BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Hebei Key Laboratory of Animal Physiology Biochemistry and Molecular Biology Hebei Collaborative Innovation Center for Eco Environment College of Life Sciences Hebei Normal University Shijiazhuang 050024 Hebei Province People's Republic of China

Hebei Research Center of the Basic Discipline Cell Biology Ministry of Education Key Laboratory of Molecular and Cellular Biology Shijiazhuang 050024 Hebei Province People's Republic of China

Muzeum Komenského 5 Přerově 750 02 Přerově Czech Republic

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Skrjabin KI, Schikhobalova NP, Mozgovoi AA. Key to parasitic nematodes. Volume II: Oxyurata and Ascaridata. Moscow: Izdatei'stvo Akademii Nauk SSSR; 1951.

Mozgovoi AA. Ascaridata of animals and man and the diseases caused by them. Volume 2: Part 1 of Osnovy nematodologii. Moscow: Izdatel’stvo Akademii Nauk SSSR; 1953.

Yamaguti S. Systema helminthum. The nematodes of vertebrates, vol. 3. New York: Interscience; 1961.

Hartwich G. Keys to genera of the Ascaridoidea. In: Anderson RC, Chabaud AG, Willmott S, editors. CIH keys to the nematode parasites of vertebrates. Farnham Royal: Commonwealth Agricultural Bureaux; 1974. p. 15.

Bruce NL, Adlard RD, Cannon LRG. Synoptic checklist of ascaridoid parasites (Nematoda) from fish hosts. Invertebr Taxon. 1994;8:583–674.

Hodda M. Phylum Nematoda Cobb 1932. In: Zhang ZQ, editor. Animal biodiversity: an outline of higher-level classification and survey of taxonomic richness (Addenda 2013) Auckland: Magnolia Press; 2011. pp. 63–95. PubMed

Li L, Lü L, Nadler SA, Gibson DI, Zhang LP, Chen HX, et al. Molecular phylogeny and dating reveal a terrestrial origin in the early carboniferous for ascaridoid nematodes. Syst Biol. 2018;67:888–900. PubMed

Gibson DI. The systematics of ascaridoid nematodes—a current assessment. In: Stone R, Platt HM, Khalil LF, editors. Concepts in nematode systematics. Systematics Association special volume 22. London: Academic Press; 1983. p. 321–38.

Fagerholm HP. Systematic implications of male caudal morphology in ascaridoid nematode parasites. Syst Parasitol. 1991;19:215–218.

Gibbons LM, Jacobs DE, Sani RA. Toxocara malaysiensis n. sp. (Nematoda: Ascaridoidea) from the domestic cat (Felis catus Linnaeus, 1758) J Parasitol. 2001;87:660–665. PubMed

Anderson RC. Nematode parasites of vertebrates their development and transmission. 2. Wallingford: CABI Publishing; 2000.

Macpherson CN. The epidemiology and public health importance of toxocariasis: a zoonosis of global importance. Int J Parasitol. 2013;43:999–1008. PubMed

Ma GX, Holland CV, Wang T, Hofmann A, Fan CK, Maizels RM, et al. Human toxocariasis. Lancet Infect Dis. 2017;18:14–24. PubMed

Han L, Yang YL, Li HM, Zhou XY, Zhou MC, Liu TL, et al. Gene rearrangements in the mitochondrial genome of ten ascaris species and phylogenetic implications for Ascaridoidea and Heterakoidea families. Int J Biol Macromol. 2022;221:1394–1403. PubMed

Nadler SA, Hudspeth DS. Ribosomal DNA and phylogeny of the Ascaridoidea (Nemata: Secernentea): implications for morphological evolution and classification. Mol Phylogenet Evol. 1998;10:221–236. PubMed

Nadler SA, Hudspeth DS. Phylogeny of the Ascaridoidea (Nematoda: Ascaridida) based on three genes and morphology: hypotheses of structural and sequence evolution. J Parasitol. 2000;86:380–393. PubMed

Xie Y, Wang LD, Chen YJ, Wang Z, Zhu PC, Hu Z, et al. The complete mitogenome of Toxocara vitulorum: novel in-sights into the phylogenetics in Toxocaridae. Animals. 2022;12:e3546. PubMed PMC

Cram E. Bird parasites of nematode suborders Strongylata, Ascaridata and Spirurata. Bull US Nat Mus. 1927;140:135–146.

