Monophyly of clade III nematodes is not supported by phylogenetic analysis of complete mitochondrial genome sequences
© Park et al; licensee BioMed Central Ltd. 2011
Received: 28 December 2010
Accepted: 3 August 2011
Published: 3 August 2011
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.
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.
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.
KeywordsMitochondrial genome Molecular phylogeny Ascaridida Spirurida Oxyurida Chromadorea Nematoda
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 . 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 . 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 ). 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 ;), evaluation of traditional orders or suborders (Cephalobina ; Strongylida ; Tylenchina [13, 14]), and examination of particular genera [15–17]. The first phylum-wide molecular phylogeny of nematodes  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 . 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 characters judged to be ambiguous with respect to positional (alignment) homology .
Analyses of complete mitochondrial genome sequences have provided an alternative hypothesis for relationships among clade III taxa [19–21]. These mitochondrial trees supported a closer relationship between Rhabditida and Ascaridida, with the single oxyurid species in the analysis sister to Rhabditida+Ascaridida, and this latter clade sister to Spirurida. The mtDNA tree topology conflicts with results based on SSU rDNA, which strongly supports monophyly of taxa representing the sampled superfamilies and orders . The recent development of high-throughput approaches for sequencing and annotating mitochondrial genomes  holds great promise for increasing the representation of nematode mtDNA genomes for analysis, and has already yielded several new strongylid sequences analyzed herein. However, high-throughput approaches have not yet been applied to increase taxon sampling for clade III nematodes, which remain sparse at most taxonomic levels, including the absence of orders (e.g., clade III, Rhigonematida). The great majority of mitochondrial genome sequences for chromadorean nematodes are derived from the Rhabditida (24 out of 36 species). In contrast, Oxyurida is represented by one species, and although Spirurida and Ascaridida are represented by multiple species, each of these orders includes a single superfamily (Filarioidea and Ascaridoidea). Improving the comparative utility of mtDNA phylogenetics requires broadened taxonomic sampling of nematode mitochondrial genomes.
In this study, we determined the complete mitochondrial genome sequences of three zooparasitic clade III nematode species: Cucullanus robustus, the first representative of the superfamily Seuratoidea (Ascaridida), Wellcomia siamensis, the second representative of the Oxyurida, and Heliconema longissimum, the first representative of the superfamily Physalopteroidea (Spirurida). We also used these data and complete mtDNA sequences from GenBank to infer phylogenetic relationships among major groups of enoplean and chromadorean nematodes.
Results and discussion
Gene organization and content
The mitochondrial genome organization of Cucullanus robustus(Cr), Wellcomia siamensis(Ws) and Heliconema longissimum(Hl)
Positions and lengths of
nucleotide sequences (bp)
Positions and lengths of
nucleotide sequences (bp)
Positions and lengths of
nucleotide sequences (bp)
1 - 1578 (1578)
1 - 1558 (1558)
1 - 1593 (1593)
1577 - 1633 (57)
1564 - 2424 (861)
1644 - 1703 (60)
1637 - 1690 (54)
2429 - 3029 (601)
1708 - 2145 (438)
1693 - 1748 (56)
3030 - 3084 (55)
2146 - 2201 (56)
1750 - 1803 (54)
3093 - 3145 (53)
2202 - 2256 (55)
1804 - 2499 (696)
3165 - 3987 (823)
2257 - 3343 (1087)
2501 - 2555 (55)
3988 - 4049 (62)
3344 - 3397 (54)
2556 - 3513 (958)
4050 - 4106 (57)
3398 - 4174 (777)
3514 - 3846 (333)
4108 - 4162 (55)
4243 - 4300 (58)
3850 - 5433 (1584)
4170 - 4224 (55)
4301 - 4354 (54)
5558 - 5992 (435)
4228 - 4282 (55)
4355 - 4631 (277)
5995 - 6228 (234)
4283 - 5235 (953)
4632 - 4685 (54)
6229 - 6284 (56)
5236 - 5297 (62)
4686 - 4738 (53)
6285 - 6338 (54)
5350 - 5784 (435)
4739 - 4796 (58)
6340 - 6393 (54)
5793 - 5846 (54)
4797 - 5030 (234)
6394 - 7070 (677)
5851 - 5909(59)
5031 - 5712 (682)
7071 - 7122 (52)
5912 - 5967 (56)
5713 - 5768 (56)
7123 - 7780 (658)
5970 - 7043 (1074)
5769 - 6636 (868)
7781 - 7834 (54)
7098 - 7859 (762)
6637 - 6693 (57)
