BACKGROUND
mitochondrial genomes are physically separate from the nuclear genome.
for animals, they are typically circular with a compact arrangement of an identical set of  <dig> genes  <cit> .
for some animals, all genes are on the same strand, whereas for others they are divided between the two.
the arrangement of these genes can remain stable for long periods of time; for example, human  <cit>  and shark  <cit>  mtdnas have the same gene arrangement, and do those of fruit fly  <cit>  and shrimp  <cit> .
however, in other lineages, rearrangements have occurred much more rapidly.
many of the same processes that occur in large and complex nuclear genomes also take place in these diminutive genomes, so comparisons among mtdnas can address general questions of genome evolution, but in a model system that is much more tractable for a large number of taxa.
toward this end, this article describes the complete mtdna sequence of the fat innkeeper worm, urechis caupo, the first example from the phylum echiura.
echiurans comprise about  <dig> species and are commonly called spoon worms because of the shape of their extensible proboscis.
unlike annelids, they have no overt segmentation, but they develop via trochophore larvae, very similar to those of annelids.
u. caupo is a pink, sausage shaped worm that lives in u-shaped burrows in the mud or sand of the intertidal or subtidal zones.
unlike other echiurans, it feeds on plankton by filtering using an elaborate mucus net.
RESULTS
gene content and organization
the mtdna of urechis caupo is  <dig>  nts in length  and contains the same  <dig> genes found for nearly all animal mtdnas .
all genes are transcribed from the same strand , as is the case for the two studied annelid mtdnas, the polychaete platynereis dumerilii  <cit>  and the oligochaete lumbricus terrestris  <cit>  and for several other animal mtdnas.
the arrangement of the genes is substantially similar to those of the two annelids, and shares short regions of similarity with several other mtdnas, as can be seen in table  <dig>
base composition and codon usage
the u. caupo mtdna is 62% a+t, about the same as for annelid mtdnas .
as is typical, all homodinucleotides and homotrinucleotides are greatly over represented relative to a random distribution and cg is the least frequent dinucleotide, both in absolute number and in the ratio of observed to expected.
the gene-coding strand has a strong skew against g vs. c but about equal amounts of a vs. t; g-skew  is –  <dig>  and t-skew  is –  <dig>   <cit> .
these values show no striking variation across the length of the mtdna.
codon usage  reflects these values, with those ending in a or t being most frequent.
in all cases except for two, where they are synonymous, nnc codons are in greater abundance than nng codons, as is consistent with the coding strand being rich in c relative to g. the two exceptions are cgg and ggg codons, which are each in greater abundance than their respective synonyms, cgc and ggc.
this invites the speculation that there is something favored about the gg dinucleotide created when g appears in the second codon position.
however, this is not consistently seen, since in the remaining case, agc codons outnumber agg codons two-to-one.
this effect has been shown to be very strong for codon usage pattern of the mtdna of the brachiopod terebratalia transversa  <cit> .
gene initiation and termination
mitochondrial genes commonly use several alternatives to atg as start codons.
however,  <dig> of the  <dig> proteins coding genes of u. caupo mtdna use atg.
the only exceptions are cox <dig>  which uses gtg and nad <dig> which uses atc.
in the case of cox <dig>  there is an in frame stop only three codons upstream and neither of the intervening codons is atg.
also, this inference of starting on gtg specifies a set of amino acids well matched to those at the beginning of other cox <dig> proteins.
the situation for nad <dig> is nearly identical, with an in frame stop only four codons upstream and no intervening atg codons.
however, downstream of the inferred start are several ata codons that can not be ruled out as alternatives.
the commonality of using atg as a start codon has also been noted for mitochondrial genes of four annelids, platynereis dumerilii  <cit> , lumbricus terrestris  <cit> , helobdella robusta and galathealinum brachiosum   <cit>  and a sipunculid, phascolopsis gouldii  <cit> .
a complete stop codon without overlap of the downstream gene is found for all except cox <dig>  nad <dig>  nad <dig>  cob, and nad <dig> .
in each of these cases, it appears that an abbreviated stop codon is generated by cleavage of a downstream trna from the polycistronic transcript, which is then completed to a taa stop codon by polyadenylation.
however, in two of these cases , a complete stop codon could be formed by including only the next two nucleotides, and two other cases , there is an in frame stop codon just one or two codons downstream, respectively.
