BACKGROUND
mitochondria were most likely acquired by the common ancestor of eukaryotes  <cit> .
presumably, these organelles originated from incorporated α-proteobacteria and still carry their own, reduced genome  <cit> .
mitochondrial  genomes show very diverse organizations and are of a very broad range of sizes  <cit> .
in comparison to many protists and plants, metazoans possess an even more reduced set of mt genes and fewer non-coding regions  <cit> .
typical metazoan mt genomes are circular dna molecules of 16– <dig> kb  <cit> .
remarkable exceptions are the linear mt genomes of medusozoan cnidarians .
linear mt genomes have not been found in other metazoan taxa, but in various other eukaryotes .
the linear structure of cnidarian mt genomes is known from the work of warrior  <cit> , who separated the dna of isolated mitochondria via electrophoresis, as well as from bridge et al.
<cit>  and ender and schierwater  <cit> , who applied rnl-probes to electrophoretically separated dna extracts.
most of the medusozoan mt genomes from these studies were encoded on one ~ <dig> kb molecule, which has been verified by the first two sequences of such linear metazoan genomes   <cit> , and hydra oligactis   <cit> ).
however, in some hydra species, and apparently in some cubozoa, the mt dna is divided onto at least two different molecules  <cit> .
in the genus hydra, mt genomes are organized on one ~ <dig> kb molecule or two ~ <dig> kb molecules  <cit> , making this genus an excellent candidate in which to examine changes due to fragmentation of mt genomes from one to two chromosomes.
the hydra oligactis mt genome, a  <dig>  kb linear dna molecule, was published recently  <cit> .
pont-kingdon et al.
<cit>  sequenced a terminal section  of one of the two mt chromosomes from hydra vulgaris , and previous hybridization experiments have shown that in this species all four termini possess a 150– <dig> bp identical sequence with unknown orientation to one another  <cit> .
by providing the complete mt genome sequence of hydra magnipapillata, encoded on two mt chromosomes, we now present in detail the organization of such a fragmented linear mt genome from early diverging metazoa.
methods
we assembled the two mt chromosomes by using publicly available sequences from the hydra magnipapillata whole-genome shotgun sequencing project by conducting blast searches  <cit>  of several mt protein-coding genes against the traces of h. magnipapillata .
hits were used to initiate local genome assembly in a bioinformatical pipeline  to obtain the two mt chromosome sequences.
the chromosomes will be referred to as mt <dig>  and mt <dig>  .
mt <dig> and mt <dig> of hydra share almost identical but inverted sequences of 191– <dig> bp at each 5' and 3' end , figs.
1a, b, see below).
warrior  <cit>  previously reported that about  <dig> bp were identical  at the ends of the mt chromosomes of hydra vulgaris, and the 5' end of one chromosome  of this species was sequenced by pont-kingdon et al.
<cit> .
by comparing our sequences with the experimentally verified 5' end of mt <dig> from h. vulgaris  <cit> , we were able to infer the ends of the h. magnipapillata mt chromosomes and found that these predicted ends coincide with an abrupt decrease in coverage in our assemblies .
this in combination with the reported sizes of one  or both  mt chromosomes from the same or a closely related hydra species suggests that the excess sequences are artifacts, and consequently they were omitted.
to exclude the possibility that other observations in our assemblies originated from methodological artifacts, we conducted additional experimental procedures and tests as follows:
<dig>  pcr experiments
pcr experiments were done with a closely related hydra species.
we obtained specimens of hydra sp.
from the schulbiologie-zentrum hannover.
dna from one polyp was prepared with the chelex method ,  <dig> μl of the undiluted supernatant or  <dig> μl of a 1: <dig> dilution was used as template.
phylogenetic analysis with partial cox <dig> data verified that our hydra sp.
specimen is very closely related to hydra magnipapillata .
primers  were designed to confirm our bioinformatically derived observations via pcr.
the fragments shown in fig.
<dig> were amplified and sequenced .
sequences have been submitted to genbank .
<dig>  additional local genome assembly experiments
to exclude the possibility that we had amplified a nuclear mt pseudogene  of the nd <dig> and partial cox <dig> fragment, we started different assemblies with the pipeline originating from blast hits of a  <dig> bp fragment , as well as two assemblies starting from the last  <dig> bp 3' of nd <dig>  and the first  <dig> bp of the incomplete copy of cox <dig>  in the first two cases, only one assembly was received, each time consistent with our former assemblies.
by starting with  <dig> bp of the partial cox <dig>  we recovered different assemblies, which were compatible with one chromosomal assembly or the other.
in no case did we observe any inconsistencies or any presence of nuclear genes, which would have indicated that the nd5-cox <dig> arrangement is part of a numt.
phylogenetic analyses
the h. magnipapillata and hydra.
sp cox <dig> sequences from our assembly and sequencing were manually aligned with additional sequences from other hydra and outgroup species available from genbank in the seaview editor  <cit> .
the final dataset contained  <dig> characters.
a maximum likelihood analysis was carried out in paup*  <dig> b <dig>  <cit>  under a model of nucleotide evolution suggested by the hierarchical likelihood ratio test in modeltest  <dig>   <cit> ; for the bootstrap analysis  we applied the same model.
the dataset was also analyzed with mrbayes v <dig> .
<dig>  <cit>  .
parameter stabilization of the chains in mrbayes was monitored with tracer  <dig>   <cit> , and convergence of chains was examined with the diagnostics provided by the awty server  <cit> .
base compositions of cnidarian mt genomes
sequences of  <dig> additional cnidarian genomes were downloaded from genbank  <cit>  .
the sequences of the  <dig> respiratory chain protein genes shared between all  <dig> mt genomes and the sequences of rns and rnl were extracted from the genbank format using the artemis software v. <dig>  <cit> .
in some mt genomes the rrna genes were not entirely annotated in their full length.
we therefore considered the non-coding regions around the apparently too small genes as rrnas.
the corresponding taxa and positions in each sequence were: nematostella sp.
; mussa angulosa  ; astrangia sp.