7835 - 8707 (873)
7840 - 7897 (58)
6694 - 7272 (579)
8712 - 9311 (600)
7901 - 7963 (63)
7273 - 7325 (53)
9312 - 9372 (61)
7969 - 9192 (1224)
7326 - 7384 (59)
9375 - 9429 (55)
9195 - 9252 (58)
7385 - 8080 (696)
9430 - 9482 (53)
9253 - 9486 (234)
8083 - 8140 (58)
9483 - 10325 (843)
9508 - 9570 (63)
8141 - 9113 (973)
10326 - 10384 (59)
9571 - 9904 (334)
9114 -9440 (327)
10387 - 10441 (55)
9905 - 11488 (1584)
9441 - 9495 (55)
10442 - 10496 (55)
11494 - 11548 (55)
9496 - 9547 (52)
10498 - 10553 (56)
11572 - 11632 (61)
9548 - 9605 (58)
10554 - 11652 (1099)
11648 - 11703 (56)
9609 - 9665 (57)
11653 - 11708 (56)
11707 - 12457 (751)
9666 - 9719 (54)
11709 - 12474 (766)
12498 - 12550 (53)
9725 - 11314 (1590)
12475 - 12529 (55)
12551 - 13282 (732)
11315 - 11372 (58)
12530 - 13759 (1230)
13283 - 13345 (63)
11403 - 11454 (52)
13762 - 13815 (54)
13490 - 13544 (55)
11470 - 12318 (849)
13860 - 13916 (57)
13545 - 14055 (511)
12321 - 12374 (54)
13917 - 13972 (56)
14056 - 14112 (57)
12377 - 13603 (1227)
Nucleotide composition of Cucullanus robustus (Cr), Wellcomia siamensis (Ws), Heliconema longissimum (Hl) mtDNAs
Protein coding sequence
Non coding region
Protein-coding genes and codon usage
Twelve protein-coding genes were identified for each of the three species (Table 1), ranging in size from 234 bp (nad4L) for C. robustus, W. siamensis and H. longissimum to 1,593 bp (cox1) for H. longissimum. For the 12 protein-coding genes of C. robustus, five (atp6, cob, nad4L, nad5 and nad6) are inferred to use ATT as the start codon, whereas the other seven (cox1, cox2, cox3, nad1, nad2, nad3 and nad4) use TTG. In H. longissimum, six genes (cox1, cox2, cox3, nad5, cob and atp6) are inferred to start with ATT, four others (nad3, nad4, nad4L and nad6) start with ATA, and nad1 and nad2 use TTG as the start codon. In W. siamensis, eight genes (cox3, nad2, nad3, nad4L, nad5, nad6, atp6 and cob) are inferred to use TTG as the start codon, three genes (cox2, nad1 and nad4) start with ATT, and cox1 uses ATA as the start codon. For C. robustus, eight genes are inferred to use TAA as the termination codon (cox2, nad2, nad3, nad4, nad4L, nad5, nad6 and atp6) and two use TAG (cox1 and nad1). In H. longissimum, TAA is also the most commonly used termination codon (cox1, cox2, nad2, cox3, nad4, nad4L and nad6) whereas TAG is used for three genes (nad3, nad5 and atp6). For W. siamensis, TAG is used as the termination codon for six genes (cox3, nad1, nad4, nad4L, nad5 and nad6), whereas TAA is used only for the cob gene. Termination with the incomplete codon T is inferred for cox3 and cob of C. robustus and nad1 and cob of H. longissimum. The incomplete termination codon T is used in high frequency (five of 12 genes) for W. siamensis (cox1, cox2, nad2, nad3 and atp6). Start and termination codons are shown for each species in Table 1. The protein-coding genes of the three mitochondrial genomes are composed of amino acids that are encoded by T-rich codons, as has been previously documented for all major clades of chromadorean nematodes (Onchocerca volvulus, Spirurida ; Strongyloides stercoralis, Rhabditida ; Anisakis simplex, Ascaridida ; Toxocara spp., Ascaridida ; Enterobius vermicularis, Oxyurida ). The six most frequently used codons are all T-rich (having more than two Ts in a triplet): TTT (13.1%), TTA (6.91%), TTG (6.56%), ATT (5.71%), GTT (5.1%), and TCT (4.48%) in C. robustus; TTT (18.88%), TTA (8.78%), ATT (7.55%), GTT (7.11%), TTG (6.08%), and TAT (5.02%) in W. siamensis; TTT (18.0%), TTA (9.41%), ATT (8.82%), TAT (6.01%), GTT (3.96%), and TCT (3.69%) in H. longissimum. These T-rich codons account for almost half of all codons used (Additional file 2) in these three species (43.3% in C. robustus, 53.4% in W. siamensis, and 49.9% in H. longissimum). In addition, unequal use of synonymous codons, especially bias against codons with C in the third position is very prominent in four-fold and two-fold degenerate codon families. For instance, the relative frequency of phenylalanine encoded by TTC is strikingly decreased in each species (1.26% in C. robustus, 0.06% in W. siamensis, and 0.35% in H. longissimum). These factors (higher frequency of T-rich codons and unequal usage of synonymous codons with bias against C-rich codons) correlate with the high percentage of A+T content in the nucleotide composition of protein-coding genes (A+T content of 69.6%, 76.9%, and 77.8% for C. robustus, W. siamensis, and H. longissimum, respectively; Table 2).
