respectively, and both contain 36 genes, including 12 PCGs (missing atp8) (cox1–3, cytb, nad1–6, nad4L and atp6), 22 tRNA genes and two rRNA genes (rrnL and rrnS) (Fig. 4; Tables 3, 4). There are three non-coding regions in the mitogenome of P. moraveci sp. n.: NCR1, which is 1173 bp and located between tRNA-Ser2 and tRNA-Asn; NCR2, which is 101 bp and located between tRNA-Thr and nad4; and NCR3, which is 117 bp and located between nad4 and cox1. In comparison, in the mitogenome of P. reticulatum there are only two non-coding regions: NCR1, which is 860 bp and located between tRNA-Ser2 and tRNA-Asn; and NCR2, which is 113 bp and located between nad4 and cox1) (Fig. 4; Tables 3, 4). All genes are transcribed from the same DNA strand. The nucleotide contents of P. moraveci sp. n. and P. reticulatum mitogenomes are provided in Table 4. The overall A+T content in the mitogenomes of P. moraveci sp. n. and P. reticulatum is 69.95% and 67.22%, respectively, with both showing a strong nucleotide compositional bias toward A+T (Table 5).
The 12 PCGs of the mitogenomes of P. moraveci sp. n. and P. reticulatum are 10,185 bp and 10,284 bp in length (excluding termination codons) and ranged in size from 234 bp (nad4L) to 1584 bp (nad5), which encoded 3395 and 3428 amino acids, respectively (Tables 3–5). Among the 12 PCGs of P. moraveci sp. n., seven genes (cox1, cox2, cox3, cytb, nad1, nad4 and nad6) used TTG as the start codon, whereas three genes (nad5, nad4L and atp6) used ATT; GTG was used as the start codon by the nad2 and nad3 genes. TAG was the most commonly used termination codon (cox1, cox2, cox3, nad1, nad3, nad4L, nad5 and nad6); two genes (atp6 and nad4) used TAA, and the incomplete termination codon T was inferred for the nad2 and cytb genes (Table 3). Among the 12 PCGs of P. reticulatum, seven genes (cox1, cox2, cytb, nad1, nad2, nad4 and nad6) used TTG as the start codon, whereas four genes (atp6, nad4L, nad5 and cox3) used ATT; GTG was used as the start codon by only the nad3 gene. TAG was the most commonly used termination codon (cox1, cox2, cox3, nad3, nad4 and cytb); four genes (nad1, nad4L, nad6 and atp6) used TAA, and the incomplete termination codon T was inferred only for the nad2 and nad5 genes (Table 4). The components and usages of codons in the mitogenomes of P. moraveci sp. n. and P. reticulatum are shown in Fig. 5 and in Tables 3, 4.
In the mitogenomes of P. moraveci sp. n. and P. reticulatum, 22 tRNAs were identified. The length of these 22 tRNAs and their anticodon secondary structures are shown in Tables 3 and 4 and in Figs. 6 and 7. Two rRNAs (rrnL located between tRNA-His and nad3, and rrnS located between tRNA-Glu and tRNA-Ser2) were identified in the mitogenomes of P. moraveci sp. n. and P. reticulatum (Fig. 4); the length of each gene is provided in Tables 3 and 4.
The gene arrangement of 36 genes in the mitogenomes of P. moraveci sp. n. and P. reticulatum are both in the following order: cox1, tRNA-Cys, tRNA-Met, tRNA-Asp, tRNA-Gly, cox2, tRNA-His, rrnL, nad3, nad5, tRNA-Ala, tRNA-Pro, tRNA-Val, nad6, nad4l, tRNA-Trp, tRNA-Glu, rrnS, tRNA-Ser2, tRNA-Asn, tRNA-Tyr, nad1, atp6, tRNA-Lys, tRNA-Leu2, tRNA-Ser1, nad2, tRNA-Ile, tRNA-Arg, tRNA-Gln, tRNA-Phe, cytb, tRNA-Leu1, cox3, tRNA-Thr, nad4 (Fig. 4). This follows the GA3 type of gene arrangement.