it is not clear how gene overlaps could be resolved from a polycistronic transcript , but the presence of these stop codons seems beyond coincidence.
it could be that they serve as a "back up" in case translation should begin in the absence of transcript cleavage.
transfer rnas
twenty-two regions can be folded into the typical cloverleaf structures of the expected set of trnas .
there are several mismatched nucleotide pairs within stems; nearly all of these are flanked by multiple g-c pairs, suggesting that they may provide compensatory stability for these arms.
t precedes the anticodon and a purine follows it for all trnas.
the two serine trnas lack potential for folding a dhu arm, as has been found for a number of other animal mtdnas.
there is an alternative folding possible for trna with a six-member anticodon stem and only one nucleotide separating the acceptor and dhu stems; this unusual folding has been found for the homologous genes of some mammals.
trna also does not have potential for a normally paired dhu arm, although there are three potential nucleotide pairs if two  nucleotides were between the dhu and anticodon stems.
however, this potential pairing could, alternatively, be a coincidence, with the dhu arm having no paired stem for this trna.
those with paired dhu arms have stems of three to five nucleotide pairs and loops of three to eight nts.
all trnas have potential for stems of three to six nucleotide pairs for their tΨc arms with loops of three to seven nts.
one of the trnas for serine has the anticodon tct; although this is often found, the alternative of gct is otherwise common.
ribosomal rnas
as has been the case for all studied animal mtdnas to date, two rrna genes are identified, one for each of the small and large mitochondrial ribosomal subunits.
determining the precise ends of the rrna transcript requires experimentation, but if it's assumed that they extend to the boundaries of the adjacent genes, then rrns is  <dig> nucleotides and rrnl is  <dig> nucleotides in length.
these genes are arranged sequentially, but without an intervening trna gene as is otherwise commonly found.
non-coding regions
the largest non-coding region is only  <dig> nts long.
the region is 71% a+t and contains one palindrome of an  <dig> nt sequence , but otherwise no large repeat elements.
obviously, this has potential for forming a stem-loop structure, and it may be significant that a short sequence a few nucleotides upstream, tcaaaa, has the potential for competing with this to form a short hairpin with the ttttga at the end of the palindrome.
there has been previous speculation that regions with potential for competing, mutually exclusive hairpins may play a role in regulating transcription and/or replication .
there are four other potential hairpins in this region with stems of 5– <dig> bp and loops of 5– <dig> nt.
all four nucleotides occur in homopolymers with much greater frequency than expected by chance, often in runs of four or five.
the second largest non-coding region is  <dig> nt between trns <dig> and cox <dig>  this has no repeat elements and the base composition is unremarkable.
what role, if any, these sequences have in the regulation of transcription and/or replication awaits further study.
aside from these  <dig> and  <dig> nt regions, there are only  <dig> total intergenic nucleotides scattered among  <dig> regions.
in seven cases these are 2– <dig> nts long .
for the other seven cases, genes are separated by a single nucleotide, and in six of these, that nucleotide is a c.  the prevalence of c is consistent with the measured g-skew between the strands, although it is possible that this otherwise indicates some function of these nucleotides.
CONCLUSIONS
this is the first description of a complete mitochondrial genome sequence of a representative of the phylum echiura.
the genome contains the same  <dig> genes most commonly found in animal mtdnas.
many features are most similar to those found for annelid mtdnas, including a+t content, use of protein initiation codons, size and potential secondary structures of the largest non-coding region, and the relative arrangement of many genes.
as in annelids examined to date, all genes are found on the same dna strand.
as noted for brachiopod mtdna, there is a preference for g nucleotides to appear in tandem, without obvious explanation.
further description and comparison of complete mtdna sequences will continue to produce a picture of genome evolution, particularly once sampling includes representatives of each animal phylum.