Ribosomal RNA and Transfer rRNA genes
The small subunit ribosomal RNA (rrnS) and large subunit ribosomal RNA (rrnL) of each mtDNA were initially identified by comparison with related species of the same orders. It was not possible to confirm the exact starting and ending positions of the two ribosomal RNA genes, therefore the entire flanking regions between the boundaries of their respective adjoining genes were estimated as rrnS and rrnL in each species (Table 1). The rrnS of C. robustus, W. siamensis and H. longissimum is 677 bp, 732 bp, and 682 bp in size, respectively. The total length of rrnL is 958 bp in C. robustus, 953 bp in W. siamensis, and 973 bp in H. longissimum. The predicted secondary structures of 22 tRNAs for each of the three species (Additional file 3) are similar to those reported from a variety of other nematode species (except Trichinella spiralis; ). For example, all these tRNAs, except for trnS1 and trnS2, lack a TΨC and instead are equipped with a TV-replacement loop; in addition, trnS1 and trnS2 lack a dihydrouridine (DHU) arm, but have a TΨC stem-loop structure [19, 20, 24, 26–29].
The AT-rich non-coding regions (NCR) for C. robustus, W. siamensis and H. longissimum are, respectively, 658 bp (located between trnS2 and trnY), 511 bp (located between trnS2 and trnN), and 277 bp (located between trnL2 and trnN) in length (Table 1). These NCR regions have much higher A+T content than any other regions of these mitochondrial genomes (Table 2), with A+T contents of 87.4% (C. robustus), 96.7% (H. longissimum) and 92.3% (W. siamensis).
Nematode Mitochondrial phylogeny
Some chromadorean taxa also showed variation in phylogenetic position among analyses and between datasets. Some of these differences likely reflect differences in potential resolution of different datasets (e.g., amino acid characters versus nucleotide data). For example, relationships within superfamilies of Rhabditida often received higher ML bootstrap values in analyses of all nucleotides than the dataset that excluded 3rd positions of codons. ML and BI analyses of the nucleotide datasets recovered all Ascaridida (including C. robustus) as monophyletic (99% BP), whereas in the BI of amino acid data (Figure 2), C. robustus (Seuratoidea) was sister (with high posterior probability; 0.99 BPP) to a group of Ascaridida and Rhabditida. Notably, one molecular phylogeny based on nuclear SSU rDNA also yielded an unexpected relationship for a different seuratoid genus, indicating more recent common ancestry with members of Rhabditida and Diplogasterida . Similarly, although the position of Steinernema carpocapsae (an entomopathogenic nematode used in biological control) was strongly supported in Bayesian analysis of amino acids (Figure 2), it was unstable and poorly supported in analyses of nucleotide data. Steinernema has also been of unstable or conflicting phylogenetic position in analyses of SSU and LSU nuclear ribosomal DNA [7, 11, 33]. In some recent classifications reflecting certain SSU trees , Steinernema and Strongyloides have been placed in the same superfamily (Strongyloidoidea), and this taxonomic convention was followed in this study. However, Steinernema plus Strongyloides was never recovered as monophyletic in analyses of mitochondrial genome data, and instead Steinernema was more closely related to other Rhabditida plus Ascaridida (Figures 1, 3 and 4). Analysis of nuclear LSU rDNA also depicts Steinernema as more closely related to Rhabditida such as Caenorhabditis and Heterorhabditis than to Strongyloides. Although some studies based on nuclear SSU rDNA have depicted Steinernema as sister to Strongyloides and closely related to free living Panagrolaimoidea [7, 8], other studies have found the position of this genus to be unresolved by SSU sequences . Perhaps factors influencing the unstable phylogenetic position of Steinernema are common to ribosomal DNA and mtDNA genes, and resolving the position of this important entomopathogenic genus will require sequences from other loci.