.
the at contents of rrnas and protein codon positions of the mt genomes are shown in additional file  <dig>
RESULTS
genes, base composition and codon usage
the two chromosomes of the h. magnipapillata mt genome each carry one rrna gene .
each of the assembled contigs of mt <dig> and mt  <dig> is represented by >  <dig>  single sequence reads from the trace archive .
our consensus sequence for mt <dig> is  <dig>  bp long.
this matches the length reported by bridge et al.
<cit>  for this h. magnipapillata mt chromosome.
the sequence of mt <dig> is shorter .
the h. magnipapillata mt genome includes  <dig> protein-coding genes of the respiratory chain usually found in other metazoa.
mt <dig> contains  <dig> protein-coding genes, rnl and two trna genes; mt <dig> contains  <dig> protein-coding genes, rns and one trna gene .
all genes are unidirectionally encoded on each of the two molecules and densely arranged along the chromosomes.
as in h. oligactis, the longest non-coding intergenic region is  <dig> bp between cox <dig> and nd <dig>  <cit> .
otherwise, subsequent genes are separated by 0– <dig> bp or overlap for up to  <dig> bp .
like many other cnidaria  <cit> , the h. magnipapillata mt genome possesses only the two trna genes for methionine  and tryptophan .
trnw is only found on mt <dig>  whereas identical copies of trnm are present on both chromosomes .
six amino acid codons are not used in the  <dig> protein-coding genes , and all genes are terminated by taa.
apparently synonymous codons that posses an a or t, instead of a g or c, at the third codon position are preferred in h. magnipapillata.
to test whether this observation is caused by mechanisms that affect base composition in the whole mt genome, we analyzed codon usage in the  <dig> respiratory protein-coding genes in  <dig> mt genomes of cnidaria.
we plotted the at content at each of the three codon positions against the at contents of the rrna genes for every genome, as rrna coding genes represent a different part of the mt genomes in terms of functional constraints compared to protein-coding genes.
remarkably, h. magnipapillata showed the highest values for at content at the third codon positions  and in the rrna genes .
moreover, a high at content in rrna genes generally correlates with the usage of a and t at third codon positions in all cnidaria , suggesting that codon usage might be the result of a general selection for base composition on the mt genome caused by interaction of mutational, repair, replication and translational mechanisms  <cit> .
the at content at the first and second codon positions also correlates with that of the rrna genes , but here at content rise at a lower rate than the increasing at content of the rrnas .
this is likely the result of selection on certain amino acids.
cnidarians posses a lower at content at the first codon position than at the second , with h. magnipapillata and h. oligactis being the only exceptions .
codon frequency among the  <dig>  codons of the  <dig> protein-coding genes in h. magnipapillata.
gene arrangement and inverted terminal repeats
compared to the gene arrangement of a. aurita and h. oligactis, only a few changes can be observed in h. magnipapillata.
neglecting the positions of trnas, two blocks  of genes are identical across the three genomes, occurring on mt <dig> or mt <dig>  respectively, in h. magnipapillata .
the mt genomes of h. oligactis and of h. magnipapillata are entirely alignable and display a sequence divergence of  <dig> % .
as mentioned before, we found 191– <dig> bp of itr at both ends of mt <dig> and mt <dig>  in the linear mt genomes of h. oligactis and a. aurita, itr were also present but were longer .
unlike itr in aurelia  <cit> , itr in h. magnipapillata have a higher gc content than the rest of the molecule .
we found that a smaller part of 3' cox <dig>  is included in all itr of h. magnipapillata.
probably because the 3' end of cox <dig> is not very conserved, pont-kingdon et al.
<cit>  missed this feature in their mt <dig> fragment of h. vulgaris.
the itr regions of h. oligactis contain a larger cox <dig> fragment .
the remaining sequenced 3' region of itr in h. oligactis is very similar to those found in h. magnipapillata and h. vulgaris , but longer.
between h. magnipapillata and h. vulgaris, the major difference is that a stretch of gs  is significantly shorter in h. magnipapillata .
in h. magnipapillata mt <dig> and mt <dig>  we found additional identical sequences at the 5' and 3' ends following  and preceding  the itr.
we refer to those regions as identically oriented sequences .
after the itr, the 5' ios of both molecules contain identical copies of non-coding dna and trnm.
at the 3' ios we found a larger partial copy of the 5' region of cox <dig> on mt <dig>  as a consequence of this arrangement, mt <dig> and mt <dig> share  <dig> bp  at the 5' end and  <dig> bp  at the 3' end, giving both molecules a specific orientation.
using pcr experiments with the closely related hydra sp., we verified the following arrangements initially observed in the h. magnipapillata sequences :  the presence and orientation of the itr at all four chromosome ends could be shown, as well as the presence of partial cox <dig> sequences in the itr;  identical regions are shared at the 5' and 3' end, respectively, between mt <dig> and mt <dig> adjacent to the itr; and  within the latter regions, the 5' motif contains trnm, which therefore appears in two copies in the genome, and a larger sequence of cox <dig> forms the shared 3' motif of mt <dig> and mt <dig>
phylogenetic analysis
the tree topology derived from our phylogenetic analysis of cox <dig> shows the close relationship of hydra sp.
and h. magnipapillata , thus ensuring that we used an appropriate taxon to test our results.
h. vulgaris  is paraphyletic, which reflects the difficult taxonomy of the genus  <cit> .
the presented phylogeny, in combination with the mt genome organization, supports the view that the ancestral state of mt genome organization in the genus hydra was a single linear mt chromosome.
1syn.
: hydra viridis; h. viridissima.
discussion
linear mt genomes and fragmentation of mt chromosomes in cnidaria
linearity of mt genomes seems to have evolved once after the divergence of medusozoa from anthozoa.
fig.
3a summarizes the results of different studies  <cit> , mapped on a cnidarian phylogeny  <cit> .
a fragmentation of mt genomes has been reported from several hydra species   <cit>  and cubozoa  <cit> .
uncertainties remain for cubozoa: bridge et al.
