BACKGROUND
hypodontus macropi sensu lato  is a nematode that occurs in the terminal ileum, caecum or colon of a range of macropodid marsupials  and was first described as a hookworm-like nematode  <cit> .
various studies of h. macropi from hosts across vast geographical distances in australia  <cit>  did not reveal any significant morphological variation in the nematode among different host species, but particular predilection sites in the gut as well as host affiliations suggested that h. macropi represented a complex of morphologically indistinguishable, but genetically distinct species or groups which have a relatively high level of host specificity.
to test this proposal, chilton et al.
<cit>  conducted a multilocus enzyme electrophoretic  analysis and subsequently  <cit>  determined genetic variation in the second internal transcribed spacer  of nuclear ribosomal dna , and provided evidence of genetic differences in h. macropi.
using single-strand conformation polymorphism  and selective sequence analysis of the its- <dig>  genetic variation in the nematode was found  <cit> , with nucleotide sequence variability mainly in loops or bulges of the predicted secondary structure.
moreover, in the most extensive investigation of h. macropi to date  <cit> , a molecular-phylogenetic analysis of  <dig> specimens representing all ten known macropidid host species of this nematode from across the australian continent revealed clear genetic clades in each of macropus agilis, m. dorsalis, m. rufogriseus, m. bicolor, petrogale persephone, thylogale billardierii and t. stigmatica.
another clade was represented by all specimens from m. robustus robustus and m. rufus, together with two examples of host switching by the nematode into m. fuliginosus.
these findings showed that h. macropi, as currently defined morphologically, might represent as many as ten cryptic species.
however, to date, dna sequencing studies have been conducted exclusively using the second internal transcribed spacer  of nuclear ribosomal dna, which represents species prospecting , rather than clear evidence for the existence of separate species.
although its- <dig> has consistently provided specific identification of strongyloid nematodes  <cit> , there is a need to provide independent evidence of genetic identity and differentiation.
various studies have shown that mt genomes provide barcodes for organisms  <cit> .
although nucleotide variation within species of nematodes is relatively high for the mt genes studied to date  <cit>  and, thus, is not useful for specific identification, this is not the case for mt protein sequences.
indeed, amino acid sequence variation within species of nematodes examined to date is usually very low   <cit> .
therefore, amino acid sequences derived from the mt genome provide barcodes for species and for studying the systematics  of nematodes  <cit> .
importantly, phylogenetic analysis of mt amino acid datasets usually provides strong statistical support for the relationships of nematodes, which is not achieved using data from short sequence tracts.
in the present study, we sequenced the complete mt genomes of individuals of h. macropi, proposed to be distinct cryptic species based on mee and its- <dig> data sets  <cit>  from three different species of macropodid hosts.
we undertook a comparative analysis of the amino acid sequence datasets derived from these genomes and conducted a phylogenetic analysis of these datasets using homologous datasets for other strongyloid nematodes for comparison.
RESULTS
characteristics and comparisons of mt genomes
the three mt genome sequences of three operational taxonomic units  otu-c, otu-g and otu-j of h. macropi from m. r. robustus, m. bicolor and t. billardierii and of macropicola  ocydromi , were assembled from  <dig> ,  <dig> ,  <dig>  and  <dig>  individual sequence reads, respectively.
for individual otus, the number of contigs resulting from each assembly ranged from  <dig> to  <dig>  with the longest and shortest contigs being  <dig>  and 41 bp in length.
the three circular mt genome sequences of otu-c, otu-g and otu-j of h. macropi were 13699 bp, 13655 bp and 13634 bp in length, respectively .
the mt genome of a closely related nematode, .
consistent with all species of related nematodes sequenced to date  <cit> , the mt genomes of all three h. macropi otus and ma.
ocydromi contained  <dig> protein coding genes , two ribosomal subunits  and  <dig> transfer rna  genes .
the overall mt genome structure for all otus of h. macropi and ma.
ocydromi was consistent with the gene arrangement ga <dig>  representing all other nematodes of the order strongylida investigated to date  <cit> .