Hartwich G. Revision der vogelparasitischen Nematoden Mitteleuropas I. Die Gattung Porrocaecum Railliet and Henry, 1912 (Ascaridoidea) Mitt Zool Mus Berlin. 1959;35:107–147.

Fagerholm HP, Overstreet RM. Ascaridoid Nematodes: Contracaecum, Porrocaecum, and Baylisascaris. In: Atkinson CT, Thomas NJ, Hunter DB, editors. Parasitic diseases of wild birds. Blackwell: Wiley; 2009. pp. 413–433.

Li L, Guo YN, Zhang LP. Porrocaecum parvum n. sp. and P. reticulatum (Linstow, 1899) (Nematoda: Ascaridoidea) from birds in China. Syst Parasitol. 2015;92:141–149. PubMed

Guo N, Sitko J, Chen HX, Li L. Morphological and genetic characterization of Porrocaecum angusticolle (Molin, 1860) (Nematoda: Ascaridomorpha) from the common buzzard Buteo buteo (Linnaeus) (Accipitriformes: Accipitridae) in Czech Republic. Parasitol Int. 2021;83:1–7. PubMed

Floyd RM, Rogers AD, Lambshead PJD, Smith CR. Nematode-specific PCR primers for the 18S small subunit rRNA gene. Mol Ecol Notes. 2005;5:611–612.

Zhu XQ, D'Amelio S, Paggi L, Gasser RB. Assessing sequence variation in the internal transcribed spacers of ribosomal DNA within and among members of the Contracaecum osculatum complex (Nematoda: Ascaridoidea: Anisakidae) Parasitol Res. 2000;86:677–683. PubMed

Jin JJ, Yu WB, Yang JB, Song Y, De Pamphilis CW, Yi TS, et al. GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biol. 2020;21:e241. PubMed PMC

Meng GL, Li YY, Yang CT, Liu SL. MitoZ: A toolkit for animal mitochondrial genome assembly, annotation and visualization. Nucleic Acids Res. 2019;47:e63. PubMed PMC

Gruber AR, Bernhart SH, Lorenz R. The viennaRNA web services. In: Picardi E, editor. RNA bioinformatics, methods in molecular biology. New York: Springer; 2015. pp. 307–326. PubMed

Bernt M, Merkle D, Ramsch K, Fritzsch G, Perseke M, Bernhard D, et al. Inferring genomic rearrangements based on common intervals. Bioinformatics. 2007;23:2957–2958. PubMed

Reuter JS, Mathews DH. RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinform. 2010;11:e129. PubMed PMC

Lee BD. Python implementation of codon adaptation index. JOSS. 2018;3:905.

Katoh K, Standley DM. Mafft multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–780. PubMed PMC

Criscuolo A, Gribaldo S. BMGE (block mapping and gathering with entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments. BMC Evol Biol. 2010;10:e210. PubMed PMC

Zhang D, Gao F, Jakovlić I, Zou H, Zhang J, Li WX, et al. PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Mol Ecol Resour. 2020;20:348–355. PubMed

Minh BQ, Hahn MW, Lanfear R. New methods to calculate concordance factors for phylogenomic datasets. Mol Biol Evol. 2020;37:2727–2733. PubMed PMC

Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012;61:539–542. PubMed PMC

Wang PQ. Notes on some nematodes of the suborder Ascaridata from Fukien, China. Acta Parasitol Sin. 1965;2:366–379.

Yamaguti Y. Studies on the helminth fauna of Japan Part 36 Avian nematodes, II. Jpn J Zool. 1941;9:441–480.

Yoshino T, Yanai T, Asano M. First record of Porrocaecum depressum (Nematoda: Ascaridoidea), Craspedorrhynchus sp. and Degeeriella sp. (Insecta: Psocodea) obtained from a Hodgson's hawk eagle, Spizaetus nipalensis, in Gifu Prefecture, Japan. Biogeography. 2012;14:127–132.

Morgan BB, Schiller EL. Porrocaecum angusticolle (Nematoda) in North American hawks. Trans Am Microsc Soc. 1950;69:371–372.