<cit>  studied the same cubozoan species carybdea marsupialis as ender and schierwater  <cit> , but reported a single ~ <dig> kb linear mt genome, while in the more recent work, a ~ <dig> kb fragment was shown to carry the rnl gene.
because ender and schierwater  <cit>  were able to repeat the experiments with different dna isolates of c. marsupialis and obtained concordant results from an additional cubozoan species , an experimental error seems unlikely.
however, their conclusion of four equally-sized mt chromosomes in cubozoa is not directly supported by their identification of a  <dig> kb chromosome carrying rnl.
alternatively, one could assume the presence of a single ~ <dig> kb mt counterpart, as indicated in fig.
3a.
such an arrangement is possible, e.g., if rnl and cox <dig>  the two genes that are encoded in different orientation to the other mt genes in a. aurita  <cit> , were encoded in one chromosome in cubozoa, and the remaining genes on a second chromosome.
however, given the available data it seems reasonable to assume that fragmented linear genomes occur in both cubozoa and hydrozoa .
this suggests from an evolutionary perspective that the mt genome in the common ancestor of medusozoa was linear and then independently split into different chromosomes in hydra , and in at least some cubozoa .
a possible mechanism for the origin of linear chromosomes from a circular molecule is the integration of one or more resolution elements  <cit> .
the circular dna molecule would be split into one or more linear molecules with identical ends.
in medusozoa, the processes of linearization and the split of one linear into two linear chromosomes were obviously different processes as shown in the phylogenetic trees .
the linearization, possibly occurring in the last common ancestor of medusozoans, seems to have preceded the splitting of the chromosomes by a long time.
if the ancestral linear mt chromosome of medusozoa originated by introduction of a resolution element, one probably would not expect to observe its original motifs, which would occur as identical repeats at the two ends of the linear molecule  <cit> .
indeed, the ends of linear medusozoan mt chromosomes have inverted terminal motifs , instead of direct repeats.
the splitting of ancestral linear mt chromosomes as in h. magnipapillata  happened much later in evolutionary history, contradicting the view that the two or more linear mt chromosomes in medusozoa directly originated from one circular dna molecule.
fragmented mt genomes are present in various eukaryotic taxa, e.g., in dinoflagellates  <cit> , ichthyosporea  <cit>  and fungi  <cit> .
in metazoa, fragmented mt genomes are known from the genera globodera , dicyema  and the rotifer brachionus plicatilis  <cit> , but unlike in h. magnipapillata, in these taxa the genomes are encoded on several small circular molecules.
the mt chromosomal organization observed in h. magnipapillata supports the hypothesis of an ancestral, linear chromosome in hydra , as represented by the mt genome of h. oligactis  <cit> , which has been split in two between nd <dig> and rns.
function of itr and ios
warrior  <cit>  already suggested the presence of identical terminal sequences on both chromosomes of h. vulgaris.
we now show that these ends are arranged as itr on mt <dig> and mt <dig>  as in other medusozoans  <cit> .
in h. oligactis, which in the phylogenetic tree branches off before hydra species carrying two mt dna molecules , the single linear mt chromosome has itr containing a large copy of the 5' end of cox <dig>  only the itr at the 3' end has been completely sequenced  <cit> .
based on our findings in h. magnipapillata, we predict that the unsequenced 5' end is almost identical to the 3' motif , and we expect that about  <dig> bp remain unsequenced on the 5' end .
in hydra, partial copies of cox <dig> play a crucial role as part in itr regions at the chromosome ends .
the itr in h. magnipapillata contains only a short sequence of the 3' end of cox <dig> , suggesting that large parts of the cox <dig> copies were lost.
a simultaneous duplication of 5' itr  and the 5' ios motif seems likely to have occurred in the process of chromosome splitting.
in this case, the longer cox <dig> copy  is a duplication of the functional cox <dig> of the original 5' end of a single mt chromosome .
itr of linear mt molecules are present in other taxa besides medusozoans, e.g., in yeasts  and in the green algae chlamydomonas reinhardtii  <cit> .
furthermore, in the green algae polytomella parva, identical itr are present at all ends of the two linear mt chromosomes  <cit> , similar to what we observe in h. magnipapillata.
we report 5' and 3' ios as an additional shared feature of the two mt chromosomes.
interestingly, such an arrangement of itr and 5' and 3' ios is also seen in another, highly fragmented eukaryotic mt genome.
in the ichthyosporean amoebidium parasiticum, mt genes are distributed over several hundred different chromosomes, each of which also possesses itr and 5' and 3' ios  <cit> .
pont-kingdon et al.
<cit>  speculated that there may be a role for transcription initiation at the  <dig> bp 5' of trnm, which they found in their h. vulgaris  partial mt <dig> sequence.
considering that transcription initiation within the itr would result in energetically expensive nonsense transcripts , transcription is more likely to start in the adjacent, non-coding regions of the 5' ios.
in h. magnipapillata and h. vulgaris this region within the 5' ios is  <dig> bp long and lies between the cox <dig> copy and trnm .
in h. oligactis, the non-coding region between the itr and trnm is only  <dig> bp.
however, a striking sequence similarity can be observed near trnm between h. oligactis and h. vulgaris   <cit> .
there is a 14-bp motif  that is shared between the species and differs by the last  <dig> bp from the 3' itr+ <dig> bp counterpart in h. oligactis.
this motif might be involved in transcription initiation.
if so, the difference in the very last  <dig> bp between the 5' end and its counterpart on the reverse strand in the itr of h. oligactis prevents a functional transcription signal on the non-coding strand in this species.
a crucial function for transcription initiation would explain selective pressure for maintaining the 5' ios of both molecules after the itr in h. magnipapillata.
all mt chromosomes from amoebidium parasiticum that contain coding genes are transcribed from 5' ios to 3' ios, as in h. magnipapillata  <cit> .
this observation led burger et al.