the positions, lengths and start/stop codons of individual genes as well as amino acid sequence lengths of predicted proteins of the three otus are shown in table  <dig>  as expected for other strongyloid nematodes  <cit> , no atp <dig> subunit is present, and all genes are transcribed from the forward strand; all protein coding genes had open reading frames .
for all three otus, the lengths of the protein-coding genes were in the following order: nad5 > cox1 > nad4 > cytb > nad1 > nad2 > cox3 > cox2 > atp6 > nad6 > nad3 > nad4l .
the longest gene was nad <dig>  and the amino acid lengths of cox <dig>  nad4l and cox <dig> were the same for all otus .
the predicted nucleotide and amino acid sequences of each of the  <dig> protein-coding genes of h. macropi and ma.
ocydromi were compared.
the mt genes of these otus had att, ata and atg as initiation codons; except for atc as its initiation codon for atp <dig>  all genes had complete termination codons, i.e., taa and tag, in contrast to some nematodes, such as ascaris suum, necator americanus and trichinella spiralis, which have abbreviated stop codons  for some genes  <cit> .
hypodontus macropi
from three different hosts and
macropicola ocydromi
1
2
3
m. ocydromi
m. ocydromi
1otu-c = h. macropi from macropus robustus robustus; 2otu-g = h. macropi from macropus bicolor, 3otu-j = h. macropi from thylogale billardierii.
twenty-two trna genes were predicted from each mt genome of the three otus of h. macropi, and these genes varied from 53– <dig> nucleotides in length.
their positions in the genome were the same for all otus and for ma.
ocydromi .
the two ribosomal rna genes  were separated by nad <dig>  nad <dig>  nad <dig>  nad4l and  <dig> trn genes .
the lengths of these rrn genes were 700 bp and 959 bp, respectively, which is similar to those reported previously for various nematodes, including ancylostoma duodenale, necator americanus, onchocerca volvulus, strongyloides stercoralis and xiphinema americanum <cit> .
the at-content of the mt genomes of h. macropi was 70-78%.
within the strongylida, at-richness in the mt genome usually ranges from  <dig> - <dig> %  <cit> .
the nucleotide composition of the entire mt genome is biased toward t, and c is least favoured , which is similar to other nematodes including caenorhabditis elegans, dirofilaria immitis and onchocerca volvulus <cit> .
the at content of the rrn genes for h. macropi and ma.
ocydromi ranged from 75–77% .
given the at bias  in the mt genome of h. macropi, there was considerable bias in codon usage.
consequently, atr , aty , ttr , and tty  were the most commonly used codons  .
from three different hosts
1
2
3
ma.
ocydromi
1otu-c = h. macropi from macropus robustus robustus; 2otu-g = h. macropi from macropus bicolor; 3otu-j = h. macropi from thylogale billardierii.
genetic differences and phylogenetic relationships
pairwise comparisons of the amino acid  sequences predicted for each of the  <dig> protein coding mt genes from otu-c, otu-g and otu-j of h. macropi from the hosts m. robustus, m. bicolor and t. billardierii, respectively, are shown in table  <dig>  most variation was detected in nad <dig> between otu-c and otu-j  and between otu-g and otu-j , and in nad4l between otu-c and otu-g ; moderate variation was detected in nad <dig>  nad <dig>  nad <dig>  nad <dig> and nad <dig>  among the otus; least variation was detected in cox <dig>  cox <dig> and cox <dig> .
pairwise comparisons of concatenated amino acid sequences revealed sequence differences of  <dig> % , 18%  and  <dig> %  .
these percentages are substantially higher than those detected within species of strongyloid nematodes  <cit> .
pairwise comparisons of the concatenated amino acid sequence of ma.
ocydromi with those of otu-c, otu-g and otu-j revealed sequence differences of  <dig> %,  <dig> % and  <dig> %, respectively .
the phylogenetic analysis of the aa sequence data showed that all three otus of h. macropi and ma.
ocydromi formed a distinct group, with maximum statistical support , to the exclusion of the other strongyloid nematodes included here  .
in this analysis, otu-c from m. r. robustus grouped together with ma.
