BACKGROUND
the mitochondrion is a double-membrane enclosed organelle found in the vast majority of extant eukaryotes.
mitochondria are best known for their essential role in energy generation, but they are also the site of additional important cellular processes such as iron-sulfur  cluster assembly and the beta-oxidation of fatty acids  <cit> .
some degenerate forms of mitochondria, such as the mitosome of the diplomonad parasite giardia lamblia, have secondarily lost energy generating pathways and seem to retain only the fe-s cluster maturation function  <cit> .
all mitochondria are believed to share a single origin from an α-proteobacterial-like prokaryote  <cit> , but a wide diversity of mitochondrial genome architectures have evolved subsequent to the diversification of modern-day eukaryotes  <cit> .
for example, whereas "derived" animals possess monomeric circular mitochondrial genomes, an observation which led to the initial assumption that mtdnas are primarily circular  <cit> , many other mitochondrial genomes, such as that of the ciliate tetrahymena pyriformis  <cit> , the green alga chlamydomonas reinhardtii  <cit>  and the cnidarian metazoan aurelia aurita   <cit>  are linear  <cit> .
in addition, while some fungi and many plants have circular-mapping mtdnas, their mitochondria actually contain predominantly linear mtdna molecules with combinations of monomers and concatemers, with only a minor fraction of the molecules present in a circular form  <cit> .
a more extreme example is the mtdna of kinetoplastids, which consists of one maxi- and many different mini-circles that are interconnected to form an extensive network  <cit> .
mitochondrial gene content is also highly variable; the mtdna of the jakobid flagellate reclinomonas americana encodes  <dig> genes, the largest set of mitochondrial genes currently known  <cit> , whereas the mtdna of the malaria parasite plasmodium falciparum contains just  <dig> protein coding genes and  <dig> highly fragmented small and large subunit ribosomal rna  genes  <cit> .
the most highly derived forms of mitochondria, such as the hydrogenosome of trichomonas vaginalis  <cit>  and the giardia lamblia mitosome  <cit> , have lost their genomes entirely  <cit> .
mitochondria are also known as sites of unusual molecular biology and biochemistry.
marande and burger  <cit>  recently showed that the mtdna genes of the euglenid diplonema papillatum are fragmented into as many as nine modules, each residing on a distinct  <dig> or  <dig> kbp chromosome.
the mechanism by which these fragmented gene pieces are linked together to form contiguous transcripts is unknown.
extensive mrna editing is another example of the bizarre molecular biology of mitochondria.
kinetoplastid mitochondrial mrnas are subject to insertions and deletions of uridylate residues, sometimes > <dig> such insertions/deletions per transcript  <cit> .
mitochondrial mrna editing is also widespread in land plants  <cit>  and dinoflagellates  <cit> .
for example, ~2% of the cox <dig> and cob gene sequences in three dinoflagellate species investigated by lin et al.
<cit>  were edited at the mrna level.
we are studying the genomic diversity and evolution of cryptophytes, a ubiquitous and ecologically significant group of single-celled eukaryotes found in freshwater and marine environments.
most cryptophytes, except for members of the genus goniomonas, harbor plastids of secondary endosymbiotic origin  <cit> .
a variety of shared morphological features, such as the presence of ejectisomes, flat mitochondrial cristae, and an anterior depression, support the monophyly of cryptophytes, as do molecular phylogenetic data  <cit> .
one unique feature of cryptophyte plastids that distinguishes them from other plastids of red algal origin is the retention of the remnant nucleus of the red algal endosymbiont, referred to as the nucleomorph  <cit> .
consequently, most cryptophytes harbor four distinct genomes–nuclear, nucleomorph, mitochondrial, and plastid genomes–contained in separate compartments.
cryptophytes are thus an interesting model system with which to study endosymbiotic gene transfer, genome evolution, and protein targeting.
in this study, we report the complete mitochondrial genome sequence of the newly described cryptophyte species hemiselmis andersenii ccmp <dig>  and compare it to the only other cryptophyte mitochondrial genome described thus far, that of rhodomonas salina  <cit> .
in addition, individual and concatenated mitochondrial protein coding gene sequences were analyzed to infer the phylogenetic relationships of cryptophytes to other eukaryotes.
methods
dna preparation, sequencing, and genome assembly
hemiselmis andersenii mtdna was isolated and sequenced to ~10× coverage as described in lane et al.
<cit> .
about  <dig>  end sequences were screened for quality and vector contamination with pregap <dig> and automatically assembled using gap <dig> version  <dig>  in the staden package  <cit> .
complete automated assembly of a large intergenic space between trns and cox <dig> was unsuccessful due to the highly repetitive nature of this region.
in an attempt to manually resolve this area, short  unique sequences within the trns and cox <dig> genes were used to probe the sequence database for reads that extended from these two loci into the repeat region.
these sequences were extracted and manually aligned using macclade version  <dig>   <cit> .
sequences at the ends of the new constructs were then selected and the process was repeated.
however, due to the presence of multiple identical copies of a > <dig> bp repeat, the assembly of a single unambiguous contig was not possible.
when all available sequence reads were considered, three robust contigs were produced, each ending with similar repetitive sequences consisting of a ~ <dig> bp repeat unit.
these three contigs were joined to circularize the map.
the complete h. andersenii mtdna has been submitted to genbank under the following accession number: eu <dig>
dna secondary structure within the repeat region was predicted using mfold version  <dig>   <cit>  at a folding temperature of 37°c and the ionic conditions of  <dig>  m  and  <dig>  m .
genome size/structure determination
we used pulsed-field gel electrophoresis  to obtain an independent size estimate of the h. andersenii mitochondrial genome.
hemiselmis andersenii total dna plugs were prepared as described in lane et al.
<cit>  and digested overnight with the restriction enzymes psti or bglii .
based on the genome sequence, these enzymes were predicted to cut the mtdna only once or twice.
both untreated and enzyme-digested h. andersenii dna plugs were run on a 1% agarose gel  in  <dig> × tbe buffer at  <dig> °c for  <dig> h at a voltage of  <dig>  v/cm with a switch time between 1– <dig> s using a chef-dr iii pulsed-field gel electrophoresis system .
dna on the pulsed-field gel was transferred to a nylon membrane.
southern hybridization using a ~ <dig> bp coxi probe as in lane and archibald  <cit>  revealed that undigested mitochondrial dna molecules were trapped in the wells or found in the 'compression zone'.
the psti or bglii endonuclease treated dna plugs revealed mitochondrial molecules in a discrete band below the 'compression zone'.
the corresponding bands could not be visualized on the ethidium-bromide stained pulsed-field gel image because of nuclear and nucleomorph dna smears in the background.
in order to visualize mtdna on the pulsed-field gel, an initial pfge run was used to remove the linear nuclear and nucleomorph chromosomes from the pfge plugs.
these plugs, which still contained organellar dna, were subsequently removed from the gel and digested with the restriction enzymes psti and bglii.
digested plugs were then inserted into a fresh gel and electrophoresed under the conditions described above.
the  <dig> kbp and lambda chef dna size standard  were used to estimate the size of the enzymatically linearized h. andersenii mtdna.
genome annotation and gc content/skew analyses
annotation of the h. andersenii mtdna and the gc content and skew analyses were performed in artemis version  <dig>  <cit> .