The phylogenetic trees of ascaridoid nematodes constructed using the BI and ML methods based on the amino acid sequences of 12 PCGs of mitogenomes were found to have similar topologies, and both showed that the family Heterocheilidae (including only Ortleppascaris sinensis) is at the base of the phylogenetic trees, which formed a sister clade to the remaining Ascaridoidea (Fig. 8). The representatives of the family Anisakidae were divided into two subclades, representing the subfamilies Contracaecinae (including Contracaecum spp.) and Anisakinae (including Anisakis spp. and Pseudoterranova spp.), respectively. Phylogenetic analyses did not support the monophyly of the family Toxocaridae (including Toxocara spp. and Porrocaecum spp.), which showed the representatives of Toxocara clustered together with species of the family Ascarididae (including Ophidascaris spp., Toxascaris leonina, Baylisascaris spp., Parascaris spp. and Ascaris spp.), with strong support in the BI tree, but weak support in the ML tree (Fig. 8). In the genus Porrocaecum, both of the phylogenetic results showed that P. moraveci sp. n. is a sister to P. reticulatum with strong support (Fig. 8).
In the phylogenetic trees of Porrocaecum species constructed using BI and ML methods based on the 18S + ITS and 28S sequence data, P. moraveci sp. n. both showed a sister relationship with P. angusticolle with strong support. In the phylogenetic trees based on the 18S + ITS sequence data, P. reticulatum was clustered with P. depressum + P. moraveci sp. n. + P. angusticolle; but P. reticulatum was sister to P. depressum in the phylogenetic trees based on the 28S sequence data (Fig. 9).
The mitogenomes are very useful for understanding the epidemiology, population genetics and molecular phylogeny of ascaridoid nematodes. However, there are sequenced mitogenomes for only 30 species of ascaridoids (Table 1). In the genus Porrocaecum, only one unidentified species, Porrocaecum sp., has been genetically sequenced for the mitogenome. In the study reported here, we sequenced and assembled the complete mitogenomes of P. moraveci sp. n. and P. reticulatum for the first time.
The complete mitogenomes of P. moraveci sp. n. and P. reticulatum are 14,517 bp and 14,210 bp in length, respectively; as such, their lengths are similar to that of Porrocaecum sp. (14,568 bp) and Toxocara spp. (14,029–15045 bp) [14, 17, 40]. The lack of atp8 in the mitogenomes of P. moraveci sp. n. and P. reticulatum is typical of most of the available mitogenomes of nematodes, with the exception of Trichinella spp. and Trichuris spp., both of which have the atp8 gene [41,42,43,44,45,46]. The gene arrangement of the mitogenomes of P. moraveci sp. n. and P. reticulatum both belong to the GA3 type, agreeing well with that of Porrocaecum sp. and the other ascaridoid species [14, 17, 40, 47,48,49,50]. The overall A + T contents in the mitogenomes of P. moraveci sp. n. (69.95%) and P. reticulatum (67.22%) are distinctly lower than that of Porrocaecum sp. (71.42%). In fact, the overall A + T contents of P. reticulatum is the lowest of all available mitogenomes of ascaridoid nematodes. Additionally, comparative mitogenomics revealed that P. reticulatum and Porrocaecum sp. both have two non-coding regions in their mitogenomes, while there are three non-coding regions in the mitogenome of P. moraveci sp. n., which is different from all of the ascaridoid mitogenomes reported so far.
Although some recent phylogenies based on molecular studies have improved and challenged the traditional classification of the superfamily Ascaridoidea [7, 14,15,16,17, 51], phylogenetic relationships within several lineages of the Ascaridoidea remain unresolved due to a paucity of genetic data. In 1974, Hartwich erected the family Toxocaridae , but he subsequently degraded it as a subfamily in the Ascarididae, a change that was widely accepted in subsequent studies [8, 9, 52]. In his 1974 classification, Hartwich listed three genera in the subfamily Toxocarinae, including Toxocara, Porrocaecum and Paradujardinia . Later, Sprent (in 1983) transferred Paradujardinia into the family Heterocheilidae, a change that was supported by Gibson (in 1983) and Fagerholm (in 1991) [8, 9, 53]. In 1958,Osche considered that the family Toxocaridae was valid and erected a new subfamily Porrocaecinae for the genus Porrocaecum in the Toxocaridae . However, Osche’s proposal has received little attention since its inception, and only Chabaud (in 1965) suggested treating the Porrocaecinae as a tribe Porrocaecinea . Our phylogenetic analyses of ascaridoids based on the amino acid sequences of 12 PCGs using ML and BI inference showed that Porrocaecum and Toxocara have no close relationship and that the Toxocaridae/Toxocarinae classification proposed by Hartwich is not a monophyletic group; these findings conflict with these above-mentioned classifications but are roughly consistent with some previous molecular phylogenetics results [14, 16, 17, 56].