ocydromi  to the exclusion of otu-g  and otu-j .
while the three otus of h. macropi did form a group with c. ovina, statistical support for this grouping was moderate  .
these percentage differences are based on comparisons between three operational taxonomic units  of hypodontus macropi from three different hosts .
these percentage differences are based on comparisons between three operational taxonomic units of hypodontus macropi from three different hosts  and macropicola ocydromi  from macropus fuliginosus.
sliding window analyses
sliding window analyses were carried out to identify conserved and variable regions in mt genomes among all three otus.
results from these analyses are shown in figure  <dig> for comparison across all the three otus and also for nucleotide diversities calculated from pairwise comparisons across the mt genomes of otu-c, otu-g and otu-j.
a nucleotide diversity pattern was broadly similar across the sliding window analyses and diversity within and between genes was relatively consistent across the genera.
greatest nucleotide diversity was detected within at-rich  region, followed by peaks of variation within nad <dig>  nad <dig>  nad <dig> and nad <dig>  gene-by-gene nucleotide diversity was highly variable, but, by far, the least variation was recorded within cox <dig>  overall, the full sliding window indicates a wealth of new genes capable of providing high levels of nucleotide variation for h. macropi population genetic studies.
discussion
molecular tools offer an unprecedented opportunity to include new components in our studies on the discovery and description of parasite species.
for example, molecular markers in the identification of parasite species can be used to  link morphologically indistinguishable life stages, such as larvae, to the adult stages of known species,  elucidate life cycles by establishing the species that may serve as intermediate or paratenic hosts for larval stages of a parasite, and  search for cryptic species  <cit> .
however, nadler and pérez-ponce de león  <cit>  suggested that, in terms of explanatory power, single locus dna barcodes and the barcoding-gap are insufficient approaches to delimit species, and concordance of independent information, including other genes, is required.
in the present study, we sequenced the whole mt genomes of individuals of h. macropi, proposed to be distinct cryptic species based on mee analysis and the its- <dig> sequences of specimens  <cit>  from three different species of macropodid marsupial hosts.
based on the analysis of the complete mt genomes of three cryptic species, within the morphospecies h. macropi, the present study provides independent support that specimens of h. macropi from three species of macropodid hosts represent three genetically distinct species.
the present mt genomic results support previous findings  <cit>  from mee and nuclear dna-based analyses and indicate that h. macropi is represented by a complex of at least ten sibling species, one in each of m. agilis, m. bicolor, m. dorsalis, m. rufogriseus, p. persephone, t. billardierii and t. stigmatica, with the remaining three species occurring in m. r. robustus and m. rufus.
although the its- <dig> rdna has been shown to be a reliable genetic marker to distinguish among a variety of strongyloid nematodes  <cit>  and this marker has been useful for discovering sibling species within various cloacinine nematodes in macropodid marsupials  <cit> , hypotheses regarding such species need to be independently tested, preferably with sequence data from additional loci  <cit> .
for this purpose, we chose mtdna genes because of their relatively rapid rate of substitution, coupled to the smaller effective population size of mtdna and the provision of evidence of lineage exclusivity in a shorter period of time  following speciation  <cit> .
using mt datasets, based upon pairwise comparisons of concatenated amino acid sequences inferred herein, we found a wide range of sequence differences of among different otus  , and this magnitude is consistent with the previous findings of chilton et al.
<cit>  using the its- <dig> rdna.
these authors also found a wide variation in the its- <dig> sequences among three otus i.e., genotype   <dig> , g <dig> , and g <dig>  of h. macropi.
based on the its- <dig> sequences, the highest sequence variation detected was between otu-c  and otu-g  , followed by otu-c  and otu-j   and otu-g  and otu-j  .
in nematodes, mt dna is usually more variable in sequence within a species than the its- <dig> and other rdna regions  <cit> , indicating that mt gene regions are well suited for studying the population genetics of parasitic nematodes  <cit> .
the sliding window analysis conducted herein displayed distinct patterns of nucleotide diversity among the three mt genomes representing otus-c, -g and -j of h. macropi .