gene identification was carried out using blastx and blastn.
small and large ribosomal rrna subunit genes were identified by comparison to rrna gene sequences in the mitochondrial genome of rhodomonas salina.
transfer rnas were identified using trnascan-se version  <dig>   <cit> .
genome rearrangements between the two cryptophyte mtdna
the extent to which the h. andersenii and r. salina mitochondrial genomes are rearranged to each other was estimated using grimm  <cit> .
each genome was designated as a sequence of  <dig> units, which include a repeat region and  <dig> genes common between the two cryptophyte mtdnas.
rt-pcr of 'trnk'
trnascan-se version  <dig>   <cit>  identified a putative intron of ~ <dig> bp in the anticodon loop of the h. andersenii trnk gene.
to determine whether this prediction was correct, we performed rt-pcr using lysine-trna-specific primer pairs and h. andersenii total rna provided by h. khan.
to eliminate dna contamination,  <dig> μl of total rna was incubated for  <dig> min with rq <dig> rnase-free dnase .
rt-pcr was performed using the qiagen one-step rt-pcr kit  and with control reactions in which the reverse-transcription process was skipped.
the following two pairs of primers were used: 1) the forward primer 5'-gaaggttgctcgaatggaa-3' with the reverse primer 5'-gaaggtataggaattgaacctattc-3' 2) and the forward primer 5'-gcccagaaggttgctc-3' with the reverse primer 5'-aagaaggtataggaattgaacctat-3'.
rt-pcr was performed with the reverse transcription step for  <dig> min at 50°c and the subsequent inactivation of reverse transcriptase and activation of hotstart taq dna polymerase for  <dig> min at 95°c, followed by  <dig> cycles at 94°c for  <dig> min, 47°c for  <dig> min, and 72°c for  <dig> min, and a final extension at 72°c for  <dig> min.
the amplified pcr fragments were cloned into pcr4-topo vector in the topo ta cloning kit for sequencing .
between  <dig> and  <dig> bacterial colonies from each reaction were selected for sequencing on a beckman coulter ceq <dig> .
molecular phylogenetic analysis
from the  <dig> protein-coding genes found in the h. andersenii mtdna,  <dig> were selected for phylogenetic analyses.
eleven genes  were excluded because their sequences were poorly conserved and/or were only present in a few taxonomic groups.
h. andersenii protein sequences were aligned with their homologs from other mitochondrial genomes available from genbank.
amino acid sequences were aligned using macclade version  <dig>   <cit>  and ambiguously aligned sites were manually removed.
in addition to individual protein analyses, a concatenated protein data set containing  <dig> proteins was analyzed.
to include the maximum number of gene sequences, we combined  <dig> protein-coding gene sequences encoded in  <dig> mitochondrial genomes across diverse eukaryotic taxa.
as most mitochondrial genomes do not possess all  <dig> protein-coding genes selected for analysis, as many as  <dig> protein gene sequences were missing per taxon.
a maximum likelihood tree was produced using raxml-vi-hpc version  <dig> .
<dig>  <cit>  with the protomixjtt model of sequence evolution and the automatic tree rearrangement setting, and from  <dig> distinct randomized maximum parsimony starting trees.
bootstrap analysis was based on  <dig> re-samplings.
RESULTS
general features of hemiselmis andersenii mtdna
the mitochondrial dna of the cryptophyte hemiselmis andersenii ccmp <dig> was sequenced, assembled and manually edited to produce a circular-mapping genome  <dig>  bp in size .
genome assembly was complicated by the presence of a highly repetitive non-coding region of ~ <dig> kbp ; genome size was thus verified using pulsed-field gel electrophoresis .
several observations suggest that the h. andersenii mtdna exists primarily in a linear-branched form comprised of multiple genome units.
in pfge, the h. andersenii mtdna remains in the well or migrates within the 'compression zone' , which contains primarily linear nuclear and nucleomorph chromosomes larger than ~ <dig> kbp .
the lack of mtdna below the 'compression zone' suggests that the h. andersenii mtdna is not composed of linear monomers or dimers.
furthermore, when the h. andersenii mtdna is partially digested with psti, an enzyme predicted to cut the genome only once, it produces a discrete band of ~ <dig> kbp in size  but not a band ~ <dig> kbp in size, which would correspond to a dimeric linear form of the genome.
this result indicates that the h. andersenii mtdna is not composed of circular concatemers or linear head-to-tail concatemers consisting of three or more genomic units.
therefore, we suggest that the h. andersenii mtdna exists primarily as a branched linear molecule although monomeric circles may also exist.
further studies using transmission electron microscopy or the 'moving picture' technique  <cit>  will be necessary to confirm this hypothesis.
the h. andersenii mitochondrial genome is comprised of a gene-rich region ~ <dig> kbp in size and a large  intergenic region between trns and cox <dig> with complex repeats .
the intergenic region accounts for  <dig> % of the entire genome and  <dig> % of the total amount of non-coding dna .
the overall gc content of the genome is  <dig> %, slightly higher than that of the nucleomorph genome of this organism  <cit> .
interestingly, a ~ <dig> bp region near the start of the coding portion of the genome is very gc-rich  and is followed by a 100% at-containing region ~ <dig> bp in size .
this unusual stretch of sequence is about  <dig> bp from a palindromic sequence that is predicted to form a type ii stem-loop , and may be involved in regulating replication or transcription.
the h. andersenii mitochondrial genome encodes  <dig> genes with predicted functions and  <dig> hypothetical protein-coding genes, a total somewhat higher than the average for eukaryotes   <cit> .
ten genes–orf <dig>  orf <dig>  rps <dig>  rps <dig>  nad <dig>  rps <dig>  tatc, 'trnk ', rps <dig>  and rps7–overlap by up to  <dig> bp, emphasizing the extreme compactness of the coding portion of the genome.
the genome encodes small and large rrna subunit genes and  <dig> trnas, one of which may be a pseudogene .
of the  <dig> identifiable protein-coding genes,  <dig> encode ribosomal proteins,  <dig> are involved in oxidative phosphorylation, and one gene encodes a membrane translocase protein .
comparison of the mtdna gene order in h. andersenii to other genomes reveals the presence of five gene clusters shared among distantly related protists: two ribosomal protein clusters  and three nadh dehydrogenase clusters .
these gene clusters have been suggested to represent vestiges of bacterial operons  <cit> .
interestingly, all  <dig> genes in the h. andersenii mitochondrial genome are encoded on the same strand.
while the evolution of such an arrangement seems improbable, absolute strand polarity has been observed in the mitochondrial genomes of diverse eukaryotes such as the amoeba acanthamoeba castellanii , the fungus penicillium marneffei , and the green alga chlamydomonas eugametos   <cit> .
in addition, strikingly similar mtdna architectures–gene-dense regions, a single large repetitive intergenic region, and all genes encoded on one strand–are seen in diverse protists such as the stramenopile thraustochytrium aureum  and the green alga pedinomonas minor  <cit> .
understanding the biological significance of such convergence at the level of genome architecture will require comparative molecular and biochemical studies of mitochondria in these organisms.