Li MW, Lin RQ, Song HQ, Wu XY, Zhu XQ. The complete mitochondrial genomes for three Toxocara species of human and animal health significance. BMC Genomics. 2008;9:e224. PubMed PMC

Lavrov DV, Brown WM. Trichinella spiralis mtDNA: a nematode mitochondrial genome that encodes a putative ATP8 and normally structured tRNAs and has a gene arrangement relatable to those of coelomate metazoans. Genetics. 2001;157:621–637. PubMed PMC

Liu GH, Gasser RB, Su A, Nejsum P, Peng L, Lin RQ, et al. Clear genetic distinctiveness between human- and pig-derived Trichuris based on analyses of mitochondrial datasets. PLoS Negl Trop Dis. 2012;6:e1539. PubMed PMC

Liu GH, Wang Y, Xu MJ, Zhou DH, Ye YG, Li JY, et al. Characterization of the complete mitochondrial genomes of two whipworms Trichuris ovis and Trichuris discolor (Nematoda: Trichuridae) Infect Genet Evol. 2012;12:1635–1641. PubMed

Liu GH, Gasser RB, Nejsum P, Wang Y, Chen Q, Song HQ, et al. Mitochondrial and nuclear ribosomal DNA evidence supports the existence of a new Trichuris species in the endangered François' leaf-monkey. PLoS ONE. 2013;8:e66249. PubMed PMC

Mohandas N, Jabbar A, Podolska M, Zhu XQ, Littlewood DT, Jex AR, et al. Mitochondrial genomes of Anisakis simplex and Contracaecum osculatum (sensu stricto)–comparisons with selected nematodes. Infect Genet Evol. 2014;21:452–462. PubMed

Hawash MB, Andersen LO, Gasser RB, Stensvold CR, Nejsum P. Mitochondrial genome analyses suggest multiple Trichuris species in humans, baboons, and pigs from different geographical regions. PLoS Negl Trop Dis. 2015;14:e0004059. PubMed PMC

Yamada A, Ikeda N, Ono H. The complete mitochondrial genome of Anisakis pegreffii Campana-Rouget and Biocca, 1955, (Nematoda, Chromadorea, Rhabditida, Anisakidae)—clarification of mitogenome sequences of the Anisakis simplex species complex. Mitochondrial DNA B Resour. 2017;2:240–241. PubMed PMC

Zhao Q, Abuzeid AMI, He L, Zhuang T, Li X, Liu J, et al. The mitochondrial genome sequence analysis of Ophidascaris baylisi from the Burmese python (Python molurus bivittatus) Parasitol Int. 2021;85:e102434. PubMed

Gao JF, Zhang XX, Wang XX, Li Q, Li Y, Xu WW, et al. According to mitochondrial DNA evidence, Parascaris equorum and Parascaris univalens may represent the same species. J Helminthol. 2019;93:383–388. PubMed

Jabbar A, Littlewood DT, Mohandas N, Briscoe AG, Foster PG, Müller F, et al. The mitochondrial genome of Parascaris univalens—implications for a "forgotten" parasite. Parasit Vectors. 2014;7:e428. PubMed PMC

Liu GH, Nadler SA, Liu SS, Podolska M, D'Amelio S, Shao RF, et al. Mitochondrial phylogenomics yields strongly supported hypotheses for Ascaridomorph nematodes. Sci Rep. 2016;6:e39248. PubMed PMC

Hartwich G. Die Vorderdarmstrukturen, das Exkretionsystem sowie der Kopfbau der Ascariden und ihre taxonomische Bedeutung. Wiss Z-Martin-Luther-Univ. 1954;3:1171–212.

Sprent JFA. Observations on the systematics of ascaridoid nematodes. In: Stone AR, Platt HM, Khalil LF, editors. Concepts in nematode systematics. London: Academic Press; 1983. pp. 303–319.

Osche G. Beiträge zur morphologie, okologie und phylogenie der Ascaridoidea (Nematoda); parallelen in der evolution von parasit und wirt [morphology, ecology and phylogeny of Ascaridoidea (Nematoda); parallels in the evolution of parasite and host] Z Parasitenkd. 1958;18:479–572. PubMed

Chabaud AG. Ordre des Ascaridida. In: Grassé PP, editor. Traité de Zoologie. Tome IV, fascicule 3. Paris: Masson et Cie; 1965. p. 932–1025.