in the present study, cox <dig> was revealed to be the gene with the lowest nucleotide diversity among these three otus, whereas nad <dig>  nad <dig>  nad <dig> and nad <dig> showed higher nucleotide diversity, which can be targeted to design primers for studying the population genetics of h. macropi.
given that cryptic species have been detected within the morphospecies h. macropi <cit> , low sequence variability is useful for the design of oligonucleotide primers that flank mt regions with high variability.
utilizing such primer sets, pcr-based sscp analysis  <cit>  could be applied to screen large numbers of individual specimens representing different host species and populations for cryptic species.
previous studies  <cit>  have shown the merit of sscp for exploring the genetic variation in various populations of h. macropi, and this approach could be applied to large-scale studies of h. macropi specimens representing distinct otus.
it is hoped that major advances in the next-generation sequencing technology   <cit>  will allow selective whole genome sequencing of cryptic species of nematodes, thereby helping to re-classify them by employing detailed phylogenetic and comparative genetic analyses.
CONCLUSIONS
the present analyses of mt proteomic sequence datasets clearly supported the hypothesis that h. macropi from m. robustus robustus, m. bicolor and t. billardierii represent distinct species.
this study emphasizes the importance and utility of the mt genomic datasets for parasites from macropodid marsupials as a basis for systematic, ecological and biological studies, and the specific diagnosis of infections.
in particular, studying cryptic species within the morphospecies h. macropi provides a stimulus to re-explore the relationships of sibling species using complete mt genomic and proteomic sequence data sets.
although the present study focused on h. macropi, the approach employed here has major implications for the study of a wide range of invertebrates and vertebrates.

 hypodontus macropi sensu lato is a nematode that occurs in the terminal ileum, caecum or colon of a range of macropodid marsupials and was first described as a hookworm-like nematode. various studies of h. macropi from hosts across vast geographical distances in australia did not reveal any significant morphological variation in the nematode among different host species, but particular predilection sites in the gut as well as host affiliations suggested that h. macropi represented a complex of morphologically indistinguishable, but genetically distinct species or groups which have a relatively high level of host specificity. to test this proposal, chilton et al. conducted a multilocus enzyme electrophoretic analysis and subsequently determined genetic variation in the second internal transcribed spacer of nuclear ribosomal dna, and provided evidence of genetic differences in h. macropi. using single-strand conformation polymorphism and selective sequence analysis of the its- genetic variation in the nematode was found, with nucleotide sequence variability mainly in loops or bulges of the predicted secondary structure. moreover, in the most extensive investigation of h. macropi to date, a molecular-phylogenetic analysis of specimens representing all ten known macropidid host species of this nematode from across the australian continent revealed clear genetic clades in each of macropus agilis, m. dorsalis, m. rufogriseus, m. bicolor, petrogale persephone, thylogale billardierii and t. stigmatica. another clade was represented by all specimens from m. robustus robustus and m. rufus, together with two examples of host switching by the nematode into m. fuliginosus. these findings showed that h. macropi, as currently defined morphologically, might represent as many as ten cryptic species. however, to date, dna sequencing studies have been conducted exclusively using the second internal transcribed spacer of nuclear ribosomal dna, which represents species prospecting, rather than clear evidence for the existence of separate species. although its- has consistently provided specific identification of strongyloid nematodes, there is a need to provide independent evidence of genetic identity and differentiation. various studies have shown that mt genomes provide barcodes for organisms. although nucleotide variation within species of nematodes is relatively high for the mt genes studied to date and, thus, is not useful for specific identification, this is not the case for mt protein sequences. indeed, amino acid sequence variation within species of nematodes examined to date is usually very low. therefore, amino acid sequences derived from the mt genome provide barcodes for species and for studying the systematics of nematodes. importantly, phylogenetic analysis of mt amino acid datasets usually provides strong statistical support for the relationships of nematodes, which is not achieved using data from short sequence tracts. in the present study, we sequenced the complete mt genomes of individuals of h. macropi, proposed to be distinct cryptic species based on mee and its- data sets from three different species of macropodid hosts. we undertook a comparative analysis of the amino acid sequence datasets derived from these genomes and conducted a phylogenetic analysis of these datasets using homologous datasets for other strongyloid nematodes for comparison.  