comparison of the mitochondrial genomes of hemiselmis andersenii and rhodomonas salina
h. andersenii is only the second cryptophyte, after r. salina  <cit> , for which a mitochondrial genome has been completely sequenced and annotated.
comparative analyses of the two genomes revealed a number of similarities.
both genomes feature a compact gene arrangement and a single large repeat region   <cit> , although the size of the large intergenic region in h. andersenii  is more than four times as large as that of r. salina .
all of the  <dig> predicted protein-coding genes in the h. andersenii mitochondrial genome are present in the r. salina mtdna.
four r. salina mitochondrion-encoded genes–rps <dig>  atp <dig>  tata, and sdh4–are not found in h. andersenii, although two open reading frames, orf <dig> and orf <dig>  in the h. andersenii mtdna show marginal sequence similarity to the r. salina tata and sdh <dig> genes, respectively.
additionally, while two group ii introns are present in r. salina mtdna, the h. andersenii mtdna is devoid of introns   <cit> .
with respect to conservation of gene order,  <dig> % of the shared genes between the two cryptophyte mitochondrial genomes ,  <dig> rrna genes) are present in thirteen syntenic blocks, each consisting of 2– <dig> genes.
these include: 1) cox1-cob-nad <dig>  2) nad4l-nad <dig>  3) atp1-trnp, 4) rps8-rpl6-rps13-rps <dig>  5) trnc-atp <dig>  6) trni-trnq-trnr-trne-trnw-nad10-nad <dig>  7) nad4-nad <dig>  8) trnr-trng, 9) trnmf-trns, 10) trny-trnl, 11) tatc-'trnk'  /trns -nad <dig>  12)cox3-rps12-rps7-rps <dig>  and 13) rps3-rpl16-rpl14-rpl5-rps <dig>  as noted earlier, some of the conserved gene clusters, such as nad4l-nad <dig>  are found in distantly related eukaryotes and appear to be vestiges of bacterial operons.
analysis using grimm  <cit>  suggests that the observed difference in gene order between the two cryptophyte mitochondrial genomes can be explained by at least  <dig> instances of genome reversal events.
repeat structure of the h. andersenii mitochondrial genome
the r. salina mtdna is characterized by a pair of ~ <dig>  kbp inverted repeats that are joined by  <dig> bp of sequence  <cit> .
in contrast, repeats in the h. andersenii mitochondrial genome are not inverted, but are instead dispersed or arranged in tandem throughout the large non-coding region, with individual repeat units ranging from  <dig> to  <dig> bp and occurring up to  <dig> times .
given that r. salina and h. andersenii are distantly related to one another  <cit> , the large repeat region presumably arose during or prior to the early diversification of cryptophytes.
while there is no obvious sequence similarity between the two repeat regions, both contain multiple copies of palindromic sequences, which are predicted to form stable stem-loop dna structures  <cit> .
in h. andersenii, two types of stem-loop structures were identified–i and ii–using the dna mfold program  <cit> .
the type i structure has two slight variations, i-a and i-b, which occur  <dig> and  <dig> times, respectively .
type i-a and i-b structures have  <dig> and  <dig> base pairings in their stems, respectively, and occur adjacent to tandem repeats .
one copy of the type ii stem-loop structure is located within a ~ <dig> bp segment that is devoid of any discernable repeat units, but close to the high and low gc regions noted earlier .
as was suggested for r. salina by hauth et al.
<cit> , tandem repeats and multiple stem-loop structures in h. andersenii mtdna might be involved in the regulation of transcription and replication, a hypothesis that needs to be tested further.
hauth et al.
<cit>  demonstrated that the repeat region of the r. salina mtdna roughly coincides with a change in the direction of 'cumulative gc skew'  and suggested that the repeat corresponds to the origin of replication.
we investigated the gc skew in the h. andersenii mitochondrial genome to see whether a similar pattern exists.
unlike r. salina, however, the h. andersenii gc skew does not change direction near the repeat region.
instead, in both the h. andersenii and r. salina mtdna, observed gc skew patterns strongly correlate with transcriptional orientations, where the coding strand tends to be g-rich .
therefore, the gc skew patterns of the two cryptophyte mitochondrial genomes do not seem to be the result of replication-associated mutational bias, but rather the non-random distribution of the protein coding genes, as has been observed in some other genomes  <cit> .
nevertheless, based on the presence of other features such as stem-loop structures, it seems reasonable to assume that the repeat region in both cryptophyte mitochondrial genomes corresponds to the origin of replication.
codon usage and transfer rnas
the h. andersenii mtdna encodes  <dig> trnas,  <dig> of which are predicted to form standard cloverleaf secondary structures.
one trna gene, 'trnk', shows atypical structure in the anticodon loop and the variable region, and is probably a pseudogene .
allowing for wobble pairings and some base modifications,  <dig> trnas are the theoretical minimum required to cover all codons in bacteria.
for some mitochondria, even smaller sets of trnas, as few as 22– <dig>  are possible by adopting several additional strategies  <cit> .
the h. andersenii mitochondrial genome lacks only one trna gene, trnk, which is minimally required in order to recognize all  <dig> codons .
it is thus predicted that nuclear-encoded cytosolic lys-trna is imported into h. andersenii mitochondria.
mitochondrial trna import has been demonstrated in apicomplexans and trypanosomatids where trna genes are completely missing in their mitochondrial genomes  <cit> , as well as in ciliates and plants where mitochondrial genomes encode fewer than the 22– <dig> minimally required trna genes  <cit> .
although most animals and some fungi do not import trnas into mitochondria  <cit> , the fungus saccharomyces cerevisiae has been shown to import one specific cytosolic trna even though its mitochondrial genome encodes the full complement of trnas  <cit> .
analyses of the trna repertoire of mitochondrial genomes suggest that a number of other protist taxa across the eukaryotic tree also import one or more trnas into their mitochondria  <cit> .
it is thus reasonable to assume that h. andersenii imports at least lys-trna, although it is possible that trna editing makes up for the lys-trna deficit by changing the identity of an existing trna, as has been shown in marsupials  <cit> .
the frequency of each codon is shown and dots indicate the presence of a corresponding trna gene in the mtdna.
†: 'trnk' identified in the genome may be a pseudogene .
another possible mechanism to account for the missing trna is that the structurally abnormal 'trnk' gene  forms a functional lys-trna to decode the codons aaa and aag.
several cases of atypically-structured trnas are known from animal and ciliate mitochondria  <cit> .
interestingly, trnascan-se  <cit>  predicted the existence of a  <dig> bp intron within the h. andersenii 'trnk', and we conducted further experiments to test whether this is indeed the case.
rt-pcr experiments using primer sets specific for 'trnk' indicated that the putative intron was not removed in the mature trna.
this results is not unexpected, given that the 20-bp putative intron is too short to be a self-splicing group i or ii intron, which are the only known types of introns reported in mitochondrial genomes  <cit> .
sequencing of ~ <dig> clones also did not reveal any evidence for rna editing within the 'trnk'.
these results suggest that if 'trnk' is indeed expressed to form a functional lys-trna, it is predicted to have an unusually au-rich stem in the codon loop and a long variable region, atypical for lys-trna .
long variable regions ranging from  <dig> to  <dig> nucleotides are generally restricted to trna-leu, trna-ser, and bacterial trna-tyr  <cit> .
