Monophyly of clade III nematodes is not supported by phylogenetic analysis of complete mitochondrial genome sequences

Joong-Ki Park 0 Tahera Sultana 2 Sang-Hwa Lee 1 Seokha Kang 6 Hyong Kyu Kim 5 Gi-Sik Min 2 Keeseon S Eom 4 Steven A Nadler 3 0 Graduate Program in Cell Biology and Genetics and Department of Parasitology, College of Medicine, Chungbuk National University , Cheongju 361-763 , Korea 1 Graduate Program in Cell Biology and Genetics, College of Medicine, Chungbuk National University , Cheongju 361-763 , Korea 2 Department of Biological Sciences, Inha University , Incheon 402-751 , Korea 3 Department of Nematology, University of California , Davis, CA 95616 , USA 4 Department of Parasitology, College of Medicine, Chungbuk National University , Cheongju 361-763 , Korea 5 Department of Microbiology, College of Medicine, Chungbuk National University , Cheongju 361-763 , Korea 6 Department of Biotechnology and Bioinformatics, College of Science and Technology, Korea University , Chungnam , Korea Background: The orders Ascaridida, Oxyurida, and Spirurida represent major components of zooparasitic nematode diversity, including many species of veterinary and medical importance. Phylum-wide nematode phylogenetic hypotheses have mainly been based on nuclear rDNA sequences, but more recently complete mitochondrial (mtDNA) gene sequences have provided another source of molecular information to evaluate relationships. Although there is much agreement between nuclear rDNA and mtDNA phylogenies, relationships among certain major clades are different. In this study we report that mtDNA sequences do not support the monophyly of Ascaridida, Oxyurida and Spirurida (clade III) in contrast to results for nuclear rDNA. Results from mtDNA genomes show promise as an additional independently evolving genome for developing phylogenetic hypotheses for nematodes, although substantially increased taxon sampling is needed for enhanced comparative value with nuclear rDNA. Ultimately, topological incongruence (and congruence) between nuclear rDNA and mtDNA phylogenetic hypotheses will need to be tested relative to additional independent loci that provide appropriate levels of resolution. Results: For this comparative phylogenetic study, we determined the complete mitochondrial genome sequences of three nematode species, Cucullanus robustus (13,972 bp) representing Ascaridida, Wellcomia siamensis (14,128 bp) representing Oxyurida, and Heliconema longissimum (13,610 bp) representing Spirurida. These new sequences were used along with 33 published nematode mitochondrial genomes to investigate phylogenetic relationships among chromadorean orders. Phylogenetic analyses of both nucleotide and amino acid sequence datasets support the hypothesis that Ascaridida is nested within Rhabditida. The position of Oxyurida within Chromadorea varies among analyses; in most analyses this order is sister to the Ascaridida plus Rhabditida clade, with representative Spirurida forming a distinct clade, however, in one case Oxyurida is sister to Spirurida. Ascaridida, Oxyurida, and Spirurida (the sampled clade III taxa) do not form a monophyletic group based on complete mitochondrial DNA sequences. Tree topology tests revealed that constraining clade III taxa to be monophyletic, given the mtDNA datasets analyzed, was a significantly worse result. Conclusion: The phylogenetic hypotheses from comparative analysis of the complete mitochondrial genome data (analysis of nucleotide and amino acid datasets, and nucleotide data excluding 3rd positions) indicates that nematodes representing Ascaridida, Oxyurida and Spirurida do not share an exclusive most recent common ancestor, in contrast to published results based on nuclear ribosomal DNA. Overall, mtDNA genome data provides reliable support for nematode relationships that often corroborates findings based on nuclear rDNA. It is anticipated that additional taxonomic sampling will provide a wealth of information on mitochondrial genome evolution and sequence data for developing phylogenetic hypotheses for the phylum Nematoda. - Background The phylum Nematoda (roundworms) is an ecologically diverse clade, that has significant impact as parasites of animals and plants, along with providing a diverse array of ecosystem services as free-living species in many environments [1-3]. Parasitic nematodes include many species that negatively impact human health, agricultural production, wildlife, and companion animals [4]. Free-living nematode species occur in almost every environment, particularly soils and aquatic sediments and serve important roles in food webs, particularly as decomposers and predators [5]. Despite their biodiversity, ubiquity, and important impacts, molecular phylogenetic hypotheses for major groups of nematodes, including zooparasitic species, are somewhat lacking when compared to recent advances for other organisms. In addition, prior to the application of molecular data, hypotheses of evolutionary relationships for nematodes relied heavily on interpretation of a limited number of key morphological characters or placed excessive emphasis on ecological features; as a result, different investigators recovered different relationships, with resulting debate concerning nematode classification systems (see [6]). During the last 20 years, molecular sequence data have been employed for nematode phylogenetics and have provided testable hypotheses at different levels, from across the phylum to within genera. The vast majority of these studies have been based on gene sequences representing a single nuclear locus, ribosomal DNA (e.g., 18S, 28S, and ITS genes). Such ribosomal DNA studies have included phylum-wide investigations [7-9], studies focused on particular clades identified in phylum-wide investigations (Clade III sensu [7];[10]), evaluation of traditional orders or suborders (Cephalobina [11]; Strongylida [12]; Tylenchina [13,14]), and examination of particular genera [15-17]. The first phylum-wide molecular phylogeny of nematodes [7] recognized five major clades. Only one of these groups (clade III) was exclusively composed of animal parasites. This clade represents four traditional orders (Ascaridida, Spirurida, Rhigonematida and Oxyurida), and includes many species of veterinary and medical importance. A revised classification system, based on interpretation of SSU (18S) rDNA phylogenetic trees, has been proposed with each traditional clade III order given infraordinal status within Rhabditida, suborder Spirurina [6,18], and renamed Ascaridomorpha, Spiruromorpha, Rhigonematomorpha and Oxyuridomorpha. Relatively comprehensive sampling and analysis of clade III taxa (103 taxa) using SSU sequences generally supported clade III monophyly (102/103 taxa were monophyletic) and that of Oxyurida, but not monophyly of Ascaridida or Spirurida as traditionally defined [10]. However, relationships among the major clade III orders were not reliably resolved by SSU data, with major differences among phylogenetic trees according to inference method, and inclusion or exclusion of charac (...truncated)