<cit>  to the conclusion that the ios in amoebidium are responsible for transcription initiation  and termination .
while in h. magnipapillata we expect the same function for 5' ios, the role of the additional partial cox <dig> copy within the 3' ios of mt <dig> and mt <dig>  if any, remains unknown; considering that the end of cox <dig> is part of the itr, transcription can only be terminated in itr and not in the 3' ios.
the sequence homologies of itr and ios within or between mt <dig> and mt <dig> are probably not the result of a relatively recent origin from ancestral sequences, as a first duplication of partial cox <dig> is already observed in h. oligactis and therefore predates the separation process.
the substitutions between itr  of the two species are found in all itr copies in each mt genome.
considering this and the fact that similar arrangements are found in other eukaryotes  <cit> , it seems more likely that concerted evolution maintains the almost identical sequences, probably caused or influenced by the yet unknown mt genome replication mechanism.
terminal sequences of linear dna molecules play a crucial role in mt replication  <cit> .
the main problem in the process of linear chromosome replication is the maintenance of the 5' ends.
in nuclear  chromosomes this is normally achieved by telomerase, an enzyme that adds short sequence motifs in tandem repeats at the end of each molecule to compensate for loss at the 5' end that occurs in each replication cycle  <cit> .
consequently, in hydra as in most metazoa, the motif n is found at the end of nc chromosomes  <cit> .
the termini of hydra mt chromosomes are much more complex, and their maintenance during replication is not yet understood but is most likely telomerase independent  <cit> .
warrior  <cit>  suggested an mt replication mechanism for the two h. vulgaris mt chromosomes similar to that of t <dig> bacteriophages.
his conclusion was based upon observations from hybridization experiments, which showed the presence of identical terminal sequences that he assumed to have the same orientation at the 5' and 3' ends.
according to this model, intermediate concatamers are formed, and via ligation, site-specific nicking and elongation, the 5' ends are finally filled.
based on our data we can reject this model, because we showed that the terminal sequences in h. magnipapillata are inverted  and therefore do not allow the necessary concatamerization in the proposed way.
similarly, ephemeral circularization as in the phage lambda is not possible when terminal sequences are not direct repeats, but inverted.
different replication mechanisms for linear chromosomes have been reported or proposed .
solutions for maintaining the terminal structure in sequences with itr include  covalently bound proteins, which also could serve as primers for a 'racket frame' replication ,  5' and 3' ends of chromosomes that are connected by a hairpin-loop  and  single-stranded 3' overhangs .
warrior  <cit>  showed that the first two possibilities are not realized in h. vulgaris.
the mt-replication mechanism in chlamydomonas requires an internal repeat of the single-stranded 3' overhangs  <cit> .
we cannot entirely rule out the existence of short single-stranded 3' overhangs, as it is possible they might have been missed due to our methods.
the outermost sequence at least cannot be part of a repeat motif, as our pcr amplification of hydra sp.
did not yield fragments of different sizes.
furthermore, neither in mt <dig> of h. vulgaris nor in the mt genome of h. oligactis were additional sequences or repeats found  <cit> .
therefore, although a similarity to the mt replication of c. reinhardtii cannot be excluded, we find that mechanisms for linear mt chromosome replication are too diverse and that too many details are still unknown  <cit>  for us to draw further conclusions about the replication process in h. magnipapillata from the presence of the itr alone.
considering that itr are a shared feature among all three available sequences of medusozoan mt genomes , it is very likely that the mechanisms for mt replication are similar in all medusozoans and are still the same after the fragmentation of the mt genome.
keeping in mind that similar arrangements of itr and ios are found in amoebidium parasiticum, the mt replication mechanisms of h. magnipapillata and medusozoa are probably not unique among eukaryotes.
CONCLUSIONS
the h. magnipapillata mt genome represents the first complete sequence of a linear metazoan mt genome that consists of two separate molecules.
the gene arrangements and our phylogenetic analysis suggest that mt <dig> and mt <dig> originated from an ancestral linear mt genome, as found in h. oligactis, that at some point divided in two between nd <dig> and rns .
of most interest is the organization at the ends of the two mt chromosomes .
we show that h. magnipapillata has itr, which include a part of cox <dig>  or partial copies of the 5' end of cox <dig> .
we conclude that mechanisms for mt replication in hydra species are different from the previously proposed ones and probably are shared among all medusozoans.
in addition to itr, both mt chromosomes of h. magnipapillata have identical motifs on their 5' and 3' ends, called respectively the 5' ios and 3' ios .
the 5' ios includes trnm and a non-coding region, including a motif that may play a role in transcription initiation.
the 5' ios is probably the result of a duplication during the separation process of a single ancestral mt chromosome.
the organization of the itr and 5' and 3' ios is not unique among eukaryotes with fragmented linear mt genomes.
itr most likely play a role in mt replication, while the duplication of the 5' end of an single ancestral linear and unidirectionally-encoded mt chromosome  and its concerted evolution ensure that transcription of all mt genes is maintained after fragmentation of linear mt chromosomes.
a similar arrangement of itr and ios regions can therefore be expected in the apparently fragmented mt genomes of cubozoa and other eukaryotes with two or more linear mt dna molecules.
authors' contributions
ov contributed to the conception and design of the study, acquired, analyzed and interpreted the data, and wrote the manuscript.
de and gw contributed to the interpretation of data and critically reviewed the manuscript.
all authors read and approved the final version of the manuscript.
supplementary material
additional file 1
trace ids of sequences included in the h. magnipapillata assemblies.
click here for file
additional file 2
coverage of mt <dig> and mt <dig> assemblies.
click here for file
additional file 3
primer sequences.
click here for file
additional file 4
taxa, genbank accession numbers and at contents of protein-coding genes and rrna genes in  <dig> cnidarian mt genomes.
click here for file
acknowledgements
this work was financially supported by the german research foundation  and is a contribution from the dfg priority programme spp <dig> "deep metazoan phylogeny".
d.e.
acknowledges financial support from the european union under a marie curie outgoing fellowship .
all authors acknowledge the j. craig venter institute for providing h.magnipapillata wgs traces via ncbi's trace archive, and the national human genome research institute for funding the h. magnipapillata genome project.
we thank bernie degnan and ben woodcroft  for providing access to their 'reefedge' pipeline for local genome assembly and the schulbiologiezentrum hannover for providing hydra specimens for our pcr experiments.
ov thanks catherine vogler for help with r and many useful discussions and remarks on the manuscript.