the d- and t-loops of the 'trnk' sequence show sequence similarity to one of the two mitochondrion-encoded trna-ser genes , both of which have a long variable region.
in addition, comparative analysis with the r. salina mtdna revealed genomic position conservation between the h. andersenii trns-like 'trnk' gene and the trns gene of r. salina, flanked by the tatc and nad <dig> genes.
the h. andersenii 'trnk' and r. salina trns genes both overlap tatc by  <dig> bp and  <dig> bp, respectively.
this strongly suggests that the h. andersenii 'trnk' is indeed derived from an ancestral gene that encoded trna-ser, explaining the origin of its long variable region.
the overlap between the h. andersenii 'trnk' and tatc suggests that 'trnk' may play a role in processing the 3' end of the tatc gene transcript.
this hypothesis could explain why the 'trnk' gene still remains in the genome and retains conserved secondary structure in the stem loop and d- and t-loops, even if it does not form a functional trna.
comprehensive molecular and biochemical experimentation will be necessary to confirm or refute the existence of mitochondrial trna import in h. andersenii and the functionality of the unusual 'trnk' gene.
when the h. andersenii trna genes were compared to those of r. salina,  <dig> homologous pairs of trnas were identified, leaving only four h. andersenii trna and three r. salina trna genes not unambiguously matched to each other.
each of the trna pairs possess identical anticodons except for the h. andersenii 'trnk' and r. salina trns pair, despite their common derivation.
the trns of h. andersenii, having sequence homology to the 'trnk', probably originated from a recent gene duplication event.
of the three remaining h. andersenii trna genes that are unmatched in r. salina, two–trnl and trng–are redundant because trnl and trng can decode all of their respective four-codon families  <cit> .
these redundant copies might have been lost in an ancestor of r. salina after it diverged from h. andersenii.
lastly, the h. andersenii trni is somewhat similar to the trnk of the r. salina and only marginally resembles the r. salina trni at the 3' end.
it is possible that the h. andersenii trni originated through recombination between ancestral trni and trnk genes, which would explain the lack of an obvious trnk homolog in h. andersenii comparable to the r. salina trnk.
substantial sequence divergence among the three genes, however, makes it difficult to accurately trace the origin of the trni and the loss of the original trnk gene in h. andersenii.
on the other hand, the unusual trni gene reported from r. salina is not found in h. andersenii.
it was suggested that the r. salina trni is derived from trnf through a recent gene duplication event  <cit> .
overall, the two cryptophyte mitochondrial genomes use similar trna sets to recognize codons.
however, unlike h. andersenii, which may need to import at least trnk from cytosol, the r. salina mtdna does possess the minimal required set for trna autonomy.
molecular phylogenetic analyses
cryptophytes are a well-established eukaryotic lineage, supported by both molecular and morphological features  <cit> .
however, their relationship to other eukaryotic groups, particularly those containing plastids of secondary endosymbiotic origin, has been the subject of considerable debate.
the cryptophyte plastid is the product of a secondary endosymbiosis involving a red algal cell, the same process which accounts for plastid origins in haptophytes, dinoflagellates, and stramenopiles  <cit> .
cavalier-smith  <cit>  suggested that plastids in these four algal lineages arose from a single secondary endosymbiosis in a common ancestor that these organisms shared, to the exclusion of other eukaryotic groups.
however, this "chromalveolate" hypothesis is controversial  <cit> .
recent molecular studies have shown that the katablepharids, an enigmatic collection of plastid-less flagellates, are a sister group to cryptophytes  <cit> , and large-scale concatenated analyses of nuclear genes suggest that cryptophytes and haptophytes are also related  <cit> .
to gain insight into the phylogenetic relationship of the cryptophytes h. andersenii and r. salina to other eukaryotes, and more specifically, to test the hypothesis that cryptophytes and haptophytes are related to one another, phylogenetic analyses of mitochondrial protein sequences were performed .
unlike the cryptophyte plastid genome, in which several cases of lgt have recently been discovered  <cit> , individual analyses of  <dig> mitochondrial proteins did not reveal any obvious instances of lgt between prokaryotes and eukaryotes or within eukaryotes .
however, the possibility of ancient lgts cannot be ruled out, as the backbones of individual protein phylogenies were generally very poorly supported.
as expected, a close relationship between the two cryptophytes h. andersenii and r. salina was well supported in the mitochondrial protein phylogenies, with twenty of twenty-five individual protein phylogenies showing this relationship.
five individual gene phylogenies–nad <dig>  rpl <dig>  rpl <dig>  rps <dig>  rps14–did not recover a h. andersenii-r. salina clade, although alternative topologies were not supported with >50% bootstrap support values.
additionally, single protein phylogenies were not, for the most part, able to resolve the relationship of cryptophytes to other eukaryotes.
the position of cryptophytes was highly variable from protein to protein and the group did not regularly associate with other taxonomic clades with >50% bootstrap support values, except for in the cob and nad <dig> gene trees, where cryptophytes branch with haptophytes  and jakobids , respectively.
we subsequently analyzed a set of  <dig> concatenated proteins to assess the phylogenetic position of cryptophytes.
in this analysis, the h. andersenii-r. salina clade received 100% bootstrap support .
other well-established eukaryotic groups including opisthokonts, rhodophytes, stramenopiles, and viridiplantae, were also strongly recovered, but the relationships among major lineages were not.
the jakobid reclinomonas branched as the sister group to the viridiplantae with moderate support , and malawimonas showed an affinity for these two groups in two of the three data sets, as was previously inferred from a concatenate of ten mitochondrial proteins  <cit> .
it is not clear whether the jabokid -viridiplantae affinity is a phylogenetic artifact or reflects the true evolutionary history of mitochondrial genes.
though growing evidence supports a relationship between cryptophytes and haptophytes  <cit> , our extensive mitochondrial protein analyses did not reveal this relationship with reasonable bootstrap support, other than in a single protein gene tree .
in summary, while mitochondrial gene sequences are able to resolve some of the eukaryotic lineages determined using other markers, they are at present incapable of resolve the deepest branches of the eukaryotic tree using current phylogenetic methods and with the present level of taxon sampling.
CONCLUSIONS
we have sequenced the mitochondrial genome of the cryptophyte h. andersenii and compared it to that of the distantly related cryptophyte r. salina.
our analyses reveal that both genomes are characterized by a gene dense region and a single large intergenic space that includes numerous repeats and palindromic sequences predicted to form stable dna stem and loop structures.
despite the overall similarities in content and architecture between the two genomes, their modes of regulating dna replication and transcription seem to differ.
unlike r. salina, all  <dig> genes in the h. andersenii mtdna are located on the same strand, a relatively rare observation in mitochondrial genomes.
phylogenic analysis of multiple mitochondrial gene sequences indicated a clear affiliation between the two cryptophytes but was not able to resolve the position of cryptophytes relative to other eukaryotic groups.
authors' contributions
ek participated in genome assembly, carried out genome analysis and drafted the manuscript.
cel isolated h. andersenii dna and participated in the initial genome assembly.
bac, ck, and sb performed the h. andersenii mitochondrial genome sequencing.
jma coordinated the study and helped draft the manuscript.
all authors read and approved the manuscript.