characteristics and comparisons of mt genomes the three mt genome sequences of three operational taxonomic units otu-c, otu-g and otu-j of h. macropi from m. r. robustus, m. bicolor and t. billardierii and of macropicola ocydromi, were assembled from,, and individual sequence reads, respectively. for individual otus, the number of contigs resulting from each assembly ranged from to with the longest and shortest contigs being and 41 bp in length. the three circular mt genome sequences of otu-c, otu-g and otu-j of h. macropi were 13699 bp, 13655 bp and 13634 bp in length, respectively. the mt genome of a closely related nematode,. consistent with all species of related nematodes sequenced to date, the mt genomes of all three h. macropi otus and ma. ocydromi contained protein coding genes, two ribosomal subunits and transfer rna genes. the overall mt genome structure for all otus of h. macropi and ma. ocydromi was consistent with the gene arrangement ga representing all other nematodes of the order strongylida investigated to date. the positions, lengths and start/stop codons of individual genes as well as amino acid sequence lengths of predicted proteins of the three otus are shown in table  as expected for other strongyloid nematodes, no atp subunit is present, and all genes are transcribed from the forward strand; all protein coding genes had open reading frames. for all three otus, the lengths of the protein-coding genes were in the following order: nad5 > cox1 > nad4 > cytb > nad1 > nad2 > cox3 > cox2 > atp6 > nad6 > nad3 > nad4l. the longest gene was nad and the amino acid lengths of cox nad4l and cox were the same for all otus. the predicted nucleotide and amino acid sequences of each of the protein-coding genes of h. macropi and ma. ocydromi were compared. the mt genes of these otus had att, ata and atg as initiation codons; except for atc as its initiation codon for atp all genes had complete termination codons, i.e., taa and tag, in contrast to some nematodes, such as ascaris suum, necator americanus and trichinella spiralis, which have abbreviated stop codons for some genes. hypodontus macropi from three different hosts and macropicola ocydromi 1 2 3 m. ocydromi m. ocydromi 1otu-c = h. macropi from macropus robustus robustus; 2otu-g = h. macropi from macropus bicolor, 3otu-j = h. macropi from thylogale billardierii. twenty-two trna genes were predicted from each mt genome of the three otus of h. macropi, and these genes varied from 53– nucleotides in length. their positions in the genome were the same for all otus and for ma. ocydromi. the two ribosomal rna genes were separated by nad nad nad nad4l and trn genes. the lengths of these rrn genes were 700 bp and 959 bp, respectively, which is similar to those reported previously for various nematodes, including ancylostoma duodenale, necator americanus, onchocerca volvulus, strongyloides stercoralis and xiphinema americanum. the at-content of the mt genomes of h. macropi was 70-78%. within the strongylida, at-richness in the mt genome usually ranges from - %. the nucleotide composition of the entire mt genome is biased toward t, and c is least favoured, which is similar to other nematodes including caenorhabditis elegans, dirofilaria immitis and onchocerca volvulus. the at content of the rrn genes for h. macropi and ma. ocydromi ranged from 75–77%. given the at bias in the mt genome of h. macropi, there was considerable bias in codon usage. consequently, atr, aty, ttr, and tty were the most commonly used codons. from three different hosts 1 2 3 ma. ocydromi 1otu-c = h. macropi from macropus robustus robustus; 2otu-g = h. macropi from macropus bicolor; 3otu-j = h. macropi from thylogale billardierii. genetic differences and phylogenetic relationships pairwise comparisons of the amino acid sequences predicted for each of the protein coding mt genes from otu-c, otu-g and otu-j of h. macropi from the hosts m. robustus, m. bicolor and t. billardierii, respectively, are shown in table  most variation was detected in nad between otu-c and otu-j and between otu-g and otu-j, and in nad4l between otu-c and otu-g; moderate variation was detected in nad nad nad nad and nad among the otus; least variation was detected in cox cox and cox. pairwise comparisons of concatenated amino acid sequences revealed sequence differences of %, 18% and %. these percentages are substantially higher than those detected within species of strongyloid nematodes. pairwise comparisons of the concatenated amino acid sequence of ma. ocydromi with those of otu-c, otu-g and otu-j