 mitochondria were most likely acquired by the common ancestor of eukaryotes. presumably, these organelles originated from incorporated α-proteobacteria and still carry their own, reduced genome. mitochondrial genomes show very diverse organizations and are of a very broad range of sizes. in comparison to many protists and plants, metazoans possess an even more reduced set of mt genes and fewer non-coding regions. typical metazoan mt genomes are circular dna molecules of 16– kb. remarkable exceptions are the linear mt genomes of medusozoan cnidarians. linear mt genomes have not been found in other metazoan taxa, but in various other eukaryotes. the linear structure of cnidarian mt genomes is known from the work of warrior, who separated the dna of isolated mitochondria via electrophoresis, as well as from bridge et al. and ender and schierwater, who applied rnl-probes to electrophoretically separated dna extracts. most of the medusozoan mt genomes from these studies were encoded on one ~ kb molecule, which has been verified by the first two sequences of such linear metazoan genomes, and hydra oligactis ). however, in some hydra species, and apparently in some cubozoa, the mt dna is divided onto at least two different molecules. in the genus hydra, mt genomes are organized on one ~ kb molecule or two ~ kb molecules, making this genus an excellent candidate in which to examine changes due to fragmentation of mt genomes from one to two chromosomes. the hydra oligactis mt genome, a kb linear dna molecule, was published recently. pont-kingdon et al. sequenced a terminal section of one of the two mt chromosomes from hydra vulgaris, and previous hybridization experiments have shown that in this species all four termini possess a 150– bp identical sequence with unknown orientation to one another. by providing the complete mt genome sequence of hydra magnipapillata, encoded on two mt chromosomes, we now present in detail the organization of such a fragmented linear mt genome from early diverging metazoa. methods we assembled the two mt chromosomes by using publicly available sequences from the hydra magnipapillata whole-genome shotgun sequencing project by conducting blast searches of several mt protein-coding genes against the traces of h. magnipapillata. hits were used to initiate local genome assembly in a bioinformatical pipeline to obtain the two mt chromosome sequences. the chromosomes will be referred to as mt and mt. mt and mt of hydra share almost identical but inverted sequences of 191– bp at each 5' and 3' end, figs. 1a, b, see below). warrior previously reported that about bp were identical at the ends of the mt chromosomes of hydra vulgaris, and the 5' end of one chromosome of this species was sequenced by pont-kingdon et al.. by comparing our sequences with the experimentally verified 5' end of mt from h. vulgaris, we were able to infer the ends of the h. magnipapillata mt chromosomes and found that these predicted ends coincide with an abrupt decrease in coverage in our assemblies. this in combination with the reported sizes of one or both mt chromosomes from the same or a closely related hydra species suggests that the excess sequences are artifacts, and consequently they were omitted. to exclude the possibility that other observations in our assemblies originated from methodological artifacts, we conducted additional experimental procedures and tests as follows: pcr experiments pcr experiments were done with a closely related hydra species. we obtained specimens of hydra sp. from the schulbiologie-zentrum hannover. dna from one polyp was prepared with the chelex method, μl of the undiluted supernatant or μl of a 1: dilution was used as template. phylogenetic analysis with partial cox data verified that our hydra sp. specimen is very closely related to hydra magnipapillata. primers were designed to confirm our bioinformatically derived observations via pcr. the fragments shown in fig. were amplified and sequenced. sequences have been submitted to genbank. additional local genome assembly experiments to exclude the possibility that we had amplified a nuclear mt pseudogene of the nd and partial cox fragment, we started different assemblies with the pipeline originating from blast hits of a bp fragment, as well as two assemblies starting from the last bp 3' of nd and the first bp of the incomplete copy of cox in the first two cases, only one assembly was received, each time consistent with our former assemblies. by starting with bp of the partial cox we recovered different assemblies, which were compatible with one chromosomal assembly or the other. in no case did we observe any inconsistencies or any presence of nuclear genes, which would have indicated that the nd5-cox arrangement is part of a numt. phylogenetic analyses the h. magnipapillata and hydra. sp cox sequences from our assembly and sequencing were manually aligned with additional sequences from other hydra and outgroup species available from genbank in the seaview editor. the final dataset contained characters. a maximum likelihood analysis was carried out in paup* b under a model of nucleotide evolution suggested by the hierarchical likelihood ratio test in modeltest; for the bootstrap analysis we applied the same model. the dataset was also analyzed with mrbayes v.. parameter stabilization of the chains in mrbayes was monitored with tracer, and convergence of chains was examined with the diagnostics provided by the awty server. base compositions of cnidarian mt genomes sequences of additional cnidarian genomes were downloaded from genbank. the sequences of the respiratory chain protein genes shared between all mt genomes and the sequences of rns and rnl were extracted from the genbank format using the artemis software v.. in some mt genomes the rrna genes were not entirely annotated in their full length. we therefore considered the non-coding regions around the apparently too small genes as rrnas. the corresponding taxa and positions in each sequence were: nematostella sp.; mussa angulosa; astrangia sp.. the at contents of rrnas and protein codon positions of the mt genomes are shown in additional file  genes, base composition and codon usage the two chromosomes of the h. magnipapillata mt genome each carry one rrna gene. each of the assembled contigs of mt and mt is represented by > single sequence reads from the trace archive. our consensus sequence for mt is bp long. this matches the length reported by bridge et al. for this h. magnipapillata mt chromosome. the sequence of mt is shorter. the h. magnipapillata mt genome includes protein-coding genes of the respiratory chain usually found in other metazoa. mt contains protein-coding genes, rnl and two trna genes; mt contains protein-coding genes, rns and one trna gene. all genes are unidirectionally encoded on each of the two molecules and densely arranged along the chromosomes. as in h. oligactis, the longest non-coding intergenic region is bp between cox and nd. otherwise, subsequent genes are separated by 0– bp or overlap for up to bp. like many other cnidaria, the h. magnipapillata mt genome possesses only the two trna genes for methionine and tryptophan. trnw is only found on mt whereas identical copies of trnm are present on both chromosomes. six amino acid codons are not used in the protein-coding genes, and all genes are terminated by taa. apparently synonymous codons that