 the mitochondrion is a double-membrane enclosed organelle found in the vast majority of extant eukaryotes. mitochondria are best known for their essential role in energy generation, but they are also the site of additional important cellular processes such as iron-sulfur cluster assembly and the beta-oxidation of fatty acids. some degenerate forms of mitochondria, such as the mitosome of the diplomonad parasite giardia lamblia, have secondarily lost energy generating pathways and seem to retain only the fe-s cluster maturation function. all mitochondria are believed to share a single origin from an α-proteobacterial-like prokaryote, but a wide diversity of mitochondrial genome architectures have evolved subsequent to the diversification of modern-day eukaryotes. for example, whereas "derived" animals possess monomeric circular mitochondrial genomes, an observation which led to the initial assumption that mtdnas are primarily circular, many other mitochondrial genomes, such as that of the ciliate tetrahymena pyriformis, the green alga chlamydomonas reinhardtii and the cnidarian metazoan aurelia aurita are linear. in addition, while some fungi and many plants have circular-mapping mtdnas, their mitochondria actually contain predominantly linear mtdna molecules with combinations of monomers and concatemers, with only a minor fraction of the molecules present in a circular form. a more extreme example is the mtdna of kinetoplastids, which consists of one maxi- and many different mini-circles that are interconnected to form an extensive network. mitochondrial gene content is also highly variable; the mtdna of the jakobid flagellate reclinomonas americana encodes genes, the largest set of mitochondrial genes currently known, whereas the mtdna of the malaria parasite plasmodium falciparum contains just protein coding genes and highly fragmented small and large subunit ribosomal rna genes. the most highly derived forms of mitochondria, such as the hydrogenosome of trichomonas vaginalis and the giardia lamblia mitosome, have lost their genomes entirely. mitochondria are also known as sites of unusual molecular biology and biochemistry. marande and burger recently showed that the mtdna genes of the euglenid diplonema papillatum are fragmented into as many as nine modules, each residing on a distinct or kbp chromosome. the mechanism by which these fragmented gene pieces are linked together to form contiguous transcripts is unknown. extensive mrna editing is another example of the bizarre molecular biology of mitochondria. kinetoplastid mitochondrial mrnas are subject to insertions and deletions of uridylate residues, sometimes > such insertions/deletions per transcript. mitochondrial mrna editing is also widespread in land plants and dinoflagellates. for example, ~2% of the cox and cob gene sequences in three dinoflagellate species investigated by lin et al. were edited at the mrna level. we are studying the genomic diversity and evolution of cryptophytes, a ubiquitous and ecologically significant group of single-celled eukaryotes found in freshwater and marine environments. most cryptophytes, except for members of the genus goniomonas, harbor plastids of secondary endosymbiotic origin. a variety of shared morphological features, such as the presence of ejectisomes, flat mitochondrial cristae, and an anterior depression, support the monophyly of cryptophytes, as do molecular phylogenetic data. one unique feature of cryptophyte plastids that distinguishes them from other plastids of red algal origin is the retention of the remnant nucleus of the red algal endosymbiont, referred to as the nucleomorph. consequently, most cryptophytes harbor four distinct genomes–nuclear, nucleomorph, mitochondrial, and plastid genomes–contained in separate compartments. cryptophytes are thus an interesting model system with which to study endosymbiotic gene transfer, genome evolution, and protein targeting. in this study, we report the complete mitochondrial genome sequence of the newly described cryptophyte species hemiselmis andersenii ccmp and compare it to the only other cryptophyte mitochondrial genome described thus far, that of rhodomonas salina. in addition, individual and concatenated mitochondrial protein coding gene sequences were analyzed to infer the phylogenetic relationships of cryptophytes to other eukaryotes. methods dna preparation, sequencing, and genome assembly hemiselmis andersenii mtdna was isolated and sequenced to ~10× coverage as described in lane et al.. about end sequences were screened for quality and vector contamination with pregap and automatically assembled using gap version in the staden package. complete automated assembly of a large intergenic space between trns and cox was unsuccessful due to the highly repetitive nature of this region. in an attempt to manually resolve this area, short unique sequences within the trns and cox genes were used to probe the sequence database for reads that extended from these two loci into the repeat region. these sequences were extracted and manually aligned using macclade version. sequences at the ends of the new constructs were then selected and the process was repeated. however, due to the presence of multiple identical copies of a > bp repeat, the assembly of a single unambiguous contig was not possible. when all available sequence reads were considered, three robust contigs were produced, each ending with similar repetitive sequences consisting of a ~ bp repeat unit. these three contigs were joined to circularize the map. the complete h. andersenii mtdna has been submitted to genbank under the following accession number: eu dna secondary structure within the repeat region was predicted using mfold version at a folding temperature of 37°c and the ionic conditions of m and m. genome size/structure determination we used pulsed-field gel electrophoresis to obtain an independent size estimate of the h. andersenii mitochondrial genome. hemiselmis andersenii total dna plugs were prepared as described in lane et al. and digested overnight with the restriction enzymes psti or bglii. based on the genome sequence, these enzymes were predicted to cut the mtdna only once or twice. both untreated and enzyme-digested h. andersenii dna plugs were run on a 1% agarose gel in × tbe buffer at °c for h at a voltage of v/cm with a switch time between 1– s using a chef-dr iii pulsed-field gel electrophoresis system. dna on the pulsed-field gel was transferred to a nylon membrane. southern hybridization using a ~ bp coxi probe as in lane and archibald revealed that undigested mitochondrial dna molecules were trapped in the wells or found in the'compression zone'. the psti or bglii endonuclease treated dna plugs revealed mitochondrial molecules in a discrete band below the'compression zone'. the corresponding bands could not be visualized on the ethidium-bromide stained pulsed-field gel image because of nuclear and nucleomorph dna smears in the background. in order to visualize mtdna on the pulsed-field gel, an initial pfge run was used to remove the linear nuclear and nucleomorph chromosomes from the pfge plugs. these plugs, which still contained organellar dna, were subsequently removed from the gel and digested with the restriction enzymes psti and bglii. digested plugs were then inserted into a fresh gel and electrophoresed under the conditions described above. the kbp and lambda chef dna size standard were used to estimate the size of the enzymatically linearized h. andersenii mtdna. genome annotation and gc content/skew analyses annotation of the h. andersenii mtdna and the gc content and skew analyses were performed in artemis version. gene identification was carried out using blastx and blastn. small and large ribosomal rrna subunit genes were identified by comparison to rrna gene sequences in the mitochondrial genome of rhodomonas salina. transfer rnas were identified using trnascan-se version. genome rearrangements between the two cryptophyte mtdna the extent to which the h. andersenii and r. salina mitochondrial genomes are rearranged to each other was estimated using grimm. each genome was designated as a sequence of units, which include a repeat region and genes common between the two cryptophyte mtdnas. rt-pcr of'trnk' trnascan-se version identified a putative intron of ~ bp in the anticodon loop of the h. andersenii trnk gene. to determine whether this prediction was correct, we performed rt-pcr using lysine-trna-specific primer pairs and h. andersenii total rna provided by h. khan. to eliminate dna