Certain older classifications emphasizing vertebrate parasites [34, 35] have included Strongyloides within Rhabditoidea. In analyses of mtDNA genome data, Strongyloides stercoralis was recovered as the sister taxon to the Ascaridida+Rhabditida clade with very robust nodal support in all analyses (Figures 1, 2, 3 and 4). This position of S. stercoralis does not conflict with results obtained from analysis of nuclear ribosomal DNA, although trees inferred from LSU rDNA showed instability of topology that may result from compositional bias or long-branch attraction for S. stercoralis. It is interesting to note that the gene order of S. stercoralis mtDNA is unique, differing substantially from those of other rhabditid taxa whose complete sequence has been reported so far . Additional complete mitochondrial genome sequences of rhabditids, including species from the suborder Cephalobina (e.g., Panagrolaimoidea, Cephaloboidea) will be needed to assess if phylogenies based on mitochondrial genes are consistent with hypotheses obtained from nuclear genes, and to better understand mitochondrial genome evolution within Chromadorea. Representation of mitochondrial genomes from as yet unsampled clades (e.g., Diplogasterida, Tylenchida) may prove important for stabilizing or modifying relationships in parts of the mitochondrial gene tree; taxa that break long branches are believed to be particularly valuable additions.
Of eight superfamiles within Chromadorea represented by two or more species, five were monophyletic in all analyses (Trichostronyloidea, Rhabditoidea, Ascaridoidea, Oxyuroidea, and Filarioidea). Strongyloidoidea and Ancylostomatoidea (hookworms), were not monophyletic in any analysis, whereas Strongyloidea was monophyletic in three of four analyses (not in Bayesian analysis of AA data; Figure 2). Relationships among the five filarioid species were identical among the different inference methods and datasets, and these groups received strong nodal support. Tree topology for relationships among clade III orders was not consistent among the analyses, but in no case was clade III monophyletic. In addition, the four Ascaridida species were nested within Rhabitida in all analyses. Comparison of the best tree for all nucleotide data (Figure 1) with the optimal ML tree for this dataset constrained for clade III monophyly (tree topology provided in Methods), indicated that monophyly of clade III was a significantly worse interpretation of these data (Shimodaira-Hasegawa test, P < 0.05). Similarly, the S-H test revealed that the best ML tree consistent with clade III monophyly for the dataset excluding 3rd positions was significantly worse (P < 0.05) than the best ML tree (Figure 3) for these data. The deepest node including all Rhabditida and Ascaridida had 100% bootstrap support (ML analyses) or 1.00 posterior probability (Bayesian analyses). In three of four analyses (Figures 1, 2 and 3), oxyurids were sister to Ascaridida plus Rhabditida but with strong nodal support only for analyses of the amino acid dataset. In the Bayesian analysis of nucleotide data excluding 3rd positions (Figure 4), oxyurids were sister to spirurids, but with low posterior probability (0.83 BPP). This difference in position of the oxyurids, was the most marked difference between the analyses of complete data (nt or AA) versus nucleotide data excluding 3rd positions. An alternative tree topology test based on ML and the dataset excluding 3rd positions showed that the best ML tree constrained to require a sister taxon relationship between oxyurids and spirurids was significantly worse (S-H test, P < 0.05). This result confirms that there is a significant difference between inference methods (ML versus Bayesian) for the same dataset (nt excluding 3rd positions) when considering the position of oxyurids. Although one analysis (Bayesian AA; Figure 2) showed strong nodal support for higher-level relationships involving oxyurids, a conservative interpretation is that additional investigations are required to confirm the relationship of oxyurids among Chromadorea based on mtDNA genomes. Phylogenetic analyses of SSU sequences did not provide reliable support for the relationship of oxyurids to other nematode taxa , suggesting that data from other loci may be necessary to resolve this question.