posses an a or t, instead of a g or c, at the third codon position are preferred in h. magnipapillata. to test whether this observation is caused by mechanisms that affect base composition in the whole mt genome, we analyzed codon usage in the respiratory protein-coding genes in mt genomes of cnidaria. we plotted the at content at each of the three codon positions against the at contents of the rrna genes for every genome, as rrna coding genes represent a different part of the mt genomes in terms of functional constraints compared to protein-coding genes. remarkably, h. magnipapillata showed the highest values for at content at the third codon positions and in the rrna genes. moreover, a high at content in rrna genes generally correlates with the usage of a and t at third codon positions in all cnidaria, suggesting that codon usage might be the result of a general selection for base composition on the mt genome caused by interaction of mutational, repair, replication and translational mechanisms. the at content at the first and second codon positions also correlates with that of the rrna genes, but here at content rise at a lower rate than the increasing at content of the rrnas. this is likely the result of selection on certain amino acids. cnidarians posses a lower at content at the first codon position than at the second, with h. magnipapillata and h. oligactis being the only exceptions. codon frequency among the codons of the protein-coding genes in h. magnipapillata. gene arrangement and inverted terminal repeats compared to the gene arrangement of a. aurita and h. oligactis, only a few changes can be observed in h. magnipapillata. neglecting the positions of trnas, two blocks of genes are identical across the three genomes, occurring on mt or mt respectively, in h. magnipapillata. the mt genomes of h. oligactis and of h. magnipapillata are entirely alignable and display a sequence divergence of %. as mentioned before, we found 191– bp of itr at both ends of mt and mt in the linear mt genomes of h. oligactis and a. aurita, itr were also present but were longer. unlike itr in aurelia, itr in h. magnipapillata have a higher gc content than the rest of the molecule. we found that a smaller part of 3' cox is included in all itr of h. magnipapillata. probably because the 3' end of cox is not very conserved, pont-kingdon et al. missed this feature in their mt fragment of h. vulgaris. the itr regions of h. oligactis contain a larger cox fragment. the remaining sequenced 3' region of itr in h. oligactis is very similar to those found in h. magnipapillata and h. vulgaris, but longer. between h. magnipapillata and h. vulgaris, the major difference is that a stretch of gs is significantly shorter in h. magnipapillata. in h. magnipapillata mt and mt we found additional identical sequences at the 5' and 3' ends following and preceding the itr. we refer to those regions as identically oriented sequences. after the itr, the 5' ios of both molecules contain identical copies of non-coding dna and trnm. at the 3' ios we found a larger partial copy of the 5' region of cox on mt as a consequence of this arrangement, mt and mt share bp at the 5' end and bp at the 3' end, giving both molecules a specific orientation. using pcr experiments with the closely related hydra sp., we verified the following arrangements initially observed in the h. magnipapillata sequences: the presence and orientation of the itr at all four chromosome ends could be shown, as well as the presence of partial cox sequences in the itr; identical regions are shared at the 5' and 3' end, respectively, between mt and mt adjacent to the itr; and within the latter regions, the 5' motif contains trnm, which therefore appears in two copies in the genome, and a larger sequence of cox forms the shared 3' motif of mt and mt phylogenetic analysis the tree topology derived from our phylogenetic analysis of cox shows the close relationship of hydra sp. and h. magnipapillata, thus ensuring that we used an appropriate taxon to test our results. h. vulgaris is paraphyletic, which reflects the difficult taxonomy of the genus. the presented phylogeny, in combination with the mt genome organization, supports the view that the ancestral state of mt genome organization in the genus hydra was a single linear mt chromosome. 1syn.: hydra viridis; h. viridissima. discussion linear mt genomes and fragmentation of mt chromosomes in cnidaria linearity of mt genomes seems to have evolved once after the divergence of medusozoa from anthozoa. fig. 3a summarizes the results of different studies, mapped on a cnidarian phylogeny. a fragmentation of mt genomes has been reported from several hydra species and cubozoa. uncertainties remain for cubozoa: bridge et al. studied the same cubozoan species carybdea marsupialis as ender and schierwater, but reported a single ~ kb linear mt genome, while in the more recent work, a ~ kb fragment was shown to carry the rnl gene. because ender and schierwater were able to repeat the experiments with different dna isolates of c. marsupialis and obtained concordant results from an additional cubozoan species, an experimental error seems unlikely. however, their conclusion of four equally-sized mt chromosomes in cubozoa is not directly supported by their identification of a kb chromosome carrying rnl. alternatively, one could assume the presence of a single ~ kb mt counterpart, as indicated in fig. 3a. such an arrangement is possible, e.g., if rnl and cox the two genes that are encoded in different orientation to the other mt genes in a. aurita, were encoded in one chromosome in cubozoa, and the remaining genes on a second chromosome. however, given the available data it seems reasonable to assume that fragmented linear genomes occur in both cubozoa and hydrozoa. this suggests from an evolutionary perspective that the mt genome in the common ancestor of medusozoa was linear and then independently split into different chromosomes in hydra, and in at least some cubozoa. a possible mechanism for the origin of linear chromosomes from a circular molecule is the integration of one or more resolution elements. the circular dna molecule would be split into one or more linear molecules with identical ends. in medusozoa, the processes of linearization and the split of one linear into two linear chromosomes were obviously different processes as shown in the phylogenetic trees. the linearization, possibly occurring in the last common ancestor of medusozoans, seems to have preceded the splitting of the chromosomes by a long time. if the ancestral linear mt chromosome of medusozoa originated by introduction of a resolution element, one probably would not expect to observe its original motifs, which would occur as identical repeats at the two ends of the linear molecule. indeed, the ends of linear medusozoan mt chromosomes have inverted terminal motifs, instead of direct repeats. the splitting of ancestral linear mt chromosomes as in h. magnipapillata happened much later in evolutionary history, contradicting the view that the two or more linear mt chromosomes in medusozoa directly originated from one circular dna molecule. fragmented mt genomes are present in various eukaryotic taxa, e.g., in dinoflagellates, ichthyosporea and fungi. in metazoa, fragmented mt genomes are known from the genera globodera, dicyema and the rotifer brachionus plicatilis, but