contamination, μl of total rna was incubated for min with rq rnase-free dnase. rt-pcr was performed using the qiagen one-step rt-pcr kit and with control reactions in which the reverse-transcription process was skipped. the following two pairs of primers were used: 1) the forward primer 5'-gaaggttgctcgaatggaa-3' with the reverse primer 5'-gaaggtataggaattgaacctattc-3' 2) and the forward primer 5'-gcccagaaggttgctc-3' with the reverse primer 5'-aagaaggtataggaattgaacctat-3'. rt-pcr was performed with the reverse transcription step for min at 50°c and the subsequent inactivation of reverse transcriptase and activation of hotstart taq dna polymerase for min at 95°c, followed by cycles at 94°c for min, 47°c for min, and 72°c for min, and a final extension at 72°c for min. the amplified pcr fragments were cloned into pcr4-topo vector in the topo ta cloning kit for sequencing. between and bacterial colonies from each reaction were selected for sequencing on a beckman coulter ceq. molecular phylogenetic analysis from the protein-coding genes found in the h. andersenii mtdna, were selected for phylogenetic analyses. eleven genes were excluded because their sequences were poorly conserved and/or were only present in a few taxonomic groups. h. andersenii protein sequences were aligned with their homologs from other mitochondrial genomes available from genbank. amino acid sequences were aligned using macclade version and ambiguously aligned sites were manually removed. in addition to individual protein analyses, a concatenated protein data set containing proteins was analyzed. to include the maximum number of gene sequences, we combined protein-coding gene sequences encoded in mitochondrial genomes across diverse eukaryotic taxa. as most mitochondrial genomes do not possess all protein-coding genes selected for analysis, as many as protein gene sequences were missing per taxon. a maximum likelihood tree was produced using raxml-vi-hpc version. with the protomixjtt model of sequence evolution and the automatic tree rearrangement setting, and from distinct randomized maximum parsimony starting trees. bootstrap analysis was based on re-samplings.  general features of hemiselmis andersenii mtdna the mitochondrial dna of the cryptophyte hemiselmis andersenii ccmp was sequenced, assembled and manually edited to produce a circular-mapping genome bp in size. genome assembly was complicated by the presence of a highly repetitive non-coding region of ~ kbp; genome size was thus verified using pulsed-field gel electrophoresis. several observations suggest that the h. andersenii mtdna exists primarily in a linear-branched form comprised of multiple genome units. in pfge, the h. andersenii mtdna remains in the well or migrates within the'compression zone', which contains primarily linear nuclear and nucleomorph chromosomes larger than ~ kbp. the lack of mtdna below the'compression zone' suggests that the h. andersenii mtdna is not composed of linear monomers or dimers. furthermore, when the h. andersenii mtdna is partially digested with psti, an enzyme predicted to cut the genome only once, it produces a discrete band of ~ kbp in size but not a band ~ kbp in size, which would correspond to a dimeric linear form of the genome. this result indicates that the h. andersenii mtdna is not composed of circular concatemers or linear head-to-tail concatemers consisting of three or more genomic units. therefore, we suggest that the h. andersenii mtdna exists primarily as a branched linear molecule although monomeric circles may also exist. further studies using transmission electron microscopy or the'moving picture' technique will be necessary to confirm this hypothesis. the h. andersenii mitochondrial genome is comprised of a gene-rich region ~ kbp in size and a large intergenic region between trns and cox with complex repeats. the intergenic region accounts for % of the entire genome and % of the total amount of non-coding dna. the overall gc content of the genome is %, slightly higher than that of the nucleomorph genome of this organism. interestingly, a ~ bp region near the start of the coding portion of the genome is very gc-rich and is followed by a 100% at-containing region ~ bp in size. this unusual stretch of sequence is about bp from a palindromic sequence that is predicted to form a type ii stem-loop, and may be involved in regulating replication or transcription. the h. andersenii mitochondrial genome encodes genes with predicted functions and hypothetical protein-coding genes, a total somewhat higher than the average for eukaryotes. ten genes–orf orf rps rps nad rps tatc,'trnk', rps and rps7–overlap by up to bp, emphasizing the extreme compactness of the coding portion of the genome. the genome encodes small and large rrna subunit genes and trnas, one of which may be a pseudogene. of the identifiable protein-coding genes, encode ribosomal proteins, are involved in oxidative phosphorylation, and one gene encodes a membrane translocase protein. comparison of the mtdna gene order in h. andersenii to other genomes reveals the presence of five gene clusters shared among distantly related protists: two ribosomal protein clusters and three nadh dehydrogenase clusters. these gene clusters have been suggested to represent vestiges of bacterial operons. interestingly, all genes in the h. andersenii mitochondrial genome are encoded on the same strand. while the evolution of such an arrangement seems improbable, absolute strand polarity has been observed in the mitochondrial genomes of diverse eukaryotes such as the amoeba acanthamoeba castellanii, the fungus penicillium marneffei, and the green alga chlamydomonas eugametos. in addition, strikingly similar mtdna architectures–gene-dense regions, a single large repetitive intergenic region, and all genes encoded on one strand–are seen in diverse protists such as the stramenopile thraustochytrium aureum and the green alga pedinomonas minor. understanding the biological significance of such convergence at the level of genome architecture will require comparative molecular and biochemical studies of mitochondria in these organisms. comparison of the mitochondrial genomes of hemiselmis andersenii and rhodomonas salina h. andersenii is only the second cryptophyte, after r. salina, for which a mitochondrial genome has been completely sequenced and annotated. comparative analyses of the two genomes revealed a number of similarities. both genomes feature a compact gene arrangement and a single large repeat region, although the size of the large intergenic region in h. andersenii is more than four times as large as that of r. salina. all of the predicted protein-coding genes in the h. andersenii mitochondrial genome are present in the r. salina mtdna. four r. salina mitochondrion-encoded genes–rps atp tata, and sdh4–are not found in h. andersenii, although two open reading frames, orf and orf in the h. andersenii mtdna show marginal sequence similarity to the r. salina tata and sdh genes, respectively. additionally, while two group ii introns are present in r. salina mtdna, the h. andersenii mtdna is devoid of introns. with respect to conservation of gene order, % of the shared genes between the two cryptophyte mitochondrial genomes, rrna genes) are present in thirteen syntenic blocks, each consisting of 2– genes. these include: 1) cox1-cob-nad 2) nad4l-nad 3) atp1-trnp, 4) rps8-rpl6-rps13-rps 5) trnc-atp 6) trni-trnq-trnr-trne-trnw-nad10-nad 7) nad4-nad 8) trnr-trng, 9) trnmf-trns, 10) trny-trnl, 11) tatc-'trnk' /trns -nad 12)cox3-rps12-rps7-rps and 13) rps3-rpl16-rpl14-rpl5-rps as noted earlier, some of the conserved gene clusters, such as nad4l-nad are found in distantly related eukaryotes and appear to be vestiges of bacterial operons. analysis using grimm suggests that the observed difference in gene order between the two cryptophyte mitochondrial genomes can be explained by at least instances of genome reversal events. repeat structure of the h. andersenii mitochondrial genome the r. salina mtdna is characterized by a pair of ~ kbp inverted repeats that are joined by bp of sequence. in contrast, repeats in the h. andersenii mitochondrial genome are not inverted, but are instead dispersed or arranged in tandem throughout the large non-coding region, with individual repeat units ranging from to bp and occurring up to times. given that r. salina and h. andersenii are distantly related to one another, the large repeat region presumably arose during or prior to the early diversification of cryptophytes. while there is no obvious sequence