 mitochondrial genomes are physically separate from the nuclear genome. for animals, they are typically circular with a compact arrangement of an identical set of genes. for some animals, all genes are on the same strand, whereas for others they are divided between the two. the arrangement of these genes can remain stable for long periods of time; for example, human and shark mtdnas have the same gene arrangement, and do those of fruit fly and shrimp. however, in other lineages, rearrangements have occurred much more rapidly. many of the same processes that occur in large and complex nuclear genomes also take place in these diminutive genomes, so comparisons among mtdnas can address general questions of genome evolution, but in a model system that is much more tractable for a large number of taxa. toward this end, this article describes the complete mtdna sequence of the fat innkeeper worm, urechis caupo, the first example from the phylum echiura. echiurans comprise about species and are commonly called spoon worms because of the shape of their extensible proboscis. unlike annelids, they have no overt segmentation, but they develop via trochophore larvae, very similar to those of annelids. u. caupo is a pink, sausage shaped worm that lives in u-shaped burrows in the mud or sand of the intertidal or subtidal zones. unlike other echiurans, it feeds on plankton by filtering using an elaborate mucus net.  gene content and organization the mtdna of urechis caupo is nts in length and contains the same genes found for nearly all animal mtdnas. all genes are transcribed from the same strand, as is the case for the two studied annelid mtdnas, the polychaete platynereis dumerilii and the oligochaete lumbricus terrestris and for several other animal mtdnas. the arrangement of the genes is substantially similar to those of the two annelids, and shares short regions of similarity with several other mtdnas, as can be seen in table base composition and codon usage the u. caupo mtdna is 62% a+t, about the same as for annelid mtdnas. as is typical, all homodinucleotides and homotrinucleotides are greatly over represented relative to a random distribution and cg is the least frequent dinucleotide, both in absolute number and in the ratio of observed to expected. the gene-coding strand has a strong skew against g vs. c but about equal amounts of a vs. t; g-skew is – and t-skew is –. these values show no striking variation across the length of the mtdna. codon usage reflects these values, with those ending in a or t being most frequent. in all cases except for two, where they are synonymous, nnc codons are in greater abundance than nng codons, as is consistent with the coding strand being rich in c relative to g. the two exceptions are cgg and ggg codons, which are each in greater abundance than their respective synonyms, cgc and ggc. this invites the speculation that there is something favored about the gg dinucleotide created when g appears in the second codon position. however, this is not consistently seen, since in the remaining case, agc codons outnumber agg codons two-to-one. this effect has been shown to be very strong for codon usage pattern of the mtdna of the brachiopod terebratalia transversa. gene initiation and termination mitochondrial genes commonly use several alternatives to atg as start codons. however, of the proteins coding genes of u. caupo mtdna use atg. the only exceptions are cox which uses gtg and nad which uses atc. in the case of cox there is an in frame stop only three codons upstream and neither of the intervening codons is atg. also, this inference of starting on gtg specifies a set of amino acids well matched to those at the beginning of other cox proteins. the situation for nad is nearly identical, with an in frame stop only four codons upstream and no intervening atg codons. however, downstream of the inferred start are several ata codons that can not be ruled out as alternatives. the commonality of using atg as a start codon has also been noted for mitochondrial genes of four annelids, platynereis dumerilii, lumbricus terrestris, helobdella robusta and galathealinum brachiosum and a sipunculid, phascolopsis gouldii. a complete stop codon without overlap of the downstream gene is found for all except cox nad nad cob, and nad. in each of these cases, it appears that an abbreviated stop codon is generated by cleavage of a downstream trna from the polycistronic transcript, which is then completed to a taa stop codon by polyadenylation. however, in two of these cases, a complete stop codon could be formed by including only the next two nucleotides, and two other cases, there is an in frame stop codon just one or two codons downstream, respectively. it is not clear how gene overlaps could be resolved from a polycistronic transcript, but the presence of these stop codons seems beyond coincidence. it could be that they serve as a "back up" in case translation should begin in the absence of transcript cleavage. transfer rnas twenty-two regions can be folded into the typical cloverleaf structures of the expected set of trnas. there are several mismatched nucleotide pairs within stems; nearly all of these are flanked by multiple g-c pairs, suggesting that they may provide compensatory stability for these arms. t precedes the anticodon and a purine follows it for all trnas. the two serine trnas lack potential for folding a dhu arm, as has been found for a number of other animal mtdnas. there is an alternative folding possible for trna with a six-member anticodon stem and only one nucleotide separating the acceptor and dhu stems; this unusual folding has been found