revealed sequence differences of %, % and %, respectively. the phylogenetic analysis of the aa sequence data showed that all three otus of h. macropi and ma. ocydromi formed a distinct group, with maximum statistical support, to the exclusion of the other strongyloid nematodes included here. in this analysis, otu-c from m. r. robustus grouped together with ma. ocydromi to the exclusion of otu-g and otu-j. while the three otus of h. macropi did form a group with c. ovina, statistical support for this grouping was moderate. these percentage differences are based on comparisons between three operational taxonomic units of hypodontus macropi from three different hosts. these percentage differences are based on comparisons between three operational taxonomic units of hypodontus macropi from three different hosts and macropicola ocydromi from macropus fuliginosus. sliding window analyses sliding window analyses were carried out to identify conserved and variable regions in mt genomes among all three otus. results from these analyses are shown in figure  for comparison across all the three otus and also for nucleotide diversities calculated from pairwise comparisons across the mt genomes of otu-c, otu-g and otu-j. a nucleotide diversity pattern was broadly similar across the sliding window analyses and diversity within and between genes was relatively consistent across the genera. greatest nucleotide diversity was detected within at-rich region, followed by peaks of variation within nad nad nad and nad gene-by-gene nucleotide diversity was highly variable, but, by far, the least variation was recorded within cox overall, the full sliding window indicates a wealth of new genes capable of providing high levels of nucleotide variation for h. macropi population genetic studies. discussion molecular tools offer an unprecedented opportunity to include new components in our studies on the discovery and description of parasite species. for example, molecular markers in the identification of parasite species can be used to link morphologically indistinguishable life stages, such as larvae, to the adult stages of known species, elucidate life cycles by establishing the species that may serve as intermediate or paratenic hosts for larval stages of a parasite, and search for cryptic species. however, nadler and pérez-ponce de león suggested that, in terms of explanatory power, single locus dna barcodes and the barcoding-gap are insufficient approaches to delimit species, and concordance of independent information, including other genes, is required. in the present study, we sequenced the whole mt genomes of individuals of h. macropi, proposed to be distinct cryptic species based on mee analysis and the its- sequences of specimens from three different species of macropodid marsupial hosts. based on the analysis of the complete mt genomes of three cryptic species, within the morphospecies h. macropi, the present study provides independent support that specimens of h. macropi from three species of macropodid hosts represent three genetically distinct species. the present mt genomic results support previous findings from mee and nuclear dna-based analyses and indicate that h. macropi is represented by a complex of at least ten sibling species, one in each of m. agilis, m. bicolor, m. dorsalis, m. rufogriseus, p. persephone, t. billardierii and t. stigmatica, with the remaining three species occurring in m. r. robustus and m. rufus. although the its- rdna has been shown to be a reliable genetic marker to distinguish among a variety of strongyloid nematodes and this marker has been useful for discovering sibling species within various cloacinine nematodes in macropodid marsupials, hypotheses regarding such species need to be independently tested, preferably with sequence data from additional loci. for this purpose, we chose mtdna genes because of their relatively rapid rate of substitution, coupled to the smaller effective population size of mtdna and the provision of evidence of lineage exclusivity in a shorter period of time following speciation. using mt datasets, based upon pairwise comparisons of concatenated amino acid sequences inferred herein, we found a wide range of sequence differences of among different otus, and this magnitude is consistent with the previous findings of chilton et al. using the its- rdna. these authors also found a wide variation in the its- sequences among three otus i.e., genotype, g, and g of h. macropi. based on the its- sequences, the highest sequence variation detected was between otu-c and otu-g, followed by otu-c and otu-j and otu-g and otu-j. in nematodes, mt dna is usually more variable in sequence within a species than the its- and other rdna regions, indicating that mt gene regions are well suited for studying the population genetics of parasitic nematodes. the sliding window analysis conducted herein displayed distinct patterns of nucleotide diversity among the three mt genomes representing