similarity between the two repeat regions, both contain multiple copies of palindromic sequences, which are predicted to form stable stem-loop dna structures. in h. andersenii, two types of stem-loop structures were identified–i and ii–using the dna mfold program. the type i structure has two slight variations, i-a and i-b, which occur and times, respectively. type i-a and i-b structures have and base pairings in their stems, respectively, and occur adjacent to tandem repeats. one copy of the type ii stem-loop structure is located within a ~ bp segment that is devoid of any discernable repeat units, but close to the high and low gc regions noted earlier. as was suggested for r. salina by hauth et al., tandem repeats and multiple stem-loop structures in h. andersenii mtdna might be involved in the regulation of transcription and replication, a hypothesis that needs to be tested further. hauth et al. demonstrated that the repeat region of the r. salina mtdna roughly coincides with a change in the direction of'cumulative gc skew' and suggested that the repeat corresponds to the origin of replication. we investigated the gc skew in the h. andersenii mitochondrial genome to see whether a similar pattern exists. unlike r. salina, however, the h. andersenii gc skew does not change direction near the repeat region. instead, in both the h. andersenii and r. salina mtdna, observed gc skew patterns strongly correlate with transcriptional orientations, where the coding strand tends to be g-rich. therefore, the gc skew patterns of the two cryptophyte mitochondrial genomes do not seem to be the result of replication-associated mutational bias, but rather the non-random distribution of the protein coding genes, as has been observed in some other genomes. nevertheless, based on the presence of other features such as stem-loop structures, it seems reasonable to assume that the repeat region in both cryptophyte mitochondrial genomes corresponds to the origin of replication. codon usage and transfer rnas the h. andersenii mtdna encodes trnas, of which are predicted to form standard cloverleaf secondary structures. one trna gene,'trnk', shows atypical structure in the anticodon loop and the variable region, and is probably a pseudogene. allowing for wobble pairings and some base modifications, trnas are the theoretical minimum required to cover all codons in bacteria. for some mitochondria, even smaller sets of trnas, as few as 22– are possible by adopting several additional strategies. the h. andersenii mitochondrial genome lacks only one trna gene, trnk, which is minimally required in order to recognize all codons. it is thus predicted that nuclear-encoded cytosolic lys-trna is imported into h. andersenii mitochondria. mitochondrial trna import has been demonstrated in apicomplexans and trypanosomatids where trna genes are completely missing in their mitochondrial genomes, as well as in ciliates and plants where mitochondrial genomes encode fewer than the 22– minimally required trna genes. although most animals and some fungi do not import trnas into mitochondria, the fungus saccharomyces cerevisiae has been shown to import one specific cytosolic trna even though its mitochondrial genome encodes the full complement of trnas. analyses of the trna repertoire of mitochondrial genomes suggest that a number of other protist taxa across the eukaryotic tree also import one or more trnas into their mitochondria. it is thus reasonable to assume that h. andersenii imports at least lys-trna, although it is possible that trna editing makes up for the lys-trna deficit by changing the identity of an existing trna, as has been shown in marsupials. the frequency of each codon is shown and dots indicate the presence of a corresponding trna gene in the mtdna. †:'trnk' identified in the genome may be a pseudogene. another possible mechanism to account for the missing trna is that the structurally abnormal'trnk' gene forms a functional lys-trna to decode the codons aaa and aag. several cases of atypically-structured trnas are known from animal and ciliate mitochondria. interestingly, trnascan-se predicted the existence of a bp intron within the h. andersenii'trnk', and we conducted further experiments to test whether this is indeed the case. rt-pcr experiments using primer sets specific for'trnk' indicated that the putative intron was not removed in the mature trna. this results is not unexpected, given that the 20-bp putative intron is too short to be a self-splicing group i or ii intron, which are the only known types of introns reported in mitochondrial genomes. sequencing of ~ clones also did not reveal any evidence for rna editing within the'trnk'. these results suggest that if'trnk' is indeed expressed to form a functional lys-trna, it is predicted to have an unusually au-rich stem in the codon loop and a long variable region, atypical for lys-trna. long variable regions ranging from to nucleotides are generally restricted to trna-leu, trna-ser, and bacterial trna-tyr. the d- and t-loops of the'trnk' sequence show sequence similarity to one of the two mitochondrion-encoded trna-ser genes, both of which have a long variable region. in addition, comparative analysis with the r. salina mtdna revealed genomic position conservation between the h. andersenii trns-like'trnk' gene and the trns gene of r. salina, flanked by the tatc and nad genes. the h. andersenii'trnk' and r. salina trns genes both overlap tatc by bp and bp, respectively. this strongly suggests that the h. andersenii'trnk' is indeed derived from an ancestral gene that encoded trna-ser, explaining the origin of its long variable region. the overlap between the h. andersenii'trnk' and tatc suggests that'trnk' may play a role in processing the 3' end of the tatc gene transcript. this hypothesis could explain why the'trnk' gene still remains in the genome and retains conserved secondary structure in the stem loop and d- and t-loops, even if it does not form a functional trna. comprehensive molecular and biochemical experimentation will be necessary to confirm or refute the existence of mitochondrial trna import in h. andersenii and the functionality of the unusual'trnk' gene. when the h. andersenii trna genes were compared to those of r. salina, homologous pairs of trnas were identified, leaving only four h. andersenii trna and three r. salina trna genes not unambiguously matched to each other. each of the trna pairs possess identical anticodons except for the h. andersenii'trnk' and r. salina trns pair, despite their common derivation. the trns of h. andersenii, having sequence homology to the'trnk', probably originated from a recent gene duplication event. of the three remaining h. andersenii trna genes that are unmatched in r. salina, two–trnl and trng–are redundant because trnl and trng can decode all of their respective four-codon families. these redundant copies might have been lost in an ancestor of r. salina after it diverged from h. andersenii. lastly, the h. andersenii trni is somewhat similar to the trnk of the r. salina and only marginally resembles the r. salina trni at the 3' end. it is possible that the h. andersenii trni originated through recombination between ancestral trni and trnk genes, which would explain the lack of an obvious trnk homolog in h. andersenii comparable to the r. salina trnk. substantial sequence divergence among the three genes, however, makes it difficult to accurately trace the origin of the trni and the loss of the original trnk gene in h. andersenii. on the other hand, the unusual trni gene reported from r. salina is not found in h. andersenii. it was suggested that the r. salina trni is derived from trnf through a recent gene duplication event. overall, the two cryptophyte mitochondrial genomes use similar trna sets to recognize codons. however, unlike h. andersenii, which may need to import at least trnk from cytosol, the r. salina mtdna does possess the minimal required set for trna autonomy. molecular phylogenetic analyses cryptophytes are a well-established eukaryotic lineage, supported by both molecular and morphological features. however, their relationship to other eukaryotic groups, particularly those containing plastids of secondary endosymbiotic origin, has been the subject of considerable debate. the cryptophyte plastid is the product of a secondary endosymbiosis involving a red algal cell, the same process which accounts for plastid origins in haptophytes, dinoflagellates, and stramenopiles. cavalier-smith suggested that plastids in these four algal lineages arose from