Nadler SA, D'Amelio S, Fagerholm HP, Berland B, Paggi L. Phylogenetic relationships among species of Contracaecum Railliet & Henry, 1912 and Phocascaris Høst, 1932 (Nematoda: Ascaridoidea) based on nuclear rDNA sequence data. Parasitology. 2000;121:455–463. PubMed

Sprent JFA. Some ascaridoid nematodes of fishes: Paranisakis and Mawsonascaris ng. Syst Parasitol. 1990;15:41–63.

Wolstenholme DR, Okimoto R, Macfarlane JL. Nucleotide correlations that suggest tertiary interactions in the TV-replacement loop-containing mitochondrial tRNAs of the nematodes, Caenorhabditis elegans and Ascaris suum. Nucleic Acids Res. 1994;22:4300–4306. PubMed PMC

Wang BJ, Gu XB, Yang GY, Wang T, Lai WM, Zhong ZJ, et al. Mitochondrial genomes of Heterakis gallinae and Heterakis beramporia support that they belong to the infraorder Ascaridomorpha. Infect Genet Evol. 2016;40:228–235. PubMed

Liu SS, Liu GH, Zhu XQ, Weng YB. The complete mitochondrial genome of Pseudoterranova azarasi and comparative analysis with other anisakid nematodes. Infect Genet Evol. 2015;33:293–298. PubMed

Park YC, Kim W, Park JK. The complete mitochondrial genome of human parasitic roundworm, Ascaris lumbricoides. Mitochondrial DNA. 2011;22:91–93. PubMed

Chen Y, Wang L, Zhou X, Tang R, Li Y, Liu Y, et al. The mitochondrial genome of the sheep roundworm Ascaris ovis (Ascaridida: Nematoda) from southwest China. Mitochondrial DNA B Resour. 2021;6:410–412. PubMed PMC

Xie Y, Zhang Z, Wang C, Lan J, Li Y, Chen Z, et al. Complete mitochondrial genomes of Baylisascaris schroederi, Baylisascaris ailuri and Baylisascaris transfuga from giant panda, red panda and polar bear. Gene. 2011;482:59–67. PubMed

Xie Y, Zhang Z, Niu L, Wang Q, Wang C, Lan J, et al. The mitochondrial genome of Baylisascaris procyonis. PLoS ONE. 2011;6:e27066. PubMed PMC

Jin YC, Li XY, Liu JH, Zhu XQ, Liu GH. Comparative analysis of mitochondrial DNA datasets indicates that Toxascaris leonina represents a species complex. Parasit Vectors. 2019;12:e194. PubMed PMC

Zhou CY, Ma J, Tang QW, Zhu XQ, Xu QM. The mitogenome of Ophidascaris wangi isolated from snakes in China. Parasitol Res. 2021;120:1677–1686. PubMed

Zhao JH, Tu GJ, Wu XB, Li CP. Characterization of the complete mitochondrial genome of Ortleppascaris sinensis (Nematoda: Heterocheilidae) and comparative mitogenomic analysis of eighteen Ascaridida nematodes. J Helminthol. 2018;92:369–378. PubMed

Kijewska A, Rokicki J, Sitko J, Wegrzyn G. Ascaridoidea: a simple DNA assay for identification of 11 species infecting marine and freshwater fish, mammals, and fish-eating birds. Exp Parasitol. 2002;101:35–39. PubMed

Nadler SA, Carreno RA, Mejía-Madrid H, Ullberg J, Pagan C, Houston R, Hugot JP. Molecular phylogeny of clade III nematodes reveals multiple origins of tissue parasitism. Parasitology. 2007;134:1421–1442. PubMed

Zhu X, Gasser RB, Jacobs DE, Hung GC, Chilton NB. Relationships among some ascaridoid nematodes based on ribosomal DNA sequence data. Parasitol Res. 2000;86:738–744. PubMed

Honisch M, Krone O. Phylogenetic relationships of Spiruromorpha from birds of prey based on 18S rDNA. J Helminthol. 2008;82:129–133. PubMed

He X, Lü MN, Liu GH, Lin RQ. Genetic analysis of Toxocara cati (Nematoda: Ascarididae) from Guangdong province, subtropical China. Mitochondrial DNA A DNA Mapp Seq Anal. 2018;29:132–135. PubMed

Li Y, Niu L, Wang Q, Zhang Z, Chen Z, Gu X, et al. Molecular characterization and phylogenetic analysis of ascarid nematodes from twenty-one species of captive wild mammals based on mitochondrial and nuclear sequences. Parasitology. 2012;139:1329–1338. PubMed