Nematodes in the orders Ascaridida, Oxyurida, and Spirurida are a major component of zooparasitic diversity. Evolutionary hypotheses and classification schemes for these orders have differed substantially, depending on the types of data considered and the taxonomic authority. For example, Chitwood  and Maggenti  both suggested that Ascaridida was more closely related to Rhabditida than to Spirurida. Specifically, Maggenti's  classification placed special emphasis on the morphology of the esophagus and life-history traits, assigning the orders Rhabditida, Strongylida and Ascaridida under the subclass Rhabditia. In contrast, other classifications emphasizing different features suggested a sister-group relationship between Ascaridida and Spirurida [35, 39], and proposed that members of this clade are more closely related to Strongylida than to Oxyurida and Rhigonematida. In other work based on traditional approaches, Inglis [40, 41] proposed a sister-group relationship between Oxyurida and Strongylida, with Ascaridida and Spirurida belonging to a separate clade. These hypotheses predate the understanding, inferred from molecular phylogenetics [7, 8], that the vertebrate parasites of the classical order Strongylida are phylogenetically nested within and closely related to free-living Rhabditoidea. Phylum-wide phylogenies based on SSU rDNA have indicated that representatives of the animal-parasitic orders Ascaridida, Spirurida, Rhigonematida and Oxyurida form a monophyletic group with strong support (clade III sensu ; ), with the exception of a single species in one study . In contrast to this overall monophyly for clade III taxa, an analysis of SSU sequences for > 100 clade III species revealed that three of the component orders (Ascaridida, Spirurida, and Rhigonematida) each lacked monophyly for their sampled species . Finally, SSU data provide inconclusive resolution for relationships among most major clade III lineages . For clade III taxa, mitochondrial genome data is inconsistent with clade III monophyly, and instead depicts Ascaridida as nested within Rhabditida. The mitochondrial phylogenetic hypothesis conflicts with the evolutionary proposals by Inglis  and Anderson [35, 39], but is more consistent with the proposal of Maggenti [37, 38] in that the Rhabditida, Strongylida, and Ascaridida are more closely related to each other than any of these orders are to Spirurida. Maggenti  also treated oxyurids as a superfamily within Ascaridida, but our mtDNA trees do not support a close relationship between ascaridids and oxyurids. Previous results based on substantially fewer mitochondrial genome sequences are consistent with the current conclusions based on broader taxon sampling, for example, support for a sister-group relationship between Ascaridida and Rhabditida  and absence of monophyly for representative clade III species . In the context of mtDNA phylogenetics, characterizing the relationships among parasitic nematodes and understanding their relationships to free-living species will require sampling of many additional lineages, which is becoming feasible with high-throughput sequencing methods. However, the current mtDNA genome phylogeny is not consistent with an evolutionary hypothesis suggesting two separate origins of zooparasitism, one for Ascaridida and Spirurida and a separate origin for Oxyurida and Strongylida (Strongyloidea) .
In this study, we tested phylogenetic relationships of nematodes using complete mitochondrial genome information, with special emphasis on species from clade III, which includes major components of zooparasitic nematode diversity. For this study, we determined the complete mitochondrial genome sequences of three nematode species, Cucullanus robustus (13,972 bp) from Ascaridida, Heliconema longissimum (13,610 bp) from Spirurida, and Wellcomia siamensis (14,128 bp) from Oxyurida, and used sequences from the protein coding genes to investigate phylogenetic relationships, focusing on chromadorean orders. Phylogenetic analysis of these new mitochondrial genomes corroborates the close relationship between Ascaridida and Rhabditida and confirms that Ascaridida is nested within Rhabditida. The Oxyurida and Spirurida are each monophyletic, and are sister groups to the Ascaridida plus Rhabditida clade. This lack of monophyly for clade III, as inferred from mtDNA genomes, conflicts with phylogenetic trees based on nuclear rDNA. These mtDNA hypotheses suggest that zooparasitic nematodes represented by Ascaridida, Oxyurida and Spirurida do not share a most recent common ancestor. Ultimately, resolution of this and other conflicts between nuclear rDNA gene trees and mtDNA gene trees will require hypotheses based on additional independent loci. However, regions of topological concordance between mtDNA and nuclear rDNA phylogenetic hypothesis indicate that continued efforts to obtain complete mitochondrial genomes for unrepresented nematode lineages will prove useful for understanding the evolution of mtDNA genomes and developing phylogenetic hypotheses for the phylum Nematoda.