unlike in h. magnipapillata, in these taxa the genomes are encoded on several small circular molecules. the mt chromosomal organization observed in h. magnipapillata supports the hypothesis of an ancestral, linear chromosome in hydra, as represented by the mt genome of h. oligactis, which has been split in two between nd and rns. function of itr and ios warrior already suggested the presence of identical terminal sequences on both chromosomes of h. vulgaris. we now show that these ends are arranged as itr on mt and mt as in other medusozoans. in h. oligactis, which in the phylogenetic tree branches off before hydra species carrying two mt dna molecules, the single linear mt chromosome has itr containing a large copy of the 5' end of cox only the itr at the 3' end has been completely sequenced. based on our findings in h. magnipapillata, we predict that the unsequenced 5' end is almost identical to the 3' motif, and we expect that about bp remain unsequenced on the 5' end. in hydra, partial copies of cox play a crucial role as part in itr regions at the chromosome ends. the itr in h. magnipapillata contains only a short sequence of the 3' end of cox, suggesting that large parts of the cox copies were lost. a simultaneous duplication of 5' itr and the 5' ios motif seems likely to have occurred in the process of chromosome splitting. in this case, the longer cox copy is a duplication of the functional cox of the original 5' end of a single mt chromosome. itr of linear mt molecules are present in other taxa besides medusozoans, e.g., in yeasts and in the green algae chlamydomonas reinhardtii. furthermore, in the green algae polytomella parva, identical itr are present at all ends of the two linear mt chromosomes, similar to what we observe in h. magnipapillata. we report 5' and 3' ios as an additional shared feature of the two mt chromosomes. interestingly, such an arrangement of itr and 5' and 3' ios is also seen in another, highly fragmented eukaryotic mt genome. in the ichthyosporean amoebidium parasiticum, mt genes are distributed over several hundred different chromosomes, each of which also possesses itr and 5' and 3' ios. pont-kingdon et al. speculated that there may be a role for transcription initiation at the bp 5' of trnm, which they found in their h. vulgaris partial mt sequence. considering that transcription initiation within the itr would result in energetically expensive nonsense transcripts, transcription is more likely to start in the adjacent, non-coding regions of the 5' ios. in h. magnipapillata and h. vulgaris this region within the 5' ios is bp long and lies between the cox copy and trnm. in h. oligactis, the non-coding region between the itr and trnm is only bp. however, a striking sequence similarity can be observed near trnm between h. oligactis and h. vulgaris. there is a 14-bp motif that is shared between the species and differs by the last bp from the 3' itr+ bp counterpart in h. oligactis. this motif might be involved in transcription initiation. if so, the difference in the very last bp between the 5' end and its counterpart on the reverse strand in the itr of h. oligactis prevents a functional transcription signal on the non-coding strand in this species. a crucial function for transcription initiation would explain selective pressure for maintaining the 5' ios of both molecules after the itr in h. magnipapillata. all mt chromosomes from amoebidium parasiticum that contain coding genes are transcribed from 5' ios to 3' ios, as in h. magnipapillata. this observation led burger et al. to the conclusion that the ios in amoebidium are responsible for transcription initiation and termination. while in h. magnipapillata we expect the same function for 5' ios, the role of the additional partial cox copy within the 3' ios of mt and mt if any, remains unknown; considering that the end of cox is part of the itr, transcription can only be terminated in itr and not in the 3' ios. the sequence homologies of itr and ios within or between mt and mt are probably not the result of a relatively recent origin from ancestral sequences, as a first duplication of partial cox is already observed in h. oligactis and therefore predates the separation process. the substitutions between itr of the two species are found in all itr copies in each mt genome. considering this and the fact that similar arrangements are found in other eukaryotes, it seems more likely that concerted evolution maintains the almost identical sequences, probably caused or influenced by the yet unknown mt genome replication mechanism. terminal sequences of linear dna molecules play a crucial role in mt replication. the main problem in the process of linear chromosome replication is the maintenance of the 5' ends. in nuclear chromosomes this is normally achieved by telomerase, an enzyme that adds short sequence motifs in tandem repeats at the end of each molecule to compensate for loss at the 5' end that occurs in each replication cycle. consequently, in hydra as in most metazoa, the motif n is found at the end of nc chromosomes. the termini of hydra mt chromosomes are much more complex, and their maintenance during replication is not yet understood but is most likely telomerase independent. warrior suggested an mt replication mechanism for the two h. vulgaris mt chromosomes similar to that of t bacteriophages. his conclusion was based upon observations from hybridization experiments, which showed the presence of identical terminal sequences that he assumed to have the same orientation at the 5' and 3' ends. according to this model, intermediate concatamers are formed, and via ligation, site-specific nicking and elongation, the 5' ends are finally filled. based on our data we can reject this model, because we showed that the terminal sequences in h. magnipapillata are inverted and therefore do not allow the necessary concatamerization in the proposed way. similarly, ephemeral circularization as in the phage lambda is not possible when terminal sequences are not direct repeats, but inverted. different replication mechanisms for linear chromosomes have been reported or proposed. solutions for maintaining the terminal structure in sequences with itr include covalently bound proteins, which also could serve as primers for a'racket frame' replication, 5' and 3' ends of chromosomes that are connected by a hairpin-loop and single-stranded 3' overhangs. warrior showed that the first two possibilities are not realized in h. vulgaris. the mt-replication mechanism in chlamydomonas requires an internal repeat of the single-stranded 3' overhangs. we cannot entirely rule out the existence of short single-stranded 3' overhangs, as it is possible they might have been missed due to our methods. the outermost sequence at least cannot be part of a repeat motif, as our pcr amplification of hydra sp. did not yield fragments of different sizes. furthermore, neither in mt of h. vulgaris nor in the mt genome of h. oligactis were additional sequences or repeats found. therefore, although a similarity to the mt replication of c. reinhardtii cannot be excluded, we find that mechanisms for linear mt chromosome replication are too diverse and that too many details are still unknown for us to draw further conclusions about the replication process in h. magnipapillata from the presence of the itr alone. considering that itr are a shared feature among all three available sequences of medusozoan mt genomes, it is very likely that the mechanisms for mt replication are similar in all medusozoans and are still the same after the fragmentation of the mt genome. keeping in mind that similar arrangements of itr and ios are found in amoebidium parasiticum, the mt replication mechanisms of h. magnipapillata and medusozoa are probably not unique among eukaryotes.  