otus-c, -g and -j of h. macropi. in the present study, cox was revealed to be the gene with the lowest nucleotide diversity among these three otus, whereas nad nad nad and nad showed higher nucleotide diversity, which can be targeted to design primers for studying the population genetics of h. macropi. given that cryptic species have been detected within the morphospecies h. macropi, low sequence variability is useful for the design of oligonucleotide primers that flank mt regions with high variability. utilizing such primer sets, pcr-based sscp analysis could be applied to screen large numbers of individual specimens representing different host species and populations for cryptic species. previous studies have shown the merit of sscp for exploring the genetic variation in various populations of h. macropi, and this approach could be applied to large-scale studies of h. macropi specimens representing distinct otus. it is hoped that major advances in the next-generation sequencing technology will allow selective whole genome sequencing of cryptic species of nematodes, thereby helping to re-classify them by employing detailed phylogenetic and comparative genetic analyses.  the present analyses of mt proteomic sequence datasets clearly supported the hypothesis that h. macropi from m. robustus robustus, m. bicolor and t. billardierii represent distinct species. this study emphasizes the importance and utility of the mt genomic datasets for parasites from macropodid marsupials as a basis for systematic, ecological and biological studies, and the specific diagnosis of infections. in particular, studying cryptic species within the morphospecies h. macropi provides a stimulus to re-explore the relationships of sibling species using complete mt genomic and proteomic sequence data sets. although the present study focused on h. macropi, the approach employed here has major implications for the study of a wide range of invertebrates and vertebrates. 
pairwise comparisons of the amino acid sequences predicted from these three mt genomes revealed differences of % to 18%. phylogenetic analysis of the amino acid sequence data sets using bayesian inference showed that h. macropi from the three different host species formed distinct, well-supported clades. the present analyses of inferred mt protein sequence datasets clearly supported the hypothesis that h. macropi from m. robustus robustus, m. bicolor and t. billardierii represent distinct species. to test this proposal using independent molecular data, we sequenced the whole mitochondrial genomes of individuals of h. macropi from three different species of hosts as well as that of macropicola ocydromi, and undertook a comparative analysis of the amino acid sequence datasets derived from these genomes. in addition, sliding window analysis of the mt genomes defined variable regions for future population genetic studies of h. macropi in different macropodid hosts and geographical regions around australia. hypodontus macropimitochondrial genomekangaroowallabysliding window analysisnext-generation sequencinggeneticssystematics hypodontus macropi is a common intestinal nematode of a range of kangaroos and wallabies. the mt genomes sequenced by next-generation technology from h. macropi from the three host species varied from  bp to  bp in size. based on previous multilocus enzyme electrophoresis and nuclear ribosomal dna sequence data sets, h. macropi has been proposed to be complex of species.
 hypodontus macropi is a common intestinal nematode of a range of kangaroos and wallabies. based on previous multilocus enzyme electrophoresis and nuclear ribosomal dna sequence data sets, h. macropi has been proposed to be complex of species. to test this proposal using independent molecular data, we sequenced the whole mitochondrial genomes of individuals of h. macropi from three different species of hosts as well as that of macropicola ocydromi, and undertook a comparative analysis of the amino acid sequence datasets derived from these genomes.  the mt genomes sequenced by next-generation technology from h. macropi from the three host species varied from  bp to  bp in size. pairwise comparisons of the amino acid sequences predicted from these three mt genomes revealed differences of % to 18%. phylogenetic analysis of the amino acid sequence data sets using bayesian inference showed that h. macropi from the three different host species formed distinct, well-supported clades. in addition, sliding window analysis of the mt genomes defined variable regions for future population genetic studies of h. macropi in different macropodid hosts and geographical regions around australia.  the present analyses of inferred mt protein sequence datasets clearly supported the hypothesis that h. macropi from m. robustus robustus, m. bicolor and t. billardierii represent distinct species. hypodontus macropimitochondrial genomekangaroowallabysliding window analysisnext-generation sequencinggeneticssystematics 