for the homologous genes of some mammals. trna also does not have potential for a normally paired dhu arm, although there are three potential nucleotide pairs if two nucleotides were between the dhu and anticodon stems. however, this potential pairing could, alternatively, be a coincidence, with the dhu arm having no paired stem for this trna. those with paired dhu arms have stems of three to five nucleotide pairs and loops of three to eight nts. all trnas have potential for stems of three to six nucleotide pairs for their tΨc arms with loops of three to seven nts. one of the trnas for serine has the anticodon tct; although this is often found, the alternative of gct is otherwise common. ribosomal rnas as has been the case for all studied animal mtdnas to date, two rrna genes are identified, one for each of the small and large mitochondrial ribosomal subunits. determining the precise ends of the rrna transcript requires experimentation, but if it's assumed that they extend to the boundaries of the adjacent genes, then rrns is nucleotides and rrnl is nucleotides in length. these genes are arranged sequentially, but without an intervening trna gene as is otherwise commonly found. non-coding regions the largest non-coding region is only nts long. the region is 71% a+t and contains one palindrome of an nt sequence, but otherwise no large repeat elements. obviously, this has potential for forming a stem-loop structure, and it may be significant that a short sequence a few nucleotides upstream, tcaaaa, has the potential for competing with this to form a short hairpin with the ttttga at the end of the palindrome. there has been previous speculation that regions with potential for competing, mutually exclusive hairpins may play a role in regulating transcription and/or replication. there are four other potential hairpins in this region with stems of 5– bp and loops of 5– nt. all four nucleotides occur in homopolymers with much greater frequency than expected by chance, often in runs of four or five. the second largest non-coding region is nt between trns and cox this has no repeat elements and the base composition is unremarkable. what role, if any, these sequences have in the regulation of transcription and/or replication awaits further study. aside from these and nt regions, there are only total intergenic nucleotides scattered among regions. in seven cases these are 2– nts long. for the other seven cases, genes are separated by a single nucleotide, and in six of these, that nucleotide is a c. the prevalence of c is consistent with the measured g-skew between the strands, although it is possible that this otherwise indicates some function of these nucleotides.  this is the first description of a complete mitochondrial genome sequence of a representative of the phylum echiura. the genome contains the same genes most commonly found in animal mtdnas. many features are most similar to those found for annelid mtdnas, including a+t content, use of protein initiation codons, size and potential secondary structures of the largest non-coding region, and the relative arrangement of many genes. as in annelids examined to date, all genes are found on the same dna strand. as noted for brachiopod mtdna, there is a preference for g nucleotides to appear in tandem, without obvious explanation. further description and comparison of complete mtdna sequences will continue to produce a picture of genome evolution, particularly once sampling includes representatives of each animal phylum. 
this is the first description of a complete mitochondrial genome sequence of a representative of the phylum echiura, that of the fat innkeeper worm, urechis caupo. their comparison serves as a model system for understanding the processes of genome evolution. it contains the genes that are typical for animal mtdnas in an arrangement somewhat similar to that of annelid worms. urechis caupo mtdna shares many features with those of the few studied annelids, including the common usage of atg start codons, unusual among animal mtdnas, as well as gene arrangements, trna structures, and codon usage biases. the largest non-coding region is only nts long, is 71% a+t, and has potential for secondary structures. this mtdna is nts in length and 62% a+t. all genes are encoded by the same dna strand which is rich in a and c relative to the opposite strand.
 mitochondria contain small genomes that are physically separate from those of nuclei. their comparison serves as a model system for understanding the processes of genome evolution. although hundreds of these genome sequences have been reported, the taxonomic sampling is highly biased toward vertebrates and arthropods, with many whole phyla remaining unstudied. this is the first description of a complete mitochondrial genome sequence of a representative of the phylum echiura, that of the fat innkeeper worm, urechis caupo.  this mtdna is nts in length and 62% a+t. it contains the genes that are typical for animal mtdnas in an arrangement somewhat similar to that of annelid worms. all genes are encoded by the same dna strand which is rich in a and c relative to the opposite strand. codons ending with the dinucleotide gg are more frequent than would be expected from apparent mutational biases. the largest non-coding region is only nts long, is 71% a+t, and has potential for secondary structures.  urechis caupo mtdna shares many features with those of the few studied annelids, including the common usage of atg start codons, unusual among animal mtdnas, as well as gene arrangements, trna structures, and codon usage biases. mtdnaevolutiongene rearrangementannelidstrand skew 