Mozgovoi  erected the subgenus Laymanicaecum in Porrocaecum based on the criterion of presence of gubernaculum in the male, and two species P. (Laymanicaecum) laymani Mozgovoi, 1950 and P. (Laymanicaecum) reticulatum (Mozgovoi, 1953) were assigned to the subgenus Laymanicaecum. However, P. (Laymanicaecum) laymani was subsequently transferred into the genus Mawsonascaris ; thus, P. (Laymanicaecum) reticulatum is the only species with gubernaculum in the male in Porrocaecum. In fact, as an important generic criterion, the gubernaculum is most often absent in the Anisakidae, Ascarididae, Toxocaridae and Raphidascarididae. Consequently, the systematic status of P. reticulatum and the subgenus Laymanicaecum in the Ascaridoidea is very puzzling. The present molecular phylogenetic analyses based on the 18S + ITS, 28S sequence data and 12 PCGs all showed that P. (Laymanicaecum) reticulatum nested in the representatives of the subgenus Porrocaecum, which supports the invalidity of the classification of Laymanicaecum as a subgenus and also indicates that care should be taken when using the gubernaculum as an important morphological character for delimitation of some genera within the Ascaridoidea.
Towards the integration of the present phylogenetic results and the traditional classification, we propose (i) to resurrect the subfamily Porrocaecinae including only the genus Porrocaecum; and (ii), and to degrade the Toxocaridae as a subfamily of the Ascarididae including only the genus Toxocara. Consequently, the Ascarididae should include four subfamilies, namely Ascaridinae, Porrocaecinae, Toxocaridae and Angusticaecinae. The present phylogenetic study represents a substantial step toward clarifying the evolutionary relationships of the subfamilies and families in the Ascaridoidea. However, we do not propose making any immediate systematic changes in the Ascaridoidea because a more rigorous study with broader representation of the Ascarididae and Ascaridoidea is required.
A new species of Porrocaecum, P. moraveci n. sp., was described based on the integration of morphological and genetic evidence from specimens collected from C. aeruginosus in the Czech Republic. The genetic characterization of the complete mitochondrial genomes of P. moraveci n. sp. and P. reticulatum was reported for the first time. Comparative mitogenomics revealed that P. moraveci n. sp. represents the first species with three non-coding regions and P. reticulatum has the lowest overall A + T content in the available mitogenomes of ascaridoid nematodes reported so far. Our phylogenetic results challenge the monophyly of the Toxocaridae and show that Porrocaecum and Toxocara do not have an affinity. The mitogenomic phylogenies determined here suggest (i) to degrade the Toxocaridae as a subfamily of the Ascarididae including only the genus Toxocara; and (ii) to resurrect the subfamily Porrocaecinae including only the genus Porrocaecum. The validity of the subgenus Laymanicaecum in Porrocaecum was also rejected. The present study enriches the database of ascaridoid mitogenomes and provides a new insight into the systematics of the superfamily Ascaridoidea.
Availability of data and materials
The nuclear and mitochondrial DNA sequences of Porrocaecum moraveci n. sp. and P. reticulatum obtained in the present study were deposited in GenBank database (sequences of Porrocaecum moraveci n. sp. under the accession numbers: OQ858491, OQ858492 (18S), OQ858562, OQ858563 (28S), OQ858560, OQ858561 (ITS), OQ863051 (mitochondrial genome); sequences of P. reticulatum under the accession numbers: OQ851895 (18S), OQ863745 (28S), OQ857284 (ITS), OQ863050 (mitochondrial genome). Type specimens of Porrocaecum moraveci n. sp. were deposited in the College of Life Sciences, Hebei Normal University, Hebei Province, China (under the accession numbers R: HBNU–N–B20220021GL, HBNU–N–B20220022GL and HBNU–N–B20220023GL).
Bayesian information criterion
Internal transcribed spacer
Scanning electron microscopy
Small ribosomal subunit
Large ribosomal subunit
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The authors are grateful to Dr. David Gibson (Natural History Museum, UK) and Vitaliy Kharchenko (I. I. Schmalhausen Institute of Zoology, National Academy of Sciences of Ukraine, Ukraine) for providing important literature.
This study was supported by the National Natural Science Foundation of China (Grant No. 32170442).
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Gu, XH., Guo, N., Chen, HX. et al. Mitogenomic phylogenies suggest the resurrection of the subfamily Porrocaecinae and provide insights into the systematics of the superfamily Ascaridoidea (Nematoda: Ascaridomorpha), with the description of a new species of Porrocaecum. Parasites Vectors 16, 275 (2023). https://doi.org/10.1186/s13071-023-05889-9