Sample collection and molecular techniques
Nematode specimens were isolated from their host animals (C. robustus from the conger eel Conger myriaster, H. longissimum from the Japanese eel Anguilla japonica, and W. siamensis from the Malayan porcupine Hystrix brachyura) and kept in 70% ethanol until they were used for total genomic DNA extraction. Total genomic DNA was extracted using a commercial kit (Qiagen Co.) according to the manufacturer's protocol. Partial fragments from different regions of the mitochondrial genome were initially amplified and directly sequenced using universal primer combinations or primer sets designed directly from the conserved regions of nematode mitochondrial gene sequences (; see Additional file 4 for primer details). Partial fragments of cox1, rrnS and rrnL were amplified for C. robustus; partial fragments of rrnS, rrnL, cob and nad4 were amplified for H. longissimum, and partial fragments of rrnS, rrnL, cox1 and nad4 were amplified for W. siamensis. PCR reactions were carried out in a 50 μl reaction volume consisting of 10 units of Taq polymerase (Roche), 2.5 mM dNTP mixture, 2.5 mM MgCl2, and 20 pmole of each primer with the following amplification conditions: one cycle of the initial denaturation step at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 30 s, primer annealing at 43~48°C for 30 s and elongation at 72°C for 1 min. The final step was extension at 72°C for 10 min.
The nucleotide sequences obtained from each of these gene fragments were then used to design species-specific primer sets for long PCR reactions. These overlapping long PCR products (ranging from ~1.5 kb to 8 kb in size), covering the entire mitochondrial genome, were amplified using the long PCR primer sets (Additional file 4) and the Expand Long Template PCR System (Roche) with the following amplification conditions: 1 cycle of initial denaturation (2 min at 93°C), 30 cycles of denaturation - primer annealing - elongation (15 s at 93°C, 30 s at 50~60°C, and 10 min at 68°C), and 1 cycle of the final extension (10 min at 68°C). The amplified long PCR products were gel-isolated, and extracted using the TOPO Gel Purification reagents supplied with the TOPO XL cloning kit (Invitrogen Co.). After gel purification, each of the long PCR products was ligated using the TOPO XL cloning kit and then transformed into competent E. coli. Cycle sequencing reactions for each of the long PCR products were performed in both directions by the 'primer walking' method using a Big Dye Terminator Cycle-Sequencing Kit (Applied Biosystems). A complete strand of the entire mtDNA sequence from each of the three species was then assembled by double-checking the sequences of overlapping regions of the long PCR fragments and partial sequences obtained from separate gene fragments.
Gene annotation and phylogenetic analyses
Twelve mitochondrial protein-coding genes and two ribosomal RNA genes of each species were identified by finding gene boundaries based on comparison with other nematode mitochondrial DNA sequences. Putative secondary structures of 22 tRNA genes from each of the three mtDNAs were identified using the tRNAscan-SE program  or by manually finding potential secondary structures and anticodon sequences. Thirty-six complete nematode mitochondrial genomes including three newly sequenced in the present study were used for phylogenetic analysis with two arthropod species (Lithobius forficatus and Limulus polyphemus) as outgroups. A complete list of species, their taxonomy and GenBank accession numbers are provided in Additional file 5. For phylogenetic analysis, both nucleotide and amino acid sequence datasets from the 12 protein-coding genes were used. For multiple alignment of amino acid sequences, the nucleotide sequences of each of 12 protein-coding genes were first translated into amino acids using the invertebrate mitochondrial genetic code. The resulting amino acid sequences were then aligned for each gene using Clustal × with default options . The nucleotide sequences of the 12 protein-coding genes were aligned based on the framework of their corresponding amino acid alignment using RevTrans, a web-based program for placing gaps in coding DNA based on amino acid alignments . Alignments of individual genes were concatenated for phylogenetic analysis. A nucleotide dataset excluding 3rd positions of codons was also constructed. Phylogenetic analyses for the concatenated datasets (full nucleotide, nucleotide excluding 3rd positions, amino acids) were performed using two different tree-building methods. Bayesian inference was used for the amino acid dataset and conducted using the codon model for MrBayes version 3.1.2 . MrBayes was run using four MCMC chains for 106 generations, and sampled every 1,000 generations. Each of the 12 genes was treated as a separate unlinked data partition. For the amino acid dataset, Bayesian posterior probability (BPP) values were determined after discarding the initial 200 trees (the first 2 × 105 generations) as burn-in. With the nucleotide dataset excluding 3rd positions, each of the 12 genes was treated as a separate unlinked data partition. MrBayes was executed on the Cipres Portal for the nucleotide datasets and using four MCMC chains for 4 × 106 generations, sampled every 4,000 generations. Bayesian posterior probability (BPP) values were determined after discarding the initial one-third of trees as burn-in. Maximum likelihood analysis was used for the two nucleotide datasets and conducted using RAxML 7.0.3  and the CIPRES web portal. For RAxML, each of the 12 genes was treated as a separate partition (with gamma rate heterogeneity and all gamma model parameters estimated for each partition by the program). Bootstrap ML analysis was performed using the rapid bootstrapping method (RAxML) with 1,000 replicates. Statistical tests for comparing alternative phylogenetic hypotheses were performed using both the complete nucleotide dataset and the dataset excluding 3rd positions of codons; alternative trees were evaluated using the likelihood-based Shimodaira-Hasegawa test  as implemented in RAxML 7.0.3. Alternative trees for comparison were found based on RAxML searches (with gene partitions as detailed previously), but with the tree topology constrained to reflect the alternative hypothesis of choice (e.g., constrained for clade III monophyly).