the h. magnipapillata mt genome represents the first complete sequence of a linear metazoan mt genome that consists of two separate molecules. the gene arrangements and our phylogenetic analysis suggest that mt and mt originated from an ancestral linear mt genome, as found in h. oligactis, that at some point divided in two between nd and rns. of most interest is the organization at the ends of the two mt chromosomes. we show that h. magnipapillata has itr, which include a part of cox or partial copies of the 5' end of cox. we conclude that mechanisms for mt replication in hydra species are different from the previously proposed ones and probably are shared among all medusozoans. in addition to itr, both mt chromosomes of h. magnipapillata have identical motifs on their 5' and 3' ends, called respectively the 5' ios and 3' ios. the 5' ios includes trnm and a non-coding region, including a motif that may play a role in transcription initiation. the 5' ios is probably the result of a duplication during the separation process of a single ancestral mt chromosome. the organization of the itr and 5' and 3' ios is not unique among eukaryotes with fragmented linear mt genomes. itr most likely play a role in mt replication, while the duplication of the 5' end of an single ancestral linear and unidirectionally-encoded mt chromosome and its concerted evolution ensure that transcription of all mt genes is maintained after fragmentation of linear mt chromosomes. a similar arrangement of itr and ios regions can therefore be expected in the apparently fragmented mt genomes of cubozoa and other eukaryotes with two or more linear mt dna molecules. authors' contributions ov contributed to the conception and design of the study, acquired, analyzed and interpreted the data, and wrote the manuscript. de and gw contributed to the interpretation of data and critically reviewed the manuscript. all authors read and approved the final version of the manuscript. supplementary material additional file 1 trace ids of sequences included in the h. magnipapillata assemblies. click here for file additional file 2 coverage of mt and mt assemblies. click here for file additional file 3 primer sequences. click here for file additional file 4 taxa, genbank accession numbers and at contents of protein-coding genes and rrna genes in cnidarian mt genomes. click here for file acknowledgements this work was financially supported by the german research foundation and is a contribution from the dfg priority programme spp "deep metazoan phylogeny". d.e. acknowledges financial support from the european union under a marie curie outgoing fellowship. all authors acknowledge the j. craig venter institute for providing h.magnipapillata wgs traces via ncbi's trace archive, and the national human genome research institute for funding the h. magnipapillata genome project. we thank bernie degnan and ben woodcroft for providing access to their'reefedge' pipeline for local genome assembly and the schulbiologiezentrum hannover for providing hydra specimens for our pcr experiments. ov thanks catherine vogler for help with r and many useful discussions and remarks on the manuscript.
we show that identical inverted terminal repeats occur on both chromosomes; these itr contain a partial copy or part of the 3' end of cox. the h. magnipapillata mt chromosomes contain the typical metazoan set of genes for respiratory proteins, the two rrna genes and two trna genes. in h. magnipapillata, the arrangement of itr and 5' ios and 3' ios suggest that these regions are crucial for mt dna replication and/or transcription initiation. with our data, we can reject a model of mt replication that has previously been proposed for hydra. here, we present the whole mt genome sequence from the hydrozoan hydra magnipapillata, comprising the first sequence of a fragmented metazoan mt genome encoded on two linear mt chromosomes. this raises new questions regarding replication mechanisms probably employed by all medusozoans, and also has general implications for the expected organization of fragmented linear mt chromosomes of other taxa. in the genus hydra, the mt genome comprises one or two mt chromosomes. strong correlations between the at content of rrna genes and the at content of protein-coding genes among cnidarian genomes imply that base composition is mainly determined by mt genome-wide constraints. the gene arrangement suggests that the two mt chromosomes originated from one linear molecule that separated between nd and rns. additionally, both mt chromosomes possess identical oriented sequences at the 5' and 3' ends adjacent to the itr. medusozoa are exceptional in that their mt genomes are linear and sometimes subdivided into two to presumably four different molecules. itr are also documented in the two other available medusozoan mt genomes. the 5' ios contains trnm and non-coding sequences, whereas the 3' ios comprises a larger part with a larger partial copy of cox.
 animal mitochondrial genomes are characteristically circular molecules of ~16– kb. medusozoa are exceptional in that their mt genomes are linear and sometimes subdivided into two to presumably four different molecules. in the genus hydra, the mt genome comprises one or two mt chromosomes. here, we present the whole mt genome sequence from the hydrozoan hydra magnipapillata, comprising the first sequence of a fragmented metazoan mt genome encoded on two linear mt chromosomes.  the h. magnipapillata mt chromosomes contain the typical metazoan set of genes for respiratory proteins, the two rrna genes and two trna genes. all genes are unidirectionally oriented on mt and mt and several genes overlap. the gene arrangement suggests that the two mt chromosomes originated from one linear molecule that separated between nd and rns. strong correlations between the at content of rrna genes and the at content of protein-coding genes among cnidarian genomes imply that base composition is mainly determined by mt genome-wide constraints. we show that identical inverted terminal repeats occur on both chromosomes; these itr contain a partial copy or part of the 3' end of cox. additionally, both mt chromosomes possess identical oriented sequences at the 5' and 3' ends adjacent to the itr. the 5' ios contains trnm and non-coding sequences, whereas the 3' ios comprises a larger part with a larger partial copy of cox.  itr are also documented in the two other available medusozoan mt genomes. in h. magnipapillata, the arrangement of itr and 5' ios and 3' ios suggest that these regions are crucial for mt dna replication and/or transcription initiation. an analogous organization occurs in a highly fragmented ichthyosporean mt genome. with our data, we can reject a model of mt replication that has previously been proposed for hydra. this raises new questions regarding replication mechanisms probably employed by all medusozoans, and also has general implications for the expected organization of fragmented linear mt chromosomes of other taxa. 