a single secondary endosymbiosis in a common ancestor that these organisms shared, to the exclusion of other eukaryotic groups. however, this "chromalveolate" hypothesis is controversial. recent molecular studies have shown that the katablepharids, an enigmatic collection of plastid-less flagellates, are a sister group to cryptophytes, and large-scale concatenated analyses of nuclear genes suggest that cryptophytes and haptophytes are also related. to gain insight into the phylogenetic relationship of the cryptophytes h. andersenii and r. salina to other eukaryotes, and more specifically, to test the hypothesis that cryptophytes and haptophytes are related to one another, phylogenetic analyses of mitochondrial protein sequences were performed. unlike the cryptophyte plastid genome, in which several cases of lgt have recently been discovered, individual analyses of mitochondrial proteins did not reveal any obvious instances of lgt between prokaryotes and eukaryotes or within eukaryotes. however, the possibility of ancient lgts cannot be ruled out, as the backbones of individual protein phylogenies were generally very poorly supported. as expected, a close relationship between the two cryptophytes h. andersenii and r. salina was well supported in the mitochondrial protein phylogenies, with twenty of twenty-five individual protein phylogenies showing this relationship. five individual gene phylogenies–nad rpl rpl rps rps14–did not recover a h. andersenii-r. salina clade, although alternative topologies were not supported with >50% bootstrap support values. additionally, single protein phylogenies were not, for the most part, able to resolve the relationship of cryptophytes to other eukaryotes. the position of cryptophytes was highly variable from protein to protein and the group did not regularly associate with other taxonomic clades with >50% bootstrap support values, except for in the cob and nad gene trees, where cryptophytes branch with haptophytes and jakobids, respectively. we subsequently analyzed a set of concatenated proteins to assess the phylogenetic position of cryptophytes. in this analysis, the h. andersenii-r. salina clade received 100% bootstrap support. other well-established eukaryotic groups including opisthokonts, rhodophytes, stramenopiles, and viridiplantae, were also strongly recovered, but the relationships among major lineages were not. the jakobid reclinomonas branched as the sister group to the viridiplantae with moderate support, and malawimonas showed an affinity for these two groups in two of the three data sets, as was previously inferred from a concatenate of ten mitochondrial proteins. it is not clear whether the jabokid -viridiplantae affinity is a phylogenetic artifact or reflects the true evolutionary history of mitochondrial genes. though growing evidence supports a relationship between cryptophytes and haptophytes, our extensive mitochondrial protein analyses did not reveal this relationship with reasonable bootstrap support, other than in a single protein gene tree. in summary, while mitochondrial gene sequences are able to resolve some of the eukaryotic lineages determined using other markers, they are at present incapable of resolve the deepest branches of the eukaryotic tree using current phylogenetic methods and with the present level of taxon sampling.  we have sequenced the mitochondrial genome of the cryptophyte h. andersenii and compared it to that of the distantly related cryptophyte r. salina. our analyses reveal that both genomes are characterized by a gene dense region and a single large intergenic space that includes numerous repeats and palindromic sequences predicted to form stable dna stem and loop structures. despite the overall similarities in content and architecture between the two genomes, their modes of regulating dna replication and transcription seem to differ. unlike r. salina, all genes in the h. andersenii mtdna are located on the same strand, a relatively rare observation in mitochondrial genomes. phylogenic analysis of multiple mitochondrial gene sequences indicated a clear affiliation between the two cryptophytes but was not able to resolve the position of cryptophytes relative to other eukaryotic groups. authors' contributions ek participated in genome assembly, carried out genome analysis and drafted the manuscript. cel isolated h. andersenii dna and participated in the initial genome assembly. bac, ck, and sb performed the h. andersenii mitochondrial genome sequencing. jma coordinated the study and helped draft the manuscript. all authors read and approved the manuscript. 
mitochondrial protein coding gene phylogenies strongly support a variety of previously established eukaryotic groups, but fail to resolve the relationships among higher-order eukaryotic lineages. cryptophytes are unusual in that they possess four genomes–a host cell-derived nuclear and mitochondrial genome and an endosymbiont-derived plastid and'nucleomorph' genome. thus far, a single complete mitochondrial genome sequence has been determined for the cryptophyte rhodomonas salina. here, the second complete mitochondrial genome of the cryptophyte alga hemiselmis andersenii ccmp is presented. comparison of the h. andersenii and r. salina mitochondrial genomes reveals a number of cryptophyte-specific genomic features, most notably the presence of a large repeat-rich intergenic region. the h. andersenii mtdna shares a number of features in common with the genome of the cryptophyte rhodomonas salina, including general architecture, gene content, and the presence of a large repeat region. adjacent to these repeats are copies of palindromic sequences predicted to form stable dna stem-loop structures. the h. andersenii mtdna is bp in size and encodes structural rnas and protein-coding genes, all located on the same strand. however, the h. andersenii mtdna is devoid of inverted repeats and introns, which are present in r. salina. comparative analyses of the suite of trnas encoded in the two genomes reveal that the h. andersenii mtdna has lost or converted its original trnk gene and possesses a trns-derived'trnk', which appears unable to produce a functional trna. however, unlike r. salina, the h. andersenii mtdna does not possess introns and lacks a lys-trna, which is presumably imported from the cytosol.
 cryptophytes are an enigmatic group of unicellular eukaryotes with plastids derived by secondary endosymbiosis. cryptophytes are unusual in that they possess four genomes–a host cell-derived nuclear and mitochondrial genome and an endosymbiont-derived plastid and'nucleomorph' genome. the evolutionary origins of the host and endosymbiont components of cryptophyte algae are at present poorly understood. thus far, a single complete mitochondrial genome sequence has been determined for the cryptophyte rhodomonas salina. here, the second complete mitochondrial genome of the cryptophyte alga hemiselmis andersenii ccmp is presented.  the h. andersenii mtdna is bp in size and encodes structural rnas and protein-coding genes, all located on the same strand. a prominent feature of the genome is the presence of a ~ kbp long intergenic region comprised of numerous tandem and dispersed repeat units of between 22– bp. adjacent to these repeats are copies of palindromic sequences predicted to form stable dna stem-loop structures. one such stem-loop is located near a gc-rich and gc-poor region and may have a regulatory function in replication or transcription. the h. andersenii mtdna shares a number of features in common with the genome of the cryptophyte rhodomonas salina, including general architecture, gene content, and the presence of a large repeat region. however, the h. andersenii mtdna is devoid of inverted repeats and introns, which are present in r. salina. comparative analyses of the suite of trnas encoded in the two genomes reveal that the h. andersenii mtdna has lost or converted its original trnk gene and possesses a trns-derived'trnk', which appears unable to produce a functional trna. mitochondrial protein coding gene phylogenies strongly support a variety of previously established eukaryotic groups, but fail to resolve the relationships among higher-order eukaryotic lineages.  comparison of the h. andersenii and r. salina mitochondrial genomes reveals a number of cryptophyte-specific genomic features, most notably the presence of a large repeat-rich intergenic region. however, unlike r. salina, the h. andersenii mtdna does not possess introns and lacks a lys-trna, which is presumably imported from the cytosol. 