The best ML tree recovered for the complete nt dataset constrained for clade III monophyly was: [((Lithobius forficatus ,((Strongyloides stercoralis,(Steinernema carpocapsae,((((Enterobius vermicularis, Wellcomia siamensis),(Heliconema longisimum,(Setaria digitata,(Brugia malayi,(Dirofilaria immitis, Onchocerca volvulus))))),(Cucullanus robustus,((Anisakis simplex, Toxocara malaysiensis), Ascaris sum))),(((((Trichostrongylus axei, Cooperia oncophora),((Mecistocirrus digitatus, Haemonchus contortus), Teladorsagia circumcincta)),(Necator americanus,((Ancylostoma duodenale, Ancylostoma caninum),((Syngamus trachea, Strongylus vulgaris),(Chabertia ovina, Oesophagostomum dentatum))))),Metastrongylus pudendotectus),(Heterorhabditis bacteriophora,(Caenorhabditis elegans, Caenorhabditis briggsae)))))),(((((Agamermis sp., Hexamermis agrotis),(Romanomermis culicivorax, Strelkovimermis spiculatus)), Thaumamermis cosgrovei), Xiphinema americanum), Trichinella spiralis))), Limulus polyphemus);].
The best ML tree recovered for the dataset excluding 3rd positions and constrained for clade III monophyly was: [((Lithobius forficatus,((Trichinella spiralis,((Thaumamermis cosgrovei,((Agamermis sp., Hexamermis agrotis),(Romanomermis culicivorax, Strelkovimermis spiculatus))), Xiphinema americanum)),(Strongyloides stercoralis,(Steinernema carpocapsae,((((Enterobius vermicularis, Wellcomia siamensis),((((Onchocerca volvulus, Dirofilaria immitis), Brugia malayi), Setaria digitata), Heliconema longisimum)),(Cucullanus robustus,(Anisakis simplex,(Ascaris sum, Toxocara malaysiensis)))),(((((Ancylostoma caninum, Ancylostoma duodenale),((Chabertia ovina, Oesophagostomum dentatum),(Syngamus trachea, Strongylus vulgaris))), Necator americanus),(Metastrongylus pudendotectus,(Trichostrongylus axei,(( Cooperia oncophora, Teladorsagia circumcincta),(Haemonchus contortus, Mecistocirrus digitatus))))),(Heterorhabditis bacteriophora,(Caenorhabditis briggsae, Caenorhabditis elegans)))))))), Limulus polyphemus);].
- atp6 :
and atp8: genes for ATP synthase subunits 6 and 8
Bayesian posterior probability
- cob :
gene for cytochrome oxidase b
genes for cytochrome oxidase c subunit 1-3
large subunit nuclear ribosomal DNA
- nad1-6 :
and nad4L: genes for NADH dehydrogenase subunits 1-6 and 4L
polymerase chain reaction
- rrnS :
and rrnL: genes for small and large mitochondrial ribosomal RNA subunits
small subunit nuclear ribosomal DNA
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST)(2007-0052998), the Ministry of Education, Science and Technology (2010-0021692), and the Korea Forest Research Institute to J.-K. Park. S. Nadler was supported by the US National Science Foundation PEET award (DEB-0731516). We thank J.P. Hugot for the W. siamensis specimens.
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